EA002808B1 - Betaines as adjuvants to susceptibility testing and antimicrobial therapy - Google Patents

Abstract

1. A method for testing a susceptibility of microorganism to a composition comprising an antibiotic, said method comprising exposing a microorganism, which has mycolic acid structures on the outer membrane thereof, to a composition comprising an antibiotic and a betaine-like detergent, and characterizing the susceptibility of said microorganism to said antibiotic based upon the viability of said microorganism in said composition. 2. The method of Claim 1, wherein said betaine-like detergent is selected from the group consisting of a CB-Like, SB-like, HSB-like, PB-like, CtB-like, PhB-like, SoB-like, RevB-like, AO-like, cAB-like, and ImB-like detergent. 3. The method of Claim 2, wherein said CB-like detergent has the structure wherein R1 is C8-C22; &alpha; is -CH2-,-CH(OH)-, -(CO)-NH-CH2 CH2 CH2-, -O-, or -C(O)-; n is 0 or 1; &beta; is -N<(+)>-, -P<(+)>-, or -S<(+)>-; R2 is -H, -CH3-, -C2H5-, -C3H7-, or -C4H9-; R3 is -H, -CH3-, -C2H5-, -C3H7-, or -C4H9-; R4 is -CH2-, -C2H4-, -C3H6-, -C4H8-, -C5H10-, -C6H12-, -CH2-C6H4-, -CmH2m-, -CH(OH)CH2 CH2-, -CH2 CH(OH) CH2-, or -CmH2m-1(OH)-, wherein m >=1; &gamma; is -COO<(+)>. 4. The method of Claim 3, wherein said betain-like detergent is selected from the group consisting of N-(carboxymethyl)-N,N-dimethyl-1-hexadecanaminium, inner salt (CAS<(RTM)> No. 693-33-4), cococarboxymethylbetaine and CAS(RTM) No. 68424-94-2), N-(carboxymethyl)-N,N-dimethyl-9-octadecen-aminium, inner salt (CAS(RTM) No.871-37-4), N-(carboxymethyl)-N,N-dimethyl-3-((1-oxooctadecyl)amino)-1-propanaminium, inner salt (CAS(RTM) No.6179-44-8), 3-amino-N(carboxymethyl)-N,N-dimethyl-1-propanaminium N-C8-C22 acyl derivatives, inner salt (CAS(RTM) No.840-44-0), N-(carboxymethyl)-3-((12-hydroxy-1-oxo-9-octadecenyl)amino)-N,N-dimethyl-1-propanaminium, inner salt (CAS(RTM) No.71850-81-2), cocoamidopropyl carboxymethylbetaine (CAS(RTM)No.61789-39-7 and CAS(RTM) No.61789-40-0), N-(2-carboxyethyl)-N,N-dimethyl-1-dodecanaminium, inner salt (CAS(RTM) No. 16527-85-8), N-(2-carboxyethyl)-N,N-dimethyl-1-tridecanaminium, inner salt (CAS(RTM) No. 132621-79-5), N-(2-carboxyethyl)-N,N-dimethyl-1-tetradecanaminium, inner salt (CAS(RTM) No.69725-38-3), N-(2-carboxyethyl)-N,N-dimethyl-1-hexadecanaminium, inner salt (CAS(RTM) No.42416-43-3), N-(2-carboxyethyl)-N,N-dimethyl-1-octadecanaminium, inner salt (CAS(RTM) No.30612-73-8), N-dodecyl-beta-alanine (CAS(RTM) No.1462-54-0), N-(3-carboxypropyl)-N,N-dimethyl-1-undecanaminium, inner salt (CAS(RTM) No.150147-53-8), N-(3-carboxypropyl)-N,N-dimethyl-1-dodecanaminium, inner salt (CAS(RTM) No.15163-30-1), N-(3-carboxypropyl)-N,N-dimethyl-1-tetradecanaminium, inner salt (CAS(RTM) No.146959-90-2), N-(3-carboxypropyl)-N,N-dimethyl-1-pentadecanaminium, inner salt (CAS(RTM) No.146959-91-3), N-(3-carboxypropyl)-N,N-dimethyl-1-hexadecanaminium, inner salt (CAS(RTM) No.71695-32-4), N-(3-carboxypropyl)-N,N-dimethyl-1-octadecanaminium, inner salt (CAS(RTM) No.78195-27-4), N-(4-carboxybutyl)-N,N-dimethyl-1-dodecanaminium, inner salt (CAS(RTM) No.120139-51-7), N-(5-carboxypentyl)-N,N-dimethyl-1-dodecanaminium, inner salt (CAS(RTM) No.76392-97-7), N-(5-carboxypentyl)-N,N-dimethyl-1-hexadecanaminium, inner salt (CAS(RTM) No.73565-98-7), N-(6-carboxyhexyl)-N,N-dimethyl-1-dodecanaminium, inner salt (CAS(RTM) No.132621-80-8), 4-carboxy-N-dodecyl-N,N-dimethyl-benzenemethanaminium, inner salt (CAS(RTM) No.71695-31-3), 2-carboxy-N-dodecyl-N,N-dimethyl-benzenemethanaminium, inner salt (CAS(RTM) No.71695-34-6), 4-carboxy-N-hexadecyl-N,N-dimethyl-benzenemethanaminium, inner salt (CAS(RTM) No.71695-33-5), 2-carboxy-N-hexadecyl-N,N-dimethyl-benzenemethanaminium, inner salt (CAS(RTM) No.71695-35-7), tallow glycinate (CAS(RTM) No.707-46-8), soyamidopropyl carboxymethylbetaine, and babassuamidopropyl carboxymethylbetaine. 5. The method of Claim 4, wherein said carboxybetaine is N-(3-carboxypropyl)-N,N-dimethyl-1-octadecanaminium, inner salt (CB-18) (CAS(RTM) No.78195-27-4). 6. The method of Claim 2, wherein said SB-like detergent is selected from the group consisting of SB-18, SB-16, SB-14 and SB-12. 7. The method of any one of Claims 1-6, wherein said composition comprises two or more betaine-like detergents. 8. The method of Claim 1, wherein said antibiotic is a member of a class selected from the group consisting of a &beta;-lactam antibiotic, an aminoglycoside, an aminocyclitol, a quinolone, a tetracycline, a macrolide, a lincosamide, a glycopeptide, a lipopeptide, a polypeptide antibiotic, a sulfonamide, trimethoprim, chloramphenicol, isoniazid, a nitroimidazole, a rifampin, a nitrofuran, methenamine, and mupirocin. 9. The method of Claim 8, wherein said &beta;-lactam antibiotic is selected from the group consisting of penicillin, cephalosporin, monobactam and carbapenem antibiotics. 10. The method of Claim 9, wherein said penicillin is selected from the group consisting of azlocillin, methicillin, nafcillin, cloxacillin, dicloxacillin, oxacillin, ampicillin, bacampicillin, carbenicillin, ticarcillin, mezlocillin and piperacillin. 11. The method of Claim 9, wherein said cephalosporin is selected from the group consisting of cefoxitin, cefoperazone, ceftazidime, ceftriaxone, cefadroxil, cefazolin, cephalexin, cephaloridine, cephalothin, cephapirin, cephradine, cefaclor, cefamandole, cefonicid, ceforanide, cefprozil, cefuroxime, loracarbef, cefmetazole, cefotetan, cefixime, cefotaxime, cefpodoxime, and ceftizoxime. 12. The method of Claim 9, wherein said monobactam is aztreonam. 13. The method of Claim 9, wherein said carbapenem is selected from the group consisting of imipenem, meropenem, panipenem, and biapenem. 14. The method of any one of Claims 8-13, wherein said composition further comprises &beta;-lactamase inhibitor. 15. The method of Claim 14 wherein said &beta;-lactamase inhibitor is selected from the group consisting of clavulanic acid, sulbactam, and tazobactam. 16. The method of Claim 8, wherein said antibiotic is said aminoglycoside or said aminocyclitol. 17. The method of Claim 16, wherein said aminoglycoside or aminocyclitol is selected from the group consisting of streptomycin, kanamycin, gentamicin, tobramycin, amikacin, sisomicin, netilmicin, neomycin, framycetin and paromomycin. 18. The method of Claim 8, wherein said quinolone is selected from the group consisting of naladixic acid, oxolinic acid, cinoxacin, flumequine, miloxacin, rosoxacin, pipemidic acid, norfloxacin, enoxacin, ciprofloxacin, ofloxacin, lomefloxacin, temafloxacin, fleroxacin, pefloxacin, amifloxacin, sparfloxacin, levofloxacin, clinafloxacin. 19. The method of Claim 8, wherein said macrolide is selected from the group consisting of erthromycin, oleandomycin, spiramycin, josamycin, rosaramicin, clarithromycin, azithromycin, dirithromycin, roxithromycin, flurithromycin, and rokitamycin. 20. The method of Claim 8, wherein said composition two or more antibiotics. 21. The method of Claim 20, wherein said antibiotics are a sulfonamide and trimethoprim. 22. The method of Claim 21, wherein said sulfonamide is sulfamethoxazole. 23. The method of Claim 8 wherein said rifampicin is selected from the group consisting of rifampin, rifamycin SV, rifamycin B (rifamide) and rifabutin. 24. The method of Claim 1 wherein said antibiotic is selected the group consisting of amikacin, azithromycin, any β-lactam in combination with any of the β-lactamase inhibitors, capreomycin, cefmetazole, cefoxitin, ciprofloxacin, clarithromycin, clofazamine, cycloserine, dapsone, erythromycin, ethambutol, ethionamide, imipenem, isoniazid, kanamycin, minocycline, ofloxacin, paraamino salicylic acid, prothionamide, pyrazinamide, rifampin, rifabutin, sparfloxacin, sulfamethoxazole with trimethoprim, streptomycin, tetracycline, thiacetazole and viomycm. 25. A method of antimicrobial therapy for a patient infected with, or at risk of being infected with, a microorganism, which has mycolic acid structures on the outer membrane thereof, said method comprising co-administering a betaine-like detergent and an antibiotic to said patient in an amount and for a length of time sufficient to kill said microorganism. 26. The method of Claim 55, wherein said betaine-like detergent is selected from the group consisting of a CB-like, SB-like, HSB-like, PB-like, CtB-like, PhB-like, SoB-like, RevB-like, AO-like, cAB-like, and ImB-like detergent. 27. The method of Claim 26, wherein said CB-like detergent has the structure wherein R1 is C8-C22; &alpha; is -CH2-,-CH(OH)-, -(CO)-NH-CH2 CH2 CH2-, -O-, or -C(O)-; n is 0 or 1; &beta; is -N<(+)>-, -p<(+)>- or -S<(+)>-; R2 is -H, -CH3-, -C2H5-, -C3H7-, or -C4H9-; R3 is -H, -CH3-, -C2H5-, -C3H7-, or -C4H9-; R4 is -CH2-, -C2H4-, -C3H6-, -C4H8-, -C5H10-, -C6H12-, -CH2-C6H4-, -CmH2m-, -CH(OH) CH2 CH2-, -CH2 CH(OH) CH2-, or -CmH2m-1 (OH)-, wherein m>=1; &gamma; is -COO<(+)>. 28. The method of Claim 27, wherein said CB-like detergent is selected from the group consisting of N-(carboxymethyl)-N,N-dimethyl-1-hexadecanaminium, inner salt (CAS(RTM) No. 693-33-4), cococarboxymethylbetaine and CAS(RTM) No. 68424-94-2), N-(carboxymethyl)-N,N-dimethyl-9-octadecen-1-aminium, inner salt (CAS(RTM) No.871-37-4), N-(carboxymethyl)-N,N-dimethyl-3-((1-oxooctadecyl)amino)-1-propanaminium, inner salt (CAS(RTM) No.6179-44-8), 3-amino-N(carboxymethyl)-N,N-dimethyl-1-propanaminium N-C8-C22 acyl derivatives, inner salt (CAS(RTM) No.840-44-0), N-(carboxymethyl)-3-((12-hydroxy-1-oxo-9-octadecenyl)amino)-N,N-dimethyl-1-propanaminium, inner salt (CAS(RTM) No. 71850-81-2), cocoamidopropyl carboxymethylbetaine (CAS(RTM) No.61789-39-7 and CAS(RTM) No.61789-40-0), N-(2-carboxyethyl)-N,N-dimethyl-1-dodecanaminium, inner salt (CAS(RTM) No. 16527-85-8), N-(2-carboxyethyl)-N,N-dimethyl-1-tridecanaminium, inner salt (CAS(RTM) No. 132621-79-5), N-(2-carboxyethyl)-N,N-dimethyl-1-tetradecanaminium, inner salt (CAS(RTM) No. 69725-38-3), N-(2-carboxyethyl)-N,N-dimethyl-1-hexadecanaminium, inner salt (CAS(RTM) No. 42416-4

Description

The present invention relates to compositions and methods for characterizing the sensitivity of bacteria containing mycolic acid structures. The compositions and methods claimed in the invention are particularly applicable as adjuvants in sensitivity testing and antimicrobial therapy, and are most applicable in testing and therapy using the family (3 lactam antibiotics.

State of the art

Modern methods of treating patients infected with bacteria include the choice of antibiotics. Usually, the treatment is based on the experience of the doctor. Simply put, the doctor believes that a wide range of antibiotics should be effective against most common infectious agents that cause certain symptoms in the patient. However, in some cases, the choice of an appropriate therapeutic agent is based on sensitivity testing. Sensitivity testing gives the practitioner the opportunity to determine which therapeutic agent is most likely to be effective in treating a particular pathogen, and which antimicrobial agents are likely to be useless. Sensitivity testing is an important tool, especially when the patient has confirmed mycobacterial infection, and, in particular, tuberculosis (TB: caused by bacteria of the Musoacobacterium tuberculosis complex (MTB)). The current federal rules provide for testing sensitivity for all new cases of TV (Teiouet, RS e! A1., 1ig. C1sh.M | sg. 31: 767-770 (1993)). Sensitivity testing is an invaluable tool that almost all doctors rely on when choosing the appropriate antibiotic therapy for their patients.

Methods of treating bacterial infections are limited by the activity spectrum of a particular compound. For example, not all bacteria are sensitive to this antimicrobial compound. Different classes of bacteria are resistant to antibiotics of various classes. Generally speaking, the activity spectrum of this antibiotic falls into separate groups depending on the class of the organism on which it acts (e.g., anti-fungal against antibacterial, acting on gram-positive bacteria against acting on gram-negative bacteria).

Antibiotics show their effect, disrupting a variety of cellular functions. For example, it is known that different classes of antibiotics act at different stages of cell wall synthesis, RNA / DNA synthesis, DNA replication, or protein synthesis. Bacteria are resistant to various antibiotics for various reasons. Ουίηΐίΐίαηί. B. e! A1., 1p: Miggau, R.V. e! A1., ebz. Mapia1 oG Syssha1 M1stoyu1odu, A8M Repts, ^ aZnpd! Op, Ό.Ο (1995) pp. 1308-1326 consider some of these mechanisms of resistance and note that the antibiotic must first penetrate the cell and only then it can show its effect at the site (site) of the action. The basis of stability may lie in the permeability of the body, the molecular configuration of the site of action may be unacceptable, or it may not exist at all. In addition, bacteria can modify, destroy or remove the agent.

Sustainability can be original or acquired. Acquired resistance may be due to the acquisition of genetic material (e.g., transposons) or inherited DNA replication disorders (e.g., point mutations). Apparently, mycobacteria have an additional mechanism of resistance. Neye! S, b.v. 1p: Aegis SiserObPyu ίη 1st SyetoSetaru og Musoob! Epa1 1pGesiopz, Neye! Z, b.V. fuck SHS Rgezz, Voz! Op, MA (1991), pp. 13-57 classifies the subpopulations of infectious MTV cells as actively growing or at different stages of the sleeping state. A sleeping state allows these subpopulations to survive during treatment of the patient.

The treatment of mycobacterial infections is also complicated by the complex nature of sensitivity. For example, while MTV infections are usually effectively treatable with isoniazid (ΙΝΗ) and / or pyrazinamide (ΡΖΑ), MAS isolates are usually resistant to these drugs, and M. Goyiyit and M. syeopope isolates are usually resistant to all first-line anti-tuberculosis agents.

Today, tuberculosis is the most prevalent infectious disease in the world, it infects approximately a third of the world's population, i.e. about 1.7 billion people (KOSY, A. Tuberger 72: 1-6 (1991)). In addition, tuberculosis kills more people worldwide (approximately 3 million annually) than any other infectious disease (Motybyu apb Mobyuyuuyuu Vera 42: 961-964 (1993)). The vast majority of cases of TB occur in developing countries, however, the drug-resistant (MEW) strains of MTV (MOB-TV) have become a serious global problem (\ Uog1b Nea11y Ogdashkhaoop (bositep! \ UNO / TV / 96.198) Otoirz a! V1zk. \ UNO By faith, Opée Théeuxiucos Epilbetis 1996 (1996)). If you do not stop the rise of MOB-TV, then it will be inevitable to return to the past when tuberculosis was the most common cause of death in both developing and industrial countries.

Other mycobacteria, such as the Muso bacillus aetus (MAS) complex, M. raga! Bilgium losum, M. and lsetap8, M. largae, M. capzazp and the complex Μ.ίοΓίιιίΙιιιη. are also common pathogens (see: Aaupe. L.O. C1T. M1sgo. Beu. 5: 1-25 (1992) or Pa1ksh1at. Ι.Θ. C1i. Mugo. Keu.9: 177-215 (199). where various mycobacterial pathogens are considered). MAS causes disseminated disease in more than half of patients with advanced AIDS in the late stages (No. dyyida1e. 8.Ό. e! A1 .. 1oit. 1nGes1. Όίδ. 165: 1082-1085 (1992)). The World Health Organization estimates that by the year 2000, the number of people infected with the human immunodeficiency virus ()ν) could exceed 40 million (Aog1y Nea1! 1 Ogdashhabop (yositep! ANO / ORACIR ^ A93.1) 01oBa1 Rogodgat op OSh8 (1993). Raga! and Leggi1o818 (a subspecies of M. auscht) causes ruminant John's disease. It is estimated that John's disease costs about $ 1.5 billion annually in U.S. agriculture (i.e., meat and dairy products) due to reduced productivity and fertility (AY ! 1osk. V. Rtosey1pd8 oG 1ye TYgy 1p1etpay PA1 So11odshit op iegsi1o818 rr.514-522 Raga (1991);!. Abl 1osk B. e a1 .. Rgoseeytdz og 1f 89 ¹¹ 'Appia1 Meeypd og 1f Ish and her 8 deg Ashta1 NeaIy!.!!!!!! A55oaaup. Pp. 484-490 (1985). Mycobacterial diseases cause huge social costs.

The most widely used today and the best characterized class of antibiotics are β-lactams. Due to the wide spectrum of action, the treatment of mycobacterial infections with these agents provides serious advantages. The use of β-lactams in therapeutic regimens for the treatment of mycobacterial infections has given limited success (Sjatbetk. N.R. e! A1 .. Aptkto. Adep! § C1eto. 39: 2620-2624 (1995)). The ability to expand the sensitivity of mycobacteria, in particular to β-lactams, taking into account resistance mechanisms, has significant potential for increasing the effectiveness of the treatment of mycobacterial infections.

The invention described in this application relates to new methods and compositions by which the sensitivity of mycobacteria can be characterized. These methods and compositions alter the sensitivity of these bacteria, increasing the effectiveness of antibiotics, especially β-lactam antibiotics. These methods and compositions can serve as a component of effective therapy and / or diagnosis of such infections.

SUMMARY OF THE INVENTION

In an effort to suppress the growth of contaminating bacteria in liquid cultures obtained from biological samples and processed according to the method T1yugp1op AO 95/27076 and i.8. 5.658.749. the authors added third-generation cephalosporin, ceftazidime (CA8®) to the standard antimicrobial composition (in particular, RAITA)

No. 72558-82-8). The authors unexpectedly found that C ₄₈ -carboxypropylbetaine (CB-18) used as a processing reagent according to T1yugp1op AO 95/27076 and i.8. 5.658.749 in combination with this antimicrobial composition leads to a significant and sharp decrease in sensitivity in liquid cultures. To realize the advantages in diagnostic sensitivity according to TNogSchop AO 95/27076 and и.8. 5.658.749 the authors conducted studies to characterize and prevent this decrease in sensitivity in liquid cultures. These studies have led to the development of methods and compositions using which the sensitivity of mycobacteria to antibiotics, especially antibiotics of the β-lactam family, can be altered through the use of betaine-like compounds described by T1yugp1op AO 95/27076 and i.8. 5.658.749. The methods are applicable for characterizing and changing the sensitivity of bacteria, in particular, mycobacteria, and especially bacteria of the complex M.! Bacillus8, to antimicrobial compounds used as adjuvants in antimicrobial therapy.

A subject of the invention is also a composition for testing sensitivity, moreover, this composition contains one or more antibiotics mixed with one or more betaine-like detergents.

The subject of the invention is also a kit for determining the sensitivity of a microorganism, moreover, this kit includes one or more betaine-like detergents and one or more antibiotics in close proximity or proximity.

Brief Description of the Figures

In FIG. 1A and 1B, a diagram of experiments conducted to obtain important growth characteristics of isolates ATCC 27294 and 571/573-ВА MusoBas! Ergst! Andbegs1o818 in the culture system CB-18 / 12B / RAITacah is presented.

In FIG. 2A-2H show the growth curves when testing the ATCC 27294 isolate MusoBac! Epitus bilbacidae according to the circuit of FIG. 1A and 1B. Asterisks: RAITA; squares: R / sah.

FIG. 2A: ATCC 27294. L-1, buffer treatment, buffer growth.

FIG. 2B: ATCC 27294. L-1. processing in buffer, growing in SV-18 (final concentration of SV-18 17 μg / ml).

FIG. 2C: ATCC 27294. L-1. processing in SV-18 (383 μg / ml), growing in buffer.

FIG. 2Ό: ATCC 27294. L-1. processing in SV-18 (383 μg / ml), growing in SV-18 (final concentration of SV-18 17 μg / ml).

FIG. 2E: ATCC 27294. selective 7H11, buffer treatment, buffer growth.

FIG. 2P: ATCC 27294, selective 7H11, buffer treatment, growth in SV-18 (final SV-18 concentration of 17 μg / ml).

FIG. 26: ATCC 27294, selective 7H11, treatment in CB-18 (383 μg / ml), growing in buffer.

FIG. 2H: ATCC 27294, selective 7H11, treatment in CB-18 (383 μg / ml), growing in CB-1 8 (final concentration of CB-1 8 17 μg / ml).

In FIG. 3A-3H show growth curves during testing of isolate 571/573-BAB Musoxabit! Bilobacter according to the scheme shown in FIG. 1A and 1B.

Asterisks: ΡΑΝΤΑ; squares: R / sah.

FIG. 3A: 571/573-BAB, b-1, processing in the buffer, growing in the buffer.

FIG. 3B: 571/573-BAB, b-1, buffer treatment, growing in CB-18 (final concentration of CB-18 17 μg / ml).

FIG. 3C: 571/573-BAB, L-1, treatment in CB-18 (383 μg / ml), growing in buffer.

FIG. 3Ό: 571/573-BAB, b-1, treatment in CB-18 (383 μg / ml), growing in CB-18 (final concentration of CB-1 8 17 μg / ml).

FIG. 3E: 571/573-BAB, selective 7H11, buffer treatment, buffer growth.

FIG. 3P: 571/573-BAB, selective 7H11, buffer treatment, growing in CB-1 8 (final concentration of CB-1 8 17 μg / ml).

FIG. 36: 571/573-BAB, selective 7H11, treatment in CB-18 (383 μg / ml), growing in buffer.

FIG. 3H: 571/573 -BAB, selective 7H11, treatment in CB-18 (383 μg / ml), growing in CB-18 (final concentration of CB-18 17 μg / ml).

In FIG. 4 is a diagram of experiments carried out with cultivation with lecithin in small volume inoculums. The purpose of the experiment was to test the ability of lecithin to neutralize the effect of CB-18. The aim of the experiment was also to test the effect of CB-1 8 in inoculums of various sizes.

In FIG. 5A-5P show growth curves when testing isolate 571/573-BAB Musoxabit! Bilobacter according to the scheme shown in FIG. 4.

FIG. 5A and 5B reflect the results of experiments with dilution of bacterial runoff 5,000 times (approximately 165 ± 54 cfu).

FIG. 5C and 5Ό reflect the results of experiments with dilution of bacterial runoff 25,000 times (approximately 33 ± 11 cfu).

FIG. 5E and 5P reflect the results of experiments with dilution of bacterial runoff 100,000 times (approximately 8 ± 3 cfu). Asterisks: buffer in K..R .; squares: buffer in P / sah; triangles: lecithin in K..R .; x: lecithin in R / sah.

FIG. 5A: growth in buffer or buffer / lecithin.

FIG. 5B: growth in CB-18 (17 μg / ml) or CB-18 / lecithin (17 μg / ml @).

FIG. 5C: growth in buffer or buffer / lecithin.

FIG. 5Ό: growing to growing in SV-18 (17 μg / ml) or SV-18 / lecithin (17 μg / ml @).

FIG. 5E: growing to growing in buffer or buffer / lecithin.

FIG. 5P: growth in in SV-18 (17 μg / ml) or SV-18 / lecithin (17 μg / ml @).

In FIG. Figure 6 shows the experimental design for titration of SV-18 and TMA-18 with the aim of comparing the effect of SV-1 8 with the action of the quaternary ammonium salt of trimethyl octadecyl ammoniumumbromide (TMA-18).

In FIG. 7A-7C show growth curves when testing the ATCC isolate 27294 MusoBac! 6. Asterisks: K..R .; squares: RAITA; triangles: P / sah.

FIG. 7A: selective 7H11, growing in buffer.

FIG. 7B: selective 7H11, cultivation in CB-18 (final concentration of CB-18 17 μg / ml).

FIG. 7C: selective 7H11, growth in TMA-18 (final concentration 3.4 μg / ml).

In FIG. 8A-8C show growth curves during testing of isolate 571/573-BAB M.! And Liucoi818 according to the circuit of FIG. 6. Asterisks: K..R .; squares: RAITA; triangles: P / sah.

FIG. 8 A: selective 7H11, growing in buffer;

FIG. 8B: selective 7H11, growth in CB-1 8 (final concentration of CB-1 8 17 μg / ml).

FIG. 8C: selective 7H11, growth in TMA-18 (final concentration 3.4 μg / ml).

In FIG. Figure 9 shows a diagram of experiments conducted with the aim of titration of SV-1 8 to test the effect of SV-1 8 at various concentrations on various types of mycobacteria listed in Table 1. 7.

In FIG. 10A and 10B show the growth curves when testing the ATCC isolate 27294 M.! Biluxi8S with titration CB-18 according to the scheme shown in FIG. 9. Asterisks: K..R .; squares: P / sah; triangles: 3 μg / ml CB-18; x: 7 μg / ml CB-18; “13 μg / ml CB-18; circles: 27 mcg / ml SV-18; vertical risks: 54 mcg / ml SV-18; horizontal risks: 109 mcg / ml SV-18.

FIG. 10A, titration without P / sah.

FIG. 1 0V, each titration was carried out with P / sah.

In FIG. 11A and 11B show the growth curves when testing isolate 571/573-BAB M.! Biecci8O8 with titration CB-18 according to the circuit shown in FIG. 9. Asterisks: K..R .; squares: P / sah; triangles: 3 μg / ml CB-18;

x: 7 μg / ml CB-18; “13 μg / ml CB-18; circles: 27 mcg / ml SV-18; vertical risks: 54 mcg / ml SV-18; horizontal risks: 109 mcg / ml.

FIG. 11A, titration without P / sah.

FIG. 11B, each titration was performed with P / sax.

In FIG. 12A and 12B show the growth curves of the isolate ATCC 25291 M.Ausht with titration of SV-18 according to the scheme shown in FIG. 9. Asterisks: V.R .; squares: P / sah; triangles: 3 μg / ml CB-18; x: 13 μg / ml CB18; “27 μg / ml CB-18.

FIG. 12 A, titration without P / eax.

FIG. 1 2B, each titration was performed with P / sax.

In FIG. 13A and 13B show growth curves of the isolate of the M.Aust 802-BAB complex with titration of CB-18 according to the scheme shown in FIG. 9. Asterisks: V.R .; squares: P / sah; triangles: 7 μg / ml CB-18; x: 13 μg / ml CB-18; “27 μg / ml CB-18.

FIG. 13 A, titration without P / eax.

FIG. 13B, each titration was performed with P / eax.

In FIG. 14A and 14B show the growth curves of the ATCC isolate 5841 M £ oiit with titration of SV-18 according to the scheme shown in FIG. 9. Asterisks: R.R .; squares: R-s; triangles: 7 μg / ml CB-18; x: 13 μg / ml CB18; “27 μg / ml CB-18; circles: 54 mcg / ml CB18; vertical risks: 109 mcg / ml SV-18.

FIG. 14 A, titration without Rs.

FIG. 1 4B, each titration was performed with P-th.

In FIG. 15A and 15B show the growth curves of isolate 495-1HN M. Goyiyit with titration of CB-1 8 according to the scheme shown in FIG. 9. Asterisks: R.R .; squares: R-s; triangles: 7 μg / ml CB-18; x: 13 μg / ml CB-18; “27 μg / ml CB-18; circles: 54 μg / ml CB-18; vertical risks: 109 mcg / ml SV-18.

FIG. 15 A, titration without Rs.

FIG. 15B, each titration was performed with P-th.

In FIG. 16 is a diagram of experiments conducted on various MTV isolates with the aim of screening antibiotics and checking the effect of CB-1 8, together with various antibiotics, on various M. 1bettsi818 isolates, listed in Table 8.

In FIG. 17A and 17B show the growth curves of the ATCC isolate 27294 M. 1begi1O818 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Squares:

RAITA; triangles: P / sah; x: Pc £ p; ": P / sah; Mugs: Rs.

FIG. 17A: selective 7H11, growing in buffer.

FIG. 1 7B: selective 7H11, cultivation in CB-18 (final concentration of CB-18 17 μg / ml).

In FIG. Figures 18A and 18B show the growth curves of isolate 571/573-BAB M. 1begsi818 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Squares: RAITA; triangles: P / sah; x: R-s1p; •: P / sah; Mugs: Rs.

FIG. 18A: selective 7H11, growing in buffer. FIG. 18B: selective 7H11, growth in CB-1 8 (final concentration of CB-1 8 17 μg / ml).

In FIG. 19A and 19B show the growth curves of isolate 573-BAB M. Ibegsi818 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: R.R .; squares: RAITA; triangles: P / sah; x: Pc £ p; "R / sah; Mugs: Rs.

FIG. 19A: selective 7H11, growing in buffer.

FIG. 19B: selective 7H11, growth in CB-1 8 (final concentration of CB-1 8 17 μg / ml).

In FIG. Figures 20A and 20B show the growth curves of isolate 535-BAB M. 1begsiO818 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: R.R .; squares: RAITA; triangles: P / sah; x: Pc £ p; "R / sah; Mugs: Rs.

FIG. 20A: selective 7H11, growing in buffer.

FIG. 20B: selective 7H11, growing in CB-1 8 (final concentration of CB-1 8 17 μg / ml).

In FIG. Figures 21A and 21B show the growth curves of isolate 896-BAB M. Ibegsi818 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: R.R .; squares: RAITA; triangles: P / sah; x: Pc £ p; ": P / sah; Mugs: Rs.

FIG. 21 A: selective 7H11, growing in buffer.

FIG. 21B: selective 7H11, growth in CB-1 8 (final concentration of CB-1 8 17 μg / ml).

In FIG. 22A and 22B show the growth curves of the 040-TCE isolate. M. l. &Lt; 1 &gt; 818 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: R.R .; squares: RAITA; triangles: P / sah; x: RsGy; ": P / sah; mugs: R-s;

FIG. 22A: selective 7H11, growing in buffer.

FIG. 22B: selective 7H11, growth in CB-1 8 (final concentration of CB-1 8 17 μg / ml).

In FIG. 23A and 23B show the growth curves of the 061-TBP isolate M.1begi1O818 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: R.R .; squares: RAITA; triangles: P / sah; x: Pc £ p; ": P / sah; Mugs: Rs.

FIG. 23 A: selective 7H11, growing in buffer.

FIG. 23B: selective 7H11, cultivation in CB-18 (final concentration of CB-18 17 μg / ml).

In FIG. 24A and 24B show the growth curves of isolate 512-1HH M4uBe1Si515 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: K..R .; squares: ΡΑΝΤΑ; triangles: P / sah; x: RsGy; ^p, p / sa; mugs: R-s;

FIG. 24A: selective 7H11, growing in buffer.

FIG. 24B: selective 7H11, cultivation in CB-18 (final concentration of CB-18 17 μg / ml).

In FIG. 25A and 25B show the growth curves of isolate 538-1HN M. 1begsi5155 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: J.R .; squares: ΡΑΝΤΑ; triangles: P / sah; x: RsGy; ^p, p / sa; Mugs: Rs.

FIG. 25A: selective 7H11, growing in buffer.

FIG. 25B: selective 7H11, grown in CB-18 (final concentration of CB-18 17 μg / ml).

In FIG. 26A and 26B show the growth curves of isolate 52-96-BOB M. 1begsi5155 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: J.R .; squares: ΡΑNΤΑ; triangles: P / sah; x: RsGy; : P / sah; Mugs: Rs.

FIG. 26A: selective 7H11, growing in buffer.

FIG. 26B: selective 7H11, cultivation in CB-18 (final concentration of CB-18 17 μg / ml).

In FIG. 27A and 27B show the growth curves of isolate 57-96-BOB M. 1begsi5155 in antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: R.R .; squares: ΡΑNΤΑ; triangles: P / sah; x: RsGy; ^p, p / sa; Mugs: Rs.

FIG. 27A: selective 7H11, growing in buffer.

FIG. 27B: selective 7H11, cultivation in CB-18 (final concentration of CB-18 17 μg / ml).

In FIG. 28A and 28B show the growth curves of isolate 572/573-ΒΑΤ M4iBegsi5155 in a modified version of antibiotic screening experiments, the scheme of which is shown in FIG. 16. Asterisks: J.R .; squares: P / sah; stars: erythromycin; x: ceftriaxone.

FIG. 28A: antibiotic screening, cultivation in K..P.

FIG. 28B: antibiotic screening, cultivation in SV-18.

In FIG. 29A and 29B show the growth curves of isolate 061-ΤΒΚ. M.1begsi5155 in a modified version of antibiotic screening experiments, the scheme of which is shown in FIG. 16. In these experiments, non-β-lactam antibiotics were additionally tested. Asterisks: J.R .; squares: P / sah; triangles: polymyxin B; x: oleandomycin; ·: Lincomycin; mugs: nalidixic acid; vertical risks: penicillin C; horizontal risks: ceftriaxone.

FIG. 29A: antibiotic screening, cultivation in K..P.

FIG. 29B: antibiotic screening, cultivation in SV-18.

In FIG. Figures 30A and 30B show the growth curves of isolate 57-96-BOB M.1bieuxi515 in a modified version of antibiotic screening experiments, the scheme of which is shown in FIG. 16. In these experiments, non-β-lactam antibiotics were additionally tested. Asterisks: J.R .; squares: P / sah; triangles: nalidixic acid; x: penicillin C; ·: Ceftazidime; Mugs: Ceftriaxone.

FIG. 30A: antibiotic screening, cultivation in K..P.

FIG. 30B: antibiotic screening, cultivation in SV-18.

In FIG. 31 is a diagram of experiments aimed at screening betaine-like detergents and other detergents shown in Tab. 10, the ability to induce induced sensitivity is similar to that for CB-18.

