Emergence of vanA gene among vancomycin-resistant enterococci in a tertiary care hospital of North - East India

Enterococci have emerged as the leading causes of multiple drug resistant hospital-acquired pathogens especially with the emergence of glycopeptide-resistant enterococcus (GRE) species. It is the second most common pathogen causing mortality and morbidity1 and the third leading cause of hospital acquired bloodstream infection2. These not only pose challenge to the clinicians but also result in treatment failure, selection pressure and spread of resistant strains in the health care institutes.

GRE strains were observed after almost 25 years of vancomycin use since 1980s. In the United States, vancomycin-resistance Enterococcus faecium accounted for 4 per cent of healthcare-associated infections3. In Europe, prevalences of VRE is diverse ranging from <1 to >40 per cent4. Resistance to glycopeptides is mediated by the van gene clusters, which produce resistance by altering the drug target from D-alanine-D-alanine to D-alanine-D-lactate5. So far, eight genotypes of glycopeptide resistance, which differ in the level and range of resistance and in transferability to glycopeptides, have been described for enterococci. Five of the van genes are acquired (vanA, B, D, E, G) and three (vanC1, C2, C3) are intrinsic. Of these, vanA is the most prevalent and is predominantly found in E. faecium and E. faecalis, the enterococcal species responsible for most infections in human6. This study was undertaken to detect the presence of vancomycin resistant enterococci (VRE) and that of vanA among the enterococcal isolates in a tertiary care hospital in North-East (NE) India.

Material & Methods

A total of 67 consecutive, non-repetitive clinical isolates of Enterococcus species from different clinical samples (urine, blood, sputum, pus and throat swabs) were obtained in the department of Microbiology and this study was carried out in the Genetic Laboratory, under department of Microbiology, Gauhati Medical College and Hospital and National Institute of Pharmaceutical and Education and Research, Guwahati. The approval from the institutional ethical committee was obtained prior to the study.

Enterococcus species were identified in accordance with the standard procedures5.

The isolates were stored for further processing in Luria Bertani (LB) broth with 20 per cent glycerol at -80°C.

Antibiotic susceptibility testing: Antibiotic susceptibility testing was done using Kirby-Bauer disk diffusion method as per the CLSI (Clinical Laboratory Standards Institute) guideline7 against vancomycin(30 μg), teicoplanin (30 μg), linezolid (30 μg), penicillin G (10 U), tetracycline (30 μg), ciprofloxacin (30 μg), chloramphenicol (30 μg) and doxycycline (30 μg). The quality control organisms were E. faecalis ATCC 51299 (VRE) and E. faecalis ATCC 29212. (Hi-media, Mumbai).

Phenotypic detection of vancomycin resistant Enterococcus: Vancomycin resistance in all enterococcal isolates was detected by disk diffusion method and VRE agar dilution screening test7. Vancomycin supplement (6 μg, Hi-media, Mumbai) was added to 1 ml of sterile distilled water and then mixed to 1000 ml of the autoclaved brain heart infusion (BHI) agar at 45°C and the medium was pour in a petridish and the bacteria was inoculated and incubated at 37°C for 48 h. Growth of even single colony was considered as vancomycin-resistant Enterococcus (VRE).

MIC for vancomycin-resistant Enterococcus: Minimum inhibitory concentration (MIC) value for vancomycin was determined using E-test strip (Hi-media, Mumbai). Any Enterococcus was considered VRE if the MIC was ≥ 16 μg/ml⁷.

Polymerase chain reaction (PCR) for vanA gene: The isolates resistant to vancomycin were taken for plasmid DNA isolation and amplification. A single colony was picked from a freshly streaked blood agar plate and inoculated in 3 ml L-B broth where it was grown at 37°C for 12-16 h with constant shaking. The culture was incubated for 1 h at 37°C with lysozyme (5 mg/ml). The bacterial cells were harvested by centrifugation at 6000 x g for 15 min at 4°C. The pellet was taken and resuspendend in tris-acetate-EDTA (TAE) buffer and heated in a heat block at 95°C for 5 min. It was further centrifuged at in ‘g’ 15000×g for 5 min at 4°C and the supernatant containing DNA was used as a source of template for amplification.

The PCR amplification for vanA was performed8 with some modifications. The reaction mixture with a final volume of 50 μl contained 3 μl of purified plasmid DNA, 1 × PCR buffer (20 mM Tris-HCl/50 mM KCl, pH 8.4), 7 mM MgCl₂, 0.2 mM each deoxynucleoside triphosphate, 0.5 nM each primer, and 2.5 units of Taq polymerase (Qiagen, Mumbai). The PCR conditions were 95° C for 5 min for the first cycle; 95°C for 1 min, 50°C for 1 min and 720C for 1 min for the next 30 cycles and final extension at 72°C for 10 min.

