Antimicrobial Resistance in Enterococci of Dairy Origin—A Review

Abstract

Enterococci are ubiquitous lactic acid bacteria frequently detected in dairy environments, where they represent an important component of the non-starter lactic acid bacteria community, particularly in artisanal cheeses produced from raw milk. Due to their metabolic versatility, enterococci may contribute to cheese ripening and the development of characteristic sensory attributes; however, their technological relevance is accompanied by growing concern regarding their role as opportunistic pathogens and reservoirs of antimicrobial resistance. This review critically summarizes current knowledge on antimicrobial resistance in enterococci isolated from milk and dairy products, with emphasis on both intrinsic and acquired resistance traits and their reported prevalence across different dairy matrices and geographical regions. Particular attention is given to artisanal cheeses, in which heterogeneous and region-specific resistance patterns have been described. Advances in whole-genome sequencing have substantially improved understanding of the genetic basis of antimicrobial resistance in dairy enterococci and have largely corroborated earlier findings obtained through phenotypic antimicrobial susceptibility testing combined with targeted resistance gene detection. Nevertheless, available data remain fragmented due to variability in study design, analytical approaches, and reporting practices. Overall, the evidence highlights the need for harmonized surveillance strategies integrating phenotypic and genomic data within a One Health framework to improve risk assessment and to better understand the role of dairy enterococci in the dissemination of antimicrobial resistance along the food chain.

1. Introduction

Enterococci are Gram-positive, catalase-negative, aerotolerant, fermentative lactic acid bacteria naturally present in the gastrointestinal tract of humans and animals, but are also frequently detected in soil, surface water, plants, food and food-related environments, including raw milk and dairy processing facilities [1,2,3,4,5]. Their broad ecological distribution is attributed to their physiological robustness. Enterococci exhibit remarkable tolerance to diverse environmental stressors, including high salt concentrations, acidic conditions, and reduced water activity, while being capable of growth across a broad pH range, including neutral and mildly alkaline conditions. They are capable of growing across a broad temperature range (10–45 °C) and can withstand exposure to detergents and disinfectants. This resilience enables them to persist throughout the entire dairy production chain—from milking equipment and raw milk to dairy products and processing environments [1,3,5].

Historically, enterococci were included among serogroup D streptococci, due to their morphological, physiological and biochemical similarities, but later were reclassified as a distinct genus based on DNA–DNA hybridization studies, multilocus sequence analyses, and whole-genome phylogenies. Today, the genus comprises nearly 60 described species, and ongoing genomic surveillance suggests that additional species remain to be formally described [6,7,8]. Among these species, Enterococcus faecalis and Enterococcus faecium are the most frequently isolated from humans, animals and foods. Dairy environments commonly harbor E. faecalis, E. faecium and Enterococcus durans, along with lower frequencies of Enterococcus casseliflavus, Enterococcus hirae, Enterococcus italicus, Enterococcus gallinarum, and Enterococcus gilvus, depending on local production conditions [4,7,9]. Surveys of artisanal cheeses from Southern and Eastern Europe, including the Balkan region, consistently show that enterococci are a substantial part on the non-starter lactic acid bacteria (NSLAB) community in artisanal products made of raw milk without added industrial starter cultures [4,6,9]. Enterococcal metabolic activities, proteolysis, lipolysis, citrate metabolism, and the production of volatile flavor compounds (diacetyl, acetoin, butanediol) may significantly contribute to the characteristic sensory attribute development of artisanal cheeses [4,6,9,10,11,12,13,14,15,16,17,18]. Moreover, some strains produce antimicrobial peptides, bacteriocins (enterocins), which can inhibit spoilage or pathogenic microorganisms’ growth and enhance the microbial stability and safety of ripened cheese [4,19]. For these reasons, enterococci have historically been regarded as technologically important microorganisms in certain artisanal cheese, especially in the Mediterranean and Balkan regions [4,11,12].

However, the genus Enterococcus also includes species and strains with clinically relevant traits. Their genomes exhibit high plasticity, with a substantial accessory repertoire comprising plasmids, transposons, pathogenicity islands, and integrative conjugative elements, which enable the rapid acquisition of virulence determinants and antimicrobial resistance genes [5,20]. High-risk clinical lineages of E. faecium may harbor vancomycin resistance operons (vanA, vanB), cytolysin clusters, aggregation factors, and biofilm-associated genes (esp, hyl), raising safety concerns. Enterococci exhibit intrinsic resistance to several antimicrobial agents and harbor mobile genetic elements carrying additional resistance determinants [21,22]. Owing to their ability to acquire and disseminate these transferable antimicrobial resistance genes, and their marked genomic adaptability, enterococci are commonly used as indicator bacteria for monitoring antimicrobial resistance in Gram-positive bacteria [23]. Their role as reservoirs and facilitators of gene exchange also makes them critical contributors to the future dissemination of antimicrobial resistance.

Phylogenomic analyses of the genus have refined our understanding of interspecies relationships. Comparative genomics has revealed a highly structured population of E. faecium with two major evolutionary clades: hospital-associated lineages characterized by a high burden of antimicrobial resistance and virulence genes (Clade A) and a food-, animal- and community-associated lineage (Clade B). Genome-based species studies have shown that Clade B isolates meet the threshold for classification as separate species, E. lactis, which is largely associated with dairy and other food environments and characterized by the absence of key hospital-associated virulence factors [5,7,24]. In contrast, E. faecalis does not demonstrate such a multi-clade population structure [5]. From a food safety perspective, the taxonomic distinction between E. faecium and Enterococcus lactis may have practical implications for the dairy industry. Historically, isolates associated with dairy products classified as E. faecium have often been interpreted as posing a clinical risk, despite originating from food or animal environments. Genome-based analyses have now shown that the majority of Clade B isolates, reclassified as E. lactis, lack key hospital-associated virulence determinants and carry a significantly lower antimicrobial resistance gene load [24]. Lack of differentiation between E. lactis and E. faecium in routine surveillance may therefore lead to an overestimation of the public health risk associated with enterococci in dairy products. Integrating genome-based species identification into safety assessments would enable a more accurate risk evaluation by clearly distinguishing food-adapted enterococcal populations from clinically relevant high-risk lineages. Such differentiation is particularly relevant for the interpretation of antimicrobial resistance data, as both the prevalence of resistance and genetic context differ significantly between hospital-associated and dairy-associated E. lactis populations.

