Prevalence of High level Gentamicin Resistance among the Clinical isolates of Enterococci Species

Enterococci infections, have drawn attention of clinicians due to rapid increase in high level aminoglycoside resistance (HLAR). This resistance predicts failure of β-lactam antibiotic and aminoglycoside combination, which is the current treatment of choice for serious enterococci infections. This led us to investigate about the prevalence of HLAR enterococci in our hospital setting in Shimoga, Karnataka. All the clinical samples received at Microbiology laboratory , McGan hospital, Shivamogga between June 2016 and December 2016 was considered for the study. Enterococci were identified and further speciated by following conventional method. Antibiotic susceptibility pattern of all the isolates was determined according to ClSi guidelines. High level gentamicin resistance was detected by disc diffusion method using 120µg gentamicin disc. A total of 64 Enterococci species were isolated from the clinical specimens. On speciation, Enterococcus faecalis ( E. faecalis ) accounted for 72% (46) of the total isolates, 24%(16) of the isolates were Enterococcus faecium ( E. faecium )and 4% (2) isolates were Enterococcus durans ( E. durans ). High level gentamicin resistance was noted in 42% (27) among the total isolates. E. faecium was found to be more resistant compared to E. faecalis. E. durans was a sensitive strain. Our study reconfirms the high prevalence of HLAR in the clinical setting. The study stresses on the need for regular screening of HlAR in all Enterococci infections and revise the battery of drugs for its treatment according to the report.


iNtRODUCtiON
Enterococci, previously classified as group D Streptococci is now placed under a new family; Enterococcaceae.These Gram positive cocci reside in human intestine and are considered as normal bowel flora.Though they are considered to be less virulent than Streptococcus pyogenes, they are one of the most feared nosocomial pathogens isolated.Common nosocomial infections caused by this genus are urinary tract infections, intraabdominal infections, peritonitis, bacteremia and endocarditis. 1,2ompared to other clinically important Gram positive cocci, this genus is intrinsically resistant to the antimicrobial agents commonly used in hospitals.In addition, Antibiotic resistance has been acquired, and disseminated via horizontal transfer of mobile genetic elements mediated mainly by conjugative plasmids.Though all acquired resistance of this organism is not significant clinically, high level aminoglycoside resistance greatly affects Enteroccoci therapy.This is because; severe enterococci infections are treated with combination of cell wall acting agent like vancomycin and an aminoglycoside.And high level aminoglycoside resistance predicts resistance to this combination therapy. 1,3,4his resistance pattern seen in genus Enterococci has become an issue of concern since decades but remains so even now.Hence this study was conducted to investigate the same so as to design the guidelines regarding measures to prevent spread of the resistant strains in the hospital and also to prevent the emergence of vancomycin resistance which is the treatment of choice for HLAR Enterococci.

MAteRiAlS AND MetHODS
This cross-sectional study was carried out in the Microbiology laboratory, McGan hospital, Shivamogga, between June 2016 and December 2016.The study was approved by the Institutional Ethical Committee of Shimoga Institute of Medical Sciences, Shivamogga.
All the pus, urine, blood and sterile body fluid samples received in our laboratory during the study period was included for the study.Faecal samples, and samples which were leaked were excluded .
The pathogen isolated by growth on blood agar after 24 hour aerobic incubation at 37°C was identified as enterococci species by Gram's staining, catalase test and bile esculin hydrolysis test.However, for blood samples, subculture for 7 days was done before reporting negative.Further, speciation was done by arginine decarboxylation, pyruvate utilization, and fermentation of arabinose, raffinose, mannitol and ribose tests. 5,6usceptibility pattern of the isolated enterococci species were carried out according to CLSI guidelines using disc diffusion method.Detection of High level gentamicin resistance was performed using120µg gentamicin antibiotic disc (Himedia). 7n HLGR detection by disc diffusion method, zone of inhibition of 6 mm was considered as resistant, 7-9 mm as inconclusive and ≥ 10 mm as Susceptible. 7

ReSUltS
A total of 64 enterococci species were isolated during our seven month study period.Out of these 64 isolates, 27 were from pus, 21 from urine and 16 from blood samples.
On speciation, Enterococcus faecalis accounted for 72% (46) of the total isolates, 24% (16) of the isolates were Enterococcus faecium and 4% (2) isolates were Enterococcus durans.Distribution of these three enterococci species in various clinical samples is documented in Table 1.
The antibiotic sensitivity pattern of the isolates is documented in Table 2. Species wise resistance pattern to various antibiotics is depicted in Fig. 1

