Enterococci are part of the microbiota of the female vaginal tract and the gastrointestinal tract [1,2]. Despite this, they are an important nosocomial pathogen that are, along with few other organisms are classified as ESKAPE pathogens because of the multidrug resistance exhibited by these pathogens [3,4]. They cause a variety of infections in patients that encompass multiple systems ranging from urinary tract infections (UTI), intra-abdominal infections, bloodstream infections (BSI) and skin and soft tissue infections [5-7]. These organisms can also cause community-acquired infections which include endocarditis, intraabdominal and skin/soft tissue infections [8]. Around 95% of human enterococcal infections are caused by the two species, Enterococcus faecalis and Enterococcus faecium [9]. Enterococci are responsible for an estimated half a million annual deaths globally, mainly due to BSIs and intra-abdominal infections.

Vancomycin-Resistant Enterococci (VRE) have become one of the most significant healthcare-associated (HA) multidrug-resistant (MDR) microorganisms causing invasive infections, particularly in critically ill and immunocompromised patients. Patients with prolonged hospital stay, those on broad spectrum antibiotics such as piperacillin tazobactam combination and those undergoing dialysis are at higher risk of acquiring VRE infection [10]. VRE has been identified as a significant healthcare issue. The strain quickly became a menace in western countries and has recently emerged as a problem for Indian healthcare system as well [11]. Genotypic assessments have shown that vancomycin resistance is mediated by nine distinct genes named vanA to vanE, vanG, vanL to vanN [12,13]. Among these, the two most important genes include the vanA and vanB as these genes are plasmid borne and induce high level resistance in enterococci [14]. Additionally, vanA gene also confers resistance to teicoplanin [15].

Considering the increasing burden of VRE in Indian healthcare set up, a study was conducted in a tertiary care institute with an aim of characterizing the phenotypic and genotypic resistance to vancomycin isolates along with an analysis of the antibiogram to estimate the drug resistance to different antimicrobials at a tertiary care center from Northern Rajasthan.

The present study was an observational, cross-sectional study conducted at a tertiary care center from North India from June 2023 to May 2024 in which Enterococci isolates from different clinical specimen such as blood, pus, urine and other body fluids were included in the study. The specimens were processed as per standard techniques using conventional Microbiological culture methods. Briefly, specimens other than urine and blood were inoculated on 5% sheep blood agar (HiMedia Laboratories, India), MacConkey agar (HiMedia Laboratories, India). Additionally, bile esculin azide agar was used as a selective medium for the growth of Enterococcus spp. The culture media was incubated aerobically upto 48-72 hours depending upon the specimen. Urine culture was performed using Cysteine Lactose Electrolyte Deficient (CLED) medium (HiMedia Laboratories, India) and incubated aerobically for upto 24 hours. For blood culture, 5-10ml blood was inoculated in brain heart infusion (BHI) broth (HiMedia Laboratories, India) and incubated aerobically for upto 5 days.

Microscopic examination of the clinical specimen was performed for correlation with the culture isolates by performing Gram’s staining for all specimens expect urine that was subjected to wet mount examination to ascertain the type and number of cells such as pus cells (≥104 CFU/ml correlated with pyuria). On Gram’s staining, enterococci were seen as Gram positive cocci arranged in pairs.

Identification of the isolates was performed by using conventional biochemical tests as per established standard protocols [16]. Colonies identified as Gram positive cocci were subjected to 3% hydrogen peroxide to demonstrate lack of effervescence consistent with a negative result indicating the presence of Streptococci/ Enterococci. Bile esculin hydrolysis test and growth in 6.5% NaCl were employed for biochemical identification of enterococci. The isolates were also sub-cultured onto potassium tellurite agar (HiMedia Laboratories, India) to distinguish between Enterococcus faecalis (produces black colonies) and Enterococcus faecium/ other spp. (negative or no black colonies).

Antimicrobial Susceptibility test (AST) was performed using Muller Hinton agar (HiMedia Laboratories, India) by Kirby Bauer disk diffusion method as per CLSI standards M100 [17]. For disc diffusion testing, the antimicrobial discs used were as follows: Ampicillin (10μg), High level gentamicin (120μg), Erythromycin (15μg), Vancomycin (30μg), Teicoplanin (30μg) and Linezolid (15μg). For urine isolates, antimicrobial discs of Ampicillin (30μg), High level gentamicin (120μg), Levofloxacin (5μg), Norfloxacin (10μg), Nitrofurantoin (300μg), Vancomycin (30μg), Teicoplanin (30μg), and Linezolid (30μg) were used. The plates were held for full 24 hours at 37°C and read in transmitted light. The isolate was considered as resistant to Vancomycin of the zone size around vancomycin was ≤14mm. Additionally, vancomycin resistance was tested using vancomycin screen agar, prepared by adding 6 μg/ml vancomycin to brain heart BHI agar. The growth of one or more colony of Enterococcus spp. was regarding as an evidence of vancomycin resistance. Suitable controls were used during culture and AST by using E. faecalis ATCC 29212 and E. faecium ATCC 51559 strains available in the laboratory.