In FIG. 32A shows the total growth curves in the presence of all controls used in the experiment, which is reflected in FIG. 31.

In FIG. 32B summarizes growth curves in the presence of several selected detergents, highlighting differences in results.

FIG. 32A: detergent controls: asterisks: buffer / Ram .; squares: buffer / ΡΑΝΤΑ; triangles: buffer / P-max;

FIG. 32B: selected detergents: sprockets: twin 80; squares: stearic acid; triangles: detail RV; x: 8B18; =: Crosultain E30; open circles: Velveteks OVV; filled circles: C ₄₈ -carboxyethyl betaine.

In FIG. Figure 33 compares the results obtained with SV-18 with those obtained for EDTA when comparing their effects in the culture system.

In FIG. 34A and 34B show the growth curves of the ATCC isolate 27294 M 1begsi5155 when growing it according to the scheme shown in FIG. 33.

FIG. 34A: seeded in J.R., P-sax or in SV-18 (17 μg / ml), as indicated: stars: buffer in J.R .; squares: buffer in P-sah; triangles: SV-18 in J.R .; x: SV-18 in R-sah.

FIG. 34B: inoculation of CBB-18 (17 μg / ml) with 85 μg / ml MDC1 ₂ or 17 μg / ml EDTA, as indicated: asterisks: CB-18 and MDC1 ₂ in K..E., squares: CB18 and MDC1 ₂ in P-sah; triangles: EDTA in K...E .; x: EDTA in R / sah.

In FIG. Figures 35A and 35B show the growth curves of isolate 571/573-BAB M.1bieuxi818 when grown according to the scheme shown in FIG. 33.

FIG. 35A: inoculation in K..E., P-sah or in SV-18 (17 μg / ml), as indicated: stars: buffer in K..E .; squares: buffer in P-sah; triangles: SV-18 V.E .; x: SV-18 in R-sah.

FIG. 35B: inoculation in SV-18 (17 μg / ml) with 85 μg / ml MDC1 ₂ or 17 μg / ml EDTA, as indicated: asterisks: CB-18 and MDC1 ₂ in K..E., squares: CB18 and MDC1 ₂ in P-sax; triangles: EDTA in K...E .; x: EDTA in R / sah.

In FIG. 36 is an experimental mycobacterial sensitivity testing scheme used to compare current mycobacterial sensitivity testing practices with the betaine sensitivity test of the present invention.

In FIG. 37 shows the growth curves of the isolate ATCC 27294 M.sub.LegsiO8SC when growing it according to the scheme shown in FIG. 36.

In FIG. 37A shows the results of a betaine sensitivity test obtained using the 0.5 Mac Farland standard (6.28 ± 0.57 x 10 ⁵ cfu) as inoculum.

In FIG. 37B, 37C, 37Ό, and 37E show growth curves using 10X (62,800 ± 5,700 CFU), 100X (6,280 ± 570 CFU), 1,000X (628 ± 57 CFU), and 10,000 X (63 ± 6 CFU) dilutions as inoculum Mac Farland standard, respectively. Asterisks: K..E .; squares: P-sax; triangles: 15 μg / ml CB-18; x: 30 μg / ml CB18; ": 60 μg / ml CB-18; circles: 15 μg / ml CB18 with P / sax; vertical risks: 30 μg / ml CB1 8 with P / sax; horizontal risks: 60 mcg / ml CB-18 with R / sah.

In FIG. 38 shows the growth curves of isolate 571/573-BAB M.1bieGi1O818 when growing it according to the scheme shown in FIG. 36.

In FIG. 38A presents the results of the betaine sensitivity test obtained using the 0.5 Mac Farland standard (1.1 ± 0.18 x 10 ⁶ cfu) as inoculum.

In FIG. 38B, 38C, 38Ό and 38E show growth curves using 10X (111,000 + 17,900 CFU), 100X (11,100 ± 1,790 CFU), 1,000X (1,110 + 179 CFU), and 10,000 X (111 ± 18 CFU) dilutions as inoculum Mac Farland standard, respectively. Asterisks: K..E .; squares: P-sax; triangles: 15 μg / ml CB18; x: 30 μg / ml CB-18; ": 60 μg / ml CB-18; circles: 15 μg / ml CB-1 8 with P / sax; vertical risks: 30 μg / ml CB-18 with R / sah; horizontal risks: 60 μg / ml CB-1 8 with R / sah.

FIG. 39 (A, B, C).

FIG. 39A illustrates the enzymatic mechanism for the modification of mycolic acids according to Waii Υ. e1 a1., Proc. Ka11. Asab. 8s1. 93: 12828-12833 (1996).

In FIG. 39B presents a mechanism by which thietetracosanoic acids can act as inhibitors of the same enzymes. The stable analogue of the transition state formed in this case will be sulfonium carboxy betaine.

In FIG. 39C presents a possible mechanism for synthesizing such a sulfonium carboxy betaine.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In the following description, many terms are widely used in pharmaceuticals and in testing the sensitivity of ίη x'Igo. In order to provide a more complete and correct understanding of the description and claims, including the meaning included in certain terms, the definitions of the following terms are given.

The term CB-18 effect means an increase in the sensitivity of individual bacteria, in particular mycobacteria, to antibiotics in the presence of a betaine-like detergent. This effect is manifested in the cultivation of a microorganism, such as an infectious agent, a clinical isolate or a viable extract capable of growth, ίη νίΙΐΌ in the presence of effective amounts of one or more antibiotics with or without the addition of one or more betaine-like detergents, in particular CB-18. The presence of a betaine-like detergent increases the sensitivity of certain bacteria, in particular mycobacteria, to antibiotics. Bacterial growth can be characterized quantitatively and qualitatively. An example of a qualitative result is a simple registration of growth or its absence. In a quantitative assessment, a growth index is recorded depending on the number of cultivation days, as is understood from the prior art. Examples of growth indices can be simple conventional symbols (i.e.-, ±, 1 +, 2 +, 3 + and 4+) or digital indicators (i.e. from 0 to 999), as provided for by the WASTES 12V cultural system (Veskop O | k1P5op. SoskeuzuShe, MO, I8A). The SV-18 effect may manifest itself in the complete suppression of growth or not affect the growth of reg 8e, but may affect the suppression, delay, or decrease in the slope of the growth curve (i.e., a decrease in the growth rate) during the exponential growth phase, as is understood from the level technicians. An essential aspect of the effect of CB-1 8 is that it manifests itself in a change in one or more growth characteristics of a microorganism, an infectious agent, or a clinical isolate, and this change is implemented in the context of the claimed betaine sensitivity test. This is illustrated in Example 10, where various antibiotics are used in combination with various betaine-like detergents. The observed change coincides with the increased sensitivity of the isolate to the present antibiotic.

The term “betaine sensitivity test” means the use of one or more betaine-like detergents in combination with one or more antibiotics in the ίη νίίτο test to determine the sensitivity of a microorganism (for example, an infectious agent or a clinical isolate), or a mixture of different microorganisms. When performing a betaine sensitivity test, a sample, for example, a homogeneous population of microorganisms or a mixture of types / variants / isolates, etc. microorganisms are brought into contact with a composition containing an antibiotic and a betaine-like detergent, and the sensitivity of the microorganism in the sample to the specified antibiotic is determined based on the viability of the microorganism in the sample. The betaine sensitivity test is the first embodiment of the methods of the invention. Such testing of betaine sensitivity can be carried out in a microtiter version, in culture bottles or plates with a dense medium, as is understood from the prior art. Examples of standard liquid media are WASTES (VeskUp ΩίΚιηδοη. Soskeu Ше, ΜΌ), Е8Р Мусо 8у81ет II ™ (SESO BaobaUpskh. Esίtοίΐ, M1) or MT / VasT ™ (Ogdopop Tekshka, Eig11a t. YS). Examples of standard solid media are Mueller-Hinton agar or other equivalent media known in the art. Such culture systems must necessarily include the appropriate antibiotic (s) and betaine-like detergent (s) in the appropriate combinations and corresponding concentrations, which are discussed in the present description. As with any other sensitivity testing, the purpose of such testing is to identify the antibiotic (s) that is most likely to successfully cure the patient of the infection.

A microorganism, such as a clinical isolate or an infectious agent, is considered sensitive if the microorganism (for example, Musobacilli) is adversely affected by the antibiotic so that the given clinical isolate or infectious agent becomes incompetent, non-infectious or non-viable, as is understood from the prior art ( Wao, 1.E.S. e1 a1., Ιη: Miggau, R.K. e1 a1., Nek.Mapia1 oG Sish sa1 MugoYuyuodu, A8M Rgekk, ^ a§Ypd! Op, Ts.S. (1995) pp .1281-1307 (cited by reference)). In this context, the term sensitive is synonymous with sensitivity. When determining the sensitivity of a microorganism, such as a clinical isolate or infectious agent, to a particular antibiotic, it is considered that the antibiotic is active or active against that clinical isolate or infectious agent.

The term sensitivity testing means the test w νίΙΐΌ. which determines the sensitivity of a microorganism, such as a clinical isolate or an infectious agent, to a number of antimicrobial compounds, as is understood from the prior art Tsogdepkep, 1. N. e1 a1., 1p: Miggau, R.K. e1 a1., nyk. Mapia1 oG C11ssa1 M1gGuyyuodu, A8M Przekk, ^ a§Ypd1op, C.S. (1995) pp. 1277-1280; Aoosk S.b. e1 a1., 1p: Miggau, R.K. e1 a1., nyk. Mapia1 oG CHssa1 MugoYuyodu, A8M Rheck, ^ akPdFp Ts.S. (1995) pp. 1327-1404; No. 10op1 Hundred! Her Gog baobaogo 81apyagy§. Meyoyog Gog O11yNop Ap11t1sgoYa1 8i5ser11b11yu Tei Gog Vas1epa Sha1 Sgo \ u AegOysa 11-Eoig1y Eyyup. Arr ^ ονei 81apyagy М7-А4 V ^ 11аηονа, RA (1997); No. 1, 1 The Supreme Gog Lebogogu 81apyagy §. RegGogtaps 81apyag§ Gog Apit1sgoYa1 That way 8i5ser11b11yu Te515-81x1y Ey11yup. Arr ^ ονei 81apyagy М2-А6 V ^ 11аηονа, RA (1997); The Greatest Gog L'Lablogo 81apyagyk. е ν ο р р ΐ 1 1 п п п У У 8 8 8 8 8 8 сер сер сер а а а 1 1 1 1 1 1 1 8 8 8 8 8 8. Arrgsui 81apyagy M23-A V ^ 11apoc, RA (1994); No. 1, 1 The Supreme Gog Lebogogu 81apyagy §. Apstusoaclapa1 8i5ser11b11uu TeSpd Gog Musoacaslgst 1iegsi1o8ta; TeiaShe 81apyagy M24-A. V ^ 11th April, RA (1995) (all works are referred to as references). The goal of any sensitivity testing is to more accurately predict the success of a therapeutic intervention.

The term antibiotic refers to any compound known in the art that has a negative effect on the viability, integrity, infectivity, or competence of an infectious agent, as understood in the art (see Miggau, R.K. e1 a1., Nek. Mapia1 oG Syssha1 Muguyuyodu, A8M Rheck, ^ a8Y1pd1op, C.S. (1995) pp. 1281-1307 and Kiseta, A. e1 a1., T1e Ike oG Apyyuysk 4 ' ¹ ' ee.E. Yrrshsoi So. PY1aye1rya, RA (1987); and og1ap, V. The s. ApNyuysk w aoga1ogu Meyyute 2 ^py Eyyyup HUyNap & VaShtoge, MC, all the works mentioned in the reference. examples of antibiotics The different classes include β-lactam antibiotics, β-lactamase inhibitors, aminoglycosides and aminocyclitols, quinolones, tetracyclines, macrolides and lincosamides, as well as glycopeptides, lipopeptides and polypeptides, sulfonamides and trimethoprim, chloramphenicol, isoniazidine, nitroiminofinamide nitroiminofinamide nitrimide nitrimide nitrofen moreover, all of these antibiotics may be useful in the implementation of the claimed invention. The term antibiotic in this context is synonymous with the terms therapeutic agent or medicine. All antibiotics are drugs or therapeutic agents, but not all drugs or therapeutic agents are antibiotics.

The term adjuvant means a chemical compound that may or may not have antimicrobial activity, but used in combination (e.g. simultaneously) with one or more antibiotics, this compound acts synergistically and enhances the effect of this antibiotic (or antibiotics). Adjuvant (s) can be used to increase the effectiveness of sensitivity tests or for antimicrobial therapy. An example of a therapeutic adjuvant is a β-lactamase inhibitor (W £ ha). The methods and compositions described in this application do not imply the use of β-lactamase inhibitors as adjuvants reg 8e, we are talking about the use of betaine-like detergents as adjuvants, however, betaine-like detergents can be used alone or with other adjuvants, including β-inhibitors lactamases.

The term betaine-like is synonymous with the term $ B-18-like, as they are interpreted in No. 0 95/27076 and in 8.5.558.749 (both works are referred to as references). According to No. 0 95/27076 and I.8.5,658,749, betaine-like detergents have the ability to disperse strands (and accumulations) of mycobacteria and / or reduce the buoyancy of mycobacteria. Dispersion of the strands of mycobacteria, such as, for example, the organisms of the MusoBac1epish 1begsi8O818 (MTB) complex, facilitates their detection, since it increases the likelihood that the aliquots taken for analysis will be as representative as the sample as a whole. Betaine-like detergents capable of dispersing mycobacterial strands have an alkyl chain of more than 16 carbon atoms, and a chain of more than 18-20 carbon atoms is most preferred. Betaine-like detergents also facilitate the collection of mycobacteria, such as, for example, organisms of the Musobacillus austust (MAC) complex, which grow without forming clusters, to some extent reducing the natural buoyancy of these organisms. This reduction occurs through a mechanism by which the detergent enters the bacterial cell. Betaine-like detergents capable of reducing buoyancy preferably have an alkyl chain with a length of more than 12 atoms uganion γ.

carbon, and the most preferred chain length of 16-20 carbon atoms. Thus, the term betaine-like, used in this context, includes the structures described in table. 2 and 3 No. 0 95/27076 and i.8.5,658,749 (both works are mentioned as references), including, for example, CB-like, 8B-like, HB-like, PB-like, 81B-like, RyB-like , 8oB-like, PCB-like, AO-like, CAB-like and 1tB-like compounds that have 8B-18-like activity, as described in No. 0 95/27076 and i.8.5,658,749. Betaine-like detergents include zwitterionic compounds having the structure shown in table. 1. It is believed that these structures are most applicable to the implementation of the claimed invention.

Table 1. The most applicable betaine-like detergents.

The general structure of the most applicable betaines is given. B ₁ is a hydrophobic alkyl chain, and α is a bond connecting B ₁ to a cation, β. If necessary, K, ₂ and P ₃ modify the cation. B ₄ serves as a bridge connecting the cation to

Kg I ^κ ιΤ <4.-β®-κ ₄ - [γ] ^θ I K,

-CH ₂ -, -0-P®-, -5®-H, -CH ₃ -, -C ₂ H ₅ -, - C ₃ H ₇ -n, -CH ₃ -, -c ₂ n ₅ -, -s ₃ n ₇ -CH ₂ -, - С ₂ Н ₄ -, - С ₃ Н ₆ -, -С ₄ Н ₈ -, -С ₅ Н ₁₀ -, - ^с b ⁿ 12 --- ^СН 2- ^С 6 ^H 4 --- ^C t ^H 2sh-> ^where “g ¹ -8O3®, -080e®, -COO®, -OPO3®, -PO3®, -PO3® The term CB-like means such a betaine-like detergent that contains carboxylate essence (-COO ⁰ ) as an anion (i.e., carboxy betaine-like). The term 8B-like means a betaine-like detergent that contains a sulfonate essence (-803 ^Θ ) as an anion (i.e. sulfobetaine-like). The term H8B-like means a betaine-like detergent that contains a sulfonate entity as an anion and a hydroxyl group (—OH) as a bridge (i.e., hydroxysulfobetaine-like). The term RVpodobny means such betainopodobny detergent which contains phosphate (-ORO3 ⁰⁾ fosfanatnuyu (-RO3 ⁰⁾ or phosphinate (-PO ₂ ⁰⁾ as the nature of the anion (i.e. fosfobetainopodobny). The term 81B-like means a betaine-like detergent that contains a sulfate (-0803 ⁰ ) entity as an anion (i.e., sulfate-betaine-like). The term AO similar means such betainopodobny detergent which comprises an oxide radical (-Ο ^Θ) as anion (i.e. aminoksidopodobny). The term PEB-like means a betaine-like detergent that contains a phosphonium (-P®-) entity as a cation (i.e., a phosphoniumumbetain-like). The term 8oB-like means such a betaine-like detergent that contains a sulfonium (-8®-) entity as a cation (i.e., sulfoniumumbetaine-like). The term palkyl betaine means a betaine-like detergent that contains an ammonium (-Ν®-) entity as a cation (i.e., p-alkyl betaine-like). The term 1TB-like means such a betaine-like detergent that contains an imidazolinium entity as a cation (i.e., imidazoliniumumbetaine-like). The term KeuB-like means a betaine-like detergent that contains an alkyl chain covalently attached to the anion, and not to the cation (i.e., the reverse betaine-like). The term cAB-like means such a betaine-like detergent, contains an alkyl chain covalently attached to the bridge, and not to the cation or anion (i.e., c-alkyl betaine-like).

The term CB-18 means Y- (3-carboxypropyl) -Y, M-dimethyl-1-octadecanaminium, an internal salt. CB-18 is also known as the internal salt of X # dimethyl-Y- (p-octadecyl) M- (3-carboxypropyl) ammonium or Cn-carboxypropyl betaine. CB-1 8 is designated CA8® Νο.78195-27-4. The term 8B-18 means M-octadecyl-S, M-dimethyl-3-ammonio-1-propane sulfonate (CA8® Νο. 13177-41-8).

The term 8B-16 means Ν-hexadecylΝ, Ν-dimethyl-3-ammonium-1-propanesulfonate (CA8® Νο.2281-11-0).

The term 8B-14 means Ν-tetradecylΝ, Ν-dimethyl-3-ammonium-1-propanesulfonate (CA8® 14ο. 14933-09-6) and the term 8B-12 means No. dodecyldecyl ^ No. dimethyl-3-ammonio1-propanesulfonate (CA8 ® Νο. 14933-08-5).

The term clinical isolate means a purified strain of a bacterial agent that causes infection, and this clinical isolate is obtained from a patient infected with this infectious agent. One or several clinical isolates can be obtained from one patient, or one and the same clinical isolate can be obtained from different patients, as is the case with nosocomial outbreaks (Р1йе !, Ό. Е! А1, АсСЫтек ο £ 1 и1егп1 Меююте 155: 1177 1184, (1995)). Such clinical isolates are usually purified by a combination of culture methods with sample processing methods. All such clinical isolates are viable and therefore suitable for further analysis and testing with respect to sensitivity to antibiotics in ίη νίΐτο tests such as a sensitivity test. Methods for isolating such clinical isolates include methods known from the prior art, in particular those described by Kep1, R.T. e! a1., RiBs NeaIu Mu ^ ba ^ eπο1ο§ν: And here we have Beta1 III ^ ai ^ a! ο ^ y, I8 OePtegi ο £ Neai ai Nitap 8e1U1se, Sep! , CA (1985) pp. 31-70 and No. 11e. R.8. e! A1., 1p: Miggau, R.R. e! A1., eDK. Mapia1 ο £ ш ш са 1 1 М 1 1 1 1 1 1 1 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 1995 43 43 1995 1995 1995 1995 (1995) pp. 400-437 for isolating Mu ^ ba ег ш т или or the methods given by ап ап Д де ТЕ, TE. e! A1., 1p: Miggau, R.R. e! A1., eDK. Mapia1 ο £ СБшса1 М ^ с ^ οЬ ^ ο ^ 1оду, А8М РГекк, ^ akРт ^ й Ό.Ο (1995) pp. 357-378 and Veatap, V.B. e! A1., 1p: Miggau, R.R. e! A1., eDK. Mapia1 ο £ C11tsa1 M ^ s ^ οЬ ^ ο ^ 1οu, А8М РГекк, ^ аКЫпдЫп, Ό.Ο (1995) pp. 379-399 to highlight Coguebas! Ergst and No. sagD | a, respectively (all works are mentioned as references )

The term “infectious agent” means an infectious microorganism, in particular an infectious bacterium, as understood in the art. Infectious agents of particular interest in the light of the methods claimed in the invention include those that contain mycolic acid structures, for example, mycobacteria, and, in particular, bacteria of the complex Mu ^ ba ^ eummy! .Ό. E! A1, 1n: Miggau, R.R. e! A1., EDc. Mapia1 ο £ Syssha1 M ^ s ^ οЬ ^ ο ^ 1οde, A8M Rgeck, ^ aKpdYup, E.S. (1995) pp .5-18 (reference is made by reference) It is believed that a person or animal suffering from a disease caused by a given infectious agent is transmitting an infection caused by a given or are infected with this agent.The infectious agent that causes the disease is considered pathogenic. Bacteria that are usually non-pathogenic and are part of the patient’s normal flora are considered saprophytes. Under certain conditions, when the patient’s immunity is weakened or suppressed (for example, with HIV infection , with AIDS, or after an organ transplant), such saprophytic microorganisms can cause infection, and the patient may be infected with one or more infectious agents.

Under the structure of mycolic acid is understood β-hydroxy acid with a substitution in position of the aliphatic chain of moderate length, as is understood from the prior art (Сο ^ еη, M.V. Vaskret. 36: 33-64 (1966) (reference is made to the reference) An example of an organism having cinnemicolenic acid is 6.0 ^ 1 ^^ 1 ^ 111.1111 D1pH (Hepaa; an example of an organism having nocardomicoleic acid is # sagD1a and WholeDec; and an example of an organism having mycolic acid is Mu ^ ba ^ т Ш си си си си Ц Ц (cm . also Ripke, S. e! a1., SBP. Mugo, Rev. 10: 125-159 (1997), where personal coryneform bacteria (reference is made by reference). Such mycous-like molecules are collectively referred to as mycolic acid structures. Additional tables of representative structures of mycolic acid, including unsaturated, cyclopropanoic, methoxylated and ketonic acids, can be found, for example, in the works of Lebeget, E. C11st Ryuk. Yr1bk 1: 294-315 (1967); Ebetet, E. Riga Arr1. Siet. 25: 135-165 (1971) (both works are referred to as references). The structures of mycolic acid are acid resistant molecules.

The term antimicrobial therapy means treating a patient, whether it is a human or animal, ίη νίνο, in effective amounts of an antibiotic-containing composition, for example, administered orally, as an injection, externally or as an inhalation. Delivery of the claimed compositions, including an antibiotic, can be carried out, without limitation, by intravenous injection, in the form of an active component of ointments or orally. The goal of antimicrobial therapy is to impair the viability, integrity or competence of the infectious agent in such a way that an infected person or animal recovers or overcomes the infection. Antimicrobial therapy can be carried out using one or more classes of drugs, one or more drugs within a given class of antibiotics, applied individually or in combination, i.e. simultaneously or sequentially. The term antimicrobial therapy is synonymous with antibiotic therapy or simply therapy.

An effective amount is understood to mean that amount of the drug that is sufficient to achieve the final goal. For example, in the context of antimicrobial therapy, an amount is considered effective whose use leads to a cure of microbial infection. Such a cure may be the result of a direct effect on the microorganism or an indirect effect, when an effective amount of the drug acts on the microorganism in such a way that it increases the sensitivity of the microorganism to the second agent.

A material is considered to be substantially free of contaminants if it is substantially free of the materials with which it was associated prior to such purification, to the extent necessary to carry out the necessary procedures or analyzes. Thus, such contaminants are either completely absent or present in such low concentrations that their presence (1) does not interfere with the desired therapeutic effect when a drug containing such materials is administered to the patient and (2) does not harm the patient after administration of such a drug.

The term administration to a patient means the administration of the desired substance to or to the desired location of the patient, whether it is a person or an animal to whom it is shown, in any way known from medical art that is sufficient to achieve the intended purpose, including (without limitation listed) enteral, parenteral (e.g. intravenous) and iontophoretic administration. The introduction can also be carried out in the form of a dressing placed at the site of infection, while the dressing provides an effective release of antibiotic (s) and betaine-like detergents.

Co-administration of two or more agents, such as an antibiotic and a betaine-like detergent, to a patient means the administration of these agents either together as a single drug, or separately, as individual drugs.

The term pharmaceutically acceptable salt combines salts formed by pharmaceutically acceptable acids or bases, such as, but not limited to, sulfuric, hydrochloric, nitric, phosphoric and other acids, or bases such as alkali or alkaline earth metal hydroxides, ammonium hydroxides, alkylammonium hydroxides, etc.

The term pharmaceutically acceptable carrier combines pharmaceutically acceptable solvents, carriers, diluents and the like, which are used as additives to the claimed preparations for the administration of such compounds.

The term “treatment” means the administration of effective amounts of one or more therapeutic agents to a subject or object that is shown to administer such agents in order to prevent, alleviate, prevent or cure a disease, or to eradicate a microorganism that is considered sensitive to these agents.

The term contacting a sample containing a microorganism means mixing the microorganism and the composition, or providing contact between the microorganism and the composition in some other way.

The authors unexpectedly found that when combining at least one betaine-like detergent, such as C ₁₈ carboxypropyl betaine (CB-18), with antimicrobial compounds, the clinical isolates of mycobacteria can be differentiated based on the induced sensitivity. The authors recognized that this phenomenon may be useful for characterizing such clinical isolates, as well as microorganisms and infectious agents in general, with respect to their sensitivity to such antimicrobial compounds. In addition, the authors also believe that this phenomenon should be useful for enhancing in a synergistic manner the sensitivity of such mycobacteria to similar antimicrobial compounds in the treatment of ίη νίνο.

Thus, in its broadest implementation, the invention is directed to a method for characterizing the sensitivity of microorganisms to antimicrobial compounds. In a preferred embodiment, the test microorganism is an infectious agent or a clinical isolate. According to another embodiment of the invention, the microorganism to be tested is obtained from a sample taken from a patient who is suspected of being infected or is actually infected with unwanted bacteria containing mycolic acid structures. The sensitivity test of the present invention is referred to as a betaine sensitivity test. The result of sensitivity testing is the characterization of the studied microorganism present in a sample such as a clinical isolate or an infectious agent, in relation to the described effect of CB-18. That is, the claimed sensitivity test makes it possible to determine whether the microorganism (microorganisms) present in the sample is sensitive to the antibiotic being tested (antibiotics). Preferably, one or more antibiotics, or a combination thereof with other effective agents, for example betaine-like detergents, to which the microorganism present in the sample is sensitive, are determined by sensitivity testing. However, it is also important that, as a result of sensitivity testing, one or more antibiotics are determined, or their combination with other effective agents, for example, betaine-like detergents, to which the microorganism present in the sample is insensitive. The result allows a medical professional to more effectively choose the appropriate antimicrobial therapy for the treatment of patients or, on the other hand, to sterilize the place from which the sample was obtained with respect to this microorganism.

In a second preferred embodiment, the invention is directed to a method of treating patients during antibiotic therapy using betaine-like detergents as adjuvants, wherein the patient is suspected of being infected, at risk of being infected or indeed infected with unwanted bacteria. According to a preferred embodiment of the invention, such undesirable bacteria contain mycolic acid structures. Antibiotic therapy can be developed to treat this infection by using a combination of one or more antibiotics with one or more betaine-like detergents. The combination of an antibiotic with a betaine-like detergent more effectively cures a patient than antibiotic-only therapy, affecting the integrity of bacteria in such a way that infectious organisms become incompetent, non-infectious or non-viable more quickly and more effectively than would happen if an antibiotic without a betaine-like detergent was used.

In a third preferred embodiment, the invention is directed to a method of sterilizing or preventing the growth of unwanted microorganisms by using a combination of one or more antibiotics with one or more betaines at the site of infection and in an environment in which the presence of such microorganisms is suspected.

The sensitivity test of the present invention is particularly useful for determining and establishing the sensitivity of a microorganism, in particular a clinical isolate and / or infectious agent, to antimicrobial agents. The disease caused by such microorganisms can be treated with effective amounts of one or more antibiotics with one or more betaine-like detergents, which, based on the results of the sensitivity test, are selected as rational components of the therapeutic course to cure a similar disease caused by these microorganisms.

When conducting sensitivity tests, the application of the concentration spectrum of a given betaine-like detergent will provide the necessary information about the effectiveness of this detergent, but it is preferable to test more than one betaine-like detergent. You can test any number of betaine-like detergents or combinations thereof. Preferably, at least five are tested, although any other number, for example 10, 15, 20.25 or 30, and even more can easily be tested in various dilutions. A sensitivity test provides additional information regarding the effect of combining such concentrations of a betaine-like detergent with the selected antibiotic (s). The working or applicable concentration of a given betaine-like detergent will depend on the particular detergent used. In general, working concentrations range from 0.1 μg / ml for the strongest compounds to 1 μg / ml for the weakest compounds, while for most betaine-like detergents, the concentration range from 1 to 100 μg / ml will be most applicable. When setting up sensitivity tests, such betaine-like detergents can be used on their own or in combination with other betaine-like detergents.

While the use of even one antibiotic in the claimed methods will provide the necessary information about the sensitivity, it is preferable to use several antibiotics in the test for betaine sensitivity. You can test any number of antibiotics or their combinations. Preferably, at least five are tested, although any other number, for example 10, 15, 20, 25 or 30, and even more can easily be tested in various dilutions. The concentration of this antibiotic will depend on the particular antibiotic used. In general, working concentrations range from 0.1 μg / ml for the strongest antimicrobial agents to 100 μg / ml for the weakest compounds, with a concentration range of 0.5 to 64 μg / ml being most applicable to most antibiotics. When implementing the claimed methods, such antibiotics can be used on their own or in combination with other antibiotics.

As in the case of other sensitivity tests (AOOSH.S.K e! A1., Ιη: Miggau, R.V. e! A1., Eb§. Mapiao £ C11n1ca1 M1stoyuodu, L8M Pte88, Aazypdop, L.S. (1995) pp. 1327-1404), it can be expected that variations in the volume of the inoculum will affect the results of the betaine sensitivity test. The inoculum is obtained in a standard manner, including colony pricking or comparison with the Mac Farland standard known in the art. The experimental results shown in FIG. 5L-5P and in Example 8, show that the effect of CB-18 is comparable for inoculum sizes ranging from several cells to several thousand cells. In general, for these tasks, we apply an inoculum from 1 cell to 10 ⁵ cells in size, an inoculum from 100 to 10,000 cells is more preferable, and an inoculum of 1,000 cells is most preferred. With an inoculum of less than 10 cells, a pipetting error can distort the test results, and with an inoculum of more than 10 ⁵ cells, the system will be overloaded, which will also distort the results. An inoculum ranging in size from 1,000 to 10,000 cells makes it possible to observe the growth of controls over time. However, we can use any inoculum that makes it possible to observe the effect of CB-1 8, and in this regard, the claimed betaine sensitivity test is similar to other ίη νίΙΐΌ sensitivity tests.

The data obtained as a result of setting the described test for betaine sensitivity are the result of the dynamic interaction of the isolate, betaine-like detergent (s) and antibiotic (antibiotics). The betaine sensitivity test combines these three components, which makes it possible for a professional to determine and select the concentration of antibiotic (s) and betaine-like detergent (s) with the inoculum in such a way as to obtain the CB-18 effect, thereby collecting the most useful information regarding the nature and / or sensitivity of the studied clinical isolate.

From example 7 it is seen that betaine-like detergents may be more useful than other detergents in the claimed sensitivity tests and therapeutic methods. For example, structures having modifications in association exhibit reduced activity in sensitivity testing and therapeutic methods. In addition, betaine-like detergents having modifications in the bridge also exhibit reduced activity in sensitivity testing and therapeutic methods. Table 1 shows the structures that are most applicable for the implementation of the claimed invention.

The most useful betaine-like detergents are those in which there are no modifications, for example, when B! is simple alkyl, bond (α) is simple methylene, B ₂ and B ₃ are hydrogen or methyl groups (depending on the cation used), and there are no substitutions in B4.

The toxicity of betaine-like detergents for bacteria containing mycolic acid depends on the anion (γ), the length of the bridge (CC and the length of the alkyl chain (B!). In general, the balance between the surface-active nature of betaine and its properties as an adjuvant must be taken into account. For example , surface active properties are dependent on the nature of the anion kislotnoosnovnogo and alkyl chain length. most ionic detergents are phosphobetaines (PO ₄₎ and most non-ionic detergents are sulfatobetainy (8o _4). it can be expected that fosfobetai It will turn out to be extremely toxic, and solubility problems will be associated with sulfatobetaines. Therefore, sulfobetaines and carboxybetaines are preferred, and carboxybetaines are most preferred. Based on the discussion of permeability (Example 9), it can be assumed that carboxybetaines have the most preferred anion, and despite the fact that CB- 1 8 contains an ammonium cation, it is reasonable to assume that sulfoniumbetaines should possess the most preferred cation. Thus, preferred structures include η-alkyl sulfobetaines, particularly preferred η-alkyl carboxybetaines, and most preferred are alkyl sulfonium carboxybetaines.

The surface-active properties of betaines change with increasing chain length. Longer alkyl chains have lower critical micellar concentrations (CMC), and vice versa, for shorter alkyl chains, higher concentrations are required to achieve SMS. The length of the alkyl chain may vary depending on the application. For example, with shorter alkyl chains, higher concentrations are required to achieve SMS and therefore, in the second embodiment of the invention (i.e., with antimicrobial therapy), shorter chains are preferable, especially when administered intravenously. Alternatively, for therapeutic use by inhalation, compositions that are less dependent on the relationship between concentration and SMS can be used; therefore, longer alkyl chains can be used. In the first embodiment of the invention (i.e., in the sensitivity test ίη νίίτο), the length of the alkyl chain can be varied so as to determine a detergent capable of maximizing the observed effect of CB-18. Betaine-like detergents with an alkyl chain length of 8-22 carbon atoms are preferred, a chain length of 12-18 carbon atoms is particularly preferred, and a chain length of 16-18 carbon atoms is most preferred.

A bridge of minimum length, which guarantees salting out, will be a propylene bridge. Toxicity to the microorganism is also a function of the length of the bridge (Tklope, K. e1 a1., 1ig.Ryagt. § 80: 441444 (1991)). For example, ethylene and butylene bridges cause greater toxicity than the propylene bridge. When using an ethylene bridge, solubility problems may occur, depending on the ions used. Therefore, it is necessary to take into account the necessary coincidence of the corresponding length of the alkyl chain with the ions and the structure of the bridge in order to avoid such problems. For example, longer alkyl chains require a combination of ions with strong polarity (8O ₄ <8O ₃ <CO ₃ <PO ₄ ) and a bridge structure that facilitates salting out. A bridge structure of the type —C _t H _2t - (where m> 1) is preferred, and a bridge with the same structure with 6>m> 3 is most preferred. According to TkLope, K. e1 a1., 1oig. Ryagt. 8s1. 80: 441 - 444 (1991), the components used to modify the cation also contribute to toxicity (i.e., toxicity is inversely proportional to the length of K. ₂ and K. ₃ (see Table 1).

The length of the bridge and cationic components may vary depending on use. For example, in the second embodiment of the invention (i.e., antimicrobial therapy), shorter alkyl chains can be used and bridge structures with m> 2 are also applicable since solubility is less of a problem in the case of short chains. Alternatively, inhalation delivery necessarily requires the solubility of a betaine-like detergent: therefore, bridge structures with m> 3 may be required. As discussed above, the polarity of betaine can be changed by varying the anion. Again, the ratio of the length of the bridge to the components used to modify the cation may vary to maximize the observed effect of CB-1 8 in the first embodiment of the invention (i.e., in the ίη νίίτο test).

