JPH11169196A - Detection of vancomycin-resistant bacterilim and culture medium for detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and rapidly detect the subject resistant bacterium by utilizing opposing reaction of vancomycin with a β-lactam agent, putting a test bacterium solution into a culture medium containing them, culturing the bacterium and observing growth of vancomycin resistant bacterium. SOLUTION: Vancomycin and a β-lactam agent such as a penicillin-based antibacterial agent, a cephem-based antibacterial agent, a monobactam-based antibacterial agent, a carbapenem-based antibacterial agent or a β-lactamase inhibitor are added to a liquid culture medium or a plate agar in which gram- positive bacterium grows so that vancomycin concentration in the culture medium is 3-6 μg/ml and a test bacterial solution is put in the above liquid culture medium or applied onto the above plate agar and the bacterium is cultured for a time enough to grow vancomycin resistant bacterium and growth of vancomycin resistant bacterium is observed to simply, rapidly and surely detect vancomycin-resistant bacterium such as hetero type vancomycin-resistant Staphylococcus aureus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting vancomycin-resistant bacteria useful in the clinical field and a medium for detection. More specifically, the present invention relates to a method for detecting vancomycin-resistant Staphylococcus aureus and a culture medium for detection. The present invention also relates to a kit for detecting vancomycin-resistant Staphylococcus aureus comprising such a detection medium.

[0002]

BACKGROUND OF THE INVENTION Over the past few decades, a number of antibiotics of various structures have been developed and used to treat bacterial infections. Important among these antibiotics are β-
Lactams, glycopeptides, macrolides, quinolones,
Includes tetracyclines and aminoglycosides.

[0003] However, with the use of antibiotics,
The frequency of emergence of mutant strains of bacteria that are resistant to antibiotics has also been increasing, and in recent years, MRSA (Methicillin-resistant Staphyloco
ccus aureus) is a major problem as an infectious disease.
This is so-called as methicillin, a penicillin that is hardly hydrolyzed by penicillinase, which was first developed as an antibacterial agent effective against penicillin-resistant Staphylococcus aureus. However, M
The essence of RSA is not only methicillin, but also antibacterials called β-lactams, that is, all penicillins, including first, second, third, and fourth generations, including penicillins for resistant staphylococci. It is resistant to almost all currently marketed β-lactam drugs, such as all of the cephem, so-called monobactams and carbapenems.

[0004] Vancomycin (hereinafter sometimes referred to as VCM) is a cyclic glycopeptide antibiotic produced by Streptomyces orientalis and is composed of two sugars (glucose and vancosamine) and seven amino acids. ing. Due to its molecular weight, it is difficult to pass through the outer membrane, so it has no effect on Gram-negative bacteria, but has a strong effect on Gram-positive bacteria.

[0005] Vancomycin binds to D-Ala-D-Ala at the terminal of murine monomer, which is a component of cell wall, and inhibits the step of incorporation of murine monomer into the cell wall (Ra
bindra K. Sinha et al., J. Bacteriol. 96: 374-382,
1968; Nieto, M. et al., Biochem. J. 123: 773-787,
1971; Nieto, M. et al., Biochem. J. 123: 789-803,19
71; Joel P. Mackay et al., J. Am. Chem. Soc. 116: 3
481-4590, 1994)). As a result, the cell wall of the bacterium is weakened and died because it cannot withstand the intracellular pressure.

The clinical use of vancomycin is old and 195
It was used in the treatment of penicillin-resistant Staphylococcus aureus infections in six years, but has been less used due to the development of penicillinase-stable semisynthetic penicillins and the renal toxicity of vancomycin itself. In Japan, 1
In 981, oral vancomycin hydrochloride was approved for bone marrow transplantation only for sterilization of the digestive tract. After that, MRSA
Due to the spread of infectious diseases, intravenous vancomycin was approved in 1991.

Although vancomycin has been in clinical use for 30 years, no resistant bacteria have emerged, and
After the emergence of SA, it has been widely used as its silver bullet.
However, avopalcin-resistant enterococci emerged due to large doses of the same line of avoparcin administered to livestock. This strain is also vancomycin-resistant Ent.
erococcus: VRE). This proved that antimicrobial drugs always produced resistant bacteria.

[0008] To date, vancomycin-resistant bacteria isolated from the clinic are the enterococci described above (Enterococcus).
Genus) and CNS Staphylococcus epidermid
es) (Sanyal, D. et al., J. Antimicrob. Chemother. 3
2: 267-278, 1993) and S. haemolyticus (Veach, LA.
l., J. Clin. Microbiol. 28: 2064-2068, 1990; Auber
t, G. et al., J. Antimicrob. Chemother. 25: 491-493,
1990), but not S. aureus.

