Antibiotic resistance profiling of Enterococcus species isolated from animal-origin foods marketed in rural settings

Introduction

Enterococcus species are Gram-positive bacteria and are among the ESKAPE organisms highlighted by the World Health Organization. There are over 73 known Enterococcus species, and two species of public health importance are E. faecalis and E. faecium (Fahim et al., 2025) [6] . Enterococci are highly resilient and present as commensals in the gut of birds, animals, and humans (Said et al., 2025) [15] . They are primarily transmitted through the fecaloral route and excreted in human and animal fecal matter. This bacterium is a significant cause of nosocomial infections, often due to virulent, antibiotic-resistant strains. The role of livestock and poultry in harboring antibiotic-resistant Enterococci and in the fecal excretion of these organisms, as well as their prevalence in raw animal-origin foods in the Indian context, is not well documented, except for a few studies (Singh and Garg, 2005; Parmar et al., 2022; Kumar et al., 2024) [17,11,14] . Enterococci are among the priority bacteria being monitored for the development and spread of antimicrobial resistance (AMR) in developed countries. Enterococci can colonize different ecological niches and could spread through contaminated animal-origin foods within the food chain. They are intrinsically resistant to several antibiotics and also can acquire and transfer AMR determinants to other species (Iweriebor et al., 2015) [9] . Fresh meat is preferred in India; unhygienic dressing, cutting, and handling operations may cross-contaminate it. Thus, it is of paramount concern to study Enterococcus species for their prevalence and antibiotic resistance. This study was undertaken to determine the prevalence of Enterococci in retail meat and eggs commonly sold in the local markets.

Materials and Methods

Sampling

In this cross-sectional study, the prevalence of Enterococcus species was estimated from randomly collected samples from a local market. The sample size was calculated using Epitools (https://epitools.ausvet.com.au/) assuming a prevalence of 40%, a desired precision of 0.05, and a 95% Confidence Interval (CI). A total of 480 samples of raw chicken, mutton, chevon, and table eggs were collected from local chicken and meat shops. The distribution of samples was kept uniform, i.e., 120 samples each of chicken, mutton, chevon, and table eggs.

Isolation and identification of Enterococcus species

Samples were initially diluted to a 1:10 ratio in peptone water, and 1 mL of the diluted sample was enriched in azide dextrose broth (HiMedia) at 37 °C for 24 hr. Enriched samples were plated on bile esculin agar (HiMedia) and incubated at 37 °C for 24 hr. Presumptive identification of Enterococci was done based on colony characteristics, Gram's staining, catalase, oxidase, sorbitol fermentation, and bile esculin hydrolysis. The pure isolated colonies of Enterococci were further sub-cultured on the selective medium (bile esculin agar) and confirmed by polymerase chain reaction (PCR).

Molecular detection

A pure bacterial colony was subjected to DNA extraction using the boiling-and-snap-chilling method. The snapchilled samples were centrifuged at 10000 g for five minutes (Sorvall ST 8, Thermo Scientific), and the supernatant was used as a DNA template. The DNA concentration was examined using a NanoDrop Lite spectrophotometer (Thermo Scientific). Bacterial DNA was further analyzed by PCR to confirm the presence of Enterococci at the genus and species levels. The oligonucleotide sequences used and the PCR protocol used in the study were as described earlier (Kariyama et al., 2000) [10] . The multiplex PCR was performed in a 30 μl reaction containing 12.5 μl 2x PCR Master Mix (Takara), 0.5 μl (10 pmol/μl) of forward and reverse primers of E. faecalis, 0.25 μl (10 pmol/μl) of forward and reverse primers of 16 rDNA and 0.125 μl (10 pmol/μl) of forward and reverse primers of E. faecalis, 13.25 μl nuclease-free water, and 2.5 μl DNA. PCR conditions were set as initial denaturation at 94 ºC for 5 min, followed by 30 cycles of denaturation at 94 ºC for 1 min, annealing at 54 ºC for 1 min, extension at 72 ºC for 1 min, and final extension was performed at 72 ºC for 10 min and holding at 4 ºC (Applied Biosystems VeritiPro). The hyl gene was detected as per the method described by Vankerckhoven et al. (2004) [19] . The uniplex PCR was performed in a 25 μl reaction containing 12.5 μl 2x PCR Master Mix (Takara), 1 μl (10 pmol/μl) of forward and reverse primers, 5.5 μl nuclease-free water, and 5 μl DNA. PCR conditions were set as initial denaturation at 94 ºC for 5 min, followed by 30 cycles of denaturation at 94 ºC for 1 min, annealing at 56 ºC for 1 min, extension at 72 ºC for 1 min, and a final extension at 72 ºC for 10 min, with a final hold at 4 ºC. The PCR products were run on a 1.5% agarose gel stained with ethidium bromide, and images were captured using the gel documentation system (IBright CL750, Thermo Scientific). Details of PCR primers are depicted in Table 1.