Several carboxybetaines were tested that exhibited the effect of CB-18 in the methods of the invention. These include: C1 _8- carboxymethyl betaine (CA8® .6ο.693-334), C1 _8- carboxyethyl betaine (CA8® 30ο. 3061273-8), C; _8: 1-carboxymethyl betaine (CA8® Νο. 871-37-4) and C1 ₈ -carboxypropyl betaine (CA8® .ο. 78195-27-4).

Examples of the most useful carboxybetaines that use a methylene bridge (carboxymethyl betaines: I ₄ = -CH ₂ -), a methylene bond (α = -CH ₂ -), and which differ only in the length of the alkyl chain, are: C ₁₀ (CA8® Νο. 2644-45-3), C _p (CA8® .ο. 2956-38-9), C ₁₂ (CA8® .ο. 683-10-3), C ₁₃ (CA8® Νο. 23609-76-9), C ₁₄ (CA8® Νο. 2601-33-4), C ₁₅ (CA8® Νο. 23609-77-0), C ₁₆ (CA8® Νο. 693-334) and C ₁₈ (CA8® Νο. 820-66- 6). FROM; _2- carboxymethyl betaine (CA8® Νο. 6232-16-2) serves as an example with Ν, Ν-diethyl (Z ₃ = Z ₄ = -CH ₂ CH ₃ ); and an example with a double alkyl bond is C _{18: 1} (CA8® Νο.871 -37-4). Examples of the most useful carboxy betaines in which an ethyl bridge is used (carboxyethyl betaines: K.4 = -CH ₂ CH ₂ -), methylene bond (α = -CH ₂ -), and which differ only in the length of the alkyl chain, are: C ₁₂ (CA8 ® Νο. 16527-85-8), C ₁₃ (CA8® Νο. 132621-79-5), C ₁₄ (CA8® Νο. 69725-38-3), C ₁₆ (CA8® Νο. 424-433) and C ₁₈ (CA8® Νο. 30612-73-8). An example of carboxyethyl betaine, in which K. ₂ and K ₄ are hydrogen atoms under appropriate conditions, is CA8® Νο. 1462-54-0 (C _{! 2} beta alanine). Examples of the most useful carboxy betaines that use a propyl bridge (carboxypropyl betaines: P4 = -CH2CH2CH2-), methylene bond (α = -CH2-), and which differ only in the length of the alkyl chain, are: Cu (CA8® Νο. 150147-53- 8), C ₁₂ (CA8® Νο. 15163-30-1), C ₁₄ (CA8® .ο. 146959-90-2), C ₁₅ (CA8® .ο. 146959-91-3), C ₁₆ (CA8® 7ο. 71695-32-4) and C ₁₈ (CA8® Νο. 7819527-4). An example of a useful carboxybetaine using a butyl bridge (carboxybutyl betaine: K. ₄ = —CH ₂ CH ₂ CH ₂ CH ₂ -) and methylene bond (α = —CH ₂ -) is: C ₁₂ (CA8® Νο. 120139- 51-7). Two examples of the most useful carboxybetaines in which the pentyl bridge is used (carboxypentyl betaines: Kd = -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -), methylene bond (α = -CH ₂ -), are: C ₁₂ (CA8® Io. 76392-97-7) and C ₁₆ (CA8® Jo. 7356598-7). An example of a carboxy betaine in which a hexyl bridge is used (carboxyhexyl betaine: Kd = —CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -) and a methylene bond (α = —CH ₂ -) is C ₁₂ (CA8® Io. 132621- 80-8). There are several examples of carboxybetaines in which the benzyl group (K ₄ = —CH ₂ —C ₆ H ₄ -) is used as a bridge. There are two C ₁₂ examples in which the carboxy function is in position 4 or the para position (CA8® Io. 7195-313) and one in which the carboxy function is in position 2 or the ortho position (CA8® Io. 7195-34 -6). There are two examples of C ₁₆ in which the carboxy function is in position 4 or the para position (CA8® Io.7195-33-5) and one in which the carboxy function is in position 2 or the ortho position (CA8® Io.7195 -35-7). Thus, the term carboxybetaine-like (CB-like) includes not only those structures in which the carboxyl group (y = COO) is used as the anion, as defined in \ UO 95/27076 and I.8. 5,658,749, but also those betaine-like structures that are given in table. 1 and which include all possible combinations of K _b α, K ₂ , K ₃ , β, and K4 as defined in this context.

In addition to carboxybetaines, there are other available betaines that can be used for the purposes of the present invention, such as sulfobetaines, for example, highly purified (i.e. reagent grade) 8B-series detergents and, in particular, 8B-18 (Ioctadecyl-I, I-dimethyl-3-ammonio-1-propanesulfonate (CA8® Io. 13177-41-8)), 8B-16 (Ihexadecyl-Ν, Ν-dimethyl-3-ammonium-1-propanesulfonate (CA8® Io.2281- 11-0), 8B-14 (Itetradedecyl-IH-dimethyl-3-ammonio-1-propanesulfonate (CA8® Io. 14933-09-6) and 8В-12 (Ideodedecyl-Ν, Ν-dimethyl-3-ammonio- 1 propane sulfonate (CA8® Io. 14933-08-5)).

Most commercially available betaines are used in the manufacture of detergents, shampoos, cosmetics, etc. These betaines are originally derived from natural fats, such as coconut oil, animal fat, wheat germ, babasse oil, castor oil, canola oil, soybean oil and rapeseed oil. There is reason to believe that all these betaine-like detergents will be useful in implementing the methods claimed in the invention.

The present invention is particularly useful for characterizing and verifying clinical isolates or treating diseases caused by infectious microorganisms, in particular microorganisms that are lipophilic or encapsulated in a wax capsule, and characterized by the presence of mycolic acid structures on their outer wall (outer membrane), such as like cinnamicoleic acids, nocardomoleic acids and mycolic acids among others.

The methods claimed in the invention provide for testing a microorganism, such as Musobacterium, for sensitivity to antibiotics. In a preferred embodiment, the microorganism is a clinical isolate. The methods claimed in the invention are directed to therapeutic use when a patient (human or animal) is subjected to antibiotic therapy, in particular, using betaine-like detergents in combination with antibiotics. The choice of betaines used for testing or treatment according to the present invention will depend on which microorganism, preferably a bacterium, is present in the sample.

Similar test procedures or therapeutic regimens are applicable to any particular microorganism, and in particular to a particular bacterium. In a particularly preferred embodiment of the invention, the microorganism is a Musocobacillus complex or is a member of the complex. For example, the bacteria to be tested can belong to any group of MusoBas! Epit, or to any type of Musobac! And, most preferably, to the M.! Bilbaco1818 (MTB) complex, M.Ausht complex (MAC), the MA18 complex and the complex M. Goyiyit. The bacteria to be tested can include both fast-growing and slow-growing mycobacteria, including identified and unidentified photochromogens, non-photochromogens and scotochromogens. Any mycobacteria can be characterized or processed according to the present invention, including M.adp, M. aksekks, M. ace! Atljujisit, M. apsaiit, M. ausheike, M. aaiysit, M. aigit, M. aikgoa Gpsaiit, M. ausht, M. Louk, M. Lousch (BC6), M. cl1e1oiae, M.cjeae, M.cBiiee, M. sooky, M.SzegnNoGep. M.buua1i, M.Ga11ah, M.Gagstodeeyek, M.yauekseik, M.Goyiyit, M.dabsht, M.dayp, M.duyt, M.dogbopae, M.BaitorShit, M.t! Gasse11iate, M.kaikakp, M.kotosekeike, M.1ergee, M.1ergipit, M.tapiit, M.t1toyee8e, M.tkgoi, M.topokaize, M.ieoaigit, M.ioisgododetsit, M.oeieuiza, M.rata Goiiyit, M. raga! M. regedpiit, M. r.Sher M. rogstit, M.rotgGegae, M. ri1uep8, M.tBobeyee, M. 8sgo & 1seit, M. zeyedaeye 8e, M.51pto1bek M.yt1ae, M.ctedtayk, M.zradsh, M.8kh1da1, Mbetgae, M. Shetottey8Bl1e, M.! Oka1ei8e, M.! Pu1a1e, M.!

M.1 & Lsi1o818, M. Iipsapit, Μ.ϋονίδ, Μ.ϋονίδ (В СО) and M. Tyutyu are members of the MusoBaslgst.1 and Letsi1o818 (MTV) complex. Mbeggae, M.jMa1e and M.popsygotodesit are members of the complex M.1eggae. M.Acht and M.Tlgase11i1age are members of the MusoBas1egst complex ηνίιιιη (MAC); there are at least three different serological groups of M. auscht and more than 25 serovars of M. t1gase11i1age. The MA13 group (M. acht1n1gase11i1age-8sg0i and iaseite) includes mycobacteria with the biochemical properties of the M.aust complex and the mycobacteria M. 8suGi1aseit, but does not hybridize with nucleotide probes (for example, Assirgoe (Oep-Prgoe, Zap Ergeo). homologous to the mycobacteria of the M.Aust complex.

M. capzazp, M. tappit, M.81t1ae and M.a81aysit are examples of photochromogens. M.8sgoGi1aseit, M.khkhi Tsak M.khepor1, M.dogbopae and M.jauesseps are examples of scotochromogens. M.Aust, M.thyase11i1age, M.da8y1, M.ta1toep8e, Mbegge and M.yMa1e are all examples of non-photochromogens.

M.aipsapit, M.a81isit, M.au1it,

M. Bouy, M. Bou18 (VCO), M. sooki, M. da Chgk M. Dogbopae, M. yayetor Yyit, M. tyase11i1age, M. kapzazi, M.1ergae, M.1ergaygsht, M.tappit, M.t1toep8e, M. tkgoy, M. popskgotodepuit, M. rutiu bisiu818, M.8 bruiu, M.81 bae, M. bisu, M. mulet, M.1 bisi818, M. bisu8 and M. hepore are slow growing growing takes more than seven days). M.adp, M. abbesziz, M. acse1at1bo1uysit, MtsShepze, M.aiigit, M.ai8goaGpsapit, M.sNe1ope, M.sByaye, M.skiepze, M.SzhepioGep, M.b ^ a1n, M.Ga11ah, M.Gagstod M. Pauehsepkh, M. Goyiyit, M. Dabst, M. Dyuit, M. Kotozzepze, M. Topokaepze, M. Peaoigit, M. Regedppit, M. Oiepze, M. Raga Goyyyit, M. рі1е1, M. rogspit, M .rogyegae, M. pterulidae 18, M. goboe81ae, M.8epedaep8e, M.ztedtayz, M.8pepadsh, M. Shegtoge818Bp1e, M.1ocaepepe and M.uassae are rapidly growing species of mycobacteria (it takes less than seven days to grow).

Examples of diseases and painful conditions in which mycobacteria of various types are involved and which can be treated in accordance with the methods proposed in the inventions are, first of all, tuberculosis (complex M.1begsi818), leprosy (M.1ergae (human leprosy) and M.1ergyipit (leprosy rodents)), diseases of birds caused by bacteria of the Muso bac1epit out complex. opportunistic infections and superinfection of AIDS patients and other diseases caused by Mdait (No. dB1tda1e, 3.Ό. e1 a1., 1ig.1pGes1.P18. 165: 1082-1085 (1992)), and any infections caused by specific mycobacterial agent, such such as M. lou18 (of particular importance for veterinary medicine), M. goyiit (soil bacterium isolated from damage in animals and humans), Mlpyase11iage (causes opportunistic infections, especially in patients infected with the AIDS virus), M. plaguebella818 ( of particular interest in the diagnosis of disease K rona (regional ileitis) in humans), MusoBas1epit cap8a8i (a rare but destructive agent, usually associated with lung diseases), Musoabaspu tappit (infects cold-blooded animals and fish; isolated from subcutaneous granule limbs in humans), John in calves; grows very slowly in culture, it takes 16 weeks before you can be sure of a negative result) and M. and Segapsa (the cause of Buruli ulcer). Many of the listed species of mycobacteria, as well as their other species, are discussed. e1 a1., C1t M | sg.K.e. 5: 1-25 (1992) and Pa1kshyat, O., C1t M1sgo. Yeh. 9: 177215 (1996) (both works are cited as references).

Betaine-like detergents can be used as adjuvants on their own (if the detergent has antimicrobial activity) or in combination with antibiotics. For example, according to one preferred embodiment of the invention, the betaine-like detergent (s) are a component of a sensitivity test panel (as described in Example 10), and such detergents are used alone or in combination with antibiotics or other betaine-like detergents to characterize the sensitivity of the clinical isolate. In another preferred embodiment, the betaine-like detergents are used alone or in combination with antibiotics for antimicrobial therapy. While betaine-like detergents can be used on their own, w Bey antibiotics, it is more preferable to use them in combination with such antibiotics. In any embodiment, betaine-like detergents are used alone or in combination with other adjuvants, simultaneously or sequentially. For example, betaine-like detergents can be used in combination with other betaine-like detergents or with other adjuvants, such as β-lactamase inhibitors.

According to a second embodiment of the invention, the claimed methods are used to treat a patient, whether it is a person or an animal to which it is shown, effective amounts of the claimed compositions containing a betaine-like detergent alone or an antibiotic in combination with a betaine-like detergent. The concept of antimicrobial therapy also includes the use of such combinations. The delivery of betaine-like detergents can be carried out by any method that ensures that the patient receives effective amounts of the drug, for example, by oral or intramuscular injection, or, in particular, by an intravenous dropper, and, most effectively, by inhalation or external use of these compositions.

The antibiotic can be administered in a separate way and in another solution, or it can be administered together with a betaine-like detergent. Obviously, betaine-like detergents should be well tolerated by oral administration and intramuscular injection, intravenous droppers can also serve as an important delivery method; however, precautions should be taken regarding the concentration of betaine in the blood. While the administration of betaine at a dose in excess of the critical micellar concentration (SMS) can be tolerated, complications may arise if its total blood level is exceeded above SMS due to the solubilization of blood components. Injection at the site of infection is obviously an effective delivery method, however, this is only possible in rare cases. Preferably, the composition or at least one betaine-like detergent is delivered in the most direct way or, if possible, directly to the site of infection. In this regard, for mycobacterial infections that, like tuberculosis, are respiratory infections, inhalation is the most preferred delivery method. An example of an inhalation device for delivering the betaine-like detergents of the invention is the inhaler currently used to deliver albuterol, a beta-blocking agent for asthmatics (i.e. Mycobacterial infections caused, for example, by M. and Segap8 (Buruli ulcer) are preferably treated by applying a betaine-like detergent directly to the lesion (with or without antibiotics) as an ointment.

Dosages and the schedule for the administration of this betaine-like detergent to the patient can be determined by a clinician experienced in the treatment of microbial infections of this kind. Typically, dosages of antibiotic and betaine-like detergent will vary depending on the following considerations: type of antibiotic and betaine-like detergent used: patient's age: state of health; disease to be treated; type of possible competitive therapy; frequency of treatment and nature of the desired effect; the degree of tissue damage, the gender of the patient, the duration of symptoms and possible contraindications among other variable factors should be considered by the doctor. To achieve the desired result, the dose can be administered once or repeatedly. In general, working concentrations can range from 0.1 μg / ml for the strongest compounds (for example, carboxyethyl betaines) to 1 μg / ml for the weakest compounds (for example, those carboxy betaines that are marked in Table 10 as having no effect ), while for most betaine-like detergents, the concentration range from 1 to 100 μg / ml will be most applicable. When using betaine-like detergents in the form of an ointment, working concentrations can vary from 0.1 μg / ml for the strongest compounds to 1 μg / ml for the weakest compounds, while for most betaine-like detergents the concentration range is from 1 to 100 μg / ml most applicable. When implementing the claimed therapeutic methods, such betaine-like detergents can be used on their own or in combination with other betaine-like detergents. Betaine is preferably administered at the same time as the antibiotic.

The concentration of this antibiotic will depend on which antibiotic is used. According to the claimed methods, antibiotics can be used in therapeutically effective doses known from the prior art, or in concentrations that are defined as therapeutically effective in the claimed sensitivity tests. Examples of various classes of antibiotics applicable in the implementation of the claimed method include β-lactam antibiotics, aminoglycosides, aminocyclitols, quinolones, tetracyclines, macrolides, lincosamides, glycopeptides, lipopeptides, polypeptide antibiotics, sulfonamides, trimethoprim, chloramphenicol nitrofiramidi rimonofenamide , methenamine and mupirocin.

Antibiotics that have significant activity against mycobacteria include, without limitation, amikacin, azithromycin, any β-lactam in combination with any β-lactamase inhibitor, capreomycin, cefmetazole, cefoxitin, ciprofloxacin, clarithromycin, clofazamine, cycloserine, dapone ethambutol (EMB), ethionamide, imipenem, isoniazid (ΙΝΗ), kanamycin, minocycline, ofloxacin, paraaminosalicylic acid, protionamide, pyrazinamide (ΡΖΑ), rifampin (CRM), rifabutin, sparfloxacin, sulfamethoxazole in combination with IOM, streptomycin (8M), tetracycline, triacetazole and viomycin (Chpbegeeb. S.V. e1 a1., Ιη: Miggau, P.K e1 a1., ebk. Mapiao £ Syssa1 M1cGo1o1o§u, A8M Eqgkk. S. (1995), pp. 1385-1404), and Kisegk A. e1 a1., TNE Ike about £ 4 ApbYobsk ^'s fucked. B.V. Yrrtsob Co. РЫ1абе1рЫа, РА (1987) pp. 1352-1437). All of these antibiotics can be used in the implementation of the claimed methods.

The term β-lactam means any penicillin, cephalosporin, monobactam or carbapenem antibiotic whose structure includes a β-lactam ring, as is understood from the prior art (Wao, Yu.S. e! A1., Ιη: Miggau, R.K. e! a1., eb 8. Mapia1 οί С11шса1 М1egoyoodu, Ά8Μ Рge88, AaChppfop. Ό.Ο (1995) pp. 1281-1282); Kiseg8, A. e! A1., THAT Ike οί ΑηΐίЬюИс8 4 ^! TV.Yrrtsoy So. RybabeRya, RA (1987) pp. 3-584). All of these β-lactams can be used in the implementation of the claimed methods.

The term penicillin means an antibiotic that has 6 aminopennicillic acid as a chemical core, as is understood from the prior art (Wao, Yu.S. e! A1., Ιη: Miggau, R.K. e! A1., Eb8. Mapia1 οί C11n1ca1 M1goyuodu , А8М Рge88, АакЫпдоп, Ό.Ο (1995) pp. 1282-1285). Examples of penicillins, without limitation, are methicillin, nafcillin, cloxacillin, dicloxacillin, oxacillin, ampicillin, bacipicillin, carbenicillin, tricacillin, meslocillin, piperacillin and, in particular, azlocillin. There is reason to believe that penicillins with a chemical structure homologous to those of the above penicillin compounds will also be applicable in the implementation of the claimed invention.

The term cephalosporin means an antibiotic having 7 aminocephalosporanilic acid as a chemical core, as is understood from the prior art (Wao, Yu.S. e! A1., Ιη: Miggau, R.K. e! A1., Eb8. Mapia1 οί С11шса1 М1сгёю1оду , A8M Prg88, Aaztdosch Ό.Ο (1995) pp. 1282-1285). Examples of cephalosporins applicable to the implementation of the claimed methods include, but are not limited to, cefadroxil, cefazolin, cephalexin, cephaloridine, cephalotin, cefapirin, cefradine, cefaclor, cefamandole, cefoncimetzefefetfolefil, cefprosimefetol, cefrefosfetolef, cefrefosfetol ceforfetzolefetor, cefrefosfetol ceforfetzolefetor, cefrefosfetol ceforfetzolefetor, cefrefosfetol ceforfetzolefetor, cefrefosfetol ceforfetzolefetzefetorfilfetzefetorfefefelot, cefprosimetol, cefprosimetol cefprodoxime, ceftidoxime and, in particular, ceftriaxone. There is reason to believe that cephalosporins with a chemical structure homologous to that of the above cephalosporin compounds will also be applicable in the implementation of the claimed invention.

The term monobactam means an antibiotic having a β-lactam ring as a chemical nucleus, as is understood from the prior art (Wao, Ι.Ό.Ο e! A1., Ιη: Miggau, R.K. e! A1., Eb8. Masha1 οί С '1 letter 1 М1сгоЬю1оду, А8М Рге88, АакIддощ Ό.Ο (1995) p.1285). An example of monobactam applicable to the implementation of the claimed methods is, without limitation, aztreonam. There is reason to believe that monobactam with a chemical structure homologous to that of the above monobactam will also be applicable in the implementation of the claimed invention.

The term carbapenem means an antibiotic having a β-lactam ring as a chemical nucleus, a side hydroxyethyl chain at position 6 (in a trans configuration) and without a sulfur or carbon atom in the nucleus, as is understood from the prior art (Wao, Yu.S. e! A1 ., Ιη: Miggau, R.K. e! A1., Eb8. Masha1 ίί Syu! Sa1 M1sgoyuuodu, A8M Prge88, ^ 8 ^^^, E.S. (1995) pp. 1285-1286). Examples of carbapenems applicable to the implementation of the claimed methods are, without limitation, imipenem, meropenem, panipenem and biapenem. There is reason to believe that carbapenems with a chemical structure homologous to those of the carbapenem compounds listed above will also be applicable in the implementation of the methods claimed in the invention.

The term β-lactamase inhibitor means an antibiotic having a modified β-lactam structure as a chemical core, as is understood from the prior art (Wao, TOC e! A1., Ιη: Miggau, R.K. e! A1., eb8. It is known that these compounds, possessing in their pure form weak antibacterial activity, act synergistically with β-lactams. Β-lactamase inhibitors interfere with enzymes that degrade β-lactams (i.e., β-lactamases). For example, β-lactamases destroy β-lactams. With this function, the microorganism effectively avoids the action of β-lactams, which determines the stability of the infectious agent. Therefore, β-lactamase inhibitors are useful in combination with β-lactam antibiotics as adjuvants in β-lactam therapy. Examples of β-lactamase inhibitors applicable to the implementation of the claimed methods along with β-lactam are, without limitation, clavulanic acid, sulbactam and tazobactam. There is reason to believe that β-lactamase inhibitors with a chemical structure homologous to that of the β-lactamase inhibitors listed above will also be applicable to the implementation of the methods of the invention.

The terms aminoglycoside and aminocyclitol mean an antibiotic having an amino sugar linked by glycosidyl bonds with the aminocyclitol core, as is understood from the prior art (Wao, 1. I.C. e! A1., Ιη: Miggau, R.K. e! A1., eb8. Masha1 οί S'1yisa1 M1goyu1odu, A8M Prge88, ^ kIp ^ oi, Ό.Ο (1995) pp. 12871288); and Kiseg8, A. e! a1., Tye I8e οί Up! 1Yoys8 4 ^'s fucked. EV.Yrrshso !! With. РЫ1абе1рЫа, РА (1987) pp. 585-750). Examples of aminoglycosides and aminocyclitols applicable to the implementation of the claimed methods are, without limitation, streptomycin, kanamycin, gentamicin, tobramycin, amikacin, sisomycin, netilmicin, neomycin, framycetin and paromomycin. There is reason to believe that aminoglycosides and aminocyclitols with a chemical structure homologous to those of the above aminoglycoside and aminocyclitol compounds will also be applicable in the implementation of the claimed invention.

The terms quinolone or fluoroquinolone mean an antibiotic having a naphthyridine nucleus with different side chains, as is understood from the prior art (Wao, Yu.S. e! A1., Ιη: Miggau, R.K. e! A1., Ebk. Mapia1 o С Syssha1 1 Msgobyuodu, A8M Przekk, ак akЫdop, E.S. (1995) pp. 1288-1290); and Kisegk, A. e! a1., Thy ike o £ Apbshbsk 4 ' ¹ ' eb. EV.Yrrshso !! With. P1n1abe1rYa, RA (1987) pp. 1203-1275). Examples of the quinolones useful for the inventive methods include, without limitation, oxolinic acid, cinoxacin, flumekvin, miloksatsin, rozoksatsin, pipemidilovaya acid, norfloxacin, enoxacin, ciprofloxacin, ofloxacin, lomefloxacin, temafloxacin, fleroxacin, pefloxacin, amifloksatsin, sparfloxacin, levofloxacin , cinafloxacin and, in particular, nalidixic acid. There are reasons to believe that quinolones or fluoroquinolones with a chemical structure homologous to those of the above quinolone or fluoroquinolone compounds will also be applicable in the implementation of the methods claimed in the invention.

The term tetracycline means an antibiotic having a hydronaphthacene structure as a core, as is understood from the prior art (Wao, I. Ό. C. e! A1., Ιη: Miggau, RK e! A1., Ebk. Mapia1 o £ С11шса1 М1сгоОо1оду, А8М Ргекк, ^ аКЫпдоп, ΌΌ. (1995) pp. 1290-1291); and Kisegk, A. e! a1., Thie ike o £ Apbyobsk 4 ' ¹ ' eb. E.V. Yrrshsob Co. PY1abe1pYa, RA (1987) pp. 9791044). Examples of tetracyclines useful for practicing the claimed methods include, but are not limited to, tetracycline, chlortetracycline, oxytetracycline, dimethylchlorotetracycline, demeclocycline, metacycline, limycycline, clomocycline, doxycycline and minocycline. There is reason to believe that tetracyclines with a chemical structure homologous to those of the above quinolone or tetracycline compounds will also be applicable in the implementation of the claimed invention.

The term macrolide means an antibiotic having a macrocyclic lactone ring with two attached sugars, decosamine and cladinose, and various substitutions, as is understood from the prior art (Wao, Yu.S. e! A1; 1p: Miggau, R.K. e! A1 ., ebk. Mapiao £ 11 са ш са 1 1 М 1 с Ь ш оду оду,, А 8 М Р г ек к, ^ й й д д! оп,,,, E.S. (1995) pp. 1291-1292); and Kisegk, A. e! a1., Thie ike o £ Apbyobsk 4 ' ¹ ' eb. E.V. Yrrshsob Co. Rybaberala, RA (1987) pp. 851-892). Examples of macrolides useful in carrying out the claimed methods include, but are not limited to, erythromycin, oleandomycin, spiramycin, josamycin, rosaramycin, clarithromycin, azithromycin (also known as azalide), dirithromycin, roxithomycin, flurithromycin and roquitamycin. There is reason to believe that macrolides with a chemical structure homologous to that of the macrolide compounds listed above will also be applicable in the implementation of the methods claimed in the invention.

The term linkcosamide means an antibiotic having an amino acid linked to an amino sugar, as is understood from the prior art (Wao, 1.O.C. e! A1., 1p: Miggau, R.K. e! A1., Ebk. Mapia1 o £ Syssha1 M1SgOb1uod, A8M Rheck, ^ akPdop, O.S. (1995) pp. 1292-1293); and Kisegk, A. e! a1., Thie ike o £ Apbyobsk 4 ' ¹ ' eb. EV.Yrrshso !! With. PY1abe1rYa, RA (1987) pp. 819850). Examples of lincosamides useful for practicing the claimed methods are, but are not limited to, lincomycin and clindamycin. There is reason to believe that lincosamides with a chemical structure homologous to those of the above lincosamide compounds will also be applicable in the implementation of the claimed invention.

The terms glycopeptidyl or lipopeptide mean an antibiotic having a combination of a peptide with a hydrocarbon or lipid component, as is understood from the prior art (Wao, 1.O.C. e! A1., 1p: Miggau, R.K. e! A1., Ebk. Mapiao £ шка шка шка с 1 1 М 1 1 Ы оду оду оду оду, А М М М г ек к, ^ ак Ы Ы д д оп оп,. and Kisegk, A. e! a1., Thie ike o £ Apbyobsk 4 ' ¹ ' eb. E.V. Yrrshoy Co. РЫ1абе1рЫа, RA (1987) p. 1045-1072). Examples of glycopeptides and lipopeptides applicable to the implementation of the claimed methods include, but are not limited to, vancomycin, teicoplanin, daptomycin (also known as YB 146032) and ramoplanin (also known as MO 62198). There is reason to believe that glycopeptides and lipopeptides with a chemical structure homologous to that of the above glycopeptide and lipopeptide compounds will also be applicable in the implementation of the claimed invention.

The term polypeptide antibiotic means an antibiotic having a cyclic polypeptide structure, or amino acids connected to a peptide, as is understood from the prior art (Wao, Yu.S. e! A1., 1p: Miggau, R.K. e! A1., Ebk .Mapia1 o £ C11shsa1 М1сгоОо1од,

Α8Μ Рge55, \ Уа51ppдУп, Ό.Ο. (1995) pp. 12951296); and Kiseg5, Α. e! a1., T1e and 5e oG Αηί ^ b ^ o! ^ C5 4 ' ¹ ' eb. L.V. Irrtsob Co. РЫ1абе1рЫа, ΡΑ (1987) pp. 899-913 and 751-753). Examples of polypeptide antibiotics applicable to the implementation of the claimed methods are, without limitation, polymyxins A, B, C, Ό and E, bacitracin and gramicidin. There is reason to believe that polypeptide antibiotics with a chemical structure homologous to that of the above polypeptide antibiotics will also be applicable in the implementation of the claimed invention.

The term sulfonamide means an antibiotic having a nuclear structure similar to paraaminobenzoyl acid, as is understood from the prior art (Wao, I. Ό. C. e! A1., Ιη: Miggau, R.K. e! A1., Eb5. Mapia1 oG С11шса1 М1сгоБ1о1оду, Α8Μ Рge55, \ Уа51ппд1оп. S. S. (1995) pp. 1293-1295); and Kiseg5, Α. e! A1., T1e and 5e оГ ΑηбЫобс5 4 ' ¹ ' еб. I. V. Irrtsoi So. РЫ1абе1рЫа, ΡΑ (1987) pp. 1075-1117). Examples of sulfonamides applicable to the implementation of the claimed methods are, without limitation, sulfonamide, sulfacetamide, sulfapyridine, sulfathiazole, sulfadiazine, sulfamerazine, sulfasalazine, manefide, sulfamethoxazole, sulfamethoxypyridazin sulfulfazinazidazinazidazinazidazinazidazinazidazinazidazinazidazinazidazinazidazin and phthalisulfathiazole. Trimethoprim is applicable to the implementation of the claimed methods alone or in combination with any of the sulfonamides. There is reason to believe that sulfonamides and analogues of trimethoprim with chemical structures homologous to that of the sulfonamide compounds listed above will also be applicable in the implementation of the methods claimed in the invention.

The term nitroimidazole antibiotic means an antibiotic having a nitroimidazole core, as is understood from the prior art (Wao, 1.O.C. e! A1., Ιη: Miggau, R.K. e! A1., Eb5. Mapia1 oG C11ssa1 M1sgoOo1o§ y, Α8Μ Рge55, ^ а5Ыпд! оп, Ό.Ο (1995) p.1297); and Kiseg5, Α. e! a1., T1e and 5e Г Rn1 | b1o11s5 4 ' ¹ ' eb. L.V. Irrtso !! With. РЫ1абе1рЫа, ΡΑ (1987) pp. 1290-1343). Examples of nitroimidazole antibiotics applicable to the implementation of the claimed methods are, without limitation, metronidazole, tinidazole, nimorazole, ornidazole, carnidazole and secnidazole. There is reason to believe that nitroimidazoles with chemical structures homologous to those of the above nitroimidazole compounds will also be applicable in the implementation of the claimed invention.

The term chloramphenicol antibiotic means an antibiotic having a nitrobenzene ring as a structural core, as is understood from the prior art (Wao, I. Ό. C. e! A1., Ιη: Miggau, R.K. e! A1., Eb5. Mapia1 оГ С11П1с1 М1сгоОо1о§у, Α ^ Prge55, ^ a5b ^ ηдιоη, E.S. (1995) pp. 1296-1297); and Kiseg5, Α. e! a1., T1e and 5e Г Αη! ^ b ^ oί ^ C5 4 ' ¹ ' eb. I. V. Irtsob Co. PY1abe1pYa, ΡΑ (1987) pp. 757-807). Examples of chloramphenicol applicable to the implementation of the claimed methods are, without limitation, chloramphenicol and thiamphenicol. There is reason to believe that chloramphenicol with chemical structures homologous to that of the above chloramphenicol compounds will also be applicable in the implementation of the claimed invention.

The term rifampicin means an antibiotic having an anza- or macrocyclic structural core (ansamycin antibiotics), as is understood from the prior art (Wao, I. Ό. C. e! A1., Ιη: Miggau, R.K. e! A1., eb5. Magsha1 oG Syssha1 M1sgoyo1o§u, Α ^ Prge55, ^ a5b ^ ηдιоη, Ό.Ο (1995) pp. 1298); and Kiseg5, Α. e! A1., T1e and 5e Г Αη ^ ώ! ^ 4 ' ¹ ' eb. I. V. Irshsho !! With. РЫ1абе1рЫа, ΡΑ (1987) pp. 914-970). Examples of rifampicins applicable to the implementation of the claimed methods are, without limitation, rifampin, rifamycin 8U, rifamycin B (rifamide) and rifabutin. There is reason to believe that rifampicins with chemical structures homologous to those of the above rifampicin compounds will also be applicable in the implementation of the claimed invention.

The term nitrofuran means an antibiotic having a heterocyclic ring with a nitro group, as is understood from the prior art (Wao, 1E.C. e! A1., Ιη: Miggau, R.K. e! A1., Eb5. Masha1 oG C1 | ssa1 M1sgoOo1o §Y, Α8Μ Рge55, Ш5Ып§! Op, Ό.Ο (1995) pp. 1276-1289); and Kiseg5, Α. e! A1., T1e and 5e Г Αη ^ ώ! ^ 4 ' ¹ ' eb. TV.Yrrtsob Co. РЫ1абе1рЫа, ΡΑ (1987) pp. 1276-1289). Examples of nitrofurans applicable to the implementation of the claimed methods are, without limitation, nifuratel, nitrofurazon, furazolidone and nitrofurantoin. There is reason to believe that nitrofurans with chemical structures homologous to those of the nitrofuran compounds listed above will also be applicable in carrying out the methods claimed in the invention.

The term methenamine means an antibiotic having a Quaternary amine, as is understood from the prior art (Wao, 1.O.C. e! A1., Ιη: Miggau, R.K. e! A1., Eb5. Masha1 oG C1ipsa1 McgoLuodu, Α8Μ Prge55, ^ a5b ^ ηd! Oη, Ό.Ο (1995) p. 1299); and Kiseg5, Α. e! a1., T1e and 5e Г Αη! ^ b ^ oί ^ C5 4 ' ¹ ' eb. TV.Yrrshso !! With. РЫ1абе1рЫа, ΡΑ (1987) pp. 1344-1348). Examples of methenamines useful for practicing the claimed methods are, but are not limited to, methenamine, mandelate, methenaminagippurate. There is reason to believe that quaternary amines with chemical structures homologous to those of the above-mentioned methenamine compounds will also be applicable in the implementation of the methods claimed in the invention.

The term mupirocin (also known as pseudo-monic acid) means an antibiotic that has the unique essence of 9-hydroxynonanoylic acid, as is understood from the prior art (Wao, 1.I. e! A1., 1p: Miggau, R.V. e! a1., ebz Mapia1 oG CHssa1 M1sgOo1odu, A8M Proc, IZnp1iop, I.S. (1995) pp. 12991300); and Kisegs, A. e! A1., Thieu Ise OG ApNojsz 4 ^{! b} eb. L.V. Irrtsoy Co. RybabeRya, RA (1987) pp. 754-756). There is reason to believe that compounds with chemical structures homologous to that of the above mupirocin compound will also be applicable in the practice of the methods of the invention.