The present inventors have found that despite receiving vancomycin administration for about one month, not only the symptoms were not improved, but also the heart of a 4-month-old boy with pulmonary atresia who was gradually worsening. M in the midline incision
Mu50, a Staphylococcus aureus strain resistant to vancomycin, was detected from the RSA wound infection site and isolated in pure culture (Hiramatsu, K. et al., J. Antimicrob. Chem.
other. 40: 135-136, 1997). Since vancomycin cannot be treated with vancomycin, the antibacterial activity of vancomycin against Mu50 was examined. NCCLS (National Comm.
The MIC (minimum inhibitory concentration: the minimum concentration of the antibacterial agent that completely inhibits growth) according to the ittee for Clinical Laboratory Standards method was 8 μg / ml. This value is classified as vancomycin mild resistance according to NCCLS criteria (National Committee for Clinical Labora
tory Standards villanova.Pa. 1993). This fungus, VRE
The vanA, vanB, vanC gene group observed in
It had a completely new resistance mechanism.

From the population curve obtained from the vancomycin concentration and the number of surviving cells, the Mu50 strain having an MIC value of 8 μg / ml was considered to be a strain having uniformly high resistance to vancomycin.

[0011]

The present inventors have now determined that MRSA having a MIC value of less than 8 μg / ml for vancomycin.
However, they found that there were cases of vancomycin treatment failure. The MIC value of this strain (Mu3 strain) isolated from a case of MRSA pneumonia that did not respond to vancomycin treatment was 2 μg / m
1 and classified as a susceptible bacterium according to NCCLS criteria. However, this strain has a frequency of about 10 ^-6 or more, and the vancomycin-resistant Staphylococcus aureus (VRSA: MIC value of 8 μg / m
l).

The population curve suggests that the Mu3 strain is a heterogeneous population having various degrees of resistance (FIG. 1), and the present inventors have named it the hetero VRSA (Hirama).
tsuet al., Lancet, in Press).

As described above, this strain grows into MIC when grown.
It is very important to detect at an early stage, as it produces a value of 8 μg / ml VRSA, causing vancomycin-ineffective infection. However, heterogeneous VRSA
The C value is low and cannot be detected by ordinary detection methods. Therefore, there is a need for a simple, rapid, and reliable method for detecting such vancomycin mildly resistant Staphylococcus aureus.

[0014]

SUMMARY OF THE INVENTION As noted above, vancomycin has been used in combination with β-lactams to treat MRSA infections. This is because vancomycin and β-lactam were thought to have a synergistic effect on MRSA infections.

However, in the Mu3 strain, which is a hetero VRSA, the present inventors have surprisingly found that vancomycin antagonizes a β-lactam agent. This finding completely reverses the conventional wisdom of the medical community.

The present invention has been made based on such novel findings.
An object of the present invention is to provide a method, a medium and a kit for simply, rapidly and reliably detecting hetero-type vancomycin-resistant Staphylococcus aureus.

That is, the present invention provides a method for detecting hetero vancomycin-resistant Staphylococcus aureus, a detection medium, and a detection kit utilizing an antagonistic reaction between vancomycin and a β-lactam agent.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The detection method of the present invention can be implemented in various modes. Includes liquid culture method and agar plate culture method.

In a first embodiment, which is carried out by a liquid culture method, for example, the present invention comprises the following steps: a) placing a test bacterial solution in a liquid medium containing gram-positive bacteria containing vancomycin and a β-lactam agent; b) culturing for a time sufficient for the growth of vancomycin-resistant bacteria, and d) observing the growth of vancomycin-resistant bacteria in a liquid medium.

In a second embodiment, which is carried out by agar plate culture, for example, the present invention comprises the following steps: a) A test bacterial solution is placed on an agar plate medium containing gram-positive bacteria containing vancomycin and a β-lactam agent. Smearing; b) culturing for a time sufficient for the growth of vancomycin-resistant bacteria; and d) observing the growth of vancomycin-resistant bacteria on agar plates.

The detection method of the present invention in the third embodiment, which is carried out by an agar plate culture method, comprises, for example, the following steps: a) smearing a test bacterial solution on an agar medium containing vancomycin-containing gram-positive bacteria; A) placing the disc containing the β-lactam agent on the medium prepared in a), c) culturing for a time sufficient for the growth of vancomycin resistant bacteria, and d) observing the growth of vancomycin resistant bacteria around the disc. .