These evolutionary insights explain the dual nature of enterococci. On one hand, strains originating from raw milk cheese exhibit desirable technological or probiotic traits. On the other hand, hospital-associated strains, particularly vancomycin-resistant E. faecium, represent a globally significant pathogen [5,20]. Because horizontal gene transfer can occur across niches and habitats, these phylogenetic patterns do not eliminate safety concerns. Current evidence indicates that the pathogenesis of enterococcal infections is strain-dependent, and appears to be more strongly associated with enterococci originating from hospital environments than with those derived from food [25,26]. Enterococci are not classified as Generally Recognized as Safe (GRAS) [27] organisms, nor are they included in the Qualified Presumption of Safety (QPS) list [28]. However, the absence of GRAS or QPS status does not imply the prohibition of their presence or use in foods, as enterococci are traditionally associated with fermented products and may be considered acceptable following thorough, strain-specific safety assessment. This regulatory status, combined with the fact that enterococci play an important role in traditional fermented foods [4], highlights the need to closely monitor and understand antimicrobial resistance in food-derived strains.

2. Methodology

This review included full-text articles published in peer-reviewed journals between 2015 and 2025 in the English language. Literature searches were conducted in Google Scholar, PubMed, and Scopus using a combination of index terms. The search strategy was structured as follows: (“Enterococcus” OR “enterococci”) AND (“antimicrobial resistance” OR “antibiotic resistance” OR “safety assessment” OR “AMR”) AND (“milk” OR “dairy” OR “cheese”). Both original research articles and reviews were considered. Only studies analyzing enterococci from raw milk intended for human consumption (bulk tank milk) or dairy products were included. Articles focusing on milk from cows with mastitis, or enterococci isolated solely from the farm environment but not from milk, were excluded.

3. Role and Prevalence of Enterococci in Dairy Products

In artisanal raw milk cheeses, enterococci are frequently among the key NSLAB members, which can significantly influence the ripening process. Numerous studies report that E. faecalis, E. lactis/E. faecium (dairy-associated isolates historically classified as E. faecium but now reclassified as E. lactis) and E. durans display proteolytic and peptidolytic activities that contribute to casein degradation and accumulation of peptides and free amino acids, precursors for further flavor-forming reactions [4]. In addition, many dairy strains exhibit lipolytic activity and can metabolize citrate into diacetyl and other aromatic compounds, thereby determining the characteristic sensory profile of traditional cheeses [4,14]. Enterococci have a long history of use as indicators of fecal contamination [14]. Their high abundance in human and animal feces, combined with superior environmental resistance compared to Escherichia coli and relatively simple cultivation, are key reasons why many national and international guidelines recommend the enumeration of enterococci as part of hygiene criteria [29,30]. However, environmental and ecological studies have demonstrated that enterococci can also persist and even multiply in non-fecal habitats such as soil, sediments, aquatic biofilms and plant material, which is why their presence may not reflect fecal contamination [30]. In the dairy environment, several species commonly found in raw milk cheeses, such as E. durans, E. italicus and E. lactis strains, are better interpreted as members of raw milk microbiota than indicators of fecal contamination [4,6].

Taking all of the above-mentioned roles into account, the interpretation of enterococci detected in dairy products becomes highly context-dependent. Their detection in raw milk and at early production stages can still provide useful information about milking hygiene and equipment sanitation [6,31]. In contrast, their presence in ripened raw milk cheeses is more related to their technological role [6,11,32,33].

Enterococci are considered part of raw milk microbiota and they are commonly isolated from raw cow, sheep and goat milk [4]. Reported counts are in the range of 2 and 4 log₁₀ CFU/mL in raw milk samples, although higher numbers may occur in association with poor milking hygiene and inadequate equipment sanitation [2,3,6,34].

Studies from different European countries show that enterococci are detected in a substantial proportion of raw milk samples, usually at low to moderate levels. Authors Dobranić et al. [34] reported enterococcal counts ranging from 1.3 to 5 log₁₀ CFU/mL, with higher values more frequently observed in milk from cows with a history of mastitis [34]. In a study with raw ewes, enterococci were present at relatively low but highly variable counts (3.65 ± 2.93 log₁₀ CFU/mL) [2], while a study with cows’ raw milk samples showed enterococci at level 2 to 3 log₁₀ CFU/mL [35]. In a study with 211 bulk tank milk samples, enterococci were detected in 96% of samples, with a count ranging from 1 to 6.8 log₁₀ CFU/mL (mean 2.48 log₁₀ CFU/mL) [33]. Most samples had counts below 3 log₁₀ CFU/mL. Species-level analyses consistently show that E. faecalis and E. faecium/E. lactis are predominant among milk isolates, with E. durans and E. casseliflavus occurring less frequently [2,6,34]. More recently, whole-genome and other next-generation sequencing approaches have largely confirmed these observations, reporting a predominance of E. faecalis and E. faecium/E. lactis among raw milk enterococci, while simultaneously providing detailed insights into their genomic diversity and associated antimicrobial resistance determinants [4,36].

Their ability to tolerate high salt concentrations, acidification and low temperature during ripening enables them to proliferate in cheese. Thus, the number of enterococci in some artisanal cheeses at intermediate ripening stages can go up to 6 to 8 log₁₀ CFU/g [4,9]. Comprehensive literature reviews on artisanal cheese indicate that up to 19 enterococcal species have been isolated from different traditional cheeses worldwide, although E. faecalis, E. lactis/E. faecium and E. durans remain the most prevalent [37].

4. Antibiotic Resistance in Dairy Enterococci

Enterococci possess intrinsic resistance to several antimicrobial classes, including cephalosporins, trimethoprim–sulfamethoxazole, and low concentrations of penicillins and aminoglycosides [38]. Two of the clinically most relevant species, E. faecalis and E. faecium [20], possess a large proportion of accessory genomes, which contributes to their extensive genomic plasticity [39]. Therefore, the evolution of enterococci was mainly driven by the recombination and proficient acquisition of novel genes through horizontal gene transfer (HGT) [39,40]. This plasticity can be explained by the absence of complete clustered regularly interspaced short palindromic repeats (CRISPRs) loci, which normally protect bacterial cells from foreign DNA and limit horizontal gene transfer [40]. Consequently, enterococci are well-recognized for their remarkable capacity to acquire antimicrobial resistance determinants, primarily via mobile genetic elements, including plasmids and transposons [39], genomic islands, and prophages [41]. The acquisition of numerous antimicrobial resistance genes includes high-level ampicillin resistance, high-level aminoglycoside resistance (HLAR), and glycopeptide resistance, as well as resistance to fluoroquinolones, tetracycline, macrolides, oxazolidinones, and chloramphenicol [5]. The main classes of antibiotics discussed in this review, their mechanism of action and resistance mechanisms are shown in

Table 1. Mechanism of action, mechanism of resistance and main genes encoding antibiotic resistance in enterococci.

