CN1264430A - Betaines as adjuvants to susceptibility testing and antimicrobial therapy - Google Patents

Abstract

The present invention is related to method and compositions for susceptibility testing of bacteria containing mycolic acid structures using betaine-like detergents, and inducing the susceptibility of such bacteria using the same.

Description

Be used as the trimethyl-glycine of the adjuvant of sensitivity test and antimicrobial therapy

Invention field

The present invention relates to be used to determine contain susceptibility combination of features thing and the method for the bacterium of mycolic acid structure.The compositions and methods of the invention especially can be used as adjuvant and are used for sensitivity test and antimicrobial therapy, relate to antibiotic test of beta-lactam family and treatment the most in particular.Background of invention

The patient's of treatment bacterial infection contemporary method comprises the microbiotic that screening is used for the treatment of.The most conventional treatment is the experience according to the doctor.Too simply, the doctor just selects a kind of it is believed that the common transmittable thing effective Broad spectrum antibiotics relevant with patient's symptom.Yet in some cases, the selection of appropriate therapeutic agent is based on sensitivity test.Sensitivity test by determine which kind of therapeutical agent have the high likelihood of successfully treating a kind of special pathogen and which kind of biocide most likely invalid instruct the practitioner.Sensitivity test is a kind of important instrument, especially when the patient is confirmed as having mycobacterial infections, when the patient suffers from tuberculosis (TB: caused by mycobacterium tuberculosis (Mycobacterium tuberculosis) complex body bacterium (MTB)), all the more so the most especially.Current federal government's suggestion comprises that all new cases to TB carry out sensitivity test (Tenover, people such as F.C., clinical microbiology magazine (Jour.Clin.Micro.) 31:767-770 (1993)).Sensitivity test is a kind of priceless instrument, and in fact all doctors rely on it constantly and come to select suitable antibiotic therapy for the patient.

The method of treatment infectation of bacteria is limited by the activity profile of specific compound.For example, not every bacterium is all to specific Antimicrobe compound sensitivity.Different types of bacterium can tolerate the antibiotic effect of different sorts.Generally speaking, the activity profile of certain antibiotics belongs to discrete group (for example, fungi is to bacterium, and perhaps gram-positive microorganism is to Gram-negative bacteria) with regard to the kind of the microorganism of its effect.

Microbiotic is brought into play its effect by disturbing the various kinds of cell function.For example, known different types of microbiotic disturbs that cell walls is synthetic, RNA/DNA is synthetic, the many aspects of dna replication dna or protein synthesis.Owing to multiple reason bacterium have resistance to different antibiosis.Quintiliani, R. wait the people, at Murray, P.R. " the clinical microbiology handbook (Manual of ClinicalMicrobiology) that waits the people to compile, ASM press, Washington has summarized several such resistance mechanisms in D.C. (1995) the 1308-1326 pages or leaves and has pointed out, microbiotic must at first enter cell, only in this way could bring into play its effect at action site.The basis of resistance may be because biological permeability, and perhaps the molecular configuration of action site is perhaps inconsistent or do not existed.In addition, medicament can be modified, destroys or be eliminated to bacterium.

Resistance can be inborn or obtain.Obtain not fidelity (for example point mutation) of inherent that resistance can pass through the acquisition of genetic material (for example transposable element) or pass through dna replication dna.As if mycobacterium have a kind of other resistance mechanism.Heifets, L.B. wait the people, at " drug susceptibility in the mycobacterial infections chemotherapy " (Drug Susceptibility in theChemotherapy of Mycobaterial Infections), Heifets, L.B. compile CRC press, Boston, MA (1991), the 13-57 page or leaf is categorized as different steps active growth or that be in dormancy with the subgroup of infectivity MTB cell.Dormancy is survived these subgroups during patient treatment.

The complex patterns of susceptibility makes the treatment mycobacterial infections further complicated.For example, although treating MTB effectively, common available vazadrine (INH) and/or pyrazinoic acid amide (PZA) infect, but the MAC strain isolated generally tolerates these medicines, and mycobacterium fortutitum (M.fortuitum) and Mycobacterium chelonei (M.chelonae) strain isolated tolerate all line antitubercular agents usually.

Tuberculosis is most popular transmissible disease in the world today, has infected the about 1/3rd of world population, about 1,700,000,000 people (Kochi, A., tuberculosis (Tubercle) 72:1-6 (1991)).In addition, compare the transmissible disease of other any single-shot, tuberculosis worldwide makes more people (annual about 300 ten thousand) (incidence and mortality weekly (Morbidity and Mortality WeeklyReport) 42:961-964 (1993)) that die.Most of TB cases are in developing country, yet, the multidrug resistance of MTB (MDR) bacterial strain (MDR-TB) has become a major issue (cohort that the World Health Organization (file WHO/TB/96.198) is dangerous, WHO tuberculosis epidemiological report (WHO Report on the Tuberculosis Epidemic 1996) (1996) in 1996) in the whole world.Stop the increase of MDR-TB unless take measures, otherwise tuberculosis become in developing country and the industrialized country that the history of the common cause of the death reappears will be inevitable.

Other mycobacterium such as mycobacterium avium (M.avium) complex body (MAC), mycobacterium paratuberculosis (M.paratuberculosis), mycobacterium buruli (M.ulcerans), Mycobacterium leprae (M.leprae), mycobacterium kansasii (M.kansasii) and mycobacterium fortutitum complex body also are that common pathogenic agent is (referring to Wayne, L.G., clinical microbiology summary (Clin.Micro.Rev.) 5:1-25 (1992) or Falkinham, J.O., clinical microbiology summary 9:177-215 (199) is about the summary of different mycobacterium pathogenic agent).MAC causes transmissible disease (Nightingale, people such as S.D., transmissible disease magazine (Jour.Infect.Dis.) 165:1082-1085 (1992)) in late period in AIDS patient's in all almost half.The World Health Organization estimates, the number of infected person immunodeficiency virus (HIV) will surpass 4,000 ten thousand (the global AIDS of the World Health Organization (file WHO/GPA/CNP/EVA/93.1) plans (1993)) till 2000.Mycobacterium paratuberculosis (subspecies of mycobacterium avium) causes johne's disease in ruminating animal.According to estimates, johne's disease makes the annual loss of U.S.'s farming industry (for example, milk and beef) above 1,500,000,000 dollars, this is because (Whitlock due to lower productivity and the reproductivity, R, the 3rd international johne's disease discussion proceedings, 514-522 page or leaf (1991); Whitlock, people such as R., the 89th U.S. animal health association annual meeting proceedings, 484-490 page or leaf (1985)).Mycobacterial diseases has expended a large amount of social costs.

Antibiotique composition kind the most frequently used up to now and best sign is a beta-lactam.Because these antibiotic deep-going and popularity, will have significant advantage with the ability of these pharmaceutical treatment mycobacterial infectionses.Limitedly successfully attempted the application (Chambers, people such as H.F., biocide chemotherapy (Antimicrob.Agents Chemo.) 39:2620-2624 (1995)) of beta-lactam in being used for treating the treatment plan of mycobacterial infections.The susceptibility that enlarges mycobacterium by the location resistance mechanism especially effectively has important potentiality in the treatment mycobacterial infections to the ability of the susceptibility of beta-lactam.

The present invention described herein has summarized novel method and the composition that mycobacterium susceptibility wherein can be characterized.These method and compositions change the susceptibility of these bacteriums, to strengthen the especially effectiveness of beta-lactam antibiotics of microbiotic.These method and compositions will make its part as effectively treatment be used for determining and/or treat this class and infect.Summary of the invention

Use Thornton WO 95/27076 and United States Patent (USP) 5 deriving from, 658, in the liquid culture bottle of the biological samples that 749 method is handled, in order to attempt to control the growth of undesirable polluted bacteria, the inventor adds third generation cephalosporin, ceftazidime (CAS in standard anti-microbial agents (being PANTA) ^No.72558-82-8).The inventor is surprised and be surprised to find that, when according to Thornton WO 95/27076 and United States Patent (USP) 5,658,749 use C ₁₈When-carboxylic CAB (CB-18) was used in combination as treatment agent and with this anti-microbial agents, liquid culture susceptibility significantly and shockingly reduced.In order to manage to keep Thornton WO 95/27076 and United States Patent (USP) 5,658, the advantage of 749 diagnostic sensitivities that provided, the inventor studies the reduction that characterizes and eliminate this liquid culture susceptibility.These researchs have produced class methods and composition, wherein can use the trimethyl-glycine sample compound of Thornton W0 95/27076 and United States Patent (USP) 5,658,749 change mycobacterium to microbiotic especially to the antibiotic susceptibility of beta-lactam family.These methods can be used for characterizing and change bacterium, especially mycobacterium, the susceptibility of mycobacterium tuberculosis complex body bacterium combating microorganisms compound the most in particular, and as the adjuvant of antimicrobial therapy.

The present invention further comprises a kind of composition that is used for sensitivity test, and said composition contains and one or more one or more microbiotic of trimethyl-glycine sample stain remover blended.

The present invention further comprises a kind of test kit that is used for determining microorganism susceptibility, and this test kit contains next-door neighbour or adjacent one or more trimethyl-glycine sample stain removers and one or more microbiotic.The accompanying drawing summary

Figure 1A and Figure 1B have summarized the experimental design that is used for characterizing mycobacterium tuberculosis isolate A TCC 27294 and the important growth/processing parameter of 571/573-BAL in the CB-18/12B/PANTA/caz culture systems.

Fig. 2 A-2H shows the growth curve when using experimental design shown in Figure 1A and Figure 1B to detect mycobacterium tuberculosis isolate A TCC 27294.Rhombus: PANTA; Square: P/caz.Fig. 2 A:ATCC 27294, L-J handles in damping fluid, and transferred species is in damping fluid; Fig. 2 B:ATCC 27294, L-J handles in damping fluid, and transferred species is in CB-18 (17 μ g/ml CB-18 final concentration); Fig. 2 C:ATCC 27294, L-J handles in CB-18 (383 μ g/ml), and transferred species is in damping fluid; Fig. 2 D:ATCC 27294, L-J handles in CB-18 (383 μ g/ml), and transferred species is in CB-18 (17 μ g/mlCB-18 final concentration); Fig. 2 E:ATCC 27294, the 7Hll-selectivity is handled in damping fluid, and transferred species is in damping fluid; Fig. 2 F:ATCC 27294, the 7H11-selectivity is handled in damping fluid, and transferred species is in CB-18 (17 μ g/ml CB-18 final concentration); Fig. 2 G:ATCC 27294, the 7H11-selectivity is handled in CB-18 (383 μ g/ml), and transferred species is in damping fluid; Fig. 2 H:ATCC 27294, the 7H11-selectivity is handled in CB-18 (383 μ g/ml), and transferred species is in CB-18 (17 μ g/ml CB-18 final concentration).

Fig. 3 A-3H shows the growth curve when using experimental design shown in Figure 1A and Figure 1B to detect mycobacterium tuberculosis strain isolated 571/573-BAL.Rhombus: PANTA; Square: P/caz.Fig. 3 A:571/573-BAL, L-J handles in damping fluid, and transferred species is in damping fluid; Fig. 3 B:571/573-BAL, L-J handles in damping fluid, and transferred species is in CB-18 (17 μ g/ml CB-18 final concentration); Fig. 3 C:571/573-BAL, L-J handles in CB-18 (383 μ g/m1), and transferred species is in damping fluid; Fig. 3 D:571/573-BAL, L-J handles in CB-18 (383 μ g/ml), and transferred species is in CB-18 (17 μ g/ml CB-18 final concentration); Fig. 3 E:571/573-BAL, the 7H11-selectivity is handled in damping fluid, and transferred species is in damping fluid; Fig. 3 F:571/573-BAL, the 7H11-selectivity is handled in damping fluid, and transferred species is in CB-18 (17 μ g/ml CB-18 final concentration); Fig. 3 G:571/573-BAL, the 7H11-selectivity is handled in CB-18 (383 μ g/m1), and transferred species is in damping fluid; Fig. 3 H:571/573-BAL, the 7H11-selectivity is handled in CB-18 (383 μ g/ml), and transferred species is in CB-18 (17 μ g/ml CB-18 final concentration).

Fig. 4 has summarized the experimental design that is used for lower concentration inoculum transferred species in Yelkin TTS.This design is used for checking in the Yelkin TTS and the ability of CB-18 effect.CB-18 effect when the different vaccination body is also checked in this experimental design.

Fig. 5 A-5F shows the growth curve when using experimental design shown in Figure 4 to detect mycobacterium tuberculosis strain isolated 571/573-BAL.Fig. 5 A and Fig. 5 B detect the bacterium stoste (about 165 ± 54cfu) of 5,000 times of dilutions.Fig. 5 C and Fig. 5 D detect the bacterium stoste of 25,000 times of dilutions, and (about 33 ± 11cfu), Fig. 5 E and Fig. 5 F detect the bacterium stoste (about 8 ± 3cfu) of 100,000 times of dilutions.Rhombus: the damping fluid among the R.F.; Square: the damping fluid among the P/caz; Trilateral: the Yelkin TTS among the R.F.; " x ": the Yelkin TTS among the P/caz.Fig. 5 A: transferred species is in damping fluid or damping fluid/Yelkin TTS; Fig. 5 B: transferred species is in CB-18 (17 μ g/ml) or CB-18/ Yelkin TTS (17 μ g/ml@); Fig. 5 C: transferred species is in damping fluid or damping fluid/Yelkin TTS; Fig. 5 D: transferred species is in CB-18 (17 μ g/ml) or CB-18/ Yelkin TTS (17 μ g/ml@); Fig. 5 E: transferred species is in damping fluid or damping fluid/Yelkin TTS; Fig. 5 F: transferred species is in CB-18 (17 μ g/ml) or CB-18/ Yelkin TTS (17 μ g/ml@).

Fig. 6 has summarized the experimental design of CB-18 and TMA-18 titration experiments, and it is used for the effect of comparison CB-18 and the effect of quaternary ammonium salt bromination trimethylammonium octadecane ammonium (TMA-18).

Fig. 7 A-7C shows the growth curve when using experimental design shown in Figure 6 to detect mycobacterium tuberculosis isolate A TCC27294.Rhombus: R.F.; Square: PANTA; Trilateral: P/caz.Fig. 7 A:7H11-selects, and transferred species is in damping fluid.Fig. 7 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).Fig. 7 C:7H11-selects, and transferred species is in TMA-18 (3.4 μ g/ml final concentration).

Fig. 8 A-8C shows the growth curve when using experimental design shown in Figure 6 to detect mycobacterium tuberculosis strain isolated 571/573-BAL.Rhombus: R.F.; Square: PANTA; Trilateral: P/caz.Fig. 8 A:7H11-selects, and transferred species is in damping fluid.Fig. 8 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).Fig. 8 C:7H11-selects, and transferred species is in TMA-18 (3.4 μ g/ml final concentration).

Fig. 9 has summarized and has been used for the titrating experimental design of CB-18, the CB-18 effect when its application table 7 listed different mycobacterium kinds are checked different CB-18 concentration.

Figure 10 A and Figure 10 B show the growth curve when using CB-18 titration experiments shown in Figure 9 to detect mycobacterium tuberculosis isolate A TCC 27294.Rhombus: R.F.; Square: P-cax; Trilateral: 3 μ g/ml CB-18; " x ": 7 μ g/m1 CB-18; " * ": 13 μ g/ml CB-18; Circular: 27 μ g/ml CB-18; Vertical hachure: 54 μ g/ml CB-18; Horizontal dotted line: 109 μ g/ml CB-18.Titration when Figure 10 A, no P-cax.Figure 10 B, every kind of titration in the presence of P-cax, carrying out.

Figure 11 A and Figure 11 B show the growth curve when using CB-18 titration experiments shown in Figure 9 to detect mycobacterium tuberculosis strain isolated 571/573-BAL.Rhombus: R.F.; Square: P-cax; Trilateral: 3 μ g/ml CB-18; " x ": 7 μ g/ml CB-18; " * ": 13 μ g/ml CB-18; Circular: 27 μ g/ml CB-18; Vertical hachure: 54 μ g/ml CB-18; Horizontal dotted line: 109 μ g/ml CB-18.Titration when Figure 11 A, no P-cax.Figure 11 B, every kind of titration in the presence of P-cax, carrying out.

Figure 12 A and Figure 12 B show the growth curve when using CB-18 titration experiments shown in Figure 9 to detect mycobacterium avium isolate A TCC 25291.Rhombus: R.F.; Square: P-caz; Trilateral: 7 μ g/ml CB-18; " x ": 13 μ g/ml CB-18; " * ": 27 μ g/ml CB-18.Titration when Figure 12 A, no P-caz.Figure 12 B, every kind of titration in the presence of P-caz, carrying out.

Figure 13 A and Figure 13 B show the growth curve when using CB-18 titration experiments shown in Figure 9 to detect mycobacterium avium complex body strain isolated 802-BAL.Rhombus: R.F.; Square: P-caz; Trilateral: 7 μ g/ml CB-18; " x ": 13 μ g/ml CB-18; " * ": 27 μ g/ml CB-18.Titration when Figure 13 A, no P-caz.Figure 13 B, every kind of titration in the presence of P-caz, carrying out.

Figure 14 A and Figure 14 B show the growth curve when using CB-18 titration experiments shown in Figure 9 to detect mycobacterium fortutitum isolate A TCC 6841.Rhombus: R.F.; Square: P-cft; Trilateral: 7 μ g/ml CB-18; " x ": 13 μ g/ml CB-18; " * ": 27 μ g/ml CB-18; Circular: 54 μ g/ml CB-18; Vertical hachure: 109 μ g/ml CB-18.Titration when Figure 14 A, no P-cft.Figure 14 B, every kind of titration in the presence of P-cft, carrying out.

Figure 15 A and Figure 15 B show the growth curve when using CB-18 titration experiments shown in Figure 9 to detect mycobacterium fortutitum complex body strain isolated 495-JHH.Rhombus: R.F.; Square: P-cft; Trilateral: 7 μ g/ml CB-18; " x ": 13 μ g/ml CB-18; " * ": 27 μ g/ml CB-18; Circular: 54 μ g/ml CB-18; Vertical hachure: 109 μ g/ml CB-18.Titration when Figure 15 A, no P-cft.Figure 15 B, every kind of titration in the presence of P-cft, carrying out.

Figure 16 has summarized the MTB strain isolated to the design of microbiotic screening experiment, and it is used for checking the CB-18 effect of using different microbiotic his-and-hers watches 8 listed mycobacterium tuberculosis strain isolateds.

Figure 17 A and Figure 17 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis isolate A TCC 27294.Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 17 A:7H11-selects, and transferred species is in damping fluid.Figure 17 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 18 A and Figure 18 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 571-BAL.Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 18 A:7H11-selects, and transferred species is in damping fluid.Figure 18 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 19 A and Figure 19 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 573-BAL.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 19 A:7H11-selects, and transferred species is in damping fluid.Figure 19 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 20 A and Figure 20 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 535-BAL.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 20 A:7H11-selects, and transferred species is in damping fluid.Figure 20 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 21 A and Figure 21 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 896-BAL.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 21 A:7H11-selects, and transferred species is in damping fluid.Figure 21 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 22 A and Figure 22 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 040-TBR.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 22 A:7H11-selects, and transferred species is in damping fluid.Figure 22 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 23 A and Figure 23 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 061-TBR.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 23 A:7H11-selects, and transferred species is in damping fluid.Figure 23 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 24 A and Figure 24 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 512-JHH.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 24 A:7H11-selects, and transferred species is in damping fluid.Figure 24 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 25 A and Figure 25 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 538-JHH.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 25 A:7H11-selects, and transferred species is in damping fluid.Figure 25 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 26 A and Figure 26 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 52-96-BOL.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 26 A:7H11-selects, and transferred species is in damping fluid.Figure 26 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 27 A and Figure 27 B show the growth curve when using antibiotic-screening experiment shown in Figure 16 to detect mycobacterium tuberculosis strain isolated 57-96-BOL.Rhombus: R.F.; Square: PANTA; Trilateral: P-caz; " x ": P/cfp; " * ": P/cax; Circular: P/cft.Figure 27 A:7H11-selects, and transferred species is in damping fluid.Figure 27 B:7H11-selects, and transferred species is in CB-18 (17 μ g/ml final concentration).

Figure 28 A and Figure 28 B show the growth curve when the improvement project of using antibiotic-screening experiment shown in Figure 16 detects mycobacterium tuberculosis strain isolated 572/573-BAL.In these experiments, detected other non--beta-lactam antibiotics.Rhombus: R.F.; Square: P-caz; Trilateral: erythromycin; " x ": ceftriaxone.Figure 28 A: antibiotic-screening, transferred species is in R.F..Figure 28 B: antibiotic-screening, transferred species is in CB-18.

Figure 29 A and Figure 29 B show the growth curve when the improvement project of using antibiotic-screening experiment shown in Figure 16 detects mycobacterium tuberculosis strain isolated 061-TBR.In these experiments, detected other non--beta-lactam antibiotics.Rhombus: R.F.; Square: P-caz; Trilateral: PXB; " x ": romicil; " * " lincomycin; Circular: nalidixic acid; Vertical hachure: penicillin G; Horizontal dotted line: ceftriaxone.Figure 29 A: antibiotic-screening, transferred species are in R.F., and Figure 29 B: antibiotic-screening, transferred species is in CB-18.

Figure 30 A and Figure 30 B show the growth curve when the improvement project of using antibiotic-screening experiment shown in Figure 16 detects mycobacterium tuberculosis strain isolated 57-96-BOL.In these experiments, detected other non--beta-lactam antibiotics.Rhombus: R.F.; Square: P-caz; Trilateral: nalidixic acid; " x ": penicillin G; " * ": ceftazidime; Circular: ceftriaxone.Figure 30 A: antibiotic-screening, transferred species are in R.F., and Figure 30 B: antibiotic-screening, transferred species is in CB-18.

Figure 31 has summarized stain remover screening experiment design, and it is used for just being similar to the ability that causes induced sensitivity of CB-18, several trimethyl-glycine sample stain removers that screening table 10 is listed and other stain remover.

Figure 32 A is presented at the growth curve of trooping of all contrasts of using in the described experiment of Figure 31, and Figure 32 B shows the growth curve of trooping of selected several stain removers, with outstanding result's diversity.Figure 32 A: stain remover contrast: rhombus, damping fluid/R.F.; Square, damping fluid/PANTA; Trilateral, damping fluid/P-cax; Figure 32 B: selected stain remover: rhombus, tween 80; Square, stearic acid; Trilateral, detaine PB; " x ", SB-18; "=" crosultaine E30; Open loop shape, velvetex OLB; Closed-loop shaped, C ₁₈-propyloic trimethyl-glycine.

Figure 33 has summarized CB-18 to the EDTA experimental design, and it is used for the effect of CB-18 and the effect of EDTA in more herein the culture systems.

Figure 34 A and Figure 34 B are presented at the growth curve when detecting mycobacterium tuberculosis isolate A TCC27294 in the experiment of Figure 33.Figure 34 A: transferred species in shown in R.F., P/cax or 17 μ g/ml CB-18 in: rhombus, the damping fluid among the R.F.; Square: the damping fluid among the P/cax; Trilateral: the CB-18 among the R.F.; CB-18 among " x " P/cax.Figure 34 B: transferred species in shown in contain 85 μ g/ml MgCl ₂17 μ g/ml CB-18 in or among the 17 μ g/ml EDTA: rhombus, CB-18 among the R.F. and MgCl ₂Square: CB-18 among the P/cax and MgCl ₂Trilateral: the EDTA among the R-F.; EDTA among " x " P/cax.

Figure 35 A and Figure 35 B are presented at the growth curve when detecting mycobacterium tuberculosis strain isolated 571-573-BAL in the experiment of Figure 33.Figure 35 A: transferred species in shown in R.F., P/cax or 17 μ g/mlCB-18 in: rhombus, the damping fluid among the R.F.; Square: the damping fluid among the P/cax; Trilateral: the CB-18 among the R.F.; CB-18 among " x " P/cax.Figure 35 B: transferred species in shown in 17 μ g/mlCB-18 and 85 μ g/ml MgCl ₂Or 17 among the μ g/ml EDTA: rhombus, CB-18 among the R.F. and MgCl ₂Square: CB-18 among the P/cax and MgCl ₂Trilateral: the EDTA among the R.F.; EDTA among " x " P/cax.

Figure 36 has summarized the experimental design of mycobacterium sensitivity test, and it is used for the current practice of mycobacterium sensitivity test is associated with trimethyl-glycine sensitivity test of the present invention.

Figure 37 is presented at the growth curve when detecting mycobacterium tuberculosis isolate A TCC 27294 in the experiment of Figure 36.Figure 37 A shows that (detection is 6.28 ± 0.57 * 10 as inoculum with 0.5 MacFarland standard ⁵Cfu) the trimethyl-glycine susceptibility result the time.Figure 37 B, 37C, 37D and 37E show respectively with 10 * (62,800 ± 5,700 cfu), 100 * (6,280 ± 570 cfu), 1, the growth curve of the MacFarland standard of 000 * (628 ± 57 cfu) and 10,000 * (63 ± 6 cfu) dilution during as inoculum.Rhombus: R.F.; Square: P-cax; Trilateral: 15 μ g/ml CB-18; " x ": 30 μ g/ml CB-18; " * " 60 μ g/ml CB-18; Circular: the 15 μ g/mlCB-18 that contain P/cax; Vertical hachure: the 30 μ g/ml CB-18 that contain P/cax; Horizontal dotted line: the 60 μ g/ml CB-18 that contain P/cax.

Figure 38 is presented at the growth curve when detecting mycobacterium tuberculosis strain isolated 571/573-BAL in the experiment of Figure 36.Figure 38 A shows that (detection is 1.11 ± 0.18 * 10 as inoculum with 0.5 MacFarland standard ⁶Cfu) the trimethyl-glycine susceptibility result the time.Figure 38 B, 38C, 38D and 38E show respectively with 10 * (111,000 ± 17,900 cfu), 100 * (11,100+1,790 cfu), 1,000 * (1,110 ± 179 cfu) and the growth curve of the MacFarland standards of 10,000 * (111 ± 18 cfu) dilutions during as inoculum.Rhombus: R.F.; Square: P-cax; Trilateral: 15 μ g/mlCB-18; " x ": 30 μ g/ml CB-18; " * " 60 μ g/ml CB-18; Circular: the 15 μ g/ml CB-18 that contain P/cax; Vertical hachure: the 30 μ g/ml CB-18 that contain P/cax; Horizontal dotted line: the 60 μ g/ml CB-18 that contain P/cax.

Figure 39 (A, B, C).Figure 39 A shows as people such as Yuan Y., the described enzyme mechanism that is used for modifying mycolic acid of newspaper (Proc.Natl.Acad.Sci.) 93:12828-12833 (1996) of institute of NAS.Figure 39 B shows the mechanism that the sulfo-tetracosanoic acid works as the enzyme inhibitors of these same enzyme: the stable transition state analog of formation is the sulfonium carboxybetaine.Figure 39 C has summarized a kind of possible synthesis mechanism of this sulfonium carboxybetaine synthetic.DESCRIPTION OF THE PREFERRED

In the following description, widespread use the many terms that in pharmaceutical field and extracorporeal maleate sensibility test, use.For provide to this specification sheets and claims comprise these terms clear with the consistent understanding of given scope, following definition is provided.

" CB-18 effect " meaning be in the presence of trimethyl-glycine sample stain remover some bacterium especially mycobacterium to the raising of antibiotic susceptibility.The method that shows this effect is, in the presence of one or more microbiotic of level of significance, having or not one or more trimethyl-glycine sample stain removers especially under the situation of CB-18, a kind of microorganism of vitro culture, the extract alive that for example a kind of infective agent or clinical separation strain or its can be grown.The existence of trimethyl-glycine sample stain remover improved some bacterium particularly mycobacterium to antibiotic susceptibility.Bacterial growth can be described with qualitative or quantitative mode.An example of qualitative results is to point out simply to grow or do not grow.An example of quantitative result is will cultivate fate as known in the art to compare with growth index.The example of growth index comprises simple classification symbol (for example, ", ±, 1+, 2+, 3+ and 4+ ") and numeral indication (for example 0-999), is produced as BACTEC 12B culture systems (Becton Dickinson, Cockeysville, MD, the U.S.).The CB-18 effect can show with the termination fully of growth, perhaps the total growth of self is not had influence and represent, can be expressed as known in the art reduction (being the decline of growth velocity) yet at inhibition, delay or the growth curve slope of the exponential phase of growth.The importance of CB-18 effect is to have observed variation in one or more growth characteristics of microorganism such as infective agent or clinical separation strain, and this variation shows in the process of trimethyl-glycine sensitivity test of the present invention.Embodiment 10 illustrations this point, the different microbiotic with the combination of multiple trimethyl-glycine sample stain remover wherein are provided.Observed variation improves consistent with strain isolated to the antibiotic susceptibility that exists.

" trimethyl-glycine sensitivity test " meaning is, for a kind of model of the antibiotics sensitivity of the mixture of setting up microorganism (for example infective agent or clinical separation strain) or different microorganisms, with one or more trimethyl-glycine sample stain remover application in vitro tests of one or more microbiotic combinations.In the trimethyl-glycine sensitivity test, make contain microorganism for example all the sample of type/variant/mixtures such as strain isolated of the microorganism of a group or microorganism contact with the composition that contains a kind of microbiotic and a kind of trimethyl-glycine sample stain remover, and according to microorganism in the viability mensuration sample of microorganism in the sample to this antibiotic susceptibility.The trimethyl-glycine sensitivity test is first kind of embodiment of the inventive method.As known in the art, this trimethyl-glycine sensitivity test can be at a kind of microtitration form, in culturing bottle or containing in the flat board of solid medium and finish.The example of standard liquid culture medium form comprise BACTEC (Becton-Dickinson, Cockeysville, MD), ESP Myco System II ^TM(DIFCO Laboratories, Detroit, MI) or MT/BacT ^TM(Organon Teknika, Durham, NC).The example of standard solid substratum comprises Mueller-Hinton agar known in the art or other suitable substratum.These are cultivated in the form must add the suitable microbiotic and the trimethyl-glycine sample stain remover of appropriate combination, proper concn as described here.The same with any sensitivity test, the purpose of this test is to determine to have the microbiotic of the high likelihood of successfully eliminating the patient infection.

As known in the art, microorganism is that " sensitivity " meaning is as clinical separation strain or infective agent, this microorganism (for example mycobacterium) is influenced nocuously by a kind of microbiotic, make so this clinical separation strain or infective agent anergy, non-infection maybe can not survive (Yao, people such as J.D.C. are in Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1281-1307 pages or leaves (being hereby incorporated by)).In this used " sensitivity " and " susceptibility " synonym.When definite a kind of microorganism such as clinical separation strain or infective agent are responsive to a kind of certain antibiotics, claim this microbiotic that this strain isolated or infective agent are had " activity " or be " active " it.

As known in the art, " sensitivity test " meaning is a kind of in vitro tests, take this to determine a kind of microorganism such as clinical separation strain or infective agent susceptibility (Jorgensen to a series of Antimicrobe compounds, J.H. wait the people, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1277-1280 pages or leaves; Woods, people such as G.L, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1327-1404 pages or leaves; Standard committee of National Laboratory, " for the method for the dilution anti-microbial sensitivity test of aerobic growth bacterium ", the 4th edition, the standard M7-A4 Villanova of permission, PA (1997); Standard committee of National Laboratory, " operative norm of antimicrobial dull and stereotyped sensitivity test ", sixth version, the standard M2-A6 Villanova of permission, PA (1997); Standard committee of National Laboratory, " development of extracorporeal maleate sensibility test standard and quality-controlling parameters ", the standard M23-A Villanova of permission, PA (1994); Standard committee of National Laboratory, " for the anti-mycobacterium sensitivity test of mycobacterium tuberculosis ", tentative standard M24-Tvillanova, PA (1995) (all being incorporated herein by reference) at this).The purpose of all sensitivity tests is all to predict more accurately that successful therapeutic gets involved.

As known in the art, " microbiotic " meaning is that viability, integrity, infectivity or irritability to infective agent known in the art any compound with detrimental action is (referring to Murray, P.R. wait the people to compile, " clinical microbiology handbook, ASM press, Washington, D.C. (1995), 1281-1307 page or leaf and 1385-1404 page or leaf; Kucers, people such as A., " antibiotic application " (The Use of Antibiotics) the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987); And Lorian, V. compiles, " laboratory medicine microbiotic " (Antibiotics inLaboratory Medicine) second edition, Williams ﹠amp; Wilkins, Baltimore, MD all is incorporated herein by reference at this).The antibiotic example of different sorts comprises: beta-lactam antibiotics, beta-lactamase inhibitor, aminoglycoside and aminocyclitol, quinolone, tsiklomitsin, macrolide and lincosamide (lincosamide), and glycopeptide, lipopeptid and polypeptide, sulfanilamide (SN) and trimethoprim, paraxin, vazadrine, nitroimidazole, Rifampin, nitrofuran, urotropine and mupirocin, it can be used in combination with method of the present invention all.Term microbiotic and " biocide ", " therapeutical agent " or " medicine " synonym when this uses.All microbiotic all are medicine or therapeutical agent, but not every medicine or therapeutical agent all are microbiotic.

" adjuvant " meaning is a kind of chemical compound, itself can have or perhaps not have antimicrobial acivity, but when with one or more microbiotic combinations (while), this compound synergy has strengthened these antibiotic effects.Adjuvant can be used to improve sensitivity test or antimicrobial therapy.An example of treatment adjuvant is beta-lactamase inhibitor (seeing below).Method and composition described herein is not to say the application of beta-lactamase inhibitor as adjuvant itself, but trimethyl-glycine sample stain remover is as the application of adjuvant; Yet trimethyl-glycine sample stain remover can use separately or comprise that with other adjuvant beta-lactamase inhibitor is used in combination.

