Antimicrobial Resistance of Enterococcus Isolates from Poultry Farms in the Republic of Serbia (Autonomous Province of Vojvodina)

Enterococcus species are significant intestinal commensals of animals, including poultry. However, they have emerged as important opportunistic infective agents in both veterinary and human medicine as well as major nosocomial pathogens, owing to their increasing antimicrobial resistance. This research aimed to investigate the prevalence and antimicrobial resistance patterns of Enterococcus spp. isolated from poultry farms in the north of Serbia. A total of 40 samples of overshoes or feces were collected from 40 poultry farms and analyzed for the presence of Enterococcus spp. using PCR or MALDI-TOF mass spectrometry for their identification. The number of isolates was 40 and included 11 isolates from laying hens, 2 isolates from turkeys, 3 from broiler breeders, and 24 from broilers. The Kirby–Bauer disk diffusion method was used to test for antibiotic susceptibility in accordance with the Clinical and Laboratory Standards Institute and EUCAST guidelines. The results showed that Enterococcus faecalis was isolated from 37.5% farms, and E. faecium from 42.5%. E. hirae was identified in 15% of poultry establishments, and E. durans and E. thialandicus on 2.5%. Notably, resistance to erythromycin, streptomycin, fluoroquinolones, and tetracyclines among the frequently used antibiotics was found. Furthermore, 35% of the isolates had multidrug resistance (MDR). In order to prevent the spread of antibiotic resistance in chicken farming and protect the health of the public and animals alike, our findings highlight the critical need for improved surveillance and control measures. To effectively establish a containment strategy for Enterococcus spp. isolated from poultry farms, more research into the processes behind their antibiotic resistance is required.


Introduction
Enterococcus spp.are among the most important nosocomial pathogens worldwide [1][2][3].As harmless commensals, they colonize the intestines of humans and animals, while poultry, in particular, are their permanent reservoirs [4].Humans may be infected with pathogenic enterococci, but the route of infection is not always known.It is considered that the most common source of pathogenic Enterococcus spp. is food of animal origin, and people can contract the infection when handling poultry meat in slaughterhouses or food factories [5].
Antimicrobial resistance (AMR) is a global public health concern that threatens the effectiveness of antimicrobials used to treat bacterial infections [6].Recently, it has been determined that the most significant poultry bacteria resistant to antibiotics in the European Union are Escherichia coli, Enterococcus faecalis, and Enterococcus cecorum [7].Therefore, in many countries, the antimicrobial resistance monitoring of Enterococcus spp.has been initiated, although currently it is not mandatory in the EU [8].There are several reasons why these bacteria require special attention.Namely, Enterococcus spp.survive in different environments and hosts, owing to their specific resistance phenotypes, virulence and good adaptability, withstanding high temperatures, salinity, and acidic conditions [9].Enterococci are easily transmitted in chicken flocks and survive well in the environment [10].The isolates can become pathogenic for chickens and their embryos [8].Furthermore, in poultry farming, where antimicrobials are commonly used for disease prevention but also for growth promotion [11], Enterococcus spp.have garnered attention as potential reservoirs and vectors for the dissemination of antimicrobial resistance genes [12].These are perfectly capable of exchanging resistance and virulence genes between themselves and other bacteria [13].
Certain strains, such as Enterococcus cecorum, which are pathogenic for poultry [14], do not typically colonize human guts and seldom cause bacteriemia [15].However, E. cecorum was isolated from a patient's pleural fluid and the intestines were thought to be the portals of entry [16].
E. faecalis is an opportunistic pathogen that may cause outbreaks in poultry flocks if they are infected concomitantly with other microorganisms or suffer from immunosuppression.Some clones such as sequence type (ST) 82 may cause amyloid arthropathy in broiler breeders and are considered pathogenic [8,17].
The Autonomous Province of Vojvodina, situated in the north of Serbia, is a significant area for poultry production in the region.Alongside the intensification of poultry farming and the widespread use of antimicrobials, concerns have been raised regarding the emergence and spread of AMR among Enterococcus spp.isolates in poultry production.Understanding the prevalence and patterns of antimicrobial resistance in Enterococcus spp.from poultry farms in Vojvodina is crucial for developing strategies aimed to mitigate the risk of AMR transmission within the food chain and to human populations.To the best of our knowledge, based on literature search, this study presents the first investigation into Enterococcus spp.resistance on poultry farms in Serbia.Moreover, there is a lack of data pertaining to the occurrence and antimicrobial resistance of Enterococcus spp. in surrounding countries.
In this work, the presence of Enterococcus spp. in the environment of the poultry farms in two districts in Vojvodina was investigated.The research goals were to reveal antimicrobial resistance phenotype and estimate the risks of antibiotic overuse in primary production farms in Serbia.Furthermore, the data about the levels of antimicrobial resistance in Enterococcus spp.will enable the comparison of isolates from different laboratories and, ultimately, from different countries.The results may help enhance therapeutic efficacy, monitor clonal dissemination, and determine the possible role of food contamination with Enterococcus spp. in public health.In addition, the acquired knowledge is essential for the development of targeted interventions and programs to preserve the effectiveness of antimicrobial therapy and safeguard public health.