Antibiotic/Class	Mechanism of Action	Mechanism of Resistance	Main Resistance Genes and Type of Resistance	Reference
Aminoglycosides	Inhibition of protein synthesis by binding to 30S ribosomal subunit	Aminoglycoside-modifying enzyme	aac(6′)-Ii, aph(3′)-IIIa (intrinsic, low-level) aac(6′)-Ie-aph(2″)-Ie, ant(3″)-Ia (acquired, HLAR)	[42,43]
		Ribosome-modifying methyltransferase	efmM (intrinsic)
Chloramphenicol	Inhibition of peptidyl transferase (50S subunit)	Enzymatic inactivation (acetylation)	cat (acquired)	[7]
Macrolides (erythromycin)	Inhibition of elongation by binding 50S ribosomal subunit	23S rRNA methylation	ermA, ermB, ermC (acquired)	[44,45,46]
		Efflux pumps	msrC, mefA/E (acquired)
Glycopeptides	Inhibition of cell wall synthesis by binding to the terminal D-alanine-D-alanine (D-Ala-D-Ala) of peptidoglycan precursors and preventing cross-linking of peptidoglycan chains	Change in the terminal amino acids of the peptidoglycan precursor	vanA, vanB (acquired) vanC (intrinsic, chromosomally encoded in E. gallinarum and E. casseliflavus)	[43,45,47]
Tetracyclines	Binding to the ribosome and interfering with the docking of aminoacyl-tRNA	Ribosomal protection proteins	tetM, tetO, tetS (acquired)	[43,45]
		Efflux pumps	tetK, tetL (acquired)

.

Although a wide range of Enterococcus species have been recovered from dairy products, this review primarily addresses E. faecalis and E. faecium, as they are clinically the most relevant species associated with human infections. Other species of enterococci have also been associated with clinical infections; however, such occurrences are infrequent and predominantly opportunistic in nature [48]. Nonetheless, the involvement of other species should not be overlooked, particularly within the One Health framework, where the dissemination of antimicrobial resistance across humans, animals, food, and the environment is of increasing concern.

4.1. Glycopeptide Resistance

Glycopeptides constitute a class of antibiotics commonly used for the treatment of severe infections caused by Gram-positive bacteria [7]. Nine types of vancomycin resistance have been described, designated as A–E, G, L, M, and N. Each resistance type corresponds to a specific gene cluster denoted by the same letters: vanA, vanB, vanC, vanD, vanE, vanG, vanL, vanM, and vanN [49]. The principal mechanism of glycopeptide resistance in enterococci involves the alteration of the peptidoglycan synthesis pathway, specifically the substitution of the terminal D-alanine–D-alanine (D-Ala-D-Ala) dipeptide with either D-alanine–D-lactate (D-Ala-D-Lac) or D-alanine–D-serine (D-Ala-D-Ser). These modifications significantly reduce the binding affinity of glycopeptide antibiotics, thereby conferring resistance [47,50]. Particular attention has been paid to the vanA and vanB gene operons, as the majority of human vancomycin-resistant enterococci (VRE) outbreaks are associated with these operons. VanA is currently the most widely distributed globally, although prevalence patterns may shift over time [51]. Both vanA and vanB operons are horizontally transferable, typically located on chromosomally integrated transposons such as Tn1546 for vanA and Tn1549 or Tn5382 for vanB [52]. Recently, vancomycin-variable enterococci (VVE) were described; while they show no resistance phenotype, they carry the vanA gene and can become resistant upon exposure to glycopeptide antibiotics [53,54]. Therefore, determining phenotypical resistance alone is not enough to rule out the presence of vancomycin resistance in enterococci. In contrast, vanC-type resistance is generally considered intrinsic and non-transferable, conferring low-level glycopeptide resistance in E. gallinarum, E. casseliflavus, and Enterococcus flavescens [55].

Findings from several studies suggest that bulk tank milk [56] and dairy products are unlikely to act as a reservoir for vancomycin resistance, as vancomycin-resistant enterococci have not been detected in analyzed dairy products. No VRE were detected in traditional Sicilian cheeses [57], French traditional cheeses [58], Slovak fresh cheese bryndza [59], traditional cheeses from Serbia and Slovenia [60], Turkish cheeses [61,62], or in other dairy products such as yogurt [61].

A low-to-moderate prevalence of VRE has been reported in several studies on enterococci from cheese. A low prevalence of vancomycin resistance was reported in the study by Chajęcka-Wierzchowska et al. [63], in which two E. faecalis isolates out of 189 enterococci isolated from different dairy products were resistant to vancomycin. In the study of eleven types of Turkish cheeses obtained from supermarkets, retail shops, and open-air markets, only three of the 85 E. faecalis isolates displayed phenotypic resistance to vancomycin, yet none harbored the analyzed van genes, including vanA and vanB [64]. Similar findings were reported for artisanal Portuguese Pico cheese, where 2 of 27 E. faecalis isolates were phenotypically resistant to vancomycin, but lacked vanA and vanB genes [65]. In the study by Terzić-Vidojević et al. [9], out of 636 enterococci isolates originating from autochtonous dairy products from West Balkan countries, 40 isolates (6.3%) were phenotypically resistant to vancomycin. A moderate prevalence of VRE (5%) was likewise reported in Egyptian fresh karish cheese. However, molecular screening did not reveal the presence of vanA, vanB, or vanC resistance determinants [66].

