A Study On High and Low level Drug Resistance Pattern among Clinical Isolates of Enterococci

The combined abilities of colonisation and both inherent and acquired resistance have made Enterococci a significant human pathogen. Aims & Objectives: This study was done to determine the Minimum Inhibitory Concentration of various antibiotics against Enterococci and to correlate the phenotypic and genotypic characteristics of Enterococci with low level and high level drug resistance. A total of 774 isolates of Enterococci obtained from various clinical samples were subjected to antimicrobial susceptibility testing by Kirby Bauer Disk Diffusion method. The Minimum Inhibitory Concentration of various antibiotics were determined by Vitek 2 automated system, agar dilution and E test. Results: 15 out of 774 isolates showed the presence of vancomycin resistant genes by Multiplex PCR. 10 (90.91 %) isolates out of 11 E. faecalis with van A gene showed high level resistance to Penicillin (16-64 μg/ml). 8 (72.73 %) out of 11 isolates showed high level resistance to Gentamicin (512-1024 μg/ml). 6 (54.55 %) , out of 11 isolates were resistant to â lactams. One isolate of E. faecalis from urine with van B gene showed showed high level resistance to Penicillin (32 μg/ml), Linezolid (e” 8μg/ml), high level resistance to Gentamicin (1024 μg/ml), Fluoroquinolones (e” 8μg/ml) and Macrolides (e” 8μg/ml). Conclusion: Isolates of Enterococci resistant to glycopeptides, penicillin, Betalactams and aminoglycosides have important clinical implications in the treatment for infection.

The genus Enterococci have exhibited the potential to harbour and transfer drug resistant genes and have become an important pathogen in clinical settings. In Enterococci, lower affinity of Penicillin Binding Proteins (PBP's) are responsible for decreased susceptibility to Penicillin's. The Minimum Inhibitory concentration (MIC) of penicillin for Enterococci are higher than that for Streptococci and inhibition of PBP's does not result in bactericidal activity in Enterococci 1 .
Enterococci display low to moderate level resistance to aminoglycosides due to slow uptake or permeability of these agents. All Enterococci have innate low level resistance to aminoglycosides with minimal inhibitory concentration ranging from 4 µg/ml to as high as 256 µg/ml. High level resistance to Gentamicin is associated with bifunctional enzyme possessing acetylase (6 ' ) and phosphotransferase (2 ' ) activities conferring resistance to all aminoglycosides except Streptomycin 2 . Enterococci also exhibits acquired resistance via mutations in existing DNA or through acquisition of new DNA and high level resistance are usually due to the transferable plasmid mediated production of aminoglycoside inactivating enzymes 3 .
Beta lactamase producing Enterococci exhibits inducible and constitutive low level resistance 4 . In clinical isolates of E. faecium, â lactamase resistance is associated with mutations or overproduction of PBP5 with Ampicillin MIC of >256 mg/L in some strains. Isolates of E. faecium with MIC of Ampicillin d"64 mg/L may respond to high dose Ampicillin therapy 5 .
Quinolone resistance occurs by mutation in the quinolone resistance determining regions of the genes that encodes gyrase and topoisomerase IV and have been observed in clinical isolates of Enterococci. These mutations prevent efficient binding of the antibiotics to the enzymes which enable DNA replication to continue despite the presence of antibiotics. The second mechanism contributes to quinolone resistance are Multidrug resistance efflux pumps (MDRs) on bacterial chromosomes. Inactivation of homolog of quinolone resistance (Qnr) identified in E. faecalis resulted in modest decrease in resistance to quinolones and over expression of gene results in increased resistance 6 .
The most common form of acquired resistance to macrolides is production of an enzyme (erm B gene) that methylates a specific adenine in the 23 S rRNA of the 50S ribosomal subunit there by reducing the binding affinity of this drug for the ribosome, which also reduces the binding of Lincosamide and Streptogramin to the ribosome. An efflux pump encoded by transferrable mefA gene is known to pump macrolides out of the cell and confers low level resistance in Enterococci. 7 Linezolid has been reported to cure VRE endocarditis and other serious intravascular infections, bacteremia, UTI and skin and soft tissue infections 8  One of the major concerns that physicians face during treatment of Enterococcal infection is the probability of developing resistance during therapy, which may lead to therapeutic failures and contributes to patient mortality. Monitoring the resistance pattern of clinical isolates of Enterococci is a useful tool to obtain information on the prevalence of multidrug resistant isolates and thereby limiting the spread of bacterial resistance.

