JP2001070000A - Culture medium for detection of vancomycin-resistant enterococcus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject culture medium capable of growing entercococcus and carrying out detection of vancomycin-resistant enterococcus and identification, etc., of vancomycin-resistant type by including component type and induction type glycopeptide-based antibiotics therein. SOLUTION: A component type glycopeptide-based antibiotic (e.g. teicoplanin) and an induction type glycopeptide-based antibiotic (e.g. vancomycin) are added to a culture medium such as Enterococcsel culture medium so as to become 0.1-50 μg/ml final concentration to provide the objective culture medium for detection of vancomycin-resistant enterococcus useful for detection method, etc., of vancomycin-resistant enterococcus bacterium capable of detecting vancomycin-resistant enterocuccus without detecting vanC-retaining enterococcus bacterium and surely carrying out identification of vanA and vanB and capable of identifying vanA, vanB and vanC genotype resistance expression type of enterococcus bacterium according to the growing state.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a vancomycin-resistant enterrococci, VR
E, vanA, and vanB-containing enterococci), and at the same time, distinguish the vancomycin-resistant types (vanA, vanB phenotype) using the expression system for vancomycin of the vanA, vanB genes possessed by vancomycin-resistant enterococci. On how to do it.

[0002]

2. Description of the Related Art Vancomycin-resistant enterococci are detected in various parts of the world such as Europe, the United States and Asia including Japan, and sometimes cause lethal infections as hospital-acquired bacteria.
There is no effective treatment at present if the disease is caused by infection with this bacterium. Therefore, understanding the carrier status of vancomycin-resistant enterococci in patients and the status of colonization in hospitals is essential for preventing the onset of vancomycin-resistant enterococcal infections and preventing hospital-acquired infections. Need to be established. In addition, food contamination is also a problem, and not only reports of foods contaminated with VRE, but also contamination of foods with humans have been reported. Therefore, it is necessary to inspect foods contaminated with VRE, and it is necessary to establish an effective detection method.

As a method for detecting vancomycin-resistant enterococci, Bile esculine azid containing 6 μg / ml of vancomycin is used.
E agar (Difco) and E containing vancomycin 8 μg / ml
nterococcosel agar (BBL) etc. are already on the market, but all commercially available vancomycin-resistant enterococci detection media are not certified as vancomycin-resistant enterococci
It detects vanC-carrying enterococci and is not strictly a vancomycin-resistant enterococcus detection medium. In addition, vancomycin-resistant enterococci (resistance by vanB gene) showing mild resistance to vancomycin may not be detected in some cases, which is a major problem. Further, a growth medium for VRE that is commercially available for testing VRE-contaminated foods also has the same problems as described above.

[0004] Furthermore, in the case of infection with vancomycin-resistant enterococci, it is necessary to perform treatment according to the degree of resistance (resistance by the vanA and vanB genes). Not only is there no way to confirm the difference in resistance phenotypes, but even vanC-carrying strains are detected as vancomycin-resistant enterococci, which involves the risk of erroneous treatment. Also, VR
ERE-contaminated foods have also been reported to cause human contamination, including the risk of community-acquired to hospital-acquired infections, so VRE-contaminated foods need to be tested.