In contrast to these results, very high and extremely high prevalences of vancomycin-resistant enterococci were reported in several studies. Vyrostkova et al. [67] reported an extremely high prevalence of vancomycin resistance in enterococci from Slovak goat and sheep cheeses: 44 of 52 isolates (84.6%), with the vanA gene confirmed in 21 isolates. Available data on antimicrobial usage in food-producing animals in Slovakia [68] do not support such a high prevalence of resistance. However, this finding should be interpreted with caution, as the limited number of isolates analyzed does not allow definitive conclusions to be drawn regarding the true prevalence of vancomycin-resistant dairy enterococci. Interestingly, vancomycin-resistant enterococci (VRE) appear to be more prevalent in sheep- and goat-derived cheeses. Salamandane et al. [69] reported vancomycin resistance in 14 of 16 enterococcal isolates recovered from ewe’s milk in Portugal. All resistant isolates harbored the vanA gene, and one E. faecalis isolate additionally carried the vanB gene. The authors suggested that the observed vancomycin resistance likely resulted from horizontal gene transfer, potentially occurring within the sheep grazing environment or during the cheese production process, particularly given that all cheeses analyzed originated from the same geographical region. An extremely high VRE prevalence was observed in Iranian dairy products (raw milk cheese, yogurt, cream, butter, buttermilk, and kashk), where 79.9% of 343 isolates across 10 enterococcal species were resistant to vancomycin [70]. Although comprehensive data on antimicrobial usage in food-producing animals in Iran are, to our knowledge, not available, the reported high prevalence of antibiotic residues in dairy products [71] suggests widespread antibiotic use and may indicate insufficient regulation in this sector. However, it is worth noting that the study also took into account prevalence for E. caselliflavus and E. gallinarum, which are intrinsically resistant to vancomycin. Resistance to vancomycin in these two species is encoded by a cluster of genes located on the chromosomes, which encode alternative pathways of the peptidoglycan biosynthesis. In E. gallinarum and E. casseliflavus, alternative ligases encoded by vanC1 and vanC2/3, respectively, replace the terminal D-Ala–D-Ala dipeptide of the peptidoglycan precursor with D-Ala–D-Ser, which weakens vancomycin binding to the modified pentapeptide [55]. Vancomycin resistance was observed with high prevalence among enterococcal isolates obtained from artisanal cheese produced in four dairy plants in Slovakia, with 14 isolates (31.8%) exhibiting a resistant phenotype. It is important to note that these determinations were based on Clinical and Laboratory Standards Institute (CLSI) criteria; however, when resistance was interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) rules, all isolates were classified as susceptible [31]. A different interpretation of phenotypical resistance (CLSI vs. EUCAST guideline) was also reported for enterococcal isolates from Portuguese Azeitão and Nisa PDO-cheeses. When EUCAST breakpoints were applied, all enterococcal isolates were classified as susceptible. In contrast, according to CLSI criteria, a small proportion of isolates exhibited resistance, although the overall prevalence remained low [72]. Direct comparison between EUCAST [73] and CLSI [74] interpretive criteria may be inappropriate, as the two standards recommend different disk potencies for vancomycin in disk-diffusion testing (5 µg according to EUCAST and 30 µg according to CLSI), and in both studies, only 30 µg vancomycin disks, as recommended by CLSI, were used. In addition, EUCAST defines only susceptible and resistant categories, whereas the CLSI standard includes an intermediate category. Owing to these methodological and interpretative differences, results obtained by disk-diffusion should be interpreted using a single guideline to avoid misclassification. This limitation can be addressed by the use of quantitative susceptibility testing methods, such as determination of minimum inhibitory concentration (MIC) values. Unlike qualitative assays, such as the disk-diffusion method, quantitative methods provide a measure of antimicrobial susceptibility, offering greater clinical and epidemiological relevance. MIC data allow assessment of the degree of susceptibility and enable evaluation of the existence of resistant subpopulations. Moreover, the use of numerical MIC values reduces ambiguity in distinguishing between susceptible and resistant isolates, thereby improving the reliability of resistance interpretation [75]. Nevertheless, subsequent confirmation of the genetic basis of resistance is recommended to ensure accurate characterization of vancomycin-resistant enterococci.

Calonico et al. [76] analyzed 75 E. faecalis and 22 E. faecium cheese isolates collected over a 14-year survey in Italy to assess phenotypic antibiotic resistance. The long sampling period allowed the authors to track resistance trends, particularly having in mind the ban on avoparcin in food-producing animals in 1997. Vancomycin-resistant strains were found in both E. faecium (5.3%) and E. faecalis (22.7%) from cheese. Moreover, authors pointed out that the notably high proportion of vancomycin-resistant E. faecalis in the final survey year was of a special concern, since it potentially marks a trend in raising numbers of VRE of dairy origin. As pointed out by Ramos et al. [77], the persistence of vancomycin-resistant enterococci in food-producing animals and associated environments can also result from co-selection mediated by other antimicrobial agents, coupled with the circulation of particular resistance genes that facilitate their survival. Silvetti et al. [78] examined forty E. faecalis isolates from traditional Italian raw milk cheeses to assess their antibiotic resistance profiles. The authors intentionally selected strains collected between 1997 and 2009, a period marked by higher antibiotic use in animal production and the years following the avoparcin ban. Despite this context, no vancomycin-resistant strains were detected.

However, the genetic mechanisms underlying vancomycin resistance in enterococcal isolates of dairy origin remain only partially understood. It is noteworthy that neither vanA nor vanB were identified as determinants of vancomycin resistance in several studies investigating enterococci isolated from dairy products [63,64,65,66]. These findings suggest that alternative vancomycin resistance genes may be more prevalent in dairy-associated enterococci, in contrast to clinical isolates, where vanA and vanB remain the predominant resistance determinants. Moreover, as pointed out by Gundong et al. [61], the possible occurrence of VVE in dairy products cannot be ruled out, as the vanA gene was identified in 15 E. faecalis and E. faecium isolates (37.5%), despite the absence of a vancomycin-resistant phenotype. This underscores the need to evaluate the presence of resistance genes even when phenotypic resistance is not expressed, as such isolates may still function as reservoirs of vancomycin resistance within the food chain.

4.2. Aminoglycoside Resistance

Aminoglycosides are broad-spectrum antibiotics that exert their activity by inhibiting protein synthesis or disrupting cell membrane integrity [7]. Enterococci exhibit intrinsic low-level resistance to all aminoglycosides due to their limited uptake of these drugs [42] and inactivation by covalent modification of the hydroxyl or amino groups of the aminoglycoside molecule carried out by naturally occurring enzymes [43]. Although E. faecium and E. faecalis are intrinsically resistant to clinically achievable concentrations of aminoglycosides [42], when administered with antibiotics that inhibit cell wall synthesis, such as β-lactams or glycopeptides, they synergistically exert bactericidal activity [79]. Intrinsic, enzyme-mediated high-level resistance to either gentamicin (HLGR) or streptomycin (HLSR) has not been documented in enterococci. Consequently, these antibiotics, together with β-lactams or glycopeptides, are used in the treatment of severe infections caused by E. faecium and E. faecalis [80].

Resistance to aminoglycosides in enterococci arises through several mechanisms, including target-site modifications, active efflux, and, most notably, the production of aminoglycoside-modifying enzymes (AMEs). Among AMEs, aminoglycoside acetyltransferases are reported to be the most prevalent determinants of resistance [7]. Enterococci easily acquire genes encoding AMEs, which confer resistance even to high concentrations of this antibiotic class [81].

In the study by Kurekci et al. [64], an extremely high prevalence of streptomycin resistance (84.2% of isolates) and a high prevalence of gentamicin resistance (51.1% of isolates) were observed, consistent with expectations for enterococci. However, HLGR was present with low prevalence, since only two E. faecalis and one E. gallinarum were resistant to high concentrations of gentamycin. HLSR had moderate prevalence, since eight isolates (5.8%) were resistant to high levels of streptomycin. The only aminoglycoside resistance gene which was identified in E. faecalis isolates was the aph(3′)-IIIa gene, but since this gene encodes only low-level resistance to kanamycin [82], the genetic basis for high-level gentamicin resistance remained undetermined. Nonetheless, given that the aph(3′)-IIIa gene is acquired, the role of dairy enterococci as a potential reservoir for kanamycin resistance should be taken into consideration. In the same study, aminoglycoside resistance in E. gallinarum isolate was attributed to aac(6′)-Ie-aph(2″)-Ia, the most common acquired determinant of high-level gentamicin resistance [83]. Similarly, Hammad et al. [66] reported a single E. faecium isolate exhibiting high-level gentamicin resistance, and it carried the aph(3′) gene encoding an aminoglycoside phosphotransferase. High-level streptomycin resistance was also presented with low prevalence in the study by Terzić-Vidojević et al. [9], where only nine isolates (1.4%) out of 636 enterococci showed HLGR and HLSR phenotypes. In the study by Gaglio et al. [57], only one E. faecalis isolate from cheese showed HLSR, associated with the aad gene. However, because only eight isolates originated specifically from cheese, the dataset is too limited to draw reliable conclusions about the true prevalence of high-level streptomycin resistance in enterococci in Sicilian cheeses.

