Introduction

Foodborne illnesses frequently occur after consuming contaminated food, particularly animal-derived products like meat1. Enterococci of animal origin are found in animal-derived foods that are consumed by humans2. Enterococci are a component of the natural microbiota in the digestive systems of animals and humans, particularly Enterococcus faecalis and Enterococcus faecium, which have been shown to be significantly important. These nosocomial pathogens are recognized as the causative agents of various animal ailments, including mastitis, endocarditis, diarrhea, and septicemia in cattle, domesticated animals, swine, and poultry3,4,5. The species Enterococcus durans, Enterococcus hirae, Enterococcus gallinarum, Enterococcus cassseliflavus, Enterococcus faecium, and Enterococcus faecalis are frequently present in the gastrointestinal systems of livestock6.

Enterococci’s pathogenesis is linked to a diverse range of virulence factors. Virulence factors contribute to the development of enterococcal infections by facilitating the attachment, colonization, and invasion of host tissues. They also affect the host’s immune response and produce enzymes and toxins outside of cells, which worsen the severity of the illness. The key adhesion factors involved in biofilm development include Ebp (endocarditis and biofilm-associated pili), Asa (aggregation substance), EfaA (E. faecalis antigen A), Esp (extracellular surface protein), Ace (collagen-binding cell wall protein), cylA (hemolysin), efm (E. faecium-specific cell wall adhesin), cad1 (pheromone cAD1 precursor lipoprotein), sagA (secreted antigen), and cpd1 (pheromone cPD1 lipoprotein)7,8,9.

The excessive use of antibiotics in animals is associated with the emergence of antimicrobial resistance, and mechanisms of antibiotic resistance can readily disseminate among all pathogenic, commensal, and environmental bacteria through horizontal gene transfer of mobile genetic elements10,11. Both human and animal enterococci possess intrinsic resistance to several antimicrobial drugs, and they also have the ability to develop resistance to additional antimicrobial agents, such as glycopeptides, quinolones, tetracyclines, macrolides, and streptogramins1,12,13. Although food-producing animals may not always directly transmit enterococci to humans, they can nonetheless facilitate the transfer of resistance genes from these animals to humans. Hence, the occurrence of resistant enterococci, particularly vancomycin-resistant enterococci, in animals used for food production has emerged as a significant issue12.

The emergence of antimicrobial resistance in zoonotic bacteria poses a substantial risk to public health, mainly owing to the heightened likelihood of treatment failures. Furthermore, the emergence of resistance, particularly through the acquisition of transmissible genetic components, might also impact other characteristics, such as the capacity to inhabit an animal host or endure in agricultural or food-processing settings14,15. The presence of antibiotic-resistant enterococci in meat, animal-related sources, and habitats linked to animals, food-handling equipment, and healthy humans emphasizes the importance of evaluating enterococci in slaughterhouses as well. The main source of zoonotic pathogens is the GI tract of healthy food animals. Most food-related diseases are spread by feces during the slaughtering process or by cross-contamination during processing14,16. The transfer of harmful microorganisms from one part of an animal’s body to another during the slaughtering process poses a substantial risk to the safety of the meat. Carcass tissues primarily become contaminated with fecal particles during the evisceration and skinning processes17,18,19. The aim of this study was to assess and characterize the prevalence, types, virulence determinants, and antimicrobial resistance profiles of enterococci from healthy cattle, sheep, and goat carcasses to highlight their zoonotic importance.