An agar plate using a β-lactam agent disk, because the growth of vancomycin-resistant bacteria around the disk can be visually observed and easy to judge, and the test method is simple and uniform results can be obtained. A simple detection method by a culture method is preferred. In this simple detection method, since a gradient of the concentration of the β-lactam agent is formed around the disk, it is possible to detect resistant bacteria in a wide concentration range, and therefore, even when a new type of resistant bacteria appears, it is easy. This can be detected.

The present invention also provides a medium for detecting vancomycin-resistant Staphylococcus aureus, which contains vancomycin and optionally a β-lactam agent in a medium in which gram-positive bacteria grow, which is used in the above-mentioned detection method. In the first and second embodiments described above, the medium contains a β-lactam agent,
In the third embodiment using a lactam agent disk, the medium does not contain a β-lactam agent.

The medium in which the gram-positive bacteria grow in the method for detecting vancomycin-resistant Staphylococcus aureus of the present invention is not particularly limited.
y Broth), TSA (Tripticase Soy Agar), MHB (Muller
Hinton Broth), MHA (Muller Hinton Agar), Columbia agar medium, MSA (Mannitol Salt Agar) and the like can be used. Particularly preferred is HI (Heart Infusio
n) Medium, HIA (Heart Infusion Agar) medium, BHI (Brai
n Heart Infusion) medium, BHIA (Brain Heart Infusio)
n Agar) medium.

The concentration of vancomycin in the medium is 3 to 6 μg.
/ Ml, and most preferably 4 μg / ml.

The pH of the medium is preferably from 6.0 to 8.0, more preferably from 6.5 to 7.5, and most preferably pH 7.0. The pH can be adjusted using a buffer such as a sodium phosphate-phosphate system.

[0027] In a further preferred embodiment of the present invention, the medium further contains one or more substances supporting a cell wall synthesis system. The substances that support the cell wall synthesis system are amino acids, sugars,
It is preferably selected from the group consisting of a peptide, a murein monomer precursor, an inorganic salt, a vitamin, a nucleic acid component, and ATP. As the amino acid, for example, lysine, glutamic acid, glycine, alanine, glutamine and the like are preferable, and the amino acid may be any of D-form, L-form and DL-form. Examples of sugar include glucose, glucosamine,
Muramic acid and the like are preferable, and peptides such as Al
a-Glu, Ala-Glu- Lys, Ala-Glu-Lys-Ala-Ala, are preferred, such as Ala-Ala, as inorganic salts such as MnCl _2, MgCl _2, Fe
Cl _{2 and the} like are preferable, and as the vitamins, for example, thiamine and nicotinamide are preferable, and as the nucleic acid component, for example, uracil, uridine, uridine monophosphate and the like are preferable.

The added amount of the substance supporting the cell wall synthesis system is, for example, 0.0% for amino acids as the concentration in the whole medium.
It is preferably from 0.01 to 10%, more preferably from 0.01 to 1%. The added amount of sugar is preferably 0.1 to 20%, and 1 to 10
% Is more preferred. 0.001 to 1
0% is preferable and 0.01 to 1% is more preferable. The amount of the inorganic salt added is preferably 0.00001 to 1.0%,
0.0001-0.1% is more preferable. The amount of added vitamins is preferably 0.00001 to 0.1%,
001 to 0.01% is more preferable. The addition amount of the nucleic acid component is preferably 0.001 to 1.0%, and 0.01 to 1.0%.
0.1% is more preferable. 0.001 to 10% is preferable, and 0.01 to 1%
Is more preferred. The amount of ATP added is 0.00001-
1.0% is preferable, and 0.001 to 0.1% is more preferable.

An additional component solution can be prepared by appropriately combining these substances supporting the cell wall synthesis system, and added to the hetero VRSA detection medium of the present invention.
Examples of preferred additional component solutions are shown in the examples, but are not limited thereto, and various combinations are possible.