In contrast to these findings, Amidi-Fazli and Hanifan [70] documented a high prevalence of acquired genes conferring high-level resistance to both gentamicin and streptomycin among enterococci isolated from artisanal dairy products in Iran. Similarly, a high prevalence of HLAR (30.2%) was detected in enterococci originating from dairy products in Poland. However, HLAR was detected only in E. faecium and E. faecalis, while all the other species (E. gallinarum, E. caselliflavus, and unidentified species of Enterococcus spp.) were sensitive to aminoglycosides. Resistance to streptomycin was most frequently associated with the ant(6′)-Ia gene, while resistance to other aminoglycosides was encoded by to aac(6′)-Ie-aph(2″)-Ia and aph(3″)-IIIa genes [63].

Few studies addressed specifically enterococcal isolates from bulk tank and quarter milk of healthy cows and they revealed a higher prevalence of high-level aminoglycoside resistance in raw milk compared to dairy products. In the study by Bae et al. [84], 86 chloramphenicol-resistant strains of E. faecalis originating from 1584 bulk tank milk samples were analyzed for antimicrobial resistance profiles. Most of the isolates (92.9%) were phenotypically resistant to aminoglycosides. The genetic basis was revealed in 63 (74.1%) isolates, with gene ant(6′)-Ia being most prevalent. Notably, 38 isolates harbored two aminoglycoside resistance genes, with the combination of aac(6″)-Ie-aph(2″)-Ia and ant(6′)-Ia detected in 26 of these isolates. Kang et al. [85] analyzed 301 E. faecalis strains originating from bulk tank milk from dairy companies in Korea and found a high prevalence (61.5%) of HLAR isolates. The genetic basis of the resistance was predominantly associated with the aac (6′)Ie-aph(2″)-la gene (38.9% isolates) and ant(6)-Ia genes (19.5% isolates). High-level streptomycin resistance was highly prevalent among enterococci isolated from quarter milk samples of healthy cows, as reported by Morandi et al. [86]. In their study, 86.7% of the 45 identified enterococcal biotypes exhibited HLSR-all E. faecalis (n = 14) and E. lactis (n = 8) strains, as well as 13 of the 14 E. faecium strains.

Since intrinsic low-level resistance to aminoglycosides is a natural characteristic of enterococci, relatively few studies have comprehensively examined aminoglycoside resistance, both phenotypically and genotypically. However, high-level aminoglycoside resistance is acquired and transmissible [23], and the ecological role of dairy enterococci in disseminating this type of resistance should be examined; however, it largely remains inconclusive.

4.3. Tetracycline Resistance

Tetracyclines are broad-spectrum antibiotics that exert their antibacterial effect by binding to the ribosome and interfering with aminoacyl-tRNA docking [43]. Tetracycline resistance in enterococci is mediated by three main mechanisms: efflux pumps, ribosomal protection proteins, and enzymatic inactivation [7].

The most frequently detected tetracycline resistance genes in non-clinical E. faecalis and E. faecium are tetM, which encodes ribosomal protection proteins, and tetL, which encodes an efflux pump [3,87,88,89,90,91,92]. These genes are often co-located on mobile genetic elements such as the pM7M2 plasmid [93] and the transposons Tn659 [94] and Tn916 [88]. Although tetracyclines are not drugs of choice for treating enterococcal infections, tetracycline resistance remains relevant due to its ecological significance, as tet genes—particularly tetM—can be transferred to a wide range of bacterial genera [66].

A high prevalence of tetracycline resistance has been documented in enterococci isolated from cheeses and other dairy products across multiple regions: Turkey (11.7% of E. faecalis and E. faecium) [62] and 33.8% in a study including additional species (E. gallinarum, E. casseliflavus, E. durans, and Enterococcus avium) [64]; the West Balkans (17.5%, 111/636 isolates) [9]; Serbia and Slovenia (25.5%) [60]; Portugal (57% and 50% respectively) [65,69]; Slovakia (29.8%) [59]; and Poland (11.6%) [63].

Resistance was mainly associated with tetM and tetL genes [57,63,66], although tetK [63,78], tetS [78] and tetW [63] were also reported. Overall, research consistently shows that tetM and tetL are the dominant tetracycline resistance determinants in dairy enterococci, and their co-occurrence is frequently reported [63,64,66].

It is worth noting that the high prevalence of tetracycline-resistant enterococci was present not only in the final product, but also in the dairy environment. Studies by Gaglio et al. [57] and Gundong et al. [61] detected tetracycline-resistant enterococci on dairy surfaces, equipment, bulk tank milk, and personnel swabs, highlighting dissemination within the processing environment.

Morandi et al. [86] reported tetracycline resistance in 73.3% of enterococcal biotypes isolated from healthy bovine quarters, mediated by tetM and tetL. All resistant E. faecalis carried tetM only, whereas E. faecium and E. lactis harbored both genes simultaneously. Furthermore, E. faecalis biotypes were capable of transferring tetM to Lactobacillus delbrueckii subsp. lactis VC107, likely facilitated by its association with transposon Tn916-1545.

Silent tetracycline resistance genes also appear to occur in dairy enterococci. Silvetti et al. [78] demonstrated that, although 25 out of 36 E. faecalis strains carried tetM, these isolates were phenotypically susceptible, reinforcing the need to detect resistance genes even in susceptible isolates. This is of particular importance given the mobility of the tetM gene [21].

4.4. Macrolide Resistance

Macrolides are a group of antibiotics that inhibit protein synthesis by blocking the activity of peptidyl transferase on the 50S ribosomal subunit, ultimately leading to impaired translation and cell lysis [95]. E. faecium and E. faecalis possess the msrC gene, which confers intrinsic low-level macrolide resistance. However, acquired resistance also occurs and is predominantly linked to methylation of the 23S rRNA subunit. Among enterococci of various origins, acquired macrolide resistance is most frequently associated with the ermB gene [96]. This gene is commonly located on mobile genetic elements, together with other antimicrobial resistance determinants, including vancomycin and tetracycline resistance operons [7]. The ermB gene can be co-transferred with vancomycin resistance gene vanA, and tetracycline resistance genes tetM and tetL [97,98]. Resistance to macrolides can also arise from mutations in 23 sRNA and through efflux pumps [43]. Resistance to macrolide antibiotics may also be linked to tolerance to metals. Zinc and copper are routinely added to cattle feed as essential micronutrients, and previous research has demonstrated an association between metal resistance and resistance to macrolides and glycopeptides in enterococci [99].