Materials and methods

Sample collection

Between November 2019 and December 2020, the researchers obtained carcass samples from a total of 20 cattle, 80 sheep, and 50 goats from a slaughterhouse located in Van, a city in the eastern region of Turkey. The animals that were sampled were chosen in a randomized way. Sampling was completed by visiting the slaughterhouse twice a year, depending on its slaughtering capacity. The selected slaughterhouse was visited at two occasions to obtain 600 surface swab samples from 150 animals (visit I, 10 cattle, 40 sheep, and 25 goats; visit II, 10 cattle, 40 sheep, and 25 goats) and carcasses during the pre-chilling stage of the slaughtering process. A total of 600 samples from different parts of beef, sheep, and goat carcasses were collected using swabs. The sampling locations were situated in the brisket, flank, hind leg, and rectal regions of the carcass. The sampling region was meticulously surveyed for 1 min using cotton swabs that were swiped in both vertical and horizontal directions. Four 100 cm2 areas, measuring 10 cm2 × 10 cm2, were swabbed on each beef, sheep, and goat carcass. Carcass swabs were collected pre-chilling using sterile cotton swabs soaked in 10 ml of buffered peptone water, following the protocols set by the International Organization for Standardization20. The sampler was pressed firmly and evenly as it was inserted vertically onto the peripheral surfaces, with this process repeated approximately 10 times. Then, the sampler was turned and used to swipe horizontally and diagonally, each motion being repeated around 10 times. The samples were promptly delivered to the laboratory of Espiye Vocational School, Giresun University, within 24–48 h of being collected, using refrigerated containers21.

Enterococcus species isolation

Swab samples were homogenized in a blender (Waring, New Hartford, Conn.) with 90 ml of buffered peptone water (BPW) (Lab M, Lancashire, UK). After incubation at 37 °C for 24 h, 0.1 ml was streaked onto Slanetz and Bartley Agar (Lab M, Lancashire, UK) and incubated for 24 ± 2 h at 37 ± 1 °C under the same condition3. After incubation period, pink or dark red colonies with a narrow, whitish border were observed. After the incubation period, five colonies that showed characteristics of Enterococcus species were selected from each petri dish and transferred to Tryptone Soya Agar (Lab M, Lancashire, UK) agar for purification. The agar plates were then incubated at a temperature of 37 ± 1 °C for 24 ± 2 h. The suspected isolates were biochemically identified using Gram staining and catalase activity. All strains were stored in skimmed milk powder stocks at − 80 °C for further testing22. The Enterococcus species were identified through MALDI-TOF MS (BioMérieux Inc., Marcy l’Etoile, France), which was performed only on Gram-positive and catalase-negative cocci23.

DNA isolation protocols

The QIAsymphony, a magnetic particle-based automated extraction system, was used to extract genomic DNA. The extraction was carried out using the QIAamp DNA micro kit (Qiagen, Hilden, Germany) following the instructions provided by the manufacturer. The isolated DNA was utilized as a template for PCR using the specified methods.

Screening for confirmation and virulence genes

All Enterococcus isolates were screened for the confirmation genes and the presence of virulence genes. These were 16S rDNA24, E. faecalis25, E. faecium25, E. casseliflavus25, E. gallinarum25, E. hirae26 identification genes and virulence genes; asa1 (aggregation substance)27, ace (collagen-binding protein)28, cylA (cytolysin activator)27, efaA (endocarditis antigen)29, esp (enterococcal surface protein)28, gelE (gelatinase)28, hyl (hyaluronidase)28. The methods reported Jahan et al.24 were modified and used for genotyping the Enterococcus isolates.

Antimicrobial susceptibility testing

The susceptibility of Enterococcus isolates to antibiotics was assessed using the disc diffusion method, following the protocols outlined by the Clinical and Laboratory Standards Institute30. To determine antibiotic resistance in the isolates, 10 µg ampicillin (AM), 5 µg ciprofloxacin (CIP), 30 μg chloramphenicol (C), 15 µg erythromycin (E), 200 µg fosfomycin (FF), 300 μg high-level streptomycin-resistant (HLSR), 120 μg high-level gentamicin-resistant (HLGR), 10 μg imipenem (IPM), 5 µg levofloxacin (LEV), 30 μg linezolid (LNZ), 300 µg nitrofurantoin (F), 10 units penicillin (P), 15 μg quinupristin-dalfopristin (QD), 5 µg rifampin (RA), 30 µg vancomycin (VA), 30 µg teicoplanin (TEC), 30 µg tetracycline (TE), 15 μg tigecycline (TIG) and 5 μg vancomycin (VA) antibiotic discs were used (all purchased from Liofilchem, Roseto degli Abruzzi, Italy). After incubation, the resulting diameters of the inhibition zones that formed around the discs of AM, P, VA-30 µg, TEC, E, TE, CIP, LEV, F, RA, FF, C, QD, LNZ, HLSR, and HLGR were classified as susceptible, intermediate, or resistant according to the diameters and breakpoints reported in CLSI documents30. For the remaining antimicrobial agents (TIG, VA-5 µg, and IPM), the critical values were evaluated according to the zone table provided by the European Committee on Antimicrobial Susceptibility Testing document31. For quality control purposes, Staphylococcus aureus ATCC 25,923 and E. faecalis ATCC 29,212 were utilized as control strains.