Poultry Farms
The poultry farms investigated in this research are situated in the northern part of Serbia, the Autonomous Province of Vojvodina.The epizootiological area in our study encompasses two districts: South Backa and Srem (Figure 1).We made sure to include all representative farms from these districts to ensure comprehensive coverage of the region.A total of 40 poultry farms, including 3 broiler breeder farms, 11 layer chicken farms, 24 broiler farms, and 2 turkey farms, were examined for the presence of Enterococcus spp. in the farm environment.
Regarding the type of feed, all farms included in our study used commercial feed formulated according to the age of the birds.This ensured that the nutritional requirements of the birds were met throughout their growth stages.All birds were vaccinated according to specific vaccination programs tailored to their needs.These programs are designed to protect against common diseases and ensure the health and well-being of the poultry.The exact vaccination schedule for each flock and category of poultry is created individually; some of the diseases included in the vaccination program are infectious bronchitis, Gum-boro disease, and Newcastle disease.To the best of our knowledge, no growth promoters were administered on any of the farms surveyed.This aligns with current best practices and regulatory standards aimed at promoting safe and sustainable poultry production.All of the farms included in our study are commercial closed poultry farms that adhere to high biosecurity measures.As part of these measures, there are no other animals, such as hogs or cattle, present on or near these farms.This strict biosecurity protocol is implemented to ensure the health and safety of the poultry.All farms are single-age farms.
The samples were overshoes or feces collected during the compulsory monitoring of Salmonella spp.according to the Rulebook on determining measures for early detection, diagnosis, prevention of spread, control, and eradication of poultry infection with certain Salmonella serovars [18], which is almost identical to the respective EU regulations.Also, we used the accompanying Salmonella spp.sampling manual for poultry flocks issued by the Serbian Veterinary Directorate (https://www.vet.minpolj.gov.rs/aktuelnosti/samoneloza%20zivine/98.pdf,accessed on 15 May 2024).
The number of samples collected and analyzed from each farm was one composite environmental sample per farm (feces or overshoes, as described in the manual).This sampling process was conducted strictly in accordance with established regulations to ensure consistency and representativeness.
All samples were collected following standardized procedures to ensure their integrity and reliability.Once collected, the samples were immediately transported to the laboratory under controlled conditions to maintain their quality.All samples were processed the same day for bacteriological analysis according to established protocols.
ments of the birds were met throughout their growth stages.All birds were vaccinated according to specific vaccination programs tailored to their needs.These programs are designed to protect against common diseases and ensure the health and well-being of the poultry.The exact vaccination schedule for each flock and category of poultry is created individually; some of the diseases included in the vaccination program are infectious bronchitis, Gumboro disease, and Newcastle disease.To the best of our knowledge, no growth promoters were administered on any of the farms surveyed.This aligns with current best practices and regulatory standards aimed at promoting safe and sustainable poultry production.All of the farms included in our study are commercial closed poultry farms that adhere to high biosecurity measures.As part of these measures, there are no other animals, such as hogs or cattle, present on or near these farms.This strict biosecurity protocol is implemented to ensure the health and safety of the poultry.All farms are single-age farms.
The samples were overshoes or feces collected during the compulsory monitoring of Salmonella spp.according to the Rulebook on determining measures for early detection, diagnosis, prevention of spread, control, and eradication of poultry infection with certain Salmonella serovars [18], which is almost identical to the respective EU regulations.Also, we used the accompanying Salmonella spp.sampling manual for poultry flocks issued by the Serbian Veterinary Directorate (https://www.vet.minpolj.gov.rs/aktuelnosti/samoneloza%20zivine/98.pdf,accessed on 15 May 2024).
The number of samples collected and analyzed from each farm was one composite environmental sample per farm (feces or overshoes, as described in the manual).This sampling process was conducted strictly in accordance with established regulations to ensure consistency and representativeness.
All samples were collected following standardized procedures to ensure their integrity and reliability.Once collected, the samples were immediately transported to the laboratory under controlled conditions to maintain their quality.All samples were processed the same day for bacteriological analysis according to established protocols.

