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ORIGINAL ARTICLE
Year : 2022 | Volume
: 21
| Issue : 3 | Page : 193-197
Enterococcal infections in a tertiary care hospital, North India
Rajesh Kumar Yadav, Loveleena Agarwal
Department of Microbiology, Prasad Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Correspondence Address:
Loveleena Agarwal
Department of Microbiology, Prasad Institute of Medical Sciences, Lucknow, Uttar Pradesh
India
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/aam.aam_110_20
Abstract
Background: The emergence of Enterococcus as an important nosocomial pathogen is mainly attributed to its inherent resistance to commonly used antibiotics and now in recent times, it has acquired resistance to other available therapeutic options as well. Materials and Methods: Enterococcus isolates from clinical samples received in the department of microbiology over a period of 1 year were included in the study. Isolates were identified and species determined by standard methods. Antibiotic susceptibility test was done by Kirby Bauer disc diffusion test. Epsilometer test was done to determine the minimum inhibitory concentration for vancomycin. Results: A total of 145 Enterococcus isolates were obtained; 73 (50.3%) isolates were identified as Enterococcus faecalis, 69 (47.5%) Enterococcus faecium, 2 (1.3%) Enterococcus durans and 1 (0.68%) as Enterococcus gallinarum. Most of the isolates were from urine samples (125, 86.2%); followed by pus (16, 11.03%). Vancomycin resistance was seen in 14 (9.6%) isolates while linezolid resistance was seen in 8 (5.5%) isolates. Conclusion: E. faecalis is the most common clinical species isolated from clinical samples and the emergence of linezolid-resistant enterococci from the hospital is a matter of concern as till now it is considered to be the last resort for treatment in patients infected with vancomycin-resistant enterococci.
Abstract in French Résumé
Contexte: L'émergence d'Enterococcus en tant qu'agent pathogène nosocomial important est principalement attribuée à sa résistance inhérente aux antibiotiques couramment utilisés et, ces derniers temps, il a également acquis une résistance à d'autres options thérapeutiques disponibles. Matériels et Méthodes: des isolats d'entérocoques provenant d'échantillons cliniques reçus au service de microbiologie sur une période d'un an ont été inclus dans l'étude. Les isolats ont été identifiés et les espèces déterminées par des méthodes standard. Le test de sensibilité aux antibiotiques a été effectué par le test de diffusion sur disque de Kirby Bauer. Un test Epsilomètre a été effectué pour déterminer la concentration minimale inhibitrice de la vancomycine. Résultats: Au total, 145 isolats d'Enterococcus ont été obtenus ; 73 (50,3 %) isolats ont été identifiés comme Enterococcus faecalis, 69 (47,5 %) Enterococcus faecium, 2 (1,3 %) Enterococcus durans et 1 (0,68 %) comme Enterococcus gallinarum. La plupart des isolats provenaient d'échantillons d'urine (125 ; 86,2 % ); suivi de pus (16 ; 11,03 %). La résistance à la vancomycine a été observée dans 14 (9,6 %) isolats, tandis que la résistance au linézolide a été observée dans 8 (5,5 %) isolats. Conclusion: E. faecalis est l'espèce clinique la plus courante isolée à partir d'échantillons cliniques et l'émergence d'entérocoques résistants au linézolide à partir de l'hôpital est préoccupante car jusqu'à présent, elle est considérée comme le dernier recours pour le traitement des patients infectés par la vancomycine. entérocoques résistants.
Mots-clés: Enterococcus faecalis, Enterococcus, entérocoques résistants au linézolide, entérocoques résistants à la vancomycine
Keywords: Enterococcus faecalis, Enterococcus, linezolid resistant enterococci, vancomycin-resistant enterococci
Introduction 
Enterococcus, initially known as an uncommon cause of infection has now established itself as a serious nosocomial pathogen. The two most common pathogenic species Enterococcus faecalis and Enterococcus faecium cause various infections like urinary tract infection, soft tissue, intra-abdominal, pelvic infection, bacteremia, and endocarditis.
Treatment of enterococcal infection is challenging as the organism is intrinsically resistant to many antibiotics and also can acquire resistance towards many other antimicrobials through plasmid transfer/transposon.[1] Vancomycin has been the drug of choice when treatment with other antibiotics has failed. However, in recent times, there has been a steady increase in vancomycin-resistant enterococci (VRE) strains in clinical isolates across the globe which is worrisome as treatment options of such infections are limited.[2] The mainstay treatment of multiple antibiotic-resistant Gram-positive pathogen infections as methicillin-resistant staphylococci, multidrug-resistant pneumococci, and VRE is Linezolid; resistance to this antibiotic is now not uncommon and in fact appears to be increasing.[3] Early detection of antibiotic resistance profile may help in determining a worthy alternative treatment and prevent the spread of VRE.