PCR detection of genes for antimicrobial resistance

In the present study, various PCR assays were used for the detection of antibiotic resistance genes (AGRs) in Enterococcus isolates. All isolates were tested for the presence of aminoglycoside modifying enzyme (AME) genes [aac(6ʹ)-Ie–aph(2ʺ)-Ia, aph(2ʺ)-Ib, aph(2ʺ)-Ic, aph(2ʺ)-Id, ant(3ʺ)-Ia, and ant(6)-Ia], phenicol resistance genes (cfr, fexA, and optrA), tetracycline resistance genes [tet(L), tet(M), and tet(O)], macrolide resistance genes [ermA, ermB, and mef], and efflux pump genes [efr(A), emeA, and lsa] by PCR using specific primers as described by a previous study32, with some modifications.

Statistical analysis

Descriptive statistics for the categorical variables in this study, namely animal species, animal carcass sites, and Enterococcus species are expressed as a number (n) and a percentage (%). Chi-square and Fisher’s exact tests were used to determine the relationships between the factors animal species, carcass sites, and Enterococcus species. The SPSS (IBM SPSS for Windows, ver. 26) statistical package program was used for the analyses and the statistical significance level was set to p < 0.05.

Ethical approval and consent to participate

Ethical review and approval were waived for this study, since all sample materials were carcass surface swab samples and were collected during authorized slaughtering in registered slaughterhouses for human consumption. No animals were slaughtered for research purposes.

Results

Prevalence of enterococci

The overall prevalence of enterococci in sheep, goat, and cattle animals from a slaughterhouse in Van, Turkey was 60% (90/150). The species distribution is shown in Table 1. The predominant species detected were E. hirae (n = 34, 37.7%) and E. casseliflavus (n = 30, 33.3%). A smaller number of E. faecium (n = 14, 15.5%), E. faecalis (n = 11, 12.2%), and E. gallinarum (n = 1, 1.1%) were also evaluated. The number of Enterococcus species in the brisket, flank, hind leg, and rectal samples of carcasses is shown in Table 1. Enterococcus species contamination was not significantly different in animal species in comparison to carcass sites (p < 0.05). E. casseliflavus and E. faecium were isolated from 10 and 6 out of 150 carcass surface samples from the brisket and hind leg, respectively, whereas E. hirae, E. faecalis, and E. gallinarum were isolated from 13, 5, and 1 out of 150 carcass surface samples from rectal swabs, respectively.

Table 1 Prevalence of Enterococci in brisket, flank, hind leg, and rectal swab samples.
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Enterococci were detected in a total of 150 animal species, comprising 60% sheep, 36.6% goats, and 3.3% cattle. There was significant difference in prevalence (p < 0.010) for the different Enterococcus species across different animal species.

Virulence of Enterococci

The distribution of virulence genes among Enterococcus species is presented in Table 2. The different species of Enterococcus showed variability in their virulence gene profiles. The hyaluronidase virulence factor hyl, enterococcal surface protein esp, and cytolysin activator gene cylA were absent in all 90 of the Enterococcus isolates. Among the E. faecalis isolates, six (54.5%) isolates tested positive for the efaA gene. The aggregation substance gene, asaI, was found in two (18.8%) E. faecalis isolates. The collagen-binding protein gene, ace, and the gelatinase gene, gelE, were possessed by one (9.9%) and one (9.9%) E. faecalis isolates, respectively. Two, two, and one of five E. hirae isolates tested positive for gelE, efaA, and asaI genes, respectively, whereas none of them possessed the ace, esp, cylA, or hyl genes. For E. faecium and E. casseliflavus isolates, 3/14 (21.4%) and 1/30 (3.3%) harbored efaA and ace genes, respectively. Table 3 displays the incidence of virulence genes in enterococci from sheep, goats, and cattle. The genes ace, gelE, efaA, and asaI were isolated from sheep species, whereas efaA and asaI were isolated from goat species.