The vanA gene primer sequence (5’-3’) used was: forward primer - A1 GGGAAAACGACAA TTGC and reverse primer -A2 GTACAATGCGGCCGTTA9. Amplification of gene was carried out by DNA thermal cycler with the help of specific primers for vanA gene. E. faecalis ATCC 51299 was used as positive control for vanA and the negative control consisted of all reagents but no DNA template. PCR product was analyzed by electrophoresis with 1.5 per cent agarose gel and 0.5 mg ethidium bromide added separately. Gel was photographed on an ultraviolet light transilluminator by gel documentation system (Perkin Elmer, USA). 100 bp molecular marker was used and a final product of 732 bp was considered as vanA gene.

Results & Discussion

A total of 67 enterococcal isolates were collected from 896 culture-positive samples screened over the one year period. The majority of isolates were recovered from urine specimens followed by isolates from blood, sputum, pus and throat swab; 49 (73%) of the enterococcal isolates were obtained from hospitalized patients and 18 (27%) were from outpatient department. Among these 67 enterococcal isolates, 16 (24%) VRE and 51 (76%) vancomycin sensitive enterococcus (VSE) were detected (Table I); 54 (81%) were E. faecalis and 13 (19%) E. faecium and among the VRE, nine (56.3%) E. faecalis and seven (43.7%) E. faecium were detected.

Table I Distribution of enterococcal isolates in various clinical specimens

Of the 67 isolates, 46 (68.6%) were multiple drug resistant to more than one class of antibiotics, three (4.5%) were resistant to linezolid (Table II) and were found to be VRE. The linezolid resistance was found in two E. faecalis and one E. faecium VRE isolates. The VRE isolates were resistant to ciprofloxacin (69%), tetracycline (56%), penicillin (58%) and erythromycin (100%). Most of the VRE were found to be sensitive to tecoplanin and linezolid (Fig. 1). Among the VRE, MIC to vancomycin ranged between 16-32 µg/ml.

Table II Antibiogram of enterococcal isolates from the clinical samples (n=67)

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Fig. 1

Antibiogram sensitivity of VRE isolates. TEI, teicoplanin; LZ, linezolid; CIP, ciprofloxacin; DO, doxycycline; TE, tetracycline; P, penicillin; E, erythromycin; C, chloramphenicol.

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Nine of 16 VRE isolates were found to possess VanA gene with a 732 bp PCR product (Fig. 2) of which six were E. faecium and three E. faecalis.

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Fig. 2

Agarose gel electrophoresis showing amplification of 723 bp fragment for vanA of VRE from the clinical samples. Lanes 1-3, 5-6: VRE strains represents the vanA amplified 732 bp fragment, Lane 7 - E. faecium ATCC 29212 (negative control), Lane 8-100 bp ladder marker.

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In the present study, 81 per cent E. faecalis and 19 per cent E. faecium were detected. Earlier studies from various parts of India had shown 55-87 per cent of E. faecalis as the predominant species followed by 10-20 per cent of E. faecium10111213. In some studies a relatively high proportion of E. faecium has been reported1415. In our study maximum isolation was from the urine followed by blood. This finding was consistent with other studies121617.

A study from Lucknow reported VRE in 55.17 per cent of the isolates18. Other Indian studies reported VRE isolation between 0-5 per cent101113. Studies from Indore and Nagpur reported 14.29 and 11.38 per cent VRE, respectively1920. In our study the VRE isolation was 24 per cent.

Studies from New Delhi reported MIC range of vancomycin >32 μg/ml10, from Chandigarh 8-16 μg/ml11 and from Mumbai 8-128 µg/ml13. Studies from Italy21, Japan22 and Turkey23 showed MIC range of vancomycin 16-32 μg/ml similar to our study.

The vanA gene was found in 56.25 per cent of the VRE isolates in our study. Mathur et al10 reported 80 per cent of vanA phenotype which was higher than our findings. Studies from Taiwan24 and Brazil25 reported vanA gene in all the VRE isolates. The steady rise in the proportion of vancomycin-resistant E. faecalis isolates in the United States from 2.7 per cent in 1999 to 3.9 per cent in 2010 has been reported26.

In conclusion, emergence of VRE and vanA gene among the isolates in a tertiary care hospital setting in NE India points towards implementing prevention and control policies.