Enterococci are common inhabitants of the gastrointestinal tract of humans and a wide variety of animals. In this study, the presence of enterococci in milk samples obtained from different mammalian species was investigated. Enterococci were isolated from all the porcine milk samples and from 7 out of 8 human samples, while they were less frequent in the canine, ovine and feline samples. All the strains were identified as E. faecalis, E. faecium, E. hirae, E. casseliflavus or E. durans. The number of different species in each milk sample was low, ranging from 1 to 3. Similarly, the number of strains was also low and, in fact, each of the canine and human samples contained only one enterococcal strain. PFGE profiling revealed that only some of the porcine samples shared a given strain, which indicates that spread is facilitated in intensive farming settings.
Globally, the results showed that milk from different mammalian species may contain enterococci and, therefore, may constitute a natural source of such microorganisms for the infant/offspring. The KAA counts (<1.16 × 103 CFU/mL) were similar to those reported for hygienically-obtained human milk on MRS plates, a medium also suitable for isolation of enterococci [6, 7]. As previously reported for lactobacilli in porcine and canine milk [8, 9], the enterococcal pattern observed in the milk samples seems to be restricted to a low number of species and strains, and also to have a high degree of individual variability. To our knowledge, this is the first description of enterococci isolated from fresh milk of healthy canine, feline and porcine hosts. Some E. faecium and E. faecalis strains from colostrum and milk of healthy women have been described previously [14–16, 47]. In relation to ewe’s milk, a pilot study showed that enterococci were present in excess of 2 × 102 CFU/ml in 15% of the samples of unpasteurized milk from goats and ewes in England and Wales . Other study focused on the identification of indigenous lactic acid bacteria in four samples of fresh ewe’s raw milk and four samples of derived artisanal cheese from Argentina revealed that 48% and 59%, respectively, of the isolates obtained belonged to the genus Enterococcus.
The E. faecalis strains analyzed in this work possessed some potential virulence determinants, including all the sex pheromone determinants, but the gene encoding cytolysin (cylA) could only be detected in 7 strains. The results for the rest of the enterococcal genes were variable depending on the strains. On the other hand, only the efaA
gene could be detected among the E. faecium isolates. These results are similar to those obtained in previous studies with enterococcal strains isolated from human colostrum and milk [14–16]. The role of adhesin EfaA
in virulence has not yet been demonstrated, in contrast to the Esp surface protein. In the absence of other virulence determinants, presence of efaA
seems to have no value as a risk indicator since this gene was also found in 100% of starter E. faecium strains with a long record of safe use in food . The results also agree with those obtained in other studies focused on foodborne enterococci in the sense that E. faecalis strains harbor multiple virulence determinants with a much higher incidence than in other enterococcal species .
A great diversity of E. faecalis and E. faecium clones were detected circulating in the milk environments of different origins including three that have not been described previously. Some of the clones were common in different animal species as it was the case of E. faecalis-ST21, which was detected among porcine and feline isolates, or E. faecalis-ST9 among porcine and ovine ones. The sequence types found among the human isolates were only observed in milk samples of this origin. It is of interest to remark that two of the STs detected among E. faecalis strains of porcine or feline origin are included in clonal complexes (CC16 and CC21) that are frequently detected in human infections in Europe . In addition, it should be highlighted that the hospital-associated lineages of E. faecalis (ST21 and ST16) and E. faecium (ST5), identified in milk of porcine origin in this study, have also been detected in the pig farm environment in a recent study .
Several food and human isolates belonging to different species of the genus Enterococcus had been previously described as BA producers . In fact, tyramine production and a variable ability to produce putrescine is a very common finding among enterococci . However, to our knowledge, no histamine-producing enterococci strains have been described so far and have not been found in this work, either. Although it has been generally assumed that the ability to produce BAs is a strain-dependent characteristic, it has been recently described that tyramine biosynthesis is a species-level characteristic in E. faecalis, E. faecium and E. durans. The same work suggests that putrescine biosynthesis by the agmatine deiminase pathway is also a species-level characteristic in E. faecalis. Since all the strains tested in this study showed ability to synthesize tyramine, and all the E. faecalis strains produced putrescine (Table 4), the results obtained are consistent with the fact that they are species-level characteristics. Moreover, all E. hirae and E. casseliflavus strains were also tyramine producers. Although further work is required, tyramine-production could also be a species-level characteristic of these species. In any case, the ability to produce tyramine is widespread in the genus Enterococcus. With respect to putrescine, the results are more variable. While all the E. faecalis were putrescine producers, only some E. faecium and E. hirae strains and none E. casseliflavus produced it. Genomic studies on E. faecium suggest that such ability could have been acquired through horizontal gene transfer .