[0005] The resistance mechanism of vancomycin-resistant enterococci has already been studied in detail by Couvalin et al. ¹⁾ . Enterococci harboring the vanA gene are of a constitutive type for vancomycin and teicoplanin (vanA is also of an inducible type, but is referred to as a constitutive type for convenience because it has high to moderate resistance to vancomycin). Enterococci with vanB gene show resistance to moderate to high inducible resistance to vancomycin but non-induced mild to moderate resistance to teicoplanin
²⁾ Enterococci harboring the vanC gene show constitutive mild resistance to vancomycin and teicoplanin2 ⁾ . From the above, it is understood that the strains carrying the vanA, vanB, and vanC genes have different sensitivities to these antibacterial agents and different resistance induction.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to detect vanC-carrying enterococci (enterococci that are not vancomycin-resistant enterococci), which have been erroneously detected by the conventional method, and to sufficiently detect them. It can detect vancomycin-resistant enterococci harboring vanB, which has a relatively low level of resistance, and uses a vanA, vanB genotype resistant phenotype that was not classified at all in the conventional vancomycin-resistant enterococcus detection medium. In addition, it is an object of the present invention to provide a method for detecting and classifying them, and a medium used for the method. Still another object of the present invention is to provide a method in which a conventional test technique in a clinical laboratory (bacterial laboratory) or the like can sufficiently cope with the problem. Further, vancomycin-resistant enterococci can be directly isolated from a sample without separating and culturing the sample. An object of the present invention is to provide a method for detecting and distinguishing resistant enterococci and a medium used in the method.
Another object of the present invention is to provide a method for detecting VRE from a VRE-contaminated foodstuff by a growth culture without reconfirmation, and a medium used in the method.

[0007]

In order to achieve the above object,
The present inventors have conducted intensive studies, and for enterococci having acquired vancomycin resistance, among glycopeptide antibiotics, glycopeptide antibiotics showing constitutive resistance and inducible glycopeptides Detection of vancomycin-resistant enterococci and van
We thought that it was possible to distinguish resistance by A and vanB genes. That is, utilizing the respective susceptibility of teicoplanin to vancomycin-resistant enterococci and vanC-containing enterococci, particularly the induction of vancomycin resistance to vanB gene, and the susceptibility of vancomycin and teicoplanin to vanAmycin, vanB, and vanC gene-bearing enterococci strains. As a result, the present invention has been completed.

That is, the present invention provides a solid medium or liquid medium capable of growing enterococci containing a constitutive and an inducible glycopeptide antibiotic, a medium containing a constitutive glycopeptide and not containing an inducible glycopeptide, This is a medium for detecting vancomycin-resistant enterococci, which comprises a combination of mediums containing both type-type and derivative-type glycopeptides.
Preferably, for detecting vancomycin-resistant enterococci, a combination of a solid medium or a liquid medium capable of growing enterococci containing vancomycin and teicoplanin, and a medium containing teicoplanin but not containing vancomycin, and a medium containing both teicoplanin and vancomycin. It is a medium. Further, another invention of the present invention is to culture enterococci in a medium containing vancomycin and teicoplanin and a medium containing teicoplanan and not containing vancomycin, and the type of vanA, vanB, vanC genotype resistance expression type of the enterococci depending on the growth state. Is a method of identifying

When a solid medium or a liquid medium containing a glycopeptide antibiotic showing constitutive and inducible resistance to vancomycin-resistant enterococci is inoculated with enterococci, the vancomycin-resistant enterococcus harboring vanA and vanB genes is obtained. Only cocci can grow, and vancomycin-sensitive enterococci and enterococci carrying the vanC gene cannot. Also, on the other hand, by inoculating enterococci on a solid medium and a liquid medium containing a constitutive glycopeptide antibiotic, only enterococci having vanA gene can grow, and enterococci having vanB and vanC can be grown. Vancomycin-sensitive enterococci cannot grow. Specific detection of only vancomycin-resistant enterococcus is possible by the method only above,
By combining the following media, vanA, vanB, vanC
The genotype resistance expression type can also be distinguished.

As a glycopeptide antibiotic showing constitutive resistance, teicoplanin is preferable, and as a glycopeptide antibiotic showing inducible resistance, vancomycin is preferable.