The β-lactam agent used in the detection method of the present invention includes antibacterial agents such as penicillin, cephem, monobactam and carbapenem, and examples of penicillin antibacterial agents include MZPC (mesulocillin), TIPC
(Ticarcillin), MCI-PC (cloxacillin),
AMPC / CVA (Augmentin), AMPC (Amoxycillin), ABPC / SBT (Sultamicin), T
IPC / CVA (ticarcillin / clavulanic acid), PC
-G (penicillin), ABPC (ampicillin), CB
Examples include PC (carbenicillin), SBPC (sulbenicillin), PIPC (piperacillin), and MPI-PC (oxacillin). Examples of cephem antibacterial agents include CPZ (cefoperazone), CZX (ceftizoxime), CXM (cefuroxime), and LMOX ( Moxalactam), CCL (cefachlor), CFPM (cefepime), CAZ (ceftazidime), CTT (cefotetan), CTRX (ceftriaxone), CPR (cefoprome), CDZM (cefodizim), FMOX (flomoxef), CFIX (cefixime), CBPZ (Cefbuperazone), CFTM (Cefteram), CMNX (Cefminox), CPM (Cefpyramide), CPZ / SB
T (Cefoperazone / Sulbactam), CPDX (Cefpodoxime), CETB (Ceftibutene), CFDN
(Cefdinir), CEMT (Cefetamet), CET
(Cephalotin), CEZ (cefazoline), CEX
(Cephalexin), CMZ (cefmethazole), CT
M (Cefotiam), CFS (Cefsulosin), CMX
(Cefmenoxime), CFX (cefoxitin), CT
X (cefotaxime) and CMD (cefamandol). Examples of the monobactam antibacterial agent include CRM.
N (carmonam) and AZT (aztreonam), and examples of the carbapenem antibacterial agent include IPM / C
S (imipenem / cilastatin), PAPM / BP (carbenine), MEPM (meropenem), BIPM (biapenem). As will be shown in the examples below, the method of the present invention was able to detect using any of these β-lactam agents. It is preferred to use multiple of these β-lactam agents to ensure detection.

The concentration of the β-lactam agent in the disc varies depending on the type of the bacterium and the type of the β-lactam agent. Can be easily determined by simple preliminary tests. Further, as described later, it was also revealed that various β-lactam agent disks commercially available as susceptibility test disks can be used in the method of the present invention.

For example, CNX30 (30 μg of CMNX)
/ Disc abbreviation including disc), CFM5 (disc abbreviation including 5 μg of CFIX / disc) and ATM30
(Abbreviation of a disk containing AZT at 5 μg / disk).

A preferred method for detecting hetero VRSA of the present invention comprises the following steps: (1) A test bacterium is added to an overnight culture of Tripto soy broth (TSB).
Alternatively, a single colony is suspended in fresh TSB, and a bacterial solution of 0.3 (Mac Farland # 1) is prepared at OD 578 nm in physiological saline. (2) Immediately using a sterile cotton swab as a medium for vancomycin resistance test (At this time, it is preferable to paint from at least two directions), (3) β-
Place a lactam disk and gently press the disk with tweezers. (4) Incubate at 35-37 ° C. for 24-48 hours. (5) Observe the presence or absence of growth of bacteria around the disk.

In the above test, when growth is observed around the disk, it is judged as hetero VRSA. If growth is observed not only around the disk but also on the entire surface of the medium, it is judged as homo VRSA. If no growth is observed in the medium, it is determined as a susceptible bacterium.

The present invention provides a kit for detecting vancomycin-resistant Staphylococcus aureus, in addition to the above-described method and medium for detecting vancomycin-resistant Staphylococcus aureus. The detection kit includes at least a medium for detecting vancomycin-resistant Staphylococcus aureus of the present invention,
The detection medium can be of any format. The kit may further include one or more types of β-lactam agent-containing discs, a cotton swab, a standard bacterial solution, instructions for use, and the like.

The Mu3 strain, which is a hetero VRSA, is about 10 ^-6
It was confirmed that a homo VRSA (MIC value: 8 μg / ml) was produced at the above frequency. Hetero-VRSA is also found in about 10% of MRSA detected in Japanese university hospitals, and may be involved in many vancomycin-ineffective cases.

[0037] Vancomycin is an excellent treatment for MRSA infection. However, as found by the present inventors, vancomycin and a β-lactam agent show an antagonistic effect in hetero VRSA. Thus, if heterozygous VRSA is detected, the combination of vancomycin with a β-lactam should be avoided and continued abuse of vancomycin will undoubtedly increase vancomycin-resistant bacteria.

Therefore, simple and reliable detection of hetero-VRSA at an early stage is very important, so that a therapeutic agent for MRSA can be determined. The clinical significance of the present invention based on novel findings is extremely important.

The present invention will be described in more detail in the following examples, but the scope of the present invention is not limited thereto. Various changes and modifications can be made by those skilled in the art based on the description in the present specification, and these are also included in the scope of the present invention.

[0040]

EXAMPLES Example 1 Isolation and Characterization of Hetero-VRSA Isolation of Mu3 Strain Mu3 strain was clinically isolated from a patient (64-year-old Japanese male) infected with MRSA pneumonia after surgery for lung cancer. The patient was treated with vancomycin (42 mg / kg / day) for 12 days but was ineffective, and the pneumonia had worsened during the last 4 days of vancomycin treatment. The patient was then treated with ampicillin /
10 by combination of sulbactam and arbekacin
Successfully treated for days. Immediately before the end of the combination treatment, Mu3 was separated from the patient's purulent sputum.