The presence of BA-producing enterococci in human milk evidences the need to research if they can produce BAs in the milk, or subsequently in the gastrointestinal tract, and therefore be considered a health risk. In fact, it has been shown that tyramine-producing E. durans strain isolated from cheese is able to produce tyramine under conditions simulating transit through the gastrointestinal tract . The milk used for the production of fermented dairy products (cows, ewes and goats) deserves also further research, since the presence of BA-producing enterococci may be responsible for the accumulation of toxic BAs concentrations in foods .
The E-test was used to determine the resistance pattern of the enterococcal strains against 10 clinically-relevant antimicrobials. The antibiotic resistance spectrum was wider among the E. hirae, E. faecium and, particularly, E. faecalis strains. In relation to the source of the samples, those isolated from porcine milk seemed to be of particular concern. Antibiotic resistance is an important factor for the safety evaluation of enterococci because it can be acquired and/or transferred to other bacteria by gene transfer. The major differences in the rate of resistant enterococci in porcine herds among different countries are most probably due to differences in the usage of antimicrobial agents .
Vancomycin-resistant enterococci (VRE) initially emerged as a relevant Public Health threat due to the use in the past of the glycopeptide avoparcin as growth promoter in animal feed. Once avoparcin was banned, the persistence of VRE was associated to co-selection of van genes and genes conferring resistance to other antibiotics (such as erythromycin) due to the intensive use of other antibiotics, such as tylosin . After the ban of antibiotics as growth promoters in all European Union countries (July 1999), Aarestrup  speculated that occurrence of VRE among pigs would decrease in the following years. In this study, none of the strains was resistant to vancomycin, an antibiotic commonly used for infections caused by multidrug-resistant bacteria, although most of the E. faecalis strains isolated from porcine milk were resistant to erythromycin.
All our E. faecalis, E. faecium and E. hirae strains of food animals (porcine and ovine) were resistant to tetracycline, which has been widely used for therapy in food animals in many countries, including Spain; this usage also could have contributed to the successful persistence of tet genes. A comparison between antibiotic resistance among enterococci isolated from pigs in Sweden, Denmark and Spain showed that tet (L) and tet (S) genes were more frequently found among isolates from Spain .
Globally, frequent occurrences of antibiotic-resistant enterococci have been observed among food animals, and it has been suggested that these animals may be a reservoir of resistant enterococci and resistance genes capable of transferring to humans through the food chain . Antimicrobial resistance genes appear to spread freely between enterococci from different reservoirs, irrespective of their apparent host association .
Therefore, continuous surveillance of antimicrobial resistance in enterococci from humans, animals and foods of animal origin is essential to detect emerging resistance and new infections . As an example, an outbreak of infective mastitis due to E. faecalis was recently reported in an intensive sheep farm in Italy. Forty-five out of the 48 E. faecalis isolates showed the same multi-drug resistance pattern and had a clonal origin. This was the first reported case of ewe’s mastitis caused by E. faecalis. Such strains could arrive to the human food chain through the consumption of cheeses elaborated with raw ewe’s milk.
Pets can also be a source of enterococci and enterococcal resistance genes to humans and other animals and vice versa. Recent results suggest that direct and frequent contact with dogs may significantly shape the composition of our microbial communities . The widespread occurrence of ampicillin-resistant clones in dogs is worrying since these animals may spread such clones among humans due to the close relationships that are usually established between dogs and humans [61, 62]. Due to this risk of zoonotic transfer, it has been suggested that pets used to promote the recovery of patients (pet therapy) may pose a risk to such patients if the dogs are not previously screened for the presence of such enterococcal clones . Similarly, it has been reported that dogs leaving the veterinary intensive care unit (ICU) carry a very large multi-drug resistant enterococcal population with capacity for horizontal gene transfer . As a consequence, the authors recommended restriction of close physical contact between pets released from ICUs and their owners to avoid potential health risks .