Therapy of patients infected with bacteria of the MusoBac! Egst! Ibci1oz1z complex (MTB) involves the use of a single drug or combination of drugs. Preferred primary drugs (first-line drugs) for treating TB include isoniazid (ΙΝΗ), rifampin (BMP), pyrazinamide (ΡΖΑ), streptomycin (8M) and ethambutol (EMB); and secondary drugs (second-line drugs) include ciprofloxacin, ofloxacin, ethionamide and cycloserine. Additional drugs that are currently being tested include amikacin, rifabutin, rifapentin, and sparfloxacin (1pbegNeb, S.V. e! A1., 1p: Miggau, R.V. e! A1., Ebz. , A8M Proc., Azimpnop, I.S. (1995) pp. 1385-1404). Any drug with activity against bacteria containing mycolic acid structures will be applicable in the practice of the methods of the invention.

Antimicrobial therapy for patients infected with bacteria of the MusoBac! Epit au1it complex (MAC) usually involves the use of a single drug or a combination of a limited number of drugs. First-line drugs for the treatment of MAC infections are azithromycin, clarithromycin and EMB; second-line drugs include amikacin, clofazamine, ciprofloxacin and rifabutin. Alternative medicines include cycloserine and 8M (1beg11eb, C. e. . (1995) pp. 1385-1404). All of these antibiotics are applicable in the implementation of the claimed invention.

Infections caused by other important mycobacterial pathogens, such as M. kappazi, are usually treated with a single drug or a combination of drugs used to treat TB or MAS infections, as discussed above. Infections caused by rapidly growing organisms (e.g. M. Goisch! Um and M. sye lopae) can be treated with amikacin or clarithromycin, as well as β-lactams and, in particular, cephalosporins (1beg11eb, C. B. e! A1., 1p: Miggau, R.V. e! A1., Ebz.Mapia1 oG CHshsa1 M1sgoYo1odu, A8M Proc. All of these antibiotics are applicable in the implementation of the claimed invention.

Betaine-like detergents and / or antibiotics useful in implementing the methods of the invention, in particular therapeutic, can be administered in any pharmacologically or pharmaceutically acceptable form, including, if necessary, a pharmaceutically acceptable salt or carrier. They can be administered in any form that has a prophylactic, palliative, preventive or therapeutic effect in microbial infections of humans or animals. Doses of antibiotics applicable to the implementation of the claimed methods are usually recommended by the manufacturer. These dosages can be found, for example, in the Doctor's Handbook (RVE), Meb1ca1 Esopotkz Sotrapu, Mop1ua1e, No. \ ν 1egzeu, I8A. Dosages for veterinary use can be found, for example, in Thieu Megg Utetag Mapia1, Megsk & Co., 1ps., Wai ^ ay, No. \ ν 1egzeu, I8A.

When an antibiotic (antibiotics) and a betaine-like detergent (detergents) are administered to the patient in the form of individual preparations, it is preferable that the concentrations of each agent be effective, i.e., coincide in time. That is, the microorganism at the site of infection in this patient should be exposed at the same time to the effective concentrations of both the antibiotic (s) and the betaine-like detergent (s), regardless of how these agents were individually administered to the patient. Therefore, for example, a patient may be given a joint administration of an antibiotic and a betaine-like detergent, for example, an oral antibiotic, and a betaine-like detergent by inhalation or intravenously. If the patient is prescribed the joint administration of an antibiotic (antibiotics) and a betaine-like detergent (detergents), then they can be used simultaneously in the same preparation, for example, in the form of an ointment or dressing that provides both antibiotic (antibiotics) and a betaine-like detergent (detergents).

In addition, the patient may be prescribed pretreatment with either an antibiotic or a betaine-like detergent in the absence of another component, as well as treatment (treatment) with an antibiotic or betaine-like detergent, using the component that was not present during the pretreatment, be it an antibiotic or betaine-like detergent, on the length of time for which the pre-treatment effect on the microorganism is not lost. Therefore, for example, a patient can be pretreated with a betaine-like detergent in order to disrupt the permeability and / or viability of the microorganism before antibiotic administration with or without a betaine-like detergent. Alternatively, the patient may be pretreated with an antibiotic prior to administration of a betaine-like detergent with or without antibiotic.

When mixed, each antibiotic (antibiotics) and betaine-like detergent (detergent) can be in solution, or each of them can be in solid form (in particular, in the form of a suspension). Alternatively, one or more antibiotics and / or one or more betaine-like detergents may be present in solid form in a solution of other antibiotics or detergents.

Examples of compositions according to the present invention intended for parenteral administration are sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of useful non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and organic esters for injection. Aqueous solvents include water, alcohol solutions in water, emulsions or suspensions, including sodium chloride solution, Ringer's dextrose solution, sodium chloride solution with dextrose, Ringer's solution containing lactose or stabilized oils. Examples of carriers for intravenous administration are liquid substituents, electrolyte substituents based on Ringer's solution with dextrose and the like.

Injectable preparations, such as oil solutions, suspensions or emulsions, may be prepared according to the prior art using suitable dispersing, moisturizing or suspending agents, as appropriate. When the active compounds are water-soluble, for example, exist as water-soluble salts, non-toxic parenteral diluents or solvents such as sterile non-pyrogenic water or 1,3 butanediol can be used to prepare sterile injectable preparations. Other acceptable carriers and solvents that may be used include 5% dextrose for injection, Ringer's solution for injection, and isotonic sodium chloride solution for injection (as described in υ8Ρ / ΝΕ - USP). When the active compounds are in an insoluble form, sterile oil suspensions are used containing acceptable lipophilic solvents or carriers such as fatty oils, for example, sesame oil or synthetic fatty acid esters, for example ethyl oleate or triglycerides. Alternatively, aqueous injectable suspensions may be used containing viscosifiers such as sodium carboxymethyl cellulose, sorbitol and / or dextran in combination with stabilizers.

Pharmaceutical preparations for oral administration can be obtained by combining the active compounds with solid excipients, by further granulating the resulting mixture. After adding acceptable additional components, the mixture or granules, if desired or necessary, can be coated and shaped into tablets or dragees.

Acceptable carriers are, in particular, excipients such as sugars, for example, lactose and sucrose, mannitol or sorbitol, cellulose preparations and / or calcium phosphates, for example tricalcium phosphate or calcium hydroxyphosphate, as well as binders made using starches and pastes, such as, for example, corn starch, wheat starch, rice starch or potato starch, gelatin, acacia, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone, and / or, optionally, disintegrir binding agents such as the starches mentioned above, carboxymethyl starches, crosslinked polyvinylpyrrolidone, agar or alginic acid or its salts, for example sodium alginate. Additional components include, in addition to the substances listed above, flow control agents and lubricants, for example, silicon, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate, with an appropriate coating that can be resistant to gastric juice, and for this purpose, among other things, concentrated sugar solutions may be introduced, which may additionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, varnish solutions and acceptable organic solvents or a mixture of solvents. In order to obtain a coating resistant to the action of gastric juice, solutions of appropriate cellulose preparations, such as cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate, are used. Paints or pigments may be added to the tablet or dragee coating, for example, for identification or to characterize various combinations of the active compound in dosage form.

Other pharmaceutical forms for oral administration include precast capsules made from gelatin, as well as soft, fused capsules made from gelatin with a plasticizer such as glycerol or sorbitol. Prefabricated capsules may contain the active compounds in the form of granules, mixed, for example, with excipients such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate, and, optionally, with stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in appropriate lipids, such as fatty acids, liquid paraffin or liquid polyethylene glycols; in this case, the addition of stabilizers is also possible.

Suppositories for rectal administration of the compounds of the present invention can be prepared by mixing the preparation with an appropriate suppository base, which includes non-irritating excipients such as coconut oil, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols, and especially bases that remain solid. at normal temperature, but become liquid at body temperature and therefore melt in the rectum, releasing the drug. In addition, it is possible to use gelatin rectal capsules consisting of a combination of an active compound and a base; Possible materials in this case include liquid triglycerides, polyethylene glycols or paraffin hydrocarbons.

Solid oral dosage forms include capsules, tablets, pills, troches, lozenges, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert excipient, such as sucrose, lactose or starch. Such dosage forms may be coated with an appropriate coating to control the release of active ingredients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert non-toxic solvents commonly used for such purposes, such as water and alcohol. Such compositions may also contain adjuvants, such as wetting agents, emulsifiers, suspending agents, sweetening agents, flavoring agents and perfumes.

The compositions of the present invention may be administered by micropumps or in the form of sustained release forms. Compositions of the present invention can be delivered to specific organs at high concentrations using appropriate catheters, or by introducing such molecules into the composition of chimeric molecules (or complexes) intended to target a specific organ.

The introduction in the form of sustained release forms is more convenient for the patient when he is shown repeated injections over a long time.

Betaine-like detergents or antibiotics used in the compositions and methods of the present invention may be administered in dosage forms such as tablets, capsules containing ointment powder, powders, or liquid solutions for oral administration if the biological activity of the material does not break down during digestion and if the characteristics of the compound make its absorption in the gastrointestinal tract possible.

The pharmaceutical compositions of the present invention can be prepared by a method known in the art, for example, by conventional mixing, granulation, dragee preparation, dissolution, lyophilization or similar processes.

The methods of the present invention, in particular a sensitivity test, are also conveniently implemented if the components used in such a test are supplied as a kit. Such a kit preferably contains appropriate buffers, salts, betaine-like detergents or a combination thereof, an antibiotic (s) or a combination thereof and, if necessary, water of an appropriate degree of purity. In a preferred embodiment, a collection of various betaine-like detergents and a collection of various antibiotics are provided. Betaine-like detergents and / or antibiotics in the collection may be those that are specifically selected for a particular microorganism, or the collection may be compiled without taking into account the specifics of a particular microorganism. Preferably, at least five different betaine-like detergents are supplied, although in a preferred embodiment of the invention a wider selection is provided, i.e. more than 5, for example, 10, 15, 20, 25 or 30, or any number within these limits. Preferably, at least five different antibiotics are supplied, although in a preferred embodiment of the invention a wider selection is provided, i.e. more than 5, for example, 10, 15, 20, 25 or 30, or any number within these limits. Specific kits, if desired, contain, inter alia, a specific mycobacterium, in particular MusobaNepit, for use as a standard. In such a kit, if the non-bacterial components have not yet been mixed, such components are in close proximity to each other, even in different containers or packages, and in close proximity to any microbiological samples included in the kit.

Pilot study of SV-18

The described invention is rooted in the observations made during the implementation of the invention TNopDop AO 95/27076 and I8A 5.658.749 (the method described in example 1). Example 2 shows the results of experiments with the described treatment procedure, showing that the sensitivity of liquid cultures is strongly influenced by the combination of CB18 with the RAITA antimicrobial additive enhanced with ceftazidime (sah) (Table 6). From the table. 6 it can be seen that the sensitivity of the IAIS / IAOI cultures in a liquid and dense medium was 98.4 and 75.4%. respectively, whereas the sensitivity in a liquid and dense medium of the same set of samples treated with SV-18 was 64.0 and 83.1%. respectively. In other words, the sensitivity in liquid culture with SV-18 was suppressed even when SV-18 provided a 46% increase in overall sensitivity (Table 3). More significant was the observation that the difference in sensitivity in liquid cultures of positive and negative samples was only 4% for IAIS / IAOI precipitation (i.e. 100% versus 95.8%). and the same comparison with respect to SV-1 8 showed a difference of 65% (i.e. 75.0% versus 45.4%). Since the difference in sensitivity on solid media for positive and negative samples treated in two different ways was comparable (34% versus 39% for IAIS / IAON and SV-18, respectively), it was initially assumed that the increase in RAITA by adding sah is completely responsible for the loss sensitivity in liquid cultures. Example 3 (Fig. 3A-3H), however, revealed that CB-1 8 has the ability not only to influence the growth characteristics of the tested isolate (571/573-BAB: see table 8). but that RAITA alone or in combination with sah also had the ability to influence the growth characteristics of this isolate. This effect, the so-called CB-18 effect, was synergistic and stepwise when CB-18 was added in combination with antibiotics.

RAITA contains polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin. The purpose of amphotericin B is to prevent contamination with fungi, and all other antibiotics have antibacterial activity. Polymyxin B is a polypeptide that interacts with phospholipids, leading to changes in permeability. Nalidixic acid is a quinolone that interferes with DNA synthesis (at the level of DNA gyrase). Trimethoprim is an analogue of pyrimidine, usually used in combination with sulfonamides, which also affects DNA synthesis (at the level of dehydrofolate reductase). Azlocillin is a penicillin antibiotic belonging to the family of β-lactam antibiotics and exerts its effect at the level of synthesis of cell wall structures. Nalidixic acid, Trimethoprim and azlocillin have been shown to be active against various types of mycobacteria. The fact that CB-1 8 can influence growth in combination with RAITA without sugars (Example 3. Fig. 3B and 3 H) shows that this phenomenon is not completely determined by the presence of ceftazidime, but is widely applicable to various classes of antibiotics (Kirg ) FIG. 28-30 illustrate the breadth of application of various classes of antibiotics. There is reason to believe that this phenomenon will be observed when using various classes of antibiotics.

Ceftazidime was initially introduced into the CB-18 / 12V / RAITA detection system to prevent contamination (Example 1). but not at the expense of the viability of mycobacteria. This conclusion was made after experiments to test the effect of various cephalosporins on the viability of mycobacteria. Proven combinations of ΡΑΝΤΑ-cephalosporin with the addition of ceftazidime include cefoperazone (sGy), cefotaxime (sG!) And ceftriaxone (sah) in various combinations (for example, sGy / si. SGy / sah and sGy / si / sah).

These experiments were carried out using CB-18 at a concentration of 7-15 μg / ml and various ATCC strains of M.! Berci1O818 (ATCC 27294). M.Ausht (ATCC 25291). M. Kaikakp (ATCC 12478). M. Goyiyit (ATCC 6841). M.hepor1 (ATCC 19250). M. Dogopyopa (ATCC 14470). The combination of SV-18 / 12V / RAITA / sah was the only composition that had a minimal effect on these isolates during preliminary studies. For example, all combinations of RAITA with sgr / sg !. cGy / sah and cGy / sH! / sah caused a noticeable growth retardation of all tested isolates. RAITA / sah caused a growth retardation of the tested isolate M. auscht and a noticeable growth retardation of the tested isolate M. dogyopae.

In a pilot study of SV-1 8 in the SV-18 / 12V / RAITA / sah system, 14 positive smear isolates were tested (Table 6). Analysis of these 14 positive isolates showed that 5 of them belonged to MTV, and the other 9 to different types of ILO, including 4 MAS. 2 M. Goyiyit and one by one M. s1e1opae. M.5hi1da1 and M. Kapkaky. The significant conclusion following from these results is that the SV-18 / 12V / RAITA / sah system affects a wide range of mycobacteria, both MTV and ILO.

The effect of SV-18 was observed in the absence of ceftazidime (example 3). those. other antibiotics (i.e., both cephalosporins, other RAITA components, and additional antibiotics) could cause growth retardation of various mycobacteria and the apparent sensitivity of the system to B-18 concentrations led to the formulation of experiments described in examples 5 and 6. The experiments of example 5 were directed for testing isolates M.1 and Lacti1.515, the M.Ausht complex and M. Goyiyit complex in relation to the concentration of CB-18, and Example 6 is devoted to determining the nature of the sensitivity of the isolates Mlbegi1ok1k. In the table. 7 summarizes the results of the experiments of example 5, in FIG. 10-15 show the results of experiments with various types of mycobacteria. In the table. 8 summarizes the results of the experiments of example 6, in FIG. Figures 17-27 present the results of experiments with the tested isolates M4iLegsiok1k1k. From FIG. 28-30 shows that this phenomenon is not limited to proven β-lactams. Despite the fact that in the experiments described in examples 5 and 6, there were significant differences between species and isolates, all tested species and isolates showed synergism of the action of CB-18 and antibiotics to one degree or another. Isolates M.1 and Lacti1ok1k turned out to be the most sensitive of all mycobacterial species tested, and for fast-growing species, they were characterized by less sensitivity, but the highest synergism when using CB-18 in combination with antibiotics. Isolates of the M. auscht complex showed an intermediate response. Basically, all isolates can be differentiated based on their growth characteristics in the presence of CB-18 and various antibiotics. Thus, the effect of CB-1 8 depends on the isolate and the antibiotic used, and the presence of CB-1 8 in the appropriate concentration is necessary for differentiation of the isolates.

This observation led to the conclusion that various betaines may provide a higher degree of sensitivity to differentiable isolates. In example 7, various detergents were used in combination with a reconstitution solution (B.E.), ΡΑΝΤΑ or ΡΑΝΤΑ / sah. Table 10 summarizes the results of these experiments and in FIG. 32B presents the results obtained with several different detergents (in the presence of only P-sax). Thus, the effect of CB1 8 depends on the dynamic interaction of the isolate, detergent and antibiotic, and it is in this interaction that the possibility of using the effect of CB-18 lies.

Antimicrobial treatment, drug resistance and permeability

Antibiotics vary with respect to their site of action. For example, Wao, Yu.S. e1 a1., 1p: Miggau, R.V. e1 a1., e <k. Mapia1 oG CHssa1 M1sgoL1o1odu, A8M Przekk, No. acNp1op, I.S. (1995) pp. 1281-1307) considers various classes of antibacterial agents and notes that the spectrum of action sites varies greatly from exposure or interference with various aspects of the synthesis and integrity of the cell wall and cell membrane to protein synthesis, nucleic acid synthesis (i.e., RNA precursors and DNA), DNA replication, and some drugs simply facilitate mutagenesis.

Bacteria are insensitive (i.e. resistant) to various antibiotics for a number of reasons. RshpyNash, V. e1 a1., 1p: Miggau, R.V. e1 a1., e <k. Mapia1 оГ С11шса1 М1стоОо1о§у, А8М Ргекк, №аКп§1оп, I.S. (1995) pp. 1308-1326) (reference is made by reference) considers some of these mechanisms. In general, an antibiotic must first enter the cell and then have an effect at the site of action. The basis of resistance can be: (a) the permeability of the body, (b) the molecular configuration of the antibiotic action site may not be suitable or this site may not exist at all in a particular clinical isolate, or (c) bacteria can modify, destroy or remove the antibiotic from the intracellular space. In the first case, if the bacterium is impervious to the drug, the antibiotic cannot reach the site of action and have an effect. In the second case, if the drug can penetrate the bacterium, but the site of the action (i.e., the three-dimensional structure of the target site) is such that the antibiotic cannot communicate, or the site of the action does not exist at all (i.e., the address structure or enzyme is not part of the expressed components), and therefore this drug does not affect the bacterium. In the latter case, the antibiotic can penetrate the cell, and its target exists, but the bacterium can effectively avoid the action of the drug by degrading the antibiotic, modifying it with reduced toxicity, or removing the antibiotic from the intracellular environment.

The treatment of mycobacterial infections is very limited (kirga), stronger than in the case of other microorganisms (1n <et11eB, S.V. e1 a1., 1n: Miggau, R.V. e1 a1., E <K. Mapia1 oG CHssa1 M1gb1i1odu, A8M Rgekk, No. acNp1op, I.S. (1995) pp. 1385-1404). The insensitivity of these microorganisms is partly due to the impenetrable nature of this class of bacteria: mycobacteria are 1,000 to 10,000 times less permeable than Exxepysosoi (LagIeg. V., e1 a1., Loyto.Vas !. 172: 1418-1423 (1990) and Iysso, N ., e1 a1., Century M1stoyu1. 142: 437-443 (1991)). Soppe11, Ν.Ό., e1 a1., 1n: Tybegi1ok1k. RashodepekE, Prgoesop ap <Soito1. V1oot, V.V. e <. A8M Rgekk, No. acNp1op, I.S. (1994) pp. 333-352 considers the characteristics of the permeability of mycobacteria and states that the low permeability of the cell wall of M. syeopen completely explains the level of resistance of this organism to cephalosporin.

Low permeability is partly responsible for the complex sensitivity characteristic of mycobacteria. For example, while MTV infections can be effectively treated with ΙΝΗ and / or P2A, MAC isolates are usually resistant to these drugs. In addition, M. [og1sh1it and M.cHeНe are isolated, are usually resistant to all antituberculous drugs of the anterior margin (1beg11eb, S.V. Sbssa1 MkGyuTsdu, A8M Rgehkh, \ ν; · ΐ51ιίη§1οη. E.S. (1995) pp. 133851404). Suηatοη, M.N. ί a1., Ιη: Aegis 8ihserb1111uu ίη 1Not Οκιηοΐίκηιρ'τ οΓ MusoBasse ^^ a1 1пГссίίοηχ, He1 £ her, b.V. fuck SNF Rgehh, Βοκίοη, MA (1991), pp. 147-159 discusses the treatment of infections caused by rapidly growing microorganisms in light of their varying sensitivity. For example, for each category of antibiotics considered (β-lactams, sulfonamides, macrolides, aminoglycosides, etc.), a sensitivity spread of several percent to 100% is described, and an interval of 30 to 60% is considered normal (depending on the drug and its concentration ) NebeK b.v. Ιη: Aegis 8ihserb1111u ίη Not Οκιηο11κηρ \ · οΓ MusoBasse ^^ a1 1n £ ssίίοηχ, Nebey, b.V. fuck SyaS Rgehkh, Βοκίοη, MA (1991), pp. 13-57 summarizes similar studies performed on MTV and MAS, discusses the diversity in sensitivity and notes that the nature of sensitivity, which is very diverse, is not very variable.

While permeability contributes significantly to the sensitivity of mycobacteria, these microorganisms have two additional mechanisms that play an important role in their resistance. First, MTV infections are caused by several subpopulations of cells. These subpopulations can be classified as (a) actively growing, (b) half-asleep due to the low pH of the macrophage, (c) half-asleep with sporadic bursts of metabolism, and (d) sleeping (Nebe ^, L.V. It’s not Sjötgörg οΓ Mutobas1epa1 Ιη ^ ύοηχ, NebeKb.V. eb.SYaS Rgehkh, Βο8ίοη, MA (1991), pp. 13-57). The sleeping state allows these subpopulations to survive during treatment. Another mechanism, apparently, is genetic instability: The formation of point mutations causes molecular variability in a potential target therapeutic agent. In general, the variability in the sensitivity of the character is a combination of these mechanisms, and that a mechanism is dominant depends on the species and even isolate a microorganism.

In general, the resistance mechanisms most significant for mycobacteria include permeability, regulation of proliferation (i.e., the sleeping state), and structural variability of the target site (s). Approaches aimed at overcoming resistance should use a modification of permeability, an understanding of the mechanisms of proliferation and the effect on them, or a modification of the chemical structure of an antibiotic so that it more closely matches the target site.

In the light of such prospects, five considerations presented in the examples should be considered. First, the mechanism of action of CB-1 8 is different from that for quaternary ammonium salts. This group of detergents has a general surface-active effect, leading to rupture of the cell membrane and denaturation of cellular proteins (Nide, V. B. Ιη: 8. C. I. Mönodgar ηο.19: 8gGase-Ac1bie Adehyh ίη Mckgoju. Γοη6οη 8ο ^ No Ιηbihbu, Γοη6οη (1964) pp. 69-82). In FIG. 7A-7C and 8A-8C, the action of TMA-18 and SV-18 is compared, which confirms that the effect of CB18 is different from the action of quaternary detergents. However, in high concentrations, betaines resemble Quaternary detergents and cause a general negative effect.

Secondly, there is no post-antibiotic effect (PAE). For example, a 90-minute treatment of SV-18 at a concentration of 383 μg / ml did not have a clear effect on growth characteristics (Figs. 3C and 36). If the effect of CB18 is specific, that is, it does not cause a general negative effect, but rather, its minimum concentration is required against the background of active metabolism, then PAE should be minimal. Isoniazid is an anti-tuberculosis agent that does not have RAE (Nebeck b.v. Ιη: Aegis 8i8ser (1LPIu ίη no Sétéίegaru οΓ Musobasse ^^ a1 &η & 6ίοηχ, Heb. S.E. 57).

The third consideration is due to the fact that, for some isolates, the SV-18 effect is observed in the presence of only reconstitution fluid (I.E.) (i.e., in the absence of antibiotics) (Fig. 10-15). The fact that this effect depends on the isolate (Figs. 17-27) suggests that there is a specific site of action for SV-18. In other words, the effect of CB-18 is a reflection of microheterogeneity in the nature of sensitivity among mycobacteria, in particular among complex mycobacteria.

Fourthly, the effect of CB-1 8 is different from the action of EDTA (Figs. 34-35). EDTA modifies permeability by extracting and chelating divalent metal cations that stabilize cell wall structures. YaahYudg Ν. eί a1., ΛηΙίιιιΚίΌό. Adehyh SNeito. 34: 759-764 (1990) report that growing in the presence of 1 mM EDTA (372.5 μg / ml) had such a devastating effect that the results could not be used to analyze the Χ / Υ coefficient used by these authors.

EDTA and SV-18 were used in the experiments described in Example 7 at a concentration of 17 μg / ml (approximately equimolar amounts) and it was shown that they behave differently in the claimed tests. In addition, Τχιιόοικ. K., ^ oi ^ .Pa ^ t. 8c ^. 50: 1051-1054 (1991) shows that there is practically no correlation (or it is very small) between the chelating activity of various phosphobetaines and antimicrobial activity. In addition, in table. 10 reviewed various betaines tested. Those carboxybetaines that contained an amidopropyl bond proved to be ineffective compared to carboxybetaines with the same bridge, but without an amidopropyl bond (compared with Khetain (He1ate СЬА) or Sherkotain (8сбегсо1аше / УОАВ) with DeTaine (ЕеТate РВ) and Velveex ( ) The amidopropyl bond should not interfere with chelation between charge centers, but should interfere with the CB-18 effect at the site of action, physiological in nature (i.e. enzyme).

Fifth, in Example 7 (FIG. 32B), Tween 80 was tested in this test: it did not have an effect on isolate 571/573-BAB at a concentration of 17 μg / ml (13 μM). This is a rather remarkable result, since several authors reported that the introduction of Tween 80 into the culture medium also induces sensitivity (Hw, 1., e! A1., Apbshkgo. Adebb Sbeto. 11: 773-779 (1977); Υato ^ ^, 8. e! A1., M1cgoOo1. 1tipo1. 35: 921-926 (1991)). The results presented by us suggest that the mechanism by which Tween 80 exerts its effect is different from that for SV-18. For example, at high concentrations of Tween 80 (i.e., 1% (10 mg / ml), the growth is really optimal and even a concentration of 10% did not exert an overwhelming effect (8bp§op, M. \ U. e! A1., At.Keu.Ke8r.P18.104: 717727 (1971)) In contrast, SV-18 at a concentration of 37 μg / ml did not affect growth, but at a concentration of 54 μg / ml completely inhibited the growth of ATCC isolates 27294 and 571/573 -BAB (Fig. 1 0A and 11A). The effect of CB-1 8 was observed in a narrow concentration range (approximately between 13 μg / ml and 27 μg / ml, depending on the isolate).

Finally, lecithin reversed the effect of CB-18. This fact contradicts the results of Waggu, S.E. e! A1., (υ.8. 5,610,198). Waggu, S.E. e! A1., (υ.8. 5, 610, 198) cite lecithin as an adjuvant in the implementation of its invention. According to the invention described herein, this phospholipid must act either by neutralizing CB-1 8 (similar to its electrostatic interaction with quaternary salts), or as a competitive inhibitor (interfering with CB-18 at the site of action). Since SV-1 8 has a total neutral charge, the latter hypothesis looks more plausible (i.e., stable electrostatic interaction should be minimal). If lipids such as lecithin act as competitive inhibitors, this may explain the absence of PAE.

If CB-1 8 is retained in lipid bodies, as suggested by Tbogyop \ UO 95/27076, then betaine-like detergents cannot act as surface-active agents. If mycobacteria are heterogeneous with respect to the site of the realization of the CB-18 effect, then different clinical isolates should behave differently in the presence of different betaine-like detergents (Example 7). In addition, if the change in permeability facilitates access of the therapeutic agent to target sites, and the population of the clinical isolate is heterogeneous with respect to the target site of the antibiotic, then variations at this level can also be expected (as can be seen from examples 5 and 6). Combinations of betaine-like detergents and various antibiotics can be expected to exhibit significant variability in behavior. Thus, a betaine sensitivity test can provide information on two different action sites.

The invention may be directed to one or more resistance mechanisms (i.e., permeability, dormancy, or molecular compatibility). Without attempting to limit ourselves to the hypothesis set forth, the following explanations are provided to illustrate the applicability of the invention. Growth retardation may be a manifestation of the stimulation of the mechanism responsible for the sleeping state. For example, since most antibiotics have a natural half-life, if the effect of CB-1 8 causes a decrease in the metabolism of mycobacteria until the toxic levels of the antibiotic decrease, then the effect of CB-18 can be used to determine the nature of the clinical isolate in relation to its proliferative characteristics. This information is useful to the clinician when choosing a drug and determining the duration of treatment.

Another mechanism of resistance is due to the permeability of mycobacteria. Without attempting to limit ourselves to the hypothesis set forth, the following explanations are provided to illustrate the applicability of the invention. Betaine-like detergents can alter the permeability of mycobacteria by modifying mycolic acid conformations. The end result will be induced antibiotic sensitivity. This information is useful to the clinician when choosing a drug for the most effective therapy.

The concept that the CB-1 8 effect may be the result of changes in the modification of mycolic acid is partially based on the work of Υιη-ιι-γΥ. e! A1., Proc. No. 111. Asab. 8s1. 93: 1282812833 (1996) and tested in detail in Example 9. In general, modifications of mycolic acid can be directly related to the fluidity of the cell wall. Certain modifications reduce cell wall fluidity, which correlates with reduced permeability. It is believed that the low permeability of mycobacteria plays a significant role in antimicrobial resistance (Wiap.U. e! A1., Prgos.Ma11. Lsab. 8s1.92: 6630-6634 (1995); Vgeppap, R.1. E! A1 ., Appy. Wew. Wuset. 64: 29-63 (1995)). Recall that Soppe11, N. Ό. e! A1., 1n: Thiegsi1o515. RaShodepezK, Rgo! Esbop apb Sop! Go1. V1oot, V.V. fuck L8M Rgezz, AazYpdFp, L.S. (1994) pp. 333-352 attribute the resistance of M. cep1opae to cephalosporins exclusively permeability.

Mycolic acid modifications occur through a series of 8-adenosyl methionine (8AM) dependent enzyme reactions (Fig. 39A). Waggu, S.E. e! A1., (I.8. 5,610,198) note that thiolated fatty acid derivatives (thietetracosanoic acids) can be used to inhibit these 8LM-dependent modifications of mycolic acids (Fig. 39B), i.e. for the treatment of diseases caused by pathogenic mycobacteria. The structure of the transition state of thietetracosanoic acids resembles a betaine-like detergent (Wiap, ¥. Е! А1., Proc. No.! 1. Asab. Δα. 93: 12828-12833 (1996)), in particular based on carboxybetaine sulfonium (Table 1 and FIG. 39B). Enzymatic catalysis is not required for the activation of sulfonium carboxybetaines and they will inhibit these 8AM-dependent enzymes.

Unlike betaine-like detergents, thietetracosanoic acids are extremely insoluble. In other words, the Kraft temperatures of the thietetracosanoic acids will be higher than physiologically normal (i.e. 37 ° C). Sulfoniumkarboksibetainy are significantly more soluble (even if B ₁ = 18-20, but especially for B _4> 3 (Table 1; aidYt, B., apdtshg 7:. 842-847 (1991)) Betainopodobnye structure should facilitate solubility problem and. , to some extent, the problem of administration, for example, thietetracosanoic acids are likely to remain in the solid phase upon intravenous administration, since betaines are more soluble, small doses intravenous will be simpler due to the lower Kraft temperature of the betaines.

Waggu, S.E. e! A1., (I.8. 5,610,198) indicate that thietetracosanoic acids will be specific for slowly growing pathogenic mycobacteria. Waggu, S.E. e! A1., (I.8. 5,610,198) also note that only those antibiotics that disrupt the synthesis of mycolic acid will act synergistically with these thiolated fatty acid derivatives. In the present description, it is stated that betaine-like detergents are applicable to a wide range of bacteria containing mycolic acid structures (i.e., corynebacteria, Sagb1a, as well as mycobacteria (Example 9)), and that a wide range of antibiotics can be used in accordance with by the methods claimed in the present invention (examples 5 and 6).

The synthesis of sulfonium carboxybetaine can be carried out according to the scheme shown in FIG. 39C, however, any method that leads to the same end result using methods known from the prior art is also acceptable (Mabs, Abuapseb Ogdashs SztGu. Weasbopz, Mesbtz apb 81gis! Ike5, Ebb Eb., 1bn Abeu & 8opz, No. \ vν Wagk, ΝΥ (1992) and Ezeg, e! A1., Weadepz ог og Ogdats ZupSezK yo. 1-16, 1oNp Abeu & 8opz, No. \ ν Υo ^ k, ΝΥ (1992) both works are referred to as references) .

Without striving to confine ourselves to the following explanations, we can conclude that the SV-18 effect is apparently a consequence of the interaction between the betaine-like detergent and the specific site of action. If this interaction modifies the permeability of bacteria containing mycolic acid structures, the end result will be an increase in the effective concentration of the antibiotic at the site of action. For example, with regard to the isolates used in the pilot study of CB-1 8 (Tables 7 and 8). If these mycobacteria exhibit heterogeneity both in relation to the target site (i.e., peptidoglycan synthesis) and in relation to the β-lactamase site, then an even greater degree of discrimination in the betaine sensitivity test can be expected. For example, a scenario is possible where a particular isolate must be sensitive to a given β-lactam, but due to the low permeability, this isolate can avoid the action of an antibiotic. If betaine-like detergents can increase the permeability of this isolate, β-lactamase may or may not have the ability to destroy the antibiotic before it exerts its effect. If β-lactamase of the test isolate is deficient in its ability to destroy a particular β-lactam structure, changes in permeability may be sufficient to exhibit a noticeable negative effect, observed as the effect of CB-18. There are reasons to believe that different bacteria containing mycolic acid structures have significant differences in terms of the site of action of the three different molecules discussed in this example (β-lactam, beta-like detergent and β-lactamase). Therefore, the greater the number of available antibiotics (especially β-lactams) used in combination with the large number of available betaine-like detergents, the higher the degree of discrimination.

The sensitivity test of the present invention opens up the possibility of using β-lactams as therapeutic adjuvants in anti-tuberculosis therapy, which is extremely important because the number of the most used and well-characterized antibiotics is much wider than just the class of β-lactams. Due to the wide spectrum of action of these antibiotics, the treatment of mycobacterial infections with these agents should have significant advantages. The use of β-lactams in therapeutic regimens aimed at treating mycobacterial infections has so far had limited success. For example, Syathega, N.R. e! A1., Liitkgo. Ldey S1eto. 39: 26202624 (1995) tested five MTV clinical isolates and several different classes of β-lactams. Most isolates were resistant to penicillins and most cephalosporins; however, imipenem (carbopenem) showed some activity. To achieve significant sensitivity, a combination of almost all β-lactams with a β-lactamase inhibitor is necessary.

As discussed above, β-lactamase inhibitors affect the resistance mechanism by which the body destroys the antibiotic. This is an example of antibiotic therapy aimed at the mechanism of resistance and therefore enhances any chemotherapy using β-lactams. The ability to expand the sensitivity of mycobacteria to antibiotics, in particular, β-lactam antibiotics, with the goal of stability mechanisms, is extremely important for the effective treatment of mycobacterial infections. In the described invention, molecules are used that affect the mechanism of resistance of bacteria in a way that has not been previously described. The analysis of isolates 512-1HN (Fig. 24) and 53 8-SHN (Fig. 25), as well as the understanding that CB-18 has a significant effect on the isolate subjected to the anti-tuberculin therapy ίη νίνο (Table 9), emphasizes the therapeutic applicability of betaine-like detergents.