Table 2 Distribution of virulence genes profiles among enterococci.
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Table 3 Distribution of virulence genes in enterococci from sheep, goat and cattle.
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Antibiotic resistance

The resistance patterns of enterococci towards the tested antimicrobial agents are presented in Table 4. Resistance to QD was the most common (28.8%), followed by TE (21.1%), AM (18%), and RA (15.5%). High rates of resistance to QD were found in E. hirae (21.1%) and E. faecium (7.7%). Tetracycline resistance was common in E. faecalis (6.6%), E. casseliflavus (6.6%), E. hirae (4.4%), and E. faecium (3.3%). Ampicillin and rifampin resistance were frequent in E. faecium (8.8% and 4.4%, respectively). Resistance to VA (7.7%), CIP (6.6%), F (4.4%), FF (4.4%), C (4.4%), P (2.2%), LEV (1.1%), and TIG (1.1%) was relatively low. HLSR (5.5%) resistance was found in a few of the isolates from E. faecalis, E. faecium, and E. casseliflavus, but not in any from E. gallinarum. A total of 90 isolates from four tested species were resistant to at least one antibiotic (64.4%, 58/90), with 17 isolates (18.8%, 17/90) from 90 Enterococcus isolates displaying multidrug resistance (Tables 4 and 5).

Table 4 Antimicrobial resistance pattern of Enterococus species.
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Table 5 Characteristics of the 17 multidrug-resistant strains of enterococci.
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Genotyping of antibiotic resistance

The distribution of antibiotic resistance genes amongst Enterococcus species is presented in Table 6 and Fig. S1. Of the three isolates showing resistance to E, one (1.1%) carried the ermA gene, and two (2.2%) carried aac(6’)Ie-aph(2")-la. Three efflux pump genes, efr(A), emeA, and lsa, were observed in 31 Enterococcus isolates. Glycopeptide genes VanC1 and VanC2 were present in 1.1% and 1.1% of E. faecium and E. hirae isolates in goat and sheep carcasses, respectively.

Table 6 The presence of antibiotic resistance genes profiles among enterococci.
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Discussion

Enterococci, being a component of the normal microorganisms found in the GI of animals, can be present in meat during the slaughtering process. The prevalent species include E. hirae, E. faecium, E. faecalis, E. casseliflavus, E. mundtii, E. durans, and E. gilvus33. Transmission from other people, the environment, and foods contaminated with livestock intestinal microflora are just a few of the ways that enterococci can infect humans34. The objective of this study was to determine the frequency of Enterococcus species, analyze their patterns of antibiotic resistance, and identify the presence of resistance and virulence genes in the Enterococcus species collected from a slaughterhouse in Van, Turkey. This research is particularly relevant because of the high consumption of meat by a significant portion of the local population. Wide variation (0–90.6%) in the prevalence of enterococci in food-producing animals has been reported in this study and different countries1,4,5,12,16,35,36,37. In the present study, the speciation of the isolates confirmed that E. hirae was the most prevalent species identified from sheep and goat carcass samples. Other studies reported the prevalence of Enterococcus spp. on cattle in a slaughterhouse with a recovery rate of E. hirae, ranging from 8 to 92%13,38. Enterococci, especially E. faecalis and E. faecium, are known to be nosocomial pathogens and have become a major clinical concern38. Although E. faecalis and E. faecium isolates identified in this study have been found at low levels by Ramos et al.1, Holman et al.36 and Güngör et al.39 both species were isolated from slaughtered animals (12.2%, 15.5% and 7.2% respectively). E. casseliflavus and E. gallinarum were also isolated from sheep and cattle carcasses, supporting similar findings reported by Ramos et al.1 and Smoglica et al.5, respectively. The data regarding the occurrence of enterococci in cattle, sheep, and goat carcasses exhibits wide variation. Differences in the occurrence rates of enterococci in cattle, sheep, and goat carcasses may be attributed to variances in geographical regions, hygiene conditions, livestock management practices, agro-climatic factors, detection and sampling methods, animal breeds, and age. Potential factors influencing the variability in the results include the quality levels of the farms supplying the animals and the health and sickness conditions of the killed animals.