DiSCUSSiON
Genus Enterococci, once neglected group; considering them as commensals have gained great attention these days due to high rate of severe nosocomial infections caused by them.Two most common species isolated from clinical samples are E. faecalis and E. faecium.These are the organisms of low pathogenicity but with a property of inborn resistance to various antibiotics like relative resistance to most of the β-lactam antibiotics because of low affinity penicillin binding proteins (PBP).Enterococci also shows tolerance to penicillin i.e., penicillin acts only as bacteriostatic.Enterococci also show intrinsic low level resistance to aminoglycosides due to lack of oxidative uptake of the drug. 8,9owever, serious enterococci infections like bacterial endocarditis are treated with   combination of β-lactam antibiotic and an aminoglycoside.To a bacteriostatic beta lactam, the addition of aminoglycoside results in synergistic, bactericidal effect and beta lactam antibiotic indeed enhances the uptake of aminoglycoside resulting in synergistic killing of the organism.Aminoglycoside warrantied for usage in this combination are only gentamicin and streptomycin.
But the current detriment for the effective treatment is emergence of High level aminoglycoside resistance.It is defined by growth at concentration of 2000mg/L and 500 mg/L of streptomycin and gentamicin respectively on brain heart infusion agar.Presence of this high level resistance abolishes the synergistic effect of these agents.HLR to gentamicin is mainly due to aminoglycoside modifying enzyme and that to streptomycin is because of mutation in 30S ribosomal subunit and presence of streptomycin adenyltransferase. 8,9n our study, E. faecalis was recovered the most, followed by E. faecium.][12] It is glad to note that no vancomycin resistant enterococci (VRE) were isolated in our setting.][18] High level gentamicin resistance was noted in 42% of the isolates and E. faecium was found to be more resistant (62% of the total E. faecium isolated) compared to E. faecalis ( 37% of the total E. faecalis isolated).E. durans was found to be a sensitive strain.
Our study report is almost in line with that of various studies carried out in different parts of our country like study by Dadfarna N et al., 19 who have reported 43.7% HLAR, Barman et al., 20 who have reported 56.7% HLAR, Jain S et al., 15 and Fernandes et al., 18 who have noted 54% and 53% HLAR in their studies respectively.

Alternative treatment options for serious HLAR Enterococci infections
Vancomycin is the drug of choice in these cases of HLAR Enterococci infections.But however resistance to vancomycin has also been noticed since 1986.The best available evidence suggests that the emergence and spread of these pathogens are promoted by poor infection control techniques and by antibiotic selective pressure.Antibiotic selective pressure favouring the emergence and spread of Vancomycin Resistant Enterococci(VRE) may involve more than simply the extent of vancomycin use.Specifically, extended-spectrum cephalosporins and similarly active beta-lactams and drugs with potent activity against anaerobes appear to predispose to Vancomycin resistant Enterococci colonization and infection.Hence this should be taken care of by following strict antibiogram policy in all medical setup. 21aptomycin is the drug of choice in VRE.Daptomycin, a cyclic lipopeptide has a unique mechanism of action that results in destruction of the membrane potential. 22Daptomycin -Ceftarolin combination is opted in daptomycin non susceptible strains as ceftarolin is proved to restore daptomycin activity by promoting its surface binding. 23uinupristin-Dalfopristin, a protein synthesis inhibitor is the drug of choice in HLAR, VR E. faecium infections.However, it has no action in E. faecalis infections due to intrinsic resistance. 21inezolid, an oxazolidinone antibiotic with broad spectrum activity against Gram-positive bacteria,that inhibits protein synthesis is not used in serious HLAR Enterococcal infections like endocarditis inspite of its low resistance rate due to its bacteriostatic nature. 21

CONClUSiON
Increased prevalence of HLAR has become a problem of reality.Combination of βlactam antibiotics and an aminoglycoside, which stood first among the choice for the treatment of serious enterococci infection, can no more help in killing these pathogens.This is a grave development and the fact that enterococci have great potential for the dissemination of acquired resistant gene is a matter to be considered seriously.A must screening for HLAR using gentamicin and/or streptomycin by disk diffusion/ agar dilution /broth microdilution should be practiced for all serious enterococci infections.Judicious use of Vancomycin, the drug of choice for HLAR enterococci infections is also advisable.

table 1 .
. For urinary isolates, apart from the antibiotics listed in the table, nitrofurantoin (300µg) and norfloxacin (10µg) antibiotic discs were used.And out of 21 urinary isolates, 9 (43%) Sample wise distribution of different

table 2 .
Antibiotic sensitivity pattern of Enterococci isolates