While the invention has been fully described, it will be understood by a person skilled in the art that the invention can be practiced within wide and equivalent ranges of conditions, parameters, etc., without violating the idea or scope of the invention or any of its embodiments. All cited works are given as references.

Examples

Example 1

The described invention was largely the result of observations made during the development of new methods for processing clinical samples in order to detect mycobacteria. These procedures are based on ΤΗοπιΙοη methods. described in νθ 95/27076 and aimed at the primary methodology for the isolation of bacteria containing the structure of mycolic acid, in particular mycobacteria, from clinical samples, in particular from respiratory samples. Method T1yugp! Οη (νθ 95/27076) is described below, and the preparation of reagents for this procedure is described at the end of this example.

(1) Place 1-10 ml of sputum or bronchial flush in a 50 ml conical tube.

Note: while respiratory samples are the predominant type of samples that will be used in the procedure described, samples of a different type, such as water, soil, tissues, feces and others, can also be used. Some of these samples must be preliminarily clarified by suspension in water or buffer and passing the mixture through a column of δρίη-Χ II filled with a 2060 μm glass mixture (Sotd SoDag, Βοκΐοη, MA). Such a column may also contain a matrix, such as 8eryabeh® (8eryabeh 0-50®: Ryagtas, Ρίδsa1 atau, N1), or another equivalent resin to enhance cleaning. Any sample can be processed as described below.

(2) Add to the sample an equal volume of the fluidizing solution of 0.5% ΝΑΡί. ’(See below: 0.5% ^ ^^ mm sodium citrate) and mix.

(3) Incubate at room temperature for 10 minutes (mix after 5 minutes and before the next step).

(4) Open the tube and add sterile, filtered water to the sample to a final volume of about 35 ml (Note: use sterile filtered water (e.g. O1BCO / BBB, Sayekshd, ΜΌ, Ca !. # 15230022)).

(5) Add 4 ml of a concentrated solution containing a betaine-like detergent, buffer 10Χ SV-18, for example (see below): 10Χ SV-18 = 10 mM SV-18, 0.5M TP8-HC1, pH 8.0, 50 mM ΝΑ ^ and 1 mM No. C1.

(6) Mix the sample well.

(7) Incubate at 37 ° C for 90 min with shaking (140 rpm).

(8) Stir the sample and centrifuge at 4,000 x d 20 min at 30 ° C.

(9) Decant the supernatant and add 500 µl of sterile water or buffer to the sample.

(10) Fully resuspend the pellet and prepare a sample for bacteria detection by methods such as rapid acid staining (i.e., microscopy), cultivation, amplification of nucleic acids, or immunodiagnostics.

In preliminary experiments aimed at determining the nature of the contaminants growing on WASTES 12B liquid medium with the addition of ΡΑΝΤΑ, respiratory samples (n = 277) from Opey ΟίηηοδΙχδ - VaStoge were used. (Note: the WASTES 1 2B liquid culture system is one of the standard methods for culturing mycobacteria, and RAITA is an antimicrobial supplement containing polymyxin B, azlocillin, nalidixic acid, trimethoprim and amphotericin B: this culture system was designated 12B / RAIT (Beskop Skit , SosheyuShe, MO.)). A sample was taken and treated with CB-18 in accordance with the procedure described above. Approximately 400-500 μl of each pellet was seeded on 12V / RAIT. All contaminants were identified morphologically and by Gram stain, and then differentiated as positive or negative for oxidase / catalase. Then, the type of contaminants and their sensitivity to antibiotics were determined using the М1сго8сап® panels (Pube, Uek1 8asgateio, CA). The results are shown in table. 2.

table 2

Identification of contaminants in the WASTES 12V / RAIT system (n = 277)

Group # % G-negative 48 84.2% R rampositive 5 8.8% Yeast 3 5.3% Mushrooms one 1.8% Total: 57

From 40 patients: contamination = 14.4%

From the table. Figure 2 shows that out of 40 samples, 57 contaminants were isolated. The contamination rate per sample was 14.4% (40-277). These data give reason to believe that the presence of gram-negative organisms (> 84%) is the main problem when processing respiratory samples in accordance with \ ¥ О 95/27076 (i.e., SV-18). Approximately 31 (64%) of the 48 gram-negative isolates were Epilogacacepaseae. The most frequent isolates were Protebepsia aiayi (n = 13), species Rkebotopopak (n = 11) and Proteus tibaibi (n = 7). Therefore, a significant reduction in the frequency of contamination requires a significant suppression of gram-negative organisms. Sensitivity data show that 40 out of 48 gram-negative bacteria were sensitive to ceftazidime (at a final concentration of 8 μg / ml) when gram-negative organisms were inhibited by contamination (the 12B / PAIT system with the addition of ceftazidime (sah) is designated 12B / RAITA / sukh). Based on parallel experiments using several ATCC mycobacterial strains, it was shown that ceftazidime should reduce the frequency of gram-negative contamination without significantly affecting the viability of mycobacteria.

Preparation of 10X CB-18 buffer (1) 20X buffer salts: 1M Tris-HC1 pH 8.0.2mM NaCl

Tris base (121.14 g / mol) 54.27 g

Tris-HC1 (157.64 g / mol) 87.02 g

NaC1 0.117g

Add water to 1 liter (ί). Pour approximately 250 ml of water into the 1 liter measuring cylinder.

(ίί) Add Tris base (81dta, 8koshk, Mo, Sak #: Т 1503), Tris-НС1 (81dta, 8ΐ. oshkok, Mo, Sak #: Т3253) and NaS1 (81dta, 8k oshoshk, Mo, Sak # : 8 7653) and mix (note: use sterile filtered water (e.g. СВВСО / ВкЬ, Sayyegkigd, MO, Sak # 15230-022)).

(w) Bring the volume to 1 liter and make sure that the salts are completely dissolved.

(ίν) Check the pH in an aliquot. The pH should be 8.0 ± 0.2.

(ν) Sterilize by filtration (0.22 micron filter), divide into 50 ml aliquots and store at room temperature.

(2) 100X stock solution SV-18: 100mM SV-18 * SV-18 (383 g / mol) 1.915 g

Isopropanol: water (1 :) up to 50 ml * CB-18: X, X-dimethyl-Y- (p-octadecyl) -Y (3-carboxypropyl) ammonium, internal salt (CA8® Но.78195-27-4) .

(ί) Mix 25 ml of analytical grade isopropanol (Bax1eg, Msate Ragk, 1b, Sac #: 3043-1 ΝΥ) with 25 ml of water in a graduated cylinder and get 50 ml of a 1: 1 mixture of isopropanol: water (note: use sterile filtered water (e.g. C1BCO / ON, Sayyegkigd, MP, Sak # 15230022)).

(ίί) Transfer 25 ml of isopropanol: water (1: 1) to a 50 ml conical tube.

(ΐϊϊ) Weigh 1.915 g of SV-18 and transfer to the graduated cylinder with the remaining 25 ml of isopropanol: water (1: 1). Shuffle by rotation.

(ίν) Add more isopropanol: water mixtures (1: 1), up to about 40 ml and mix gently by rotation. Let the solution stand for about 20 minutes, stirring gently every 5 minutes.

(ν) When SV-18 dissolves (after approximately 30 minutes), bring the final volume to 50 ml with isopropanol: water (1: 1) and mix by inverting the cylinder.

(νί) Pour the solution into two 50 ml sterile plastic conical tubes and store at room temperature.

(3) Buffer 10X CB-18 (ί) Determine the number of samples to be tested and enter this number in the table below (add one to this number to have sufficient buffer volume), calculate the final amount of each component needed for application the supply of an appropriate amount of 10X SV-18 as described below.

Component Multiplication factor After actual amount 20X buffer salts 2ml x Quantity = 100HSV-18 400 μl x samples = AA1.C (163.2 g / mol) 0.033 g x plus = Add water to: 4ml x one =

(ίί) Immediately before use, mix 20X buffer salts, HABS (P1ika, ^ n ^ pksta, ΝΥ, Ca!. #: 01039), 100X CB-18 and bring the volume with sterile filtered water (note: use sterile filtered water (e.g. , C1BCO / ON, Sayyegkigd, MO, Ca!. # 15230-022)).

Note: If a precipitate has formed in the buffer at any time during the procedure, do not use this buffer. The solution during storage and use should be warm (i.e. above 20 ° C). Do not store this solution in the refrigerator.

Preparation of a fluidizing solution of 0.5% NА ^ С (1) 10X stock solution of Να-citrate: 0.25 mM sodium citrate dihydrate.

Trisodium citrate dihydrate (294.1 g / mol) 7.35 g

Add water to: 100 ml (ί) Pour about 25 ml of water into a 100 ml graduated cylinder (note: use sterile filtered water (e.g. C1BCO / ON, Sayyegkigd, MO, Ca! .15230-022)).

(ίί) Add trisodium citrate dihydrate (81dta, 8 !. Εοω ^ MO, Ca!. #% C-3434) and mix by rotation. Add the remaining water to 10 ml and mix by turning.

(w) Filter sterilize (0.22 micron filter), divide into 50 ml aliquots in conical tubes and store at room temperature.

(2) A fluidizing solution of 0.5% NА ^ С (daily prepared anew).

(ί) Determine the approximate volume of the required fluidizing solution ΝΛΕΟ (ίί) Mix 10X stock solution of Νι citrate and NABS (Р1ика, ^ п ^ пкста, ΝΥ, Са!. #: 01039) in a 50 ml conical tube or graduated cylinder and bring the final volume with water (note: use sterile filtered water (e.g. C1BCO / ON, Sayyegkigd, MO, Ca!. # 15230022)).

(w) Use immediately and discard unused solution.

Approximate number of samples

3-10 7-25 12-37 15-50

10X stock solution

Να-citrate \ A1. C (163.2 g / mol) Add water to:

2.5ml 5 ml 7.5ml 10 ml 0.12 g 0.25 g 0.38 g 0.5 g 25 ml 50ml 75 ml 100ml

Preparation of stock solution of ceftazidime and PB / RAJAH / sah (1) Stock solution of ceftazidime (36 mg / ml).

Ceftazidime 72mg

1M Να-bicarbonate 85.6 μl

Add water to 2ml (ί) In a 2 ml volumetric flask, mix 1 ml of water and 1M Να-bicarbonate (81dta, 8 !. Εοω ^ MO, Ca!. #: 8 6297) (dissolve 8.40 g of Να-bicarbonate in 100 ml water, sterilized by filtration and stored frozen in 10 ml and 1 ml aliquots) (note: use sterile filtered water (e.g. C1BCO / BPA Sayyegkigd, MO, Sa!. # 15230-022)).

(ίί) Add ceftazidime (81dta, 8 !. Εοω ^ MO, Ca!. #: C 3809) and immediately bring the volume with water to 2 ml.

(w) Gently mix by turning until the solution is clear. Do not heat the solution above room temperature (i.e. do not warm the solution in your hands).

(ίν) Immediately divide into 50 μl servings in 1.5 ml microcentrifuge tubes and store at -70 ° C until use.

(2) Strengthening and application of ΡΑΝΤΑΑαζ.

(ί) Remove lyophilized RAITA and reconstitution fluid (KB.) from the refrigerator, and one 50 μl aliquot of the ceftazidime stock solution (36 mg / ml) from the freezer.

(ίί) When ceftazidime is thawing, add 1 ml of K.R. using a 5 ml syringe. to the stock of ceftazidime. Shuffle the kit into the syringe once. Using a syringe, transfer the entire solution to the vial with RAETA.

(w) Add another 4 ml K.R. in a bottle with RAETA (final volume = 5 ml). Designate PANTA / saζ.

(ίν) Add 100 µl of RAITA / sah to each 12V vial prior to use (antibiotic addition should take place within 2 hours of adding the sample).

(ν) Store unused solution at -20 ° C for not more than 48 hours (i.e. do not freeze and thaw more than 1 time).

Example 2. Pilot study of SV-18.

CB-18 was used in the treatment of respiratory samples to detect mycobacteria (acidic fast-growing bacilli, ac1D Cancer! Bac1Sh: ARV) in order to evaluate the methods of ΤΙιοπιΙοπ XV O 95/27076. Respiratory samples (n = 573) were collected in the TV lab Quest Diagnostics-Baltimore (VAB), Quest Diagnostics-Teterboro (TCE), Washington, D.S.

Laboratory Bureau (BOC), John Hopkins Hospital (GNN), and the University of Maryland at Baltimore (IIW). In the laboratories where the sampling was carried out, each sample was divided into two portions and one portion was treated on the spot using the standard IAbC / IAon method (Kep !. RTT e! A1 .. RbNs Nea1! 1 MusoBas! Epood. W A. Sshye Gog Do not bite 1 III baoga! V. 8. Bereptep! OG Hea1! 1 apy Nitap 8egu1se. Sep! Egk Gog b | keake Sop1go1. (1985) pp.31-46) and inoculated into a liquid medium (WASTES 12B or BBB M61T ) and on a solid medium (7H11, L-1 or Septicheck (8percSec)), and another portion of each sample was sent to the Quest Diagnostics Baltimore laboratory, where the samples were treated daily with SV-18, as described in Example 1. O the treated SV-18 sediments were seeded in 12V / RAITA / sah and on 7H11-selective jambs as described above. All smear analyzes were carried out in accordance with the recommended procedures (Kep !. R.T. e! A1. RiBnS NeaI MusObas! Epoodu. W A Szhye Gog Ne Bieu1 III Baaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. 8egu1se. Sep! Egk Gog O1keake Sop! Go1. (1985) pp. 57-69). The results of this study are designated as Pilot Study SV-18.

Of the 573 processed samples, 106 were ΑΓΒ-positive by all methods (i.e., using IAon or CB-18, in liquid or solid cultures). There were 8 M.dogyopae isolates. They turned out to be inconsistent, negative in smears and were distributed evenly between the two methods (i.e., 4 isolates recognized by the IAON method were not recognized when processing SV-18, and vice versa). Since M.dogyopae isolates are considered contaminants of limited clinical value (Aaupe. L. S. e! A1 .. Cl. MugoGyuGweu. 5: 1-25 (1992)). they were omitted in further analysis: i.e. 98 AGV-positive samples were analyzed. In the table. 3 summarizes the results obtained in cultures excluding M.dogyopae isolates.

There were 52 concordant positive samples, 467 concordant negative samples and 46 discordant ones. JaON identified a total of 61 positive samples with a sensitivity of 62.2%. SV-18 identified a total of 89 positive samples with a sensitivity of 90.8%. SV-18 increased the total sensitivity in cultures by approximately 40%.

Table 3

The total results of sensitivity in cultures in the Pilot study of SV-18 IAIS / IAON p = 573 + + 52 37 90.8%

SV-18 - 9,467

62.2% Total AGV = 98

Of the 98 culture-positive samples, 69 contained mycobacteria other than tuberculosis (MOTT), and 29 MTV. JaON identified 33 of the ILOT positive samples, but only 25 MTV samples. Sensitivity in cultures for samples treated with IAOH. accounted for 47.8% and 96.6% for ILO and MTV, respectively. Sensitivity in cultures for samples treated with CB-18. amounted to 92.8% and 86.2% for ILO and MTB, respectively. CB-1 8 increased the sensitivity in cultures for ILO by 94.1%. but reduced the sensitivity in cultures for MTV by 12.1%.

In the table. 4 shows the results of the analysis of smears. Of the 61 culturally positive samples, all 61 gave positive smears with any treatment method. 18 samples gave the same values for both processing methods, and 19 samples for both processing methods gave positive smears, but the value obtained with SV-18 was higher. There were no cases where higher values were obtained among positive smears during the treatment of IAOH. There were 23 cases when, when processing SV-18, the smear was considered positive, and when processing IaON, it was considered negative. There was only 1 case when the smear was considered positive during the treatment of IAOH, and negative when treated with CB-1 8. IaON identified 38 smears from 98. positive by culture with a sensitivity of 38.8%. and SV-18 identified 60 smears with a sensitivity of 61.2%. From the table. Figure 4 also shows that an increase in the sensitivity of smears is a consequence of an increase in the detection of both tuberculous mycobacteria (MTB) and mycobacteria other than tuberculous (MOTT).

Table 4

Smear analysis in a pilot study of SV-18

Total ILO MTV Identical smears eighteen 8 10 Smear performance SV-18> nineteen nine 10 smears of IAon JaON Smear Indicators> 0 0 0 smear indicators SV-18 Smears of SV-18 f and smears of IAON Θ 23 sixteen 7 Smears of IAON f and smears of SV-18 Θ one one 0 General f culture and f smears 61 34 27 General f agv culture in experience 98 69 29th Smear sensitivity 38.8% 26.1% 69.0% Sensitivity of smears SV-18 61.2% 42.8% 93.1%

While an increase in the sensitivity of smears with SV-18 was noted, the specificity of the smear analysis was 99.8% and 96.8% for IAIS / IAon and SV-18, respectively. There were a large number of samples with SV-18. which were evaluated as a ± smear, and for which there were no confirmatory cultural results with SV-18. According to the recommendations of the SES (Kep !. RT.E! A1 .. RiNs Nea1! 1 Musoobas! Epo1odu. 1n And Szhye Gog! 1e Lebee1 III Leboga! Oh. And. 8.

Oeraytip! OG Nea1! ny apb Nitap 8eguye, Sep! kogz Ezease Sopio1, (1985) pp. 57-69), laboratories should consider such ± smears doubtful in the absence of confirming cultural results.

When processing samples of SV-1 8, an extremely high number of ± smears was noted. Analysis independently in each way instead of taking into account the results obtained using alternative processing methods, and taking into account the results of these ± smears, gave a completely different picture. For example, the data table. 5 show that 37 of the 61 samples that were positive in culture when treated with LiAc / MaOH also gave positive smears when treated with LiAc / MaOH. Of 89 samples treated with CB-18 and positive in culture, 56 also gave positive smears when processing CB18.

The sensitivity and specificity of smear analysis using LABC / MaOH were 60.6% and 99.6%, respectively, and in the case of SV-18, 62.9% and 91.2%, respectively.

Of the 31 ± smears (5 + 26 = 31) obtained with SV-18, for approximately 42% (n = 13) there were confirmatory cultural results obtained in another way or obtained on other samples, or this result could be associated with previous disease, or there was a positive result for DNA amplification (i.e., confirming the presence of mycobacterial DNA). (Note: according to the recommendations of SES, 16 smears with a value of 1 + and 2+ in the case of culture-negative treated CB-1 samples of 8 samples were considered smear-positive in the analysis shown in table 4 - hence the initial decrease in specificity (99.8% versus 96 , 8% (Table 4).) In general, the specificity of the smears was 99.6% and 91.2% for LiAb / MaOH and CB18, respectively, in accordance with the analysis presented in Table 5, although CB-18 provided a total sensitivity increase in cultures (tab. 3).

Presented in the table. 5 results show that the treatment in SV-1 8 did not have an effect on the smear procedure itself. For example, SV-18 did not alter the stickiness of the bacterium with respect to its contact with glass. This is based on the observation that SV-18 increased the sensitivity of smears in relation to culture-positive samples by 58% compared to the treatment with LAB / MaOH (Table 4); however, each method remained approximately equivalent with respect to culture-positive samples tested by smears using this method (Table 5: 60.6% versus 62.9%). In other words, cultivation remains a more sensitive diagnostic method, but CB-18 appears to increase the overall sensitivity of both methods equally.

Table 5

Independent evaluation when processing in two different ways

Smear rate The denial LAGS / LaON SV-18 ARV® 24 ARVO 502 ARV® 33 ARVO 434 full ± 3 0 5 26 1 + 7 one 8 14 2+ 8 one 6 2 3+ 5 0 7 0 4+ 14 0 thirty 0 TOTAL 61 504 89 476 Smear: Feel- Specific Feel- Specific bodyness nost bodyness nost 60.6% 99.6% 62.9% 91.2%

In general, the numbers of culture-negative and smear-positive samples (i.e., smears + and smears ±) identified by each method differed sharply: only 2 smears positive and culture-negative were observed with LABC / MaOH, and with SV-1 8 42 smear positives (including SV-18 samples giving ± smears, but negative during cultivation: Table 5). The question of the significance of these ± smears (i.e., the loss of specificity of smears) is important. In particular, if the above conclusion is true (i.e., SV-18 increases the overall sensitivity of both detection methods equally), then SV-1 8 reduces the specificity of the smear analysis. This fact may be significant for a laboratory researcher. If the conclusion is incorrect and the results of the smear analysis are significant (i.e., these patients can actually be infected with mycobacteria), then the sensitivity of the smear analysis with SV-18 may be higher than shown in the table. 4 and 5. This final conclusion may be important to the doctor. In order to explain these results, an analysis was carried out, the results of which are shown in table. 6.

The sensitivity of liquid and dense cultures was independently verified using two different processing methods used in the pilot study of CB-1-8 (Table 6). For each processing method (ΝηΟΝ versus CB18), the number of ARVs of culture-positive samples isolated by this culture method was analyzed. Of the 61 samples treated with ΝηΟΝ, 45 were positive in liquid and dense cultures, 15 were positive only in liquid and 1 was positive only in dense culture. Of the 89 treated SV-1, 8 culture-positive samples, 42 were positive in liquid and dense cultures, 15 were positive only in liquid and 32 were positive only in dense cultures. The sensitivity of liquid cultures when processing ΝηΟΝ and SV-18 was 98.4% and 64.0%, respectively. The sensitivity of dense crops when processing ΝηΟΝ and

SV-18 was 75.4% and 83.1%, respectively.

The expected result was obtained for samples treated with ΝηΟΝ: liquid cultures were approximately 31% more sensitive than cultures on solid media. In contrast, when the sample was treated with CB18 (as described in Example 1) and seeded on 12B / RAITA / sah and 7H11-selective jambs, cultures on solid media were approximately 30% more sensitive.

Table 6

Comparison of liquid and solid cultures. Result in culture. Sensitivity.

Liquid and dense Only liquid Only dense Total ARV Liquid Dense Yaon Smear® 31 6 0 27 one hundred% 83.8% Smear® 14 nine one 24 95.8% 62.5% Total 45 fifteen one 61 98.4% 75.4% SV-18 Smear® 38 4 14 56 75.0% 92.9% Smear® 4 eleven eighteen 33 45.4% 66.7% Total 42 fifteen 32 89 64.0% 83.1%

In the results presented, an important and unexpected dichotomy is noted. For example, despite the fact that the total sensitivity in cultures increased by approximately 46% (Table 3), this increase was due solely to an increase in the release of MOTT (i.e., there was a decrease in sensitivity in cultures with respect to MTB). The sharp opposite was the sharp increase in the overall sensitivity of smears, while this contribution was due equally to positive samples of MOTT and MTV (Table 4).

T1yugp1op XVO 95/27076 suggested that the increased sensitivities are due to (1) a decrease in the negative impact during processing compared to NaOH treatment, (ίί) increased separation efficiency during centrifugation as a result of compensation for the internal buoyancy of organisms and (w) increased detection probability due to dispersion organisms forming strands. Despite the fact that increased cultural sensitivity may be the result of a combination of all three of these aspects, increased sensitivity of smears is caused only by dispersion or increased detection (viability does not affect the smear). Since ILOT organisms usually do not form strands to the same extent as MTV bacteria, increased smear sensitivity is most likely due to increased detection. Alternatively, vitality should be an essential point in cultural sensitivity. The dichotomy is rooted in the fact that smear results indicate an increased detection of both MOTT organisms and MTV organisms, and cultural data indicate an increased viability of ILO organisms, but reduced viability of MTV organisms.

The inverse sensitivity in liquid and dense cultures for the treated SV-18 samples (Table 6) suggests that the dichotomy is a consequence of the combination of CB18 with antibiotics in the addition of RAIT / ceftazidime. For example, a comparison of the sensitivity in dense cultures of smear-positive and smear-negative samples treated in two different ways showed significant similarities. The sensitivity in dense cultures for smear-positive samples was 34% higher than for smear-negative samples treated with LABC / LNAO, and 39% higher with processing SV-18. The same comparison of data for liquid cultures showed an insignificant difference in sensitivity between smear-positive and smear-negative samples when processing LABC / LnOH (i.e. a difference of 4%), but a significant difference between the same groups when processing CB-18 ( i.e. 65% difference). The 75% sensitivity in cultures among smear-positive samples treated with SV-18 was unexpected and extremely unusual (these 14 samples were: 5-MTB and 9MOTT: 4 MAC2-M.Goish1it and one each M.c1e1opae, M. 8hi1da1 and M. Kapkaki). Not surprising was the 100% sensitivity in liquid cultures of smear-positive samples treated with LiAc / NaaOH. §! Ope, V.B. e1 a1., 1ig.S1sh-M1sgo. 35: 1030-1031 (1997) report that the sensitivity in liquid cultures of smear-positive samples (treated with LABC / LNOH) was also 99.3% (this was a much broader study, n = 439).

Two conclusions follow from the data presented. Firstly, the SV-18 / 12V / RAITA / sah system affects a wide range of mycobacteria. For example, while the 5 missing MTV isolates were of high social significance, the MTV isolates made up only 36% (514) smear-positive culture-negative samples and 30% (29-98) of all ARV isolates used in this study. Similarly, ILOT isolates accounted for 64% (9-14) of smear-positive culture-negative samples and 70% (69-98) of all ARV isolates used in this study. Since the ratio of isolates was the same, this phenomenon can be widely used: the destructive combination of SV-18 and antibiotics similarly affects MTV and ILO.

Secondly, the contribution of the SV-18 / 12V / RAIT / sah system may depend on the inoculum. For example, similarly to the results of Epd, K.K., e1 a1., ApNt1go, Ad.C NetoFeg. 26: 42-47 (1984) for Rkeyiyotopak aegidtok, the minimum inhibitory concentration (M1C) of ceftazidime increased with increasing inoculum. Since the smear directly reflects the number of organisms per unit volume, the smear-negative samples will be more susceptible to the action of the CB-18 / 12V / RAKHTA / sah system - hence the 65% difference in the sensitivity of liquid cultures of smear-positive and smear-negative samples.

In this study, CB-1 8 was used at a concentration of 1 mM (383 μg / ml). The concentration of CB-18 in samples seeded on WASTES 12B probably varied from 35 μg / ml to 7 μg / ml depending on the initial consistency of the sample, the ability of CB-1 8 to liquefy the sample, and the amount of residual wash-off remaining after decanting (i.e. e., the original processing fluid). In the worst case (i.e., dense, heavy sputum), the sample did not liquefy too well and a large volume of the initial treatment fluid remained. Under such conditions, the sample must be seeded in the solution for processing. The addition of 400-500 μl of the pellet to a WASTES 12B vial with a final volume of a 4.5 ml gave a dilution of a 11-fold (i.e. 35 μg / ml). In the best case (i.e., a light bronchial washout), about 100 μl of the treatment fluid remained. The pellet was resuspended in 500 μl of water, obtaining a 5-fold dilution. The concentration in such samples should be about 7 μg / ml. While the concentration of CB-1 8 is not known for each specific sample, it seems reasonable to assume that CB-1 8 is homogeneously dissolved in the processing medium, and since it was not washed, the concentration range for each sample is within 7- 35 mcg / ml.

Dense medium is very different from liquid. Firstly, a significantly smaller volume (and 100 μl) is added to the jamb. The smaller volume of the inoculum partially explains the difference in the sensitivity of the culture systems. Secondly, any volume of SV-18 added to the jamb will be minimized due to capillarity: the detergent will be adsorbed by the medium without coming into contact with bacteria, which minimizes its effect.

Summarizing, we can conclude that antibiotics and SV-18 have a synergistic negative effect, and this effect is widely manifested in all mycobacteria.

Example 3. Processing in SV-18 against seeding in SV-18.

To broaden the understanding of the dynamic interactions of CB-18 in the 12B / PACTA / sah culture system, the experiments shown in FIG. 1A and 1B (note: FIGS. 1A and 1B represent the same experience, two images are due to the complexity of the experimental design). Two different isolates of M.! Bersi1O818 were used: ATCC 27294 (ATCC, KoskuShe, ME) and the clinical isolate 571/573-BAB used in the pilot study of CB-18 (note: 571/573-BAB represents one of the two isolates, both isolates were obtained from the same patient, but in the form of two independent analyzes (ie 571-BAB and 573-BAB (see tab. 8 and compare Figs. 18 and 19) taken on the same day. two isolates behaved identically in every sense and were used interchangeably in the experiments described in the present and subsequent examples).

Each isolate was cultivated either on the Lovenshtein-Jensen jambs (LL) or on the jambs of the 7H11 selective medium (7H11-selective jambs were strengthened by the addition of polymyxin B, carbenicillin, amphotericin B and trimethoprim (Beskonon ^^ sk ^ η8οη, Sosukeu). Cells were scraped off from the jambs and transferred to a buffer or buffer containing 1 mM CB-1 8 (as described in Example 1), and then diluted 1,000 times with the same buffer (Figs. 1A and 1B). Then, each bacterial stock was incubated for 90 min at 37 ° C with shaking (140 rpm) before further dilutions. Then, each bacterial stock was again diluted with buffer or 0.5 mM CB-18 in order to obtain final dilutions of 25,000X, 50,000X and

100,000X (in relation to the initial drain (Fig. 1A and 1B)). Each dilution was seeded in parallel (400 μl each) in vials with WASTES 12B, with the addition of RAIT or RAIT reinforced with sah, so that the final concentration of sah was 8 μg / ml (P / sah). The concentration of CB-1 8 in these experiments was approximately 17 μg / ml (as discussed in Example 2, it ranged from 7 to 35 μg / ml in most vials). Vials were periodically checked and growth indices recorded. Growth indices for all six bottles of each series were averaged and a graph was plotted against the timing of cultivation. The results for the ATCC 27294 isolate are shown in FIG. 2A-2H, and the results for isolate 571/573-BAB are shown in FIG. 3A-3H.

The ATCC 27294 isolate was generally not affected by cultivation conditions (L vs. 7H11), antibiotics (RAITA vs. RAIT Asah), nor the treatment solution (buffer against CB18), nor the solution for inoculation (buffer against CB-18) did not significantly impact on the growth characteristics of the isolate ATCC 27294 (Fig. 2A-2H).

Alternatively, isolate 571/573-BAB was influenced by almost all components of the experimental design. For example, the behavior of isolate 571/573-BAB depended not only on how it was prepared for the experiment (LL versus 7H11 selective medium), but also on the presence of ceftazidime and the composition of the solution for inoculation (i.e. buffer against SV-18). In fact, the most significant parameter seems to be the presence of CB-1 8 in the inoculum solution (compare Fig. 3A with 3B; 3C with 3Ό; 3E with 3E; and 36 with 3 N). Cells that were treated in buffer and then seeded in SV-18 did not come into contact with SV-18 before seeding. From FIG. 3E shows that the isolate seeded in the presence of P / sah. not growing up. In fact, growth was observed in only 3 of the 12 vials used to obtain growth curves in the presence of P / sax.

Apparently, the treatment makes a small contribution to the growth characteristics. For example, the experiment was designed so that the cells treated in CB-18 were diluted to obtain a final concentration of CB-18 in a culture vial, ranging from 0.68 μg / ml (@ 25,000X), 0.34 μg / ml (@ 50,000 X) up to 0.17 μg / ml (@ 100,000X). There were no significant differences in growth characteristics between these dilutions. Comparison of FIG. 3A and 3C of FIG. 3E and 36 shows that cells can be treated in CB-18, but if the concentration of CB-18 in the culture bottle remains lower, for example, 1 μg / ml, then this does not affect growth. In other words, the post-antibiotic effect does not occur in the case of CB-1 8. NebeK b.v. 1p: Aegis 8ikserbyb 1p! Be off! Begaru OG Musobas1epa 1nGesbopk, Not! Ge1k, b.V. fuck SHS Rgekk, VokYup, MA (1991), pp. 13-57.

In general, the above results, in combination with the data from a pilot study of SV-18, show that different isolates behave differently and the way of handling it contributes to the sensitivity of this isolate (for example, previous contact with antibiotics: cells were cultured on a 7H11 selective medium (Fig. 3E-3H)). However, it is more significant that the presence of betaine (i.e. CB18) in the culture medium significantly increased the sensitivity of these isolates to antibiotics also present in the culture medium.

Example 4. The addition of lecithin overcomes the effect of SV-18: verification of small inoculums and comparison with Quaternary ammonium salts.

The experiment described in example 3 (Fig. 1A and 1B) shows that the use of CB-1 8 as a primary diagnostic tool requires further refinement. For example, is it necessary to remove CB-1 8 from the solution for inoculation, is it worth using less toxic betaine, or should the action of CB-1 8 be neutralized. These considerations led to the hypothesis that the action of CB-1 8 can be neutralized by lecithin, similarly to what occurs in the case of quaternary ammonium salts (e.g. 2erbgap® or benzalkonium chloride) described by Rabegkop, V.A. ATEG. 1st game Ribes Nea1! B 46: 1429-1430 (1956) and а aupe, b. 6. Ateg.Veu.Vekr.O1.80: 912-913 (1959).

It is believed that Quaternary ammonium salts have a general negative effect on bacterial and, in particular, mycobacterial organisms. Nido, XV.V. 1p: 8.C.1. Mopodgarb by. 19: 8gasse-Asbue Abep! To t M1sgoyoodod. Lopbop 8os.Sbet. 1pbikbu, Lopbop (1965) pp. 69-82 reviews the literature and summarizes the action of quaternary ammonium salts as causing cell membrane rupture and total denaturation (i.e. inactivation) of intracellular proteins.

Rabegkop, V.A. 1st game Rebbs Neabb 46: 1429-1430 (1956) and No. Aupe, b.6. ATEG. Wow. Vekr. Όίκ. 80: 912-913 (1959) showed that the negative effect of quaternary ammonium salts in mycobacterial culture can be overcome by lecithin (phosphatidylcholine, phospholipid). In FIG. Figure 4 shows an experimental design designed to test the hypothesis that lecithin can overcome the effects of CB-1 8 with respect to increasing the sensitivity of isolate 571/573-BAB;

In this experiment, isolate 571/573-BAB was cultured in a 7H11-selective medium and subsequently diluted with LABC / citrate at approximately 1,000X. In order to check what contribution the inoculum volume makes to the results of this test, three different drains were prepared, one series of dilutions being prepared in a buffer and the other in SV-18. Accordingly, subsequent dilutions (2,500X; 12,500X and 50,000X) were prepared in buffer or 1.0 mM SV-18. A seeding solution was prepared by mixing these three stock solutions of 1: 1 with a buffer or buffered lecithin solution (81dta, 8ΐ. Loshk, MO; Ca!. #: P5394 (7.5% lecithin was prepared as 100X concentrate in 100% ethanol (3 grams in 40 ml) and diluted with the Tris buffer used in Example 1, immediately before use). The final dilutions of the three different inoculum solutions were approximately 5,000X, 25,000X and 100,000X. The final concentration of CB-1 8 and lecithin in the respective solutions for inoculation was approximately 0.5 mM. Each dilution was seeded in four parallels (400 μl each) in vials with WASTES 1 2B supplemented with reconstitution fluid (B.E.) or RACHA enhanced with ceftazidime at a concentration of 8 μg / ml (P / sa). Final concentration of lecithin and CB-1 8 a during incubation was approximately 17 μg / ml, the Vials were periodically checked and recorded growth indices. Growth indices for each series were averaged and then plotted growth indices on the time of cultivation.