High levels of erythromycin resistance have been reported in dairy products worldwide. In Iran, Amidi-Fazli and Hanifan [70] detected frequent macrolide resistance, with strains carrying ermA, ermB, or both. In traditional Turkish cheeses, erythromycin resistance was recorded in E. faecalis (18.4%) and E. faecium (19.3%), though resistance genes were not identified [62]. In another Turkish study, 33.3% of E. faecium isolates were erythromycin-resistant, while E. casseliflavus, E. durans, and E. avium remained susceptible [64]. A high prevalence of erythromycin-resistant enterococci was reported in the West Balkans, where erythromycin resistance reached 13.7% [9], and in Slovakia (27%) [59]. Similar findings were described by Chajęcka-Wierzchowska et al. [63], where 14.3% of enterococci were erythromycin-resistant. Further evidence of widespread macrolide resistance was reported in artisanal Pico cheese (25%) [65], along the yogurt and cheese production chain [61], and in the dairy environment associated with traditional Sicilian cheese processing [57]. Resistance was mainly associated with ermA/ermB, occasionally accompanied by the ermC gene [63,64,66,78,86]. Interestingly Vyrostková et al. [67] and Salamandane et al. [69] reported very high erythromycin resistance among enterococci isolated from sheep and goat milk cheeses (76.9% and 75%, respectively), implying that the occurrence of erythromycin resistance could be associated with the dairy species.

One likely explanation for the high prevalence of erythromycin-resistant enterococci is the frequent use of macrolides for treating clinical and subclinical mastitis, exerting selective pressure on the microbiota [100,101].

4.5. Chloramphenicol Resistance

Chloramphenicol is a broad-spectrum antibiotic that inhibits translation by binding to the peptidyl transferase enzyme in the 50S ribosomal subunit [7]. Due to its serious side-effects, the use of chloramphenicol is currently limited. In food-producing animals, it is completely banned in many countries, including the European Union, China, and the United States [84]. Chloramphenicol resistance in enterococci is mainly encoded by the acquired cat gene, which encodes chloramphenicol acetyl transferases [7].

In most of the studies, chloramphenicol resistance had low-to-moderate prevalence: 4.2% in E. faecalis and E. faecium originating from Turkish raw traditional cheeses [62], 1.4% in enterococci originating from cheeses from West Balkan countries [9], 1.6% in Egyptian raw milk cheese [66], 5% in Italian raw milk cheeses [78], and 2.1% in different dairy products from Poland [63].

Contrary to these results, a high prevalence of chloramphenicol resistance was present in enterococci isolated from artisanal dairy products in Iran [70] and artisanal Pico cheese from Portugal [65], but the genes encoding the resistance were not analyzed.

One study in particular analyzed the occurrence of chloramphenicol-resistant enterococci in bulk tank milk [84]. A total of 301 E. faecalis strains isolated from 1584 samples of bulk tank milk in Korea were analyzed for chloramphenicol resistance and 85 isolates (28.2%) were found to be phenotypically resistant. It is worth noting that, out of four dairy companies from which the samples were taken, one had significantly higher prevalence of chloramphenicol-resistant isolates. Although use of chloramphenicol in cattle is banned in Korea, florfenicol is still in use, and this difference could be attributed to its use. The most prevalent gene encoding chloramphenicol resistance was catA; however, for 52 (61.1%) isolates, the genetic basis of resistance was not determined.

5. Challenges in Applying the One Health Framework to the Assessment of Antimicrobial Resistance in Enterococci of Dairy Origin

The One Health concept was established as an integrated, multidisciplinary concept that acknowledges the interconnectedness of human, animal, and environmental health. Although the concept is not entirely new, its importance has significantly increased in recent years due to shifts in human–animal interactions, climate change, and broader environmental alterations [102]. Given the dual nature of enterococci as both beneficial microbiota and opportunistic pathogens, monitoring the dissemination of antimicrobial resistance determinants across the One Health continuum is essential [103]. Recent One Health-oriented reviews propose enterococci as integrative indicators of antimicrobial resistance circulation across humans, animals, foods and the environment [7].

The food chain serves as a vehicle for disseminating antimicrobial-resistant bacteria from farm to fork. Animal-derived foods such as meat, milk, and eggs are major reservoirs of AMR genes [104,105]. In particular, dairy products made from raw milk, such as traditional cheeses, can facilitate the transfer of antibiotic-resistant bacteria between animals and humans [106,107]. This is due to several factors: The absence of heat treatment preserves a high microbial diversity; manual handling during production may introduce additional resistant bacteria via poor hygiene or inadequately cleaned equipment; and the high bacterial load during fermentation creates ideal conditions for horizontal gene transfer, including conjugation. Consequently, raw milk cheeses provide a favorable environment for the spread of AMR genes among bacterial populations [108].

Several monitoring programs address enterococcal resistance—such as the Danish Integrated Antimicrobial Resistance Monitoring and Research Program (DANMAP), the U.S. National Antimicrobial Resistance Monitoring System (NARMS), and the European Food Safety Association (EFSA) surveillance initiatives. These programs collect samples from diverse sources, including human screening samples, meat, water, and soil, but enterococci of dairy products are not included. Moreover, studies that used the One Health approach to compare animal, environmental and human samples are lacking, despite the fact that there are a number of different interventions described to address the global challenge associated with AMR, of which the One Health Approach is one of the most significant [109,110]. The number of studies providing in-depth analysis of antibiotic resistance in enterococci originating from dairy products is limited. Existing data are insufficient to support a thorough assessment of the risks associated with the dissemination of antibiotic-resistant enterococci through the food chain.

Initially, antimicrobial resistance surveillance in enterococci relied primarily on phenotypic methods, such as antimicrobial susceptibility testing, accompanied by molecular assays designed to detect specific resistance genes only in phenotypically resistant isolates [111]. Some of the recent studies which applied this approach are listed in

Table 2. Summary of the studies investigating antibiotic resistance in dairy enterococci.