Used " SB-18 sample " synonym in term " trimethyl-glycine sample " and WO 95/27076 and the United States Patent (USP) 5,658,749 all is incorporated herein by reference at these two pieces.Trimethyl-glycine sample stain remover according to WO 95/27076 and United States Patent (USP) 5,658,749 has the ability of disperseing mycobacterium rope (and piece) and/or compensation mycobacterium buoyancy.Become the mycobacterium such as biological being dispersed with of mycobacterium tuberculosis complex body (MTB) of rope to be beneficial to detection, this is because it has improved the possibility that the aliquots containig of surveying is represented whole sample.Can disperse the trimethyl-glycine sample stain remover of rope to have the alkyl chain length that surpasses 16 carbon atoms, the alkyl chain that contains 18-20 carbon atom is most preferred.Trimethyl-glycine sample stain remover also has the ability that mycobacterium is collected as the mycobacterium avium complex body (MAC) that does not become the piece growth that is beneficial to, and this is because it has compensated these biological buoyancy to a certain extent.This compensation preferably takes place by a kind of mechanism that comprises that stain remover moves in bacterial cell.The trimethyl-glycine sample stain remover of compensation buoyancy preferably has the alkyl chain length that surpasses 12 carbon atoms, most preferably is 16-20 carbon atom.Therefore, the table 2 and table 3 and the United States Patent (USP) 5 that comprise WO 95/27076 at this used " trimethyl-glycine sample ", 658, structure described in 749 all is incorporated herein by reference at these two pieces, comprising, for example, have the active CB sample of SB-18 sample, SB sample, HSB sample, PB sample, StB sample, PhB sample, SoB sample, RevB sample, AO sample, cAB sample and an ImB sample compound as WO 95/27076 and United States Patent (USP) 5,658,749 are described." trimethyl-glycine sample " stain remover comprises the zwitterionic compound of structure shown in the table 1.These structures should be the most useful in the method for the invention.

Table 1: the most useful trimethyl-glycine sample stain remover has shown the universal architecture of the most useful trimethyl-glycine.R ₁Be the hydrophobic alkyl chain, α is for connecting R ₁" key " with positively charged ion β.R ₂And R ₃When needs, modify positively charged ion.R ₄For connecting this positively charged ion and negatively charged ion γ " bridge ".

??R 1	??C 8-C 22
		??α	??-CH 2-，-O-
??β	??-N -、-P -，-S -
		??R 2	??-H，-CH 3，-C 2H 5，-C 3H 2
??R 3	??-H，-CH 3，-C 2H 5，-C 3H 7
		??R 4	??-CH 2-，-C 2H 4-，C 3H 6-，-C 4H 8-，-C 5H 10-， ??-C 6H 12-，-CH 2-C 6H 4-，-C mH 2m-; M 〉=1 wherein
??γ	-SO 3 ，-OSO 3 ，-COO ，-OPO 3 ，-PO 3 ，-PO 2 -

" CB sample " means carboxylate-containing (COO ) partly as anionic trimethyl-glycine sample stain remover (for example carboxybetaine sample)." SB sample " means and contains sulfonate (SO ₃) partly as anionic trimethyl-glycine sample stain remover (for example sultaine sample)." HSB sample " means and contains the sulfonate part as (trimethyl-glycine sample stain remover OH) (for example hydroxy sulfo lycine sample) of hydroxyl in negatively charged ion and the bridge." PB sample " means phosphorous hydrochlorate (OPO ₃), phosphonate (PO ₃) or phosphinate (PO ₂) partly as anionic trimethyl-glycine sample stain remover (for example phosphoric acid betaine sample)." StB sample " means sulfur-bearing hydrochlorate (OSO ₃) partly as anionic trimethyl-glycine sample stain remover (for example sulfuric acid trimethyl-glycine sample)." AO sample " means oxycompound group (O ) as anionic trimethyl-glycine sample stain remover (for example amine oxide sample)." PhB sample " means Han Phosphonium (P -) partly as cationic trimethyl-glycine sample stain remover (Li such as Phosphonium trimethyl-glycine sample)." SoB sample " means and contains sulfonium (S -) partly as cationic trimethyl-glycine sample stain remover (for example sulfonium trimethyl-glycine sample)." positive alkyl betaine " means and contains ammonium (N -) partly as cationic trimethyl-glycine sample stain remover (for example positive alkyl betaine sample)." ImB sample " means and contains the tetrahydroglyoxaline part as cationic trimethyl-glycine sample stain remover (for example imidazolinium betaine sample)." RevB sample " means wherein covalently bound with negatively charged ion, relative with the positively charged ion trimethyl-glycine sample stain remover (for example reverse trimethyl-glycine sample) of alkyl chain." cAB sample " mean wherein alkyl chain covalently bound with bridge, with positively charged ion or the relative trimethyl-glycine sample stain remover (for example c-alkyl betaine sample) of negatively charged ion.

" CB-18 " means N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecylammonium, inner salt.CB-18 is also referred to as N, N-dimethyl-N-(Octadecane base)-N-(3-carboxylic propyl group) ammonium inner salt, or C ₁₈-carboxylic CAB.CB-18 is assigned to CAS ^No.78195-27-4.

" SB-18 " means N-octadecyl-N, N-dimethyl-3-ammonium-1-propyl group-sulfonate (CAS ^No.13177-41-8).

" SB-16 " means N-hexadecyl-N, N-dimethyl-3-ammonium-1-propyl group-sulfonate (CAS ^No.2281-11-0).

" SB-14 " means N-tetradecyl-N, N-dimethyl-3-ammonium-1-propyl group-sulfonate (CAS ^No.14933-09-6), " SB-12 " means N-dodecyl-N, N-dimethyl-3-ammonium-1-propyl group-sulfonate (CAS ^No.14933-08-5).

" clinical separation strain " meaning is the infectious bacterial isolates of causing of a kind of purifying, and this clinical separation strain derives from the patient who has infected this infective agent.One or more clinical separation strains can derive from identical patient, perhaps identical strain isolated can derive from different patients, being seen (Pittet, people such as D. during breaking out as hospital, Archives of Internal Medicine (Archives of InternalMedicine) 155:1177-1184, (1995)).These clinical separation strains generally come purifying by the combination of sample disposal and cultural method.These clinical separation strains are alive like this, therefore are used in the in vitro tests (as sensitivity test) with regard to it antibiotic susceptibility further to be analyzed and detected.The method of these clinical separation strains of purifying comprises methods known in the art and step, especially Kent, P.T. wait the people, " the public health mycobacterium is learned: III level lab guide ", U.S. sanitary and human service department, Center for Disease Control, Atlanta, GA (1985) 31-70 pages or leaves, and Nolte, F.S. wait the people,: Murray, " clinical microbiology handbook, ASM press that people such as P.R. compile, Washington, D.C. (1995) 400-437 pages or leaves, for the described method of the separation of mycobacterium, or Clarridge, J.E. wait the people,: Murray, " clinical microbiology handbook, ASM press that people such as P.R. compile, Washington, D.C. (1995) 357-378 pages or leaves, and Beaman, people such as B.L.,: Murray, P.R. " clinical microbiology handbook, ASM press, the Washington that wait the people to compile, D.C. (1995) 379-399 pages or leaves, the method for being summarized for Corynebacterium (Corynebacterium) and separating of Nocardia (Nocardia) (all being incorporated herein by reference) respectively at this.

" infective agent " meaning is a kind of infectious microorganism, a kind of infective bacterial especially known in the art.The infective agent of the method according to this invention institute special concern comprises that those contain the infective agent of mycolic acid structure, for example, can cause the mycobacterium of disease, the most in particular mycobacterium tuberculosis complex body bacterium (Isenberg, H.D. wait the people, in: Murray, people such as P.R. compile, " clinical microbiology handbook, ASM press, Washington, D.C.1995,5-18 page or leaf (being hereby incorporated by)).Suffer from the mankind of the disease that causes by this infective agent or animal patient and be called as and have " infection " that causes by this infective agent, or " infection has " this infective agent.The infective agent that causes disease be known as " morbific ".General not pathogenic and be known as saprophytic for the bacterium of patient's normal microflora part.In some cases, for example damaged by immunity or during immunosuppression (for example, infected HIV, or contained the AIDS complex body, or after standing organ transplantation), this class saprophytic microbe also can cause infection as the patient.A patient can infect one or more infective agents are arranged.

As known in the art, " mycolic acid structure " meaning is the β-hydroxy acid (bacteriology summary (Bact.Rev.) 36:33-64 (1966) is hereby incorporated by for Goren, M.B.) that is replaced by the aliphatic chain of moderate length at alpha position.An example that contains the biology of excellent bacillus mycolic acid is corynebacterium diphtheriae (Corynebacterium diphtheria); An example that contains the biology of Nocardia mycolic acid is nocardia asteroide (Nocardia asteroides); An example that contains the biology of mycolic acid is mycobacterium tuberculosis (referring to Funke, people such as G., clinical microbiology summary (Clin.Micro.Rev.) 10:125-159 (1997) is to coryneform further discussion (being hereby incorporated by)).These mycolic acid sample molecules are referred to as " mycolic acid structure " at this.At for example Lederer, E., the chemistry of lipid and physics (Chem.Phys.Lipids) 1:294-315 (1967); Lederer, E., also can find other tabulation of representative mycolic acid structure among pure and applied chemistry (Pure Appl.Chem.) 25:135-165 (1971), comprise that some is undersaturated, cyclopropane sample, methoxylation and ketone acid, two pieces all are incorporated herein by reference at this." mycolic acid structure " is the acid acceptance molecule.

" antimicrobial therapy " meaning be by any suitable method with containing of level of significance antibiotic composition treat the mankind or animal patient in vivo; For example, by oral, injection, application or suck this microbiotic.The conveying that comprises the antibiotic present composition also includes but not limited to: medicine is by the introducing of intravenously mode, as the activeconstituents of ointment or by swallowing.The purpose of antimicrobial therapy is to destroy viability, integrity or the irritability of infective agent, makes infected patient or animal break away from, eliminate or overcomes infection.Antimicrobial therapy is meant, is used singly or in combination-promptly uses simultaneously or continuously one or more medicines in-one class or a multiclass medicine or the class certain antibiotics.Antimicrobial therapy and " antibiotic therapy " or " treatment " synonym when this uses.

" significant quantity " meaning is the amount that is enough to reach desirable target.For example, when using in the antimicrobial therapy process, significant quantity is the amount that causes infected by microbes to take a turn for the better.This improvement may be the direct effect to microorganism, or a kind of indirect action, and it makes and damage this microorganism with contacting of significant quantity, improves the susceptibility of this microorganism to second kind of medicament like this.

If a kind of material is substantially purified to the associated material before purifying carries out the method for wishing or analyze required degree, so this material is called as " being substantially free of pollutent ".Therefore, these pollutents or lack fully or exist with lower concentration, what this lower concentration made pollutent exists (1) when the patient to needing said preparation of the preparation that will contain this material uses, do not hinder desirable result of treatment, and (2) can be owing to not using of this preparation patient harm.

" use " or the patient " used " and be meant, by any proper method known in the medical field, that be suitable for reaching desired purpose, the material of hope is introduced the position of wishing, as the human or animal's of this material of needs position, these methods include but not limited in the intestines, parenteral (for example intravenously) and iontophoretic injection are used.Also can place the form of the bandage of infection site to use with the part, bandage be effective release that design is used to provide microbiotic of the present invention and trimethyl-glycine sample stain remover.

" use simultaneously " or the patient " is used " two or more medicaments simultaneously,, be meant with a kind of form of preparation and use together or with these medicaments of form separate administration of each preparation as a kind of microbiotic and a kind of trimethyl-glycine sample stain remover.

" pharmacologically acceptable salts " mean and comprise the salt that is formed by acceptable acid of pharmacy or alkali, and such as but not limited to acid, as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid etc., perhaps alkali is as the oxyhydroxide of alkali or alkaline-earth metal, ammonium hydroxide, alkyl ammonium hydroxide etc.

Term " pharmaceutically acceptable carrier " means and comprises pharmacy acceptable solvent, carrier, thinner etc., and they are used as the additive of preparation of the present invention, so that provide carrier or adjuvant for using of these compounds.

Term " treatment (treatment) " or " treatment (treating) " mean the medicament that comprises one or more hope of significant quantity the patient or the object of these medicaments of needs are used, its purpose comprise a kind of disease prevention, take a turn for the better, prevent or cure, perhaps it is believed that elimination to the microorganism of these medicament sensitivities.

Make a kind of sample " contact " composition of the present invention that contains microorganism, be meant this microorganism is mixed with said composition, perhaps provide contacting between this microorganism and the said composition in other mode.

The present inventor chances on, as at least a trimethyl-glycine sample stain remover such as C ₁₈When-carboxylic CAB (CB-18) makes up with Antimicrobe compound, can distinguish the clinical separation strain of mycobacterium according to the susceptibility of bringing out.The inventor thinks, this phenomenon can be used for regard to it also characterizing microorganism and infective agent usually to the susceptibility of these Antimicrobe compounds and characterize these clinical separation strains.In addition, the inventor thinks also that this phenomenon is used in the interior therapeutic in collaborative mode and improves the susceptibility of these mycobacteriums to these Antimicrobe compounds.

Therefore, in embodiment the most widely, the present invention relates to a kind of method that characterizes the susceptibility of microorganism combating microorganisms compound.In a kind of preferred embodiment, the microorganism of trying is a kind of infective agent or strain isolated.In a kind of preferred embodiment, characterize and measure the susceptibility of microorganism to beta-lactam antibiotics.In a kind of preferred embodiment, the microorganism of trying obtains from the sample of taking from the patient, and this patient infection under a cloud has undesirable bacterium that contains the mycolic acid structure, or is in the danger of infection, or determines infected.Sensitivity test of the present invention is referred to herein as the trimethyl-glycine sensitivity test.The microorganism of the result of this sensitivity test with regard to existing in the sample in the CB-18 effect described herein aspect characterization research is as clinical separation strain or infective agent.That is, the whether antibiotic sensitive to being tried of the microorganism that exists in the sample is identified in sensitivity test of the present invention.Preferably, by sensitivity test of the present invention, identify the combination of one or more microbiotic or itself and other beneficial agents such as the trimethyl-glycine sample stain remover of the Institute of Micro-biology's sensitivity that exists in the sample.Yet, it is also important that, by sensitivity test of the present invention, identify the combination of insensitive one or more microbiotic of Institute of Micro-biology of existing in the sample or itself and other medicament such as trimethyl-glycine sample stain remover.Its result makes the professional medical-care personnel can more effectively select a kind of suitable antimicrobial therapy to treat the patient, perhaps the position of taking sample of otherwise sterilizing and wishing for this microorganism.

In second kind of preferred embodiment, the present invention relates to a kind of trimethyl-glycine sample stain remover is treated the patient as adjuvant in antibiotic therapy method of using, these patients infection under a cloud has undesirable bacterium or has the dangerous of infection or determine infected.In a kind of preferred embodiment, these undesirable bacteriums contain the mycolic acid structure.This antibiotic therapy can be used for treating this infection with the combination of one or more microbiotic of the present invention and one or more trimethyl-glycine sample stain removers.Microbiotic is more effectively treated the patient with the combination of trimethyl-glycine sample stain remover than independent antibiotic therapy, this is owing to compare application microbiotic when no trimethyl-glycine sample stain remover, this combination can faster or more effectively destroy the integrity of bacterium, causes infective agent anergy, non-infection maybe can't survive like this.

In the third preferred embodiment, the present invention relates to a kind of method of undesirable microorganism growth of sterilizing or otherwise prevent, method is the combination that the environment that infection site or suspection contain this microorganism is provided one or more microbiotic and one or more trimethyl-glycines.

Sensitivity test of the present invention especially can be used for characterizing, estimating and determine the susceptibility of particularly a kind of clinical separation strain of a kind of microorganism and/or the agent of infective agent combating microorganisms.Disease can be treated with one or more microbiotic of significant quantity and the combination of trimethyl-glycine sample stain remover due to these microorganisms, and sensitivity test of the present invention proves that this is combined as the effective constituent in the therapeutic process that treatment infects due to this microorganism.

In susceptibility method of the present invention, the application of the specific trimethyl-glycine sample stain remover of one group of concentration provides the useful information about this stain remover usefulness, and the test of using more than a kind of trimethyl-glycine sample stain remover is preferred.Can detect as many as desired trimethyl-glycine sample stain remover or its combination.Preferably detect five kinds at least, although any number for example 10,15,20,25 30 or more kinds of stain remover in different extent of dilution, also be easy to detect.Sensitivity test provides the out of Memory about the effect of this concentration trimethyl-glycine sample stain remover and the combination of selected microbiotic.The working concentration or the effective concentration of specific trimethyl-glycine sample stain remover depend on used stain remover.Usually, the effective concentration scope is the 1mg/ml from 0.1 μ g/ml of resulting structure to invalid structure, and for most of trimethyl-glycine sample stain removers, 1 μ g/ml-100 μ g/ml is the most effective concentration.In sensitivity testing method of the present invention, these trimethyl-glycine sample stain removers can use separately or be used in combination with other trimethyl-glycine sample stain remover.

Although it is necessary that at least a in the method for the invention microbiotic provides sensitive information, estimate in the trimethyl-glycine sensitivity test, will use more than a kind of microbiotic.Can detect as many as desired microbiotic or antibiotic combination.Preferably detect five kinds at least, although any number for example 10,15,20,25 30 or more kinds of microbiotic in different extent of dilution, also be easy to detect.The concentration of certain antibiotics depends on used microbiotic.Usually, the effective concentration scope is 100 μ g/ml from 0.1 μ g/ml of the most effective biocide to ineffective treatment compound, and for most antibiotics, 0.5 μ g/ml-64 μ g/ml is the most effective concentration.In the method for the invention, these microbiotic can use separately or be used in combination with other microbiotic.

As other sensitivity test (Woods, G.L. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1327-1404 pages or leaves), reasonably expection, the variation of inoculum will influence the result that the trimethyl-glycine sensitivity test is produced.Inoculum prepares by standard method, comprises picking colony or is similar to MacFarland standard known in the art.Experimental result shown in Fig. 5 A-5F and the embodiment 8 shows that several cells are comparable to the CB-18 effect of the inoculum of several thousand cells.Usually, 1 cell to 10 in this regard ⁵Individual cell is effectively, 100-10, and 000 cell is the most effective, about 1,000 cell is preferred.When being lower than 10 cells, sampling problem (for example sampling error) can make the result of test smudgy, is higher than 10 ⁵During individual cell, owing to covering system influences the result.1,000-10, the inoculation physical efficiency of 000 cell makes contrast grow in good time mode.Yet, can use any inoculum that is used to observe the CB-18 effect, and in this regard, trimethyl-glycine sensitivity test of the present invention is similar to other extracorporeal maleate sensibility test.

The information that trimethyl-glycine sensitivity test described herein is provided is the result of strain isolated, trimethyl-glycine sample stain remover and microbiotic dynamic interaction.This three kinds of compositions are made up in the trimethyl-glycine sensitivity test, and make the technician can determine and regulate the proper concn of microbiotic and trimethyl-glycine sample stain remover and inoculum, cause the CB-18 effect with this method, the useful information about characteristic of clinical separation strain in the research and/or susceptibility is provided thus.

Embodiment 7 shows, what some trimethyl-glycine sample stain remover may be than other in susceptibility of the present invention and methods of treatment is more useful.For example, comprise showing the activity that reduces in the structure sensitivity test herein of the modification of key, and thereby in therapeutic test, show active the reduction.In addition, contain in the trimethyl-glycine sample stain remover sensitivity test herein of modification of bridge and also show the activity that weakens, and thereby in therapeutic test, show active the reduction.Table 1 shows those the most useful in the method for the invention structures.

The most useful trimethyl-glycine sample stain remover is the trimethyl-glycine sample stain remover that does not contain modification, for example, and R wherein ₁Be simple alkyl, key (α) is simple methylene radical, R ₂And R ₃Be hydrogen or methyl (depending on used positively charged ion), R ₄No component.

Trimethyl-glycine sample stain remover depends on negatively charged ion (γ), bridge length (R to the toxicity of the bacterium that contains mycolic acid ₄) and alkyl chain length (R ₁).Usually, must consider the surface-active property of trimethyl-glycine and in this regard as the balance between the effect of adjuvant.For example, surface-active property depends on anionic soda acid character and alkyl chain length.Most of ionic detergents are phosphoric acid betaine (PO ₄), and most of non-ionic detergent is sulfuric acid trimethyl-glycine (SO ₄).Estimate that phosphoric acid betaine is poisonous especially, and the sulfuric acid trimethyl-glycine there is solubility problem.Therefore, sultaine and carboxybetaine are preferred, and carboxybetaine is most preferred.According to permeability discussion (embodiment 9), estimate that carboxybetaine has most preferred negatively charged ion, and CB-18 has ammonium cation, estimates that reasonably the sulfonium trimethyl-glycine has most preferred positively charged ion.Therefore, preferred construction comprises positive alkyl sultaine, and particularly preferred structure is positive alkyl carboxybetaine, and most preferred structure is an alkyl sulfonium carboxybetaine.

The surface-active property of trimethyl-glycine changes with cumulative chain length.Long alkyl chain has lower micelle-forming concentration (CMC), and perhaps on the contrary, short alkyl chain needs higher concentration to reach CMC.The length of alkyl chain can change according to purposes.For example, because short alkyl chain needs higher concentration reach CMC, when being used for second kind of embodiment (, in the antimicrobial therapy) so, short chain is preferred, particularly uses in methods of treatment of the present invention when carrying by intravenous route.In addition, can use the therapeutic through sucking to carry, the composition of this conveying is less to be limited by the relation between concentration and CMC because can be used for; Equally, can use long alkyl chain.In first kind of embodiment (that is, in extracorporeal maleate sensibility test), can change the dependency of chain length, can make observable CB-18 effect reach maximum stain remover to identify.Alkyl chain length is that the trimethyl-glycine sample stain remover of 8-22 carbon atom is preferred, and the alkyl chain length of 12-18 carbon atom is particularly preferred, and the alkyl chain length of 16-18 carbon atom is most preferred.

For guaranteeing the molten ability of salt, minimum bridge length should be propylene.Toxicity to microorganism also becomes (Tsubone, people such as K., pharmaceutical science magazine (Jour.Pharm.Sci) 80:441-444 (1991)) with bridge length.For example, ethene and butylene all show the toxicity higher than propylene.Possible etheno has solubility problem, and this depends on used ion.Therefore, further consideration need make suitable alkyl chain length and ion and bridge construction coupling, to avoid these problems.For example, long alkyl chain needs strong polar ion population (SO ₄＜SO ₃＜CO ₂＜PO ₄) and be beneficial to the molten bridge construction of salt.-C _mH _2mThe bridge construction of-(wherein m 〉=1) type is preferred, and the bridge with same structure of 6 〉=m 〉=3 is most preferred.According to Tsubone, people such as K., pharmaceutical science magazine 80:441-444 (1991), being used to modify cationic component also influences toxicity (for example, toxicity and R ₂And R ₃Length negative correlation (seeing Table 1)).

The length of bridge and cationic components can change according to purposes.For example, because short alkyl chain can be used for (being in the antimicrobial therapy) in second kind of embodiment, so the bridge construction of m 〉=2 is acceptable, because solvability is not with high than the short chain dependency.In addition, it is soluble that the conveying through sucking requires trimethyl-glycine sample stain remover, and the bridge construction of m 〉=3 is essential.As mentioned above, by changing the polarity that negatively charged ion can further change trimethyl-glycine.In addition, can change bridge length and the dependency that is used to modify cationic component, so that (being in the in vitro tests) observed CB-18 effect reaches maximum in first kind of embodiment.

Detect several carboxybetaines, and shown CB-18 effect in the method for the invention.They comprise: C ₁₆-carboxymethyl betaine (CAS ^No.693-33-4), C ₁₈-propyloic trimethyl-glycine (CAS ^No.30612-73-8), C _{18: 1}-carboxymethyl betaine (CAS ^No.871-37-4) and C ₁₈-carboxylic CAB (CAS ^No.78195-27-4).

Utilize methylene bridge (" carboxymethyl betaine ": R ₄=-CH ₂-) or methene key (α=-CH ₂-) and the example of the most useful carboxybetaine that only changes according to alkyl chain length be: C ₁₀(CAS ^No.2644-45-3), C ₁₁(CAS ^No.2956-38-9), C ₁₂(CAS ^No.683-10-3), C ₁₃(CAS ^No.23609-76-9), C ₁₄(CAS ^No.2601-33-4), C ₁₅(CAS ^No.23609-77-0), C ₁₆(CAS ^No.693-33-4) and C ₁₈(CAS ^No.820-66-6).There is a C ₁₂-carboxymethyl betaine (CAS ^No.6232-16-2) example, i.e. N, N diethyl (R ₃=R ₄=-CH ₂CH ₃); With one wherein alkyl contain the example of two keys: C _{18: 1}(CAS ^No.871-37-4).Utilize ethyl bridge (" propyloic trimethyl-glycine ": R ₄=-CH ₂CH ₂-), methene key (α=-CH ₂-) and the example of the most useful carboxybetaine that only changes according to alkyl chain length comprise: C ₁₂(CAS ^No.16527-85-8), C ₁₃(CAS ^No.132621-79-5), C ₁₄(CAS ^No.69725-38-3), C ₁₆(CAS ^No.42416-43-3) and C ₁₈(CAS ^No.30612-73-8).Under proper condition, R ₂And R ₃For an example of the propyloic trimethyl-glycine of hydrogen atom is CAS ^No.1462-54-0 (C ₁₂-beta Alanine).Utilize propyl group bridge (" carboxylic CAB ": R ₄=-CH ₂CH ₂CH ₂-), methene key (α=-CH ₂-) and the example of the most useful carboxybetaine that only changes according to alkyl chain length comprise: C ₁₁(CAS ^No.150147-53-8), C ₁₂(CAS ^No.15163-30-1), C ₁₄(CAS ^No.146959-90-2), C ₁₅(CAS ^No.146959-91-3), C ₁₆(CAS ^No.71695-32-4) and C ₁₈(CAS ^No.78195-27-4).Utilize butyl bridge (" carboxylic butyrobetaine ": R ₄=-CH ₂CH ₂CH ₂CH ₂-), methene key (α=-CH ₂-) an example of useful carboxybetaine be: C ₁₂(CAS ^No.120139-51-7).Utilize amyl group bridge (" carboxylic amyl group trimethyl-glycine ": R ₄=-CH ₂CH ₂CH ₂CH ₂CH ₂-), methene key (α=-CH ₂-) two examples of the most useful carboxybetaine be: C ₁₂(CAS ^No.76392-97-7) and C ₁₆(CAS ^No.73565-98-7).Utilize hexyl bridge (" carboxylic hexyl trimethyl-glycine ": R ₄=-CH ₂CH ₂CH ₂CH ₂CH ₂CH ₂-), methene key (α=-CH ₂-) an example of useful carboxybetaine be: C ₁₂(CAS ^No.132621-80-8).Exist with benzyl as the (R of bridge functional group ₄=-CH ₂-C ₆H ₄-) the example of several carboxybetaines.Two kinds of C are arranged ₁₂Example, a kind of wherein carboxyl functional group is positioned at 4 or contraposition (CAS ^No.71695-31-3), a kind of wherein carboxyl functional group is positioned at 2 or ortho position (CAS ^No.71695-34-6).Two kinds of C are arranged ₁₆Example, a kind of wherein carboxyl functional group is positioned at 4 or contraposition (CAS ^No.71695-33-5), a kind of wherein carboxyl functional group is positioned at 2 or ortho position (CAS ^No.71695-35-7).Therefore, " carboxybetaine sample " (" CB sample ") not only comprise those as WO 95/27076 and United States Patent (USP) 5,658,749 described with carboxyls as negatively charged ion (γ=-COO ) structure, refer to those trimethyl-glycine spline structures shown in the table 1 the most especially, and will comprise R as indicated above ₁, α, R ₂, R ₃, β and R ₄All possible combination.

Except that carboxybetaine, other trimethyl-glycine that is easy to obtain that can be used in combination with method of the present invention comprises sultaine, for example highly purified (being research grade) " SB " serial stain remover, SB-18 (N-octadecyl-N, N-dimethyl-3-ammonium-1-propyl group-sulfonate (CAS particularly ^No.13177-41-8)), SB-16 (N-hexadecyl-N, N-dimethyl-3-ammonium-1-propyl group-sulfonate (CAS ^No.2281-11-0)), SB-14 (N-tetradecyl-N, N-dimethyl-3-ammonium-1-propyl group-sulfonate (CAS ^And SB-12 (N-dodecyl-N, N-dimethyl-3-ammonium-1-propyl group-sulfonate (CAS No.14933-09-6)) ^No.14933-08-5)).

Great majority can be used to produce stain remover, shampoo, hairdressing agent and other toilet articles by the commercial trimethyl-glycine that obtains.These trimethyl-glycines are mainly derived from natural oil such as Oleum Cocois, butter, wheatgerm, babassu oil, Viscotrol C, Tower rape oil, soya-bean oil and rapeseed oil.Reasonably expection, all these trimethyl-glycine sample stain removers all can be used in combination with method of the present invention.

The present invention especially can be used for characterizing and detecting clinical separation strain, or treatment is by infectious microorganism especially lipotropy or be wrapped in the disease that the microorganism in the wax shape pod membrane causes, this microorganism is characterised in that in its outer cell walls (adventitia) and contains the mycolic acid structure, for example, especially excellent bacillus mycolic acid, Nocardia mycolic acid and mycolic acid.

Method of the present invention relates to a kind of method that detects microorganism such as mycobacterium to antibiotic susceptibility.In a kind of highly preferred embodiment, this microorganism is a clinical separation strain.Method of the present invention also relates to a kind of methods of treatment, wherein is used in combination trimethyl-glycine sample stain remover with microbiotic and treats patient (human or animal) by antibiotic therapy.The final trimethyl-glycine that uses will depend on the microorganism that exists in the sample, preferably bacterium in the test of the inventive method or treatment.

These test methods or treatment plan can be used for the microorganism of any hope, the bacterium of especially any hope.In a kind of highly preferred embodiment, this microorganism is group or complex body or its member of Mycobacterium.For example, the bacterium of trying can comprise the group or the kind of complex body or Mycobacterium of the Mycobacterium of any hope, mycobacterium complex body most preferably is as mycobacterium tuberculosis (MTB) complex body, mycobacterium avium (MAC) complex body, MAIS complex body and mycobacterium fortutitum complex body.The bacterium of trying can comprise that also speed gives birth to gentle branch bacillus estranged, comprise photochromic former, non-photochromic former, the scotochromogen strain of pointing out He do not point out.Can characterize or handle any mycobacterium according to the present invention, comprise: field mycobacterium (M.agri), mycobacterium abscessus (M.abscessus), M.acetamidolyticum, mycobacterium africanum (M.africanum), like to know mycobacterium (M.aichiense), Asia mycobacterium (M.asiaticum), golden mycobacterium (M.aurum), South Africa mycobacterium (M.austroafricanum), mycobacterium avium, Mycobacterium bovis (M.boyis), Mycobacterium bovis (BCG), Mycobacterium chelonei (M.chelonae), thousand field mycobacteriums (M.chitae), Chu cloth mycobacterium (M.chubuense), Ku Shi mycobacterium (M.cookii), Di Shi mycobacterium (M.diernhoferi), Du Shi mycobacterium (M.duvalii), crafty mycobacterium (M.fallax), produce glanders mycobacterium (M.farcinogenes), little yellow mycobacterium (M.flavescens), mycobacterium fortutitum, add ground this mycobacterium (M.gadium), mycobacterium gastri (M.gastri), pale yellow mycobacterium (M.gilyum), mycobacterium gordonae (M.gordonae), mycobacterium haemophilum (M.haemophilum), Mycobacterium intracellulare (M.intracellulare), mycobacterium kansasii, this mycobacterium of Como (M.komossense), Mycobacterium leprae, mycobacterium lepraemurim (M.lepraemurium), Mycobacterium marinum (M.marinum), Ma Ermo mycobacterium (M.malmoense), mycobacterium microti (M.microti), Mo Liaoka mycobacterium (M.moriokaense), new golden mycobacterium (M.neoaurum), mycobacterium nonchromogenicum (M.nonchromogenicum), cloth mycobacterium (M.obuense) difficult to understand, secondary mycobacterium fortuitum (M.parafortuitum), mycobacterium paratuberculosis, external mycobacterium (M.peregrinum), Mycobacterium phlei (M.phlei), pig mycobacterium (M.porcinum), porous mycobacterium (M.poriferae), dust mycobacterium (M.pulveris), Rhode Island mycobacterium (M.rhodesiae), Mycobacterium scrofulaceum (M.scrofulaceum), Senegal mycobacterium (M.senegalense), Shi Shi mycobacterium (M.shimoidei), mycobacterium habana (M.simiae), M. smegmatics (M.smegmatis), sphagnum moss mycobacterium (M.sphagni), Si Shi mycobacterium (M.szulgai), mycobacterium terrae (M.terrae), heat resistanceheat resistant mycobacterium (M.thermoresistible), East Sea mycobacterium (M.tokaiense), mycobacterium triviale (M.triviale), mycobacterium tuberculosis, mycobacterium buruli, cow mycobacterium (M.vaccae), mycobacterium littorale (M.xenopi), and serovar.

Mycobacterium tuberculosis, mycobacterium africanum, Mycobacterium bovis, Mycobacterium bovis (BCG) and mycobacterium microti are the member of mycobacterium tuberculosis complex body (MTB).Mycobacterium terrae, mycobacterium triviale and mycobacterium nonchromogenicum are the members of mycobacterium terrae complex body.Mycobacterium avium and Mycobacterium intracellulare are the members of mycobacterium avium complex body (MAC); Mycobacterium avium has three kinds of unique serogroupss at least, and Mycobacterium intracellulare has the serovar above 25 kinds.MAIS group's (mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum) comprise biochemical property with mycobacterium avium complex body and Mycobacterium scrofulaceum but not with mycobacterium avium complex body mycobacterium homologous nucleic acid probe (for example, AccuProbe (Gen-Probe, San Diego, the CA)) mycobacterium of hybridization.