[0011] As the basic medium used for the medium containing teicoplanin and vancomycin in the present invention, a medium capable of growing vancomycin-resistant enterococci is used. Brain heart infusion as solid medium
agar, Heart infusion agar, Torypto Soy agar, Mull
er hinton agar, Nutrient agar, blood agar medium, etc., and as a liquid medium, Brain heart infusion b
roth, Heart infusionbroth, Torypto Soy broth, Mull
er hinton broth, Nutrient broth, blood culture media and the like. These media may be commercially available media or original media. The culture medium of the present invention contains such a basic medium containing a glycopeptide antibiotic showing constitutive resistance and a glycopeptide antibiotic showing inducible resistance, and preferably contains teicoplanin and vancomycin. It was made. The concentration of each of the constitutive glycopeptide antibiotics and the derivative glycopeptide antibiotics is preferably in the range of 0.1 to 50 μg / ml, and more preferably in the range of 2 to 15 μg / ml. These media may contain additives such as antibacterial agents and disinfectants that do not interfere with the growth of enterococci.

The detection of vancomycin-resistant enterococci using the medium of the present invention and the identification of the resistance genes are carried out, for example, specifically as follows. First, a medium containing vancomycin and teicoplanin and a medium containing teicoplanin are prepared, and smeared directly from a sample onto a plate. In this case, when a medium containing vancomycin and teicoplanin is prepared on one side and a medium containing teicoplanin is prepared on the other side using a two-part plate, vancomycin-resistant enterococci can be detected on one plate and the resistance gene can be identified. become. After smearing the sample bacteria, 8 ~
After culturing for 48 hours, the growth status of the cultured bacteria is confirmed. The culture temperature is preferably from 27 to 42 ° C.

The medium containing teicoplanin without vancomycin can be prepared using the same basic medium as the medium containing both of the above. In this case, the concentration of teicoplanin is 0.1 to 50 for both the solid medium and the liquid medium.
It is preferably in the range of μg / ml, more preferably 2-1.
It is in the range of 5 μg / ml.

The liquid medium is also suitable for growing and culturing VRE-contaminated foods such as foods, but the preparation method is the same as described above. A medium containing vancomycin and teicoplanin and a medium containing teicoplanin are prepared, and directly from the specimen. To the liquid medium. After culturing for 8 to 48 hours, the state of growth of the cultured bacteria is confirmed. The culture temperature is preferably from 27 to 42 ° C.

Since the enterococci having the vanA gene are highly resistant to both vancomycin and teicoplanin, the growth of the bacteria can be confirmed in both the medium containing vancomycin and teicoplanin and the medium containing teicoplanin. In addition, the strain containing vanB cannot grow on a teicoplanin-containing medium because it is sensitive to teicoplanin, but since vancomycin induces resistance to teicoplanin, growth can be confirmed on a medium containing vancomycin and teicoplanin.
In addition, vanC-containing strains grown on conventional VRE detection media are teicoplanin-sensitive and do not grow on teicoplanin-containing media, and their resistance is not induced by vancomycin, and conversely, a synergistic effect occurs with vancomycin and teicoplanin. Therefore, it cannot grow on a medium containing vancomycin and teicoplanin. Therefore, no vanC-containing strain grows in any of the present detection media.

[0016]

EXAMPLES Example 1 (Comparison between vancomycin-resistant enterococci and PCR method) The test methods are described below. Plates are made with Enterococcsel medium containing 8 μg / ml vancomycin and 4 μg / ml teicoplanin. On the other hand, a plate is prepared with Enterococcsel medium containing 4 μg / ml teicoplanin. The bacterial solution cultured overnight is adjusted to 10 ⁷ CFU / ml, smeared on the front surface of the medium prepared using a sterile cotton swab, and determined after culturing at 37 ° C. for 18 hours. Liquid medium is also produced in the same manner but, when considering the VRE contamination of food, the amount of contaminants was added to a final bacterial suspension concentration 10 ³ CFU / ml for less, 3
It is determined after culturing at 7 ° C. for 18 hours.

As samples, 20 vancomycin-resistant enterococci carrying vanA, 19 vanB-carrying enterococci, 16 vanCmycin-carrying enterococci, and 3 vancomycin-sensitive enterococci were used. In each case, a strain whose van gene was confirmed by the PCR method was used. Vancomycin-sensitive enterococcus is the agar medium,
The above liquid culture medium, VRE detection medium manufactured by BBL (Enteroco
ccosel agar) was not observed. In addition, a VBL detection medium manufactured by BBL (Enterococcosel aga
Comparison with this detection method was performed using r). And NCC
The minimum inhibitory concentration (MIC) according to the LS method was measured.