An MRSA strain (Mu5) having an MIC value of 8 μg / ml of vancomycin derived from another strain used for comparison.
0) was isolated from the site of MRSA wound infection that occurred at the midline heart surgery incision in a 4-month-old boy with pulmonary atresia,
(Hiramatsu, K. et al., J. Antimicrob. Chemother.
40: 135-136, 1997).

The MRSA H1 strain was isolated from the purulent sputum of an MRSA pneumonia patient (86-year-old, Japanese male). This patient had idiopathic chronic renal failure (Ccr = 11.5 ml / min) and had pneumonia in 21 patients.
Treatment was successful with vancomycin (0.5 or 1.0 g every 4-5 days) for one day.

A strain of Staphylococcus aureus, FDA209P (ATCC
6538P) was purchased from the National Institute of Health.

The sensitivity test MIC value was measured using a BHI agar plate. 5 × 10 ⁴ CFU / sp after incubation at 37 ° C. for 16 hours
The minimum inhibitory concentration of an antibiotic that inhibits
C value. The MIC value of the Mu3 strain measured by this method is 2 μg
/ Ml, and is classified as a susceptible bacterium according to NCCLS standards.

Population analysis The bacterial resistance subpopulation analysis (population analysis) was performed by diluting a cell suspension (prepared by diluting an overnight culture cultured at 578 nm to an OD of 0.3) and 50 μl of a serial dilution thereof with various types. BHI containing vancomycin at a concentration
Performed by smearing on agar plates. Plate is 37
After incubation at 48 ° C for 48 hours, the number of mature colonies was counted. The number of resistant cells theoretically contained in 50 μl of the cell suspension was calculated and plotted on a log-log graph.

Vancomycin-resistant Staphylococcus aureus
The results of comparing the population analysis of Mu50 and Mu3 with the population analysis of vancomycin-sensitive MRSA H1 and FDA209P are shown in FIG. Mu50 cells were able to grow 100% in 4 μg / ml vancomycin, and about 0.001% of cells could grow in 10 μg / ml vancomycin.

On the other hand, Mu3 was considered to be more sensitive to Vancomycin than Mu50, since 4 μg / ml vancomycin inhibited almost 99.99% of the cells. However, Mu3, like Mu50, is 5-9 μg
V / ml vancomycin in the presence of a resistant subpopulation.

On the other hand, the growth of vancomycin-sensitive MRSA H1 and FDA209P cells was completely inhibited at 100% at 4 μg / ml.

Therefore, the Mu50 strain was obtained from the population curve obtained from the vancomycin concentration and the number of viable cells,
It is considered that the strain has a uniform high resistance to vancomycin, and the present inventors propose that it is a homo VRSA (vancomycin).
ycin-resistant Stapylococcus aureus), while the Mu3 strain was confirmed from a population curve to be a heterogeneous population containing cells of various resistance levels. This was named hetero VRSA.

Example 2: Homo VRS from hetero VRSA strain Mu3
Appearance of A It was examined whether resistant cells contained in the Mu3 strain could generate a homo VRSA such as Mu50. Various concentrations of vancomycin
A subclone of Mu3 was obtained by exposing Mu3.

As a result, a total of 82 resistant subclones (10 subclones each at a vancomycin selective concentration of 1 to 8 μg / ml and 2 subclones at a vancomycin selective concentration of 9 μg / ml) were obtained, and their MIC values were determined. did. Table 1 shows the appearance frequency and MIC distribution of resistant subclones.

[0052]

[Table 1]

As the concentration of vancomycin used for selection increased, the MIC value range of the subclone also increased. However, subclones that achieved a vancomycin resistance level of Mu50 (MIC of 8 μg / ml) were obtained only when the vancomycin concentrations used for selection were 8 and 9 μg / ml.
ml only.

Further, by analyzing Table 1, Mu3
From this, it was confirmed that homo VRSA appeared at a frequency of about 10 ⁻⁶ or more.

Example 3 Screening for Homo VRSA and Hetero VRSA A total of 129 MRSA strains obtained from seven university hospitals in Japan and a total of 97 strains obtained from 195 non-university hospitals in Japan
0 MRSA strains were tested. The test was performed by simple population analysis. That is, the bacterial solution cultured overnight was transferred to OD 5
The solution was adjusted to 0.3 at 78 nm, and the bacterial solution was spotted on a BHI agar plate containing 4 μg / ml vancomycin. The plates were incubated at 37 ° C. for 48 hours, and after 24 hours and 4 hours.
Eight hours later, cell growth was observed. If no cell growth is observed at 48 hours, it is considered sensitive to vancomycin. If confluent growth is observed within 24 hours, the strain is suspected to be a homo VRSA. If a measurable number (1-30) of colonies is observed within 48 hours, the strain is suspected to be a heterozygous VRSA. When selected at 8 μg / ml vancomycin,
A homologous VRSA is defined as a subclone that has a MIC of 8 μg / ml or greater and has a resistance level that remains stable after 9 days of culture in drug-free medium. Finally decided.