Mycobacterium kansasii, Mycobacterium marinum, mycobacterium habana and Asia mycobacterium are light chromogenic examples.The example that Mycobacterium scrofulaceum, Si Shi mycobacterium, mycobacterium littorale, mycobacterium gordonae and little yellow mycobacterium are the scotochromogen strains.Mycobacterium avium, Mycobacterium intracellulare, mycobacterium gastri, Ma Ermo mycobacterium, mycobacterium terrae and mycobacterium triviale be right and wrong light chromogenic example all.

Mycobacterium africanum, the Asia mycobacterium, mycobacterium avium, Mycobacterium bovis, Mycobacterium bovis (BCG), the Ku Shi mycobacterium, mycobacterium gastri, mycobacterium gordonae, mycobacterium haemophilum, Mycobacterium intracellulare, mycobacterium kansasii, Mycobacterium leprae, mycobacterium lepraemurim, Mycobacterium marinum, the Ma Ermo mycobacterium, mycobacterium microti, mycobacterium nonchromogenicum, mycobacterium paratuberculosis, Mycobacterium scrofulaceum, the Shi Shi mycobacterium, mycobacterium habana, the Si Shi mycobacterium, mycobacterium terrae, mycobacterium triviale, mycobacterium tuberculosis, mycobacterium buruli and mycobacterium littorale all are the examples of slow giving birth to (needing above 7 days) mycobacterium kind.The field mycobacterium, mycobacterium abscessus, M.acetamidolyticum, like to know mycobacterium, golden mycobacterium, the South Africa mycobacterium, Mycobacterium chelonei, thousand field mycobacteriums, Chu cloth mycobacterium, the Di Shi mycobacterium, the Du Shi mycobacterium, crafty mycobacterium, produce the glanders mycobacterium, little yellow mycobacterium, mycobacterium fortutitum, add this mycobacterium of ground, pale yellow mycobacterium, this mycobacterium of Como, the Mo Liaoka mycobacterium, new golden mycobacterium, external mycobacterium, cloth mycobacterium difficult to understand, secondary mycobacterium fortuitum, Mycobacterium phlei, the pig mycobacterium, the porous mycobacterium, the dust mycobacterium, the Rhode Island mycobacterium, the Senegal mycobacterium, M. smegmatics, the sphagnum moss mycobacterium, the heat resistanceheat resistant mycobacterium, East Sea mycobacterium and cow mycobacterium all are the examples that speed is given birth to (need be less than 7 days) mycobacterium kind.

The example that has multiple mycobacterium kind and the disease that can treat according to the present invention and an illness is particularly including tuberculosis (mycobacterium tuberculosis complex body), leprosy (Mycobacterium leprae (people's leprosy) and mycobacterium lepraemurim (rodent leprosy)), by the bacterial bird disease of mycobacterium avium complex body, other patient's opportunistic infection and superingection (Nightingale due to AIDS patient and the mycobacterium avium, S.D. wait the people, transmissible disease magazine 165:1082-1085 (1992)), and any infection that causes by following special mycobacterium, for example: Mycobacterium bovis (particularly important in veterinary science), mycobacterium fortutitum (from the isolating a kind of soil bacteria of animal and human's focus), Mycobacterium intracellulare (the especially opportunistic infection seen in infection AIDS virus patient), mycobacterium paratuberculosis (particularly important in the diagnosis of human Crohn's disease (Crohn disease)), mycobacterium kansasii (being a kind of rare but destructive infective agent, relevant with pulmonary disorder usually), Mycobacterium marinum (infects cold blooded animal and fish; Also from the granuloma of people's acra top layer, be separated to), the mycobacterium paratuberculosis (pathogenic agent of bovine paratuberculosis; Its growth is very slow, must continue to cultivate for 16 weeks before determining that it is feminine gender) and mycobacterium buruli (cause of disease of Buruli ulcer).Wayne, people such as L.G., clinical microbiology summary 5:1-25 (1992) and Falkinham, people such as O. have discussed above-mentioned and other multiple disease among the clinical microbiology summary 9:177-215 (1996), and two pieces all are incorporated herein by reference at this.

Trimethyl-glycine sample stain remover can use (if this stain remover has antimicrobial acivity) as adjuvant separately or be used in combination with microbiotic.For example, in first kind of preferred embodiment, trimethyl-glycine sample stain remover is the part (as described in embodiment 10) of sensitivity test table, wherein uses separately and is used in combination this stain remover with microbiotic or other trimethyl-glycine sample stain remover, characterizes the susceptibility of clinical separation strain.In second kind of preferred embodiment, in the antimicrobial therapy process, use separately or be used in combination trimethyl-glycine sample stain remover of the present invention with microbiotic.Although can replace microbiotic to use trimethyl-glycine sample stain remover separately, more preferably be used in combination with microbiotic.In arbitrary embodiment, the use of trimethyl-glycine sample stain remover can be independent or with the combination of other adjuvant, both can side by side also can be continuously.For example, trimethyl-glycine sample stain remover can be used in combination with other trimethyl-glycine sample stain remover or with other adjuvant such as beta-lactamase inhibitor.

In second kind of embodiment, method of the present invention need with the combination treatment of the present invention of significant quantity to prepare the mankind or the animal patient of this treatment, the microbiotic that said composition comprises independent trimethyl-glycine sample stain remover or makes up with trimethyl-glycine sample stain remover.Antimicrobial therapy also comprises this combination as used herein.The conveying of trimethyl-glycine sample stain remover can be by supplying with any approach of patient's level of significance, and for example, oral or intramuscular injection perhaps especially by intravenous drip, sucks or these compositions of application the most in particular.

Compare trimethyl-glycine sample stain remover and can provide microbiotic with mode and the solution that separates, perhaps they can provide together.Trimethyl-glycine sample stain remover perhaps can not tolerate oral or intramuscular injection well, and intravenous drip may be a kind of feasible transport way; Yet, must be noted that the concentration of trimethyl-glycine in the blood.Although adding trimethyl-glycine is feasible to micelle-forming concentration (CMC), if owing to the dissolving of blood ingredient the whole blood level is elevated to more than the CMC then can causes complication.The injection that reasonably is expected at infection site is feasible transportation means, yet this is possible in few cases.Preferably, carry by the most direct approach or as might be directly to infection site delivering composition or trimethyl-glycine sample stain remover at least.In this, for mycobacterial infections, especially be the tuberculosis of respiratory tract infection, suction should be most preferred carrying method.Be used to carry an example of the suction apparatus of trimethyl-glycine sample stain remover of the present invention to be similar to current device (for example, the Allen ﹠amp that is used for a kind of Beta receptor blockers-conveying of salbutamol-asthma in design; The salbutamol that Hanburys sold ^, Allen ﹠amp; Hanburys is the company of Glaxo, ResearchTriangle Park, NC).Preferably infect (Buruli ulcer) due to mycobacterial infections such as the mycobacterium buruli by directly treating to affected part application trimethyl-glycine sample stain remover (containing or do not contain microbiotic) with ointment form.

The those of ordinary skill for the treatment of the clinical field of these infected by microbes can easily determine the patient is used the amount and the scheme of specific trimethyl-glycine sample stain remover.Usually, the dosage of microbiotic and trimethyl-glycine sample compound becomes according to following consideration: the type of used microbiotic and trimethyl-glycine sample compound; Age; Healthy state; The illness of being treated; If any, kind, the frequency of treatment and the character of desirable effect for the treatment of simultaneously; The degree of tissue injury; Sex; Duration of symptoms; And if any, other variable that contraindication and each doctor are regulated.Can use the result of application dosage with one or many to obtain to wish.Usually, effective concentration comprises from for 0.1 μ g/kg of resulting structure (for example propyloic trimethyl-glycine) 1mg/kg to invalid structure (for example those listed carboxybetaines that " do not have influence " of table 10), for most of trimethyl-glycine sample stain removers, 1 μ g/kg-100 μ g/kg is the most effective concentration.When using trimethyl-glycine sample stain remover of the present invention with ointment form, effective concentration is for 0.1 μ g/ml of the resulting structure 1mg/ml to invalid structure, for most of trimethyl-glycine sample stain removers, 1 μ g/ml-100 μ g/ml is the most effective concentration.In methods of treatment of the present invention, can use separately or be used in combination these stain removers with other trimethyl-glycine sample stain remover.Preferably, the trimethyl-glycine time of application is identical with the time of administration of antibiotics.

The concentration of certain antibiotics depends on used microbiotic.Can use method of the present invention, go up effective concentration, microbiotic is provided with therapeutic dose known in the art or to be accredited as treatment through sensitivity test of the present invention.The antibiotic example of available different sorts comprises in the method for the present invention: beta-lactam antibiotics, with beta-lactamase inhibitor, aminoglycoside and the aminocyclitol of beta-lactam antibiotics combination, quinolone, tsiklomitsin, macrolide and lincosamide, and microbiotic glycopeptide, lipopeptid and polypeptide, sulfanilamide (SN) and trimethoprim, paraxin, vazadrine, nitroimidazole, Rifampin, nitrofuran, urotropine and mupirocin.

The known microbiotic that mycobacterium is had a remarkable activity includes but not limited to: Amikacin Sulphate, Azythromycin, arbitrary beta-lactam with arbitrary beta-lactamase inhibitor combination, capromycin, cefmetazole, cefoxitin, Ciprofloxacin, clarithromycin, Rimonophenazine, seromycin, dapsone, erythromycin, Tibutol (EMB), ethionamide, imipenum, vazadrine (INH), kantlex, MINOCYCLINE HCL, Ofloxacine USP 23, to Aminosalicylic, Protionamide, pyrazinoic acid amide (PZA), Rifampin (RMP), rifabutin, sparfloxacin, sulfamethoxazole and trimethoprim, Streptomycin sulphate (SM), tsiklomitsin, thiacetazole and Viothenate (Inderlied, C.B. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1385-1404 pages or leaves; And Kucers, people such as A., " antibiotic application) " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 1352-1437 pages or leaves).Can be used for all in the method for the present invention.

As known in the art, " beta-lactam " meaning is for containing any penicillin, cynnematin, monobactam and the carbapenem antibiotics (Yao of beta-lactam nucleus as its structural constituent, J.D.C. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C.1995 1281-1286 page or leaf; Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 3-584 pages or leaves).All beta-lactams all can be used in the method for the present invention.

As known in the art, " penicillin " meaning is for containing microbiotic (Yao, the people such as J.D.C. of 6-amino-penicillanic acid chemistry nuclear, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1281-1282 pages or leaves).The example of penicillin includes but not limited to: X-1497, nafcillin, cloxacillin, dicloxacillin, Oxazacillin, penbritin, bacampicillin, Pyocianil, ticarcillin, mezlocillin and piperacillin, especially azlocillin.Reasonably expection, the penicillin that has with above-mentioned any penicillin compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " cynnematin " meaning is for containing microbiotic (Yao, the people such as J.D.C. of 7-aminocephalosporinic acid chemistry nuclear, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1282-1285 pages or leaves).The example that can be used for the cynnematin of the inventive method includes but not limited to: S 578, Kefzol, Cephalexin Monohydrate Micro/Compacted, Cephaloridine, cefoxitin, Cephapirin, Cephradine, cefaclor, Cefamandole, cefonicid, BL-S 786, Prozef, cephalofruxin, Loracarbef, cefmetazole, cefotetan, Cefixime Micronized, cefotaxime, Cefpodoxime and ceftizoxime, particularly cefoxitin, cefoperazone and ceftazidime, ceftriaxone the most in particular.Reasonably expection, the cynnematin that has with above-mentioned any cephalosporin compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " monobactam " meaning examines and contains the microbiotic (Yao of different side chains for containing beta-lactam nucleus as chemistry, J.D.C. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, the 1285th page of D.C. (1995)).The example that can be used for the monobactam of the inventive method includes but not limited to aztreonam.Reasonably expection, the monobactam that has with above-mentioned monobactam compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " carbapenem " means and contains beta-lactam nucleus as chemistry nuclear and at 6 microbiotic (Yao that contain propyloic side chain (transconfiguration) and lack sulphur or Sauerstoffatom in nuclear, J.D.C. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1285-1286 pages or leaves).The example that can be used for the carbapenem of the inventive method includes but not limited to: imipenum, meropenem, the handkerchief people train south and biapenem.Reasonably expection, the carbapenem that has with above-mentioned any carbapenem compounds homologous chemical structure also can be used in the method for the present invention.

As known in the art, " beta-lactamase inhibitor " means the beta-lactam structure that the contains modification microbiotic (Yao as chemistry nuclear, J.D.C. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1286-1287 pages or leaves).Known these have the compound and the beta-lactam synergy of limited anti-microbial activity when separating.Beta-lactamase inhibitor disturbs the enzyme (for example β-Nei Xiananmei) of degraded beta-lactam.For example, beta-lactam enzyme liberating beta-lactam.In this case, microorganism avoids the effect of beta-lactam effectively, thereby gives the infective agent resistance.Therefore, the beta-lactamase inhibitor adjuvant and the beta-lactam antibiotics that can be used as beta-lactam treatment is used in combination.When also using beta-lactam, the example that can be used for the beta-lactamase inhibitor of the inventive method includes but not limited to: clavulanic acid, Sulbactam and Zosyn.Reasonably expection, the beta-lactamase inhibitor that has with above-mentioned any beta-lactamase inhibitor compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " aminoglycoside " or " aminocyclitol " means the microbiotic (Yao that contains the aminosugar that is connected with aminocyclitol nuclear by glycosidic link, J.D.C. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1287-1288 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 585-750 pages or leaves).Can be used for the aminoglycoside of the inventive method and aminocyclitol example include but not limited to: Streptomycin sulphate, kantlex, gentamicin, tobramycin, Amikacin Sulphate, sisomycin, netilmicin, Xin Meisu, Soframycin and paromycin.Reasonably expection, the aminoglycoside and the aminocyclitol that have with above-mentioned any aminoglycoside and aminocyclitol compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " quinolone " or " fluoroquinolone " means to contain has 1 of different side chains, microbiotic (the Yao of 5-naphthyridine nuclear, J.D.C. wait the people, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1288-1290 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 1203-1275 pages or leaves).The example that can be used for the quinolone of the inventive method includes but not limited to: oxolinic acid, cinoxacin, flumequine, Miloxacin, rosoxacin, pipemidic acid, norfloxicin, enoxacin, Ciprofloxacin, Ofloxacine USP 23, lomefloxacin, Temafloxacin, fleroxacin, Pefloxacin, amifloxacin, sparfloxacin, levofloxacin, Clinafloxacin, particularly nalidixic acid.Reasonably expection, the quinolone or the fluoroquinolone that have with above-mentioned any quinolone or fluoroquinolone compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " tsiklomitsin " means and contains microbiotic (Yao, the people such as J.D.C. of hydrogen tetracene structure as nuclear, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1290-1291 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 979-1044 pages or leaves).The example that can be used for the tsiklomitsin of the inventive method includes but not limited to: tsiklomitsin, Uromycin, terramycin, dimethyl chloride tsiklomitsin, demethyltetracycline, methacycline, lymecycline, clomocycline, doxycycline and MINOCYCLINE HCL.Reasonably expection, the tsiklomitsin that has with above-mentioned any tetracycline compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " macrolide " mean contain one have two additional sugar, desosamine and cladinose macrolide rings with and multiple alternate microbiotic (Yao, J.D.C. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1291-1292 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 851-892 pages or leaves).The example that can be used for the macrolide of the inventive method includes but not limited to: erythromycin, romicil, Spiramycin Base, josamycin, rosaramicin, clarithromycin, Azythromycin (being also referred to as azalide), dirithromycin, Roxithromycin, Flurithromycin and rokitamycin.Reasonably expection, the macrolide that has with above-mentioned any Macrocyclic lactone compounds homologous chemical structure also can be used in the method for the present invention.

As known in the art, " lincosamide " means and contains a kind of amino acid whose microbiotic (Yao, people such as J.D.C. who is connected with aminosugar, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1292-1293 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 819-850 pages or leaves).The example that can be used for the lincosamide of the inventive method includes but not limited to lincomycin and clindamycin.Reasonably expection, the lincosamide that has with above-mentioned any lincosamide compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " glycopeptide " or " lipopeptid " means the microbiotic (Yao that contains peptide and one of carbohydrate or lipid component or both combinations, J.D.C. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, the 1293rd page of D.C. (1995); And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphi a, PA (1987) 1045-1072 pages or leaves).Can be used for the glycopeptide of the inventive method and the example of lipopeptid includes but not limited to: vancomycin, trip wall rhzomorph, dive mycin (being also referred to as YL146032) and Ramoplanin (being also referred to as MDL 62198).Reasonably expection, the glycopeptide and the lipopeptid that have with above-mentioned any glycopeptide and lipopeptid compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " peptide antibiotics " means amino acid whose microbiotic (Yao, the people such as J.D.C. of containing cyclic polypeptide structure or peptide connection, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1295-1296 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 899-913 pages or leaves).The example that can be used for the peptide antibiotics of the inventive method includes but not limited to: aerosporin, B, C, D and E, and bacitracin and linear gramicidins.Reasonably expection, the peptide antibiotics that has with above-mentioned any peptide antibiotics compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " sulfanilamide (SN) " means the microbiotic that contains the core texture that is similar to para-amino benzoic acid, as known in the art, " trimethoprim " mean be pyrimidine analogue microbiotic (Yao, people such as J.D.C. are in Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1293-1295 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 1075-1117 pages or leaves).The example that can be used for the sulfanilamide (SN) of the inventive method includes but not limited to: sulfanilic amide, sulfacetimide, sulfapyridine, Sulphathiazole, Sulphadiazine Sodium, Sulphamerazine, sulphamethazine, sulphasomidine, sulfasalazine, marfanil, sulfamethoxazole, sulfamethoxypyridazine, sulfadimethoxine, prosymasul, sulphormethoxine, sulfalene pyrazine, Sulphaguanidine, succinylsulfathiazole and phthalylsulfathiazole.Trimethoprim can use separately or be used in combination with any sulfanilamide (SN) in the method for the invention.Reasonably expection, the sulfanilamide (SN) and the trimethoprim analogue that have with above-mentioned any amine compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " nitroimidazole " means the microbiotic that contains nitroimidazole nuclear (in Murray, people such as P.R. compiles " clinical microbiology handbook, ASM press, Washington, the 1297th page of D.C. (1995) for Yao, people such as J.D.C.; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 1290-1343 pages or leaves).The example that can be used for the nitroimidazole of the inventive method includes but not limited to: metronidazole, fasigyne, naxogin, chloromethane nitre imidazoles, card imidazoles and flagentyl.Reasonably expection, the nitroimidazole that has with above-mentioned any nitroimidazole compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " paraxin " means and contains microbiotic (Yao, the people such as J.D.C. of a nitro phenyl ring as structural core, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1296-1297 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 757-807 pages or leaves).The example that can be used for the paraxin of the inventive method includes but not limited to paraxin and thiamphenicol.Reasonably expection, the paraxin that has with above-mentioned any paraxin compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " Rifampin " means microbiotic (Ansamycin microbiotic) (Yao, the people such as J.D.C. of containing an ansa or macrocyclic structure core, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, the 1298th page of D.C. (1995); And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 914-970 pages or leaves).The example that can be used for the Rifampin of the inventive method includes but not limited to: Rifampin, Rifamycin Sodium, rifamycin B (rifamide) and rifabutin.Reasonably expection, the Rifampin that has with above-mentioned any Rifampin compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " nitrofuran " means and has heterocyclic microbiotic (Yao, a people such as J.D.C. who contains nitro, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1298-1299 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 1276-1289 pages or leaves).The example that can be used for the nitrofuran of the inventive method includes but not limited to: the nitre furan is disliked ketone, nitro sugar hydrazone, Nifurazolidone and nitrofuran holder English.Reasonably expection, the nitrofuran that has with above-mentioned any Nitrofuran compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " urotropine " means the microbiotic that contains tertiary amine (in Murray, people such as P.R. compiles " clinical microbiology handbook, ASM press, Washington, the 1299th page of D.C. (1995) for Yao, people such as J.D.C.; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 1344-1348 pages or leaves).The example that can be used for the tertiary amine of the inventive method includes but not limited to: urotropine, mandelate, methenamine hippu.Reasonably expection, the tertiary amine that has with above-mentioned any urotropine compound homologous chemical structure also can be used in the method for the present invention.

As known in the art, " mupirocin " (being also referred to as pseudomonic acid) means the microbiotic (Yao that contains unique 9-hydroxyl-n-nonanoic acid part, J.D.C. wait the people, in: Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1299-1300 pages or leaves; And Kucers, people such as A., " antibiotic application " the 4th edition, J.B.Lippincott Co.Philadelphia, PA (1987) 754-756 pages or leaves).Reasonably expection, the compound that has with above-mentioned mupirocin compound homologous chemical structure also can be used in the method for the present invention.

There is the patient's of mycobacterium tuberculosis complex body bacterium (MTB) treatment to generally comprise the combination of a kind of medicine or multiple medicine to infection.The preferred choice drug (" line " medicine) that is used for the treatment of TB comprises vazadrine (INH), Rifampin (RMP), pyrazinoic acid amide (PZA), Streptomycin sulphate (SM) and Tibutol (EMB); Secondary (or " two wires ") medicine comprises Ciprofloxacin, Ofloxacine USP 23, ethionamide and seromycin.Other medicines in the research comprise Amikacin Sulphate, rifabutin, rifapentine and sparfloxacin (Inderlied, people such as C.B. are in Murray, P.R. wait the people to compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1385-1404 pages or leaves).The activated any medicine of the bacterium that contains the mycolic acid structure all be can be used in the method for the present invention.

There is the patient's of mycobacterium avium complex body (MAC) bacterium antimicrobial therapy generally also to comprise the combination of the medicine of a kind of medicine or limited quantity to infection.The line medicine that treatment MAC infects comprises Azythromycin, clarithromycin and EMB.The two wires medicine comprises Amikacin Sulphate, Rimonophenazine, Ciprofloxacin and rifabutin.Stock of drugs comprise seromycin and SM (Inderlied, people such as C.B., in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1385-1404 pages or leaves).Can be used for all in the method for the present invention.

The mycobacterium pathogenic agent that other is important, mycobacterium kansasii for example, it is treated general use and is used for the treatment of a kind of medicine that TB or MAC infect or the combination of multiple medicine as mentioned above.The treatment of the livings bacterium of speed (for example mycobacterium fortutitum and Mycobacterium chelonei) infection can be with Amikacin Sulphate or clarithromycin, and some beta-lactam, especially cynnematin (Inderlied, C.B. wait the people, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1385-1404 pages or leaves).Can be used for all in the method for the present invention.

Method of the present invention is employed trimethyl-glycine sample stain remover and/or microbiotic in the methods of treatment especially, and it is used and can comprise pharmacologically acceptable salts or carrier when needing with any suitable pharmacology or the acceptable form of pharmacy.Can use them with any form of the infected by microbes situation that realizes preventing, alleviate, prevent or to cure people and animal.The antibiotic dosage that can be used for methods of treatment of the present invention is the dosage that the manufacturer recommends.For example (PDR) at " doctor's desk with reference to " (Physician ' sDesk Reference), Medical Economics Company, Montvale, NewJersey can find these dosage in the U.S..The dosage of using about the animal doctor as seen for example, " Merck animal doctor's handbook (Merck Veterinary Manual), Merck﹠amp; Co., Inc., Rahway, NewJersey, the U.S..

When microbiotic and trimethyl-glycine sample stain remover being administered to the patient who needs them, preferably provide every kind of medicament, so that medicament is present among this patient with level of significance simultaneously to the patient with the form of single preparation.Just, provide these medicaments individually to the patient in any case, the microorganism at this patient infection position is contacted with trimethyl-glycine sample stain remover simultaneously with the microbiotic of level of significance.Therefore, for example, use in the time of can be by microbiotic and trimethyl-glycine sample stain remover and treat the patient, for example, Orally administeredly use with suction or intravenously trimethyl-glycine sample stain remover by antibiotic.The administering therapeutic patient also can followingly be taken place by the while: in same preparation, for example, in ointment or bandage that microbiotic and trimethyl-glycine sample stain remover are provided, provide trimethyl-glycine sample stain remover and microbiotic simultaneously.

In addition, methods of treatment to the patient also can be, in the two, not only contain " pre-treatment " patient under a kind of situation with microbiotic or trimethyl-glycine sample stain remover, then with microbiotic and trimethyl-glycine sample stain remover or with " the another kind "-microbiotic that does not contain in the pre-treatment or trimethyl-glycine sample stain remover-come " treatment " patient, as long as pre-treatment does not disappear treating fashion to action of microorganisms.Therefore, for example, energy garden beet alkali sample stain remover pre-treatment patient is with permeability and/or the viability of infringement microorganism before using the microbiotic that contains or do not conform to trimethyl-glycine sample stain remover.In addition, also can use contain or do not contain antibiotic trimethyl-glycine sample stain remover before with microbiotic pre-treatment patient.

In mixture, but in microbiotic and each comfortable solution of trimethyl-glycine sample stain remover or the solid form of respectively doing for oneself (especially suspension).In addition, one or more microbiotic and/or one or more trimethyl-glycine sample stain removers can be in solution, and other any microbiotic or stain remover in the same preparation can be solid form.

The useful preparation of the present composition of parenteral administration comprises sterilized water solvent or non-aqueous solvent, suspension and emulsion.Useful examples of non-aqueous comprises: propylene glycol, polyoxyethylene glycol, vegetables oil, fish oil and injectable organic ester.The example of water carrier comprises: water, water-ethanol solution, emulsion or suspension, comprise salt solution and the medical parenteral carrier of buffering, and comprise sodium chloride solution, woods lattice glucose solution, glucose+sodium chloride solution, lactinated Ringer's solution or fixed oil.The example of intravenous vehicles comprises liquid and nutrient supplement, electrolyte replenisher, as based on supplement of woods lattice glucose etc.

According to known technology, use suitable dispersion agent or wetting agent and suspension agent when needing, can prepare injection preparation, as oily solution, suspension or emulsion.When active compound was water-soluble form such as water-soluble salt form, aseptic injection preparation can use nontoxic parenteral acceptable diluent or solvent, for example aseptic apirogen water or 1,3 butylene glycol.In other acceptable carrier and spendable solvent, be 5% glucose injection, ringer's injection and isotonic sodium chloride injection liquid (as described in USP/NF).When active compound is water-insoluble form, can use aseptic suitable oily suspension, it contains suitable lipophilic solvent or carrier, as fatty oil, for example sesame oil, or synthetic fatty acid ester, for example ethyl oleate or triglyceride level.In addition, also can use the injection water suspension, it contains the material of tackifying, for example Xylo-Mucine, Sorbitol Powder and/or dextran, and randomly also contain stablizer.

The pharmaceutical preparation that orally uses can followingly obtain: active compound is combined with solid excipient, randomly the mixture that obtains is made particle, and in hope or add suitable auxiliary in case of necessity, process this mixture or particle afterwards, to produce the tablet of sugar-coat core.

Suitable vehicle comprises, especially weighting agent such as sugar, for example lactose or sucrose, N.F,USP MANNITOL or Sorbitol Powder, cellulose preparation and/or calcium phosphate, for example tricalcium phosphate or secondary calcium phosphate, and tackiness agent, as starch, paste, W-Gum for example, wheat starch, rice starch or yam starch, gelatin, tragakanta, methylcellulose gum, HPMC, Xylo-Mucine and/or polyvinylpyrrolidone, and/or the disintegrating agent when wishing, as above-mentioned starch, and carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar or alginic acid or its salt such as sodium alginate.Auxiliary at first is flowing regulator and lubricant, for example, silica, talcum powder, stearic acid or its salt such as Magnesium Stearate or calcium stearate, it has suitable bag tegillum when wishing, can tolerate gastric juice, especially spissated thus sugar soln, it randomly contains gum arabic, talcum powder, polyvinylpyrrolidone, polyoxyethylene glycol and/or titanium dioxide, lacquer solution and appropriate organic solvent or solvent mixture.In order to produce the bag tegillum of tolerance gastric juice, use the suitable cellulose preparation such as the solution of ethanoyl phthalic ester Mierocrystalline cellulose or hydroxypropyl methylcellulose phthalate.For example, in order to distinguish or, can in tablet or sugar-coat bag quilt, to add dyestuff or pigment in order to characterize the various combination of active compound doses.

The other medicines preparation that can orally use is to agree with capsule by pushing of making of gelatin, and the soft seal capsule of being made by gelatin and softening agent such as glycerine or Sorbitol Powder.Push and agree with the active compound that capsule can contain particle form, for example, with weighting agent such as lactose, tackiness agent such as starch and/or lubricant such as talcum powder or Magnesium Stearate and randomly stablizer mix mutually.In soft capsule, active compound preferably dissolves or is suspended in suitable liquid such as fatty oil, whiteruss or the liquid macrogol, and it also is possible adding stablizer.

The suppository that is used for rectal administration compound of the present invention can be prepared as follows, hybrid medicine and suitable suppository base such as non-irritating vehicle, for example, therefore can and discharge the matrix of medicine in the internal rectum fusing for liquid down for solid body temperature under theobroma oil, natural or synthetic triglyceride level, paraffinic hydrocarbons, polyoxyethylene glycol or higher alkanols, the especially normal temperature.In addition, it also is possible using the gelatin rectal capsule that combination constituted by active compound and matrix; Possible substrate material is, for example, and liquid triglycerides, polyoxyethylene glycol or paraffinic hydrocarbons.

Orally administered solid dosage comprises capsule, tablet, pill, lozenge, lozenge, pulvis and particle.In these solid dosages, active compound can mix with at least a inert diluent such as sucrose, lactose or starch.When normally using, these formulations also can comprise pharmacy auxiliary substance, for example stearate lubricant.Also enteric solubility or other bag of the release of available adjustment activeconstituents are produced solid orally ingestible.

Orally administered liquid dosage form comprises the acceptable emulsion of pharmacy, solution, suspension, syrup and elixir, and it contains inert non-toxic thinner usually used in this field, as water and ethanol.These compositions also can comprise adjuvant, as wetting agent, emulsifying agent, suspension agent, sweeting agent, seasonings and perfume compound.

Also can use composition of the present invention by means of pump or with the slowly-releasing form.Also can be by the conduit that suitably inserts, or the part of the chimeric molecule (or mixture) by these molecules being become be used for the target certain organs, compound of the present invention carried to certain organs high density ground.

When the long duplicate injection of needs so that patient comfortable when reaching maximum, with the slowly-releasing form to use the patient be more easily.

If the biological activity of material is not destroyed by digestive process and the characteristic of compound can be absorbed when making it by intestinal tissue, can use trimethyl-glycine sample stain remover or the microbiotic that uses at the compositions and methods of the invention to be used for Orally administered formulation such as tablet, capsule, pulvis, ointment, cartridge bag or liquor so.

Can produce pharmaceutical composition of the present invention with known method, for example, by means of mixing, granulating, sugar-coat formation, dissolving, lyophilize or the similar method of routine.

Also can provide the reagent that uses in this method to implement method of the present invention, especially sensitivity test easily by form with test kit.This test kit preferably contains suitable damping fluid, salt, trimethyl-glycine sample stain remover or its combination, microbiotic or its combination, and as the water of adequate purity when wishing.In a kind of preferred embodiment, provide the set and the different antibiotic set of different trimethyl-glycine sample stain removers.Trimethyl-glycine sample stain remover in the set and/or microbiotic can not only be fit to a kind of specified microorganisms or be particularly suitable for a kind of specified microorganisms.5 kinds of different trimethyl-glycine sample stain removers preferably are provided at least,, also provide selection more than 5 kinds although in preferred embodiments, for example 10,15,20,25 or 30 kind, or any amount therebetween.5 kinds of different microbiotic preferably are provided at least,, also provide selection more than 5 kinds although in preferred embodiments, for example 10,15,20,25 or 30 kind, or any amount therebetween.If wish that special test kit especially can contain specific microorganism, particularly mycobacterium, preferably to be used as standard.In such test kit, if non-bacterium composition is together unmixed, even be restricted to so in separately the container or packing, these compositions are also very approaching mutually usually, and any microorganism sample that provides with this test kit is very approaching.The CB-18 trial test

The present invention is derived from around the observation of the application of Thornton WO 95/27076 and United States Patent (USP) 5,658,749 described inventions (embodiment 1 described method).In embodiment 2, use this Study on Processing Methods result to show, with CB-18 and the microbicidal additives PANTA combination remarkably influenced liquid culture susceptibility of strengthening with ceftazidime (caz) (table 6).Table 6 shows that the NALC/NaOH of liquid and solid medium cultivates susceptibility and is respectively 98.4% and 75.4%, and for the sample of handling with CB-18 on the same group, the susceptibility of liquid and solid medium is respectively 64.0% and 83.1%.In other words, although total CB-18 makes the culture susceptibility improve 46% (table 3), but CB-18 liquid culture susceptibility reduces.The more important thing is following discovery: precipitate for NALC/NaOH, the difference of the positive liquid culture susceptibility with the negative sample of smear of smear only is 4% (promptly, 100% pair 95.8%), and for identical 65% the difference (that is, 75.0% pair 45.4%) that relatively shows of CB-18.Because for two kinds of different methods, difference between the positive solid medium susceptibility with negative sample of smear is comparable (being respectively 34% pair 39% for NALC/NaOH and CB-18), so think that at first be the sole cause of liquid culture susceptibility reduction with caz to the reinforcement of PANTA.Yet embodiment 3 (Fig. 3 A-3H) discloses, and not only CB-18 has the ability of the influence strain isolated that tries (571/573-BAL: see Table 8) growth characteristics, and independent and also all have the ability that influences this strain isolated growth characteristics with PANTA that caz makes up.When adding CB-18 with microbiotic combination, this effect, promptly so-called CB-18 effect, be work in coordination with the fractionated effect.