FIG. 1 shows the results on an agar medium. On the left is vancomycin 8 μg / ml and teicoplanin 4 μg / m
The right side is a plate of Enterococcsel medium containing 4 μg / ml of teicoplanin, and the right side is a plate of Enterococcsel medium containing 4 μg / ml. FIG. 1 (a) shows the results of vancomycin-resistant enterococci carrying vanA, and FIG. The results are shown in FIG. 1 (c) for vanC-carrying enterococci.

Since vancomycin-resistant enterococci harboring vanA are highly resistant to both vancomycin and teicoplanin, vancomycin 8 μg / ml and teicoplanin 4 μm are used.
g / ml-containing Enterococcsel medium also contains teicoplanin 4
Growth is also observed in the Enterococcsel medium containing μg / ml. Vancomycin-resistant Enterococcus harboring vanB contains 4 μg / ml teicoplanin because it is sensitive to teicoplanin
Although it does not grow on Enterococcsel medium, its resistance is induced by vancomycin, so that vancomycin 8
Enteroco containing μg / ml and 4 μg / ml teicoplanin
Grow in ccsel medium. Since vancomycin-induced enterococci do not induce teicoplanin resistance by vancomycin, they do not grow on both media containing teicoplanin. In addition, common enterococci sensitive to vancomycin are not sensitive to both vancomycin and teicoplanin, and thus do not grow on both media like vanC. Therefore, the only enterococci that grow on these media are vancomycin-resistant enterococci (VRE).

FIG. 2 shows the results of the Enterococcsel liquid medium containing 8 μg / ml of vancomycin and 4 μg / ml of teicoplanin. After inoculation of vancomycin-resistant enterococci with vanA on the left, vancomycin-resistant enterococci with vanB in the middle and vancmycin-resistant enterococci in the middle on the right, 37 ° C. after inoculation of 10 ³ CFU / ml of vanctomycoccus with vanc.
Results after 18 cultures. The vancomycin-resistant enterococcus harboring vanA proliferates because of its strong resistance to both teicoplanin and vancomycin, decomposes esculin in the Enterococcsel liquid medium, and turns the culture solution black. vanBmycin-resistant enterococcus harboring vanB is susceptible to teicoplanin,
Because the resistance is induced by vancomycin, it grows and the culture turns black. Since vanC-carrying enterococci are sensitive to both teicoplanin and vancomycin, and appear until the synergistic action of both drugs, they do not grow in this liquid medium. Vancomycin-sensitive enterococci do not grow as well.

Table 1 shows the results of vancomycin-resistant enterococci carrying vanA. Table 2 shows the results of vancomycin-resistant enterococci carrying vanB. Table 3 shows the results for enterococci harboring vanC.

[0022]

[Table 1]

[0023]

[Table 2]

[0024]

[Table 3]

As shown in Table 1, vancomycin-resistant enterococci harboring vanA are highly resistant to vancomycin and teicoplanin, and therefore vancomycin 8 μg / ml.
All the tested strains grew on the Enterococcsel medium containing 4 μg / ml of teicoplanin and on the Enterococcsel medium containing 4 μg / ml of teicoplanin. Also, BBL
All strains also grew on the VRE detection medium (Enterococcosel agar) manufactured by the company.

As shown in Table 2, the vancomycin-resistant enterococci harboring vanB were all grown on the Enterococcsel medium containing only vancomycin 8 μg / ml and teicoplanin 4 μg / ml, and teicoplanin 4 μg / m
1 did not grow on Enterococcsel medium. Also,
VBL detection media (Enterococcosel agar) manufactured by BBL
All strains grew.