The present study was undertaken with the aim to determine the antibiotic resistance profile of Enterococcus isolates from clinical samples at our tertiary care hospital for better patient care.
Materials and Methods Clinical samples received in the Department of Microbiology for bacterial culture from June 2019 to May 2020 at our tertiary hospital were included in the study. Samples inclusive were urine, pus, blood, and body fluids whereas sputum, throat swab, stool, and vaginal swab were excluded from the study, as Enterococcus forms a part of the normal flora. Culture and identification of the isolates were performed as per the standard protocol.[4] In brief, the samples were inoculated on appropriate culture media to isolate the bacteria. All small pinpoint, cream or white, smooth, circular, convex colonies with α, β, or γ hemolysis on Blood agar or small dark-red magenta colonies on MacConkey agar were presumptively considered as Enterococcus species. Further identification was done with Gram stain, catalase production, esculin hydrolysis, salt tolerance (ability to grow in 6.5% NaCl broth) and L pyrrolidonyl β naphthylamide (PYR) testing. E. faecalis ATCC 29212 was used as the positive control. The species of Enterococcus were identified based on acid production from 1% sugars like arabinose, mannitol, pyruvate, raffinose, and sorbitol.[5]
Following the Clinical and Laboratory Standards Institute 2017 guidelines antimicrobial susceptibility testing was carried out by Kirby-Bauer disk diffusion method on Mueller Hinton agar. The antibiotics tested were: Ampicillin (10 μg), ciprofloxacin (5 μg), doxycycline (30 μg), tetracycline (30 μg), linezolid (30 μg); and for urine isolates nitrofurantoin (300 μg) in the form of commercially available antibiotic discs (HiMedia, Mumbai, India). For detection by disc diffusion method of high-level gentamicin (HLG) and high-level streptomycin (HLS) resistance, gentamicin (120 μg) and streptomycin (300 μg) discs were used.[6]
Screening for vancomycin resistance by disc diffusion method was done by using vancomycin (30 μg) and teicoplanin (30 μg). Further, the resistant isolates were further confirmed by Epsilometer test. Of the five known vancomycin resistance phenotypes, Van A and Van B are the most common and have been primarily described in E. faecalis and E. faecium. VanA-resistant strains show high-level resistance to vancomycin (minimum inhibitory concentrations [MICs], ≥64 mg/ml) and teicoplanin (MICs, ≥16 mg/ml), whereas Van B isolates show resistance to vancomycin (MICs, 4 to ≥1000 mg/ml) but are susceptible to teicoplanin.[7] The isolates were identified for the phenotype of vancomycin resistance in the absence of molecular setup. Disc diffusion test was done for linezolid resistance detection and confirmed by MIC breakpoints. For quality control Staphylococcus aureus ATCC 25923 was used.
Results A total of 145 clinical isolates of Enterococcus were studied, of which 73 (50.3%) isolates were identified as E. faecalis, 69 (47.5%) as E. faecium, 2 (1.3%) as Enterococcus durans and 1 (0.68%) was Enterococcus gallinarum. Majority of the enterococci were isolated from in-patients (116, 80%) and most isolates were from urine sample (125, 86.2%); followed by pus (16, 11.03%), blood (2, 1.3%), and body fluids (2, 1.3%) [Figure 1].
Female patients (83,57.2%) outnumbered male patients (62, 42.7%), age distribution showed a range from 17 months to 82 years with the maximum number of isolates in the age group 20–29 years (36, 24.8%) [Table 1].
Table 1: Age and sexwise distribution of the patients from which Enterococcus is isolatedThe clinical isolates of enterococci showed highest resistance to doxycycline (101, 69.6%), tetracycline (96, 66.2%), ciprofloxacin (88, 60.6%), and ampicillin (86, 59.3%) [Table 2]. Nearly half the isolates showed resistance to HLG (HLGR) i.e., 69 (47.5%), whereas 56 (38.6%) isolates were resistant to HLS (HLSR) and 62 (42.7%) isolates were both HLGR and HLSR [Table 2]. It was noted that the number of E. faecium isolates showing high-level aminoglycoside resistance (HLGR, HLSR, and both) was more as compared to E. faecalis, which was statistically significant (P < 0.05 by Z proportion test) [Table 2].