Given that enterococci are naturally found in the intestinal tract of animals, it is possible for meat to get contaminated during the slaughtering process. Various enterococcal virulence genes associated with the pathogenesis of disease in humans have been documented. It is essential to carry out genetic screening on enterococci to determine their capacity to cause disease and their ability to spread between from animals to humans, which poses a significant health risk. The genetic transmission mechanisms are closely interconnected with the virulence traits of particular enterococci40,41,42,43. The identification of virulence factors is crucial in assessing bacterial pathogenicity, as these factors enable microorganisms to invade and harm the host. In this study, virulence typing was conducted by targeting seven specific genes. The high prevalence (12.2%) of endocarditis antigen efaA in E. faecalis, E. hirae, and E. faecium was consistent with findings from previous reports, whereas the moderate presence of ace, gelE, and asaI was lower than that previously reported by Beukers et al.10, Zhang et al.44, and Güngör et al.39. Other authors have observed different values. Klibi et al.12 detected gelE, especially in 11.5% and 10% of Enterococcus isolates in fecal samples from animals in Tunisia, respectively. In Italy, Smoglica et al.5, observed the gelE, asaI, efaA, ace, and esp genes in 35.41%, 25%, 22.91%, 0.08%, and 0.04% of Enterococcus isolates, respectively. In another study, Song et al.8 reported that E. faecalis isolates were positive for gelE (88%), asa1(44%), cylA (16%), and esp (4%) virulence factor genes. The diversity in enterococci virulence genes reported from other studies might be attributable the different sampling techniques, sample types, isolation processes, environmental conditions, or geographic regions.

Enterococci are environmental organisms that have the ability to adapt to and spread antimicrobial-resistant traits45. Antimicrobial-resistant enterococci in animals are thought to serve as a reservoir for transmitting resistance genes to enterococci in humans. This transmission can occur through various means, such as human ingestion of animal-derived food, direct contact between animals and humans, or environmental factors. The preference of enterococci for certain hosts does not exclude the potential spread of antimicrobial resistance from animals to humans by enterococci2. In this investigation, Enterococcus isolates exhibited resistance to one or more antimicrobial agents, with a prevalence of 64.4%. The antibiotic resistance of Enterococcus isolates was assessed for 18 antimicrobial agents using the disk diffusion method. In this study, the high prevalence of tetracycline resistance, which was detected in enterococci other than antibiotics that belong to the natural resistance group, may be linked to the use of tetracyclines in the treatment of cattle, sheep, and goats. OTC is a tetracycline antibiotic primarily used to treat infections caused by a broad range of bacteria. However, it is important to note that resistance to OTC is frequently observed among Gram-negative bacilli of enteric origin and staphylococci46,47. One prevalent application is in the treatment of BRD in cattle, which is caused by Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni (formerly known as Haemophilus somnus). Doxycycline is the preferred treatment for Rickettsiae and Ehrlichiae in small animals, as opposed to oxytetracycline46,48. Resistance to tetracycline in Enterococcus isolates was consistent with current results, which revealed a high resistance rate1,4,49,50, but a higher prevalence of tetracycline-resistant E. faecalis was obtained in the present study51,52,53. A study conducted in Tunisia between September 2011 and December 2011 showed that E. hirae species isolated from animal stools were resistant to tetracyclines, in accordance with the findings of this study12. The E. faecium isolates were more resistant to ampicillin antimicrobial agents (20%). This result was in accordance with another study in Saudi Arabia that revealed similar result9. Resistance to rifampin in E. casseliflavus was consistent with the current result, which revealed a low resistance rate16. Compared with other antimicrobial agents, the resistance rates to ciprofloxacin were relatively low in E. faecium and E. faecalis (1.1% and 2.2%, respectively). These findings produced parallel results to those of other studies conducted in the USA and Korea8,36. The prevalence of antimicrobial-resistant Enterococcus strains in slaughtering and production can be explained by the extensive utilization of antibiotics for growth promotion, disease prevention, and infection treatment.

