A facultatively anaerobic, Gram-positive bacterium, enterococci are found in nature and in both human and animal digestive tracts. They are a significant pathogen of urethral infection, soft tissue infection, sepsis, and meningitis and the second most common cause of infections linked to healthcare1,2. These include the clinically important Enterococci, Enterococcus faecalis, and Enterococcus faecium. About 95% of human enterococcal infections are caused by Enterococcus fecium, a nosocomial pathogen—a form of bacteria that is resistant to most antibiotics and is usually acquired in a hospital

setting. It is also a major cause of hospital-acquired and multidrug-resistant infections3.Patients are seriously at risk from the rising incidence of vancomycin-resistant enterococci (VRE) in hospitals because of their multiple treatment resistance. This study is important because it clarifies the possible source of resistance genes and how they spread to different bacterial strains, which could impact and perhaps transform medical approaches. This discovery has significant ramifications for healthcare practices that call for modification and adaptability4.The medical community is now quite concerned about vancomycin-resistant enterococcal infections. Based on the degree and inducibility of resistance to vancomycin and teicoplanin, three separate glycopeptide resistance phenotypes (Van A, Van B, and Van C) can be distinguished. Whereas the VanB type has developed inducible resistance to vancomycin but not to teicoplanin, the VanA type has developed inducible resistance to both.5 one of the most important ways to stop the spread of VRE in hospitals and the community is to identify resistance profiles early.

The current investigation sought to identify the vanA gene's prevalence and ascertain the antibiotic resistance profile of enterococci isolated from clinical samples. The study places special emphasis on the phenotypic description of enterococci's vancomycin resistance. The results of this study will be extremely valuable to the field since they offer a thorough understanding of the resistance profiles, especially with regard to vancomycin.

This study was conducted at Lord Buddha Koshi Medical College and Hospital, Saharsa. A total of 150 Enterococcus strains were isolated from various clinical samples, including urine, blood, pus, pleural fluid, cerebrospinal fluid (CSF), sputum, and ascitic fluid.

Identification of Enterococcus Species

Enterococcus isolates were initially identified based on morphological characteristics, followed by Gram staining and standard biochemical tests, including:

• Arabinose utilization
• Growth in 6.5% NaCl
• Bile esculin degradation
• Pyrrolidinyl β-naphthylamide (PYR) degradation

Antibiotic Susceptibility Testing

Antibiotic susceptibility was determined using the Modified Kirby-Bauer disc diffusion method, following Clinical and Laboratory Standards Institute (CLSI) guidelines [6]. The following antibiotic discs (Himedia) were used:

• Penicillin (10U/disc)
• Ampicillin (10μg)
• High-level gentamicin (120μg)
• Ciprofloxacin (5μg)
• Vancomycin (30μg)
• Linezolid (30μg)
• Nitrofurantoin (300μg)
• Fosfomycin
• Tetracycline
• Minocycline
• Doxycycline

Minimum Inhibitory Concentration (MIC) Determination for Vancomycin

The MIC of vancomycin was determined using the micro broth dilution method, following CLSI protocol and MIC breakpoints [6].

As part of quality control, the following reference strains were included to ensure accuracy and reliability in MIC determinations:

• E. faecalis ATCC 29212
• E. faecalis ATCC 51299

The MIC testing involved preparing a stock solution and determining vancomycin’s minimum inhibitory concentration (MIC) against Enterococcus isolates using the agar dilution method.

Among the 150 Enterococcus isolates, E. faecalis was the most prevalent species, accounting for 45.3% (68 isolates), followed by E. faecium (34.6%, 52 isolates) and other Enterococcus species (20%, 30 isolates). The most common clinical sources of Enterococcus isolates were pus (24.6%), urine (21.3%), and ear swabs (15.3%), followed by fluids & other samples (14%), blood (12.6%), and sputum (12%). Among these, 11 isolates (7.3%) were identified as Vancomycin-Resistant Enterococci (VRE). The highest proportion of VRE was found in pus and urine cultures (27.2% each), followed by fluids & other samples (18.1%), while blood, ear swabs, and sputum samples each contributed 9.1% of VRE cases.