PANTA contains PXB, amphotericin B, nalidixic acid, trimethoprim and azlocillin.Amphotericin B is used for controlling fungal contamination, and all the other microbiotic all have antibacterial activity.PXB is a kind of polypeptide that changes permeability with the phosphatide interaction.Nalidixic acid is a kind of quinolone that disturbs DNA synthetic (in the dna gyrase level).Trimethoprim is a kind of pyrimidine analogue, generally uses with sulfanilamide (SN), and also known its disturbs DNA synthetic (in the Tetrahydrofolate dehydrogenase level).The azlocillin is a kind of penicillin, represents beta-lactam antibiotics, its effect of performance on the synthetic level of cell walls.Nalidixic acid, trimethoprim and azlocillin all have the certain activity not of the same race to mycobacterium according to reports.CB-18 shows that with the fact (embodiment 3, Fig. 3 F and Fig. 3 H) that the PANTA combination that does not contain caz can influence growth this phenomenon not only depends on the existence of ceftazidime, and is widely used in different types of microbiotic (seeing above) on the contrary.Figure 28-30 has given prominence to the popularity of the antibiotic application of different sorts.Reasonably expection, this phenomenon will extend to different types of microbiotic.

At first ceftazidime is mixed in the CB-18/12B/PANTA detection system and pollute (embodiment 1), rather than sacrifice the viability of mycobacterium with control.After the different cynnematins of detection are to the experiment of the influence of mycobacterium viability, carry out this detection.The PANTA-cynnematin that tries combination, except ceftazidime, also comprise cefoperazone (cfp), cefotaxime (cft) and the ceftriaxone (cax) (for example, cfp/cft, cfp/cax and cfp/cft/cax) of various combination.

Implement these experiments and will use the CB-18 of about 7-15 μ g/ml and the different ATCC type bacterial strains of mycobacterium tuberculosis (ATCC27294), mycobacterium avium (ATCC 25291), mycobacterium kansasii (ATCC 12478), mycobacterium fortutitum (ATCC 6841), mycobacterium littorale (ATCC 19250) and mycobacterium gordonae (ATCC 14470).The CB-18/12B/PANTA/caz combination is the unique preparation that these strain isolateds is shown minimum influence in preliminary study.For example, PANTA and cfp/cft, cfp/cax and cfp/cft/cax's is combined in most of obvious delays that try all to cause in the strain isolated.PANTA/caz causes the delay of the mycobacterium avium strain isolated that tries and the obvious delay of the mycobacterium gordonae strain isolated that tries.

In the CB-18 trial test, the CB-18/12B/PANTA/caz system has missed 14 smear positive samples (table 6).Analysis to the positive strain isolated of these smears shows that 5 is MTB, and other 9 is different MOTT kinds, comprises 4 MAC, 2 mycobacterium fortutitums, 1 mycobacterium gordonae, Si Shi mycobacterium and 1 mycobacterium kansasii.The important conclusion that is drawn by these data is, the CB-18/12B/PANTA/caz systematic influence large-scale mycobacterium, MTB and MOTT all are.

The CB-18 effect is observable (embodiment 3) when no cephalo Kefadim, that is to say, other microbiotic (for example, other composition of cynnematin, PANTA and other microbiotic) can cause the delay of different mycobacterium growths, system causes the experiment of embodiment 5 and embodiment 6 to the apparent susceptibility of CB-18 concentration.The experiment of embodiment 5 has detected mycobacterium tuberculosis, mycobacterium avium complex body and mycobacterium fortutitum complex body strain isolated with regard to the change of CB-18 concentration, and embodiment 6 concentrates on the susceptibility pattern of mycobacterium tuberculosis strain isolated.Table 7 has been summed up the experimental result of embodiment 5, the result of the different mycobacterium kinds of Figure 10-15 display application.Table 8 has been summed up the experimental result of embodiment 6, and Figure 17-27 shows the result of the mycobacterium tuberculosis strain isolated that tries.Figure 28-30 shows, this phenomenon expand to try outside the beta-lactam.Although in the experiment of embodiment 5 and embodiment 6, have significant difference between kind and the strain isolated, all worked in coordination with influence in a way by CB-18 and antibiotic being combined in.The mycobacterium tuberculosis strain isolated seemingly tries in the mycobacterium kind the most responsive, and the living bacterium of speed is subjected to the influence of less degree, but it shows maximum synergy when being used in combination with microbiotic.Mycobacterium avium complex body strain isolated shows the reaction of moderate.Basically, according to the growth characteristics in the presence of CB-18 and different microbiotic, can differentiate all strain isolateds.Therefore, the CB-18 effect depends on used strain isolated and microbiotic, and the existence of proper concn CB-18 is to differentiating that strain isolated is necessary.

This has produced following notion subsequently: for differentiating strain isolated, different trimethyl-glycines can provide the susceptibility of higher degree.Embodiment 7 has used in test and the multiple stain remover of rebuilding liquid (R.F.), PANTA or PANTA/cax combination.Table 10 has been summed up these result of experiment, and Figure 32 B shows the result (having only in the presence of the P-cax) of several different stain removers.Therefore, the CB-18 effect depends on strain isolated, stain remover and antibiotic dynamic interaction, the application that wherein has the CB-18 effect.Antimicrobial therapy, resistance and permeability

With regard to action site, microbiotic is not quite similar.For example, Yao, people such as J.D.C. are at Murray, P.R. " the clinical microbiology handbook that waits the people to compile, ASM press, Washington has discussed different types of biocide in D.C. (1995) the 1281-1307 pages or leaves, and report is for influence or hinder cell walls and cytolemma is synthetic or integrity, protein synthesis, nucleic acid are synthetic (for example, RNA and DNA precursor) and the different aspect of dna replication dna, and the medicine that only is beneficial to mutagenesis, the scope difference of action site.

Bacterium owing to multiple reason to different microbiotic insensitive (resistance is promptly arranged).Quintiliani, people such as R. be at Murray, people such as P.R. compile " Washington has discussed several such mechanism in D.C. (1995) the 1308-1326 pages or leaves (being hereby incorporated by) for clinical microbiology handbook, ASM press.In a word, microbiotic must at first enter cell, brings into play its effect at action site then.Drug-fast basis may be because: (a) Sheng Wu permeability, (b) molecular configuration of action site may be inconsistent or non-existent fully in the specific clinical strain isolated, and perhaps microbiotic can be modified, destroy or be pumped to (c) bacterium from intercellular space.In first kind of situation, if bacterium is impermeable for medicine, microbiotic can not arrive action site and bring into play its influence so.In second kind of situation, if medicine can enter bacterium, but action site (for example 3-D structure of target site) makes microbiotic combination with it, if perhaps action site (does not for example exist, described structure or enzyme are not the parts of the component of expression), bacterium will not be subjected to drug influence so.In in the end a kind of situation, if microbiotic can enter and target exists, bacterium can be by degraded microbiotic, modified antibiotic to reduce its toxicity or to remove from intracellular environment/to pump microbiotic and avoid the effect of medicine effectively so.

The treatment of mycobacterial infections is very limited (seeing above), compares most of microbe (Inderlied, people such as C.B. all the more so, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1385-1404 pages or leaves).This insensitivity partly is because the impervioursness of this bacterioid: mycobacterium is than intestinal bacteria (Escherichia coli) permeability low 1,000-10,000 times of (Jarlier, V. wait the people, bacteriology magazine (Jour.Bact.) 172:1418-1423 (1990) and Nikaido, H. wait the people, microbiological research (Res.Microbiol.) 142:437-443 (1991)).Connell; N.D. wait the people " tuberculosis. pathogeny, protection and control " (Tuberculosis.Pathogenesis; Protection; and Control) Bloom; B.R. compile ASM press, Washington; D.C. discussed the permeability feature of mycobacterium in (1994) the 333-352 pages or leaves, and statement " the low permeability of Mycobacterium chelonei cell walls has been explained the level of this microorganism to the cynnematin resistance fully ".

Low permeability partly causes the complex patterns of the shown susceptibility of mycobacterium.For example, MTB infects common available INH and/or PZA treats effectively, and the MAC strain isolated generally tolerates these medicines.In addition, mycobacterium fortutitum and Mycobacterium chelonei strain isolated generally all have resistance (Inderlied, people such as C.B. to all line antitubercular agents, in: Murray, people such as P.R. compile " clinical microbiology handbook, ASM press, Washington, D.C. (1995) 1385-1404 pages or leaves).Cynamon, people such as M.H. are at " drug susceptibility in the mycobacterial infections chemotherapy " Heifts, and L.B. compiles, CRC press, Boston has discussed among MA (1991) 147-159 about treating the research that these speed infect due to living bacterium, and has reported the various modes of susceptibility.For example, in described antibiotic every kind (beta-lactam, sulfanilamide (SN), macrolide, aminoglycoside etc.), the scope of susceptibility has been described, from several percentage points to 100%, 30%-60% is standard (depending on medicine and concentration thereof).Heifts, L.B. is at " drug susceptibility in the mycobacterial infections chemotherapy " Heifts.L.B. compile, CRC press, Boston has discussed the similar research to MTB and MAC in MA (1991) the 13-57 pages or leaves, and the diversity of susceptibility has been discussed, although the pattern of suggestion susceptibility is still various, but does not change.

As if though permeability has been explained the susceptibility pattern that the part of mycobacterium is important, these microorganisms have other two kinds of mechanism that play an important role in resistance.As if the first, MTB infects and is made up of the subgroup of cell.These subgroups can be classified as: (a) active growth, (b) semidormancy-because the low pH of scavenger cell, (c) semidormancy-have a metabolism outburst of distributing, (d) dormancy (Heifts, L.B. is in " drug susceptibility in the mycobacterial infections chemotherapy ", Heifts, L.B. compile, CRC press, Boston, MA (1991) 13-57 pages or leaves).Dormancy is survived these subgroups during treating.Another kind of mechanism may be genetic instability.The generation of point mutation causes the molecular change opposite sex of potential treatment target site.Fully, the variability of susceptibility pattern is the combination of these mechanism, and main mechanism perhaps depends on plants even strain isolated itself.

Generally speaking, in mycobacterium as if the most effective resistance mechanism be the adjusting (for example dormancy) of permeability, propagation and the structure variation of target site.The method that overcomes or walk around resistance must be modified the chemical structure of permeability, understanding and location propagation or modified antibiotic more suitably " to be fit to " target site.

According to this viewpoint, must be in harmonious proportion 5 information shown in the embodiment herein.The first, the mechanism of action of CB-18 effect is different from the mechanism of quaternary ammonium salt.The one group of common surfactivity of stain remover performance in back is used for destroying cytolemma and denaturing cell proteins matter (Hugo, W.B. in " S.C.I. special topic No. 19: the tensio-active agent in the microbiology " (S.C.I.Monograph no.19:Surface-Active agents in Microbiology) London Soc.Chem.Industry, London (1965) 69-82 pages or leaves).The effect that Fig. 7 A-7C and Fig. 8 A-8C have compared TMA-18 and CB-18 confirms that the CB-18 effect is different from the effect of season class stain remover.As if yet when high density, trimethyl-glycine is simulated season class stain remover and cause common deleterious effects.

The second, there is not post antibiotic effect (PAE).For example, contact 90 minutes to not obviously influence (Fig. 3 C and Fig. 3 G) of growth characteristics with 383 μ g/ml CB-18.If the CB-18 effect is special, that is to say not cause common deleterious effects, but in active metabolism, need minimum concentration that PAE may be minimum so.The vazadrine is the antitubercular agent (Heifts, L.B. be in " drug susceptibility in the mycobacterial infections chemotherapy ", Heifts, L.B. compiles, CRC press, Boston, MA (1991) 13-57 pages or leaves) of a kind of PAE of not having.

Thirdly round the following fact: in some strain isolated, only rebuilding in the presence of the liquid (R.F.) when antibiotic-free (that is, (Figure 10-15)) as seen CB-18 effect.This effect is the true strong prompting that relies on strain isolated (Figure 17-27), and the CB-18 effect has special action site, and this and global result are not inconsistent.In other words, the CB-18 effect is the reflection of susceptibility pattern microheterogeneity in the mycobacterium tuberculosis complex body the most in particular in the mycobacterium.

The 4th, the CB-18 effect is different from the effect (Figure 34-35) of EDTA.The divalent metal of chelating stabilized cell wall construction changes permeability to EDTA by extracting also.Rastogi, people such as N., biocide chemotherapy 34:759-764 (1990) report, the growth in 1mM EDTA (372.5 μ g/ml) is affected the degree that can't use these data in " X/Y merchant " that the author carries out analyzes.EDTA and CB-18 use in the experiment of embodiment 7 with the concentration (approximately equimolar amount) of 17 μ g/ml, and show different performances in test of the present invention.In addition, Tsubone, K., pharmaceutical science magazine 80:1051-1054 (1991) show, between the sequestering activity of different phosphoric acid betaines and antimicrobial acivity general not or extremely low dependency arranged.In addition, table 10 has been commented the different trimethyl-glycines that tried.With respect to containing identical bridge but there is not the carboxybetaine of amido propyl key, the carboxybetaine that contains the amido propyl key is invalid (relatively Hetaine CLA or Schercotaine WOAB and DeTaine PB and Velvetex OLB).Estimate that the amido propyl key can not disturb the chelating between the charge-site, if but action site is physiological (for example enzyme) in nature, then should be able to disturb the CB-18 effect.

The 5th, embodiment 7 (Figure 32 B) has detected in test tween 80: tween 80 does not have influence to the 571/573-BAL strain isolated when 17 μ g/ml (13 μ M).This is quite interesting result, because several author's been reported are mixed (Hui, people such as J., the biocide chemotherapy 11:773-779 (1977) of inducing that tween 80 also causes susceptibility in substratum; Yamori, people such as S., microbiology and immunology (Microbiol.Immunol.) 35:921-926 (1991)).The mechanism that the Notes of Key Data herein, tween 80 play a role is different from CB-18.For example, when the tween 80 of high density (for example 1% (10mg/ml)), it is best that growth is actually, high to 10% amount also unrestraint (Stinson, M.W. wait the people, U.S. respiratory tract disease summary (Am.Rev.Resp.Dis.) 104:717-727 (1971)).On the contrary, approximately the CB-18 of 3-7 μ g/ml does not cause the variation of growth, and the growth of two kinds of strain isolateds ( ATCC 27294 and 571/573-BAL) is all suppressed (Figure 10 A and Figure 11 A) fully when 54 μ g/ml (222 μ M).The CB-18 effect is observable (between the about 27 μ g/ml of about 13 μ g/ml-, to depend on strain isolated) in narrower concentration range.

At last, Yelkin TTS can overcome the CB-18 effect.This and Barry, people's such as C.E. (United States Patent (USP) 5,610,198) instruction is opposite.Barry, people such as C.E. (United States Patent (USP) 5,610,198) classify Yelkin TTS as the adjuvant of its invention.Among the present invention described herein, during this phosphatide can play a role and and CB-18 (being similar to the electrostatic interaction of itself and quaternary salt) or as a kind of competitive inhibitor (in action site interference CB-18 effect).As if because CB-18 has neutral net charge, a kind of hypothesis in back more likely (that is, will have minimum stable electrostatic interaction).If lipid such as Yelkin TTS is as competitive inhibitor, the shortage of this soluble PAE so.

If as Thornton WO 95/27076 is advised CB-18 is isolated from the mixtinite, trimethyl-glycine sample stain remover can't work as tensio-active agent so.If mycobacterium is allogenic at the action site of CB-18 effect, then different clinical separation strains should have different performance (embodiment 7) in the presence of different trimethyl-glycine sample stain removers.In addition, enter target site if the change permeability is beneficial to therapeutical agent, and allogenic clinical separation strain group is arranged for antibiotic target site, expection also has the variation (as embodiment 5,6 findings) on this level so.Estimate that trimethyl-glycine sample stain remover and different antibiotic are combined in the behavior aspect and show significant variability.The analysis of this information to two different action sites trimethyl-glycine sensitivity test just will provide.

The present invention can locate one or more such resistance mechanisms (that is, permeability, dormancy or molecule consistency).Following explanation is not in order to support following hypothesis, just illustrates application of the present invention as a kind of means: the delay of growth may be to be responsible for the performance that the mechanism of dormancy is stimulated.For example, because most antibiotics has the natural transformation period, metabolic termination descends up to antibiotic toxic level if the CB-18 effect causes mycobacterium, and the CB-18 effect can be used to distinguish the character of clinical separation strain with regard to the propagation feature so.This information is being valuable to the clinician aspect used medicine and treatment time.

Other mechanism is permeability-related with mycobacterium.Following explanation is not in order to support following hypothesis, just illustrates application of the present invention as a kind of means: trimethyl-glycine sample stain remover can change the permeability of mycobacterium by the conformation of modifying the mycolic acid composition.Net result be bring out to antibiotic susceptibility.The information that is obtained by the present invention is being valuable to the clinician aspect the medicine that is used for the most effective treatment.

The CB-18 effect may be that this notion as a result that mycolic acid modification pattern changes is based in part on Yuan, people such as Y., and national academy of sciences institute reports the work of 93:12828-12833 (1996), and Detailed Inspection in embodiment 9.In a word, directly relevant to the modification of mycolic acid with the flowability of cell walls.Some modifies the flowability that reduces cell walls, and the decline of cell walls flowability can be relevant with the reduction of permeability.The low permeability of mycobacterium is considered to play an important role in antimicrobial resistance, and (national academy of sciences institute reports 92:6630-6634 (1995) for Yuan, people such as Y.; Brennan, people such as P.J. state (Annu.Rev.Biochem.) 64:29-63 (1995) bioid academic year).Remember Connell, people such as N.D. " tuberculosis. pathogeny, protection and control ", Bloom; B.R. compile ASM press, Washington; D.C. in (1994) the 333-352 pages or leaves, with Mycobacterium chelonei to the resistance of cynnematin uniquely owing to permeability.

(Figure 39 A) taken place in the modification of the mycolic acid enzyme reaction by a series of relevant dependence S-adenosylmethionines (SAM).Barry, people such as C.E. (U.S.5,610,198) instruction can suppress modification (Figure 39 B) to this SAM of dependence of mycolic acid with thia fatty acid derivative (" sulfo-tetracosanoic acid "), treats the infection due to the pathogenic mycobacterium.The transition state structures of sulfo-tetracosanoic acid is similar to trimethyl-glycine sample stain remover (national academy of sciences institute reports 93:12828-12833 (1996) for Yuan, people such as Y.), especially based on the carboxybetaine (table 1 and Figure 39 B) of sulfonium.The sulfonium carboxybetaine does not need enzyme catalysis to activate, and only suppresses the enzyme that these rely on SAM.

Compare with beet sample stain remover of the present invention, the sulfo-tetracosanoic acid is extremely insoluble.In other words, the Krafft temperature of sulfo-tetracosanoic acid is on physiological standard (for example 37 ℃).Its conveying will have remarkable obstacle.The sulfonium carboxybetaine is more solublely (even to work as R ₁During=18-20, but especially work as R ₄〉=3 o'clock (tables 1; Laughlin, R., Langmuir 7:842-847 (1991))).The trimethyl-glycine spline structure directly solves solubility problem and solves transportation problem to a certain extent.For example, if intravenously is carried, sulfo-tetracosanoic acid most probable is a solid phase.Because trimethyl-glycine is more solvable, because the low Krafft temperature of trimethyl-glycine makes the intravenously of low dosage carry more feasible.

Also openly the sulfo-tetracosanoic acid is special to the slow living mycobacterium of causing a disease for Barry, people such as C.E. (United States Patent (USP) 5,610,198).Barry, people such as C.E. (United States Patent (USP) 5,610,198) further openly have only also disturb mycolic acid synthetic microbiotic could with these thia fatty acid derivatives synergy.The present invention shows that trimethyl-glycine sample stain remover is applicable to the bacterium (that is, excellent bacillus, Nocardia bacteria and mycobacterium (embodiment 9)) that contains the mycolic acid structure on a large scale, and the present invention also can use large-scale microbiotic (embodiment 5 and embodiment 6).

The synthetic of sulfonium carboxybetaine can be according to synthesizing shown in Figure 39 C, but use methods known in the art to realize that any method of identical purpose all is acceptable (March, " senior organic chemistry. reaction, mechanism and structure " (Advanced Organic Chemistry.Reactions, Mechanismsand Structures) the 4th edition, John Wiley ﹠amp; Sons, New York, NY (1992); With people such as Fieser, " organic synthesis reagent " (Reagents for Organic Synthesis) 1-16 volume, John Wiley ﹠amp; Sons, New York, NY (1992), two pieces all are incorporated herein by reference at this).

In a word, not in order to carry out following explanation, the result of CB-18 effect trimethyl-glycine sample stain remover and the interphase interaction of specific function site seemingly.If this interaction is a permeability of modifying the bacterium that contains the mycolic acid structure, then net result is to have increased the effective concentration of microbiotic at target site.For example, for the strain isolated that derives from the CB-18 trial test (table 7 and table 8), if these mycobacteriums all show heterogeneity at target site (be peptidoglycan synthetic) and β-Nei Xiananmei site, then in the trimethyl-glycine sensitivity test, be contemplated to even the rate of distinguishing of higher degree.Use a kind of particular separation strain in a kind of situation, but because this strain isolated of deficiency of permeability can be escaped antibiotic effect to specific beta-lactam sensitivity.If trimethyl-glycine sample stain remover can improve the permeability of this strain isolated, then perhaps β-Nei Xiananmei has or does not perhaps have the antibiotic ability of destruction before playing a role.If it is not enough on the ability of destroying specific beta-lactam structure to derive from the β-Nei Xiananmei of described strain isolated, then changing permeability is enough to bring into play destructive effect, is viewed as the CB-18 effect.Reasonably expection, for the action site of three kinds of differing moleculars discussing among this embodiment (beta-lactam, trimethyl-glycine sample stain remover and β-Nei Xiananmei), the different bacterium that contains the mycolic acid structure has significant textural difference.Therefore, because a large amount of obtainable microbiotic (especially beta-lactam) that can be used in combination with a large amount of obtainable trimethyl-glycine sample stain removers, the rate of distinguishing of height is possible.

A kind of sensitivity test according to the present invention makes beta-lactam become possibility as the application of adjuvant in the antituberculosis therapy, and this test is very useful, because the Antibiotique composition kind of the most frequently used up to now and best sign is a beta-lactam.Because these antibiotic deep-going and popularity, will have tangible advantage with the ability of these pharmaceutical treatment mycobacterial infectionses.Limitedly successfully attempted the application of beta-lactam in being used for treating the treatment plan of mycobacterial infections.Chambers for example, people such as H.F., biocide chemotherapy 39:2620-2624 (1995) have checked 5 kinds of MTB clinical separation strains and have checked several different types of beta-lactams.Most of strain isolateds have resistance to penicillin and most of cynnematin; Yet imipenum (a kind of carbapenem) shows certain active.With the combination of nearly all beta-lactam and a kind of beta-lactamase inhibitor is that to reach remarkable susceptibility necessary.

As mentioned above, beta-lactamase inhibitor disturbs the antibiotic resistance mechanism of biological degradation.An example is arranged, and wherein antibiotic therapy has been located a kind of resistance mechanism, and strengthens any chemotherapy based on beta-lactam thus.By the location resistance mechanism enlarge mycobacterium to microbiotic especially to the ability of the susceptibility of beta-lactam antibiotics, this effectively has important potentiality in the treatment mycobacterial infections.The present invention described herein uses the molecule of a kind of mechanism in location, and this mechanism influences the resistance of bacterium in the former mode of not describing.To the analysis of strain isolated 512-JHH (Figure 24) and 538-JHH (Figure 25), and CB-18 has the fact of remarkably influenced (table 9) to the strain isolated of anti-tubercle bacillus extract for treating in acceptor, emphasized that the treatment of trimethyl-glycine sample stain remover is used.

Narrated the present invention now fully, it will be understood by those of skill in the art that under the situation of the spirit or scope that do not influence the present invention or its any embodiment, can be at broad and quite carry out the present invention within the condition, parameter or the like of scope.All be incorporated herein by reference at this at these all reference of quoting.EXAMPLE Example 1CB-18 handles with mycobacterium and cultivates

Initial owing to handle the observation of being carried out in the Study on new method of clinical samples relating to for the mycobacterium detection, and conceived the present invention described herein.These methods are based on the method for the Thornton described in WO 95/27076, and are used for from clinical samples, separate bacterium, the especially mycobacterium that contains the mycolic acid structure from the respiratory tract sample the most especially as main methods.Narrated the method for Thornton WO 95/27076 below, and in the last preparation of narrating this method agents useful for same of present embodiment.

(1) 1-10ml phlegm or irrigation of bronchus liquid are placed the 50ml tapered tube

Attention: although the respiratory tract sample is the main sample type that expectation is used in combination with method described herein, other sample type such as water, soil, tissue, ight soil etc. also can be suitable for being used in combination with following method.Wherein some sample at first should be clarified, and method is a resuspension in water or damping fluid, and make the Spin-X II post of mixture by being equipped with 20-60 micron agglomerated material (frit) (Corning Costar, Boston, MA).This post also can contain a kind of matrix such as Sephadex ^(Sephadex G-50 ^: Pharmacia, Piscataway, NJ) or a kind of suitable resin to strengthen purifying.Any then sample can both as described belowly be handled.

(2) in sample, add isopyknic 0.5%NALC liquefaction solution (as follows: 0.5%

The NALC/25mM Trisodium Citrate) also vibration.

(3) incubation 10 minutes (vibrated about 5 minutes, and promptly carried out next step then) at room temperature.

(4) open test tube, in sample, add filtering sterilized water to final volume and be about 35ml (notes

The meaning: use filtering sterilized water (for example, GIBCO/BRL, Gaithersburg, MD,

Catalogue #:15230-022)).

(5) add the concentrated solution that 4ml contains trimethyl-glycine sample stain remover, for example 10 * CB-18 buffering

Liquid (as follows): 10 * CB-18=10mM CB-18,0.5M Tris-HCl pH8.0,50mM

NALC and 1mM NaCl.

(6) fully vibrate with complete mixing sample.

(7) shaking following (140 rev/mins) 37 ℃ of incubations 90 minutes.

(8) vibration, then in 30 ℃ with the centrifugal sample of 4,000 * g 20 minutes.

(9) abundant decantation test tube adds 500 μ l sterilized water or damping fluids in sample.

(10) fully resuspension precipitates piece, and the preparation precipitation is used for detecting, and for example passes through acid-fast bacterium

Staining (being microscopy), cultivation, nucleic acid amplification or immunodiagnosis.

Preliminary study is used for estimating to break through the character of the pollutent of the BACTEC 12B liquid nutrient medium that has added PANTA, and its uses the depleted respiratory tract sample (n=277) from Quest Diagnostics-Baltimore.(note: BACTEC 12B liquid culture system is used to one of standard method of cultivating mycobacterium in this area, PANTA is a kind of microbicidal additives, contain PXB, azlocillin, nalidixic acid, trimethoprim and amphotericin B: this culture systems is referred to herein as " 12B/PANTA " (Becton Dickinson, Cockeysville, MD.)).Make a collection of specimens and handle with CB-18 according to aforesaid method.With every kind of throw out transferred species of about 400-500 μ l on 12B/PANTA.Identify all contaminants by morphology and/or gramstaining, thereby differentiate to be oxydase/catalase, positive or negative.Make pollutent form species (speciate) then, and use MicroScan ^(Dade, West Sacramento CA) measure antibiotics sensitivity to table.The results are shown in the table 2.

The evaluation (n=277) of table 2 BACTEC 12B/PANTA pollutent
			Group	????#	????％
Gram-negative	????48	????84.2％
			Gram-positive	????5	????8.8％
Yeast	????3	????5.3％
			Fungi	????1	????1.8％
Sum:	????57
			From 40 patients: pollute=14.4%

Table 2 demonstration is separated to 57 kinds of pollutents from 40 samples.Pollution rate based on each sample is 14.4% (40 ÷ 277).This prompting, the subject matter that the respiratory tract sample of handling according to WO 95/27076 (for example CB-18) runs into is exist (＞84%) of gram-negative biological.About 31 kinds (64%) are enterobacteriaceae (Enterobacteriaceae) in 48 kinds of Gram-negative strain isolateds.Modal strain isolated be providencia stuartii (Providencia stuartii) (n=13), Rhodopseudomonas (Pseudomonas) plants (n=11) and Proteus mirabilis (Proteusmirabilis) (n=7).Therefore, the remarkably influenced of pollution rate is needed the reduction of gram-negative biological incidence.Sensitive data shows, 40 kinds of ceftazidime sensitivities to 8 μ g/ml in 48 kinds of Gram-negative bacterias.Because these data and other result of experiment, decision should be used ceftazidime (8 μ g/ml final concentration) potentiate antibiotics additive PANTA, to attempt to control this gram-negative contact scar (the 12B/PANTA system that adds ceftazidime (caz) is referred to herein as " 12B/PANTA/caz ").According to the parallel laboratory test of using several ATCC mycobacterium type bacterial strains, think that ceftazidime can reduce the incidence of gram-negative contact scar, and the viability of not remarkably influenced mycobacterium.Preparation (1) 20 * buffering salt of 10 * CB-18 damping fluid: 1M Tris-HCl pH8.0,2mM NaCl

Tris alkali (121.14 gram/mole)	54.27 gram
		Tris HCl (157.64 gram/mole)	87.02 gram
NaCl	0.117 gram
		Add water to:	1 liter

(i) in 1 liter of graduated cylinder, add about 250ml water.

(ii) add Tris alkali (Sigma, St.Louis, MO, catalogue #:T 1503), Tris-HCl (Sigma, St.Louis, MO, catalogue #:T 3253) and NaCl (Sigma, St.Louis, MO, catalogue #:S 7653), mix and (note: use filtering sterilized water (for example, GIBCO/BRL, Gaithersburg, MD, catalogue #:15230-022)).

(iii) add remaining water to 1 liter, and guarantee abundant mixing.

(iv) check pH by taking out an aliquot.PH should be ± 0.2pH unit.

(v) filtration sterilization (0.22 μ filter membrane) is divided into the 50ml equal portions, stores under the room temperature.(2) 100 * CB-18 stock solutions: 100mM CB-18

* CB-18 (383 gram/mole)	1.915 gram
		Virahol: water (1: 1) extremely:	50ml

* CB-18:N, N-dimethyl-N-(Octadecane base)-N-(3-carboxylic propyl group) ammonium inner salt

(CAS ^No.78195-27-4)

(i) with 25ml AG Virahol (Baxter, McGaw Park, IL, catalogue #:3043-1 NY) mixes in graduated cylinder with 25ml water, to prepare 1: 1 Virahol of 50ml: water (is noted: use filtering sterilized water (for example, GIBCO/BRL, Gaithersburg, MD, catalogue #:15230-022)).

(ii) with the 25ml Virahol: water (1: 1) solution is transferred in the 50ml tapered tube.

(iii) take by weighing 1.915 gram CB-18, be placed on and contain 25ml residue Virahol: in the graduated cylinder of water (1: 1).Vortex vibration mixing.

(iv) add more Virahol: water (1: 1) solution, to about 40ml, and vortex vibration gently.Left standstill solution about 20 minutes, approximately vortex vibration was once gently every 5 minutes.

(v) after the CB-18 dissolving (about altogether 30 minutes), use Virahol: water (1: 1) solution makes final volume reach 50ml, puts upside down mixing.

(vi) solution is divided in two 50ml aseptic plastic tapered tubes, store under the room temperature.(3) 10 * CB-18 damping fluids

(i) determine pending number of samples, should numeral insert in the following table (this numeral adds 1, to guarantee enough damping fluids), calculate the final quantity of required every kind of composition, and 10 an amount of * CB-18 of preparation as described below.

Composition	Multiplication factor				Final quantity
						20 * buffering salt	????2ml	??×	Number of samples+1	＝
??100×CB-18	??400μl	??×	＝
					NALC (163.2 gram/mole)	0.033 gram	??×	＝
Add water to	????4ml	??×	＝

(ii) merge 20 * buffering salt, NALC (Fluka, Ronkonkoma, NY, catalogue #01039) and 100 * CB-18, making it to reach volume required with filtering sterilized water (notes: use filtering sterilized water (for example, GIBCO/BRL, Gaithersburg, MD, catalogue #:15230-022)), use immediately.

Attention: in this method whenever,, be sure not to use if there is precipitation in the damping fluid.This solution should be incubated (for example, being higher than 20 ℃) in storing and using.Do not refrigerate this solution.Preparation (1) 10 * Trisodium Citrate stock solution: the 0.25M two hydration Trisodium Citrates of 0.5%NALC liquefaction solution

Two hydration trisodium citrates (294.1 gram/mole)	7.35 gram
		Add water to:	100ml

(i) adding about 25ml water in 100 graduated cylinders (notes: use filtering sterilized water (example

As, GIBCO/BRL, Gaithersburg, MD, catalogue #:15230-022)).

(ii) add two hydration trisodium citrate (Sigma, St.Louis, MO, catalogue #:C

3434), the vortex vibration mixes.Add remaining water to 100ml, put upside down mixing.

(iii) filtration sterilization (0.22 μ filter membrane), five equilibrium is in the 50ml tapered tube.Store under the room temperature

Deposit.(2) 0.5%NALC liquefaction solution (prepared fresh every day)

(i) determine the approximate volume of required NALC liquefaction solution.

(ii) in 50ml tapered tube or graduated cylinder, merge 10 * Trisodium Citrate stock solution and NALC

(Fluka, Ronkonkoma, NY, catalogue #01039), water add to final volume and (annotate

The meaning: use filtering sterilized water (for example, GIBCO/BRL, Gaithersburg, MD,

Catalogue #:15230-022)).