Antibiogram

Antibiotic sensitivity and resistance profiling of isolated Enterococci was performed by the disk diffusion method described previously (Bauer et al., 1966), and results were interpreted according to the CLSI (2020) [5] guidelines and the manufacturer's instructions (HiMedia). Enterococcus species were studied using 10 antibiotics from different classes: aminopenicillins & β-lactams, amphenicol, fluoroquinolones, lincosamides, tetracyclines, macrolides, aminoglycosides, and rifamycins. The antibiotic discs procured from HiMedia Lab. Pvt. Ltd., Mumbai, India, used in this study were amoxyclav (20/10 μg), ampicillin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), doxycycline (30 μg), erythromycin (15 μg), gentamicin (10 μg), rifampicin (15 μg), and tetracycline (30 μg). Pure colonies of Enterococci were first overnight enriched in Brain Heart Infusion (BHI) broth at 37 °C, and the enriched cultures were then spread onto Mueller-Hinton agar (MHA). Antibiotic discs were aseptically placed on the plates and incubated overnight at 37 °C.

Statistical analysis

The prevalence of Enterococci in food samples was calculated, and a significant association between Enterococci and antibiotic resistance and sensitivity was assessed using the Chi-square (χ2) test (p-value), at a 95% confidence level.

Results

In this study, 480 samples were collected, and the prevalence of Enterococcus species was estimated. A total of 249 Enterococci (51 chicken, 48 eggshell, 67 chevon, 83 mutton) were isolated and identified using cultural and molecular methods. The overall prevalence was 51.87%, and the distribution across samples was as follows: chicken (42.50%), eggs (40%), chevon (55.83%), and mutton (69.16%). Multiplex PCR targeting 16S rRNA, ddl E. faecalis, and ddl E. faecium genes revealed the presence of E. faecalis (28.57%) and E. faecium (82.14%) in eggshellsurface-origin isolates. Other isolates from chicken, chevon, and mutton were Enterococci harboring the 16S rRNA gene; however, they may belong to species other than E. faecalis and E. faecium (Fig 1). The hyl gene was present in 16.66%, 14.92%, and 28.91% of isolates from eggshell surface, chevon, and mutton, respectively (Fig 2). The antibiotic susceptibilities of Enterococcus species isolated from chicken, eggshell surface, chevon, and mutton are shown in Tables 2,3, 4 & 5. It was observed that Enterococci isolated from meat and chicken are multidrug-resistant, with greater resistance to clindamycin, rifampicin, erythromycin, and tetracycline. However, chicken-and egg-origin Enterococci showed sensitivity to antibiotics, viz., amoxicillinclavulanate, ampicillin, ciprofloxacin, chloramphenicol, and gentamicin. The chevon and mutton origin isolates were mostly sensitive to amoxicillin-clavulanate, chloramphenicol, ciprofloxacin, and gentamicin.