Species Investigated	Origin	Country	Phenotypical Resistance *	Phenotypical Methods and Interpretation Criteria	Genetic Determinants of Resistance	Ref.
E. faecalis (n = 301)	Bulk tank milk	South Korea	C, GEN, ERY, KAN, S, TET, VAN	Commercial broth microdilution test (CLSI)	cfr, catA, catB, fexA; aac(6′)-Ie-aph(2″)-Ia, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id; ant(3″)-Ia, ant(6′)-Ia; tetL, tetM, tetO;	[84]
E. faecalis (n = 28) E. faecium (n = 12) E. durans (n = 12)	Sheep and goat cheese	Slovakia, Hungary	ERY, VAN	Agar dilution (CLSI)	ermA, ermB, ermC, msrC; vanA	[67]
E. faecalis (n = 14) E. faecium (n = 14) E. lactis (n = 8) E. durans (n = 2) E. gallinarum (n = 2) E. malodoratus (n = 2) E. casseliflavus (n = 1) E. gilvus (n = 1) E. hirae (n = 1)	Raw milk	Italy	C, GEN, ERY, S, TET, VAN	Commercial MIC strips (CLSI, EUCAST)	ermB; tetK, tetL, tetM, tetS vanA	[86]
E. faecalis (n = 37) E. faecium (n = 13)	Karish cheese	Egypt	C, GEN, ERY, KAN, S, TET, VAN	Disk-diffusion (CLSI)	aacA-aphD, aadE, ant(6), aac(6′)-aph(2″); ermA, ermB, ermC, msrA/B; tetK, tetL, tetM; vanA, vanB, vanC	[66]
E. faecalis (n = 301)	Bulk tank milk	South Korea	C, GEN, ERY, S, TET, VAN	Disk-diffusion (CLSI)	cfr, catA, catB, fexA; aac(6′)-Ie-aph(2″)-Ia, aph(2″)-Ic, aph(2″)-Id; ant(6′)-Ia; ermA, ermB, mef; tetL, tetM, tetO;	[85]
E. faecalis (n = 37) E. faecium (n = 78) E. durans (n = 30)	Azeitão and Nisa cheeses	Portugal	C, GEN, ERY, S, TET, VAN	Disk-diffusion (CLSI, EUCAST)	n.d.	[71]
E. faecalis (n = 16) E. faecium (n = 26)	Cheese, yogurt, bulk tank milk, cow nasal swabs, farm and plant equipment, farm and plant personnel nose and hand swabs	Turkey	C, ERY, TET, VAN	Commercial broth microdilution test (CLSI)	vanA, vanB, vanC1, vanC2	[61]
E. faecalis (n = 168) E. faecium (n = 139) E. durans (n = 8) E. saccharolyticus (n = 6) E. gallinarum (n = 5) E. raffinosus (n = 4) E. hirae (n = 4) E. casseliflavus (n = 3) E. avium (n = 3) E. mundtii (n = 3)	Raw milk cheese, yogurt, cream, butter, buttermilk, kashk	Iran	C, GEN, ERY, S, TET, VAN	Disk-diffusion (CLSI)	aacA-aphD, aadE; ermA, ermB; tetK, tetL, vanA, vanB, vanC	[70]
E. faecalis (n = 85) E. faecium (n = 21) E. gallinarum (n = 18) E. casseliflavus (n = 4) E. durans (n = 7) E. avium (n = 4)	Cheeses	Turkey	C, GEN, ERY, KAN, S, TET, VAN	Disk-diffusion (CLSI)	cat aac(6′)-Ie-aph(2″)-Ia, aph(2″)-Ib, aph(2″)-Id; ant(4′)-Ia, aph(3)-IIIa; ermA, ermB, mefA/E; tetK, tetL, tetM, tetO vanA, vanB, vanC1/2, vanD, vanE, vanG	[64]
E. faecalis (n = 22) E. faecium (n = 18) E. durans (n = 9) E. casseliflavus (n = 6) E. saccharolyticus (n = 6) E. gilvus (n = 4) Enterococcus spp. (n = 9)	Bryndza cheese	Slovakia	C, ERY, TET, VAN	Disk-diffusion (CLSI)	n.d.	[59]
E. faecalis (n = 65) E. faecium (n = 101) E. gallinarum (n = 12) E. casseliflavus (n = 5) Enterococcus spp. (n = 6)	Cheese, condensed milk, powdered milk, sour cream, butter	Poland	C, GEN, ERY, TET, VAN	Disk-diffusion (CLSI)	aac(6′)-Ie-aph(2″)-Ia, ant(6′)-Ia, ant(4′)-Ia, aph(3)-IIIa; ermA, ermC, mefA/E, msrC; tetK, tetL, tetM, tetW vanA, vanB, vanC1, vanC2, vanC3	[63]
E. faecalis (n = 22) E. faecium (n = 75) E. durans (n = 22) E. avium (n = 4)	Cheese	Italy	C, ERY, TET, VAN	Disk-diffusion (EUCAST, CLSI)	n.d.	[75]
E. faecalis (n = 17) E. faecium (n = 18) E. gallinarum (n = 2) E. casseliflavus (n = 2) E. durans (n = 1)	Animal rennet for cheese making, wooden vat surfaces, PDO Vastedda della valle del Belice cheese, PDO Pecorino Siciliano cheese, Caciovallo Palermitano cheese	Italy	C, GEN, ERY, S, TET, VAN	Disk-diffusion (CLSI)	cat aadA, aadE ermA, ermC, ermC; msrC tetK, tetM vanA, vanB	[57]
E. faecalis (n = 125) E. faecium (n = 88)	Cheese	Turkey	C, GEN, ERY, KAN, S, TET, VAN	Disk-diffusion (CLSI)	n.d.	[62]
E. faecalis (n = 27) Enterococcus spp. (n = 1)	Pico cheese	Portugal	C, GEN, ERY, KAN, S, TET, VAN	Disk-diffusion (CLSI)	vanA, vanB	[65]
E. faecalis (n = 40)	Raw milk cheese	Italy	C, ERY, S, TET, VAN	Disk-diffusion (CLSI)	ermB tetK, tetL, tetM, tetO, tetS vanA	[78]
E. faecalis (n = 28) E. faecium (n = 11) E. durans (n = 5) E. casseliflavus (n =2), E. gallinarum (n =1)	Raw milk cheese, cheese curd	Slovenia, Serbia	C, GEN, ERY, TET, VAN	Commercial broth microdilution test (EUCAST)	cat aac(6′)-Ie-aph(2″)-Ia, ant(6)-Ia, aph(3)-IIIa; ermA, ermB, ermC; tetM, tetW vanA, vanB, vanC1, vanC2,	[60]

*—In some studies, more antibiotics groups were included in testing, but only antibiotics that are included in this review are shown in the table. C—chloramphenicol; GEN—gentamycin; ERY—erythromycin; KAN—kanamycin; S—streptomycin; TET—tetracycline; VAN—vancomycin.

.

However, these approaches soon demonstrated clear limitations. These limitations mainly stem from methodological heterogeneity, particularly in the methods used for detecting phenotypic antimicrobial resistance. Several approaches are applied, including broth microdilution, disk-diffusion, and agar diffusion methods. While EFSA has provided detailed guidance for meat and meat-producing animals [112], covering sampling strategies, which antibiotics should isolates be tested against, and epidemiological cut-off values, no comparable harmonized guidance exists for milk and dairy products.