Isolation and Identification of Enterococcus spp.
The total number of isolates was 40 and included 11 isolates from laying hens, 2 isolates from turkeys, 3 from broiler breeders, and 24 from broilers.Enterococcus spp.were isolated after inoculation in buffer peptone water upon incubation for 24 h at 37 • C. the next day, 100 µL of inoculated peptone was transferred to Slanetz Bartley medium (Oxoid CM0377B) to obtain single colonies.After incubation for 24-48 h, a single colony of Enterococcus sp. was transferred to Bile Aesculin agar (Oxoid CM0888) to obtain pure culture.In each case, the growth was visible after 24 h of incubation at 37 • C.
The identification of Enterococcus spp. was achieved either by PCR or MALDI-TOF.PCR assays were run with the commercial kit BioLabs (New England BioLabs) in accordance with the manufacturer's instructions using primers recommended by the Danish Technical University (DTU).The sequences of the primers were as follows: E. faecalis E1-5 ′ ATCAAGTACAGTTAGTCTT and E2-5 ′ -ACGATTCAAAGCTAACTG-3 ′ , E. faecium F1-5 ′ -GCAAGGCTTCTTAGAGA-3 ′ and F2-5 ′ -CATCGTGTAAGCTAACTTC-3 ′ .The cycles for the PCR were as follows: 94 • C for 2 min, and then the master cycler was programmed for 30 cycles at 94 • C for 1 min., 54 • C for 1 min., and 78 • C for 1 min., with the last cycle being 78 • C 10 min [19].The PCR reaction yielded products of 941 bp for E. faecalis and 550 bp for E. faecium.MALDI-TOF mass spectrometry was used for the identification of all isolates that were not possible to identify using PCR.
Isolates that demonstrated resistance to three or more antimicrobial drugs from different families were deemed multidrug-resistant (MDR) [22,23].
All isolates from broiler breeder farms were identified as E. faecium and were resistant to at least two antibiotics or one or two classes, whereas it was found that there were no MDR isolates present (Tables 1 and 2).

