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 enterococcus has emerged as a nosocomial pathogen in the last two decades , causing urinary tract infections , genital tract infections , and endocarditis due to its colonizing capacity and multidrug resistance [ 1 , 2 ] . the emergence of multidrug resistant enterococci to commonly used antibiotics , for example , aminoglycosides and cephalosporins , is because of their ability to attain and transfer the drug resistance gene , giving rise to enterococci with high level aminoglycoside ( hlar ) and glycopeptide resistance .
however , combinations of penicillins with aminoglycosides are synergistically bactericidal against enterococci in vitro and are effective in treating severe enterococcal endocarditis .
the mechanism of this synergy has been explained by the enhanced uptake of aminoglycosides in the presence of penicillins or other agents which inhibit cell wall synthesis .
an increased frequency of high level resistance to aminoglycoside antibiotics ( mic > 8,000  g / ml ) in clinical isolates of enterococci has been reported which were also resistant to synergism with the penicillins . this study was aimed at determining the antibiotic susceptibility of enterococci isolated from various clinical samples with reference to aminoglycoside and vancomycin .
so if the knowledge of hlar prevalence is available , clinicians can prescribe the various drug combination ( cell wall inhibitor + aminoglycosides ) at the very beginning of treatment avoiding the unnecessary usage of other antimicrobials .
this study was conducted on 100 isolates of enterococcus spp . from various clinical specimens including urine , pus , blood , semen , vaginal swab , and throat swab during a period of six months from july
the samples were processed immediately after collection and enterococcus isolates were identified by standard protocols based on gram 's staining , colony morphology , catalase test , bile solubility , growth in sodium chloride , bile esculin test , and sugar fermentation tests .
antibiotic sensitivity testing of enterococci was performed using kirby - bauer disc diffusion method and mueller - hinton agar supplemented with 5% sheep blood was used as per clsi guidelines .
the antibiotics discs used were penicillin ( 10  u ) , gentamicin ( 10  g ) , ciprofloxacin ( 5  g ) , linezolid ( 30  g ) , vancomycin ( 30  g ) , erythromycin ( 15  g ) , and doxycycline ( 30  g ) , and nitrofurantoin ( 300  g ) was also added in urinary isolates .
, high level ( 120  g ) gentamicin and streptomycin ( 300  g ) discs were placed on the agar medium .
plates were incubated at 37c for 24 hours , and diameter of zone of inhibition was measured .
resistance was indicated by no zone and susceptibility by a zone of diameter 10  mm .
the test was quality controlled using e. faecalis atcc 29212 ( susceptible ) and e. faecalis atcc 51299 ( resistant ) .
vancomycin resistance was determined by using e - strip ( hi - media ) on blood agar for those isolates which were resistant by disc diffusion test .
out of a total of 100 isolates , 41% from urine and semen and 25% from blood followed by 19% from pus and 8% from body fluid drains were included in the study .
the majority of clinical samples from which enterococcus spp . were recovered were from indoor ( 60% ) in comparison to outdoor ( 40% ) patients . 

enterococcus isolates from blood samples were resistant to penicillin ( 64% ) , and urinary isolates were resistant to fluoroquinolones ( 53.6% ) .
commonly used antimicrobials were found to be sensitive in enterococcus spp . recovered from vaginal swab samples ( table 1 ) .
high level gentamicin resistance ( hlgr ) was more common in urine samples ( 41.5% ) followed by blood ( 36% ) samples .
high level streptomycin resistance ( hlsr ) was more common in pus samples ( 52.6% ) followed by blood samples ( 36% ) ( table 2 ) . in table 3 , more resistance to nitrofurantoin in enterococcus isolates from urine samples was not noticed in indoor ( 17% ) patients in comparison to opd ( 7.3% ) .
similarly resistant to glycopeptides ( vancomycin ) , fluoroquinolones were more common in hospitalized patients . also , hlar was more common in indoor ( 39% ) versus outdoor patients ( 25% ) ( table 3 ) .
it was more common in ipd patients ( 28% ) as compared to opd patients ( 4% ) .
high level aminoglycoside resistance was more common in vancomycin resistant enterococci ( vre ) isolates than vancomycin sensitive enterococci ( vse ) isolates .
five out of nine of the enterococcus isolates resistant to vancomycin by disk diffusion test were found resistant to these glycopeptides by e - strip test ( table 4 ) .
over the last few years , they have become important nosocomial pathogens probably due to inherent resistance to antibiotics ( cephalosporins ) , ability to adhere to indwelling medical devices , and ability to survive in adverse environmental conditions .
antimicrobial resistance in enterococcus has been increasing mainly in hospitalized patients [ 911 ] . in our study , out of the total 100 enterococcus isolates from different clinical specimens , the majority were recovered from hospitalized ( 60% ) patients in comparison to outdoor ( 40% ) patients , similar to other studies .