Out of the 14 (9.6%) isolates showing vancomycin resistance 9 isolates were E. faecium, 4 E. faecalis isolates, and one E. durans. Among the 14 VRE isolated, 10 (71.4%) were of Van A phenotype and 4 (28.5%) were of Van B phenotype. Sample-wise distribution and MIC of Van A and Van B is described in [Table 3]. Many of the VRE isolates12 (85.7%) were HLGR and HLSR.
Table 3: Speciation of vancomycin-resistant enterococci isolates with respect to the minimum inhibitory concentration of vancomycin and teicoplaninResistance to linezolid was seen in 6 E. faecium and 2 E. faecalis isolates with MIC ≥8 μg/mL which were isolated from clinical samples (seven urine samples, one blood sample) of inpatient department patients.
Discussion Enterococci a nosocomial pathogen is responsible for 10%–20% of all hospital infections and now is a major cause of hospital-acquired infections.[8]
In our study, the maximum number of isolates were obtained from urine (86.2%), followed by pus (11.03%) as seen in other studies where the urine isolates were maximum as compared to the isolates from pus, however, some studies have shown a maximum of 43% isolates from pus as well.[9],[10]
Although more than a dozen of species have been identified for Enterococcus, most of the human infections are caused by E. faecalis accounting for nearly 80%–90% of the isolates followed by E faecium which is responsible for nearly 5%–10%[11] of the infections. However, the latest trends show an increase in the isolation rate of E faecium[12] which is worrisome as its intrinsic resistance may lead to a treatment failure; the finding in this study E. faecalis (50.3%) and E. faecium (47.5%).
Our study showed that 59.3% of isolates were resistant to ampicillin, 60.6% to ciprofloxacin, and 47.5% to high-level gentamicin which is in agreement with other studies as study of Chakraborty et al., 2015[13] The recent literature shows a drastic increase in the resistance pattern of the commonly used drugs, an increase in the penicillin resistance to 95%, an increase in the ampicillin resistance to 95% and an increase in the HLGR to nearly 50%.[14]
The prevalence of vancomycin resistance in the tertiary care hospitals of India has been reported to vary between 1.7% and 20%; in fact, in the past decade, it has been shown to be on the rise.[15] The prevalence of VRE was 9.6% in the present study. Similar findings of VRE have been reported in studies by Praharaj I et al., 8.7% and Yadav et al., 7%. Praharaj I et al., showed the prevalence of Van A and Van B as 90.6% and 6.25% respectively, and Yadav et al. reported VanA 78.5% 21.4% van B prevalence which was similar to the present study.[16],[17] As compared to a study done by Tripathi A et al., the isolation rate of VRE was 7.9% and all were of Van A phenotype.
The first case of linezolid resistance among Enterococcus isolates from India was reported in 2014 by Kumar et al.[18] Thereafter many more cases of VRE resistance to linezolid were reported and the numbers are increasing since then. A study conducted at Children's Hospital Bagga reports reduced sensitivity to linezolid among Enterococcus isolates from 98% in 2007–46% in 2014.[19] In a meta-analysis done in Turkey antimicrobial resistance of Enterococcus isolates was studied and found mean resistance rates of E. faecalis to linezolid was 1.9 ± 2.6%[20] Resistance to linezolid among clinical isolates in this study was found to be 5.5%, a little higher than reported in other studies from India.[21] These isolates were resistant to linezolid, vancomycin as well as high-level aminoglycosides making treatment of infections by such isolates difficult.
Usually, multidrug-resistant enterococcal infections are hospital-acquired. Measures like strict adherence to hand hygiene, barrier nursing of patients with multidrug-resistant organism along with judicious use and de-escalation wherever linezolid is prescribed are a few measures to prevent such infections.
Limitation of the study
We were unable to confirm VRE phenotypes by the molecular method in this study due to financial constraints. However, phenotypic identification of Van A and Van B types by MIC to vancomycin and teicoplanin in limited-resource setting helped us in treating the patients; for Van B phenotype enterococci teicoplanin is a therapeutic option.
Conclusion Vancomycin and linezolid resistance among clinical isolates of enterococci is a major therapeutic concern as the physicians are left with barely any option to treat such infections. Continuous surveillance for antibiotic resistance patterns may help in judicious use of antibiotics.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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