(iii) use immediately, abandon the not part of usefulness.

About # sample	??3-10	??7-25	??12-37	??15-50
					10 * Trisodium Citrate stock solution	??2.5ml	??5ml	??7.5ml	??10ml
NALC (163.2 gram/mole)	0.12 gram	0.25 gram	0.38 gram	0.5 gram
					Add water to:	??25ml	??50ml	??75ml	??100ml

The preparation of ceftazidime stock solution and 12B/PANTA/caz (1) ceftazidime stock solution (36mg/ml)

Ceftazidime	72mg
		The 1M sodium bicarbonate	85.6μl
Add water to:	2ml

(i) in the 2ml volumetric flask, mix 1ml water and 1M sodium bicarbonate (Sigma, St.Louis, MO: catalogue #:S 6297 (dissolving 8.40 gram sodium bicarbonates in 100ml water, sterile filtration, equal portions with 10ml and 1ml are frozen)) (note: use filtering sterilized water (for example, GIBCO/BRL, Gaithersburg, MD, catalogue #:15230-022)).(ii) add ceftazidime (Sigma, St.Louis, MO: catalogue #:C 3809),

Water makes volume reach 2ml immediately.

(iii) put upside down mixing gently, clarify up to solution.Be sure not at the above heated solution of room temperature

(for example, not warm this solution in hand).

(iv) five equilibrium 50 μ l part is stored in-70 immediately in the 1.5ml Eppendorf tube immediately

℃ up to use.(2) reinforcement of PANTA/caz and use

(i) from refrigerator, take out freeze dried PANTA and reconstruction liquid (R.F.), from refrigerated tank

The middle ceftazidime stock solution (36mg/ml) that takes out 1 part of 50 μ l equal portions.

After (ii) ceftazidime melts, use 1 5ml syringe, to cephalo thiophene first carboxylic

Add 1ml R.F. in the oxime stock solution.By discharging once in its inhalation syringe

And mix.Use syringe, entire contents is transferred in the PANTA bottle.

(iii) in the PANTA bottle, add (the final volume=5ml) of R.F. more than 4 milliliters.Label

“PANTA/caz”。

(iv) use and add 100 μ l PANTA/caz in each 12B bottle of forward direction (at the adding sample

Add microbiotic in 2 hours).

(v) the part that will not use is stored in-20 ℃.Abandon after 48 hours and (for example, do not freeze

Melt above 1 time).Embodiment 2 CB-18 trial tests

Handle the respiratory tract sample with CB-18 and be used for mycobacterium (acid-fast bacilli: AFB) detect, to attempt to estimate the method for Thornton WO 95/27076.Respiratory tract sample (n=573) is from the TB laboratory of QuestDiagnostics-Baltimore (BAL), and Quest Diagnostics-Teterboro (TBR), Washington, Maryland (Maryland) university (UMB) of D.C. laboratory office (BOL), Johns HopkinsHospitals (JHH) and Baltimore separate and collect.The source place with each sample separately, make in the locality with standard NALC/NaOH method (Kent, P.T. wait the people, " public health mycobacterium ", in " three grades of lab guides ", U.S. sanitary and human service department, Center for Disease Control, (1985) 31-46 pages or leaves) handle half of each sample, and transferred species is in liquid (BACTEC 12B or BBL MGIT) and solid medium (7H11, L-J or SeptiCheck), and second half of each sample delivered to the Quest-Baltimore facility, as being that sample is handled with CB-18 in the basis as described in the embodiment 1 with the every day.As mentioned above CB-18 is precipitated transferred species on 12B/PANTA/caz and 7H11 selectivity inclined-plane.Carry out all smear analyses according to the method (" public health mycobacterium " is in " three grades of lab guides ", U.S. sanitary and human service department, Center for Disease Control, (1985) 57-69 pages or leaves for Kent, people such as P.T.) of recommending.The result of this research is called " CB-18 trial test " at this.

In 573 samples handling, there are 106 AFB to cultivate positive sample according to all methods (that is,, cultivating) according to liquid or solid according to NaOH or CB-18.8 mycobacterium gordonae strain isolateds are arranged.They are difference, gram-negative and five equilibrium (that is, CB-18 has missed isolating 4 samples with NaOH, and vice versa) all between two kinds of methods.Owing to think that the mycobacterium gordonae strain isolated is the pollutent (Wayne with limited clinical meaning, L.G. wait the people, clinical microbiology summary (Clin.Microbiol.Rev.) 5:1-25 (1992)), so it is omitted from following analysis: therefore analyzed 98 AFB and cultivated positive sample.Table 3 has been summed up the cultivation results of no mycobacterium gordonae strain isolated.

Negative sample and 46 inconsistent samples that the positive sample of 52 unanimities, 467 unanimities are arranged.NaOH identifies 61 positive samples altogether, and susceptibility is 62.2%.CB-18 identifies 89 positive samples altogether, and susceptibility is 90.8%.CB-18 makes gathering cultivation susceptibility improve about 46%.

The gathering cultivation results of table 3 CB-18 trial test
		n＝573 CB-18	NALC/NaOH+-+52 37 90.8%-9 467 62.2% amount to AFB=98

Cultivate in the positive sample at 98,69 is the mycobacterium (MOTT) of non-tuberculosis, and 29 is MTB.NaOH identifies 33 MOTT positive samples and 28 MTB positive samples.CB-18 identifies 64 MOTT samples, but 25 MTB samples only.Cultivation susceptibility in the sample that NaOH handles is respectively 47.8% and 96.6% for MOTT and MTB.Cultivation susceptibility in the sample that CB-18 handles is respectively 92.8% and 86.2% for MOTT and MTB.CB-18 makes the cultivation susceptibility in the MOTT disease positive sample improve 94.1%, but makes the cultivation susceptibility in the MTB disease positive sample reduce by 12.1%.

Table 4 shows the result that smear is analyzed.It also is the smear male by arbitrary treatment process that 61 cultivation positive samples are arranged.Have 18 samples all to have identical smear value by two kinds of treatment processs, have 19 samples wherein two kinds of methods all reported the smear positive findings, but the value of CB-18 report is higher.There is not the example that NaOH has higher smear value in the smear positive sample.23 examples wherein CB-18 report smear positive findings and NaOH report smear negative findings are arranged.Has only NaOH report smear positive findings in the example and CB-18 report smear negative findings.Identify 98 38 of cultivating in the positive sample according to smear NaOH, susceptibility is 38.8%, and identifies 60 18 of cultivating in the positive sample according to smear CB-18, and susceptibility is 61.2%.Therefore, CB-18 makes the smear susceptibility improve about 58%.Table 4 shows that also the increase of smear susceptibility is the result that mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (MOTT) both increase.

The smear analysis of table 4 CB-18 trial test
					All	????MOTT	????MTB
Identical smear value CB-18 smear value＞NaOH smear value NaOH smear value＞CB-18 smear value CB-18 smear and NaOH smear NaOH smear and CB-18 smear	????18 ????19 ????0 ????23 ????1	????8 ????9 ????0 ????16 ????1	????10 ????10 ????0 ????7 ????0
				Total AFB cultivates in total of cultivation and the smear research	????61 ????98	????34 ????69	????27 ????29
NaOH smear susceptibility CB-18 smear susceptibility	????38.8％ ????61.2％	????26.1％ ????42.8％	????69.0％ ????93.1％

Using CB-18 smear susceptibility increases, but is respectively 99.8% and 96.8% by the specificity of NALC/NaOH and the analysis of CB-18 smear.There are a large amount of samples to be reported as " smear ± " but the conclusive evidence of not carrying out CB-18 is cultivated through CB-18.CDC guide (Kent, P.T. wait the people, " public health mycobacterium ", in " three grades of lab guides ", U.S. sanitary and human service department, Center for Disease Control, (1985) 57-69 pages or leaves) the indication laboratory is called these smear ± results in the following text in the situation of not proving conclusively cultivation results and is " suspicious ".

When handling sample, exist with CB-18 a large amount of especially smears ±.Every kind of method of independent analysis promptly replaces the result of another kind of treatment process, and comprises these smear ± results, obtains very different results.For example, the data presentation of table 5,37 that cultivate in 61 samples of male for NALC/NaOH also is the NALC/NaOH smear positive.Cultivate in the positive CB-18 sample at 89,56 also is the CB-18 smear positive.The susceptibility and the specificity of the analysis of NALC/NaOH smear are respectively 60.6% and 99.6%, and CB-18 is respectively 62.9% and 91.2%.

In 31 smear ± results that CB-18 finds (5+26=31), about 42% (n=13) cultivates through other method or from other sample conclusive evidence, perhaps can join, perhaps have positive amplification (that is, confirming the existence of mycobacterium DNA) with former disease-related.(note: according to the CDC guide, 16 smear 1+ and 2+CB-18 cultivate negative sample and are considered to the CB-18 smear positive-therefore initial specificity reduce (99.8% pair 96.8% (table 4)) in the analysis of table 4.) in a word, susceptibility improves (table 3) comprehensively although CB-18 makes cultivation, according to the analysis shown in the table 5, the smear specificity of NALC/NaOH and CB-18 is respectively 99.6% and 91.2%.

Results suggest shown in the table 5, CB-18 handles not influence of smear technique itself.For example, for the adhesion for slide glass, CB-18 does not change " viscosity " of bacterium.This is based on following observation: with respect to NALC/NaOH, CB-18 makes the smear susceptibility of cultivating positive sample improve 58% (table 4); Yet, with regard to the quantity of the cultivation positive sample chosen through smear by this method, every kind of method roughly the same (table 5:60.6% is to 62.9%).In other words, cultivate and to remain more responsive diagnostic techniques, but CB-18 seems and influences total susceptibility of two kinds of methods roughly the samely.

The independent assessment of two kinds of different treatment methods of table 5
			The smear value	????NALC/NaOH	??????CB-18
??AFB???AFB?	??AFB???AFB?
		Feminine gender ± 1+		????24????502 ????3?????0 ????7?????1	????33????434 ????5?????26 ????8?????14

????2+ ????3+ ????4+	??????8??????????????????1 ??????5??????????????????0 ??????14?????????????????O		??????6??????????????????2 ??????7??????????????????0 ??????30?????????????????0
					Sum	??????61?????????????????504		??????89?????????????????476
Smear:	Susceptibility	Specificity	Susceptibility	Specificity
					????60.6％	????99.6％	????62.9％	????91.2％

In a word, the cultivation feminine gender of identifying by every kind of method and the smear positive are (promptly, smear+with smear ± both) quantity of sample is obviously different: NALC/NaOH only reports that 2 smears are positive and cultivate negative sample, and 42 smear positive samples of CB-18 report (comprise CB-18 smear ± but cultivate negative sample: table 5).These smear ± results' correctness problem (being the specific reduction of smear) is significant.Especially, if above-mentioned hypothesis is correct (for example, CB-18 influences total susceptibility of two kinds of detection methods) roughly the samely, then CB-18 reduces the specificity that smear is analyzed.This is significant to the laboratory technician.If should hypothesis incorrect, and these are effective smear results, show that in fact these patients have infected mycobacterium, and perhaps the susceptibility of CB-18 smear analysis is higher than shown in table 4 or the table 5 so.This a kind of conclusion in back is significant to the doctor.In order to understand these results, the analysis shown in carry out table 6.

Independent check the liquid of two kinds of different treatment methods in the CB-18 trial test, using and the susceptibility (table 6) of solid culture.Analyze the quantity of cultivating positive sample by the isolating AFB of specific cultural method (liquid is to solid) for treatment process (NaOH is to CB-18).For 61 NaOH cultivated positive samples: 45 liquid and two kinds of cultivations of solid were all positive, and 15 only liquid culture is positive, and 1 only positive on solid medium.Checked that 89 CB-18 cultivate positive samples: 42 liquid and two kinds of cultivations of solid are all positive, and 15 only liquid culture is positive, and 32 only positive on solid medium.The susceptibility of liquid culture is respectively 98.4% and 64.0% when handling with NaOH and CB-18.The susceptibility of solid medium is respectively 75.4% and 83.1% when handling with NaOH and CB-18.

The sample expected result of handling for NaOH is: liquid culture is than the cultivation on the solid medium about responsive 31%.On the contrary, when handling sample (as described in embodiment 1) with CB-18, and transferred species is when 12B/PANTA/caz and 7H11 selectivity inclined-plane, solid medium approximately responsive 30%.

The comparison of table 6 liquid culture and solid medium
					Cultivation results	The AFB sum	Susceptibility
The independent solid of the independent liquid of liquid and solid	Liquid solid
		NaOH smear smear	????31???????6?????????0 ????14???????9?????????1		????37 ????24		????100％???83.8％ ????95.8％??62.5％
Sum	????45???????15????????1			????61		????98.4％??75.4％
CB-18 smear smear	????38???????4?????????14 ????4????????11????????18					????56 ????33	????75.0％??92.9％ ????45.4％??66.7％
		Sum	????42???????15????????32	????89	????64.0％??83.1％

Significant and unexpected two minutes phenomenons (dichotomy) are arranged in these results.For example, improve about 46% (table 3) although assemble the cultivation susceptibility, this increase is just because the isolating increase of MOTT (that is, MTB cultivation susceptibility reduces).Obviously opposite is comprehensive raising of smear susceptibility therewith, and wherein contribution almost comes from MOTT and two kinds of positive samples of MTB (table 4) comparably.

Thornton WO 95/27076 suggestion, the susceptibility that improves is because (i) reduction of the harmful effect of relevant processing with NaOH, (ii) owing to the centrifugal organic efficiency that improves of the biological inherent buoyancy of compensation, and (iii) because the detection possibility that the dispersion of the biology of one-tenth rope increases.The cultivation susceptibility that improves may be the combination owing to all these three aspects, and the smear susceptibility that improves may be because dispersion or enhanced reclaim (smear and viability have nothing to do).Because the MOTT biology generally can not form the strap of degree as the MTB bacterium, so the smear susceptibility that improves is most likely owing to reclaim enhancing.In addition, instinctively, viability is an importance of cultivating susceptibility certainly.Phenomenon derived from the following fact in two minutes: reclaim enhancing in two kinds of biologies of smear data presentation MOTT and MTB, but cultivate the viability that improves in the data presentation MOTT biology and the viability that reduces in the MTB biology.

Liquid-solid is cultivated reverse (table 6) prompting of susceptibility in the sample that CB-18 handles, and two fens phenomenons are results of microbiotic associating in CB-18 and the PANTA/ ceftazidime additive.For example, relatively the solid culture susceptibility of the smear of the two kinds of different treatment methods positive and the negative sample of smear shows tangible similarity.When handling with NALC/NaOH in the smear positive sample solid medium susceptibility higher by 34% than the negative sample of smear, high by 39% when handling with CB-18.The identical check of liquid culture data shows, when handling with NALC/NaOH smear positive with smear feminine gender sample between susceptibility less difference (for example, 4% difference) is arranged, and there were significant differences (for example, 65% difference) between on the same group with the phase of CB-18 processing.75% liquid culture susceptibility is unexpectedly and very uncommon (these 14 samples comprise 5 MTB and 9 MOTT:4 MAC in the smear positive sample of handling with CB-18,2 mycobacterium fortutitums, each 1 of Mycobacterium chelonei, Si Shi mycobacterium and mycobacterium kansasii).For the smear positive sample of handling with NALC/NaOH, 100% liquid culture susceptibility is not serendipitous.Stone, people such as B.L., clinical microbiology magazine 35:1030-1031 (1997) report, the liquid culture susceptibility of (handling with NALC/NaOH) smear positive sample also are that 99.3% (this is once bigger research: n=439).

Draw two conclusions from these data.The first, the large-scale mycobacterium of CB-18/12B/PANTA/caz systematic influence.For example, although 5 kinds of MTB strain isolateds that leaked because its social importance and very obvious, the MTB strain isolated only accounts for 30% (29 ÷ 98) of all AFB strain isolateds in 36% (5 ÷ 14) of the negative sample of the smear positive-liquid culture and this research.Similarly, the MOTT strain isolated accounts for 70% (69 ÷ 98) of all AFB strain isolateds in 64% (9 ÷ 14) of the negative sample of the smear positive-liquid culture and this research.Because the ratio of affected strain isolated is identical, this is a phenomenon that extensively is suitable for seemingly: CB-18 and antibiotic harmful combination are similar to the influence of MTB and MOTT.

The second, the influence of CB-18/12B/PANTA/caz system may be to rely on inoculum.For example, be similar to Eng, R.K. wait the people, biocide chemotherapy 26:42-47 (1984) is reported for Pseudomonas aeruginosa (Pseudomonas aeruginosa), and the minimum inhibition concentration of ceftazidime (MIC) increases with the inoculum that increases progressively.Because smear is the direct reflection of per unit volume biomass, thus the CB-18/12B/PANTA/caz system more can influence the negative sample of smear-so the positive liquid culture susceptibility with smear feminine gender sample of smear between 65% difference is arranged.

In this research, use CB-18 with the concentration of 1mM (383 μ g/ml).CB-18 concentration in the sample of transferred species in BACTEC 12B probably is 35 μ g/ml-7 μ g/ml, and this depends on the amount of remaining " backwash liquor " (that is liquid of original processing) behind the ability of original denseness, CB-18 method liquefaction sample of sample and the decantation.In the poorest a kind of situation (for example, thick dense phlegm), sample can not liquefy well, the original processing matrix of residual large volume.In these cases, substantially with the sample transferred species in treatment soln.Add 400-500 μ l precipitation in BACTEC 12B bottle, final volume ≈ 4.5ml provides the dilution (that is 35 μ g/ml) of 11 times of ≈.Under best situation (for example, rare irrigation of bronchus thing), the matrix of the original processing of residual ≈ 100 μ l.Then with pellet resuspended in 500 μ l water, ≈ is provided 5 times dilution.These samples are about 7 μ g/ml.Although do not know for given sample how much CB-18 concentration is, corresponding supposition CB-18 is dissolved in equably and handles in the matrix, and because CB-18 is not washed off, so for every kind of sample certain concentration range is arranged, is 7 μ g/ml-35 μ g/ml sometimes.

Solid substrate is very different with the liquid counterpart.At first, in the inclined-plane, add littler volume (≈ 100 μ l).This less inoculum has partly been explained the difference of cultivating susceptibility.Secondly, any CB-18 that adds to the inclined-plane reduces to minimum by capillary action: stain remover will be absorbed in the substratum, away from bacterium, make its effect reduce to minimum thus.

In a word, the above-mentioned Notes of Key Data, microbiotic and CB-18 are collaborative deleterious, this action range influences all mycobacteriums.Processing among the embodiment 3CB-18 is to the transferred species among the CB-18

In order to attempt to understand the dynamic interaction that is transferred to the CB-18 in the 12B/PANTA/caz culture systems, designed the experiment that Figure 1A and Figure 1B summarized (note: Figure 1A and Figure 1B are meant identical experiment-because the complicacy of experimental design provides two kinds of different designs of identical experiment).Use two kinds of different isolates: mycobacterium tuberculosis type strains A TCC 27294 (ATCC, Rockville, MD) and derive from the clinical separation strain 571/573-BAL of CB-18 trial test (note: " 571/573-BAL " is meant one of two kinds of strain isolateds, these two kinds of strain isolateds all derive from same patient, but derive from the same day two kinds of different natural specimens submitting to (promptly, 571-BAL and 573-BAL (see Table 8 and relatively Figure 18 and Figure 19))-these two kinds of strain isolateds all show in all respects identical, and in present embodiment and later embodiment, use convertibly).

Every kind of strain isolated is incubated at (7H11 selectivity inclined-plane PXB, Pyocianil, amphotericin B and trimethoprim (Becton Dickinson on Lowenstein-Jensen (L-J) inclined-plane or the 7H11 selectivity inclined-plane, Cockeysville MD) strengthens).Scrape cell from these inclined-planes, place damping fluid or contain the damping fluid (as described in embodiment 1) of 1mM CB-18, then 1000 times (Figure 1A and Figure 1B) of dilution in same buffer.Further before the dilution every kind of bacterium stoste was being shaken following (140 rev/mins) 37 ℃ of incubations 90 minutes.Then with damping fluid or every kind of bacterium stoste of 0.5mMCB-18 serial dilution, produce 25,000 *, 50,000 * and 100,000 * whole diluent (with respect to original stoste (Figure 1A and Figure 1B)).Then with each dilution series a-type double (every part 400 μ l) transferred species in BACTEC 12B bottle, be added with PANTA in the bottle or strengthen and make that the caz final concentration is the PANTA (P/caz) of 8 μ g/ml with caz.The concentration of CB-18 is about 17 μ g/ml (being estimated as between the 7 μ g/ml-3 μ g/ml) in most of bottles as described in embodiment 2 in these experiments.Make regular check on culturing bottle and write down growth index.The growth index of whole 6 culturing bottles in the particular series is average, and to cultivating the fate mapping.Fig. 2 A-2H shows the result of ATCC 27294, and Fig. 3 A-3H shows the result of 571/573-BAL.

Any condition that ATCC 27294 strain isolateds are not generally applied by this experiment example influences: culture condition (L-J is to 7H11) or microbiotic (PANTA is to PANTA/caz) or treatment soln (damping fluid is to CB-18) or transferred species solution (damping fluid is to CB-18) all have no significant effect (Fig. 2 A-2H) to the growth characteristics of ATCC 27294.

In addition, the 571/573-BAL strain isolated is by nearly all aspect remarkably influenced of experimental program.For example, how the behavior of 571/573-BAL is experiment if not only being depended on prepares strain isolated (L-J is to the 7H11 selectivity), and the existence of ceftazidime and the composition of transferred species solution (for example, damping fluid is to CB-18) are also depended in its behavior.In fact, most important parameter seemingly in the transferred species solution CB-18 have (comparison diagram 3A and a Fig. 3 B; 3C and 3D; 3E and 3F; And 3G and 3H).In damping fluid, handle and the cell of transferred species in CB-18, before lucky transferred species, just contact with CB-18.In Fig. 3 F, the strain isolated of transferred species is not grown in the presence of P/caz.In fact, be used in Fig. 3 F and Fig. 3 H generating in 12 bottles of P/caz growth curve and have only 3 actual growths.

It seems that actual treatment has minimum influence to growth characteristics.For example, contrived experiment can be handled and dilute cell in CB-18, makes that the concentration of CB-18 is about 0.68 μ g/ml (@25 in the culturing bottle, 000 *), 0.34 μ g/ml (@50,000 *) to 0.17 μ g/ml (@100,000 *).Growth characteristics does not have significant difference between these different extent of dilution.Comparison diagram 3A and Fig. 3 C and Fig. 3 E and Fig. 3 G show, can handle cell in CB-18, but as long as the CB-18 concentration in the culturing bottle is lower than for example about 1 μ g/ml, growth is just not interrupted.In other words, there be not " post antibiotic effect " (in " drug susceptibility in the mycobacterial infections chemotherapy ", Heifets, L.B. compiles, CRC press, Boston, MA (1991), 13-57 page or leaf for Heifets, people such as L.B.) with " CB-18 ".

In a word, with the above-mentioned data presentation of CB-18 trial test bonded, different isolates performance is different, and the performance of these strain isolateds influences the susceptibility of strain isolated (for example, with before microbiotic contacts: cultured cells on the 7H11 selectivity (Fig. 3 E-3H)).Yet most important ground, the existence of trimethyl-glycine in the substratum (for example CB-18) has aggravated these strain isolateds to also being present in the antibiotic susceptibility in the substratum.The adding of embodiment 4 Yelkin TTS overcomes the CB-18 effect: the detection of lower concentration inoculum reaches the comparison with quaternary ammonium salt

Embodiment 3 described experiments (Figure 1A and Figure 1B) show that CB-18 further improves as the application need of main diagnostic tool.For example, CB-18 must remove from transferred species solution, should use hypotoxic trimethyl-glycine, and perhaps the CB-18 effect must be neutralized.This causes the effect of CB-18 can be by Yelkin TTS to be similar to Patterson, R.A., American Journal of Public Health (Amer.Jour.Public Health) 46:1429-1430 (1956) and Wayne, L.G., the described quaternary ammonium salt of U.S. respiratory tract disease summary 80:912-913 (1959) (for example, Ziphiran ^Or benzalkonium chloride) mode this idea that neutralizes.

It is believed that quaternary ammonium salt has quite general deleterious effect to bacterium and fungal organism.Hugo, W.B. at " No. 19, S.C.I. special topic: the tensio-active agent in the microbiology " London Soc.Chem.Industry, summarize document in (1965) the 69-82 pages or leaves of London, and summed up the effect that quaternary salt causes membranolysis and the general sex change of intracellular protein (for example inactivation).

Patterson, people such as R.A., American Journal of Public Health 46:1429-1430 (1956) and Wayne, L.G., U.S. respiratory tract disease summary 80:912-913 (1959) shows, can overcome the deleterious effect of quaternary ammonium salt in the mycobacterium cultivation with Yelkin TTS (phosphatidylcholine, a kind of phosphatide).Fig. 4 shows a kind of experimental design, and it is used for checking Yelkin TTS whether also can overcome the CB-18 sensitization of 571/573-BAL.

In this experiment, in the 7H11 selective medium, cultivate the 571/573-BAL strain isolated, with the serial dilution of NALC/ citric acid be about 1000 *.In order to attempt to check inoculum how to influence this test-results, prepare three kinds of different stostes, a kind of series is diluted in damping fluid, and a kind of series is diluted in CB-18.Therefore, in damping fluid or 1.0mM CB-18, carry out further serial dilution to 2,500 *, 12,500 * and 50,000 *.By buffered soln (Sigma, St.Louis, MO with these three kinds of stostes and damping fluid or Yelkin TTS; Catalogue #:P5394 (in 100% ethanol, prepare 7.5% Yelkin TTS (3 restrain in 40ml),, use immediately)) is mixed with transferred species solution at 1: 1 with the Tris damping fluid dilution of embodiment 1 with 100 * concentrated solution.The final extent of dilution of three kinds of different transferred species solution is about 5,000 *, 25,000 * and 100,000 *.CB-18 and the Yelkin TTS final concentration in suitable transferred species solution is about 0.5mM.Then with quadruplicate (the every part 400 μ l) transferred species of each series in BACTEC 12B bottle, added reconstruction liquid (R.F.) or contained the PANTA (P/caz) that has added 8 μ g/ml ceftazidimes in the bottle.The final concentration of Yelkin TTS and CB-18 all is about 17 μ g/ml between incubation period.Make regular check on culturing bottle, the record growth index.The growth index of average particular series, and to cultivating the fate mapping.

Also (MD) equal portions to three kinds of stostes (that is, 2,500 *, 12,500 * and 50,000 *) carry out quantitative culture for Becton Dickinson, Cockeysville in the 7H10 flat board by transferred species.The 7H10 Analysis of Plate proves, nearly 165 ± 54 colony-forming units of each bottle (cfu) in 5,000 * series, nearly 33 ± 11 cfu of each bottle in 25,000 * series, nearly 8 ± 3 cfu of each bottle in 100,000 * series.Fig. 5 A shows transferred species in damping fluid or contain 5,000 * series in the damping fluid of Yelkin TTS, Fig. 5 B show transferred species in CB-18 or with the CB-18 of Yelkin TTS combination in 5,000 * series.Fig. 5 C is presented at damping fluid or contains 25,000 * series of transferred species in the damping fluid of Yelkin TTS, Fig. 5 D show transferred species in CB-18 or with the CB-18 of Yelkin TTS combination in 25,000 * series.Fig. 5 E shows transferred species in damping fluid or contain 100,000 * series in the damping fluid of Yelkin TTS, Fig. 5 F show transferred species in CB-18 or with the CB-18 of Yelkin TTS combination in 100,000 * series.

Fig. 5 A, 5C and 5E verify, and the interpolation of this concentration Yelkin TTS if any also has only slight influence to the susceptibility of BACTEC 12B test.In addition, Yelkin TTS has the ability of the deleterious effect that overcomes antibiotic formulations when not containing CB-18 (P/caz) to a certain extent.The check of Fig. 5 B, 5D and 5F shows, Yelkin TTS can overcome independent or with the deleterious effect of the CB-18 of microbiotic combination.When inoculum when 165 ± 54 cfu reduce to 33 ± 11 cfu to 8 ± 3 cfu, as if the ability that Yelkin TTS overcomes the deleterious effect of microbiotic, CB-18 or both combinations reduce.Yet when the composition as transferred species solution added Yelkin TTS, all parallel bottles all became the positive in all cases.On the contrary, CB-18 is negative (Fig. 5 B, 5D and 5F) with all parallel bottles of P/caz combination.Obviously, Yelkin TTS can overcome the CB-18 effect.Repeated experiments has confirmed these discoveries.

Quaternary ammonium salt

Result shown in Fig. 5 A-5F shows, the mechanism of action of CB-18 is similar to quaternary ammonium compounds (for example, general surface-active property).This conclusion is based in the Yelkin TTS energy and the notion of quaternary ammonium stain remover and CB-18.Probably, be that electrostatic interaction by two kinds of fat realizes this neutralization.Positively charged ion season the class stain remover and the interaction of anionic phospholipid seemingly rational, but trimethyl-glycine has neutral net charge.The interaction of trimethyl-glycine one Yelkin TTS is of short duration.

The result of Fig. 3 C and Fig. 3 G shows, handles but the cell of transferred species in damping fluid do not have " post antibiotic effect " with CB-18.If the mechanism of action of CB-18 is similar to quaternary salt (that is, deleterious usually), then in advance in respect of certain delay.For example, if the CB-18 effect is the result of stain remover surface-active property, the destruction of cell function will not only have non-specific long-term consequence but also will not rely on strain isolated so.In order to attempt to check the similarity of mechanism, replicate(determination) CB-18 and bromination trimethylammonium octadecyl ammonium (TMA-18 (Aldrich, Milwaukee, WI, catalogue #35,924-6): be similar among the embodiment 1 described water at 1: 1: be made as 100 * stoste in the Virahol) for CB-18.Fig. 6 has summarized this experimental design.

In this experiment, ATCC 27294 and 571/573-BAL cultivate in the 7H11 selective medium, then with the serial dilution of NALC/ citric acid be 10,000 *.By prepare transferred species solution (that is final extent of dilution (50,000 *)) with damping fluid, 0.5mMCB-18 or 0.1mM TMA-18 (all concentration are represented the stain remover ultimate density in the transferred species solution) dilution.Then with duplicate (the every part 400 μ l) transferred species of each series in BACTEC 12B bottle, be added with PANTA (the 8 μ g/ml final concentrations: P/cax) of rebuilding liquid (R.F.), PANTA or strengthening in the bottle with ceftriaxone.The final concentration of CB-18 is about 17 μ g/ml between incubation period.The final concentration of TMA-18 is about 3.4 μ g/ml between incubation period.Make regular check on culturing bottle, the record growth index.The growth index of average particular series, and to cultivating the fate mapping.Fig. 7 A-7C shows a kind of experiment, wherein with ATCC 27294 cell transferred speciess in above-mentioned different solutions, Fig. 8 A-8C shows a kind of parallel laboratory test, wherein with 571/573-BAL cell transferred species in same train.

Use the result of ATCC 27294 to show, P/cax has more deleterious effects than independent PANTA (Fig. 7 A) or PANTA/caz combination (comparison diagram 2A and Fig. 7 A).In the presence of 17 μ g/ml CB-18, less delay becomes obviously among the PANTA separately, and the P/cax preparation shows significantly delay (Fig. 7 B).Cultivation at ATCC 27294 in the presence of the 3.4 μ g/ml TMA-18 shows obviously delay (Fig. 7 C) in all bottles.

The behavior of 571/573-BAL strain isolated is similar to ATCC 27294 strain isolateds in many aspects, has delicate but evident difference.For example, in the presence of 17 μ g/ml CB-18, the delay under all conditions becomes obviously, and the P/cax preparation shows surprising delay (Fig. 8 B).Cultivation at 571/573-BAL in the presence of the 3.4 μ g/ml TMA-18 is identical with ATCC 27294: all observe tangible delay (Fig. 8 C) in all bottles.

These test prompting, and the mechanism that CB-18 plays a role is different from quaternary ammonium salt (for example TMA-18).For example, when the stain remover of no any interpolation (comparison diagram 7A and Fig. 8 A), or in the presence of lower concentration CB-18 (comparison diagram 10A and Figure 11 A), the growth curve of ATCC 27294 and 571/573-BAL is practically identical.When CB-18 concentration increases, can distinguish strain isolated (comparison diagram 7B and Fig. 8 B).As if when further improving CB-18 concentration, strain isolated has similar behavior once more: both can not grow (comparison diagram 10A and Figure 11 A once more) in the presence of high density CB-18.This obviously is different from the behavior (comparison diagram 7C and Fig. 8 C) of these two kinds of strain isolateds in the presence of TMA-18: two kinds of strain isolated behavior performances much at one.If CB-18 is identical with the action site of TMA-18, then two kinds of strain isolateds all should show similar under all conditions.If the action site difference of two kinds of stain removers expects that then the mode that two kinds of strain isolateds show is variant under culture condition separately.Importantly be to remember, this result predicts by the discussion around Fig. 3 C and Fig. 3 G: CB-18 does not show post antibiotic effect (embodiment 3).