Consequently, studies differ considerably in their methodological design. Although cheese is the most frequently used matrix, other sample types such as raw milk, bulk tank milk, and environmental surfaces have also been investigated. In addition, the antibiotic panels are not harmonized, because the choice of antibiotics relies on the study design and not guidance documents, and also, commercial test systems vary in the antimicrobials included. Further inconsistencies arise from the use of different disk potencies in disk-diffusion assays and from discrepancies in interpretative criteria, since cut-off values differ between CLSI and EUCAST standards. Interpretative criteria are generally established for E. faecalis and E. faecium, owing to their clinical relevance, whereas such criteria are lacking for other Enterococcus species that are considered clinically insignificant, but are often present in dairy products and can potentially serve as a reservoir for antibiotic resistance genes.

Moreover, because the expression of antibiotic resistance genes often imposes a substantial fitness cost on bacteria, evolutionary adaptation is the silencing of these genes in the absence of selective pressure exerted by antibiotics [113]. Enterococci are known to harbor silent resistance determinants, including vancomycin-variable enterococci [53,54]. Of particular concern in enterococci is the phenomenon of transiently silent acquired antimicrobial resistance (tsaAMR). TsaAMR refers to the presence of acquired resistance genes in bacteria that display a susceptible phenotype; however, when these determinants are present, genetic alterations can activate or modulate gene expression to levels that confer clinically relevant resistance [113]. This phenomenon has been documented for vanA-type vancomycin-resistant E. faecium in clinical settings [53]. Nevertheless, because these resistance genes are not phenotypically expressed and are typically located on mobile genetic elements, surveillance strategies relying solely on phenotypic methods, followed by gene confirmation only in resistant isolates, may substantially underestimate the role of enterococci from food and environment as reservoirs of antimicrobial resistance genes.

Increasingly, researchers are addressing this challenge through comparative genomics and advanced sequencing technologies, particularly whole-genome sequencing (WGS) [114,115,116,117,118,119,120]. These studies show that whole-genome sequencing-derived resistance profiles are consistent with earlier findings obtained through phenotypic antimicrobial susceptibility testing combined with targeted molecular detection of specific resistance genes. The most frequently detected resistance determinants included genes encoding efflux pumps, aminoglycoside and macrolide resistance, together with tetM and tetL, associated with tetracycline resistance. It is worth noting that vancomycin resistance was determined in only one study implementing WGS in only one E. faecalis isolate originating from raw sheep milk [120]. Particularly noteworthy are studies comparing enterococci of clinical origin with those isolated from dairy environments. Zhong et al. [116] reported that the E. faecium strains isolated from fermented dairy products were sequenced and compared with genomes of enterococci isolated from feces of healthy individuals and hospital patients. The genomic comparison revealed that dairy isolates clustered together with fecal isolates from healthy individuals, yet were clearly separated from isolates originating from hospitalized patients. Moreover, dairy-associated strains harbored significantly fewer antimicrobial resistance genes compared with fecal isolates from clinical settings, highlighting notable differences in genetic resistance potential between food-borne and hospital-derived enterococci. Similarly, He et al. [114] reported that E. faecalis strains originating from dairy environments possessed markedly fewer antimicrobial resistance genes compared with E. faecalis isolates recovered from blood or other clinical sources.

From a One Health perspective, antimicrobial resistance in dairy-associated enterococci should be interpreted within the ecological context of dairy ecosystems. Raw milk, dairy environment, and dairy products, especially cheeses made from raw milk, harbor complex microbial consortia in which enterococci coexist with lactic acid bacteria and environmental microorganisms. Having in mind microbial diversity and high microbial diversity, these environments can facilitate genetic exchange. However, the actual transferability within the cheese matrix and dairy environment is dependent on the genetic context of the determinants and the presence of mobile genetic elements. Distinguishing between gene presence and realistic transfer potential is therefore essential for an accurate One Health risk assessment.

In practical terms, safety assessments of enterococci in the dairy chain should increasingly rely on genome-based approaches capable of resolving species-level taxonomy and population structure. Where whole-genome sequencing is not feasible, caution is warranted when interpreting results based solely on the phenotypic identification of E. faecium. In addition, antimicrobial resistance monitoring programs should consider reporting resistance data separately for E. lactis and E. faecium, where possible, to avoid conflation of food-associated and hospital-associated populations. Finally, the growing body of genomic evidence supports a more nuanced, strain- and species-specific approach to enterococcal risk assessment in dairy products, rather than treating the genus Enterococcus as a uniform hazard category [3,5,24].

6. Conclusions

Recent taxonomic advances, particularly the reclassification of food-associated E. faecium Clade B isolates as E. lactis, underline the need for refined safety assessment frameworks in the dairy industry. Incorporating genome-based identification into antimicrobial resistance surveillance would improve risk assessment by preventing misclassification of low-risk, dairy-adapted populations as clinically relevant enterococci. Such an approach supports a more accurate, evidence-based evaluation of enterococci in dairy products and aligns with the One Health framework.

A number of limitations must be acknowledged when interpreting the findings of this review. First, relatively few studies focus specifically on antimicrobial resistance in dairy enterococci, meaning that the current body of evidence is insufficient to draw comprehensive or definitive conclusions about resistance trends within these populations. Available datasets involve only a limited number of isolates, and large-scale surveillance studies or databases on resistance in dairy enterococci are still lacking. Additionally, although organizations such as EFSA provide recommendations on which antimicrobials should be tested in enterococci, not all studies follow these guidelines, resulting in inconsistent datasets and reduced comparability between studies. Another important limitation is the incomplete connection between phenotypic and genotypic data. Many studies do not determine the genetic basis of resistance, while WGS-based studies often lack corresponding phenotypic susceptibility testing, making it difficult to assess gene expression, functionality, and the clinical relevance of detected resistance genes. Finally, heterogeneity in methodological approaches and interpretive criteria further complicate comparisons.

Although resistant enterococci are frequently detected in milk and cheese, many of the identified resistance traits are species-associated, chromosomally encoded, or linked to genetic elements with limited mobility, which reduces their realistic potential for horizontal transfer under dairy processing and ripening conditions. While the complex microbial ecology of dairy matrices is theoretically permissive to gene exchange, the presence of resistance genes does not inherently translate into a significant public health risk but instead highlights the role of dairy environments as surveillance points for resistance monitoring.

The true extent of enterococci and their antibiotic resistance in dairy products has not yet been fully clarified; however, several preventive measures can be implemented to minimize potential risks. Even if dairy products are ultimately shown to play only a minor role in the dissemination of antimicrobial resistance, proactive control measures remain valuable. Key strategies include routine monitoring of antibiotic residues in raw milk, the monitoring of antimicrobial resistance, the enforcement of strict hygiene and sanitation standards throughout production, prudent and controlled use of antibiotics in dairy farming, and strengthened general hygiene practices across the food chain. These measures collectively contribute to reducing the spread of bacteria, including antibiotic-resistant enterococci.