resistance pattern of enterococcal isolates, phenotypic characterization of vancomycin resistant isolates from various clinical samples in a tertiary care hospital

Introduction: Enterococci cause a multitude of infections and emergence of vancomycin resistance is of special concern, as it is the primary alternative drug to penicillin for treating Enterococcal infections. Objectives: The present study was taken up to know the species predominance, resistance pattern and various phenotypes of vancomycin resistant Enterococci in a tertiary care hospital in south India. Material & methods: The Data collected from the department of Microbiology at Krishna Institute of Medical Sciences, Secunderabad from January 2012 to December 2015 was retrospectively analysed. 226 Enterococci were speciated, antibiograms were analyzed and phenotyping of VRE was done based on standard guidelines. Results & discussion: The predominant species was found to be E. faecalis (51.3%) followed by E. faecium (43.3%), E. gallinarum (2.2%), E. durans (1.32%), E. casseliflavus (0.88%) and E. raffinosus (0.88). E. faecalis showed less resistance (30.39%) to antimicrobials than E. faecium (50.64%). The resistance pattern was less in out-patient samples when compared to in-patient samples. Vancomycin resistant Enterococci were 13(5.75%), all isolated from inpatients. Among the 13 isolates, 6(46.15%) were VanA, 4(30.76%) were VanB and 3(23.07%) were VanC phenotypes. VanC phenotype was observed in E. casseliflavus and E. gallinarum due to intrinsic resistance. Conclusion: The species predominance of E. faecalis and E. faecium reflects the change in recent decade. Drug resistance pattern and phenotyping is correlating with other studies, VanA being the commonest. Monitoring for vancomycin resistant Enterococci is critical in hospital infection control and for effective treatment.


introduction
The term 'Enterococcus' originates from the Greek enteron meaning 'the gut or intestine' and kokkos meaning 'a berry or kernel'. This is helpful as multidrug resistant Enterococci are associated with the gastrointestinal tract and have become 'kernels' of antimicrobial resistance [1].
Enterococci, previously classified as group D Streptococci, are normal part of enteric microbiota. On Grams stain they appear typically as Gram positive oval cocci of size approximately 1µm arranged in pairs at angles, or in short chains. They are usually nonhemolytic, grow in presence of bile, hydrolyze esculin, grow well in 6.5% NaCl and grow well between 10 o C and 45 o C (Characteristics that differentiate Enterococci from Streptococci).
Most common sites of infection are the urinary tract, wounds, biliary tract and blood. They are also associated with meningitis and bacteremia in neonates and endocarditis in adults. Among the total species of Enterococci identified till date, only one third are implicated in human disease. Although Enterococcus faecalis was the most common species isolated earlier, a shift to Enterococcus faecium is observed in the last few decades [2].
Enterococcus is an important pathogen because of its intrinsic as well as acquired antibiotic resistance. The intensive use of broad spectrum antibiotics in hospitals provides selective pressure favoring growth of intrinsically drug-resistant commensal organisms, one of them being Enterococcus. The first case of Vancomycin Resistant Enterococci (VRE) was reported in England in 1988 by Uttley et al; since then observed all over the world [3]. In India there are reports from New Delhi, Chandigarh and Mumbai [4][5][6].
Several genes, including VanA, VanB, VanC, VanD, and VanE, contribute to resistance to vancomycin in Enterococci. Acquired resistance is mediated by VanA and VanB phenotypes whereas intrinsic resistance is VanC mediated.
Newer agents such as linezolid, daptomycin, quinupristin-dalfopristin and tigecycline (among others) are used for the treatment of Vancomycin Resistant Enterococci infections [7].
The importance of VRE is because of the few options available for treatment, the difficulty to eradicate once established and the possibility of transfer of vancomycin resistance to Staphylococcus aureus and Listeria monocytogenes [8].

Material and methods
Study area: Department of Microbiology at Krishna Institute of Medical Sciences, Secunderabad Study population: All patients admitted to KIMS and visiting out-patient departments were included.
Inclusion criteria: 1) All age groups, 2) IP and OP patients of all departments, 3) Both males and females are included, 4) Percentage Identification more than 89% in VITEK 2 compact (bioMerieux).

Method of measurement of outcome of interest
All clinical samples of patients which showed growth of Enterococci were analyzed. In the study, 226 enterococcal isolates were speciated by VITEK 2 compact (with percentage identification > 89%).
Antibiogram was analysed according to Clinical Laboratory Standards Institute (CLSI) guidelines [9] prevailing at that point of time.
Isolates showing resistance to Vancomycin were phenotyped into VanA, VanB, VanC, based on MIC values of Vancomycin and Teicoplanin, according to CDC guidelines [8,10].

results
A total number of 226 isolates of Enterococci were identified during the study period, among which 13 were Vancomycin Resistant (VRE). All Vancomycin Resistant Enterococci were isolated from inpatient samples only. When vancomycin phenotypes were further analyzed, 6 were found to be VanA, 4 were VanB and 3 were VanC.    Enterococci have adequate intrinsic and acquired resistance to many antibiotics [13]. They are intrinsically resistant to penicillinase resistant penicillins and cephalosporins; have acquired resistance to chloramphenicol, erythromycin and high level resistance to aminoglycosides, penicillin, fluoroquinolones and vancomycin [14]. E. faecium and E. faecalis were more resistant when compared to other species. Among the two, E. faecalis is less resistant (30.39%) to antimicrobials than E .faecium (50.64%). Antimicrobial resistant Enterococci are being reported with increasing frequency in United States and other parts of the world [15]. Their number in Asian subcontinent is comparatively less.
The phenotypic classification scheme used usually corresponds well to the genotypic classification. It is an inexpensive method as it uses information that is derived simply in a laboratory. However there are certain limitations [10].
Thirteen Vancomycin resistant phenotypes were isolated (5.75%), majority of them from urine   samples. VanA is the commonest (6 out of 13), closely followed by VanB (4 out of 13) and then VanC (3 out of 13). When the resistance pattern is looked into, majority of the isolates were found to be sensitive to Linezolid. Nitrofurantoin though useful in vancomycin susceptible isolates, 5 out of 13 (38.46%) VRE isolates were resistant to it. VRE were highly resistant to levofloxacin, penicillin, tetracycline and ciprofloxacin.
The overall resistance of VRE isolates was higher than non-VRE isolates except for ciprofloxacin and tetracycline. Even though tigecycline is reported to have good activity against VRE infections, it could not be tested as majority isolates were from urine. Use of tigecycline for the treatment of Urinary Tract Infections (UTIs) has been questioned because of low peak serum concentrations and limited excretion into urine [16,17].

limitations of study
• The study would have been better had it been prospective • Clinical history, treatment given and outcome of patients are not known • Phenotypic classification system used in the study is not fool proof • Genotypic confirmation of Van phenotypes using PCR was not done