Multidrug resistance in enterococci can arise from either intrinsic attributes of the species or from acquired resistance mechanisms. Resistance to aminoglycosides can be attributed to both intrinsic and acquired factors. Resistance to high concentrations of gentamicin and streptomycin is usually acquired through the transfer of resistant genes, while resistance to low concentrations of amikacin, tobramycin, and kanamycin is frequently caused by intrinsic factors54,55. In this study, we have observed HLSR and multidrug resitance in about 5.5% and 18.8% of Enterococcus isolates, whereas other studies have reported higher rates, including Li et al. (50%)56 and Ngbede et al. (53.1%)49, respectively. Research conducted on cattle in South Australia16. revealed that 26.9% of the isolates were resistant to at least two different classes of antibiotics. The values recorded by those authors were higher than the values noted in the current study (18.8%).

Enterococci can gain resistance to antibiotics through chromosomal mutations and horizontal gene transfer. Enterococci are naturally resistant to a wide range of antibiotic classes15. Thus, enterococci present a major challenge to illness treatment because of their limited susceptibility to antibiotics, which is caused by both intrinsic and acquired resistance. This resistance enables them to obtain supplementary resistances on mobile genetic elements, resulting in heightened interaction with other antibiotic-resistant microorganisms57. Overall, 22 target antibiotic-resistant and efflux pump genes were detected at variable frequencies in the tested isolates of Enterococcus, where tet(M), efr(A), Isa, and eme(A) were more commonly detected compared to the ermA, ermB, vanC1, vanC2, and aac(6’)Ie-aph(2")-la genes, which were detected at lower frequency (Table 6, Fig. S1). E. faecalis harbored a greater number of tet(M) gene (5 isolates) than the other Enterococcus isolates, in agreement the results reported from a previous study9. Holman et al.36 reported that the tet(M) antimicrobial resistance-encoding gene was detected in 31.9% (15 isolates) of E. faecalis bacteria. They also found that the tet(M) gene was detected in 37.5% (3 isolates) of E. faecium. Yu et al.53 reported similar results. They showed that the distribution of tet(M) and aac(6’)Ie-aph(2")-la resistance genes in E. faecalis strains isolated from poultry in China was 91.80% and 67.21%, respectively. A study carried out in Tunisian12 reported that E. faecalis isolated from food-producing animals carried aac(6’)Ie-aph(2") antimicrobial resistance-encoding genes. A total of 31 of the 85 E. casseliflavus isolates harbored the tetM gene. This result is similar to that reported for E. casseliflavus isolates from swine farms58. Out of the three genes ermA, ermB, and mef, ermB was the most frequently detected in this investigation. It was found in 5.8% of E. hirae isolates (n = 2/34), which is consistent with findings from other studies1,10,12,41. VanC1 and VanC2 were identified in E. faecium and E. hirae isolates, respectively. These results are consistent with a previous study that identified VanC1/VanC2 resistance genes in E. faecium isolates from aquaculture and slaughterhouse facilities41. Efflux pumps play a significant role in both natural and acquired resistance to antimicrobial medicines that are currently employed for the treatment of infectious illnesses59. The finding of the present that over 34.4% of enterococcal isolates carried efflux pump genes is consistent with previous reports from bovine feces, retail chicken meat, and broiler chickens10,32,60. The changes in antibiotic use differs between nations because of the different variations use patterns. Efflux pumps and the acquisition of genetic elements such as plasmids and transposons can result in increased tolerance or resistance to antimicrobials in enterococci.

Conclusions

The isolation of virulence potential and multidrug-resistant enterococci isolates from slaughtered cattle, sheep, and goat carcasses emphasized the importance of slaughter hygiene in the transmission of pathogenic enterococci. The presence of enterococci in different parts of the carcasses and during the pre-chilling stage poses a risk of cross-contamination in the examined facility. Carcasses can be contaminated with fecal bacteria, the majority of which results from contamination during the slaughtering process, such as damage to intestinal tissue during evisceration or fecal leakage, which can increase cross-contamination of carcasses. According to the the findings in this study, enterococci may pose a potential risk to public health, considering their virulence potential and antibiotic resistance (mainly against quinupristin-dalfopristin, tetracycline, and ampicillin). Therefore, considering the slaughterhouse in Van Province included in this study, larger studies from different geographical regions and more slaughterhouses or other sources of animals are needed to fully understand the genetic diversity of enterococci in farm animals.