Aliquots of three stock solutions (i.e., 5,000X, 25,000X and 50,000X) were also used for quantitative cultivation on 7H10 7H10 plates (Vesk! Op Okktkop, SosukeuShu, MO)). Analysis of 7H10 plates showed that approximately 165 ± 54 colony forming units (cfu) were diluted in each vial at a dilution of 5,000X, approximately 33 ± 11 cfu at a dilution of 25,000X, and approximately 8 ± 3 cfu at a dilution of 100,000X. In FIG. 5A shows the results for a dilution of 5,000X seeded in buffer or in lecithin buffer, and FIG. 5B shows the results for a dilution of 5,000X seeded in SV-18 or SV-18 in combination with lecithin. In FIG. 5C shows the results for a dilution of 25,000X seeded in buffer or lecithin buffer, and FIG. 5Ό presents the results for a dilution of 25,000X seeded in SV-18 or SV-18 in combination with lecithin. In FIG. 5E presents the results for a dilution of 100,000X seeded in buffer or in lecithin buffer, and FIG. 5P presents results for a dilution of 100,000X seeded in SV-18 or SV-18 in combination with lecithin.

From FIG. 5A, 5C, and 5E, it can be seen that the addition of lecithin at these concentrations does not contribute to the sensitivity of the WASTES B 12 test. In addition, lecithin to some extent was able to overcome the negative effect of the composition of antibiotics (P / sa) in the absence of CB-18. From FIG. 5B, 5Ό and 5P shows that lecithin was able to overcome the negative effect of CB-1 8 alone or in combination with antibiotics. With a decrease in the inoculum from 165 ± 54 cfu to 33 ± 11 cfu and 8 ± 3 cf, the ability of lecithin to overcome the effect of antibiotics, CB-18, or a combination of these drugs, decreased. However, in all cases where lecithin was added to the inoculum, all repeated vials were negative (Figs. 5B, 5Ό, and 5A). Obviously, lecithin is able to overcome the effect of CB-1 8. Repeated experiments confirmed the results.

Quaternary ammonium salts

The results presented in FIG. 5A5P, give reason to believe that the mechanism of action of SV-18 is similar to that for quaternary ammonium compounds (i.e., general surface-active properties). This conclusion is based on the concept that lecithin neutralizes both Quaternary ammonium detergents and CB-18. Supposedly, neutralization occurs through the electrostatic interaction of two lipids. If the interaction of cationic quaternary detergents with anionic phospholipids seems logical, then betaines have a neutral net charge. The interaction of betainlecithin should be temporary (transient).

The results presented in FIG. 3C and 30 show that on cells treated in CB-1 8 but seeded in the buffer, there is no post-antibiotic effect. If the mechanism of action of SV-1 8 is similar to that for quaternary salts (i.e., generally destructive), then some delay would be understandable. For example, if the effect of CB-18 is a consequence of the surface-active properties of the detergent, then the violation of cellular function should not only have long-lasting nonspecific consequences, but should also not depend on the isolate. In order to check the similarity of the mechanisms of action, CB-1 8 was tested in parallel with trimethyl octadecylammonium bromide (TMA-18 (A1bpsb, Mi ^ aikee, ^ 1, Ca! # 35,924-6): 100X stock solution in water was prepared: isopropanol 1: 1, in the same way as described for CB-1 8 in example 1). The experimental design is shown in FIG. 6.

In this experiment, ATCC 27294 and 571/573-BAB isolates were cultured in 7H11 selective medium and then 10,000-fold diluted with AIbC / citrate. A seeding solution (i.e., a final dilution of 50,000X) was prepared by dilution in buffer, 0.5 mM SV-18, or 0.1 mM TMA-18 (all concentrations reflect the final concentration of detergent in the seeding solution). Each batch was then seeded in repeated inoculums (400 μl each) in bottles of WASTES 12V supplemented with reconstitution fluid (K.R.), RAITA, or RAITA reinforced with ceftriaxone (final concentration 8 μg / ml: P / sax). The final concentration of CB-18 during incubation was approximately 17 μg / ml. The final concentration of TMA-18 during incubation was approximately 3.4 μg / ml. Vials were periodically checked and growth indices recorded. Growth indices for each series were averaged and then a graph of growth indices versus cultivation time was plotted. Figures 7A-7C reflect one of the experiments in which ATCC 27294 cells were seeded in the various solutions described above, and FIG. 8A-8C reflect a parallel experiment in which cells 571/573-BAB were similarly seeded.

The results obtained with the ATCC 27294 isolate show that P / sax was more damaging than just RAITA (Fig. 7A), or the combination of RAITA / sah (compare Figs. 2A and 7A). In the presence of 17 μg / ml CB-18, slight growth retardation was noted in RAITA, and significant growth retardation was noted in P / sa (Fig. 7B). When culturing the ATCC 27294 isolate in the presence of 3.4 μg / ml TMA-18, significant growth retardation was observed in all bottles (Fig. 7C).

The behavior of isolate 571/573-BAB was in many respects similar to that for isolate ATCC 27294 with small but significant differences. For example, the presence of 17 μg / ml CB-1 8 caused obvious growth retardation in all variants, and a sharp drop in growth was noted in the composition of P / sax (Fig. 8B). A culture of 571/573-BAB in the presence of 3.4 μg / ml TMA-18 was identical to that of ATCC 27294: a significant growth retardation was observed in all bottles (Fig. 8C).

The assumption, for verification of which these experiments were performed, was that the mechanism by which CB-1 8 exerts its effect is different from that for quaternary ammonium salts (i.e., TMA18). For example, the ATCC 27294 and 571/573-ΒL ^ growth curves in the absence of any detergent (compare Fig. 7 A and 8 A) or in the presence of low concentrations of CB-18 (compare Fig. 10A and 11A) were almost identical. With increasing concentrations of CB-18, it became possible to differentiate isolates (compare Figs. 7B and 8B). When the concentration of CB-18 increased even more, the isolates again behaved similarly: none of them could grow in high concentrations of CB-18 (again, compare Figs. 10A and 11A). This was clearly different from the behavior of these two isolates in the presence of TMA-18 (compare Figs. 7C and 8C): the isolates behaved almost identically. If the place of action of SV-1 8 and TMA-18 is the same, then one can expect the same behavior of isolates in all conditions. If the sites of action of two different detergents are different, then we can expect differences in the behavior of the two isolates in the respective cultural conditions.

It is important to remember that a similar result was predicted when discussing FIG. 3C and 3C: CB-1 8 does not show a post-antibiotic effect (example 3).

Another conclusion that follows from the results obtained is that the combination of CB-18-P / sax is mainly synergistic. For example, despite the fact that CB-1 8 (Fig. 7B) and P / sah (Fig. 7 A) individually had a weak effect on the ATCC 27294 isolate, the combination of these two components led to a synergistic rather than additive effect. Alternatively to FIG. 8A and 8B show that P / sah and CB1 8 individually affect the isolate 571/573-ΒЛ ^, however, the combination of CB18-P / sah, being somewhat additive, is primarily synergistic. In contrast, TMA18 itself has a bacteriostatic effect (Figs. 7C and 8C) and, in combination with antibiotics, has an additive effect. Additional experiments with SV-1 8 and TMA-1 8 on data and other isolates confirmed subtle but significant differences in the action of the two given detergents.

Conclusion

From a comparison of FIG. 2H and 7B (isolate ATCC 27294), as well as a comparison of FIG. 3H and 8B with 5B, 5Ό, 5P (isolate 571/573-ΒЛ ^), regarding antibiotic sensitivity in the presence of CB-18, an important question follows. For example, as can be seen from FIG. 3H and 8B, isolate 571/573-ΒЛ ^ grew in the presence of CB-1 8 with antibiotics. In contrast, from FIG. 5B, 5Ό, and 5P shows that, under similar conditions, no growth was observed in the case of the ATCC 27294 isolate. A priori, these differences can be logically explained by variations in the actual concentration of CB-18, the concentration of the antibiotic, or in the volume of the inoculum used (see Example 2 and the discussion of Table 6 in the article Εη§, K.K. e! A1., Αη! ^ T ^ c ^ ob. Αd.Seto! le ^. 26: 42-47 (1984) and inoculum), or their combinations. For example, higher concentrations of CB1 8 or antibiotics could be accidentally used in experiments, the results of which are presented in FIG. 7B or 5B, 5Ό and 5P, in comparison with the experiments, the results of which are presented in FIG. 2H or 3H and 8B, respectively; or inoculum in the case of the experiments shown in FIG. 7B, 5B, 5Ό, and 5P were smaller than in the corresponding parallels. Due to the method by which CB-18, antibiotics and inoculum were introduced into each vial (i.e., using a 1 ml syringe), the variations for all three components from vial to vial are quite expected: they will be the rule rather than the exception. If the culture system used in these experiments is hypersensitive to one of the components, then differences similar to those described should be observed. In the experiment, the experimental scheme of which is shown in FIG. 4, and the results are shown in FIG. 5–5P, differences in the course of growth curves depending on inoculums were noted. These differences consisted of primary growth retardation, which is expected with small inoculums. Variations in antibiotic concentrations should be minimal compared with changes in CB-18 concentration and inoculum volume. Apparently, the culture system should be significantly more elastic with respect to changes in the concentration of CB-18.

Example 5. Titration of SV-18: Comparison of MTB, ° C and fast-growing strains.

The results of the pilot study of SV-1 8 (Example 2) gave reason to believe that the effect of SV-1 8 is widely applicable to a wide range of mycobacterial species (Table 6), and from Example 4 it followed that the system is elastic with respect to the concentration of SV-18. To test this hypothesis, an experiment was illustrated, illustrated in FIG. 9, using various mycobacterial species, some of which were also used in the pilot study of SV-18.

In this experiment, the isolates of the complex M.! Bersi-515, the complex M. auscht, and the complex M. goyiiit were tested. The tested isolates of the M.! And Lsi1155 complex included ATCC 27294 and 571/573-ΒΛ ^. The tested isolates of the M.Ausht complex included ATCC25291 and 802-ΒЛ ^; and the tested isolates of the M.Goyiyit complex included ATCC 6841 and 495-SHN. The characteristics of these isolates and the results of these experiments are summarized in table. 7.

All isolates were cultured on 7H11 selective medium, and then sequentially diluted with ILS / citrate to 1,000X. A solution for inoculation (ie, final dilution (5,000X) was obtained by dilution in buffer or in various concentrations of CB-18. These concentrations were: 0.1 mm SW-18, 0.2 mm SW-18, 0.4 mM SV-18, 0.8 mM SV-18, 1.6 mM SV-18 or 3.2 mM SV-18. All concentrations reflect the final concentration of detergent in the solution for inoculation and not all isolates were tested at all concentrations.

Each batch was then seeded in triplicated inoculum (400 μl each) in bottles of WASTES 12V supplemented with reconstitution fluid (B.R.), RAETA, or RACHA reinforced with ceftriaxone (final concentration 8 μg / ml: P / sah), ceftazidime (final concentration 8 μg / ml: P / sax) or cefoxitin (final concentration 8 μg / ml: P / cc). Isolates M.! And leucoi818 were seeded in P / sa, isolates M. auscht were seeded in P / sa and isolates M. Goisthit were seeded in P / sa. The final concentration of CB-1 8 during incubation was approximately 3 μg / ml for a series of 0.1 mm, approximately 7 μg / ml for a series of 0.2 mm, approximately 13 μg / ml for a series of 0.4 mm, approximately 27 μg / ml for a series of 0.8 mm, approximately 54 μg / ml for a series of 1.6 mm and approximately 109 μg / ml for a series of 3.2 mm. Vials were periodically checked and growth indices recorded. Growth indices for each series were averaged and then a graph of growth indices versus cultivation time was plotted.

FIG. 1 0A and 1 0B reflect one experiment in which the isolate M.! Bibliso818 ATCC 27294 was seeded in various solutions described above, and FIG. 11A and 11B depict a parallel experiment in which the isolate M.! Bibliso818 571/573-BAB was seeded in the same dilutions. FIG. 1 2A and 1 2B reflect one experiment in which the M. auscht ATCC 25291 isolate was seeded in the various solutions described above, and FIG. 13A and 13B depict a parallel experiment in which the M.Ausht 802-BAB isolate was seeded in the same dilutions. FIG. 14A and 14B depict one experiment in which the M.Joistit ATCC 6841 isolate was seeded in the various solutions described above, and FIG. 15A and 15B depict a parallel experiment in which the isolate M.! Bibliso818 495-SHN was seeded in the same dilutions.

The viability of the isolate M.! Biluxi818 ATCC 27294 decreased when the concentration of CB-18 reached 54 μg / ml (Fig. 10A), while the viability of the isolate M.! Biluxi818 571/573 BAB decreased when the concentration of CB-18 reached 27 μg / ml (Fig. 11A). With the introduction of antibiotics into the culture medium, the viability of the ATCC 27294 isolate decreased when the concentration

CB-1 8 reached 27 μg / ml (Fig. 1 0B), and the viability of isolate 571/573-BAB decreased when CB-1 8 reached a concentration of 13 μg / ml (Fig. 11B). Obviously, the system was very sensitive to changes in the concentration of CB-18. Deviations of the results from experiment to experiment are easily explained by this sensitivity.

The viability of M. auscht ATCC 25291 isolate decreased when the concentration of CB-18 reached 27 μg / ml (Fig. 12A), while the tested concentrations of exposure did not affect the viability of the M. auscht (MAC) 802-BAB complex isolate (Fig. 13 A) . When antibiotics were included in the culture medium, the viability of the ATCC 25291 isolate decreased when the CB-1 concentration of level 8 reached 13 μg / ml (Fig. 12B), and the vitality of the 802-BAB isolate decreased when the CB-18 concentration reached 27 μg / ml (Fig. 13B). At a concentration of 27 μg / ml, the curve reached a plateau, since only one of the three vials remained positive (Fig. 13B). Again, the system was sensitive to changes in the concentration of CB-18.

The viability of both isolates of M. ой oish! Um (ATCC 6841 and 495-1HH) decreased when the concentration of CB-1 8 reached 109 μg / ml (Figs. 14A and 15A, respectively). When antibiotics were introduced into the culture medium, the viability of the ATCC 6841 isolate decreased when the concentration of CB-18 level reached 54 μg / ml (Fig. 14B), and the viability of the 495-SH isolate fell when the CB-18 concentration reached 27 μg / ml ( Fig. 15B).

The fast-growing strains, as a group as a whole, turned out to be more resistant to the action of CB18 in itself, but the synergism of the action of CB-18 and antibiotics was well manifested on them. For example, isolate 495-1HH remained viable at a concentration of CB-18 equal to 109 μg / ml, but its viability decreased at a concentration of CB-18 in the range of 13-27 μg / ml in the presence of P-cf (Figs. 15A and 15B). Alternatively, isolate 571/573-BAB remained viable at a concentration of CB-18 equal to 13 μg / ml, but viability decreased at a concentration of CB-1 8 in the range of 7-13 in the presence of P-sax (Fig. 11A and 11B).

The results obtained with two fast-growing strains are of interest in the light of Soppe11, Ν.Ό. e! A1., 1n: ThiGsi1o818, RaShodepezK, PTO! esyop apb Sop! go1. V1oot, V.V. fuck A8M Rgezz, AazYpdop, L.S. (1994) pp. 3335352, in which the resistance of fast-growing strains to cephalosporins is attributed to permeability. As suggested in Example 9, the SV-18 somehow affects the permeability, so the result was quite expected.

Table 7

Screened species of mycobacteria

ΝΛΙ.ΓΝπΟΙΙ SV-18

Culture Culture

Yu Smear Liquid Dense Smear 12V 7H11 ATCC 27294 MTV t t t t t t Viability decreased in the range of concentrations of CB-1 8 alone 27-54 μg / ml and in the range of 13-27 μg / ml in the presence of antibiotics 573 - BA1. MTV 1+ F / 28 0 3+ F 0/20 Viability decreased in the range of concentrations of CB-18 alone 13-27 μg / ml and in the range of 7-13 μg / ml in the presence of antibiotics ATCC 25291 M.au. t t t t t t Viability decreased in the range of concentrations of CB-18 alone 13-27 μg / ml and in the range of 7-13 μg / ml in the presence of antibiotics 802 VA1. MAS 0 F / 10 F / 11 1+ 0 F / 20 Proven concentrations of CB-1 8 alone did not affect viability, but it fell in concentration ranges of 13-27 μg / ml in the presence of antibiotics ATCC 6841 M. Go t t t t t t Viability decreased when CB-1 reached a concentration of higher than 109 μg / ml6 and in the presence of antibiotics in the range of 2754 μg / ml. 495 - M. Go. 0 F / 9 0 ± 0 F / 14 Viability decreased upon reaching

.11III CB-18 concentrations above 109 μg / ml and in the presence of antibiotics in the range of 1327 μg / ml.

* 3 values of smears are given in accordance with the recommendations of the ATP. Positive cultures are indicated by the sign F with a number through a fraction. This number indicates the time in days it took to get a positive result. Negative cultures are indicated by -. Shoals that were lost due to contamination were indicated by sop1, and liquid cultures sent for reanalysis were indicated by 0. The ATCC strain 27294 was not obtained in the CB18 pilot study; therefore, the culture results were designated as inapplicable ('TCA). M.au. denotes M. aust. M. Go denotes M. Gog! sh1it.

Several conclusions follow from the results. Firstly, there is a minimum applicable concentration of CB-18: low concentrations of CB-18 did not have an inducing effect, and high concentrations of CB18 were bactericidal (even in the absence of antibiotics). The ability to induce depends on the type of mycobacteria, as well as on the isolate.

Example 6. Characterization of MTB isolates and antibiotics.

The principal conclusion made on the basis of examples 2, 3 and 5 is that SV-18 makes some types of mycobacteria sensitive to antibiotics, to which they were not sensitive under normal conditions. Thus, the breadth of this phenomenon in terms of isolates and antibiotics was investigated. Several isolates used in the SV-18 pilot study (Example 2) were tested for various β-lactam antibiotics in the presence and absence of CB18. In an additional series of experiments, other (i.e., non-β-lactam) antibiotics were tested.

In FIG. Figure 16 shows the experimental design used to test various MTV isolates. These isolates (Table 8) were cultured on 7H11-selective shoals and subsequently diluted with NaBC / citrate to 10,000X.

A seeding solution (ie, final dilution (approximately 50,000X) was obtained by further dilution with buffer or 0.5 mM CB-1 8 (final concentration). Each batch was then seeded in parallel inoculum (400 μl each) in VASTES vials 12B with the addition of reconstitution fluid (B.E.), PA #TA, or RAET reinforced with ceftazidime at a final concentration of 8 μg / ml (P / Ca7 '), cefoperazone at a final concentration of 16 μg / ml (P / sGy) ( 81dta, 81. Loshk, M0, Ca1. # C4292: cefoperazone was dissolved in water at a concentration of 72 mg / ml and added in the manner described for ceft ideyma (example 1)), ceftriaxone at a final concentration of 8 μg / ml (P / sah) (81dta, 81. Loshk, M0, Ca1. # C-5793: ceftriaxone was dissolved in water at a concentration of 36 mg / ml and added by the method described for ceftazidime (example 1)), or cefoxitin at a final concentration of 8 μg / ml (P / cu) (81 dta, 81. Loshk, M0, Ca1. # C-4786: cefoxitin was dissolved in water at a concentration of 36 mg / ml and was added by the method described for ceftazidime (example 1)). Again, the final concentration of CB-1 8 during incubation was approximately 17 μg / ml. Vials were periodically checked and growth indices recorded. Growth indices for each series were averaged and then a graph of growth indices versus cultivation time was plotted.

Eleven isolates were screened (including ATCC strain 27294). In the table. 8 summarizes the clinical isolates used and their test results. Representative isolates are shown in FIG. 17-27.

Significant differences were noted between these test cases.

isolates regarding behavior in this

Table 8

Screened isolates MTV IAIS / IAON SV-18

Culture

CultureΙΏ Smear Liquid Dense Smear 12V 7H11 ATCC 27294 IA IA IA IA IA IA The minimal induced SV-1 8 sensitivity to the tested antibiotics, however, the sensitivity to sah sharply increased SV-1 8 571-YOU 1+ F / 28 F / 42 1+ Θ Θ / soy! Strong induced CB-1 8 sensitivity to all proven antibiotics (similar to 573BAB). 573-YOU 1+ F / 28 Θ 3+ Θ F / 20 Strong induced CB-1 8 sensitivity to all proven antibiotics (similar to 571 VA). 535-YOU 4+ F / 3 F / 28 4+ F / 3 F / 11 Minimal induced SV-18 effects with respect to proven antibiotics, sensitivity to sah independent of SV-18 896 -VA 4+ F / 1 F / 14 4+ F / 1 F / 5 Minimum induced SV-18 sensitivity to most tested antibiotics; sensitivity to sah was slightly enhanced by SV-18. The strain has multiple drug resistance: SH, K1P & P / A. 040-TCE Θ F / 29 F / 42 1+ Φ / 14 / Θ Θ The small induced SV-18 sensitivity to most tested antibiotics, the sensitivity to sah sharply increased by SV-18. 061-TVK Θ F / 29 F / 35 2+ F / 10 F / 19 The insignificant induced SV-1 8 sensitivity to the majority of tested antibiotics, the sensitivity to sah sharply increased SV-18. 1 Strain resistant to 1IN. 512-.1HN 2+ F / 4 Θ 4+ F / 3 F / 12 Minor induced CB-1 8 sensitivity to most proven antibiotics (similar to 538-TIN) 538-1NN Θ F / 31 ® / 33 2+ Θ F / 49 Slight induced CB-1 8 sensitivity to most proven antibiotics (similar to 512-.1HH) 52-96-VOI 1+ F / 25 F / 27 1+ Θ F / 39 The insignificant induced SV-1 8 sensitivity to most tested antibiotics, however, the sensitivity to sah sharply increased SV-1 8 57-96-VOI 1+ F / 25 F / 27 3+ Θ F / 25 Moderate sensitivity to most antibiotic

tics and moderate induced SV-18 sensitivity to most antibiotics. Resistant strain (IOC).

* The values of the smears are given in accordance with the recommendations of the ATP. Positive cultures are indicated by the sign f with a number through a fraction. This number indicates the time in days it took to get a positive result. Negative cultures are indicated by -. Shoals that were lost due to contamination were indicated by the soybean symbol!., And liquid cultures sent for reanalysis were indicated by the symbol Θ. Strain ATCC 27294 was not obtained in the Pilot study CB18, therefore, the cultural results are designated as inapplicable (IA).

The ATCC 27294 isolate showed no differences (or showed slight differences) with respect to CB-18 induced sensitivity, except for slight delays when CB-18 was combined with P-sax (Figs. 17A and 17B). These results are confirmed by earlier P-sax verification data (compare FIG. 2P with Figure 1 7B) and P-sax (compare FIG. 7B with FIG. 17B). The apparent difference in the induced sensitivity to RAITA seen in FIG. 7B, but not in FIG. 17B, apparently, is the result of changes in the concentration of CB-1 8 from experience to experience, as discussed in example 5.

Isolates 571-BAB (Fig. 18A and 18B) and 573BAB (Fig. 19A and 19B) showed significant induction of sensitivity to all tested drugs. Only when antibiotics were combined with CB-18 did a synergistic negative effect on 571/573-BAB be noted.

Similarly to ATCC isolate 27294, isolate 535-BAB did not respond to CB-18 at the concentrations used, however, P-sax influenced this isolate (Figs. 20A and 20B). The behavior of isolate 896-BAB, which was resistant to 1IN, K1P, and P2A, was similar to that of isolate ATCC 27294 (compare Figs. 17A and 17B with Figs. 21A and 21B). The isolates 040TVK and 52-96-BOB SV-18 had some effect, however, the sensitivity to sax increased significantly with the presence of CB-1 8 in the case of both isolates (Figs. 22A and 22B and Figs. 26A and 26B, respectively). In contrast, the 061-TBK and 57-96-BOB isolates yielded to antibiotics in the absence of CB-18, however, the effect of antibiotics was significantly enhanced in the presence of CB-18 (Figs. 23A and 23B and Figs. 27A and 27B, respectively).

A pair of 512-1НН and 538-1НН (Figs. 24A and 24B and Figs. 25A and 25B, respectively) was of interest from this point of view since in the pilot study of CB-1 8 with exposure to CB-1 8 one of them (512-RNN ) was native, and the other was exposed to CB-1 8 against the background of antituberculin therapy (538-1HH). Four samples were obtained from this patient (table. 9). The first sample was obtained on February 8, 1996 and was qualified as smear positive for the first 24 hours (a period considered mandatory for this type of diagnosis), and for four days it was qualified as positive for LRV cultivation. A second sample from the same patient (527-1HH) was obtained ten days after the first sample, approximately 1 week after the isolation of AEB from the culture bottle and 5 days after treatment of the patient was started. All three samples obtained after the start of antibiotic treatment were CB-18 / 12V / RAITA / ca7-negative, and only two (538-1НН and 541-1НН) were positive on the 7Н11 joint. While the contamination did not play any role in these differences in relation to SV-18 / 12V / PA #TAs7, the fact that 527-1НН had the lowest smear rate of all four isolates can explain the drop-out result from 7Н11 ; however, a significant growth retardation of isolate 538ShN in a dense medium (i.e. 49 days) is unusual for a smear-positive sample. Despite the fact that growth retardation was also observed for similar samples treated with NΑ ^ C / NaOH, CB-1 8 during the treatment made a significant contribution to reducing the viability of this isolate. This fact coincides with the discussion of Example 3 and the series of experiments shown in FIG. 3 (compare FIG. 3B with 3P and FIG. 3Ό with 3H) and confirms the clinical applicability of betaine-like detergents as therapeutic adjuvants.

ΝΑΓ-C ΝπΟΠ

Table 9 *. Isolates M.1 and Lepsi1oz18 obtained from one patient

SV-18

Culture Culture

Yu Smear Liquid Dense Smear Liquid Dense A comment 512-SHN MTV 2+ ® / 4 Θ 4+ ® ® 2/8/96 Untreated 527-.1HH MTV _- ® / 24 _- ± Θ Θ 2/17/96 Therapy 538-SHN MTV ® / 31 F / 33 2+ Θ ® 2/27/96 Therapy 54TH MTV ® / 31 ® / 33 2+ Θ ® 2/27/96 Therapy

* 3 values for smears are given in accordance with the recommendations of the SOS. Positive cultures are indicated by ®, followed by a number. This number reflects the time in days that it took to achieve a positive result. Negative cultures are indicated by

The experimental design shown in FIG. 16 was also used to evaluate the breadth of the effect of CB-1 8 in relation to other, non-β-lactam antibiotics. In these experiments, antibiotics (81dta, 8ΐ. Loshk, MO) were prepared in the form of stock solutions in water or buffer, and then diluted with reconstitution liquid and added to culture vials before inoculation. Nystatin (final concentration 100 μg / ml), erythromycin (final concentration 4 μg / ml), oleandomycin (final concentration 2 μg / ml), penicillin (final concentration 8 μg / ml), nalidixic acid (final concentration 30 μg / ml) , lincomycin (final concentration 2 μg / ml), ceftriaxone (final concentration 32 μg / ml), ceftazidime (final concentration 16 μg / ml) (ceftazidime is available in 10% sodium bicarbonate) were tested on ATCC isolates 27294.571 / 573-BAB, 061 TVK, 52-96-BOB and 57-96-BOB. In FIG. 28 presents the results obtained on isolate 571/573-BAB with erythromycin and ceftriaxone. In FIG. 29 presents the results obtained on isolate 061-TBP with polymyxin B, oleandomycin, lincomycin, nalidixic acid, penicillin 0 and ceftriaxone. In FIG. 30 presents the results obtained on isolate 57-96-BOB with nalidixic acid, penicillin 0, ceftazidime, and ceftriaxone.

These results show that the effect of CB-1 8 depends on both the isolate and the antibiotic. It is noteworthy that ceftazidime alone (i.e., in the absence of ΡΑΝΤΑ) at a final concentration of 16 μg / ml turned out to be one of the less harmless drugs.

Conclusion

The obtained results emphasize the microheterogeneity of the complex M1bEsi1O818 with respect to antimicrobial sensitivity. Despite the fact that the mechanisms involved in these processes have not yet been fully determined, CB18 makes possible a high degree of discrimination of isolates M4iLegsiO818 based on sensitivity. This information may be useful in choosing the most effective therapeutic regimens for the treatment of mycobacterial infections, in particular MTV infections. In addition, the fact that CB-1 8 induces sensitivity creates the possibility of using this compound as a therapeutic adjuvant.

Effect of SV-18, i.e. the ability to induce sensitivity depends on the dynamic interaction of a betaine-like detergent (i.e. CB-18), an antibiotic (s) and an isolate. The most applicable concentrations of betaine-like detergents will depend on which particular betaine-like detergent is used and can be determined using the screening methods of the present invention.

Example 7. Screening of detergents and other betaine-like structures.

In order to expand the results obtained in example 6, a test was developed for screening SV-18 homologs. In the table. 10 lists the detergents used in the test illustrated in FIG. 31. Due to the sensitivity of isolate 571/573-BAB to sensitivity induction, this isolate was used as a reporter in these screening experiments. Isolate 571/573-BAB was cultured on 7H11 schools and sequentially diluted with ILS / citrate to approximately 10,000X. Seeding solution, i.e. final dilution (approximately 50,000X) was obtained by additional dilution in 0.5 mM CB-1 8 buffer or in the appropriate detergent. All detergents were prepared as 1 00X concentrates in a 1: 1 mixture of isopropanol: water and used as a solvent in the last stages in these experimental dilutions.

The final concentration of each detergent was such that in each vial of 1 2B this detergent was added at a final concentration of about 17 μg / ml. Each experimental series, along with the controls (buffer and CB-1 8), was seeded in parallel inoculums (400 μl each) in bottles of WASTES 12B with the addition of reconstitution fluid (B.R.), RAITA, or RAIT with ceftriaxone in the final concentration 8 μg / ml (P / sah). Five to seven detergents were simultaneously tested. Each diluted detergent containing MTB cells was seeded in parallel inoculum (400 μl each) in bottles of WASTES 12B supplemented with RAITA or P / sax. Almost all detergents were tested twice. Vials were periodically checked and growth indices recorded. Growth indices for each series were averaged and then a graph of growth indices versus cultivation time was plotted.

In the table. 10 summarizes the tested detergents and the results obtained with their use. The following detergents were obtained from A1bps11 CHet1ca1 Sotrapu (Mb ^ aikee, XVI): benzyldimethylstearyl ammonium chloride (Ca!. # 22,901-6), benzyldimethyltetradecyl ammonium chloride (Ca!. # 29,279-6) and octadecyl ammonium bromide. 35.924-6). The following detergents were obtained from (81dta Cetka1 Sotrapu, 8!. Bocz, Mo.): mixed alkyl trimethyl ammonium bromide (Ca!. # M-7635), palmitic acid (Ca!. # P0500), stearic acid (Ca!. # 8 -4751), СНАР8 (Са!. # С-3023), deoxycholic acid (Ca!. # Ό-6750), 8В-18 (С _{! 8} -sulfopropyl betaine) (Ca!. # О-8004), Bridge 97 ( oleyl (polyoxyethylene) 1 ₀ ) (Ca!. # P-6136) and Bridge 56 (cetyl (pooxyethylene) 10) (Ca!. # P-5759). Tween 80 (polyoxyethylene sorbitan ester of oleic acid) (Ca!. # P-1334 875) was obtained from Boebppdeg Mappbeth (1pb1aparobz, S). Specially prepared detergents included: C1 ₈ -carboxyethyl betaine, C1 ₈ -sulfobutyl betaine, C1 ₆ -hydroxypropylsulfobetaine, C _{! 6} -amidopropyl sulfate betaine, synthesized

Eset Weses, 1ps. (Saska, WE), and C ₁₆ ANTMAR (reverse betaine), which was received as a gift from KAO 1pzb! And! EG Ripbatep! A1 Vezeag (Tosyd1, 1arap). Hembetain-8 (soiamidopropyl-carboxymethylbetaine) was obtained as a sample from Sjetogop Sogrogabop (Razo Bojez, CA). DeTain PB (LeTate PB) (cetyl-carboxymethylbetaine) was obtained as a sample from OeRoges !, 1 ps., (Vosa Ba! Op, Pb). Hetaine CHA (He! A1pe CHA) (canolamidopropyl carboxybetaine) was obtained as a sample from He! Epepe, 1 ps. (Ra! Egzop, ΝΡ). The revoteric AMB-4 (Be \\ 'o1eps AM B-4) (fatty glycinate) was obtained in the form of a sample from 8LexecLet1 Sotrapu (Ln. OH). Sherkotainin 1AB (8cbego! Ate 1AB) (stearylmidopropylcarboxybetaine) and Shercoatin VB (8cbgoxo! VBB) were obtained in the form of samples from 8cbg CHepis, 1ps. (Sbyop, ΝΡ). Velvetex ОВВ-50 (Уе1уе! Ex ОВВ-50) (oleylcarboxymethyl betaine) was obtained as a sample from Nepke1 Sogrogabop, Etogu Sgoir Sozra (Nookep, ΝΡ). Crosultain E-30 (Sgozb! A1pe E-30) (erucamidopropyl hydroxypropyl sulfobetaine) was obtained as a sample from Sgob, 1 ps. (Ragarrapu, ΝΡ). Makaimin O2 (Muscatte O2) (oleamine oxide) was obtained in the form of a sample from MSbIuge Sgoir, bTb. (Ishuegyu Ragk, 1b).

The controls for these experiments are shown in FIG. 32 A, and representative results obtained with detergents are shown in FIG. 32B. In FIG. 32 And the results obtained in parallel vials for each control series in eight experiments were averaged and presented graphically in order to show the relative contribution of antibiotics (♦ -RANTA, and Δ -P / sax), CB-18 (x-CB-18 only), and a combination of the two components (-CB18 / RAXTA, and "-CB-18 / P / sah). The growth curves shown in FIG. 32B are based on the average values obtained for parallel vials in both experiments, when the cells were seeded in the corresponding detergent in WASTES 1 2B with the addition of P / sax.

As indicated previously, isolate 571 / 573BAB did not (or minimally succumb) to ACT or P / sax in the absence of CB-18 (compare FIGS. 3A, 3E, 8A and 18A / 19A with FIG. 32a). Alternatively, CB-18 itself had an effect on the growth of isolate 571/573-BAB, and the combination of CB-18 with antibiotics was synergistically negative, while the combination with P / sa provided the greatest growth inhibition (Fig. 32A: x-CB-18 only, -SV-18 / RAITA, and • -SV-18 / P / sah).

Again, there was a notable difference in results depending on the structure of the detergent. In general, all tested quaternary ammonium salts were extremely active and completely inhibited growth at the used concentration (Table 10). Alternatively, neither the anionic fatty acids nor the bile salts that have been tested have an effect on growth.

Only those carboxybetaines that have not been modified showed activity. For example, those carboxybetaines in which the amidopropyl group was used to bond the alkyl chain with the cation (α in Table 1) did not show activity in the test used. Alternatively, carboxybetaines without substitutions showed a strong ability to inhibit growth in combination with antibiotics, especially C ₁₈ carboxyethyl betaine (homologue of CB-18 with an ethyl bridge). Further studies of C ₁₈ carboxyethyl betaine using AECC27294 isolate showed a very narrow range of activity (approximately 7-13 μg / ml). In FIG. 32A and 32B show the variability when using simple (i.e., unmodified) carboxybetaines with methylene (Detaine PB and Velvetex OBB), ethylene (C ₁₈ -carboxyethyl betaine) and propylene (CB18) bridge in this test. This coincides with the data of K.K.K., Boig. 80: 441-444 (1991) that toxicity correlates with bridge length.