A total of 11(7.3%) isolates were found to be vancomycin-resistant using the broth microdilution technique as per CLSI guidelines. 7/68(10.2%) were vancomycin-resistant enterococcus faecalis, 4/52(7.7%) were vancomycin-resistant enterococcus faecium, and no VRE was isolated from other enterococcus species. All 11 isolates found vancomycin-resistant by the broth microdilution method showed MIC between ≥32μg/ml to ≥256μg/ml. 

The sensitivity pattern of E. faecalis and E. faecium shows that 100% were sensitive to  linezolid. Where E. faecalis shows that 67(98.5%) were sensitive to ampicillin, followed by 56(82.3%) were to gentamycin (HLG), 61 (89.7%) were to vancomycin, 19(27.9%) were to ciprofloxacin, 15(22.1%) were to penicillin. In the case of E. faecium38(92.3%) were sensitive to vancomycin followed by 42(82.7%) were to gentamycin (HLG), 14(26.9%) were to ciprofloxacin, 9(17.3%) were to penicillin.

Enterococci have emerged as a major cause of nosocomial infections in healthcare environments and are increasingly implicated in opportunistic infections among immunocompromised individuals [7–9]. These pathogens are associated with severe and life-threatening conditions, such as endocarditis, bacteremia, surgical site infections, and urinary tract infections [7–9], underscoring the necessity of early detection and targeted antimicrobial therapy. The growing prevalence of vancomycin-resistant Enterococci (VRE) has further complicated clinical management, narrowing therapeutic options for these multidrug-resistant organisms.

In this study, 11 (7.3%) of 150 Enterococcus isolates exhibited vancomycin resistance via broth microdilution, with minimum inhibitory concentrations (MICs) ranging from ≥32 μg/mL to ≥256 μg/mL. The emergence of VRE in India was first reported by Mathur et al. in 1999 [10], with subsequent studies indicating variable resistance rates. For instance, Taneja et al. identified 5.55% VRE using E-test and agar dilution methods [11], while Fernandes and Dhanashree documented an 8.6% prevalence of vancomycin resistance among 150 isolates [12]. Similarly, Shafiyabi et al. reported a 5% VRE rate in a South Indian tertiary care center [13]. Although VRE prevalence in India remains lower than in Western nations, its upward trajectory warrants concern [14].

This study analyzed 150 Enterococcus isolates from diverse clinical specimens, including urine (21.3%), pus (24.6%), blood (12.6%), ear swabs (15.3%), sputum (12%), and other fluids (14%). Among the 11 VRE isolates, the majority originated from pus (27.2%) and urine (27.2%), followed by fluids (18.1%), blood (9.1%), ear swabs (9.1%), and sputum (9.1%). Comparatively, Akhter S [15] observed a lower VRE prevalence of 4.65% in E.

faecalis, while another study [16] reported only 1.4% resistance exclusively in E. faecium. The higher resistance rate in our findings highlights the critical need for stringent antibiotic stewardship and robust infection control protocols to curb the spread of multidrug-resistant strains.

The rising incidence of VRE underscores the urgency of enforcing evidence-based antibiotic policies and vigilant surveillance in healthcare settings. These measures are essential to mitigate the emergence and transmission of resistant pathogens, emphasizing the importance of prudent antimicrobial use and implementing rigorous infection control practices

Enterococci have become a significant cause of severe nosocomial infections, with urinary tract infections (UTIs) representing the most common clinical manifestation, followed by wound infections and bloodstream infections. Enterococcus faecalis and Enterococcus faecium account for the majority of these infections. Vancomycin remains a critical therapeutic agent for multidrug-resistant Gram-positive bacteria, particularly those resistant to β-lactams and aminoglycosides. However, the rise of vancomycin-resistant Enterococci (VRE) poses a formidable clinical challenge, as treatment options are severely limited. In this study, 11 isolates demonstrated high-level vancomycin resistance (MIC ≥32 μg/mL) via broth microdilution, underscoring the urgency of addressing this resistance. These findings emphasize the need for enhanced antimicrobial stewardship, rigorous infection control protocols, and the development of novel therapeutic strategies to combat the growing threat of VRE in healthcare settings.

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