MATERIALS And METHOdS
A total of 774 isolates of Enterococci obtained from various clinical samples were subjected to antimicrobial susceptibility testing on Muller Hinton Agar by Kirby-Bauer disk diffusion method and the plates were incubated for 16-18 hrs at 37 0 C.The diameter of the zone of inhibition was measured and zone size interpreted according to CLSI standards 10 The antibiotic disks used were Penicillin (10 U), Amoxicillin Clavulanic acid (20/10ìg), Erythromycin (15ìg), High Level Gentamicin (120 ìg), Linezolid (30ìg), Teicoplanin (30 ìg), Ciprofloxacin (5ìg), Levofloxacin (5 ìg) and Nitrofurantoin (300ìg) .Zones of inhibition were measured and recorded and the organism was interpreted as sensitive or resistant as per the recommendations from CLSI guidelines using Enterococcus faecalis ATCC 29212 as control. Screening for isolates resistant to Vancomycin and High Level Gentamicin was done on Brain Heart Infusion agar containing 6µg/ml of vancomycin and 500 µg/ml of Gentamicin respectively. Inoculum of 10 ìL of 0.5 McFarland's was spot inoculated. Presence of more than 1 colony was interpreted as a resistant strain. 11 Multiplex PCR was performed to detect the presence of vancomycin resistance genes., vanA and vanB using the following primers (Sigma Aldrich, USA). Minimum Inhibitory Concentration for Gentamicin was determined by agar dilution method and Epsilometer test. Agar dilution was performed by on Brain heart infusion agar by using 500 ìg , 1000 ìg and 2000 ìg of Gentamicin. To detect Gentamicin resistance by E test,MIC strips coated with Gentamicin in a concentration gradient of 0.064-1024 ìg/ml was used . MIC was read when the ellipse intersects MIC scale on the strip. 11 Turbidometrically controlled bacterial pure growths were suspended into sterile physiological saline and this suspension was used to fill Vitek 2 Compact system and antimicrobial susceptibility testing cards. 13 For biochemical identification of isolates in Vitek system, the following parameters were used: Growth in 6.5 % NaCl, â-glucuronidase, Trehalose, Arginine dihydrolase, D-sorbitol, Urease, Raffinose, D-galactose, D-mannitol, Sucrose, â-galactosidase, Salicin, L-pyrrolidonyl arylamidase, D-xylose, D-maltose, Methyl-â-Dglycopyranoside, D-ribose, á-glucosidase, ámannosidase, Phosphatase etc. 14 Minimum inhibitory concentrations for the following antibiotics were tested: Penicillin, Erythromycin, Vancomycin, Teicoplanin, Ciprofloxacin, Levofloxacin, Tigecycline, Nitrofurantoin, Linezolid and Daptomycin.   (1) vanA 0 (0.00) 0 (0.00)  One isolate of E. faecalis from urine with van B gene showed high level resistance to Penicillin (32 µg/ml), Linezolid (e" 8µg/ml), high level resistance to Gentamicin (1024 µg/ml), Fluoroquinolones (e" 8µg/ml) and Macrolides (e" 8µg/ml). One E.faecium isolate with van A gene showed susceptibility to Penicillin with high level resistance to Gentamicin (1024 µg/ml). One E. faecalis isolate from pus with both van A and van B gene showed high level resistance to Penicillin (32 µg/ml), Linezolid (e" 8 µg/ml), high level resistance to Gentamicin (1024 µg/ ml),Fluoroquinolone(e" 8µg/ml) and intermediate resistance to macrolides(4 µg/ml).

Out
No significant association was found between Vancomycin resistant genotypes of Enterococci and their high and low levels of resistance to Penicillin, Gentamicin and â lactams (p value >0.05) [ Table:2, 3&4].

dISCUSSIOn
Enterococci possess remarkable ability to acquire antibiotic resistance determinants. The ability of these organisms to colonise the gastrointestinal tract of hospitalised patients for prolonged period is a crucial factor that influences the development of drug resistance. With the increased use of Vancomycin and wide spectrum antibiotics in the health care setting, these organism have been identified as common etiology of nosocomial infection .  17 . In the present study no significant difference in resistance to Penicillin was found between E. faecalis and E. faecium isolates (p value >0.05).

E n t e ro c o c c i e x h i b i t d e c r e a s e d susceptibility to Penicillins and Ampicillins as a result of expression of low level Penicillin
High level resistance to â lactam antibiotics are seen in E. faecium due to the over production of low affinity Penicillin Binding Proteins (PBP's). A variety of point mutation in PBP has been described in E. faecalis and E. faecium 18  The prevalence of Enterococci with high level Gentamicin resistance are increasing and varies by geographical region and are generally resistant to all aminoglycosides with the occasional exception of Streptomycin. Aminoglycoside Modifying Enzymes (AME's) are the most important mechanism mediating high level resistance. These enzymes confer high level resistance to more than one aminoglycoside. A single strain of Enterococci may acquire several AME's. Both E. faecalis and E. faecium may acquire 6'-adenyl transferase which confer HLR to Streptomycin 3 . By disk diffusion method, 53.49 % showed high level Gentamicin resistance. In a study from North India, D K Mendritta et al., 2008 have reported, 46 % isolates resistant to both high Level Gentamicin(HLG) and High Level Streptomycin(HLS) by both disk diffusion and agar dilution method and combined resistance to both HLG and HLS was higher in E. faecium compared to E. faecalis 21 .In the present study no significant difference in combined resistance to HLG and HLS was observed between E. faecalis and E. faecium (p value >0.05) where as HLGR was higher among E. faecium isolates. In our study 15 Tigecycline shows in vitro bacteriostatic activity against Vancomycin resistant Enterococci. Tigecycline was found to be the most effective drug against Enterococci in our study with 100 % susceptibility. Similar finding were reported from Indian studies 31,32 As a result of mutation of chromosomal genes, Daptomycin resistance following therapy has been observed in clinical isolates of Enterococci. Daptomycin synergy has been described in vitro with Ampicillin, Cephalosporins, Imipenem, Rifampin and Gentamicin 34 . In our study, 96.12 % isolates showed susceptibility to Daptomycin with MIC d" 4 µg/ml.
The overall antimicrobial pattern of the isolates showed significantly higher (p value < 0.05) percentage resistance to Penicillins, High level aminoglycosides, Macrolides, Fluoroquinolones and Linezolid among the clinical isolates of Enterococci.

COnCLUSIOn
The present study reports significantly higher percentage resistance to Penicillins, High level aminoglycosides, Macrolides, Fluoroquinolones and Linezolid among the clinical isolates of Enterococci. The most effective drug against Vancomycin Resistant Enterococci was found to be Tigecycline. No significant association was found between Vancomycin resistant genotypes of Enterococci and high and low levels of resistance to Penicillin, Gentamicin and â lactams.