As a result, only one specimen from a university hospital was determined to be homo VRSA, and was not detected from other Japanese hospitals. On the other hand, heterozygous VRSA was 9.3% (12/129) at university hospitals and 1.3% at non-university hospitals.
(13/970).

Example 4: Examination of the culture medium for hetero VRSA detection (1) The examination of the culture medium in the Agar dilution method was carried out as follows.

MICs were obtained using MHA (Muller Hinton Agar) and MHA, BHIA and HIA media supplemented with horse serum.
The value was determined. FIG. 2 shows the obtained results. In MHA,
0.5 μg / ml for FDA209P strain and MRSA H1 strain, 1 for Mu3 strain
μg / ml and the Mu50 strain showed 4 μg / ml. All of the horse serum-added MHA showed values almost twice. Mu3-8R obtained by selecting Mu3 strain at 8 μg / ml of vancomycin
The strain and the Mu50 strain were at 8 μg / ml. The media showing MIC similar to this MIC value were BHIA and HIA.

(2) mircobroth diluti according to the NCCLS method
The MIC was measured by the on method and compared with the result of (1). As shown in FIG. 3, the FDA209P strain was 0.5 μg / ml, the MRSA H1 strain was 1 μg / ml, the Mu3 strain was 2 μg / ml, and the Mu50 strain was 8 μg / ml.
g / ml. Agar dil in BHIA, HIA
The MIC value of the ution method reflects these results,
It was suggested that A and HIA were suitable as VRSA detection media.

(3) Population analysis of the Mu3 strain was performed using each of the STA, MHA, TSA, HIA and BHIA media, and the media was further examined. As shown in FIG. 4, STA (Sensitivity Test Agar), MHA, TS
Compared with A, HIA and BHIA showed a population curve shifted to a higher concentration side about twice as high as the vancomycin concentration, and were considered to be suitable for detection of resistant strains.

Example 5: Effect of Combination of Vancomycin and β-Lactam Agent on Hetero VRSA The effect of combination of vancomycin and β-lactam agent was performed by the Checker board method using an agar plate dilution method, and the minimum fractional inhibitory concentration was obtained from the following equation.
The index (min. FIC index) was calculated, and the synergistic effect, additive effect, incompleteness and antagonism were determined based on the criteria shown below. Table 2 shows the obtained results.

[0062]

[Table 2]

Note that Mu6, Mu7, Mu13, Mu20, Mu22, Mu2
6, Mu46 and Mu49 are M that we have separated from clinical material.
RSA strain.

As is clear from Table 2, the Mu3 strain exhibited antagonism with vancomycin with all β-lactam agents.

Example 6: Antagonism between vancomycin and β-lactam agent Vancomycin and β-lactam agent (SBT / ABPC)
Was confirmed by population analysis using a medium containing 1 μg / ml of vancomycin.

The growth of the Mu3 strain on a medium in which various concentrations of SBT / ABPC shown in FIG. 5 were added to a 1 μg / ml HIA medium was examined. As shown in FIG. 5, the residual viability in the medium containing 1 μg / ml of vancomycin alone was 1%.
Although it was 0 ³ CFU / ml, when SBT / ABPC was added, an increase in the residual survival rate was confirmed in the range of 0.125 to 8 μg / ml. This indicates a clear antagonism between SBT / ABPC and vancomycin.

Example 7: Induction of vancomycin resistance by β-lactam agent Mu, a hetero VRSA, depending on the presence or absence of β-lactam agent
Changes in vancomycin resistance of the three strains were examined. A gradient gel of BHIA medium having a concentration gradient of vancomycin of 0 to 4 μg / ml was prepared and examined. One of the gradient gels did not contain a β-lactam agent, and the other was added with 0.1 μg / ml of CZX as a β-lactam agent.

Sensitive strains FDA209P strain (ATCC6538P), MR
Mu50 of SA H1 strain and homo VRSA were used as controls.

FIG. 6 shows the obtained results. FDA209P strain (A
TCC6538P) and the MRSA H1 strain exhibited low MIC values regardless of the presence or absence of CZX, and the Mu50 strain exhibited high MIC values regardless of the presence or absence of CZX. However, Mu3
It was observed that the strain shifted to a higher MIC value in the CZX-supplemented medium than in the CZX-free medium in the strain. This indicates that vancomycin resistance was induced by the addition of the β-lactam agent.