Betaine-like detergents in the other category showed mixed results. Neither reverse betaine nor sulfatobetaine (i.e., -8O ₄ ) inhibited growth. Only one of the four sulfobetaines (-8О ₃ ) (i.e., Crosultain E-30), two of the three non-ionic detergents (Bridge 56 and Bridge 97) and one amine oxide (oleamine oxide) were able to significantly inhibit growth (Table. 10).

While the apparent bactericidal activity of amine oxide is not surprising, if we take into account the use of these compounds in antimicrobial formulations (Mkyayayk, E.V. i. 564,454 and i. 644,728), then the activity of the compounds Bridge and Crosultain E-30 turned out to be unexpected. Unlike Crosultain E-30, the only other sulfobetaine that showed significant activity in this test was 8B-18. Further studies of 8B-18 on isolate 571/573-BAB showed a very wide spectrum of activity (approximately 20-75 μg / ml). All other sulfobetaines had amidopropyl bonds or hydroxypropyl bridges, which made them similar to carboxy betaines. Modifications to the skeletal structure appear to interfere with the activity described here. Crosultain E-30 also has an amidopropyl bond and a hydroxypropyl bridge. Since Bridge compounds do not have skeleton modifications, these reagents should stimulate growth. It is historically known that non-ionic detergents are contaminated with impurities and stimulate growth only in a purified form (Oybok, Kb. E! A1., Boig. Exp! 1. Meb. 83: 409-423 (1946)). The presence of impurities may explain the results obtained with the detergents Bridge and Crosultain E-30.

From the data presented it follows the possibility that the observed effect is due to impurities. However, SV-1 8 is supplied at approximately 98% purity (private message from Bob Carlson, Esokiet Vekeagsy, 1ps., Syakka, WE). Therefore, any impurity should manifest itself in these tests at a concentration of about 0.34 μg / ml (if 2% of the added CB-18 are impurities, then 2% of 17 μg / ml will leave approximately 0.34 μg / ml). TMA-18 in this test had an inhibitory effect at ten times lower concentrations (Fig. 7C and 8C). In addition, while many commercially available betaines can be contaminated with quaternary salts formed during synthesis (i.e. precursors or synthetic by-products), the possibility that quaternary salts are formed during the synthesis of CB-18 is unlikely (personal message from Bob Carlson, Esociet Vekeagsy, 1ps., Syakka, WE). In addition, it is believed that the by-products formed during the synthesis of SV-18, carried out by modified methods ^ eegk, b. e! A1., Lapps 7: 854-867 (1991) or Κι / but (6P 8125139), must not be toxic (personal message of Bob Carlson, Esokiet Vekeagsy, 1 ps. (Syakka, WE).

These experiments (Fig. 32B) also showed that Tween 80 at a concentration of 17 μg / ml had no effect on isolate 571 / 573BAC Nsh, b. e! A1., Aptggo. AdeSch Cieto. 11: 773-779 (1977) varied the concentration of Tween 80 and showed that the induction of sensitivity to rifampin was not achieved at a concentration of approximately 0.005% (50 μg / ml)). More importantly, higher concentrations of Tween 80 (up to 0.05% (500 μg / ml (380 μM)) only stimulated growth. 8ypkop, M.№. E! A1., At.Kgu. Vekr. P1k. 104 : 717-727 (1971) reported that maximum stimulation was achieved at 1% (10 mg / ml (7.69 mmol), and concentrations as high as 10% did not give additional benefits but did not have an overwhelming effect. Adding Tween 80 to the cultural environment was the most significant diagnostic achievement of mycobacteriology in this century, since it caused rapid growth (Oybok, Kb. e! a1., boig. Exp.Meb. 83: 409-423 (1946). atogk 8. е! а1., М1сгоОо1.1ттцпо1.35: 921-926 (1991)) reported changes in the nature of sensitivity to individual drugs when titrating Tween 80 from 0 to 0.5 and 2%. Presence of OELS (culture supplement, containing BSA, No. C1, dextrose, catalase and oleic acid) causes significant changes in sensitivity in combination with Tween 80. On the other hand, CB18 at a concentration of 3-7 μg / ml does not cause growth changes, but completely inhibits growth at a concentration of 13- 27 μg / ml (Fig. 10A and 11A).

The conclusion that can be drawn on the basis of these experiments is that for the implementation of the methods claimed in the invention, a wide range of betaine-like detergents is applicable, and the induction of sensitivity varies in accordance with the structure of the betaine-like agent. Each betaine-like detergent must be optimized for use in accordance with the methods of the invention. An exceptional degree of discrimination is possible with the differentiation of isolates by combining various antibiotics with various betaine-like detergents.

Table 10

Detergent Result

Cationic detergents

Benzyldimethylstearyl Complete Inhibition of Ammonium Chloride Growth

Benzyldimethyltetradecyl Complete suppression of ammonium chloride Mixed alkyltrimethyl ammonium bromide Octadecyltrimethyl ammonium bromide

Anionic Growth Detergents

Complete growth suppression

Complete growth suppression

Palmitic acid No effect Stearic acid No effect

Bile salts

CHAP8 No effect

Deoxycholic Acid No Effect

Carboxybetaines

C18-Carboxyethyl Betaine Complete Suppression

Hembetain-8 (soiamidopropyl-carboxymethyl betaine)

De Tain RV (cetylcarboxymethyl betaine) Hetain CABA (canolamidopropyl carboxybetaine) Reioteric AM I-4 (fat glycinate) Sherkotain ILΒ (stearylamidopropylcarboxybetaine) Sherkotain VΟLΒ Velveteks OVV-50 growth

No effect

Strong growth inhibition

No effect

No effect

No effect

No effect

Very strong growth suppression

Other betaines

C1 ₆ -ANTMAR (reverse No betaine effect)

C18-sulfobutyl betaine Weak growth retardation

C16-hydroxypropyl sulfobetaine

C16-amidopropylsulfatobetaine

Crosultain E-30 (erucamidopropyl hydroxypropyl sulfobetaine) 8B-18 (C; _8- sulfopropyl betaine)

Macamine O ₂ (oleamine oxide)

Tween 80 (polyoxyethylene sorbitan oleic ester

Weak growth retardation

No effect

Strong growth inhibition

Moderate growth inhibition

Complete growth suppression

No effect

Nonionic Acid Detergents

Bridge 97 (oleyl (polyoxyethylene) ₁₀ )

Bridge 56 (cetyl (polyoxyethylene) ₁₀ )

Very strong in growth pressure

Very strong in growth pressure

Effect of SV-18 and EDTA

From example 4 it is seen that the action of SV-18 is different from that for TMA-18, and from FIG. Figure 32B shows that the effect of CB-1 8 is different from the effect of Tween 80. The results of testing various betaine-like detergents suggest that unmodified structures should be most useful (Table 10). In order to further differentiate the effect of CB-18 from the action of other agents, for example, chelating metals (i.e., EDTA), the experiments described in FIG. 33.

EDTA alters the permeability of bacteria by extracting and chelating divalent metal cations that stabilize the structure of the cell wall. YaazFts Ν. eί a1., Aηί ^ t ^ s ^ οb. Adeηί8 CHeito. 34: 759-764 (1990) reported that attempts to grow mycobacteria in 1 mM EDTA (372.5 μg / ml) showed a significant effect on viability. Given the fact that betaines are salted out on the basis of their ability to bind water (Τδπρΐ, K. eί a1., Игο ig. RNukh. SN. 82: 1610-1614 (1978), it was suggested that the effect of CB-1 8 is a result of chelating activity of this compound.

In order to differentiate the effect of CB-1 8 from the action of metal chelators, such as EDTA, a test was developed to isolate the action of CB-18 (Fig. 33). The ATCC 27294 and 571/573-BAB isolates were grown on 7H11-selective shoals, and then each was subsequently diluted with XABC / citrate approximately 10,000 times. A solution for seeding (approximately 10,000X) was obtained by further dilution in buffer, 0.5 mm SW-18. 0.5 mM CB-18 containing 2.5 mM MdC1 ₂ , or 0.5 mM EDTA (Lb Τ CesNηο1ο ^ e5. SayNehlgd, MO). The final concentration of CB-1 8 and EDTA was approximately 17 μg / ml during incubation. and the concentration of MDC12 during incubation was approximately 85 μg / ml. Each experimental series was seeded in three vials with WASTES 1 2B (400 μl each), with the addition of reconstitution fluid (V.G.) or RAITA with ceftriaxone at a final concentration of 8 μg / ml (P / sah). Vials were periodically checked and growth indices recorded. Growth indices for each series were averaged and plotted against the days of cultivation. In FIG. 34A and 34B show the results obtained with ATCC isolate 27294. and in FIG. 35A and 35B show the results obtained with isolate 571 / 573BAB.

EDTA at the concentrations used had no effect on the growth characteristics of any isolate (FIGS. 34B and 35B). The concentration of EDTA in these experiments was almost 22 times lower than that used in the Vacuum experiments1. I. e! a1 .. Apt1sto.B.Adep! to net. 34: 759-764 (1990) (372 μg / ml vs 17 μg / ml).

The most interesting aspect of these experiments was the combination of CB-18 / MDC1 ₂ . The hypothesis was that SV-18 is able to chelate MDC12 and therefore the effect of SV-18 will be neutralized by such an interaction (to some extent). While the ATCC 27294 CB-18 isolate alone (Fig. 34A: i.e., in the absence of MDC1 ₂ ) was not affected, isolate 571/573-BAB was suppressed by one CB-18 (Fig. 35A). The addition of a five-fold molar excess of MDCl ₂ had no effect on the growth pattern of isolate 571 / 573BAB (Fig. 35B). Neither ATCC 27294. nor 571 / 573BAB showed a pronounced growth retardation in the presence of only P / sax; however, P / sah in the presence of CB-1 8 acted synergistically negatively, and this was more pronounced on isolate 571/573-BAB (Figs. 34A and 35A). The addition of MDC12 to the CB-18 / P / sax combination somewhat weakened the negative effect of the CB-18 / P / sax combination (Figs. 34B and 35B), and the CB-18 / P / sax effect was more pronounced on isolate 571 / 573- YOU. If MDC1 ₂ does not mitigate the effect of CB-18 in the absence of P / sax, but MDC1 ₂ reduces sensitivity in the presence of P / sax, then MDC12 should interfere with the action of P / sax. Since ceftriaxone (sah) is supplied as a disodium salt, the antibiotic has a net negative charge (i.e. -2) and must interact with MDC1 ₂ .

Tkjope K., 1oit. Patt. 8c1. 80: 1051-1054 (1991) showed that, as a rule, there is no relationship (or it is small) between the chelating activity of various phosphobetaines and their antimicrobial activity. This conclusion is consistent with the observation that the CB-18 effect is not a consequence of the chelation of metal ions.

Example 8. Testing the sensitivity of mycobacteria and the effect of SV-18.

There are a number of methods for testing sensitivity. The standard methods currently used include: (a) a diffusion test with disks, (b) micro-dilutions in a broth, (c) an agar gradient, and (d) fast automated instrumental methods. When implementing these methods, the concentration of the drug is changed and growth characteristics are evaluated. It is known that the inoculum volume significantly affects the results of sensitivity testing.

In modern mycobacteriological tests of sensitivity, the method of 1% proportion described by Vek! A1 follows. A. b. Sep! Egk Gogekeake Sop! Go1. | Bp1. OG Nea1! 1. Eisyop apy Ae1Gagerriysyop po (SES) 76-8230] pp. 97115 (1975). It uses 100-fold dilution of the control (10X) and comparison of growth in the presence of antituberculin with the control. If more than 1% of the inoculum is resistant to the drug, then the growth of the studied isolate will be equal to or higher than the control. If the isolate is sensitive, or less than 1% of the inoculum is stable, then the growth will be less than in the control.

Gold standards for testing tuberculosis sensitivity are the BASTES® 8.L.V. tests. or WASTES® P2A (Vesk! op Okktkop. SosukeukuShe. MO). This is an automated fluid method (8shyet. 8.N. e! A1 .., 1oig. C1sh. M1sgo. 13: 908-912 (1981); pk. 6 .. e! A1 .. 1oit. Aptkto.SyetSheg. 10 : 351-354 (1982); Voeik. 6. Ό .. e! A1 .. 1ig. C1t. M1go. 18: 689-696 (1983); and Taggapy. 1. 1. e! A1 .. 1oig. C1t M1sgo. 27: 941-946 (1985)). In the description of the corporate test, it is indicated that after the growth control index (CO exceeds 30 (6ί _! = ₀ ). The bottle should be taken into account the next day (Οί _{! +} Ι) and the difference between the two days should be determined (6 ^ 1 - Of = LS1 _sop4go 1). Bottles with the test drug are taken into account on the same two days and determine the A61 _ihydride for the drug in the same way. If> A61 _ihydide isolate is considered sensitive. If ABO ^ s> A61 _ihydide isolate is considered borderline. If Abyot ^ s <A61 _ygid isolate is considered insensitive to the Neue .Apyt1sto.Adep to SNET 40:.!.!. 1759-1767 (1996) points out, 6I in the presence of drug should not exceed 50 for 8 days of culture (when the amount of inoculum in vials containing drug is from 1 0 ⁴ 1 0 ⁵ cfu / ml).

In order to correlate the results obtained in our experiments with the proportional method used in test 8.NI.E., the experiment shown in FIG. 36. This experimental scheme was developed to bring existing methods of testing mycobacterial sensitivity into line with the results obtained with betaine-like detergents.

For example, the control bottle for bottles containing the standard Mac Farland with the drug will be the bottle 100X-K.R .; the control for bottles containing 10X dilutions of the drug will be a bottle of 1,000X-K.R .; and the control for bottles containing 100X-fold dilutions of the drug will be a bottle of 10,000X-K.R.

In this experiment, isolates M.SHegsiЦkšk ATCC 27294 and 571/573-BAB were cultured on a 7H11-selective medium for 2 weeks. Cells were scraped off and resuspended in HARS / citrate, sonicated for 60 s, and then the cells were allowed to settle. The solution was adjusted to 0.5 Mac Farland standard and diluted ten times successively up to 10,000 times.

Each dilution series, with the exception of 10,000X dilutions, was seeded in parallel on 7H11 to count the colonies and inoculated in parallel into bottles containing K.R., RAITA with ceftriaxone at a final concentration of 8 μg / ml (P-sax), only CB- 18 at a concentration of 15 μg / ml, 30 μg / ml or 60 μg / ml, or CB-18 in combination with P-sax (CB-1 8 at a concentration of 15 μg / ml, 30 μg / ml or 60 μg / ml) . A dilution of 10,000X was seeded in parallel on 7H11 and inoculated in parallel in bottles containing K.R. or only P-sah.

Colony counting made it possible to determine the 0.5 Mac Farland standard for each isolate. These determinations were then used to determine the amount of cfu inoculated with each dilution in 1 2B vials. The number of seeds sown in each series is shown below. The results obtained on isolate ATCC 27294 are shown in FIG. 37A37E, and the results obtained on isolate 571/573-BAB are shown in FIG. 38A-38E.

Some sown in 1 2B Some sown in 12V Breeding ATCC 27294 571/573-VA 0.5 Mac Farland 6.28 ± 0.57x10 ⁵ 1.11 ± 0.18x10 ⁶ 10X 62,800 ± 5,700 111,000 ± 17,900 100X 6,280 ± 570 11,100 ± 1,790 1,000X 628 ± 57 1,11 ± 179 10,000X 63 ± 6 111 ± 18

At a high inoculum dose (> 1x10 ⁵ cfu), the growth curves of both ATCC 27294 and 571/573-BAB isolates showed an unusual character (Figs. 37A and 38A). Even at a CB18 concentration of 30 μg / ml, the isolates grew unhindered during the first week. At a CB-18 concentration of 30 μg / ml, in combination with P-sax, both isolates after the first week showed an unusual growth retardation. At a concentration of CB-18 of 60 μg / ml (with or without antibiotics), both isolates grew, but in an unusual way: both began to grow, but then slowed down development. Only the ATCC 27294 isolate survived.

The WASTES test uses an analysis of the release of ¹⁴ CO2. Since the vials were counted daily, the ¹⁴ CO2 released as a result of metabolism left the vials every day for the first two weeks. The difference in C1 from day to day is a reflection of the amount of new ¹⁴ СО ₂ formed from the moment of the last accounting. Since the vials were counted daily only during the first two weeks, a positive C1 reflects an increase, but does not exponentially increase C1: it indicates only the absence of new growth. After 2 weeks, the vials were counted every 3-5 days. During this time, C1 accumulated. Using the AC parameter discussed above, in none of the variants using the Mac Farland standard as an inoculum, it was impossible to determine the isolate as sensitive or to differentiate the isolates. Therefore, inoculation of vials with a sowing dose exceeding 10 ⁵ CFU will not be productive and therefore not recommended.

An analysis of 10X dilutions began to give accountable results. Both isolates were sensitive at a concentration of CB-18 equal to 60 μg / ml in the presence of P-sax. Only isolate 571/573-BAB was found to be sensitive at a concentration of CB-18 of 30 μg / ml in the presence of P-sax.

Analysis of serial dilutions showed that at 100X dilution, the ATCC 27294 isolate is sensitive to CB-18 at a concentration of 30 μg / ml in the presence of P-sax. The inoculum of this dilution of isolate 571/573-BAB was sensitive to CB-1 8 at a concentration of 15 μg / ml in the presence of P-sax. Verification of the dilutions of 1,000X yielded identical results for the dilution of 100X, but the growth curves of isolate 571/573-BAB in these last two cases (i.e., 100X and 1,000X) strongly resembled the earlier curves (Figs. 5A-5P and Figs. eleven).

Two conclusions can be drawn from these experiments. Firstly, the ATCC 27294 and 571 / 573BAB isolates can be differentiated, but not under all conditions. Secondly, the lowest limit of testing is represented by one organism, while, on the other hand, the use of a dose of 10 ⁵ bacteria overloads the system. Sowing doses between 100 and 10 ⁴ , make it possible to differentiate isolates, and an inoculum of 1 0 ³ and 10 ⁴ bacilli makes it possible to obtain reproducible results depending on time.

Example 9. The permeability of mycobacteria and the effect of SV-18.

One possible explanation for the mechanism by which SV-18 exerts its effect is the permeability of mycobacteria. Without striving to limit ourselves to the following hypothesis, we present it only for the purpose of illustrating the invention. Betaine-like detergents can alter the permeability of bacteria containing mycolic acid structures, making these bacteria more susceptible to antibiotics. In particular, the effect of CB-1 8 can be the result of changes in the distribution of conformations of mycolic acid structures.

This assumption is based primarily on the work of Wiap, Υ. e! A1., Rgos. Ya! 1.Asay.8s1. 93: 12828-12833 (1996). In general, some mycobacteria have the ability to alter the fluidity of their cell wall (Lb, 1., e! A1., 1Oig. Vu1. Sit. 271: 29545-29551 (1996)). This is achieved either by changing the length of mycolic acids, or by changing the structural conformations of mycolic acids, or both. In other words, both the length and conformation of mycolic acids of the cell wall affect the melting temperature of the cell wall. For example, with a change in the length of mycolic acids or the ratio of trans / cis double bonds, or the number and / or conformations of cyclopropanes, the degree of hydroxylation, methylation and carbonylation, the melting point of mycolic acids also changes (Valu, S.E. e! A1., 1ig. Vu1 Cl. 271: 29545-29551 (1996)). For example, in the case of M.ctedtaIc, with an increase in the temperature of growth, a corresponding increase in the proportion of trans-olefins in the proximal double bond of mycolic acids is observed, which leads to an increase in the melting temperature of these lipids (i.e., decrease in fluidity). In the case of M.! Bibliso818 and M.at. With an increase in the proportion of trans-cyclopropanes in the proximal position, the melting point of mycolic acids increases accordingly.

It is believed that the fluidity of the mycobacterial cell wall plays an important role in permeability and, accordingly, in the stability of и and,, Υ. e! A1., Proc. Oh! 1. Asai. 8cP92: 66306634 (1995); Wgeppap, R.1. e! A1., Appi. Ket. B1ocNo. 64: 29-63 (1995)). Sustainability

M. c1e1opae to cephalosporins is determined exclusively by permeability (Soppe11, Ν.Ό. e! A1., 1n: TiGeGi1o818. Pa! Upepe818, Proc! Es! Upp api Sop1go1. B1oot, V.K. et.A8M Rgekk, No. ! op, Ό. S. (1994) pp. 333-352). For example, Valu, S.E. e! A1., 1ig. Vu1. C1.271: 29545-29551 (1996) showed that when M.ctedtike's cell wall fluidity decreases (i.e., the proportion of trans-olefins increases), the penetration of norfloxacin and chenodeoxycholate also decreases. Kapeya, K., e! A1., 1oig.6ep. M1goGo1.134: 22132229 (1988) showed that in highly stable fast-growing strains, the majority of proximal double bonds are trans-olefins.

The formation of conformations of various mycolic acids occurs primarily with the help of structurally related 8-adenosylmethionine (8AM) -dependent enzymes. Molecular analysis showed that M.! Biegliso818 has four structurally related genes encoding methoxymethylic acid synthases (MMA8), which are responsible for many of these modifications (Anap, E. e! A1., Proc. Baa! 1.

Asai. 8cP 93: 12828-12833 (1996)). Products of these four genes (MMA8-1, MMA8-2,

MMA8-3 and MMA8-4) have a strong sequence homology with the cyclopropane fatty acid synthase E.coH (CPA8) and cyclopropane mycolic synthases M.1ergae and M.! Ilegs1o818 (CMA8-1 and CMA8-2). It is believed that they use the same mechanism. This mechanism involves 8AM and a carbon intermediate (Fig. 39A). Different products are obtained because different synthases have different electron donors / acceptors in their active sites, which leads to the formation of olefins, cyclopropanes, methylation and a combination of hydroxylation with methylation (Wuai Υ. E! A1., Proc. Ba! 1. Asai. 80.93: 12828-12833 (1996)).

Valu e! A1., in the patent and. 8. 5,610,198 indicate that thietetracosanoic acids (thiolated fatty acid derivatives) can be used to inhibit the cyclopropanation and oxygenation of mycolic acids of pathogenic mycobacteria by inhibiting 8AM-dependent reactions (Fig. 39B). Four thietetracosanoic acids have been tested in this regard and their effect has been shown.

In general, the mechanism of inhibition by these analogs provides for their participation as substrates in the first part of the synthase enzymatic reaction (in particular, the transfer of the methyl group from 8AM). After catalysis, a structure is formed, which is called a stable analogue of the transition state (Fig. 39B). Essentially, the compound is not released from the active site and acts as a classic competitive inhibitor. The structure of the transition state resembles a betaine-like detergent. In particular, betaine-based sulfonium (Table 1 and FIG. 29B): enzymatic catalysis is not required to activate sulfonium carboxybetaines and they will directly inhibit 8AM-dependent modifications.

Waggu e! A1., in the patent and. 8. 5,610,198 also state that thietetracosanoic acids are ineffective against saprophytic mycobacteria such as M.ctedta Ic. In contrast to the description of Valu e! A1., (I.8. 5,610,198), it follows from the present invention that such saprophytic mycobacteria are sensitive to the action of betaine-like detergents: from the tested strains M. Goisch! it showed the greatest degree of synergism between CB-18 and cephalosporin. For example, the growth of clinical isolate 495-1HH was not affected by the presence of cefoxitin and it was able to grow in the presence of CB-1 8 at a concentration of 109 μg / ml (Fig. 15). However, when cefoxitin was combined with only 13 μg / ml CB-18, the growth of this isolate was inhibited and completely inhibited at a CB18 concentration of 27 μg / ml.

If betaine-like detergents affect the permeability of mycobacteria at the level of fluidity of the cell wall, through 8AM-dependent enzymes, then shown in FIG. 14 and 15, the results can be considered quite expected. The basis for such expectations lies in the similarity of M. Goisch! Ith and

M. ctedta D.K.Kopop, S., e! A1., 1n: SoobGe11o ^, M. e! A1., ebk. S'etka1 Me! Lobk sh you! Epa1 8uk! Eta! 1sk, Asabetk Rgekk, Lopbop (1985) pp. 237265 and Wgeppap, R. 1. e! A1, Appi. Keu. 64: 29-63 (1995) summarize the distribution of mycobacterial acids of mycobacteria of various species and show that rapidly growing strains (e.g. M. Goyiyit, M. cepelope, M. ctedtike, etc.) have a similar distribution of mycolic acid: primary mycolic acid is an a 'type acid. Bh, 1., e! A1., 1ig. Vu1. Eat. 271: 29545-29551 (1996) showed that with an increase in the temperature of cultivation, the main M. myctedta DK mycolates become myolates of types a _b and a ₂ . Types a _b and a _{2 are} longer than type a '(a' = C ₆₄ , and a ₂ = C _78-79 ), and type a ₂ has a proximal trans-olefin, a structure that gives less fluidity and makes the cell wall less permeable . Type a ₂ prevails at higher temperatures (the structures a ', a ₁ and a _{2 of} mycolic acids are given by Seogde, K., e! A1., 1Oig.Vyu.Set. 270: 27292-27298 (1995)).

The significance of this data becomes apparent in light of the mechanism proposed by Wiap, Υ. e! A1., Proc. IA! 1. Asab. 8c! 93: 12828-12833 (1996) for these 8LM-dependent enzymes (Fig. 39A): the trans-olefin is a product of 8AM-dependent methylation, and fluidity is necessarily proportional to the transolefin content. The addition of CB-1 8 to the culture medium should inhibit the formation of transolefin, thereby increasing the fluidity of the cell wall and increasing the permeability of cells for antibiotics. With increased permeability, antibiotics penetrate the cell more easily. The effectiveness of this antibiotic will depend on the nature of the isolate itself (i.e., the interaction of the antibiotic with the target of action of the drug in the studied isolate). The final result will be manifested in the form of the SV-18 effect.

If 8LM-dependent modifications of fatty acids are a common mechanism by which mycobacteria regulate the fluidity of their cell walls, as suggested (bw, 1., e! A1., 1wig. Wb1.beth. 277: 29545-29551 (1996)), then betaine-like detergents should affect a wide range of mycolic acid structures, such as, for example, cinnamicolates (Oigapb, E. e! a1., Eig. 1oig. Blocet. 93: 103-112 (1979)), and therefore nocardiomycolates.

The above reasoning explains the extreme synergy observed between cefoxitin and CB-18 in the case of M. goyiit (Table 7); however, they do not explain the extreme sensitivity of M.! Biloxioc to betaine-like detergents (Table 8). For example, growth

In the absence of antibiotics, M. bilobactyl strain ATCC 27294 is slightly inhibited at 27 μg / ml CB-1 8 and completely suppressed with the addition of 54 μg / ml CB-18. In the presence of antibiotics, the growth of this isolate was insignificant at 13 μg / ml CB-18 and completely stopped at 27 μg / ml CB-1 8 (Fig. 10). In the absence of antibiotics, the growth of isolate 571/573-BAB M.! Bersi1ok1k was slightly inhibited at 13 μg / ml CB-18 and completely inhibited by the addition of 27 μg / ml CB-18. In the presence of antibiotics, the growth of this isolate was not observed even at 1 3 μg / ml CB-1 8 (Fig. 11). In contrast to M. Goyiyit (Figs. 14 and 15), M.! Biloxiok turned out to be significantly more sensitive to betaine-like detergents per se.

Obviously, slow-growing mycobacteria, such as M.! And Lacti1k1k, do not regulate the fluidity of the cell wall, similar to what happens in fast-growing mycobacteria, such as M.ctedtaDk (L1, 1., e! A1., 1. See 271: 29545-29551 (1996)). For example, Takauata, K. e! A1., At.Keu.Kekr1g.P1k. 118: 113-117 (1978) showed the extreme sensitivity of M.! Libs1ok1k to the cultivation temperature: at a temperature that differs from the optimum by only a few degrees, the synthesis of mycolic acids ceases and M.! Libs1ok1k is unable to survive. The sensitivity of M.! Bieffs1ok1k to the declared betaine-like detergents or to the tietetracosanoic acids described by Waggu, S.E. e! A1., (I.8. 5,610,198) in combination with antibiotics may be a consequence of the generic sensitivity of the isolates M.! biblins to disturbances in the synthesis of mycolic acids. For example, suppression of cyclopropanation or oxygenation of mycolic acids will have an effect on permeability and therefore on sensitivity; violation of the synthesis of mycolic acids in general can have fatal consequences. Induced sensitivity to antibiotics will be observed only at low concentrations of CB-18 and on slowly growing mycobacteria.

If the target of the action of betaine-like detergents is 8AM-dependent synthases of bacteria containing mycolic acid structures, then it can be expected that these detergents will inhibit the modification of fatty acids (i.e., mycolic acids) and, in some cases, cause the cessation of mycolic acid synthesis. Consequently, betaine-like detergents will play only an auxiliary therapeutic role in the treatment of infections caused by rapidly growing organisms.

For example, based on its structure, alkylsulfonium carboxybetaine may be an ideal compound to suppress mycolic acid modifications. Alkylsulfonium carboxybetaine can be synthesized by the modified method 21tteg, K.E. (i. 8. 3,560,393). For example, octadecyl mercaptan can be methylated and K ₁ -8-K ₂ purified by the corresponding type of halide ester (Fig. 39C). Purification by cation exchange chromatography will remove ether to give acid.

Example 10. Betaine sensitivity panels for screening clinical isolates.

Examples 5 and 6 show that different clinical isolates behave differently in tests in which one betaine is used with a number of different antibiotics. From example 6 it is seen that one clinical isolate behaves differently in the test, in which different detergents are used with the composition of the antibiotics P / sa. It follows that for the purpose of sensitivity testing, a betaine sensitivity panel can be developed and refined. In such a panel, various betaines will be combined with various antibiotics. This example describes the creation of such a panel and its use for sensitivity testing. This example is provided only to illustrate the creation of such a panel and does not limit the scope of the invention.

Using various betaines, for example, five, with a wide spectrum of action against various clinical isolates, in combination with several, for example, five, antibiotics, which have a wide spectrum of action against various clinical isolates, create a betaine sensitivity panel. For example, define five antibiotics α-1, α-2, α-3, α-4 and α-5, and five betaines β-1, β-2, β-3, β-4 and β-5. Five different betaine positions in the panel may represent different concentrations of the same detergent, or a combination of different detergents. Similarly, five different antibiotic positions in a panel can represent different concentrations of the same antibiotic, or combinations of different antibiotics. From the table. Figure 11 shows how these five betaines can be combined with these five antibiotics in order to determine the most effective betaine / antibiotic combinations.

low, but should lead to fatal consequences in the case of many slowly growing mycobacteria. For example, if the permeability of slowly growing mycobacteria with the presence of δΆΜ-dependent inhibitors will cause induced sensitivity to antibiotics, then a negative effect on survival due to interference with the synthesis of mycolic acid will be an important, possibly dominant factor. On the other hand, fast-growing mycobacteria, as a rule, will not be exposed to betaine-like detergents on their own, which are characterized by pronounced synergism with antibiotics due to the suppression of the mechanism that regulates membrane fluidity.

Valu e! A1., (and 8. 5,610,198) also claim that only antimycobacterial drugs, such as isoniazid, ethambutol, streptomycin, rifampin, dapzone, rifabutin, clarithromycin, ciprofloxacin, clofosamine, azithromycin, ethionamicin, amicin, amicin or amicin with thietetracosanoic acids. The present invention, illustrated by examples 5 and 6, shows that the presence of CB-18 in a certain way alters the physiology of bacteria, presumably through the mechanism discussed here, inducing sensitivity to a wide range of antibiotics. Therefore, according to the invention, the use of a betaine sensitivity test based on the CB-1 8 effect in combination with a variety of antibiotics to determine the natural sensitivity of the tested isolate can give the doctor more effective recommendations.

The betaine-like detergents of the present invention have significant advantages over the thietetracosanoic acids described by Val e! A1., (i.8. 5,610,198). For example, thietetracosanoic acids are extremely insoluble. In other words, the Kraft temperatures of these reagents are significantly higher than physiologically normal. The introduction of an appropriate concentration becomes impossible. In contrast, betaine-like detergents prepared according to the present invention are, in most cases, soluble. For example, Nobde, Ό. e! A1., Bandtssg 7: 878-884 (1991) synthesized C ₂₀ carboxyhexyl betaine with a Craft point of 20 ° C.

While SV-18 was used to describe the effect of CB-1 8, it is understood that other betaines are also useful in this regard.

Table 11. Betaine Sensitivity Panel Betaine Detergents

Antibiotic ^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

100 α-4 α-4 / α-4 / β-1 α-5 α-5 / α-5 / β-1

Betaine Sensitivity Panels

Use the tab. 11 panel as described below.

(1) Grow the test clinical isolate in a liquid culture (e.g., WASTES 12B) or in a solid medium (e.g. 7H11 jambs).

(2) Betaines and antibiotics can be lyophilized in an appropriate vial and then dissolved in a growth medium, or mixed as individual solutions of an antibiotic, betaine and broth to obtain the same result. The first option is preferred.

(3) In this way, prepare the bacterial stock solution so that the appropriate number of cells (i.e. 100 to 10,000 cfu) gets into each vial or tube. The number of cells in such a stock solution can be adjusted by preparing a suspension based on Mac Farland standards, as is known in the art, with subsequent dilutions.

(4) Incubate plates, tubes or vials for a predetermined time and check growth using one of the selected methods. Growth monitoring can be based on the release of ¹⁴ C (ie, VASTES 12B: VesYumPhsEhp ^ op, Soskeu5U111e, MO), the consumption of O ₂ (E8R Muso 8u5 is ΙΙ ™, ΩΙΕίΌ aoga ope5, ev goy, MC changes!!! pH (MW / BashT®, Delivered η Tekshka, Ligat, ΝΟ) or other standard methods known in the art, such as turbidity.

Such a panel can also be used to classify clinical isolates. For example, if the mechanism (s) by which betaines exert their effect on enhancing antimicrobial therapy is known, then such information will be useful in developing more effective therapeutic regimens depending (or without) on the betaine-antibiotic combination used. Thus, there are two aspects of the invention. According to a first aspect, a betaine susceptibility panel is used as a means of classifying clinical isolates. Such a classification is aimed at choosing a more effective therapeutic regimen, regardless of the use of betaine as a therapeutic adjuvant. According to a second aspect, the goal of testing betaine sensitivity is to determine the combination of a specific betaine-like detergent (s) and a specific antibiotic (s) (as described in the following example (Example 11).

α-4 / β-2 α-4 / β-3 α-4 / β-4 α-4 / β-5 α-5 / β-2 α-5 / β-Ί α-5 / β-4 α -5 / β-5

Example 11. The use of betaine-like detergents as therapeutic adjuvants.

As follows from Example 10, there are two aspects of sensitivity testing. In one aspect, the betaine panel is used as a classic sensitivity test to select a more effective therapeutic regimen for eliminating an infection, in particular one of the panel antibiotics shown in Example 10. In this case, the therapeutic regimen does not involve the use of a betaine-like detergent (s) in as a therapeutic adjuvant (adjuvants).

According to another aspect, the therapeutic regimen is based on the use of one or more antibiotic combinations: a betaine-like detergent included in the panel. In this case, the information obtained using the panel described in Example 10 indicates to the specialist a specific combination of antibiotic (s) and betaine-like detergent (s). In this case, betaine should interact with the patient. The purpose of this example is to describe the use of betaine (betaines) as therapeutic adjuvants. This example illustrates the preparation of such a panel and does not limit the scope of the invention.