a combination of penicillin and aminoglycosides is considered as treatment of choice for enterococcal infections ; therefore , resistance against these antibiotics has important clinical results effecting therapeutic prognosis . in the present study
, enterococcus isolates from blood samples were found to be penicillin resistant in 64% strains ( 16/25 = 64% ) ( mic ranges from 16 to 32  g / ml ) , which could be due to resistance mechanism involving low affinity penicillin binding proteins or production of  lactamases . in this study ,
prevalence of drug resistance to various antibiotics was as follows : ciprofloxacin ( 25% ) , penicillin ( 66.67% ) , and nitrofurantoin in urinary isolates ( 24.3% ) .
one isolate of enterococcus spp . was found to be resistant to linezolid having inhibitory zone of diameter less than 15  mm .
this type of antibiogram has been documented in earlier studies also [ 1214 ] . here a low prevalence of fluoroquinolone ( 25% ) and other antibiotic resistance was found in comparison to other studies , 72%   and 62% , respectively , which could be due to very precise and judicious use of this antimicrobial in our institute .
the present study demonstrated high prevalence of hlar ( gentamicin and streptomycin ) 29% and 35% , respectively .
hlgr was more common in urine samples ( 41.5% ) followed by blood ( 36% ) samples .
these findings are also reported in some studies . however , a higher and lower prevalence level of hlgr and hlsr have been reported in few studies , respectively [ 13 , 14 , 16 ] . in our study ,
hlar to both gentamicin and streptomycin was found in 22% isolates , specifically more in blood isolates .
hlar in these enterococcal strains nullify the efficacy of beta lactam and aminoglycoside combination therapy .
therefore , differentiation of hlar from simple intrinsic resistance is important and should be adopted as a part of routine microbiology laboratories . in this study
, it was found that hlar was more common in ipd ( 28% ) as compared to opd patients ( 4% ) , similar to other studies .
vancomycin resistance was found in nine isolates of enterococcus by disc diffusion method ; out of nine isolates five isolates were confirmed as vre on e - strip test having mic ( > 64  gm / ml ) . in india , the prevalence of vre has been reported to be between 0 and 30 per cent [ 1722 ] . in the present study , resistance to vancomycin was maximum in blood isolates , that is , 16.25% ( 4/25 ) , with more prevalence in indoor patients ( table 4 ) .
it could be explained by the facts that in hospitalized patients use of broad spectrum antimicrobials is common practice and it leads to colonization pressure for selection of vancomycin resistance strains .
it may increase the risk of cross - infection among hospitalized patients via staff members and environmental contamination with vre strains .
out of four vre strains , three were found to be sensitive to higher concentration of either of or both the aminoglycosides ( table 5 ) .
so the combination of the higher level aminoglycoside with cell wall inhibitor can be considered for treatment of vre infection after antibiotic susceptibility testing . 

control efforts . due to lack of effective therapy for multiple antibiotic resistant enterococcal infection , prevention of the dissemination of these strains
. a very precise use of antimicrobials , for example , cephalosporins , and anti aerobic drugs   should be in practice .
there are certain recommendations to reduce the cross - contamination by these organisms which include surveillance for colonization , identification of colonized and infected patients with their isolation , the use of gowns and gloves by health staff ( barrier method ) , hand washing with an antiseptic after gloves removal , and avoiding contact with environmental surfaces after gloves removal .
medical equipment , for example , stethoscopes and blood pressure cuffs , must be dedicated to hlr patients .
environmental decontamination is also required with effective disinfectants ( isopropyl alcohol , hypochlorite , and phenolic and quaternary ammonium salts ) [ 2629 ] .
deficiency of effective antimicrobial therapy and control measure for prevention of dissemination for multiple drug resistant enterococci are among the major factors for increasing prevalence of vre and hlar .
thus , it becomes important for laboratories to provide accurate antimicrobial resistance patterns for enterococci so that effective therapy and infection control measures can be initiated .
it becomes equivalently important that clinicians who are in direct contact with patients should go primarily for first / low generation of antibiotics for simple infections , for example , sore throat , rather than switching to higher class of antimicrobials .
aims . this study was aimed at knowing the prevalence of vancomycin and high level aminoglycoside resistance in enterococcal strains among clinical samples . 
 study design . 
 it was an investigational study . 
 place and duration of study . 
 it was conducted on 100 enterococcus isolates , in the department of microbiology , pt . 
 bds pgims , rohtak , over a period of six months from july to december 2014 . methodology . 
 clinical specimens including urine , pus , blood , semen , vaginal swab , and throat swab were processed and enterococcus isolates were identified by standard protocols . 
 antibiotic sensitivity testing of enterococci was performed using kirby - bauer disc diffusion method . 
 results . high level gentamicin resistance ( hlgr ) was more common in urine samples ( 41.5% ) followed by blood ( 36% ) samples . 
 high level streptomycin resistance ( hlsr ) was more common in pus samples ( 52.6% ) followed by blood samples ( 36% ) . 
 resistance to vancomycin was maximum in blood isolates . 
 conclusion . 
 enterococci resistant to multiple antimicrobial agents have been recognized . 
 thus , it is crucial for laboratories to provide accurate antimicrobial resistance patterns for enterococci so that effective therapy and infection control measures can be initiated .