Observe the another kind of mode of these data and emphasize that as if the combination of CB-18 and P/cax mainly work in coordination with.For example, although CB-18 that separates (Fig. 7 B) and P/cax (Fig. 7 A) have slight influence to ATCC 27294, the two combination is worked in coordination with, rather than addition.In addition, as if Fig. 8 A and Fig. 8 B show that P/cax that separates and CB-18 influence 571/573-BAL, yet, though the CB-18-P/cax combination is mainly working in coordination with of addition to a certain extent.On the contrary, isolating TMA-18 is fungistat (Fig. 7 C and Fig. 8 C), as if when being used in combination with microbiotic, the result mainly is addition.Use CB-18 and TMA-18 to other experiment confirm of these and other strain isolated these two kinds stain remover played a role delicate but tangible character.Conclusion

The comparison of Fig. 2 H and Fig. 7 B (ATCC 27294 strain isolateds), and the comparison (571/573-BAL strain isolated) of Fig. 3 H and Fig. 8 B and Fig. 5 B, 5D, 5F, in the presence of CB-18 for antibiotic susceptibility, produce the important point.For example, in Fig. 3 H and Fig. 8 B, the 571/573-BAL strain isolated is grown in the presence of CB-18 and microbiotic.On the contrary, in Fig. 5 B, 5D and 5F, under simulated condition, there is not independent repeated growth.Inferentially, can think logically that these differences are that used inoculum (is seen the discussion of embodiment 2 and table 6 in actual CB-18 concentration, antibiotic concentration or the particular experiment, with reference to Eng, R.K. wait the people, biocide chemotherapy 26:42-47 (1984) and inoculum) or the result of the variation of its combination.For example, with respect to the experiment that is used for producing Fig. 2 H or 3H and Fig. 8 B respectively, accidental CB-18 or the microbiotic that uses higher concentration in the experiment that is used for producing Fig. 7 B or Fig. 5 B, 5D and 5F; Perhaps the inoculum among Fig. 7 B, 5B, 5D and the 5F is lower than its counterpart separately.Owing in each bottle, introduce the mode (that is, using the 1ml syringe) of CB-18, microbiotic and inoculum, be contemplated to the difference of all three kinds of variablees between particular series bottle and bottle: be rule rather than exception.If culture systems is too responsive to any composition as used herein, then aforesaid difference will become obvious.In Fig. 4 and the described experiment of Fig. 5 A-5F, between the different vaccination body, observe the difference of growth curve.These differences mainly are the delays that expection has low input.With respect to the variation of CB-18 concentration or inoculum, antibiotic concentration be changed to minimum.As if for the variation of CB-18 concentration, culture systems has more handiness.Embodiment 5CB-18 titration: the comparison of NTB, MAC and the living bacterium of speed

The results suggest of CB-18 trial test (embodiment 2), the CB-18 effect is widely used in large-scale mycobacterium kind (table 6), embodiment 4 promptings, this system is flexibly for CB-18 concentration.In order to attempt to confirm this hypothesis, use different mycobacterium kinds to carry out the described experiment of Fig. 9, wherein several mycobacterium kinds derive from the CB-18 trial test.

In this experiment, mycobacterium tuberculosis complex body, mycobacterium avium complex body and mycobacterium fortutitum complex body strain isolated have been tested.The mycobacterium tuberculosis complex body strain isolated that tries is ATCC 27294 and 571/573-BAL.The mycobacterium avium complex body strain isolated that tries is ATCC 25291 and 802-BAL; The mycobacterium fortutitum complex body strain isolated that tries is ATCC 6841 and 495-JHH.The feature and the experimental result of these strain isolateds are summarized in the table 7.

All strain isolateds are all cultivated on the 7H11 selective medium, then with the serial dilution of NALC/ citric acid be 1,000 *.By prepare transferred species solution (that is final extent of dilution (5,000 *)) with damping fluid or a series of different CB-18 concentration dilution.Comprising 0.1mM CB-18,0.2mM CB-18,0.4mM CB-18,0.8mM CB-18,1.6mM CB-18 or 3.2mM CB-18.All concentration are represented the final detergent concentration in the transferred species solution, but not all strain isolated is all in all concentration tests.

Then with triplicate (the every part 400 μ l) transferred species of each series in BACTEC 12B bottle, added reconstruction liquid (R.F.), PANTA in the bottle or with ceftriaxone (8 μ g/ml final concentrations: P/cax), ceftazidime (8 μ g/ml final concentrations: P/caz) or cefoxitin (8 μ g/ml final concentrations: the P/cft) PANTA of Qiang Huaing.The mycobacterium tuberculosis strain isolated by transferred species in P-cax, the mycobacterium avium strain isolated by transferred species in P-caz, the mycobacterium fortutitum strain isolated by transferred species in P-cft.The final concentration of CB-18 is about 3 μ g/ml for 0.1mM series between incubation period, be about 7 μ g/ml for 0.2mM series, be about 13 μ g/ml, be about 27 μ g/ml for 0.8mM series for 0.4mM series, be about 54 μ g/ml for 1.6mM series, be about 109 μ g/ml for 3.2mM series.Make regular check on culturing bottle, the record growth index.The growth index of average particular series, and to cultivating the fate mapping.

Figure 10 A and Figure 10 B show a kind of experiment, transferred species mycobacterium tuberculosis isolate A TCC 27294 in above-mentioned different solutions wherein, and Figure 11 A and Figure 11 B show a kind of parallel laboratory test, wherein transferred species mycobacterium tuberculosis strain isolated 571/573-BAL in the same train.Figure 12 A and Figure 12 B show a kind of experiment, transferred species mycobacterium avium isolate A TCC 25291 in above-mentioned different solutions wherein, and Figure 13 A and Figure 13 B show a kind of parallel laboratory test, wherein transferred species mycobacterium avium strain isolated 802-BAL in same train.Figure 14 A and Figure 14 B show a kind of experiment, transferred species mycobacterium fortutitum isolate A TCC 6841 in above-mentioned different solutions wherein, and Figure 15 A and Figure 15 B show a kind of parallel laboratory test, wherein transferred species mycobacterium fortutitum strain isolated 495-JHHL in same train.

When the viability of the concentration of CB-18 mycobacterium tuberculosis isolate A TCC 27294 during near 54 μ g/ml descends (Figure 10 A), and when the viability decline (Figure 11 A) of the concentration of CB-18 mycobacterium tuberculosis strain isolated 571/573-BAL during near 27 μ g/ml.When containing microbiotic in the substratum, when the concentration of CB-18 during near 27 μ g/ml the viability of ATCC 27294 descend (Figure 10 B), when the concentration of the CB-18 viability of 571/573-BAL descend (Figure 11 B) during near 13 μ g/ml.Obviously, this system is very responsive to the change of CB-18 concentration.Volatility between explaining experiment easily and test with this sensitive property.

When the viability of the concentration of CB-18 mycobacterium avium isolate A TCC 25291 during near 27 μ g/ml descends (Figure 12 A), and the viability unaffected under experimental concentration (Figure 13 A) of mycobacterium avium complex body (MAC) strain isolated 802-BAL.When containing microbiotic in the substratum, when the concentration of CB-18 during near 13 μ g/ml the viability of ATCC 25291 descend (Figure 12 B), when the concentration of the CB-18 viability of 802-BAL descend (Figure 13 B) during near 27 μ g/ml.Curve begins plateau when 27 μ g/ml, because have only an one-tenth positive (Figure 13 B) in three bottles.In addition, this system is to the change sensitivity of CB-18 concentration.

As the viability of the concentration of CB-18 two kinds of mycobacterium fortutitum strain isolateds (ATCC 6841 and 495-JHH) during near 109 μ g/ml all descend (Figure 14 A and Figure 15 A respectively).When containing microbiotic in the substratum, when the concentration of CB-18 during near 54 μ g/ml the viability of ATCC 6841 descend (Figure 14 B), when the concentration of the CB-18 viability of 495-JHH descend (Figure 15 B) during near 27 μ g/ml.

The living bacterium of speed is as a kind of cohort, as if the effect to independent CB-18 more has resistance, but shows synergy when making up with microbiotic.For example, 495-JHH is survived when 109 μ g/ml CB-18, but at descend during at 13-27 μ g/ml in the presence of the P-cft (Figure 15 A and Figure 15 B).In addition, 571/573-BAL is survived when 13 μ g/ml CB-18, but in viability descend (Figure 11 A and Figure 11 B) during at 7-13 μ g/ml in the presence of the P-cax.

The result of two kinds of living bacterium of speed is the interesting result who is produced by following viewpoint: Connell; N.D. wait the people " tuberculosis. pathogeny, protection and control " Bloom; B.R. compile; ASM press; Washington, in D.C. (1994) the 333-352 pages or leaves with the living bacterium of speed to the resistance of cynnematin owing to permeability.If as described in embodiment 9, CB-18 influences permeability in some way, and this result can expect so.

The mycobacterium kind of table 7* screening
									????ID		????NALC/NaOH			????CB-18			Result and comment
	Culture			Culture
						Smear	Liquid	Solid			Smear	12 ?B	????7H11
ATCC
	27294	?MTB	?NA	?NA	??NA	??NA	NA	????NA	When the independent CB-18 of 27-54 μ g/ml, and the microbiotic existence is following when 13-27 μ g/ml, and viability descends
573- BAL										?MTB	?1+	/28	??	??3+		????/20	When the independent CB-18 of 13-27 μ g/ml, and the microbiotic existence is following when 7-13 μ g/ml, and viability descends
	ATCC 25291	?M.av 1	?NA	?NA	??NA	??NA	NA	????NA	When the independent CB-18 of 13-27 μ g/ml, and the microbiotic existence is following when 7-13 μ g/ml, and viability descends
802- BAL										?MAC	?	/10	/ll	??1+		????/20	The independent CB-18 of test level does not influence viability, and viability descended when there was to descend 13-27 μ g/ml in microbiotic
	ATCC 6841	?M.fo 2	?NA	?NA	??NA	??NA	NA	????NA	When being higher than the independent CB-18 of 109 μ g/ml levels, and the microbiotic existence is following when 27-5 μ g/ml, and viability descends
495- JHH										?M.fo	?	/	??	??±		????/14	When being higher than the independent CB-18 of 109 μ g/ml levels, and the microbiotic existence is following when 13-27 μ g/ml, and viability descends

* the smear value is reported according to the CDC guide.It is a numeral subsequently that positive culture is expressed as " " symbol.The total fate of this digitized representation positive findings.Negative culture is outstanding with " " symbol.Contaminated inclined-plane usefulness " cont. " sign flag, the liquid culture that stands to digest again usefulness " ^" sign flag.ATCC 27294 strains are not to come from the CB-18 trial test.Thereby, cultivation results invalid (" NA "). ¹M.av is a mycobacterium avium. ¹M.fo is a mycobacterium fortutitum.

Can draw some by these data.The first, as if there is the minimum effective concentration of CB-18: the effect that the CB-18 of lower concentration does not bring out, but the CB-18 of high density is bactericide (even when not having the microbiotic that adds).Inducibility depends on mycobacterium kind and strain isolated itself.Embodiment 6MTB strain isolated and antibiotic sign

The Main Conclusions that is drawn by embodiment 2,3 and 5 is, CB-18 makes that insensitive some mycobacterium becomes responsive to microbiotic under the normal circumstances.In the popularity of having checked this phenomenon aspect strain isolated and the microbiotic.Several strain isolateds have been checked having or not under the situation of CB-18 from CB-18 trial test (embodiment 2) for different beta-lactam antibioticss.In a series of additional experiments, checked other (that is non-beta-lactam) microbiotic.

Figure 16 has summarized the experimental design that is used for testing different MTB strain isolateds.On 7H11 selectivity inclined-plane, cultivate these strain isolateds (table 8), and with the serial dilution of NALC/ citric acid be about 10,000 *.By with the further dilution preparation transferred species solution (that is final extent of dilution (about 50,000 *)) of damping fluid or 0.5mM CB-18 (final concentration).Then with duplicate (the every part 400 μ l) transferred species of each series in BACTEC 12B bottle, added reconstruction liquid (R.F.) in the bottle, the ceftazidime (" P/caz ") of PANTA or the PANTA:8 μ g/ml final concentration strengthened in order to following cynnematin, the cefoperazone of 16 μ g/ml final concentrations (" P/cfp ") (Sigma, St.Louis, MO, catalogue #:C-4292: in water, prepare cefoperazone with 72mg/ml, and to be similar to) for the described mode of ceftazidime (embodiment 1) adding, the ceftriaxone of 8 μ g/ml final concentrations (" P/cax ") (Sigma, St.Louis, MO, catalogue #:C-5793: in water, prepare ceftriaxone with 36mg/ml, and add to be similar to for the described mode of ceftazidime (embodiment 1)) or cefoxitin (" P/cft ") (Sigma of 8 μ g/ml final concentrations, St.Louis, MO, catalogue #:C-4786: in water, prepare cefoxitin, and add) to be similar to for the described mode of ceftazidime (embodiment 1) with 36mg/ml.In addition, the final concentration of CB-18 is about 17 μ g/ml between incubation period.Make regular check on culturing bottle, the record growth index.The growth index of average particular series, and to cultivating the fate mapping.

Filter out 11 strain isolateds (comprising ATCC 27294 strains).Table 8 has been summed up 10 clinical separation strains and the test-results of using.Figure 17-27 shows representative strain isolated.For the behavior in the test, there is significant difference in these strain isolateds.

The MIB strain isolated of table 8* screening
								ID	NALC/NaOH			CB-18			Result and comment
	Culture			Culture
						Smear	Liquid		Solid	Smear	12B	7H11
ATCC
	27294	NA	NA	NA	NA	NA	NA	If any, the CB-18 inductive is to the antibiotic susceptibility minimum of try, yet CB-18 aggravates cax susceptibility.
571-BAL									1+	/28	/42	1+		/cont.	The CB-18 inductive is to all antibiotic susceptibility of try surprising (Pt. identical with 573-BAL).
	573-BAL	1+	/28		3+		/20	The CB-18 inductive is to all antibiotic susceptibility of try surprising (Pt. identical with 571-BAL).
535-BAL									4+	/3	/28	4+	/3	/11	The CB-18 inductive to try antibiotic effect minimum: cax susceptibility does not rely on CB-18.
	896-BAL	4+	/1	/14	4+	/1	/5	If any, the CB-18 inductive is to the antibiotic susceptibility minimum of try, yet, CB-18 slightly aggravates cax susceptibility, and bacterial strain is wide spectrum resistance: INH, RIF and PZA.
040-TBR										/29	/42	1+	/14/ 		The CB-18 inductive is less to the susceptibility of examination most antibiotics, yet C8-18 significantly aggravates cax susceptibility.
	061-1BR		/29	/35	2+	/10	/19	If any, the CB-18 inductive is less to the antibiotic susceptibility of try, yet, CB-18 aggravation cax susceptibility.Bacterial strain is the INH resistance.
512-JHH									2+	/4		4+	/3	/12	The CB-18 inductive is to the susceptibility of examination most antibiotics less (Pt. identical with 538-JHH).
	538-JHH		/31	/33	2+		/49	The CB-18 inductive is to the susceptibility of examination most antibiotics less (Pt. identical with 512-JHH).
52-96-BOL									1+	/25	/27	1+		/39	The CB-18 inductive is less to the susceptibility of examination most antibiotics, yet CB-18 significantly aggravates cax susceptibility.
	57-96-BOL	1+	/25	/27	3+		/25	Susceptibility to most antibiotics is moderate, and the CB-18 inductive is also moderate to the susceptibility of most antibiotics.Bacterial strain is MDR.

* the smear value is reported according to the CDC guide.It is a numeral subsequently that positive culture is expressed as " " symbol.The total fate of this digitized representation positive findings.Negative culture is outstanding with " " symbol.Contaminated inclined-plane " cont. " sign flag, the liquid culture that stands to digest again "  " sign flag.ATCC 27294 strains are not to come from the CB-18 trial test.Thereby, cultivation results invalid (" NA ").Except having the less delayed when CB-18 and the P/cax combination, ATCC 27294 is showing little difference or is not having difference (Figure 17 A and Figure 17 B) aspect the CB-18 inductive susceptibility.These results and the data consistent of checking P/caz (comparison diagram 2F and Figure 17 B) and P/cax (comparison diagram 7B and Figure 17 B) in the past.Observe the notable difference of inductive to PANTA susceptibility in Fig. 7 B rather than in Figure 17 B, this perhaps is the result who changes as CB-18 concentration between the experiment as described in the embodiment 5.

571-BAL (Figure 18 A and Figure 18 B) and 573-BAL (Figure 19 A and Figure 19 B) show significantly inducing the susceptibility of all medicines of try once more.Have only when microbiotic and CB-18 combination, the collaborative deleterious effect of couple 571/573-BAL is just arranged.

Be similar to ATCC 27294, the 535-BAL strain isolated is not subjected to the influence of this concentration C B-18, yet P-cax influences this strain isolated (Figure 20 A and Figure 20 B).There is the behavior of the 896-BAL strain isolated of resistance also to be similar to ATCC 27294 (comparison diagram 17A and 17B and Figure 21 A and 21B) to INH, RIF and PZA.Strain isolated 040-TBR and 52-96-BOL are influenced by CB-18 to a certain extent, yet CB-18's exists the susceptibility (respectively see Figure 22 A and 22B, Figure 26 A and 26B) of remarkably influenced to cax in these two kinds of strain isolateds.On the contrary, 061-TBR and 57-96-BOL strain isolated are influenced by all microbiotic when no CB-18, yet the existence of CB-18 aggravates antibiotic deleterious effect (seeing Figure 23 A and 23B, Figure 27 A and 27B respectively).

512-JHH and 538-JHH are interesting to (seeing Figure 24 A and 24B, Figure 25 A and 25B respectively), this is because following viewpoint: in CB-18 trial test process, a kind of when contact CB-18 is nature (512-JHH), and the another kind of CB-18 (538-JHH) that when the anti-tubercle bacillus extract for treating, contacts.Processing is from 4 samples (table 9) among this patient.First sample was submitted on February 8th, 1996, was reported as the smear positive at mandatory 24 hours in the report time, presented AFB in 4 days and cultivated positive submitting to.After initial sample 10 days, from culturing bottle, separate after about 1 week after the AFB, patient beginning pharmacological agent submission in 5 days from second sample (572-JHH) of this patient.Three samples that begin to submit to the back in pharmacological agent all are the CB-18/12B/PANTA/caz feminine genders, have only two (538-JHH and 541-JHH) positive on the 7H11 inclined-plane.Although pollution is inoperative in any of these CB-18/12B/PANTA/caz difference, 527-JHH has 4 and submits to the fact of minimum smear value in the things can explain discrepant 7H11 result; Yet the obvious delay (promptly 49 day) of 538-JHH on solid medium is unusual for the smear positive sample.Though the similar sample of handling with NALC/NaOH also postpones with respect to initial sample, CB-18 seems the viability of this strain isolated in the remarkably influenced treatment.This discussion and series shown in Figure 3 (comparison diagram 3B and Fig. 3 F, Fig. 3 D and Fig. 3 H) with embodiment 3 is consistent, and proposes the clinical application of trimethyl-glycine sample stain remover as the treatment adjuvant.

Table 9* is from a patient's mycobacterium tuberculosis strain isolated
									????ID		??????????NALC/NaOH			????CB-18			Comment
	Culture			Culture
						Smear	Liquid	Solid			Smear	????12B	?7H11
512-JHH	?MTB	??2+	?/4	?	4+									????/3	?/12	The 2/8/96-nature
						527-JHH	?MTB	??	?/24	?	±	????	?				The 2/17/96-treatment
538-JHH	?MTB	??	?/31	?/33	2+									????	?/49	The 2/27/96-treatment
						541-JHH	?MTB	??	?/31	?/33	2+	????	?/20				The 2/27/96-treatment

* the smear value is reported according to the CDC guide.It is a numeral subsequently that positive culture is expressed as " " symbol.The total fate of this digitized representation positive findings.Negative culture is outstanding with " " symbol.

The experimental design of Figure 16 also is used for checking the popularity of CB-18 effect for other non-beta-lactam antibiotics.In these experiments, (Sigma, St.Louis MO) are prepared as stock solution, dilute with rebuilding liquid then, and add in the culturing bottle before inoculation with microbiotic in water or damping fluid.To ATCC27294,571/573-BAL, 061-TBR, 52-96-BOL and 57-96-BOL test nystatin (100 μ g/ml final concentration), PXB (300 unit final concentration), erythromycin (4 μ g/ml final concentration), romicil (2 μ g/ml final concentration), penicillin G (8 μ g/ml final concentration), nalidixic acid (30 μ g/ml final concentration), lincomycin (2 μ g/ml final concentration), ceftriaxone (32 μ g/ml final concentration) and ceftazidime (16 μ g/ml final concentration) (in 10% sodium bicarbonate, preparing ceftazidime).All experiments are all used R.F. and P-caz in contrast, and all culturing bottles that add CB-18 all use the final concentration of 17 μ g/ml.The result of the 571/573-BAL strain isolated of Figure 28 display application erythromycin and ceftriaxone.The result of the 061-TBR of Figure 29 display application PXB, romicil, lincomycin, nalidixic acid, penicillin G and ceftriaxone.The result of the 57-96-BOL of Figure 30 display application nalidixic acid, penicillin G, ceftazidime and ceftriaxone.

These results show that the CB-18 effect depends on strain isolated and microbiotic.What is interesting is that the independent ceftazidime of 16 μ g/ml final concentrations (promptly not conforming to PANTA) is one of the most deleterious compound.Conclusion

These results have given prominence to the microheterogeneity of mycobacterium tuberculosis complex body about antimicrobial susceptibility.Determine as yet although observe relevant mechanism as if CB-18 makes according to susceptibility becomes possibility to the height ability to see things in their true light of mycobacterium tuberculosis strain isolated with these.This information can be used to the especially more successful treatment plan of MTB infection of predicted treatment mycobacterial infections.In addition, to induce the fact of susceptibility to point out this compound itself also may be a kind of useful treatment adjuvant to CB-18.

" CB-18 effect " promptly induces the ability of susceptibility, depends on the dynamic interaction of trimethyl-glycine sample stain remover (for example CB-18), microbiotic and strain isolated.The effective concentration of trimethyl-glycine sample stain remover should depend on trimethyl-glycine sample stain remover itself, and can determine with screening method of the present invention.Embodiment 7 screening stain remover and other trimethyl-glycine spline structures

In order to attempt to extend the discovery of embodiment 6, developed the structure homologue that CB-18 is screened in a kind of test.Table 10 has been listed the stain remover that uses in the test of Figure 31 general introduction.Because the 571/573-BAL strain isolated is to susceptibility inductive susceptibility, used as the report strain in these screening experiments.On 7H11 selectivity inclined-plane, cultivate the 571/573-BAL strain isolated, and with the serial dilution of NALC/ Citrate trianion be about 10,000 *.By with damping fluid, 0.5mM CB-18 or suitably stain remover further dilute preparation transferred species solution (that is final extent of dilution (about 50,000 *)).With all stain removers at 1: 1 Virahol: be prepared as 100 * concentrated solution in the water, in particular experiment series, be used as final thinner.

The final concentration of every kind of stain remover, the 12B bottle that obtains contain the specific stain remover of the 17 μ g/ml final concentrations of having an appointment.Carry out each experimentalists and technicians with damping fluid, and with duplicate (the every part 400 μ l) transferred species of CB-18 transferred species contrast in BACTEC 12B bottle, contain the PANTA (" P/cax ") that rebuilds liquid (R.F.) or PANTA or added the ceftriaxone of 8 μ g/ml final concentrations in the bottle.Single test 5-7 kind stain remover.Duplicate (the every part 400 μ l) transferred species of stain remover of every kind of dilution that will contain the MTB cell is in the BACTEC 12B bottle that has added PANTA or P/cax.Nearly all stain remover is all tested twice.Make regular check on culturing bottle, and the record growth index.The growth index of average particular series, and to cultivating the fate mapping.

Table 10 has been summed up the stain remover and the result thereof that detect.Following stain remover is from Aldrich chemical company (Milwaukee, WI) obtain: benzyl dimethyl-stearyl chlorination ammonium (catalogue #:22,901-6), benzyl dimethyl-tetradecyl ammonium chloride (catalogue #:29,279-6) and the octadecyl trimethylammonium bromide (catalogue #:35,924-6).(St.Louis MO) obtains: blended alkyl trimethyl ammonium bromide (catalogue #:M-7635), Palmiticacid (catalogue #:P-0500), stearic acid (catalogue #:S-4751), CHAPS (catalogue #:C-3023), Septochol (catalogue #:D-6750), SB-18 (C following stain remover from the Sigma chemical company ₁₈-azochlorosulfonate propyl lycine) (catalogue #:0-8004), Brij 97 (oleyl (polyoxyethylene) ₁₀) (catalogue #:P-6136) and Brij56 (hexadecyl (polyoxyethylene) ₁₀) (catalogue #:P-5759).(Indianapolis IN) obtains tween 80 (oleic polyoxyethylene sorbitan esters) (catalogue #:1334 875) from Boehringer Mannheim.Customized stain remover comprises: C ₁₈-propyloic trimethyl-glycine, C ₁₈-sulphur butyrobetaine, C ₁₆-hydroxypropyl sultaine, C ₁₆-amido propyl sulfuric acid trimethyl-glycine, they are by Ecochem Research, Inc. (Chaska, MN) synthetic, and the C that obtains from KAO fundamental research institute (Tochigi, Japan) as present ₁₆-AHTMAP (a kind of reverse trimethyl-glycine).Trimethyl-glycine (chembetaine)-(Paso Robles CA) obtains S (soy amide propyl group carboxymethyl betaine) chemistry from Chemron company as sample.From DeForest, (Boca Raton FL) obtains Inc. DeTaine PB (Product HDN) as sample.From Heterene, (Paterson NJ) obtains Inc. Hetaine CLA (carola amido propyl carboxybetaine) as sample.(Dublin OH) obtains Rewoteric AM R-4 (tallow glycinate) from the Sherex chemical company as sample.From Scher Chemicals, (Clifton NJ) obtains Inc. as sample for Schercotaine IAB (hard ester acyl amido propyl carboxybetaine) and Schercoatine WOAB (Fructus Hordei Germinatus amido propyl carboxybetaine).From Henkel company, (Hoboken NJ) obtains Emory Group Cospha Velvetex OLB-50 (Velvetex OLB-50) as sample.From Croda, (Parsippany NJ) obtains Inc. Crosultaine E-30 (Eruc amido propyl (erucamidopropyl) hydroxypropyl sultaine) as sample.From McIntyre Group, (University Park IL) obtains LTD. Mackamine 02 (Standamox 01) as sample.

Figure 32 A has shown the contrast of these experiments, and Figure 32 B has shown the result of representative stain remover.In Figure 32 A, will be from the double bottle mean deviation mapping of each control series of all 8 experiments, with show with microbiotic (◆-PANTA, ▲-P/cax), CB-18 ( -independent CB-18) and both combinations (-CB-18/PANTA, ●-CB-18/P-cax) the relevant result's of influence trend.Growth curve seen in Figure 32 B is based on the mean value from the double bottle of two experiments, in this experiment with in the stain remover separately of cell transferred species in the BACTEC 12B bottle that has added P/cax.

As previously shown, the existence of PANTA or P/cax does not influence or minimum influence 571/573-BAL (comparison diagram 3A, 3E, 8A and 18A/19A and Figure 32 A) when no CB-18.In addition, independent CB-18 is influential to the growth characteristics of 571/573-BAL strain, and CB-18 and antibiotic combination are collaborative deleterious, wherein P/cax provide maximum growth-inhibiting (Figure 32 A:

-independent CB-18,-CB-18/PANTA, ●-CB-18/P-cax).

In addition, in the structure results about stain remover significant diversity is arranged.In a word, all quaternary ammonium salts that tried have activity in this concentration very much, suppress in test growth (table 10) fully.In addition, negatively charged ion fat acid that is tried or cholate are to growing all without any influence.

Show that active carboxybetaine is the carboxybetaine of those unmodifieds.For example, wherein the amido propyl carboxybetaine that is used to connect alkyl chain and positively charged ion (" α " in the table 1) does not show activity in this test.In addition, the ability, the especially C that when making up, do not have the carboxybetaine demonstration intensive inhibition growth of component with microbiotic ₁₈-propyloic trimethyl-glycine (the ethyl bridge homologue of CB-18).Use C ₁₈-propyloic trimethyl-glycine further studies show that very narrow field of activity (about 7-13 μ g/ml) to ATCC's 27294.Figure 32 A and Figure 32 B demonstration contain methylene radical (Detain PB and Velvetex OLB), ethene (C when using in this test ₁₈-propyloic trimethyl-glycine) and the variability during simple (being unmodified) carboxybetaine of propylene (CB-18) bridge.This and Tsubone, people such as K., the data consistent of pharmaceutical science magazine 80:441-44 (1991), its toxic is relevant with bridge length.

The trimethyl-glycine sample stain remover of " other " kind shows the miscellaneous result.Reverse trimethyl-glycine or sulfuric acid trimethyl-glycine (for example ,-SO ₄) do not suppress to grow.Four kinds of sultaine (SO ₃) in have only and have two kinds (Brij 56 and Brij 97) and a kind of amine oxide (Standamox 01) that is tried can significantly suppress growth (table 10) in a kind of (that is Crosultaine E30), the three kinds of nonionic detergent.

Although the application in anti-microbial agents can be predicted the obvious sterilization activity (Michaels of oxidized nicotinamide according to these compounds, E.B., United States Patent (USP) 564,454 and United States Patent (USP) 641,728), but the activity of Brij compound and Crosultaine E-30 is still surprising.As if except CrosultaineE-30, other sultaine that has remarkable activity in this test has only SB-18.With SB-18 571/573-BAL further be studies show that very wide field of activity (about 20-75 μ g/ml).As if other sultaine all contains amido propyl key or hydroxypropyl bridge, shows to be similar to carboxybetaine, and the modification of backbone structure is hindered activity described herein.The existing amido propyl key of CrosultaineE-30 has the hydroxypropyl bridge again.Although the Brij compound, estimates that these reagent can stimulating growth not to the modification of main chain.In history, once reported nonionic detergent by contaminating impurity, having only could stimulating growth (Dubos, people such as R.J., The Journal of Experimental Medicine (Jour.Exptl.Med.) 83:409-423 (1946)) behind the purifying.The result of impurity soluble application Brij stain remover and Crosultaine E-30.

It is this possibility that is caused by a kind of impurity that these data have proposed observed effect.Yet, the CB-18 purity that provides be about 98% (personal communications, Bob Carlson, Ecochem Research, Inc., Chaska, MN).Therefore, any impurity when about 0.34 μ g/ml, all must work in these trials (2% of the CB-18 that supposition is added is a kind of impurity, and then 2% of 17 μ g/ml is about 0.34 μ g/ml).TMA-18 is an inhibition when lower concentration in this test, and 10 times to above concentration (Fig. 7 C and Fig. 8 C).In addition, although manyly can the commercial trimethyl-glycine that obtains to be synthesized the quaternary salt that mechanism produces and (for example to pollute, precursor or synthesising by-product), but the possibility that forms quaternary salt at CB-18 between synthesis phase is very impossible (personal communications, Bob Carlson, EcochemResearch, Inc., Chaska, MN).In addition, according to Weers, J. wait the people, the expection by product that is produced in the CB-18 building-up process of carrying out of improving one's methods of Langmuir7:854-867 (1991) or Kazuo (JP 8125139) should be estimated nontoxicity (personal communications, Bob Carlson, Ecochem Research, Inc., Chaska, MN).

These experiments (Figure 32 B) show that also tween 80 does not have influence to the 571/573-BAL strain isolated when 17 μ g/ml (13 μ M).Hui, people such as J., biocide chemotherapy 11:773-779 (1977) changes the concentration of tween 80, shows just to reach the susceptibility of Rifampin is induced up to about 0.005% (50 μ g/ml (38 μ M)).The more important thing is that the tween 80 of higher concentration (up to 0.05% (500 μ g/ml (380 μ M))) only is used for stimulating growth.Stinson, M.W. wait the people, U.S. respiratory tract disease summary 104:717-727 (1971) reaches maximal stimulation when being reported in 1% (10mg/ml (7.69mM)), and does not have other superiority up to 10% amount (100mg/ml (76.9mM)), but also unrestraint.Perhaps, adding tween 80 in substratum is most important diagnosis progress in mycobacterium is learned in this century, because it causes " fast " growth (Dubos, people such as R., The Journal of Experimental Medicine 83:409-423 (1946)).Yamori, people such as S., microbiology and immunology 35:921-926 (1991) have reported when from 0% to 0.5% to 2% titration tween 80 the change to some drugs susceptibility pattern.The existence of ODAC (a kind of cultivation additive contains BSA, NaCl, glucose, catalase and oleic acid) causes the remarkable change of susceptibility when making up with tween 80.On the other hand, CB-18 does not cause the change of growth when about 3-7 μ g/ml, but causes the inhibition fully (Figure 10 A and Figure 11 A) of growth when 13-27 μ g/ml.

The conclusion that draws is from these experiments, and multiple trimethyl-glycine sample stain remover can be used in the method for the present invention, and susceptibility is induced with the trimethyl-glycine spline structure and become.Every kind of trimethyl-glycine sample stain remover is necessary for and is used in combination with method of the present invention and optimizes.By different microbiotic are made up with different trimethyl-glycine sample stain removers, the ability to see things in their true light of the differentiation strain isolated of special height is possible.