Discussion
This work is the first attempt to determine the occurrence and resistotypes of Enterococcus species in environmental samples from poultry in Serbia.Unexpectedly, antimicrobial resistance was established in 27 isolates (67.5%), while isolates from 14 poultry farms (35%) were multidrug-resistant.Fluoroquinolone (FQ) resistance was also significant, being detected in Enterococcus spp.isolates from 15 poultry farms (37.5%).Such high-level resistance raises concerns about food safety in Serbia.In this study, the resistance against chemically improved fluoroquinolones, which exhibit better anti-bacillar activity against Gram-positive microorganisms, such as moxifloxacin, levofloxacin, and norfloxacin, is pinpointed.Newer drugs, descendants of ciprofloxacin, are important in human medicine due to their quick absorption and distribution to many tissues and body fluids.Therefore, they may be used, with caution, for the treatment of hospital-and community-acquired infections [24].Fluoroquinolones inhibit bacterial DNA synthesis by acting on target enzymes or by activating multidrug exporters.Double mutations on gyrA and parC genes in Streptococcus pneumoniae induce high-level resistance to ciprofloxacin.The order of mutations depends on the type of FQ used for the therapy in some Gram-positive bacteria.
Consequently, it is essential to carefully assess antibiotic resistance in indicator microorganisms such Enterococcus spp.Enrofloxacin (ENR) was removed from the chicken business in the United States in 2005 due to the serious health risks that resistance to ENR posed for humans infected with Campylobacter spp.[25,26].Likewise, the administration of enrofloxacin in the poultry industry in Serbia has caused resistance to nalidixic acid in Salmonella Infantis isolates [27,28] and in avian pathogenic and commensal Escherichia coli as well [29], but also in Campylobacter isolates from human patients [30].With this work, it has now been proven that resistance to FQ also occurs in Gram-positive bacteria from poultry origin such as Enterococcus spp.
Enterococcus spp.have become important pathogens in veterinary medicine because of their resistance to vancomycin [31].In poultry, vancomycin resistance is caused by the misuse of the growth promoter avoparcin, which belongs to glycopeptide antibiotics, chemically similar to vancomycin.However, in this research, resistance to vancomycin was not detected in the collection of isolates from poultry flocks.To the best of our knowledge, avoparcin was not used in Serbia as a feed additive, perhaps due its high price.Therefore, the absence of resistance to vancomycin in Enterococcus from poultry litter was not surprising.However, it is doubtful whether this absence is absolute: monitoring has not been established and there is a fair chance that isolates originating from ceca collected in slaughterhouses may provide different results and that vancomycin resistance will be found.
The Serbian poultry industry depends on the import of breeding flocks and often also layer chickens.Enterococci from these birds may carry vancomycin-resistant genes.The risks of antibiotic therapy in food-producing animals must be carefully considered, taking into account the advantages and disadvantages, because new resistance mechanisms may emerge and some of the infectious clones become well established in different environments all around the globe.Before and after the ban of avoparcin in developed countries, some controversy has arisen regarding the role of medicinal food (a homogeneous mixture of feed and veterinary medicinal products, including antibiotics) in the spread of resistance to glycopeptide antibiotics; however, it was difficult to prove the epidemiological links between enterococci in food animals and isolates from human patients [32].Similar controversy exists in evaluating the role of virginiamycin feed additive in the development of resistance to the streptogramin antibiotics quinupristin and dalfopristin in E. faecium.The transferable vat genes may support the spread of streptogramin-resistant isolates in farm animals, which could be transferred to humans via contaminated meat and environments.However, streptogramin-resistant E. faecium is well adapted to the cloaca and does not colonize the gut efficiently as it barely survives the gastric barrier [32].
Many isolates in this work were resistant to erythromycin and tetracycline.Such a resistance phenotype is a matter of concern in human medicine [33].It could be assumed that antibiotic-resistant Enterococcus spp.from the environment can colonize poultry intestines and create a food contamination hazard during processing.The dissemination of such environmental bacteria through contaminated poultry litter is another important issue, especially in developing countries, where the disposal of bedding material from poultry houses is not safe [34].Interestingly, only isolates from broilers and laying hens proved to be multidrug-resistant.This could be pure coincidence or due to the fact that the breeders and the two turkey flocks were not treated with antibiotics as frequently as broilers and layers.In our study, and in accordance with previous observations, we observed that the differential frequency of treatment among poultry types, such as broilers and turkeys, is influenced by various factors including the specific health challenges and management practices associated with each type.
The genome plasticity of enterococci spp. is characterized by their ability to acquire new genes and genetic elements and the capacity to transfer large amounts of genetic material from one strain to another [13].These properties have led to the formation of well-established clones in hospital and community settings [2].It is noteworthy that some E. faecalis isolates from poultry and humans were found to be genetically indistinguish-able in several independent studies [8].It was also discovered that the vanA gene of the vancomycin-resistant E. faecium from poultry, ingested by healthy volunteers, can transfer resistance genes to human commensal E. faecium, causing transient colonization of the intestines [8].Interestingly, genes encoding resistance to quinupristin/dalfopristin and to erythromycin were also transferable together with the vanA gene from resistant to susceptible E. faecium in the intestines of one out of six healthy volunteers [8,35].Considering all of the above-mentioned facts, Enterococcus spp.are bacteria of great importance to public health, which require regular control of their resistotype and the study of transmission routes to both humans and animals.
Owing to the possibility of acquiring resistance genes against several antibiotic classes, including glycopeptides, the significance of Enterococcus spp. in human and veterinary medicine has increased over the past few years [8].These bacteria are intrinsically resistant to low doses of aminoglycosides (gentamicin and streptomycin) and cephalosporines.In addition, E. faecalis is intrinsically resistant to quinupristin/dalfopristin. Data from the European Antimicrobial Susceptibility Surveillance in Animals (EASSA) has shown that, except for erythromycin, tetracycline, and quinupristin/dalfopristin, clinical resistance to antibiotics was not found, or was rare in enterococci originating from cattle, pigs, and poultry.In 2014, a marked decrease in resistance to vancomycin in the EU was observed in comparison to previous reports from 2002 [36].Similar results were obtained by Rebelo et al. [37].Also, although resistance to linezolid is on the rise in nosocomial isolates, animals were not yet carriers of Enterococcus spp. with resistance to critical antibiotics [37].Given that Enterococcus spp.are susceptible to transspecies transmission, antimicrobial resistance surveillance is of global importance.
The main limitation of the present study is the relatively small number of samples.The number of samples in the present study was limited, but we ensured that the sampling methodology was representative of the region under study.The epizootiological area in our study encompasses two districts: South Backa and Srem.We made sure to include all representative farms from these districts to ensure comprehensive coverage of the region.
The inclusion of various types of poultry, including broiler breeders, laying hens, broiler chickens, and turkeys, was intentional to capture a broad spectrum of the poultry farming practices in the region.This diversity provides a more comprehensive understanding of the poultry industry.Regarding the inclusion of turkey farms, it is important to note that turkey farms are quite rare, not only in the specified districts, but also throughout the entire country.We recognize that only two turkey farms were included.However, this reflects the actual distribution and prevalence of turkey farms in the region, where broiler chicken farms are more predominant.The limited number of turkey farms in our sample accurately reflects their scarcity in the region.Thus, it is proportional to the local situation.Future studies will aim to increase the sample size and to cover a larger geographic area of the study and achieve an even more balanced representation of different poultry types.

