Context: Enterococcus is considered an important nosocomial pathogen because of its intrinsic as well as acquired antibiotic resistance. It also has the potential of transferring vancomycin resistance to other organisms such as Listeria monocytogens and Staphylococcus aureus. Aims: The objective of the present study was to determine antibiotic-resistance pattern of Enterococcus with special reference to vancomycin. Settings and Design: A total of 54 clinical isolates of enterococci were collected during the study period of 1 year at a tertiary care center in Mumbai. Material and Methods: Speciation and antibiotic sensitivity testing were done by standard procedures. Minimum inhibitory concentration (MIC) to vancomycin was carried out by agar dilution method. Results: Speciation and antibiotic sensitivity testing were done by standard procedures. The MIC to vancomycin was done by agar dilution method. Conclusions: Vancomycin, Linezolid, and Teicoplanin can be safely used for the treatment of serious enterococcal infections.
Keywords: Enterococci, Minimum inhibitory concentration, Vancomycin, Vancomycin-resistant enterococci
Enterococci are Gram-positive cocci, which are normal commensals of the gastrointestinal tract, genital tract, and anterior urethra.  However, in recent years, it has gained importance as a nosocomial pathogen because of its antibiotic resistance. It is the second most common cause of urinary tract infection (UTI) and third most common cause of bacteremia. ,
Although 19 species within the genus are recognized, Enterococcus faecalis is the most commonly isolated pathogen, followed by E. faecium Species identification is useful for epidemiological investigation of an outbreak and also for clinical decisions, particularly with regard to therapy, as antimicrobial susceptibility differs by species. ,
Its acquired high-level resistance (HLR) to aminoglycoside and vancomycin resistance are the causes of concern. This resistance contributes to failure of combination therapy of aminoglycoside and β-lactam antibiotics used for severe enterococcal infections. Moreover, acquired vancomycin resistance is transferable to other organisms such as Staphylococcus aureus and Listeria monocytogenes. 
This study was undertaken in a tertiary health center in Mumbai to characterize different strains of enterococci isolated from clinical specimens, study their antimicrobial susceptibility pattern including high-level aminoglycoside resistance, vancomycin resistance, β-lactamase production, and MIC to vancomycin.
During the study period of one year, 54 isolates of enterococci were collected from 980 culture-positive samples. The samples were mainly urine, pus, blood culture, and body fluids.
From urine and pus samples, Gram smears were prepared and examined microscopically. The samples were cultured on blood agar, MacConkey agar and incubated at 37°C for 24 hr aerobically. Blood samples were cultured on blood agar, MacConkey agar on days 2, 4, and 7 and incubated at 37°C for 24 hr aerobically. Preliminary identification of enterococci was done by studying colony morphology, Gram stain, and catalase test.
Isolates were confirmed by inoculation on Bile Esculin Azide Agar, growth in 6.5% NaCl, growth at 10°C and 45°C. 
Speciation was done by detecting fermentation of arabinose, mannitol, raffinose, and sorbitol as well as motility and pigment production, if any. 
Drug susceptibility testing: The standard disc diffusion test for susceptibility to various antibiotics including gentamicin (120 μg) vancomycin, Teicoplanin, and Linezolid was done by Kirby Bauer method. Zone diameter was measured and interpreted according to standards of the Clinical and Laboratory Standards Institute. 
Detection of β lactamases: Beta-lactamase production was tested using the nitrocefin disc, which is a chromogenic cephalosporin. A positive reaction showed change in color from yellow to red in the area where the culture was applied.
Determination of MIC:  MIC of vancomycin was carried out for the 54 isolates by agar dilution method. Dilutions of vancomycin were prepared in distilled water in the range of 5,120-1.25 μg /ml.
The Mueller-Hinton Agar medium was prepared and allowed to cool to 50°C after autoclaving. Prediluted antimicrobial solutions were added to the melted and cooled medium in the ratio of 1:10 and mixed properly.
Fifty microliter of an overnight broth culture of enterococci was added to 5 ml peptone water to obtain a suspension of approximately 10 7 Colony Forming Unit (CFU)/ml. Of this, 1 μl was added to the Mueller Hinton agar plate with a calibrated loop. E. faecalis ATCC 29212 was used as organism control with an MIC of 1-4 μg /ml.
[Table 1] shows distribution of Enterococcus in various samples.
Patient characteristics: Out of 54 patients, 52 (96.29%) were hospitalized and only 2 were outpatient department patients. There were only two pediatric patients (<14 yr) in the study. There was no gender difference associated with enterococcal infection.
Risk factors: Out of 54 patients, 9 had prolonged hospitalization as a risk factor, most of whom were orthopedic and surgical ICU patients. Out of 29 UTI cases, 14 were catheterized and 12 had diabetes. Out of 18 wound infection patients, 6 had diabetic ulcer. One female child had an indwelling Ventriculo peritoneal shunt for hydrocephalus.
E. faecalis (87.03%) was the most commonly isolated species, followed by E. Faecium (9.25%) and E. durans (3.7%).
Penicillin resistance was observed in16.6% of the isolates, whereas high level resistance (HLR) to gentamicin was seen in 44.4% isolates. No resistance was found against vancomycin, Teicoplanin, and Linezolid, as shown in [Figure 1]. Also, there were no β-lactamase producing strains. All strains were sensitive to vancomycin, except one which showed intermediate sensitivity.