Table 10
		Stain remover	The result
Cationic detergent
		Benzyl dimethyl stearyl chlorination ammonium	Growth-inhibiting completely
The benzyl dimethyl tetradecyl ammonium chloride	Growth-inhibiting completely
		The blended alkyl trimethyl ammonium bromide	Growth-inhibiting completely
The octadecyl trimethylammonium bromide	Growth-inhibiting completely
		Anionic detergent
Palmiticacid	There is not influence
		Stearic acid	There is not influence
Cholate
		CHAPS	There is not influence
Septochol	There is not influence
		Carboxybetaine
C 18-propyloic trimethyl-glycine	Growth-inhibiting completely
		Chemistry trimethyl-glycine-S (soy amide propyl group carboxymethyl betaine)	There is not influence
DeTaine PB (Product HDN)	The intensive growth-inhibiting
		Hetaine CLA (canola amido propyl carboxybetaine)	There is not influence
Rewoteric AM R-4 (tallow glycinate)	There is not influence
		Schercotaine IAB (hard ester acyl amido propyl carboxybetaine)	There is not influence
Schercotaine WOAB (Fructus Hordei Germinatus amido propyl carboxybetaine)	There is not influence
		Velvetex OLB-50 (Velvetex OLB-50)	Unusual intensive growth-inhibiting
Other trimethyl-glycine
		C 16-AHTMAP (oppositely trimethyl-glycine)	There is not influence
C 18-sulphur butyrobetaine	Slight delayed growth
		C 16-hydroxypropyl sultaine	Slight delayed growth
C 16-amido propyl sulfuric acid trimethyl-glycine	There is not influence
		Crosultaine E-30 (Crosultaine E-30)	The intensive growth-inhibiting
SB-18(C 18-azochlorosulfonate propyl lycine)	The inhibition of appropriateness
		Mackamine 02 (Standamox 01)	Growth-inhibiting completely
Nonionic detergent
		Tween 80 (oleic polyoxyethylene sorbitan esters)	There is not influence
Brij 97 (oleyl (polyoxyethylene) 10)	Unusual intensive growth-inhibiting
		Brij 56 (hexadecyl (polyoxyethylene) 10)	Unusual intensive growth-inhibiting

CB-18 effect and EDTA

Embodiment 4 shows that the effect of CB-18 is different from TMA-18, and Figure 32 B shows that the effect of CB-18 is different from tween 80.Test the results suggest of different trimethyl-glycine sample stain removers, the structure of unmodified may be the most useful (table 10).(for example, EDTA), carried out the described experiment of Figure 33 in order to attempt further to distinguish CB-18 effect and other effector such as metal chelator.

EDTA changes the permeability of bacterium by the divalent metal of extraction and chelating stabilized cell wall construction.Rastogi, people such as N., biocide chemotherapy 34:759-764 (1990) report, the trial of cultivating mycobacterium in 1mM EDTA (372.5 μ g/ml) has remarkably influenced to viability.Suppose that trimethyl-glycine is according to the ability of its coordinated water and with the form dissolving (Tsujii of salt, K. wait the people, physical chemistry magazine (Jour.Phys.Chem.) 82:1610-1614 (1978)), then draw this possibility as a result that the CB-18 effect is this compound sequestering activity.

In order to attempt to distinguish the effect of CB-18 effect and metal chelator such as EDTA, developed a kind of test and analyzed the effect of CB-18 (Figure 33).On 7H11 selectivity inclined-plane, cultivate ATCC 27294 and 571/573-BAL strain isolated, with each be then with the serial dilution of NALC/ Citrate trianion about 10,000 *.By with damping fluid, 0.5mM CB-18, contain 2.5mM MgCl ₂0.5mM CB-18 or 0.5mM EDTA (Life Technologies, Gaithersburg, MD) further dilution preparation transferred species solution (that is final extent of dilution (about 50,000 *)).The final concentration of CB-18 and EDTA is about 17 μ g/ml, MgCl between incubation period between incubation period ₂Concentration be about 85 μ g/ml.Triplicate (the every part 400 μ l) transferred species of each experimentalists and technicians in BACTEC 12B bottle, is contained the PANTA (" P/cax ") that rebuilds liquid (R.F.) or added the ceftriaxone of 8 μ g/ml final concentrations in the bottle.Make regular check on culturing bottle, and the record growth index.Mapped to cultivating fate in the average back of the growth index of particular series.Figure 34 A and Figure 34 B show the result of ATCC 27294 strain isolateds, and Figure 35 A and Figure 35 B show the result of 571/573-BAL strain isolated.

EDTA does not have influence (Figure 34 B and Figure 35 B) in used concentration to the growth characteristics of arbitrary strain isolated.The EDTA concentration ratio Rastogi that in these experiments, uses, people such as N., the used concentration of biocide chemotherapy 34:759-764 (1990) is almost hanged down 22 times (372 μ g/ml are to 17 μ g/ml).

The most interesting aspect is CB-18/MgCl in these experiments ₂Combination.Support the hypothesis of this experiment to be, if CB-18 can chelating MgCl ₂, then should interact perhaps can (to a certain extent) in and the effect of CB-18.ATCC 27294 strain isolateds be not subjected to independent CB-18 influence (Figure 34 A: promptly, no MgCl ₂The time), and the 571/573-BAL strain isolated is suppressed (Figure 35 A) by independent CB-18.The MgCl that adds 5 times of molar excess ₂Improve (Figure 35 B) that does not show the 571/573-BAL growth characteristics.ATCC 27294 or 571/573-BAL do not show the delay of aggravation in the presence of independent P/cax; Yet, in the presence of CB-18 P/cax be shown as once more collaborative deleterious, for 571/573-BAL strain isolated (Figure 34 A and Figure 35 A) all the more so.In the CB-18/P-cax combination, add MgCl ₂As if alleviated the deleterious effect (Figure 34 B and Figure 35 B) of CB-18/P-cax combination to a certain extent, especially CB-18/P-cax is to the effect of 571/573-BAL.If MgCl ₂Do not alleviate the CB-18 effect when not conforming to P-cax, but MgCl ₂Weaken the susceptibility in the presence of P-cax, so MgCl ₂Certainly hinder the effect of P-cax.Because ceftriaxone (cax) is sold as disodium salt, thus this microbiotic have negative net charge (promptly-2) and should be able to MgCl ₂Interact.

Tsubone K., pharmaceutical science magazine 80:1051-1054 (1991) show, generally between the sequestering activity of different phosphoric acid betaines and antimicrobial acivity has very little dependency or do not have dependency.This is not that this discovery of consequence of chelated metal ions is consistent with the CB-18 effect.Embodiment 8 mycobacterium sensitivity tests and CB-18 effect

Carry out sensitivity test several different methods is arranged.The standard method of current use comprises: (a) dull and stereotyped diffusion test, (b) broth microdilution antifungal susceptibility test, (c) agar gradient and (d) rapid automatized instrumental method.In these methods, change drug concentrations and describe growth characteristics.The amount of known inoculum extremely influences the result of sensitivity test.

The mycobacterium in the present age is learned sensitivity test according to as Vestal, A.L. Center for Disease Control, [Ministry of Health, education and welfare publication number (CDC) 76-8230] described 1% scaling method of 97-115 page or leaf (1975).It is characterized in that also will be in the growth in the presence of the antituberculin compared with the control with contrast dilution 100 times (100 *).If 1% or more inoculum to drug resistant, then the growth of the strain isolated of being studied will be equal to or be higher than contrast.If strain isolated is responsive, or to be lower than 1% inoculum be resistance, and then its growth will be weaker than contrast.

Golden standard in the tuberculosis sensitivity test is BACTEC ^S.I.R.E or BACTEC ^The PZA test (Becton Dickinson, Cockeysville, MD).This is a kind of automatization meat soup method (Snider, people such as D.E., U.S. respiratory tract disease summary 123:402-406 (1981); Siddiqi, people such as S.H., clinical microbiology magazine 13:908-912 (1981); Vincke, people such as G., antimicrobial chemotherapy magazine (Jour.Antimicrob.Chemther.) 10:351-354 (1982); Roberts, people such as G.D., clinical microbiology magazine 18:689-696 (1983); And Tarrand, people such as J.J., clinical microbiology magazine 21:941-946 (1985)).The Product labelling explanation, contrast growth index (GI) surpasses 30 (GI _T=0) after, read culturing bottle (GI in next sky _T+1) and determine difference (GI between these two days _T+1-GI _t=Δ GI _Contrast).Read the culturing bottle that contains trial drug at identical these two days, and determine Δ GI for medicine with same procedure _MedicineIf Δ GI _Contrast＞＞Δ GI _Medicine, think that then strain isolated is responsive.If Δ GI _Contrast〉=Δ GI _Medicine, think that then strain isolated is critical.If Δ GI _Contrast＜Δ GI _Medicine, think that then strain isolated is a resistance.Heifets, L., biocide chemotherapy 40:1759-1767 (1996) illustrate that the GI under medicine exists between 8 days incubation period should not surpass 50, and (inoculum is 10 in the culturing bottle that contains medicine ⁴-10 ⁵During cfu/ml).

In order to attempt that scaling method used in herein discovery and the S.I.R.E. test is associated, carry out the described experiment of Figure 36.This experimental design is to be used for estimating the current method of mycobacterium sensitivity test and the dependency between the observed result of use trimethyl-glycine sample stain remover.For example, the contrast that contains the MacFarland apothecary jar be 100 *-the R.F. bottle; The contrast of 10 * dilution apothecary jar is 1,000 *-the R.F. bottle; The contrast of 100 * dilution apothecary jar is 10,000 *-the R.F. bottle.

In this experiment, ATCC 27294 and 571/573-BAL mycobacterium tuberculosis strain isolated were all cultivated for 2 weeks on the 7H11 selective medium.Scrape cell, it is suspended in the NALC/ Citrate trianion, supersound process 60 seconds leaves standstill then.Solution is adjusted to 0.5 MacFarland standard, with 10,000 times of 10 times of gradient serial dilutions.

Remove 10, outside 000 * extent of dilution, the duplicate transferred species of each dilution series is carried out enumeration in 7H11, be inoculated in duplicate then in the culturing bottle, contain R.F. in the bottle, contain 8 μ g/ml final concentration ceftriaxones PANTA (P-cax), contain 15 μ g/ml (or 30 μ g/ml or 60 μ g/ml) independent CB-18 or with the CB-18 (CB-18 of 15 μ g/ml, 30 μ g/ml or 60 μ g/ml) of P-cax combination.With the duplicate transferred species of 10,000 * diluent on 7H11, and be inoculated in duplicate contain R.F. or only contain P-cax the bottle in.

Enumeration makes for 0.5 MacFarland standard of every kind of strain isolated estimates to become possibility.Be approximately each extent of dilution with these estimated values then and be inoculated in cfu number in the 12B bottle.Below shown cfu number for each serial transferred species.Figure 37 A-37E shows the result of ATCC 27294 strain isolateds, and Figure 38 A-38E shows the result of 571/573-BAL strain isolated.

Extent of dilution	The cfu of transferred species in 12B
			????ATCC?27294	????571/573-BAL
	????0.5?MacFarland	????6.28±0.57×10 5			????1.11±0.18×10 6
????10×			????62,800±5,700	????111,000±17,900
	????100×	????6,280±570			????11,100±1,790
????1,000×			????628±57	????1,110±179
	????10,000×	????63±6			????111±18

When height is imported (＞1 * 10 ⁵Cfu), the growth curve of ATCC 27294 type bacterial strains and 571/573-BAL strain isolated pattern of display abnormality (Figure 37 A and Figure 38 A) all.In first week, even the growth of strain isolated is also undisturbed when 30 μ g/ml CB-18.Yet when containing the 30 μ g/mlCB-18 of P-cax, two kinds of strain isolated all delays of display abnormality after first week.When 60 μ g/ml CB-18 (have or during antibiotic-free), two kinds of strain isolateds are all grown, but with a kind of anomalous mode growth: both begin growth at first, but stop gradually then.Have only ATCC 27294 strain isolateds once to recover.

BACTEC tests use ¹⁴CO ₂Release test.Owing to is that culturing bottle is read on the basis, so in first two weeks, should from bottle, remove owing to metabolism discharges with the every day ¹⁴CO ₂It and day between the difference of GI be to read new that the back produces from for the last time ¹⁴CO ₂Reflection.Having only first two weeks is that culturing bottle is read on the basis with the every day, and positive GI represents growth, but the GI that non-exponential raises represents there is not new growth.After two weeks, read culturing bottle in every 3-5 days.GI accumulation in this section period.In any case, use above-mentioned Δ G standard, with in the situation of MacFarland standard as inoculum, none is described strain isolated to be responsive or can to distinguish them.Therefore, to surpass 10 ⁵Cfu inoculation culture bottle does not reach re-set target, that is to say, will not recommend.

10 * dilution analysis is begun to show the result of readability.Be reported in P-cax and have all sensitivities when 60 μ g/ml CB-18 of following two kinds of strain isolateds.Be reported under the P-cax existence and have only 571/573-BAL strain isolated sensitivity when 30 μ g/ml CB-18.

The continuity of dilution series shows, thinks ATCC27294 strain isolated sensitivity when 30 μ g/ml CB-18 in the presence of P-cax when 100 * extent of dilution.Use this inoculum, be reported in P-cax and have 571/573-BAL strain isolated sensitivity when 15 μ g/ml CB-18 down.1,000 * dilution check shows, the result is identical during with 100 * extent of dilution, but in the end under two kinds of conditions the growth curve of (that is, 100 * and 1,000 *) 571/573-BAL strain isolated is very similar to the curve (Fig. 5 A-5F and Figure 11) of morning.

The conclusion that draws from these experiments has two-layer.The first, can distinguish ATCC 27294 and 571/573-BAL, but not be can both distinguish under all conditions.The second, the lower limit of test is a biology, although on the other hand, surpasses 10 ⁵The bacterium dosage will make system's excess load.100-10 ⁴The input level of individual bacillus produces readable result, and it makes distinguishing of strain isolated become possibility, 10 ³-10 ⁴The inoculum of individual bacillus will provide reportable result in good time mode.Permeability of embodiment 9 mycobacteriums and CB-18 effect

A kind of possibility of the mechanism that plays a role about CB-18 and mycobacterium permeability-related.Be not in order to support following hypothesis, following explanation is just illustrated application of the present invention as a kind of means: trimethyl-glycine sample stain remover can change the permeability of the bacterium that contains the mycolic acid structure, makes this bacterium become responsive more to microbiotic thus.Especially, the CB-18 effect may be the result that mycolic acid structure conformation distributes and changes.

This is mainly based on Yuan, people such as Y., and national academy of sciences institute reports the work of 93:12828-12833 (1996).In a word, some mycobacterium has the ability (Liu, people such as J., journal of biological chemistry 271:29545-29551 (1996)) that changes its cell walls flowability.This is to realize by the composition of the length that changes mycolic acid, change mycolic acid structure conformation or by the two.In other words, the length of cell walls mycolic acid and structure conformation all influence the melting temperature (Tm) of cell walls.For example, when the length of mycolic acid changes, perhaps when trans ratio than cis-double bonds changes, perhaps when cyclopropane, hydroxylation, methylate and when the quantity of carbonyl and/or conformational change, the melting temperature (Tm) of mycolic acid also changes (Barry, C.E. wait the people, journal of biological chemistry 271:29545-29551 (1996)).For example in M. smegmatics, when growth temperature raises, the also corresponding raising of the ratio of trans alkene in the nearly terminal double bond of mycolic acid, this is associated with the rising (that is Liu Dongxing reduction) of the melting temperature (Tm) of these fat again.In mycobacterium tuberculosis and mycobacterium avium, when the ratio of trans-cyclopropane in the proximal location improves, the also corresponding raising of the melting temperature (Tm) of these mycolic acids.

The flowability that it is believed that the mycobacterium cell walls plays an important role in permeability, thereby resistance is arranged, and (national academy of sciences institute reports 92:6630-6634 (1995) for Yuan, people such as Y.; Brennan, people such as P.J. state 64:29-63 (1995) bioid academic year).The resistance of cynnematin is considered to just Mycobacterium chelonei because permeability (Connell, people such as N.D., in: " tuberculosis; pathogeny, protection and control ", Bloom, B.R. compiles; ASM press, Washington, D.C. (1994) 333-352 pages or leaves).For example, Barry, people such as C.E., journal of biological chemistry 271:29545-29551 (1996) shows, when the flowability of M. smegmatics cell walls reduces (, when the percentage ratio of trans alkene raises), the picked-up of norfloxicin and chenodesoxy cholate also all reduces.Kaneda, people such as K., general microbiology magazine (Jour.Gen.Microbiol.) 134:2213-2229 (1988) show that in the living bacterium of high resistance property of medicine speed, the nearly terminal double bond of great majority is trans alkene.

The generation of the different mycolic acid conformations mainly enzyme of the dependence S-adenosylmethionine (SAM) by a series of structurally associateds is finished.Analysis of molecules shows, has the gene of four kinds of structurally associateds in mycobacterium tuberculosis, and their codings are responsible for the methoxyl group mycolic acid synthase (MMAS) (national academy of sciences institute reports 93:12828-12833 (1996) for Yuan, people such as Y.) of many this modifications.These four kinds of gene products (MMAS-1, MMAS-2, MMAS-3 and MMAS-4) all show the strong sequence homology with intestinal bacteria cyclopropane fatty acid synthase (CFAS) and Mycobacterium leprae and mycobacterium tuberculosis cyclopropane mycolic acid synthase (CMAS-1 and CMAS-2).Hint their shared a kind of general mechanism.This mechanism comprises SAM and a kind of carbon intermediate product (Figure 39 A).Different products is produced by the following fact: different synthase have different electron donor/acceptors at avtive spot separately, produce alkene, cyclopropane, methylate and hydroxylation and methylated combination (Yuan, Y. wait the people, national academy of sciences institute reports 93:l2828-12833 (1996)).

At United States Patent (USP) 5, in 610,198, Barry, C.E. wait the people to point out, " sulfo-tetracosanoic acid " (thia fatty acid derivative) can be used for by these inhibition that relies on the reaction of SAM suppress to cause a disease Cyclopropanated and oxygenate (Figure 39 B) of mycolic acid in the mycobacterium.Four kinds " sulfo-tetracosanoic acid " have been tested as the evidence of using.In a word, the inhibition mechanism of these analogues comprises before it is as the enzyme reaction of synthase the participation (especially, the Methyl transporters from SAM) of half section substrate.After the catalysis, the structure of formation is " stable transition state analog " (Figure 39 B).In essence, this compound does not discharge from avtive spot, and as a kind of typical competitive inhibitor.Transition state structures is similar to trimethyl-glycine sample stain remover.Especially, a kind of carboxybetaine based on sulfonium (table 1 and Figure 39 B): the sulfonium carboxybetaine does not need the catalysis of enzyme to activate, and directly suppresses to rely on the modification of SAM.

At United States Patent (USP) 5,610, in 198, Barry, people such as C.E. point out that also these sulphur tetracosanoic acid are invalid to saprophytic mycobacterium as M. smegmatics.Disclosure (United States Patent (USP) 5 with people such as Barry, 610,198) opposite, the present invention's explanation described herein, these saprophytic mycobacteriums are to the effect sensitivity of trimethyl-glycine sample stain remover: mycobacterium fortutitum shows the synergy of the CB-18 and the top between the cynnematin of trying.For example, the influence that clinical separation strain 495-JHH is not existed by cefoxitin, and can in the presence of 109 μ g/ml CB-18, grow (Figure 15) be arranged to a certain degree.Yet when cefoxitin made up with the CB-18 that is low to moderate 13 μ g/ml, influence growth to a certain extent suppressed when 27 μ g/ml CB-18 fully.

If trimethyl-glycine sample stain remover is by the permeability that influences mycobacterium on the cell walls liquidity level that acts on to the enzyme that relies on SAM, then in fact the result of Figure 14 and Figure 15 is exactly expected result.The basis of this expection comes from the similarity of mycobacterium fortutitum and M. smegmatics.Dobson, G. wait the people,: Goodfellow, M. " chemical process in the systematic bacteriology " (the ChemicalMethods in Bacterial Systematics) that waits the people to compile, Academic Press, London (1985) 237-265 page or leaf and Brennan, P.J. wait the people, stating the mycolic acid of having summed up different mycobacterium kinds among the 64:29-63 (1995) bioid academic year distributes, show that the total similar mycolic acid of the living bacterium of speed (for example, mycobacterium fortutitum, Mycobacterium chelonei, M. smegmatics etc.) distributes: main mycolic acid is a α ' type.Liu, people such as J., journal of biological chemistry 271:29545-29551 (1996) report, when growth temperature raise, the main mycomycete acid esters (mycolate) in the M. smegmatics became α ₁And α ₂Type.α ₁And α ₂Type all be longer than α ' type (α '=C ₆₄, α ₁And α ₂=C _78-79), α ₂Type has the trans alkene of near-end, and this structure gives cell walls minimum mobile or minimum permeability.α ₂Type when comparatively high temps, preponderate (α ', α ₁And α ₂The structure of mycolic acid all is shown in people such as George, K., journal of biological chemistry 270:27292-27298 (1995)).

When looking back Yuan, Y. wait the people, national academy of sciences institute report 93:12828-12833 (1996) for these rely on SAM enzyme proposed when machine-processed (Figure 39 A): trans alkene is the product that relies on the methylation of SAM, content mobile and trans alkene is inversely proportional to, and it is obvious that the meaning of these observations becomes.In substratum, add the formation that CB-18 can suppress trans alkene, improve the flowability of cell walls thus, thereby improve cell antibiotic permeability.In case permeability is alleviated, microbiotic just can more easily enter cell.The usefulness of certain antibiotics depends on the character (that is the interaction of microbiotic and the strain isolated Chinese traditional medicine action site studied) of strain isolated itself.Net result is observed CB-18 effect.

If as Liu, J. wait the people, journal of biological chemistry 271:29545-29551 (1996) is described, the modification of the dependence SAM of lipid acid is a kind of general mechanism of the mycobacterium adjusting cell walls flowability of borrowing, estimate that so trimethyl-glycine sample stain remover can influence large-scale mycolic acid structure, for example, clavate mycomycete acid esters (Durand, E. wait the people, european journal of biological chemistry (Eur.Jour.Biochem.) 93:103-112 (1979)) and Nocardia mycomycete acid esters (norcardiomycolates).

Although aforementioned discussion has reasonably illustrated the extreme synergy (table 7) between the observed cefoxitin and CB-18 in mycobacterium fortutitum, do not explain the extreme sensitivity (table 8) of mycobacterium tuberculosis to trimethyl-glycine sample stain remover.For example, when antibiotic-free mycobacterium tuberculosis type strains A TCC 27294 be grown in 27 μ g/ml CB-18 the time be subjected to the influence of less degree, when 54 μ g/mlCB-18, stop fully.In the presence of microbiotic, growth is subjected to the influence of less degree when 13 μ g/ml CB-18, does not exist when 27 μ g/ml CB-18 (Figure 10).When antibiotic-free 571/573-BAL mycobacterium tuberculosis strain isolated be grown in 13 μ g/ml CB-18 the time be subjected to the influence of less degree, when 27 μ g/ml CB-18, stop fully.In the presence of microbiotic, this strain isolated be grown in 13 μ g/ml CB-18 the time do not have (Figure 11).Compare with mycobacterium fortutitum test (Figure 14 and Figure 15), mycobacterium tuberculosis is more responsive to beet sample stain remover itself.

Obviously, slow branch bacillus estranged as mycobacterium tuberculosis, does not resemble the flowability that speed branch estranged bacillus such as M. smegmatics (Liu, people such as J., journal of biological chemistry 271:29545-29551 (1996)) are regulated cell walls like that.For example, Takayama, people such as K., U.S. respiratory tract disease summary 118:113-117 (1978) show the extreme sensitivity of mycobacterium tuberculosis to growth temperature: depart from optimum temperuture during the several years, mycolic acid is synthetic to be stopped, and mycobacterium tuberculosis can not be survived.Mycobacterium tuberculosis is to replacing antibiotic trimethyl-glycine sample stain remover of the present invention or Barry, C.E. wait people (U.S.5, the susceptibility of sulfo-tetracosanoic acid 610,198) may be the consequence of interferential susceptibility between the mycobacterium tuberculosis strain isolated belongs to the mycolic acid synthetic.For example, the Cyclopropanated or oxygenate that suppresses mycolic acid will influence permeability also thereby influence susceptibility; The mycolic acid synthetic disturbs has lethal effect usually.In slow branch bacillus estranged, only when low CB-18 concentration, observe bring out to antibiotic susceptibility.

If the action site of trimethyl-glycine sample stain remover is the synthase that relies on SAM in the bacterium that contains the mycolic acid structure, then expection will suppress lipid acid (for example, mycolic acid) modification, and cause that in some cases the mycolic acid synthetic stops.Therefore, trimethyl-glycine sample stain remover only plays a kind of assisting therapy effect in the processing of the living bacterium of speed, but expection has auxiliary and the two kinds of consequences that cause death to multiple slow branch bacillus estranged.For example, when bring out with the permeability performance of the branch bacillus slow estranged of the inhibitor incubation that relies on SAM to antibiotic susceptibility the time also be tangible and may be main factor because the mycolic acid synthetic disturbs the disadvantageous effect to surviving that causes.On the other hand, the influence that speed branch estranged bacillus is not existed by isolating trimethyl-glycine sample stain remover generally, but owing to the inhibition of the mechanism of influence liquidity shows and antibiotic surprising synergy.

People such as Barry (United States Patent (USP) 5,610,198) also explanation has only anti-mycobacterium medicine such as vazadrine, Tibutol, Streptomycin sulphate, Rifampin, dapsone, rifabutin, clarithromycin, Ciprofloxacin, Rimonophenazine, Azythromycin, ethionamide, Amikacin Sulphate or resorcinomycin A to use with the sulfo-tetracosanoic acid.The present invention said and illustration in embodiment 5 and 6 shows, in the presence of CB-18, changes aspect some of bacterial physiology, and general permeability changes mechanism described herein, thus bring out susceptibility to wide range antibiotic.Therefore, according to the present invention, the trimethyl-glycine sensitivity test will utilize CB-18 effect and multiple microbiotic, identify the natural susceptibility of the strain isolated that tries, and more effectively instruct the doctor thus.

Trimethyl-glycine sample stain remover of the present invention has tangible advantage than people's such as Barry (U.S.5,610,198) sulfo-tetracosanoic acid.For example, the sulfo-tetracosanoic acid is extremely insoluble.In other words, the Krafft temperature of these reagent is higher than physiological standard far away.Conveying at proper concn almost is impossible.On the contrary, the trimethyl-glycine sample stain remover major part of using among the present invention is soluble.For example, to have synthesized a kind of Krafft value be 20 ℃ C for Hodge, people such as D., Langmuir 7:878-884 (1991) ₂₀-carboxylic hexyl trimethyl-glycine.

Although use CB-18 to characterize the CB-18 effect at this, obviously other trimethyl-glycine also is useful in this.For example, according to structure, a kind of alkyl sulfonium carboxybetaine will be to suppress the idealized compound that mycolic acid is modified.Can pass through Zimmer, a kind of the improving one's methods of R.E. (United States Patent (USP) 3,560,393) synthesized this alkyl sulfonium carboxybetaine.For example, can methylate Stearyl mercaptan and purifying R ₁-S-R ₂, the halogenation ester that is used for adequate types is modified (Figure 39 C).To remove ester with the cation-exchange chromatography purifying and produce acid.Embodiment 10 is used to screen the trimethyl-glycine susceptibility table of clinical separation strain

Embodiment 5 and embodiment 6 show, in the process of the test of a kind of trimethyl-glycine and multiple different microbiotic couplings, and different clinical separation strain behavior performance differences.Embodiment 6 shows that in the test of different stain removers and the combination of P/cax antibiotic formulations, a kind of clinical separation strain behavior performance is different.So,, develop and improve a kind of trimethyl-glycine susceptibility table for sensitivity test.This table will intersect and use different trimethyl-glycine and different microbiotic.Embodiment described herein shows this table is how to design and be used for sensitivity test.Present embodiment is to illustrate how to develop this table as a kind of means, rather than as restriction of the present invention.

Use causes wide spectrum result's different trimethyl-glycines for different clinical separation strains, for example five kinds, together with the different microbiotic that cause the wide spectrum result for different clinical separation strains, for example five kinds, forms trimethyl-glycine susceptibility table.For example, determine 5 kinds of microbiotic α-1, α-2, α-3, α-4 and α-5, and determine that 5 kinds of trimethyl-glycines are as β-1, β-2, β-3, β-4 and β-5.Five kinds of different trimethyl-glycine sample stain remover conditions can be identical stain remover or its combination of different concns.Equally, five kinds of identical microbiotic or its combinations that different microbiotic can be different concns.Table 11 shows how these 5 kinds of trimethyl-glycines are complementary to identify the most effective trimethyl-glycine-microbiotic combination with these 5 kinds of microbiotic.

Table 11 trimethyl-glycine susceptibility table
							Microbiotic	Trimethyl-glycine sample stain remover
????φ	????β-1	????β-2	????β-3	????β-4	????β-5
						????φ		????-	????β-1	????β-2	????β-3	????β-4	????β-5
????α-1	????α-1	?α-1/β-1	?α-1/β-2	?α-1/β-3	?α-1/β-4		?α-1/β-5

????α-2	????α-2	?α-2/β-1	?α-2/β-2	?α-2/β-3	?α-2/β-4	?α-2/β-5
							????α-3	????α-3	?α-3/β-1	?α-3/β-2	?α-3/β-3	?α-3/β-4	?α-3/β-5
????α-4	????α-4	?α-4/β-1	?α-4/β-2	?α-4/β-3	?α-4/β-4	?α-4/β-5
							????α-5	????α-5	?α-5/β-1	?α-5/β-2	?α-5/β-3	?α-5/β-4	?α-5/β-5

Trimethyl-glycine susceptibility table

Below shown in the narration table 11 in application (1) liquid medium within (for example BACTEC 12B) of trimethyl-glycine table or solid medium (for example 7H11 is oblique

Face) goes up the described clinical separation strain of cultivation.(2) set up the trimethyl-glycine table.For example can in microtiter plate or single bottle, set up this table.Trimethyl-glycine

Can freeze-drying in suitable hole or bottle with microbiotic, rebuild with growth medium then, perhaps

Mix with the single solution of microbiotic, trimethyl-glycine and meat soup and to reach identical purpose.The former is

Preferably.(3) use the side that in each hole or pipe, adds an amount of cell (for example, 100-10,000 cfu)

Formula prepares the bacterium stock solution.As known in the art, by answering based on the MacFarland standard

This stock solution can be adjusted to an amount of cell with producing suspension, be diluted to hope then

Terminal point.(4), and use institute's choosing method inspection growth with plate, pipe or bottle incubation preset time.Growth

Detection can based on ¹⁴C release (for example, BACTEC 12B:Becton-Dickinson,

Cockeysville, MD), O ₂Consume (ESP Myco System II ^TM, DIFCO

Laboratories, Detroit, MI), pH changes (MB/BacT ^, Organon Teknika,

Durham, NC) or other standard technique known in the art, as nephelometry.

This table also can be used for the clinical separation strain of classifying.For example, borrow it to play a role and strengthen the mechanism of antimicrobial therapy if known trimethyl-glycine, so this information will have with reference to property for improving the treatment plan relevant or irrelevant with trimethyl-glycine-microbiotic combination.Therefore, there are two kinds of distinct terminal points.In first kind of terminal point, trimethyl-glycine susceptibility table is used as a kind of means of the clinical separation strain of classifying.This classification is to be used for more effectively determining and the application irrelevant treatment plan of trimethyl-glycine as the treatment adjuvant.In second kind of terminal point, the purpose of trimethyl-glycine sensitivity test is to determine the combination (as described in next embodiment (embodiment 11)) of specific trimethyl-glycine sample stain remover and certain antibiotics.Embodiment 11 trimethyl-glycine sample stain removers are as the application of treatment adjuvant

As described in embodiment 10, sensitivity test has two kinds of terminal points.In a kind of terminal point, the trimethyl-glycine table is used as a kind of typical susceptibility sieve method and is used for the most effective treatment plan that determine to eliminate infects, is a kind of microbiotic in the table of embodiment 10 the most significantly.In this terminal point, treatment plan does not comprise the application of trimethyl-glycine sample stain remover as the treatment adjuvant.

In another kind of terminal point, treatment plan is based on one or more contained microbiotic in the table: trimethyl-glycine sample stain remover combination.In this terminal point, the understanding that obtains from the result of embodiment 10 described tables is that for example, terminal point itself: this information is specified the particular combinations of microbiotic and trimethyl-glycine sample stain remover to the technician.In the case, trimethyl-glycine must contact with the patient.How the purpose of this embodiment is to describe trimethyl-glycine as the treatment adjuvant.This embodiment illustrates how to develop this table as a kind of means, is not as restriction of the present invention.

Embodiment described herein and data presentation although trimethyl-glycine sample stain remover has limited antimicrobial usefulness when separately using, are the most effective when being used in combination with one or more microbiotic.In order to make any antimicrobial therapy effective, must carry microbiotic to microorganism.The conveying of microbiotic itself can be by the standard method that is used for the microbiotic conveying known in the art in the method for the invention.How the purpose of present embodiment carries trimethyl-glycine to infective agent if being to describe.Therefore, accept among the patient of adjuvant treatment,, can carry trimethyl-glycine to carry microbiotic by a kind of method by another kind of method if wish in the method according to this invention.

Carry the standard method of trimethyl-glycine sample stain remover to be known in the art, comprise oral, intramuscular injection, intravenous drip, in the infection site injection or suck.Yet trimethyl-glycine sample stain remover is a tensio-active agent, may not tolerate well by oral or intramuscular injection.For example, a large amount of the oral of trimethyl-glycine sample stain remover may cause gastrointestinal pain, and the injection of a large amount of trimethyl-glycines may cause cyto-architectural dissolving, causes the stimulation of injection site.When using intravenous drip, must be noted that the concentration of trimethyl-glycine in the blood.Although adding trimethyl-glycine is feasible to micelle-forming concentration (CMC), if owing to the dissolving of blood ingredient the whole blood level is increased to more than the CMC then can leads to complications.In the infection site injection also may be a kind of feasible carrying method; Unfortunately, this is only possible in situation seldom, for example tuberculosis infringement or the granuloma infringement that is caused by propagated MAC disease.In a kind of preferred embodiment, carry trimethyl-glycine by sucking.

Tuberculosis mainly is a kind of respiratory tract infection.The tuberculosis infringement generally comes across the last leaf of lung.If the surface coverage of lung has a kind of natural surface active agent, carry the effective way of trimethyl-glycine sample stain remover may be similar to the conveying of beta blocker among the asthma patient or steroid to infection site.For example, some steroid and beta blocker are generally carried with the form of micro-crystallization, as Ventolin ^(by Allen﹠amp; Hanburys produces, Allen ﹠amp; Hanburys is the company of Glaxo, ResearchTriangle Park, NC).