Discussion

In the current study, the prevalence of Enterococcus species in animal-origin raw foods, namely chicken chevon, mutton, and table eggs, was estimated. The recorded prevalence is towards the higher side, indicating unhygienic processing and cross-contamination of raw meat. A study from Anand, Gujarat, recorded an overall prevalence of 56% for Enterococci in chicken meat collected from retail chicken shops, and over 77% of Enterococci were positive for the ddl E. faecalis gene (Parmar et al., 2022) [14] . The present findings are consistent with these observations on the overall prevalence of Enterococci. Studies from Hisar, Haryana, documented an E. faecalis prevalence exceeding 79% in animal products such as chicken, chevon, and milk (Chandra and Garg, 2005; Singh and Garg, 2005) [4,17] , and confirmed the ubiquitous occurrence of Enterococci in foods of animal origin. Another study from Andhra Pradesh also detected Enterococci on pig farms and in retail pork and confirmed that the species were E. faecalis, E. faecium, E. gallinarum, and E. casseliflavus (Kumar et al., 2024) [11] . This bacterium is not very extensively studied in India, and there are no references available from the state of Maharashtra. However, several studies worldwide have reported its prevalence in animal-derived foods. Enterococci are the natural inhabitants of the gastrointestinal tract (GIT) of humans and animals. Enterococcus species, namely E. faecalis, E. faecium, and E. durans, are capable of causing human infections, and E. faecalis and E. faecium are the most abundant species in human feces. However, animal GIT also represents a predominant reservoir for the different species of Enterococci (Lebreton et al., 2014) [12] . During slaughter and dressing operations on meat animals, Enterococci, along with other gastrointestinal microbes, may contaminate meat. The species prevalent in meat animals include E. hirae, E. faecium, E. faecalis, E. casseliflavus, E. mundtii, E. durans, and E. gilvus. A study from Turkey reported the prevalence of Enterococcus species in healthy sheep and goat carcasses at 60% and 36.6%, respectively (Cebeci, 2024) [3] . In the current study, we have also recorded a higher prevalence of Enterococci in raw sheep and goat meat samples collected from retail meat shops. Consistent with our observations, Amuasi et al. (2023) [1] also reported a high prevalence of Enterococci in chevon and mutton samples collected from slaughterhouses in southern Ghana. A study conducted in the United States of America revealed that E. faecalis and E. faecium were the predominant species in chicken meat, regardless of antibiotic use (Manson et al., 2019) [13] . Predominance of E. faecium in the chicken breast (79%) was observed in the study from Maryland (Hayes et al., 2003) [8] . Over 79% of prepacked chicken samples collected from supermarkets in Turkey were contaminated with Enterococci (Sanlibaba et al., 2018) [16] . Virulent Enterococcus species may harbor specific genetic sequences that enable them to evade the host immune system. Some of the predominant virulence genes in Enterococci are gelE, esp, cylA, and hyl. We have studied Enterococci for the presence of the hyl gene, which encodes hyaluronidase, an enzyme that degrades hyaluronic acid in connective tissue. It was present in 16.66%, 14.92%, and 28.91% of isolates from eggshell surface, chevon, and mutton, respectively. The hyl gene is predominantly present in clinically important Enterococci and is mostly detected in human-origin isolates. In contrast to our observations, the hyl gene could not be detected in the Enterococci isolated from red meat in a study from Slovenia (Golob et al., 2019) [7] . The antibiotic susceptibilities of Enterococcus species isolated from chicken, eggshell surface, chevon, and mutton are shown in Tables 2,3, 4 & 5. It was observed that Enterococci isolated from meat and chicken are multidrugresistant, with greater resistance to clindamycin, rifampicin, erythromycin, and tetracycline. However, chicken-and eggorigin Enterococci showed sensitivity to antibiotics, viz., amoxicillin-clavulanate, ampicillin, ciprofloxacin, chloramphenicol, and gentamicin. The chevon and mutton origin isolates were mostly sensitive to amoxicillinclavulanate, chloramphenicol, ciprofloxacin, and gentamicin. As observed in the current study, Enterococci resistant to erythromycin and tetracycline and sensitive to gentamicin and chloramphenicol were also documented in India from Enterococci isolated from chicken meat (Parmar et al., 2022) [14] . Enterococci are intrinsically resistant to cephalosporins and exhibit reduced susceptibility to penicillins due to low-affinity penicillin-binding proteins (Torres et al., 2018) [18] . Enterococci are highly resistant to tetracycline due to their ability to readily acquire and share tet genes via mobile genetic elements, which bypass tetracycline's mechanism of action.

Conclusion

The present study highlighted the importance of raw meat and chicken in harboring antibiotic-resistant Enterococci that cross-contaminate the meat during unhygienic slaughtering and dressing operations. Our study showed a high level of phenotypic resistance among Enterococcus species isolated from eggshell surfaces, chicken, chevon, and mutton. The current study emphasizes the need for targeted antibiotic-resistance surveillance of Enterococci in food animals.

Acknowledgment

The authors are thankful to the Indian Council of Medical Research, New Delhi, for financial support (Grant No. Zon50/2020/ECDII), and the laboratory facilities developed through ICMR funding were used in the research.

Conflict of interest

The authors declare no conflict of interest

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