[0027] As shown in Table 3, vanC-carrying enterococci were:
Vancomycin 8 µg / ml and Teicoplanin 4 µg / ml
Teicoplanin 4 μg also in Enterococcsel medium containing ml
All strains tested did not grow on the Enterococcsel medium containing / ml either. However, all strains grew on the VBL detection medium (Enterococcosel agar) manufactured by BBL. The liquid medium had similar results.

[0028]

[Effect of the Invention] Enterococcosel aga which is a conventionally used medium for detecting vancomycin-resistant enterococcus (VRE) is widely used.
r (BBL) etc. are not vancomycin-resistant enterococci v
Detecting even anC-carrying enterococci, strictly speaking VRE
Not a detection medium. Previous VREs were vanA, vanB, and vanC-carrying enterococci, but vanC-carrying enterococci have been excluded from the VRE because they are not pathogenic. Enterococcosel agar (B
An existing VRE detection medium such as BL) is an earlier VRE detection medium, and is therefore made to detect even vanC-containing enterococci. Thus, vanC-bearing enterococci would be detected, which is now not VRE, but vanC.
The detection of retained enterococci will significantly reduce the accuracy of these media. However, the detection method of the present invention does not detect vanC-containing enterococci, and can reliably discriminate between vanA and vanB.
It has the advantage that it can promptly make recommendations on how to treat infected people.

It is a well-known fact that foods are contaminated with VRE, and has already been reported in Japan by the Ministry of Health and Welfare. Therefore, VRE inspection in foods will become a major problem in the future. Since the number of bacteria of VRE in foods is small, it is necessary to conduct tests after growing and culturing them. Even if the medium and the medium in which vancomycin is added after combining them, even the vanC-containing enterococcus grows, so a retest is required to test for VRE. However, if the cells are grown and cultured in the liquid medium of the present invention, only the VRE grows, which has the advantage of eliminating the need for retesting. In addition, vancomycin 8 μg /
Enterococ containing 4 ml of teicoplanin and 4 μg / ml g / ml
Entero containing csel liquid medium and 4 μg / ml teicoplanin
Combination of coccsel liquid medium allows simultaneous discrimination between vanA and vanB.

[0030]

[Literature] 1) Courvalin P. Resistance of enterococci to
glycopeptides. Antimicrob.Agent Chemother. 34: 2291
-2296,1991. 2) Arthur M., and P.Courvalin.Genetics and mechanis
ms of glycopeptide resistance in Enterococci. Anti
microb.Agents Chemother. 37: 1563-1571, 1993.

[Brief description of the drawings]

FIG. 1 is a photograph showing the results of culturing enterococci on an agar medium containing only teicoplanin and vancomycin and teicoplanin.

FIG. 2 is a photograph showing the results of culturing in a liquid medium containing vancomycin and teicoplanin.

Claims (8)

[Claims] 1. A solid medium capable of growing enterococci, comprising a constitutive and an inducible glycopeptide antibiotic. 2. A liquid medium capable of growing enterococci, comprising a constitutive and an inducible glycopeptide antibiotic. 3. A medium for detecting vancomycin-resistant enterococci, which comprises a combination of a medium containing a constitutive glycopeptide and no inducible glycopeptide and a medium containing both a constitutive and an inducible glycopeptide. 4. The medium according to claim 1, further comprising an additive that does not interfere with the growth of enterococci. 5. The concentration of each of the constitutive and inducible glycopeptide antibiotics is 0.1 to 50 at the final concentration.
The medium according to any one of claims 1 to 3, wherein the medium is µg / ml. 6. The medium according to claim 1, wherein the constitutive glycopeptide is teicoplanin. 7. The medium according to claim 1, wherein the inducible glycopeptide antibiotic is vancomycin. 8. Enterococci are cultured in a medium containing vancomycin and teicoplanin and a medium containing teicoplanin but not vancomycin, and the type of resistance to the genotypes of vanA, vanB and vanC of said enterococci is identified based on their growth state. Method.