Example 8 Induction of Vancomycin Resistance by Various β-Lactam Disks
The test was performed using lactam disks.

Mu pre-cultured overnight at 37 ° C. in TSB medium
Three strains were used as test bacteria.

BHI containing 3 μg / ml vancomycin
A medium was smeared with Mu3 strain adjusted to McFarland 1.0, and various β-lactam agent disks (PCG 10
μg / ml, CEZ 10 μg / ml, CZX 10 μ
g / ml, LMOX 100 μg / ml)
Incubated at 7 ° C for 20 hours.

FIG. 7 shows the obtained results. Any β-
A growth circle was also observed around the disc with the lactam, and β-
It was confirmed that the lactam agent induced vancomycin resistance.

Although the results are not shown, the homo VRSA Mu
In 50 strains, the bacteria grew on the entire surface of the plate.

Example 9: Optimal Induced Concentration of β-Lactam Agent The optimal induced concentration of β-lactam agent disk in the simple detection method of hetero VRSA was examined.

Mu pre-cultured overnight at 37 ° C. in TSB medium
Three strains were used as test bacteria.

BHI containing 3 μg / ml vancomycin
A3 medium was smeared with Mu3 strain adjusted to McFarland 1.0, and β-lactam agent disks (CMZ
10 μg / ml and 30 μg / ml disks) and incubated at 37 ° C. for 20 hours.

FIG. 8 shows the obtained results. 10 μg / m
1 g, uniform growth was observed around the disc, and 30 μg
At / ml, growth was observed in a ring at a certain distance around the disk. From this test, it can be seen that the β-lactam agent has an optimal induction concentration in hetero VRSA detection.

Example 10: Effect of additional components on the medium The effect of adding a substance supporting the cell wall synthesis system to the medium on the detection was tested.

The following ingredients: sodium phosphate 69.6 g glucose 25.7 g L-lysine 0.18 g L-glutamic acid 0.74 g L-glycine 0.38 g alanine 0.22 g MnCl ₂ 0.50 g uracil 0.10 g thiamine 0 0.0004 g of nicotinamide was dissolved in 500 ml of sterilized distilled water, adjusted to pH 7.0 with 0.1 N phosphoric acid, and sterilized and filtered to prepare an additional component solution.

Vancomycin 3 μg / ml and 4 μg / ml
A HIA medium containing ml was prepared as well as a medium in which the above additional component solution was added to the same medium (actually, prepared by adding the components of the HIA medium to the above additional component solution). Hetero-VRSA (Mu3 strain) was smeared over both media. CZX 10 μg / ml as a β-lactam agent,
And 30 μg / ml were used to compare the detection of heterogeneous VRSA (Mu3 strain).

FIG. 9 shows the results when vancomycin was used at 3 μg / ml (the left side shows the case where no additional component solution was added, and the right side shows the case where it was added). It can be seen that the detection can be carried out only with the HIA medium, but the addition of the additional component solution makes the detection more clear.

FIG. 10 shows the results when vancomycin 4 μg / ml was used (the left side shows the case where no additional component solution was added, and the right side shows the case where the additional component solution was added). It can be seen that it is not detected only in the HIA medium, but can be detected very clearly when the additional component solution is added.

Example 11: Measurement results using various β-lactam agents Using penicillin-based, cephem-based, and other β-lactam agent disks, strain Mu3 and heterogeneous VRSA J4- isolated separately by the present inventors. Nine strains were detected. Incubate at 35 ° C. in BHIA medium containing 4 μg / ml vancomycin (containing the additional component solution described in Example 9),
The results were determined after 4 hours and 48 hours.

The results obtained are shown in Tables 3 and 4.

[0086]

[Table 3]

[0087]

[Table 4]

In the table, "out" indicates that a colony was formed away from the disk, and "in" indicates that a colony was formed by sticking to the disk. In addition, the size of the growth was visually determined and evaluated from 1+ to 3+. The star is 2
It was not observed after 4 hours, indicating that it was observed after 48 hours.

It was found that hetero VRSA can be detected using any of the β-lactam agents.

[0090]

According to the present invention, hetero VRSA which cannot be detected by the conventional method can be detected simply and reliably. Hetero-VRSA is considered to be a precursor of homo-VRSA, and its early detection is important in selecting an appropriate therapeutic. In the method of the present invention, since a concentration gradient is formed around the β-lactam agent disk, not only the detection of the hetero VRSA strain discovered until now, but also the hetero VRSA strain newly appearing, the gradient concentration There is a high possibility that it can be detected at any of the sites.
The use of the present invention in the clinical field is extremely large.