The examples and the results show that betaine-like detergents, having limited antimicrobial efficacy when used in isolation, are most effective when used in combination with one or more antibiotics. In order for any antimicrobial therapy to be effective, an antibiotic (antibiotics) must be delivered to the microorganism. Delivery of reg 5e antibiotic in accordance with the claimed methods can be carried out by standard methods known in the art for the delivery of antibiotics. The purpose of this example is to describe how betaine (betaines) can be delivered to an infectious agent. So, if the patient is being treated with an adjuvant in accordance with the claimed methods, at the request of betaine (betaines) can be administered in one way, and an antibiotic (antibiotics) in another.

Standard methods for delivering betaine-like detergent (s) are known in the art and include oral administration, intramuscular injection, intravenous droppers, injection at the site of infection or inhalation. However, betaine-like detergents are surfactants and will not be well tolerated when taken orally or

101

102 intramuscular injections. For example, ingestion of large amounts of betaine-like detergent is likely to disrupt the gastrointestinal tract, and the injection of large amounts of betaine can solubilize cell structures, causing irritation at the injection site. When using intravenous droppers, precautions should be taken regarding the concentration of betaine in the blood. While the administration of betaine at a dose in excess of the critical micellar concentration (SMS) can be tolerated, complications may arise if its total blood level is exceeded above SMS due to the solubilization of blood components. Injection at the site of infection is also possible; however, unfortunately, this is possible only in rare cases, such as tuberculous lesions or granulomatous lesions, for example, with disseminated MAC infection. In a preferred embodiment, betaine (s) is administered by inhalation.

Tuberculosis is primarily a respiratory infection. Tuberculous lesions are usually observed in the upper lobes of the lungs. Given that the surface of the lungs is coated with a natural surfactant, the effective route for delivering betaine-like detergents to the site of infection will be similar to the delivery of β-blockers and steroids in asthmatics. For example, some steroids and β-blockers are usually produced in microcrystalline form, such as VepuBn® (manufactured by A11ep & Napbigu, a division of O1axo, Ke8eabs Tpapdie Ragk, N0).

While the invention has been fully described, it will be understood by a person skilled in the art that the invention can be practiced within wide and equivalent ranges of conditions, parameters, etc., without violating the idea or scope of the invention or any of its embodiments.

Claims (70)

CLAIM 1. A method for determining the sensitivity of microorganisms to antibiotics, comprising contacting a microorganism having mycolic acid structures on its outer membrane with a composition containing an antibiotic and betaine-like detergent, followed by an assessment of the sensitivity of said microorganism to said antibiotic based on the viability of said microorganism in said composition . 2. The method according to p. 1, characterized in that said betaine-like detergent is selected from the group consisting of CB-like, 3B-like, NZV-like, RV-like, C1B-like, RBV-like, ZOV-like, iMB-like, AO-like , CAB-like and 1T-like detergents. 3. The method according to claim 2, characterized in that said CB-like detergent has a structure k, Κ, - [α] „- β®-Κ ₄ - [γ] ^θ I K ₃ where K ₁ is C ₈ -C ₂₂ ; and is —CH ₂ -, —CH (OH) -, - (CO) —NH — CH ₂ CH2CH2-, -O- or -C (0) -; η is 0 or 1; β is -Ν®-, -P®-, or -3®-; Κ2 is —H, —CH ₃ -, —C ₂ H ₅ -, -C ₃ H ₇ - or -C ₄ H ₉ -; Κ3 is —H, —CH ₃ -, —C ₂ H ₅ -, -C ₃ H ₇ - or -C ₄ H ₉ -; Κ4 is -CH ₂ -, -C ₂ H ₄ -, -C ₃ H ₆ -, -C ₄ H ₈ -, ^- ^^ 5 ^ ^H 10 ^-, -C6H12-, -CH2-C6H4-, -StN2t-, -CH (OH) CH2 CH2-, -CH2 CH (OH) CH2- or -C _t H _2t .1 (OH) -, where t>1; γ is -COO ⁰ . 4. The method according to claim 3, characterized in that said betaine-like detergent is selected from the group consisting of No. (carboxymethyl) -H # dimethyl-1-hexadecanaminium, internal salt (CAZ® No. 693-33-4), cococarboxymethyl betaine and CAZ ® No. 68424-94-2), No. (carboxymethyl) -H # dimethyl-9-octadecenamium, inner salt (CAZ® No. 87137-4), Ν- (carboxymethyl) -Ν, Ν-dimethyl-Z - ((1 oxo-octadecyl) amino) -1-propanaminium, inner salt (CAZ® No. 6179-44-8), 3-amino-No. Carboxymethyl) -H # dimethyl 1 -propanaminium No. C ₈ -C ₂₂ and acyl derivatives, internal salt (CAZ® No. 840-44-0), No. (carboxymethyl) -3 - ((12-hydroxy-1 oxo-9-oxtadecenyl) amio) -H # dimethyl-1 propanaminium, internal salt (CAZ® No. 71850-81-2), cocoamidopropylcarboxymethyl betaine (CAZ® No. 61789-39-7 and CAZ® No. 61 789-40-0 ), No. (2-carboxyethyl) -H # dimethyl-1-dodecanamanium, internal salt (CAZ® No. 16527-85-8), No. (2-carboxyethyl) -N№-dimethyl-1-tridecanaminium, internal salt (SAZ ® No. 132621-79-5), No. (2-carboxyethyl) -Н№dimethyl-1-tetradecananaminium, internal salt (SAZ ^® No. 69725-38-3), No. (2-carboxyethyl) -H # dimethyl-1 hexadecanoaminium, internal salt (CAZ® No. 42416-43-3), No. (2-carboxyethyl) -N # dimethyl-1 octadecanoamine, inner salt (SAZ ^® No. 30612-73-8), 103 104 Ν-dodecyl-beta-alanine (CA8® No. 1462-540), N- (3-carboxypropyl) ^^ - dimethyl-1 undecanamanium, internal salt (CA8® No. 150147-53-8), N- (3-carboxypropyl) - ^ No. dimethyl-1 dodecanamine, internal salt (CA8® No. 15163-30-1), N- (3-carboxypropyl) - ^ No. dimethyl-1 tetradecanoaminium, internal salt (CA8 ^® No. 146959-90-2), N- (3-carboxypropyl) - ^ No. dimethyl-1 pentadecanane, internal salt (CA8 ^® No. 146959-91-3), N- (3-carboxypropyl) - ^ No. dimethyl-1 hexadecananamium, internal salt (CA8 ^® No. 71 695-32-4), N- (3-carboxypropyl) - ^ No. dimethyl-1 octadecanamium, internal salt (CA8 ^® No. 78195-27-4), No. (4-carboxybutyl) - ^ No. dimethyl-1 dodecanamanium, internal salt (CA8 ^® No. 120139-51-7), N- (5-carboxypentyl) ^^ - dimethyl-1 dodecanamine, internal salt (CA8 ^® No. 76392-97-7), N- (5-carboxypentyl) ^^ - dimethyl-1 hexadecanaminium, internal salt (CA8® No. 73565-98-7), No. (6-carboxypentyl) - ^ No. dimethyl-1 dodecanamanium, internal salt (CA8 ^® No. 132621-80-8), 4-carboxy-N-dodecyl- ^ N-dimethyl-1 benzenemethanaminium, internal salt (CA8 ^® No. 71695-31-3), 2-carboxy-N-dodecyl- ^ N-dimethyl-1 benzenemethanaminium, internal salt (CA8 ^® No. 71695-34-6), 4-carboxy-No.hexadecyl- ^ No. dimethyl-1-benzenemethanamium, internal salt (CA8 ^® No. 71695-33-5), 2-carboxy-№geksadetsil- ^ №dimetil 1benzenmetanaminium, inner salt (SA8 ^® №.71695-35-7), tallow glycinate (SA8® №.707-46-8), soyamidopropil babassuamidopropil carboxymethyl and carboxymethyl. 5. The method according to p. 4, characterized in that the specified carboxybetaine is No. (3-carboxypropyl) -H # dimethyl-1-octadecanane, internal salt (CB-18) (CA8® No. 78195-27-4). 6. The method according to p. 2, characterized in that the specified 8B-like detergent is selected from the group comprising 8B-18, 8B-16, 8B-14 and 8B12. 7. The method according to any one of claims 1 to 6, characterized in that said composition contains two or more betaine-like detergents. 8. The method according to p. 1, characterized in that said antibiotic is a representative of a class selected from the group consisting of β lactam antibiotic, aminoglycoside, aminocyclitol, quinolone, tetracycline, macrolide, lincosamide, glycopeptide, lipopeptide, polypeptide antibiotic, sulfonamide, trimethoprim, chloramphenicol, isoniazid, nitroimidazole, rifampicin, nitrofuran, methenamine and mupirocin. 9. The method according to claim 8, characterized in that said β-lactam antibiotic is selected from the group comprising penicillin, cephalosporin, monobactam and carbapenem antibiotics. 10. The method according to claim 9, wherein said penicillin is selected from the group consisting of azlocillin, methicillin, nafcillin, cloxacillin, dicloxacillin, oxacillin, ampicillin, bacampicillin, carbenicillin, tricarcillin, meslocillin and piperacillin. 11. The method according to claim 9, characterized in that said cephalosporin is selected from the group consisting of cefoxitin, cefoperazone, ceftazidime, ceftriaxone, cefadroxil, cefazolin, cefalexin, cephaloridine, cefalotin, cefapirin, cefradine, cefacdorf, cefacdorfon, cefacdorfil, cefacdorfil, cefacdorfil, cefacdorfil, cefacdorfil, cefacdorfon, cefacdorfon, cefacdorfon , cefuroxime, loracarbef, cefmetazole, cefotetan, cefixime, cefotaxime, cefpodoxime and ceftizoxime. 12. The method according to claim 9, characterized in that said monobactam is aztreonam. 13. The method according to claim 9, characterized in that said carbapenem is selected from the group consisting of imipenem, meropenem, panipenem and biapenem. 14. The method according to any one of claims 8 to 13, characterized in that said composition also comprises a β-lactamase inhibitor. 15. The method of claim 14, wherein said β-lactamase inhibitor is selected from the group comprising clavulanic acid, sulbactam and tazobactam. 16. The method according to claim 8, characterized in that said antibiotic is an aminoglycoside or aminocyclitol. 17. The method according to clause 16, wherein said aminoglycoside or aminocyclitol is selected from the group consisting of streptomycin, kanamycin, gentamicin, tobramycin, amikacin, sisomycin, netilmicin, neomycin, framycetin and paromomycin. 18. The method according to claim 8, characterized in that said quinolone is selected from the group consisting of naladixic acid, oxolinic acid, cinoxacin, flumequin, miloxacin, roxoxacin, pipemidic acid, norfloxacin, enoxacin, ciprofloxacin, ofloxacin, lomefloxacin, fafafacacin, temafafacin, pefloxacin, amifloxacin, sparfloxacin, levofloxacin, clinafloxacin. 19. The method according to claim 8, characterized in that said macrolide is selected from the group, including 105 106 erythromycin, oleandomycin, spiramycin, josamycin, rosaramycin, clarithromycin, azithromycin, dirithromycin, roxithromycin, flurithromycin and roquitamycin. 20. The method according to claim 8, characterized in that the composition contains two or more antibiotics. 21. The method according to claim 20, characterized in that said antibiotics are sulfonamide and trimethoprim. 22. The method according to item 21, wherein the specified sulfonamide is sulfamethoxazole. 23. The method according to claim 8, characterized in that said rifampicin is selected from the group comprising rifampin, rifamycin 8ν, rifamycin B (rifamide) and rifabutin. 24. The method according to claim 1, characterized in that the antibiotic is selected from the group consisting of amikacin, azithromycin, any β-lactam in combination with any β-lactamase inhibitors, capreomycin, cefmetazole, cefoxitin, ciprofloxacin, clarithromycin, clofazamine, cycloserine, dapzone, ethambutol, ethionamide, imipenem, isoniazid, kanamycin, minocycline, ofloxacin, paraaminosalicylic acid, protionamide, pyrazinamide, rifampin, rifabutin, sparfloxacin, sulfamethoxazole in combination with trimethoprim, streptomycin, tetracycline, th and viomycin. 25. A method for antimicrobial therapy of a patient infected with a microorganism or a person who is at risk of infection with a microorganism having a mycolic acid structure on the outer membrane, comprising administering to this patient a betaine-like detergent and an antibiotic in such quantity and for such a time that are sufficient to kill said microorganism. 26. The method according to claim 25, wherein said betaine-like detergent is selected from the group consisting of CB-like, 8B-like, HB8-like, PB-like, C1B-like, PBB-like, 8oB-like, VSu-like, AO-like, CAB-like and 1T-like detergents. 27. The method according to p. 26, wherein said CB-like detergent has a structure of ₂ I K.-I-β®-K ₄ - [γ] ^θ I And where is B! is C ₈ -C ₂₂ ; α is —CH ₂ -, —CH (OH) -, - (ί.Ό) -ΝΗ-ί.Ή _; CH ₂ CH ₂ -, —O— or —C (O) -; η is 0 or 1; β is -Ν ^φ -, -P ^Ф - or -8 ^Ф -; B ₂ is —H, —CH ₃ -, —C ₂ H ₅ -, —C ₃ H ₇ -, or —C4H9-; B ₃ is —H, —CH ₃ -, —C ₂ H ₅ -, —C ₃ H ₇ -, or —C4H9-; B ₄ is -CH ₂ -, -C ₂ H ₄ -, -C ₃ H ₆ -, -C ₄ H ₈ -, -C5H10-, -C6H12-, -CH2-C6H4-, -StH2t-, -CH ( OH) CH2CH2-, -CH2 CH (OH) CH2-, or -StH2t-1 (OH) -, where m>1; γ is -COO ⁰ . 28. The method according to item 27, wherein the specified CB-like detergent selected from the group including Ν- (carboxymethyl) -Ν, Ν-dimethyl-1-hexadecananamium, internal salt (CA8® No. 693-33-4), cococarboxymethyl betaine and CA8® No. 68424-94-2), No. (carboxymethyl) - ^ No. dimethyl-9-octadecenium, internal salt (CA8® No. 87137-4), Ν- (carboxymethyl) -Ν, Ν-dimethyl-Z - ((1-oxo-octadecyl) amino) -1-propanamine, internal salt (CA8® No. 6179-44-8), 3-amino-no. (Carboxymethyl) -H, no. Dimethyl- 1-propanaminium No. C ₈ -C ₂₂ a, cyclic derivatives, internal salt (CA8® No. 840-44-0), No. (carboxymethyl) -3 - ((12-hydroxy-1 oxo-9-okstadecenyl) amino) -H , No. dimethyl-1 propanaminium, internal salt (CA8® No. 71850-81-2), cocoamidopropylcarboxymethyl betaine (CA8® No. 61789-39-7 and CA8® No. 61789-40-0), No. (2-carboxyethyl) - And, No. dimethyl-1 dodecanaminium, internal salt (CA8® No. 16527-85-8), No. (2-carboxyethyl) -I, No. dimethyl-1-tridecanaminium, internal salt (CA8® No. 132621-79-5 ), No. (2-carboxyethyl) -I, No. dimethyl-1-tetradecanaminium, internal salt (CA8® No. 69725-38-3), No. (2-carboxyethyl) -I, No. dimethyl-1-hexadecananamine, ext morning salt (CA8® No. 42416-43-3), No. (2-carboxyethyl) -I, No. dimethyl-1-octadecanaminium, internal salt (CA8® No. 30612-73-8), Ν-dodecyl-beta-alanine (CA8® No. 1462-540), Ν- (3-carboxypropyl) -H # dimethyl-1 undecanamanium, internal salt (CA8® No. 150147-53-8), Ν- (3-carboxypropyl) -Ν, Ν-dimethyl-1 dodecanamine, internal salt (CA8®№. 15163-30-1), Ν- (3-carboxypropyl) -Ν, Ν-dimethyl-1 tetradecanonium, internal salt (CA8 ^® No. 146959-90-2), Ν- (3-carboxypropyl) -Ν, Ν-dimethyl-1 pentadecanoamine, internal salt (CA8 ^® No. 146959-91-3), 107 108 N- (3-carboxypropyl) -I ^ -dimethyl-1 hexadecananamium, internal salt (CA8® Io.71695-32-4), N- (3-carboxypropyl) -I ^ -dimethyl-1 octadecanaminium, internal salt (CA8® Io.78195-27-4), No. (4-carboxybutyl) -I ^ -dimethyl-1 dodecanamanium, internal salt (CA8® Jo. 120139-51-7), N- (5-carboxypentyl) -I ^ -dimethyl-1 dodecanamanium, inner salt (CA8® Jo. 76392-97-7), No. (5-carboxypentyl) -I ^ -dimethyl-1-hexadecanaminium, inner salt (CA8 ® Io. 73565-98-7), No. (6-carboxypentyl) -I ^ -dimethyl-1-dodecanoaminium, internal salt (CA8® Io. 132621-80-8), 4-carboxy-Y-dodecyl-H, No. dimethyl-1-benzenemethanamine, inner salt (CA8® Io. 71695-31-3), 2-carboxy-Y-dodecyl-N, dimethyl-1-benzenemethanaminium, internal salt (CA8® Io.71695-34-6), 4-carboxy-Y-hexadecyl-N, No. dimethyl-1-benzenemethanamine, internal salt (CA8® Io.71695-33-5), 2-carboxy-Y-hexadecyl-N, No. dimethyl-1-benzenemethanamine, internal salt (CA8® Io.71695-35-7), fatty glycinate (CA8® Io.707-46-8), soiamidopropyl carboxymethylbetaine and babassuamidopropylcarboxymethylbetine. 29. The method according to p. 28, characterized in that the said carboxybetaine is No. (3-carboxypropyl) -I, No. dimethyl-1-octadecanamanium, internal salt (CB-18) (CA8® Io. 78195-27-4). 30. The method according to p. 26, characterized in that the specified 8B-like detergent is selected from the group including 8B-18, 8B-16, 8B-14 and 8B12. 31. The method according to any one of paragraphs.26-30, characterized in that the patient is administered two or more betaine-like detergents. 32. The method of claim 25, wherein said antibiotic is a member of a class selected from the group consisting of β-lactam antibiotic, aminocyclitol, quinolone, tetracycline, macrolide, linkcosamide, glycopeptide, lipopeptide, polypeptide antibiotic, sulfonamide, trimethoprim, chloramphenicol, , nitroimidazole, rifampicin, nirofuran, methenamine and mupirocin. 33. The method according to p, characterized in that the specified L-lactam antibiotic is selected from the group comprising penicillin, cephalosporin, monobactam and carbapenem antibiotics. 34. The method according to claim 33, wherein said penicillin is selected from the group consisting of azlocillin, methicillin, nafcillin, cloxacillin, dicloxacillin, oxacillin, ampicillin, bacampicillin, carbenicillin, tricarcillin, meslocillin, penicillin. 35. The method according to claim 33, characterized in that said cephalosporin is selected from the group consisting of cefoxitin, cefoperazone, ceftazidime, ceftriaxone, cefadroxil, cefazolin, cephalexin, cephaloridine, cephalothin, cephapirin, cephradine, cefaclor, cefamandole, cefonicid, tseforanid, cefprozil , cefuroxime, loracarbef, cefmetazole, cefotetan, cefixime, cefotaxime, cefpodoxime and ceftizoxime. 36. The method according to claim 33, wherein said monobactam is aztreonam. 37. The method according to claim 33, wherein said carbapenem is selected from the group consisting of imipenem, meropenem, panipenem and biapenem. 38. The method according to any one of paragraphs.32-37, characterized in that said method also comprises administering a β-lactamase inhibitor to the patient. 39. The method according to § 38, wherein said β-lactamase inhibitor is selected from the group comprising clavulanic acid, sulbactam and tazobactam. 40. The method according to p, characterized in that said antibiotic is an aminoglycoside or aminocyclitol. 41. The method of claim 40, wherein said aminoglycoside or aminocyclitol is selected from the group consisting of streptomycin, kanamycin, gentamicin, tobramycin, amikacin, sisomycin, netilmicin, neomycin, framycetin and paromomycin. 42. The method according to p, characterized in that said quinolone is selected from the group consisting of naladixic acid, oxolinic acid, cinoxacin, flumequin, miloxacin, roxoxacin, pipemidic acid, norfloxacin, enoxacin, ciprofloxacin, ofloxacin, lomefloxacin, temaf, pefloxacin, amifloxacin, sparfloxacin, levofloxacin and clinafloxacin. 43. The method according to p, characterized in that said macrolide is selected from the group consisting of erythromycin, oleandomycin, spiramycin, josamycin, rosaramycin, clarithromycin, azithromycin, dirithromycin, roxithromycin, flurithromycin and rocithamycin. 44. The method according to p, characterized in that the composition contains two or more antibiotics. 45. The method according to item 44, wherein the indicated antibiotics are sulfonamide and trimethoprim. 46. The method according to item 45, wherein the specified sulfonamide is sulfamethoxazole. 109 110 47. The method according to p, characterized in that said rifampicin is selected from the group comprising rifampin, rifamycin 8U, rifamycin B (rifamide) and rifabutin. 48. The method according to claim 25, wherein said antibiotic is selected from the group consisting of amikacin, azithromycin, any β-lactam in combination with any β-lactamase inhibitors, capreomycin, cefmetazole, cefoxitin, ciprofloxacin, clarithromycin, clofazamine, cycloserine, dapzone ethambutol, ethionamide, imipenem, isoniazid, kanamycin, minocycline, ofloxacin, paraaminosalicylic acid, protionamide, pyrazinamide, rifampin, rifabutin, sparfloxacin, sulfamethoxazole in combination with trimethoprim, streptomycin, tetracycline, scrap and viomycin. 49. The method according to p. 1 or 25, characterized in that said microorganism is Musobas! 50. The method according to 49, characterized in that said Mycobacterium is selected from the group consisting of M. adp, M. aeraceticis, M. acitum ulitumus, M. aglapit, M. ayepse, M. a81a, M. aigit , M.aizgoaGpsapit, M.Aausht, M. Lousch, M. Lou18 (BC6), M.cie1opae, M.cieae, M.ciebiepze, M. sooki, M. biegpoeGe1, M. biauli, M.Ga11ah, M. Gagstodepez, M. Dauezsepz, M. Goysh! Um, M. dabsht, M. dazDt, M. dPuit, M.dogbopae, M. LjetorShsht, Mlp! Gase11i1age, M. kapzazi, M.kotozzepze, M. 1ergee, M. 1ergigigsht, M.tagtit, M.ta1toepze, M.tugoy, M.topokaepze, M.peaigit, M.pops, ready-to-go, M. biepze, M. raga Gopiyit, M. raga! M. regedgtit, M. rb1e1, M. rogstit, M.ropGegae, M. riuei8, M.gobe81ae, M.zsgogShaseit, M.zepedaepse, M.8But1be1, M.81Sh1ae, M.ztedtayz, M.zradt, M. 5 / Idak M.! Eggae, M.111egtoge518Bb1e, M.! Oka1epze, M.! Pu1a1e, M.! Boggi1o818, M.u1segap8, M.uassae, M.khepor 51. The method according to p. 50, characterized in that the specified mycobacterium is a member of the complex Musobas! Excht! Ibeci1o818 (MTB). 52. The method according to p. 51, characterized in that the microorganism is M.! 53. The method according to p. 50, characterized in that said microorganism is a member of the Musobas! Egst aust (MAS) complex. 54. The method according to p. 50, characterized in that the microorganism is a member of the MA18 group. 55. Composition for checking the sensitivity of microorganisms having mycolic acid structures on their outer membrane to antibiotics, characterized in that it contains one or more antibiotics mixed with one or more betaine-like detergents. 56. The composition according to p. 55, wherein at least one of the betaine-like detergents is selected from the group comprising CB-like, 8B-like, HB8-like, PB-like, OB-like, PBB-like, 8oB-like, VeuB-like, AO -like, SAB-like and 1tB-like detergents. 57. The composition according to p. 56, characterized in that said CB-like detergent has a structure of ₂ I β®-K <- [γ] ^θ I in, where B ₁ is C ₈ -C ₂₂ ; α is —CH ₂ -, —CH (OH) -, - (CO) —F — CH ₂ CH ₂ CH ₂ -, —O— or —C (O) -; η is 0 or 1; β is -Ν®-, -P®- or -8®-; B ₂ is —H, —CH ₃ -, —C ₂ H ₅ -, —C ₃ H ₇ -, or —C4H9-; B ₃ is —H, —CH ₃ -, —C ₂ H ₅ -, —C ₃ H ₇ -, or —C4H9-; B ₄ is -CH ₂ -, -C ₂ H ₄ -, -C ₃ H ₆ -, -C ₄ H ₈ -, -C5H10-, -C6H12-, -CH2-C6H4-, -StH2t-, -CH ( OH) CH ₂ CH ₂ -, —CH ₂ CH (OH) CH ₂ - or —C _t H _2t-1 (OH) -, where t>1; γ is -COO ⁰ . 58. The composition according to clause 57, wherein said CB-like detergent is selected from the group including Y- (carboxymethyl) -Y, Y-dimethyl-1-hexadecanaminium, internal salt (CA8® Но. 693-33-4), cococarboxymethyl betaine and CA8® No. 68424-94-2), Y- (carboxymethyl) -Y, Y-dimethyl-9-octadecenium, internal salt (CA8® No. 87137-4), Ν- (carboxymethyl) -Y, Y-dimethyl-3 - ((1 oxo-octadecyl) amino) -1-propanamine, internal salt (CA8® No. 6179-44-8), 3-amino-O (carboxymethyl) -Y, Y-dimethyl1-propanamium No. C ₈ -C ₂₂ and acyl derivatives, internal salt (CA8® No. 840-44-0), Y- (carboxymethyl) -3 - ((12-hydroxy-1oxo-9-okstadecenyl) amino) -Y, Y-dimethyl-1 propanaminium, internal salt (CA8® No. 71850-81-2), cocoamidopropylcarboxymethyl betaine (CA8® No. .61789-39-7 and CA8® No. 61789-40-0), Y- (2-carboxyethyl) -Y, Y-dimethyl-1-dodecanoamine, internal salt (CA8® No. 16527-85-8), Y- (2-carboxyethyl) -Y, Y-dimethyl-1-tridecanaminium, inner salt (CA8® No. 132621-79-5), Y- (2-carboxyethyl) -Y, Y-dimethyl-1-tetradecanaminium, internal salt (CA8® No. 69725-38-3), 111 112 Y- (2-carboxyethyl) -Y, Y-dimethyl-1-hexadecanoamine, internal salt (CA8® Νο.42416-43-3), Y- (2-carboxyethyl) -Y, Y-dimethyl-1-octadecanamanium, internal salt (CA8® Νο. 30612-73-8), Ν-dodecyl-beta-alanine (SL8® Νο.1462-540), No. (3-carboxypropyl) -H ^ dimethyl-1undecananamium, internal salt (CA8 ^® Νο. 150147-53-8), No. (3-carboxypropyl) -H ^ dimethyl-1-dodecanoaminium, inner salt (CA8 ^® Νο.146959-90-2), No. (3-carboxypropyl) -N ^ dimethyl-1-tetradecanaminium, inner salt (CA8 ^® .1ο.146959-90-2), No. ( 3-carboxypropyl) -H ^ dimethyl-1-pentadecanoaminium, internal salt (CA8 ^® .1ο.146959-91-3), No. (3-carboxypropyl) -H ^ dimethyl-1-hexadecananamium, internal salt (CA8® .7ο.71695-32-4 ), No. (3-carboxypropyl) -H ^ dimethyl-1-octadecanane, inner salt CA8 ^® Νο.78195-27-4), N- (4-carboxybutyl UMF Dimethyl-1-dodecanamium, internal salt (CA8® Νο. 120139-51-7), No. (5-carboxypentyl) -N№dimethyl-1 dodecanamium, internal salt (CA8 ^® .ο. 76392-97 -7), No. (5-carboxypentyl) - ^ No. dimethyl-1 hexadecanamanium, internal salt (CA8 ^® 7ο. 73565-98-7), No. (6-carboxypentyl) - ^ No. dimethyl-1 dodecanamanium, internal salt (CA8 ^® Νο. 132621-80-8), 4-carboxy-No. Dodecyl- ^ No. dimethyl-1 benzenemethanaminium, internal salt (CA8® Νο. 71695-31-3), 2-carboxy-no. Dodecyl-no. Dimethyl-1 benzenemethanaminium, internal salt (CA8 ^® Νο. 71695-34-6), 4-carboxy-№hexadecyl-Н№dimethyl-1-benzenemethanamium, internal salt (CA8 ^® Νο.71695-33-5), 2-carboxy-No.hexadecyl- ^ No. dimethyl-1-benzenemethanamium, internal salt (CA8 ^® Νο.71695-35-7), fatty glycinate (CA8® .7ο.707-46-8), soiamidopropyl carboxymethyl betaine and babassuamidopropyl carboxymethyl betaine. 59. The composition according to p. 58, characterized in that said carboxybetaine is Ν (3-carboxypropyl) - ^ No. dimethyl-1-octadecananamium, internal salt (CB-18) (CA8® Νο.78195-27-4). 60. The composition according to p, characterized in that the specified 8B-like detergent is selected from the group comprising 8B-18, 8B-16, 8B14 and 8B-12. 61. A composition according to any one of claims 55-60, characterized in that said composition contains two or more betaine-like detergents. 62. The composition according to p. 55, characterized in that said antibiotic is a member of a class selected from the group consisting of β-lactam antibiotic, aminocyclitol, quinolone, tetracycline, macrolide, linkcosamide, glycopeptide, lipopeptide, polypeptide antibiotic, sulfonamide, trimethoprim, islamonifenicol , nitroimidazole, rifampicin, nirofuran, methenamine and mupirocin. 63. A set for determining the sensitivity of microorganisms having an external membrane of mycolic acid structure to antibiotics, characterized in that it contains one or more betaine-like detergents and one or more antibiotics in the immediate vicinity or neighborhood. 64. The kit according to item 63, wherein at least one of the betaine-like detergents is selected from the group consisting of CB-like, 8B-like, HB8-like, PB-like, C1B-like, RIV-like, 8οΒ-like , YеνΒ-like, AO-like, CAB-like and 1tB-like detergents. 65. The kit of claim 64, wherein said CB-like detergent has a structure To ₂ I κ- ["] _π -β®-κ ₄ - [γ] ^θ I to ₃ where I] is C ₈ -C ₂₂ ; α is —CH ₂ -, —CH (OH) -, - (CO) —KH — CH ₂ CH ₂ CH ₂ -, —O— or —C (O) -; η is 0 or 1; β is -Ν®-, -P®- or -8®-; I ₂ is —H, —CH ₃ -, —C ₂ H ₅ -, —C ₃ H ₇ -, or —C4H9-; I ₃ is —H, —CH ₃ -, —C ₂ H ₅ -, —C ₃ H ₇ - or —C ₄ H 9—; The poison is -CH ₂ -, -C ₂ H ₄ -, -C ₃ H ₆ -, -C ₄ H ₈ -, -C5H10-, -C6H12-, -CH2-C6H4-, -StN ₂ t-, -CH (OH) CH ₂ CH ₂ -, -CH ₂ CH (OH) CH ₂ - or -CtH ^. / OH) -, where m>1; γ is -COO®. 66. The kit according to item 65, wherein said CB-like detergent is selected from the group consisting of No. (carboxymethyl) -H ^ dimethyl-1-hexadecanaminium, internal salt (CA8 ^® Νο. 693-33-4), cococarboxymethyl betaine and CA8 ^® Νο. 68424-94-2), No. (carboxymethyl) -H ^ dimethyl-9-octadecenamium, internal salt (CA8 ^® Νο. 871-37-4), 113 114 I- (carboxymethyl) -I, I-dimethyl-3 - ((1 oxo-octadecyl) amino) -1-propanamine, internal salt (CA8® Io.6179-44-8), 3-amino-Ν (carboxymethyl) -I, I-dimethyl1-propanamine I-C ₈ -C ₂₂ acyl derivatives, internal salt (CA8® Io.840-44-0), No. (carboxymethyl) -3 - ((12- hydroxy-1 oxo-9-okstadecenyl) amino) -X # dimethyl-1 propanaminium, internal salt (CA8® Io. 71850-81-2), cocoamidopropylcarboxymethyl betaine (CA8® Io.61789-39-7 and CA8® Io.61789- 40-0), No. (2-carboxyethyl) - ^ No. dimethyl-1-dodecanamanium, internal salt (CA8® Io. 16527-85-8), N- (2-carboxyethyl) - ^ No. dimethyl-1-tride kanaminium, inner salt (CA8® Io. 132621-79-5), No. (2-carboxyethyl) -X # dimethyl-1-tetradecananium, internal salt (CA8 ® Io. 69725-38-3), No. (2-carboxyethyl) -X # dimethyl-1-hexadecanaminium, internal salt (CA8 ^® Io.42416-43-3), No. (2-carboxyethyl) -X№dimethyl-1-octadecananium, internal salt (CA8® Io. 30612-73-8), No. dodecyl-beta-alanine (CA8® Io.1462 -540) N- (3-carboxypropyl) ^^ - dimethyl-1 undecanamanium, internal salt (CA8® Io. 150147-53-8), N- (3-carboxypropyl) -X ^ dimethyl-1 dodecanamanium internal salt (CA8® Io. 15163-30-1), N- (3-carboxypropyl) ^^ - dimethyl-1 tetradecanoaminium, internal salt (CA8 ^® Io.146959-90-2), I- (3-carboxypropyl) -X ^ dimethyl-1 pentadecanane, inner salt (CA8 ^® Io.146959-91-3), N- (3-carboxypropyl) -X ^ dimethyl-1 hexadecanaminium, internal salt (CA8 ^® Io.71695-32-4), I- (3-carboxypropyl) -I, I-dimethyl-1 octadecanaminium, inner salt (CA8 ^® Io. 78195-27-4), No. (4-carboxybutyl) -X ^ dimethyl-1 dodecanaminium, internal salt (CA8 ^® Io .120139-51-7), I- (5-carboxypentyl) -I, I-dimethyl-1-dodecanane, internal salt (CA8 ^® Io. 76392-97-7), N- (5-carboxypentyl) -H ^ dimethyl-1-hexadecananamium, internal salt (CA8 ^® Io. 73565-98-7), N- (6-carboxype ntyl) -H ^ dimethyl-1 dodecanamine, internal salt (CA8 ^® Io.132621-80-8), 4-carboxy-No dodecyl-X # dimethyl-1-benzenemethanaminium, internal salt (CA8 ^® Io.71695-31-3), 2-carboxy-No dodecyl-X # dimethyl-1-benzenemethanaminium, internal salt (CA8 ^® Io.71695-34-6), 4-carboxy-No.hexadecyl-X ^ dimethyl-1-benzenemethanamium, internal salt (CA8 ^® Io.71695-33-5), 2-carboxy-No.hexadecyl-X ^ dimethyl-1-benzenemethanamium, internal salt (CA8® Io.71695-35-7), fatty glycinate (CA8® Io.707-46-8), soiamidopropyl carboxymethyl betaine and babassuamidopropyl carboxymethyl betaine. 67. The kit according to claim 66, characterized in that said carboxybetaine is No. (3-carboxypropyl) -C # dimethyl-1-octadecanoamine, internal salt (CB-18) (CA8® Jo. 78195-27-4). 68. The kit of claim 64, wherein said §B-like detergent is selected from the group consisting of 8B-18, 8B-16, 8B-14, and 8B12. 69. A kit according to any one of claims 63-68, characterized in that said kit contains a collection of various betaine-like detergents. 70. The kit of claim 63, wherein said antibiotic is representative of a class selected from the group consisting of β-lactam antibiotic, aminoglycoside, aminocyclitol, quinolone, tetracycline, macrolide, lincosamide, glycopeptide, lipopeptide, polypeptide antibiotic, sulfonamide, trimethimenol, trimethimen, , isoniazid, nitroimidazole, rifampicin, nitrofuran, methenamine and mupirocin.