[Table 2] shows difference in the resistance pattern between E. faecalis and E. Faecium. MIC level for vancomycin in most isolates (24) was 2 μg/L. None of the isolates showed MIC level above 8 μg /ml. One strain that showed intermediate sensitivity showed an MIC of 8 μg/ml. [Figure 1] shows the resistance pattern of enterococcal isolates to different antibiotics.
Enterococci are the commensals of the human intestinal flora. Sites less often colonized by these organisms include the oral cavity, genitourinary tract, and skin, especially in the perineal area. The main sites of colonization in the hospitalized patients are soft tissue wounds, ulcers, and the gastrointestinal tract. Enterococci were traditionally regarded as low-grade pathogens, but have emerged as an increasingly important cause of nosocomial infections in recent years. 
The spectrum of disease varies from UTI, wound infection, soft tissue infection to bacteremia. It is the second most common cause of UTI and third most common cause of bacteremia. Urinary tract instrumentation or catheterization, genitourinary pathology, prior use of antibiotics, prolonged hospitalization are some of the predisposing factors for enterococcal infections.
In our study, the urinary tract was the most common site of infection (53.7%), which often occurred after instrumentation of the patient’s urinary tract. Enterococci were isolated in 9% of the total UTI cases and were culture positive, thus becoming one of the important etiological agents of UTI. The next common site of infection was wound infection followed by bacteremia.
Enterococcal infections occurred significantly more in hospitalized patients as compared to outdoor patients. In hospitalized patients, infections were more commonly seen in patients in orthopedic and surgical ICU, which is possible because of prolonged hospitalization. Enterococcal infection were more commonly seen in adults and evenly distributed by sex.
Until recently, E. faecalis had been the predominant enterococcal species, accounting for 80-90% of all clinical isolates, and E. faecium had accounted for 5-15%. Other Enterococcus species (E. gallinarum, E. casseliflavus, E. durans, E. avium, and E. raffinosis) are isolated less frequently and account for less than 5% of clinical isolates.  In recent years, a progressive decline has been witnessed in the ratio, ie, progressive increase in E. faecium infection.  E. faecium was found to be more resistant to penicillin and aminoglycosides. The reason for increased resistance to aminoglycosides is due to production of enzyme, 6-acetyl transferase and more penicillin-binding proteins. 
In our study, E. faecalis was the commonly isolated species followed by E. faecium and E. durans ratio being 9.4:1. No other species were reported. Species identification enabled us to assess species-specific antimicrobial resistance. E. faecium was found to be more resistant to penicillin and gentamicin. Penicillin resistance is attributable to either β-lactamase production or low-affinity penicillin-binding proteins.
Penicillin resistance was observed in 16.6% of the isolates. No β-lactamase producing strain was found in our study. The mechanism of penicillin resistance could be attributable to the synthesis of low affinity of penicillin-binding proteins. Although prevalence of β-lactamase producing strains is low, all isolates of enterococci should be tested for β-lactamase production.
The HLR to gentamicin was observed in 44.4% isolates with E. faecalis resistance 44.68% and E. faecium as 60%. HLR to gentamicin is demonstrated in all countries, with the prevalence in the range of 1-48% (mean, 22.6±12.3). HLR to gentamicin nullifies the efficacy of combination therapy, used for serious enterococcal infections. This helps distinguish these HLR strains from simply resistant strains, which is extremely crucial. 
Acquired resistance to vancomycin is also a cause of concern. The first case of vancomycin-resistant enterococci (VRE) was observed in Europe in 1986; since then it has been observed all over the world.  In India, there are few reports from Delhi, Chandigarh, and Mumbai. ,, VRE is important for several reasons: only few options available for treatment of VRE; VRE once established is difficult to eradicate; vancomycin resistance in enterococci is transferable to L. monocytogenes and S. aureus; VRE can spread directly from patient to patient or indirectly via transient carriage on the hands of personnel, contaminated environmental surface, or patient care equipment. ,
Considering the importance of VRE, all isolates were tested for vancomycin resistance by disc diffusion and agar dilution methods. All isolates were found sensitive to vancomycin, except one which showed intermediate zone by Kirby-Bauer disc-diffusion method. ICLS recommends performing MIC levels for the enterococcal isolates that yields intermediate zones or zones just above the susceptible limits. MIC for vancomycin was done by agar dilution to determine the MIC ranges of these isolates. It was found that maximum isolates (44.4%) showed MIC of 2 μg/ml, while only one isolate showed MIC of 8 μg/ml. Two interesting facts that none of the strains were inhibited by very low concentrations (0.25 and 0.125 μg/ml) and one strain showed intermediate zone indicating the threat of VRE in the area. The low prevalence of VRE in India is possible because of lesser use of vancomycin for treatment of patients.
It was found that all isolates were sensitive to linezolid and teicoplanin. Hence, the two antibiotics can be safely used in severe enterococcal infections.
Enterococcal infections are therapeutic challenge because of its intrinsic and acquired resistance to vancomycin as well as the ability to produce β-lactamases. Therefore, appropriate infection control measures should be implemented to decrease the transmission of these microorganisms in hospital settings. Microbiology laboratory plays an important role in the detection, timely reporting, and control of VRE. Also, rational use of antibiotics can help minimize incidence of VRE.
Authors thank Dr BG Mantur, Head of Department, Microbiology, Belgaum Institute of Medical Sciences, for reviewing the manuscript.
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]