Conclusions
Because meat, meat products, and other foodstuffs can be contaminated with enterococci in addition to poultry litter and manure, it is important to continuously examine these bacteria of human, animal, and environmental origin.The systematic analysis of antimicrobial resistance in various Enterococcus spp.sampled from poultry farms in Serbia may provide insights into the epidemiology and determinants of resistance, which could help researchers to develop adequate strategies for antimicrobial stewardship and public health interventions.

Figure 1 .
Figure 1.The map of the examined epizootiological areas (South Backa and Srem districts).Figure 1.The map of the examined epizootiological areas (South Backa and Srem districts).

Figure 1 .
Figure 1.The map of the examined epizootiological areas (South Backa and Srem districts).Figure 1.The map of the examined epizootiological areas (South Backa and Srem districts).

Table 1 .
Resistotype of Enterococcus spp.isolates from poultry and turkey farms.

Table 2 .
Multidrug-resistant (MDR) Enterococcus spp.isolates from different types of farms.High-level resistance to streptomycin was present in 11 poultry isolates (27.5%).Resistance to quinupristin-dalfopristin (QDF) in E. faecium was found in only one isolate (No. 16) (2.5%) originating from a flock of broiler chickens.One isolate of E. faecium from a broiler farm (isolate number 16) was MDR to six antibiotic classes.None of the isolates were resistant to vancomycin or linezolid antibiotics.