Once vancomycin resistance was detected among Enterococcus spp. by phenotypic method, the detection of resistance genes, i.e. VanA and VanB as well as genotypic identification of Enterococcus spp. was done using real time PCR (qPCR) using HiMedia Hi‐PCR Vancomycin Resistant Enterococci (VRE) (Multiplex) Probe PCR Kit. A cycle threshold of ≤40 was considered positive for both VanA as well as VanB genes. Appropriate positive, negative and internal controls were used as per manufacturer instructions. The nucleic acid purification was done using HiPurA Multi‐Sample DNA Purification Kit (MB554) for clinical specimens and HiPurA Bacterial Genomic DNA Purification Kit (MB505) for cultured isolates as per the supplied manufacturer protocol.

A total of 105 isolates of Enterococci were obtained from clinical specimen during the study duration. The mean age of the patients was 44.3 years (range 2-83 years), with 59% males and 41% females. Around 95.4% were hospitalized patients and the rest 4.6% were OPD patients.

Table 1: Profile of Enterococcus spp. isolates that exhibited phenotypic resistance against vancomycin

Out of these 105 isolates, 78 (74.3%) were identified as Enterococcus faecalis while the rest 27 (25.7%) were Enterococcus faecium. These enterococci isolates were obtained from various specimens, the commonest being urine (n=74, 70.5%), blood (n=16, 15.2%), pus (n=8, 7.6%), body fluids from normally sterile sites (n=3, 2.8%), pus (n=2, 1.9%) and CSF (n=2, 1.9%). Among these isolates, a total of 10 were resistant to vancomycin, indicating a prevalence of 9.5%.

The profile of the isolates showing phenotypic resistance against vancomycin is shown in table 1. Majority of the isolates were obtained from urine specimen. Three isolates were identified as Enterococcus faecium while the rest seven were Enterococcus faecalis. Vancomycin-resistance gene was detected in eight isolates by qPCR. Five of these strains carried vanA gene and three carried vanB gene.

The antibiogram was analyzed and the resistance pattern was as follows. All isolates were resistant to penicillin, ampicillin and tetracycline while 90% were resistant to ciprofloxacin. High level Gentamicin and levofloxacin exhibited 80% resistance while 60% resistance was observed against erythromycin and norfloxacin and 40% against fosfomycin and nitrofurantoin. Approximately one third isolates were resistant to linezolid, teicoplanin and tigecycline and only 20% resistance was observed against doxycycline and minocycline. All isolates were susceptible to chloramphenicol.

The present study reports a higher prevalence of Enterococcus faecalis (75%) as compared to Enterococcus faecium (25%). In a systematic review and meta-analysis by Smout et al, a similar dominance of Enterococcus faecalis as the commonest isolate from hospital based studies was observed [18]. Similar findings were reported by Phukan et al. [19] and Sivaradjy et al [20], while another study from NewDelhi showed a contrasting data as Enterococcus faecium was isolated more frequently as compared to Enterococcus faecalis [21].

While the emergence of vancomycin drug resistance among enterococci is attributable to the selection pressure exerted by the use of cell wall acting agents including vancomycin [22], there are other reasons underscoring the threat of vancomycin resistant enterococci to humans. Apart from being one of the leading causes of drug resistant organisms causing life threatening nosocomial infections, they can also transfer the gene clusters of vancomycin resistance to Staphylococcus aureus strains such as methicillin resistant Staphylococcus aureus (MRSA) via horizontal gene transfer mechanisms [20,23].

As observed in the previous studies, the isolates showed very high resistance (more than 90%) to beta lactam antibiotics (penicillin and ampicillin), tetracycline and fluoroquinolone such as ciprofloxacin and levofloxacin [12]. High level resistance was also noted against gentamicin in 80% VRE isolates which is in accordance with the previous studies [21]. In the present study, linezolid, teicoplanin, tigecycline, doxycycline and minocycline showed reasonable susceptibility patterns with more than 65% isolates of enterococci being susceptible to these antimicrobials. Previous studies have demonstrated mixed results of these antimicrobials in case of VRE isolates [12], still making these drugs the mainstay of treatment.

The prevalent isolates in the present study were E. faecalis and E. faecium that demonstrated approximately 9.5% resistance to vancomycin by phenotypic method. Majority of the isolates were found to be carrying the vanA gene that suggests a need for continuous monitoring of vancomycin resistant enterococci.

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