[Brief description of the drawings]

FIG. 1. Mu50, a homo VRSA, Mu3, a hetero VRSA,
And Population curves of vancomycin-sensitive MRSA H1 and FDA209P.

FIG. 2 shows the results of obtaining MIC values of various bacteria using each medium of MHA, horse serum-added MHA, BHIA and HIA.

Fig. 3 Microbroth dilution MIC according to NCCLS method
FIG. 3 shows the results obtained by comparing the MIC measured by the agar plate dilution method with HIA and BHIA media.

FIG. 4 STA, MHA, TSA, HIA and BHIA
Shows the results of the population analysis of the Mu3 strain using each of the mediums.

FIG. 5 shows the results of a population analysis showing the antagonism between vancomycin and a β-lactam agent.

FIG. 6 shows the results of a test using a gradient gel medium for the change in vancomycin resistance of the Mu3 strain depending on the presence or absence of a β-lactam agent.

FIG. 7 shows the results of a vancomycin resistance induction test performed using various β-lactam agent disks.

FIG. 8 shows an example of the concentration of β-lactam agent contained in a β-lactam agent disk and obtained colonies.

FIG. 9 shows the effect of addition of an additional component in a medium containing 3 μg / ml of vancomycin.

FIG. 10 shows the effect of addition of an additional component on a medium containing 4 μg / ml of vancomycin.

Continuation of the front page (71) Applicant 595117091 1 BECTION DRIVE, FRA NKLIN LAKES, NEW JE RSEY 07417-1880, UNITED STATES OF AMERICA (72) Inventor Hideaki Hanaki 560-4, Hiratsuka, Kanagawa Prefecture

Claims (15)

[Claims] 1. A method for detecting hetero vancomycin-resistant Staphylococcus aureus utilizing an antagonistic reaction between vancomycin and a β-lactam agent. 2. The method according to claim 1, wherein the β-lactam agent is selected from the group consisting of penicillin antibacterial agents, cephem antibacterial agents, monobactam antibacterial agents, carbapenem antibacterial agents and β-lactamase inhibitors. 3. The following steps: a) placing the test bacterial solution in a liquid medium in which gram-positive bacteria containing vancomycin and a β-lactam agent grow; b) culturing for a time sufficient for the growth of vancomycin-resistant bacteria; 2. The detection method according to claim 1, further comprising: d) observing the growth of vancomycin-resistant bacteria in the liquid medium. 4. The following steps: a) spreading the test bacterial solution on an agar plate medium on which a gram-positive bacterium containing vancomycin and a β-lactam agent grows; b) culturing for a time sufficient for the growth of vancomycin-resistant bacteria 2. The detection method according to claim 1, comprising: d) observing the growth of vancomycin-resistant bacteria on an agar plate. 5. The following steps: a) A test bacterial solution is spread on an agar plate medium on which a gram-positive bacterium containing vancomycin grows. B) A disc containing a β-lactam agent is placed on the medium prepared in a). 2. The detection method according to claim 1, comprising: c) culturing for a time sufficient for the growth of vancomycin-resistant bacteria; and d) observing the growth of vancomycin-resistant bacteria around the disk. 6. A medium in which a gram-positive bacterium grows is an HI medium,
The detection method according to any one of claims 3 to 5, which is a HIA medium, a BHI medium, or a BHIA medium. 7. A vancomycin concentration in a medium of 3 to 6 μg.
The detection method according to any one of claims 1 to 6, wherein the concentration is / ml. 8. A medium having a vancomycin concentration of 4 μg / m
8. The detection method according to claim 7, wherein l is 1. 9. The detection method according to claim 1, wherein the detection is carried out by combining a plurality of β-lactam agents. 10. A medium for detecting vancomycin-resistant Staphylococcus aureus, comprising vancomycin and optionally a β-lactam agent in a medium in which gram-positive bacteria grow. 11. The medium according to claim 10, wherein the medium in which Gram-positive bacteria grow is HI medium, HIA medium, BHI medium or BHIA medium. 12. The medium according to claim 1, wherein the concentration of vancomycin in the medium is 3 to 6 μm.
The medium according to claim 10 or 11, which is g / ml. 13. The method according to claim 1, wherein the concentration of vancomycin in the medium is 4 μg /
The medium according to claim 12, which is ml. 14. A kit for detecting vancomycin-resistant Staphylococcus aureus, comprising at least the medium for detecting vancomycin-resistant Staphylococcus aureus according to claim 10. 15. The detection kit according to claim 14, further comprising one or more kinds of discs containing a β-lactam agent.