Now narrate the present invention fully, it will be understood by those of skill in the art that under the situation of the spirit or scope that do not influence the present invention or its any embodiment, can within the condition of broad and equal scope, parameter etc., carry out the present invention.

Claims (143)

1. the method for a sensitivity test, this method comprises makes microorganism contact with the composition that contains microbiotic and trimethyl-glycine sample stain remover, and characterizes this microorganism to this antibiotic susceptibility according to the viability of this microorganism in said composition.

2. the process of claim 1 wherein that described trimethyl-glycine sample stain remover is selected from: CB sample, SB sample, HSB sample, PB sample, StB sample, PhB sample, SoB sample, RevB sample, AO sample, cAB sample and ImB sample stain remover.

3. the method for claim 2, wherein this trimethyl-glycine sample stain remover is a CB sample stain remover.

4. the method for claim 3, wherein this CB sample stain remover has structure

R wherein ₁Be C ₈-C ₂₂

α is-CH ₂-,-CH (OH)-,-(CO)-NH-CH ₂CH ₂CH ₂-,-O-or-C (0)-;

N is 0 or 1;

β is-N -,-P -or-S -;

R ₂For-H ,-CH ₃,-C ₂H ₅,-C ₃H ₇Or-C ₄H ₉

R ₃For-H ,-CH ₃,-C ₂H ₅,-C ₃₁H ₇Or-C ₄H ₉

R ₄For-CH ₂-,-C ₂H ₄-,-C ₃H ₆-,-C ₄H ₈-,-C ₅H ₁₀-,-C ₆H ₁₂-,-CH ₂-C ₆H ₄-,-C _mH _2m-,-CH (OH) CH ₂CH ₂-,-CH ₂CH (OH) CH ₂-or-C _mH _2m-1 (OH)-, m 〉=1 wherein; And

γ is-COO

5. the method for claim 4, wherein said CB sample stain remover is selected from:

N-(carboxymethyl)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.693-33-4),

Cocounut oil carboxymethyl betaine and (CAS ^No.68424-94-2),

N-(carboxymethyl)-N, N-dimethyl-9-octadecane-1-ammonium, inner salt (CAS ^No.871-37-4),

N-(carboxymethyl)-N, N-dimethyl-3-((1-oxygen octadecyl) amino)-1-third ammonium, inner salt (CAS ^No.6179-44-8),

3-amino-N (carboxymethyl)-N, N-dimethyl-1-third ammonium N-C8-C22 acyl derivative, inner salt (CAS ^No.84082-44-0),

N-(carboxymethyl)-3-((12-hydroxyl-1-oxygen-9-octadecyl) amino)-N, N-dimethyl-1-third ammonium, inner salt (CAS ^No.71850-81-2),

Cocamidopropyl propyl amide carboxymethyl betaine (CAS ^No.61789-39-7 and CAS ^No.61789-40-0),

N-(2-propyloic)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.16527-85-8),

N-(2-propyloic)-N, N-dimethyl-1-tridecane ammonium, inner salt (CAS ^No.132621-79-5),

N-(2-propyloic)-N, N-dimethyl-1-tetradecane ammonium, inner salt (CAS ^No.69725-38-3),

N-(2-propyloic)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.42416-43-3),

N-(2-propyloic)-N, N-dimethyl-1-octadecane ammonium, inner salt (CAS ^No.30612-73-8),

N-dodecyl-Beta-alanine (CAS ^No.1462-54-0),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-undecane ammonium, inner salt (CAS ^No.150147-53-8),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.15163-30-1),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-tetradecane ammonium, inner salt (CAS ^No.146959-90-2),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-pentadecane ammonium, inner salt (CAS ^No.146959-91-3),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.71695-32-4),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecane ammonium, inner salt (CAS ^No.78195-27-4),

N-(4-carboxylic butyl)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.120139-51-7),

N-(5-carboxylic amyl group)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.76392-97-7),

N-(5-carboxylic amyl group)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.73565-98-7),

N-(6-carboxylic hexyl)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.132621-80-8),

4-carboxy-N-dodecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-31-3),

2-carboxy-N-dodecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-34-6),

4-carboxy-N-hexadecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-33-5),

2-carboxy-N-hexadecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-35-7), tallow glycinate (CAS ^No.70750-46-8), soy amide propyl group carboxymethyl betaine, and babassu amido propyl carboxymethyl betaine.

6. the method for claim 5, wherein said carboxybetaine is N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecane ammonium, inner salt (CB-18) (CAS ^No.78195-27-4)

7. the method for claim 2, wherein said trimethyl-glycine sample stain remover is a SB sample stain remover.

8. the method for claim 7, wherein this SB sample stain remover is selected from SB-18, SB-16, SB-14 and SB-12.

9. the method for claim 8, wherein this SB sample stain remover is SB-16.

10. the method for claim 8, wherein this SB sample stain remover is SB-18.

11. the method for claim 2, wherein said trimethyl-glycine sample stain remover is a SoB sample stain remover.

12. each method of claim 1-11, wherein said composition contains two or more trimethyl-glycine sample stain removers.

13. the process of claim 1 wherein that described microbiotic is selected from: beta-lactam antibiotics, aminoglycoside, aminocyclitol, quinolone, tsiklomitsin, macrolide, lincosamide, glycopeptide, lipopeptid, peptide antibiotics, sulfanilamide (SN), trimethoprim, paraxin, vazadrine, nitroimidazole, Rifampin, nitrofuran, urotropine and mupirocin.

14. the method for claim 13, wherein this microbiotic is a beta-lactam antibiotics.

15. the method for claim 14, wherein this beta-lactam antibiotics is selected from penicillin, cynnematin, monobactam and carbapenem antibiotics.

16. the method for claim 15, wherein this beta-lactam antibiotics is a penicillin.

17. the method for claim 16, wherein this penicillin is selected from: azlocillin, X-1497, nafcillin, cloxacillin, dicloxacillin, Oxazacillin, penbritin, bacampicillin, Pyocianil, ticarcillin, mezlocillin and piperacillin.

18. the method for claim 15, wherein such microbiotic is a cynnematin.

19. the method for claim 18, wherein this cynnematin is selected from: cefoxitin, cefoperazone, ceftazidime, ceftriaxone, S 578, Kefzol, Cephalexin Monohydrate Micro/Compacted, Cephaloridine, cefoxitin, Cephapirin, Cephradine, cefaclor, Cefamandole, cefonicid, BL-S 786, Prozef, cephalofruxin, Loracarbef, cefmetazole, cefotetan, Cefixime Micronized, cefotaxime, Cefpodoxime and ceftizoxime.

20. the method for claim 19, wherein this cynnematin is a ceftriaxone.

21. the method for claim 15, wherein said beta-lactam antibiotics is a monobactam.

22. the method for claim 21, wherein this monobactam is an aztreonam.

23. the method for claim 15, wherein said beta-lactam antibiotics is a carbapenem.

24. the method for claim 23, wherein this carbapenem is selected from imipenum, meropenem, the handkerchief people and trains south and biapenem.

25. the method for claim 24, wherein this carbapenem is an imipenum.

26. each method of claim 13-25, wherein said composition further contains a kind of beta-lactamase inhibitor.

27. the method for claim 26, wherein this beta-lactamase inhibitor is selected from clavulanic acid, Sulbactam and Zosyn.

28. the method for claim 13, wherein said microbiotic are aminoglycoside or aminocyclitol.

29. the method for claim 28, wherein this aminoglycoside or aminocyclitol are selected from: Streptomycin sulphate, kantlex, gentamicin, tobramycin, Amikacin Sulphate, sisomycin, netilmicin, Xin Meisu, Soframycin and paromycin.

30. the method for claim 13, wherein said microbiotic is a quinolone.

31. the method for claim 30, wherein this quinolone is selected from: nalidixic acid, oxolinic acid, cinoxacin, flumequine, Miloxacin, rosoxacin, pipemidic acid, norfloxicin, enoxacin, Ciprofloxacin, Ofloxacine USP 23, lomefloxacin, Temafloxacin, fleroxacin, Pefloxacin, amifloxacin, sparfloxacin, levofloxacin and Clinafloxacin.

32. the method for claim 13, wherein said microbiotic is a tsiklomitsin.

33. the method for claim 13, wherein said microbiotic is a macrolide.

34. the method for claim 33, wherein this macrolide is selected from: erythromycin, romicil, Spiramycin Base, josamycin, rosaramicin, clarithromycin, Azythromycin, dirithromycin, Roxithromycin, Flurithromycin and rokitamycin.

35. the method for claim 13, wherein said microbiotic is a lincosamide.

36. the method for claim 13, wherein said microbiotic is a glycopeptide.

37. the method for claim 13, wherein said microbiotic is a lipopeptid.

38. the method for claim 13, wherein said microbiotic is a peptide antibiotics.

39. the method for claim 13, wherein said microbiotic is a sulfanilamide (SN).

40. the method for claim 13, wherein said microbiotic is a trimethoprim.

41. the method for claim 13, wherein said composition contains two or more microbiotic.

42. the method for claim 41, wherein this microbiotic is sulfanilamide (SN) and trimethoprim.

43. the method for claim 42, wherein this sulfanilamide (SN) is sulfamethoxazole.

44. the method for claim 13, wherein said microbiotic is a paraxin.

45. the method for claim 13, wherein said microbiotic is the vazadrine.

46. the method for claim 13, wherein said microbiotic is a nitroimidazole.

47. the method for claim 13, wherein said microbiotic is a Rifampin.

48. the method for claim 47, wherein this Rifampin is selected from Rifampin, Rifamycin Sodium, rifamycin B (rifamide) and rifabutin.

49. the method for claim 48, wherein this Rifampin is a Rifampin.

50. the method for claim 48, wherein this Rifampin is a rifabutin.

51. the method for claim 13, wherein said microbiotic is a nitrofuran.

52. the method for claim 13, wherein said microbiotic is a urotropine.

53. the method for claim 13, wherein said microbiotic is a mupirocin.

54. the process of claim 1 wherein that described microbiotic is selected from: Amikacin Sulphate, Azythromycin, arbitrary beta-lactam with arbitrary beta-lactamase inhibitor combination, capromycin, cefmetazole, cefoxitin, Ciprofloxacin, clarithromycin, Rimonophenazine, seromycin, dapsone, erythromycin, Tibutol, ethionamide, imipenum, the vazadrine, kantlex, MINOCYCLINE HCL, Ofloxacine USP 23, to Aminosalicylic, Protionamide, pyrazinoic acid amide, Rifampin, rifabutin, sparfloxacin, sulfamethoxazole and trimethoprim, Streptomycin sulphate, tsiklomitsin, thiacetazole and Viothenate.

55. the method for an antimicrobial therapy has been used to infect a kind of microorganism or the patient who infects this microorganism danger has been arranged, this method comprise with the amount that is enough to kill this microorganism and time to this patient betaine administration sample stain remover and microbiotic simultaneously.

56. the method for claim 55, wherein said trimethyl-glycine sample stain remover is selected from: CB sample, SB sample, HSB sample, PB sample, StB sample, PhB sample, SoB sample, RevB sample, AO sample, cAB sample and ImB sample stain remover.

57. the method for claim 56, wherein this trimethyl-glycine sample stain remover is a CB sample stain remover.

58. the method for claim 57, wherein this CB sample stain remover has structure

R wherein ₁Be C ₈-C ₂₂

α is-CH ₂-,-CH (OH)-,-(CO)-NH-CH ₂CH ₂CH ₂-,-O-or-C (O)-;

N is 0 or 1;

β is-N -,-P -or-S -;

R ₂For-H ,-CH ₃,-C ₂H ₅,-C ₃H ₇Or-C ₄H ₉

R ₃For-H ,-CH ₃,-C ₂H ₅,-C ₃₁H ₇Or-C ₄H ₉

R ₄For-CH ₂-,-C ₂H ₄-,-C ₃H ₆-,-C ₄H ₈-,-C ₅H ₁₀-,-C ₆H ₁₂-,-CH ₂-C ₆H ₄-,-C _mH _2m-,-CH (OH) CH ₂CH ₂-,-CH ₂CH (OH) CH ₂-or-C _mH _2m-1(OH)-, m 〉=1 wherein; And

γ is-COO

59. the method for claim 58, wherein said CB sample stain remover is selected from:

N-(carboxymethyl)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.693-33-4),

Cocounut oil carboxymethyl betaine and (CAS ^No.68424-94-2),

N-(carboxymethyl)-N, N-dimethyl-9-octadecane-1-ammonium, inner salt (CAS ^No.871-37-4),

N-(carboxymethyl)-N, N-dimethyl-3-((1-oxygen octadecyl) amino)-1-third ammonium, inner salt (CAS ^No.6179-44-8),

3-amino-N (carboxymethyl)-N, N-dimethyl-1-third ammonium N-C8-C22 acyl derivative, inner salt (CAS ^No.84082-44-0),

N-(carboxymethyl)-3-((12-hydroxyl-1-oxygen-9-octadecyl) amino)-N, N-dimethyl-1-third ammonium, inner salt (CAS ^No.71850-81-2),

Cocamidopropyl propyl amide carboxymethyl betaine (CAS ^No.61789-39-7 and CAS ^No.61789-40-0),

N-(2-propyloic)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.16527-85-8),

N-(2-propyloic)-N, N-dimethyl-1-tridecane ammonium, inner salt (CAS ^No.132621-79-5),

N-(2-propyloic)-N, N-dimethyl-1-tetradecane ammonium, inner salt (CAS ^No.69725-38-3),

N-(2-propyloic)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.42416-43-3),

N-(2-propyloic)-N, N-dimethyl-1-octadecane ammonium, inner salt (CAS ^No.30612-73-8),

N-dodecyl-Beta-alanine (CAS ^No.1462-54-0),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-undecane ammonium inner salt (CAS ^No.150147-53-8),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.15163-30-1),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-tetradecane ammonium, inner salt (CAS ^No.146959-90-2),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-pentadecane ammonium, inner salt (CAS1 ^No.146959-91-3),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.71695-32-4),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecane ammonium, inner salt (CAS ^No.78195-27-4),

N-(4-carboxylic butyl)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.120139-51-7),

N-(5-carboxylic amyl group)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.76392-97-7),

N-(5-carboxylic amyl group)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.73565-98-7),

N-(6-carboxylic hexyl)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.132621-80-8),

4-carboxy-N-dodecyl-N, N-trimethylammonium-benzalkonium, inner salt (CAS ^No.71695-31-3),

2-carboxy-N-dodecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-34-6),

4-carboxy-N-hexadecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-33-5),

2-carboxy-N-hexadecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-35-7), tallow glycinate (CAS ^No.70750-46-8), soy amide propyl group carboxymethyl betaine, and babassu amido propyl carboxymethyl betaine.

60. the method for claim 59, wherein said carboxybetaine are N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecane ammonium, inner salt (CB-18) (CAS ^No.78195-27-4)

61. the method for claim 56, wherein said trimethyl-glycine sample stain remover is a SB sample stain remover.

62. the method for claim 61, wherein this SB sample stain remover is selected from SB-18, SB-16, SB-14 and SB-12.

63. the method for claim 62, wherein this SB sample stain remover is SB-16.

64. the method for claim 62, wherein this SB sample stain remover is SB-18.

65. the method for claim 56, wherein said trimethyl-glycine sample stain remover is a SoB sample stain remover.

66. each method of claim 56-65, wherein said method comprises uses two or more trimethyl-glycine sample stain removers to the patient.

67. the method for claim 55, wherein said microbiotic is selected from: beta-lactam antibiotics, aminoglycoside, aminocyclitol, quinolone, tsiklomitsin, macrolide, lincosamide, glycopeptide, lipopeptid, peptide antibiotics, sulfanilamide (SN), trimethoprim, paraxin, vazadrine, nitroimidazole, Rifampin, nitrofuran, urotropine and mupirocin.

68. the method for claim 67, wherein this microbiotic is a beta-lactam antibiotics.

69. the method for claim 68, wherein this beta-lactam antibiotics is selected from penicillin, cynnematin, monobactam and carbapenem antibiotics.

70. the method for claim 69, wherein this microbiotic is a penicillin.

71. the method for claim 70, wherein this penicillin is selected from: azlocillin, X-1497, nafcillin, cloxacillin, dicloxacillin, Oxazacillin, penbritin, bacampicillin, Pyocianil, ticarcillin, mezlocillin, penicillin and piperacillin.

72. the method for claim 69, wherein this microbiotic is a cynnematin.

73. the method for claim 72, wherein this cynnematin is selected from: cefoxitin, cefoperazone, ceftazidime, ceftriaxone, S 578, Kefzol, Cephalexin Monohydrate Micro/Compacted, Cephaloridine, cefoxitin, Cephapirin, Cephradine, cefaclor, Cefamandole, cefonicid, BL-S 786, Prozef, cephalofruxin, Loracarbef, cefmetazole, cefotetan, Cefixime Micronized, cefotaxime, Cefpodoxime and ceftizoxime.

74. the method for claim 73, wherein this cynnematin is a ceftriaxone.

75. the method for claim 69, wherein said microbiotic is a monobactam.

76. the method for claim 75, wherein this monobactam is an aztreonam.

77. the method for claim 69, wherein said microbiotic is a carbapenem.

78. the method for claim 77, wherein this carbapenem is selected from imipenum, meropenem, the handkerchief people and trains south and biaapenem.

79. the method for claim 78, wherein this carbapenem is an imipenum.

80. each method of claim 68-80, wherein said method further comprises uses beta-lactamase inhibitor to the patient.

81. the method for claim 80, wherein this beta-lactamase inhibitor is selected from clavulanic acid, Sulbactam and Zosyn.

82. the method for claim 67, wherein said microbiotic are aminoglycoside or aminocyclitol.

83. the method for claim 82, wherein this aminoglycoside or aminocyclitol are selected from: Streptomycin sulphate, kantlex, gentamicin, tobramycin, Amikacin Sulphate, sisomycin, netilmicin, Xin Meisu, Soframycin and paromycin.

84. the method for claim 67, wherein said microbiotic is a quinolone.

85. the method for claim 83, wherein this quinolone is selected from: nalidixic acid, oxolinic acid, cinoxacin, flumequine, Miloxacin, rosoxacin, pipemidic acid, norfloxicin, enoxacin, Ciprofloxacin, Ofloxacine USP 23, lomefloxacin, Temafloxacin, fleroxacin, Pefloxacin, amifloxacin, sparfloxacin, levofloxacin, Clinafloxacin.

86. the method for claim 67, wherein said microbiotic is a tsiklomitsin.

87. the method for claim 67, wherein said microbiotic is a macrolide.

88. the method for claim 87, wherein this macrolide is selected from: erythromycin, romicil, Spiramycin Base, josamycin, rosaramicin, clarithromycin, Azythromycin, dirithromycin, Roxithromycin, Flurithromycin and rokitamycin.

89. the method for claim 67, wherein said microbiotic is a lincosamide.

90. the method for claim 67, wherein said microbiotic is a glycopeptide.

91. the method for claim 67, wherein said microbiotic is a lipopeptid.

92. the method for claim 67, wherein said microbiotic is a peptide antibiotics.

93. the method for claim 67, wherein said microbiotic is a sulfanilamide (SN).

94. the method for claim 67, wherein said microbiotic is a trimethoprim.

95. the method for claim 55 is wherein used two kinds of microbiotic to the patient.

96. the method for claim 95, wherein this microbiotic is sulfanilamide (SN) and trimethoprim.

97. the method for claim 96, wherein this sulfanilamide (SN) is sulfamethoxazole.

98. the method for claim 67, wherein said microbiotic is a paraxin.

99. the method for claim 67, wherein said microbiotic is the vazadrine.

100. the method for claim 67, wherein said microbiotic is a nitroimidazole.

101. the method for claim 67, wherein said microbiotic is a Rifampin.

102. the method for claim 101, wherein this Rifampin is selected from Rifampin, Rifamycin Sodium, rifamycin B (rifamide) and rifabutin.

103. the method for claim 102, wherein this Rifampin is a Rifampin.

104. the method for claim 102, wherein this Rifampin is a rifabutin.

105. the method for claim 67, wherein said microbiotic is a nitrofuran.

106. the method for claim 67, wherein said microbiotic is a urotropine.

107. the method for claim 67, wherein said microbiotic is a mupirocin.

108. the method for claim 55, wherein said microbiotic is selected from Amikacin Sulphate, Azythromycin, arbitrary beta-lactam with arbitrary beta-lactamase inhibitor combination, capromycin, cefmetazole, cefoxitin, Ciprofloxacin, clarithromycin, Rimonophenazine, seromycin, dapsone, erythromycin, Tibutol, ethionamide, imipenum, the vazadrine, kantlex, MINOCYCLINE HCL, Ofloxacine USP 23, to Aminosalicylic, Protionamide, pyrazinoic acid amide, Rifampin, rifabutin, sparfloxacin, sulfamethoxazole and trimethoprim, Streptomycin sulphate, tsiklomitsin, thiacetazole and Viothenate.

109. claim 1 or 55 each methods, wherein said microorganism has the mycolic acid structure in its adventitia.

110. the method for claim 109, wherein this microorganism is a Mycobacterium.

111. the method for claim 110, wherein this mycobacterium is selected from: the field mycobacterium, mycobacterium abscessus, M.acetamidolyticum, mycobacterium africanum, like to know mycobacterium, the Asia mycobacterium, golden mycobacterium, the South Africa mycobacterium, mycobacterium avium, Mycobacterium bovis, Mycobacterium bovis (BCG), Mycobacterium chelonei, thousand field mycobacteriums, Chu cloth mycobacterium, the Ku Shi mycobacterium, the Di Shi mycobacterium, the Du Shi mycobacterium, crafty mycobacterium, produce the glanders mycobacterium, little yellow mycobacterium, mycobacterium fortutitum, add this mycobacterium of ground, mycobacterium gastri, pale yellow mycobacterium, mycobacterium gordonae, mycobacterium haemophilum, Mycobacterium intracellulare, mycobacterium kansasii, this mycobacterium of Como, Mycobacterium leprae, mycobacterium lepraemurim, Mycobacterium marinum, the Ma Ermo mycobacterium, mycobacterium microti, the Mo Liaoka mycobacterium, new golden mycobacterium, mycobacterium nonchromogenicum, cloth mycobacterium difficult to understand, secondary mycobacterium fortuitum, mycobacterium paratuberculosis, external mycobacterium, Mycobacterium phlei, the pig mycobacterium, the porous mycobacterium, the dust mycobacterium, the Rhode Island mycobacterium, Mycobacterium scrofulaceum, the Senegal mycobacterium, the Shi Shi mycobacterium, mycobacterium habana, M. smegmatics, the sphagnum moss mycobacterium, the Si Shi mycobacterium, mycobacterium terrae, the heat resistanceheat resistant mycobacterium, East Sea mycobacterium, mycobacterium triviale, mycobacterium tuberculosis, mycobacterium buruli, the cow mycobacterium, mycobacterium littorale.

112. the method for claim 111, wherein this mycobacterium is a member of mycobacterium tuberculosis complex body (MTB).

113. the method for claim 112, wherein this microorganism is a mycobacterium tuberculosis.

114. the method for claim 111, wherein this microorganism is a member of mycobacterium avium (MAC) complex body.

115. the method for claim 111, wherein this microorganism is a member of MAIS group.

116. the method for claim 111, wherein this microorganism is a mycobacterium buruli.

117. the method for claim 111, wherein this microorganism is a mycobacterium kansasii.

118. the method for claim 111, wherein this microorganism is a Mycobacterium chelonei.

119. the method for claim 111, wherein this microorganism is a mycobacterium fortutitum.

120. a composition that is used for sensitivity test, said composition contain and one or more one or more microbiotic of trimethyl-glycine sample stain remover blended.

121. the composition of claim 121, wherein at least a trimethyl-glycine sample stain remover is selected from: CB sample, SB sample, HSB sample, PB sample, StB sample, PhB sample, SoB sample, RevB sample, AO sample, cAB sample and ImB sample stain remover.

122. the composition of claim 122, wherein this trimethyl-glycine sample stain remover is a CB sample stain remover.

123. the composition of claim 123, wherein this CB sample stain remover has structure

R wherein ₁Be C ₈-C ₂₂

α is-CH ₂-,-CH (OH)-,-(CO)-NH-CH ₂CH ₂CH ₂-,-O-or-C (O)-;

N is O or 1;

β is-N -,-P -or-S -;

R ₂For-H ,-CH ₃,-C ₂H ₅,-C ₃H ₇Or-C ₄H ₉

R ₃For-H ,-CH ₃,-C ₂H ₅,-C ₃₁H ₇Or-C ₄H ₉

R ₄For-CH ₂-,-C ₂H ₄-,-C ₃H ₆-,-C ₄H ₈-,-C ₅H ₁₀-,-C ₆H ₁₂-,-CH ₂-C ₆H ₄-,-C _mH _2m-,-CH (OH) CH ₂CH ₂-,-CH ₂CH (OH) CH ₂-or-C _mH _2m-1(OH)-, m 〉=1 wherein; And

γ is-COO

124. the composition of claim 124, wherein said CB sample stain remover is selected from:

N-(carboxymethyl)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.693-33-4),

Cocounut oil carboxymethyl betaine and (CAS ^No.68424-94-2),

N-(carboxymethyl)-N, N-dimethyl-9-octadecane-1-ammonium, inner salt (CAS ^No.871-37-4),

N-(carboxymethyl)-N, N-dimethyl-3-((1-oxygen octadecyl) amino)-1-third ammonium, inner salt (CAS ^No.6179-44-8),

3-amino-N (carboxymethyl)-N, N-dimethyl-1-third ammonium N-C8-C22 acyl derivative, inner salt (CAS ^No.84082-44-0),

N-(carboxymethyl)-3-((12-hydroxyl-1-oxygen-9-octadecyl) amino)-N, N-dimethyl-1-third ammonium, inner salt (CAS ^No.71850-81-2),

Cocamidopropyl propyl amide carboxymethyl betaine (CAS ^No.61789-39-7 and CAS ^No.61789-40-0),

N-(2-propyloic)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.16527-85-8),

N-(2-propyloic)-N, N-dimethyl-1-tridecane ammonium, inner salt (CAS ^No.132621-79-5),

N-(2-propyloic)-N, N-dimethyl-1-tetradecane ammonium, inner salt (CAS ^No.69725-38-3),

N-(2-propyloic)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.42416-43-3),

N-(2-propyloic)-N, N-dimethyl-1-octadecane ammonium, inner salt (CAS ^No.30612-73-8),

N-dodecyl-Beta-alanine (CAS ^No.1462-54-0),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-undecane ammonium, inner salt (CAS ^No.150147-53-8),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.15163-30-1),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-tetradecane ammonium, inner salt (CAS ^No.146959-90-2),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-pentadecane ammonium, inner salt (CAS ^No.146959-91-3),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.71695-32-4),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecane ammonium, inner salt (CAS ^No.78195-27-4),

N-(4-carboxylic butyl)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.120139-51-7),

N-(5-carboxylic amyl group)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.76392-97-7),

N-(5-carboxylic amyl group)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.73565-98-7),

N-(6-carboxylic hexyl)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.132621-80-8),

4-carboxy-N-dodecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-31-3),

2-carboxy-N-dodecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-34-6),

4-carboxy-N-hexadecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-33-5),

2-carboxy-N-hexadecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-35-7), tallow glycinate (CAS ^No.70750-46-8), soy amide propyl group carboxymethyl betaine, and babassu amido propyl carboxymethyl betaine.

125. the composition of claim 125, wherein said carboxybetaine are N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecane ammonium, inner salt (CB-18) (CAS ^No.78195-27-4)

126. the composition of claim 121, wherein said trimethyl-glycine sample stain remover is a SB sample stain remover.

127. the composition of claim 126, wherein this SB sample stain remover is selected from SB-18, SB-16, SB-14 and SB-12.

128. the composition of claim 127, wherein this SB sample stain remover is SB-16.

129. the composition of claim 127, wherein this SB sample stain remover is SB-18.

130. each composition of claim 120-129, wherein said composition contains two or more trimethyl-glycine sample stain removers.

131. the composition of claim 120, wherein said microbiotic is selected from: beta-lactam antibiotics, aminoglycoside, aminocyclitol, quinolone, tsiklomitsin, macrolide, lincosamide, glycopeptide, lipopeptid, peptide antibiotics, sulfanilamide (SN), trimethoprim, paraxin, vazadrine, nitroimidazole, Rifampin, nitrofuran, urotropine and mupirocin.

132. a test kit that is used to measure microorganism susceptibility, this test kit contain next-door neighbour or adjacent one or more trimethyl-glycine sample stain removers and one or more microbiotic.

133. the test kit of claim 132, wherein at least a trimethyl-glycine sample stain remover is selected from: CB sample, SB sample, HSB sample, PB sample, StB sample, PhB sample, SoB sample, RevB sample, AO sample, cAB sample and ImB sample stain remover.

134. the test kit of claim 133, wherein this trimethyl-glycine sample stain remover is a CB sample stain remover.

135. the test kit of claim 134, wherein this CB sample stain remover has structure

R wherein ₁Be C ₈-C ₂₂

α is-CH ₂-,-CH (OH)-,-(CO)-NH-CH ₂CH ₂CH ₂-,-O-or-C (O)-;

N is 0 or 1;

β is-N -,-P -or-S -;

R ₂For-H ,-CH ₃,-C ₂H ₅,-C ₃H ₇Or-C ₄H ₉

R ₃For-H ,-CH ₃,-C ₂H ₅,-C ₃₁H ₇Or-C ₄H ₉

R ₄For-CH ₂-,-C ₂H ₄-,-C ₃H ₆-,-C ₄H ₈-,-C ₅H ₁₀-,-C ₆H ₁₂-,-CH ₂-C ₆H ₄-,-C _mH _2m-,-CH (OH) CH ₂CH ₂-,-CH ₂CH (OH) CH ₂-or-C _mH _2m-1(OH)-, m 〉=1 wherein; And

γ is-COO

136. the test kit of claim 135, wherein said CB sample stain remover is selected from:

N-(carboxymethyl)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.693-33-4),

Cocounut oil carboxymethyl betaine and (CAS ^No.68424-94-2),

N-(carboxymethyl)-N, N-dimethyl-9-octadecane-1-ammonium, inner salt (CAS ^No.871-37-4),

N-(carboxymethyl)-N, N-dimethyl-3-((1-oxygen octadecyl) amino)-1-third ammonium, inner salt (CAS ^No.6179-44-8),

3-amino-N (carboxymethyl)-N, N-dimethyl-1-third ammonium N-C8-C22 acyl derivative, inner salt (CAS ^No.84082-44-0),

N-(carboxymethyl)-3-((12-hydroxyl-1-oxygen-9-octadecyl) amino)-N, N-dimethyl-1-third ammonium, inner salt (CAS ^No.71850-81-2),

Cocamidopropyl propyl amide carboxymethyl betaine (CAS ^No.61789-39-7 and CAS ^No.61789-40-0),

N-(2-propyloic)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.16527-85-8),

N-(2-propyloic)-N, N-dimethyl-1-tridecane ammonium, inner salt (CAS ^No.132621-79-5),

N-(2-propyloic)-N, N-dimethyl-1-tetradecane ammonium, inner salt (CAS ^No.69725-38-3),

N-(2-propyloic)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.42416-43-3),

N-(2-propyloic)-N, N-dimethyl-1-octadecane ammonium, inner salt (CAS ^No.30612-73-8),

N-dodecyl-Beta-alanine (CAS ^No.1462-54-0),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-undecane ammonium, inner salt (CAS ^No.150147-53-8),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.15163-30-1),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-tetradecane ammonium, inner salt (CAS ^No.146959-90-2),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-pentadecane ammonium, inner salt (CAS ^No.146959-91-3),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.71695-32-4),

N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecane ammonium, inner salt (CAS ^No.78195-27-4),

N-(4-carboxylic butyl)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.120139-51-7),

N-(5-carboxylic amyl group)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.76392-97-7),

N-(5-carboxylic amyl group)-N, N-dimethyl-1-n-Hexadecane ammonium, inner salt (CAS ^No.73565-98-7),

N-(6-carboxylic hexyl)-N, N-dimethyl-1-dodecane ammonium, inner salt (CAS ^No.132621-80-8),

4-carboxy-N-dodecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-31-3),

2-carboxy-N-dodecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-34-6),

4-carboxy-N-hexadecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-33-5),

2-carboxy-N-hexadecyl-N, N-dimethyl-benzalkonium, inner salt (CAS ^No.71695-35-7), tallow glycinate (CAS ^No.70750-46-8), soy amide propyl group carboxymethyl betaine, and babassu amido propyl carboxymethyl betaine.

137. the test kit of claim 136, wherein said carboxybetaine are N-(3-carboxylic propyl group)-N, N-dimethyl-1-octadecane ammonium, inner salt (CB-18) (CAS ^No.78195-27-4)

138. the test kit of claim 133, wherein said trimethyl-glycine sample stain remover is a SB sample stain remover.

139. the test kit of claim 138, wherein this SB sample stain remover is selected from SB-18, SB-16, SB-14 and SB-12.

140. the test kit of claim 139, wherein this SB sample stain remover is SB-16.

141. the test kit of claim 139, wherein this SB sample stain remover is SB-18.

142. each test kit of claim 132-141, wherein said test kit contains the set of different trimethyl-glycine sample stain removers.

143. the test kit of claim 132, wherein said microbiotic is selected from: beta-lactam antibiotics, aminoglycoside, aminocyclitol, quinolone, tsiklomitsin, macrolide, lincosamide, glycopeptide, lipopeptid, peptide antibiotics, sulfanilamide (SN), trimethoprim, paraxin, vazadrine, nitroimidazole, Rifampin, nitrofuran, urotropine and mupirocin.

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