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Enterococci constitute a major component of the microflora of artisanal cheeses produced in southern Europe (10) and are considered to play an important role in ripening and aroma development (1, 5, 28). This has led to the suggestion that enterococci be included in starter culture preparations for the manufacture of certain Mediterranean cheeses (1, 5, 23). However, enterococci are also major nosocomial pathogens causing a variety of infections (19, 20). Enterococcus faecalis strains clearly dominate among enterococci isolated from human infections, while Enterococcus faecium strains are associated with the majority of the remainder (15).

A specific cause for concern and contributing factor to pathogenesis of enterococci is their resistance to a wide variety of antibiotics (18, 20). However, antibiotic resistance as such cannot explain the virulence of enterococci. Although enterococci possess subtle virulence traits (25), considerable progress has recently been made in determining these. For example, studies have shown that phenotypes such as -hemolysin/bacteriocin (also called cytolysin) and aggregation substance (AS), which are encoded by E. faecalis pheromone-responsive plasmids, are related to pathogenicity and enhance the virulence of enterococci in animal models (2, 12, 14, 15, 26). AS is an adhesin which mediates the formation of cell clumps that allow the highly efficient transfer of the sex pheromone plasmid on which AS is encoded (4). An interesting variation in the generally similar AS (about 90% homology) encoded by sex pheromone plasmids such as pAD1, pCF10, and pPD1 is encoded by asa373 on plasmid pAM373. This AS is also involved in a clumping response, but it has little homology to the "classical" AS and therefore represents a rather unique type of adhesin (22). Other virulence factors include the adhesin called enterococcal surface protein (Esp) and gelatinase (Gel), which is an extracellular metalloendopeptidase (26).

This study aimed to determine the incidence of hemolysin; classical AS; and the aggregation substances Asa373, Gel, and Esp and antibiotic susceptibility among enterococci isolated mostly from cheeses. Studies formed part of the European Union (EU) project FAIR-CT97-3078, "Enterococci in Food Fermentations: Functional and Safety Aspects." As E. faecalis and E. faecium are predominantly associated with human infection and as these species were also predominant among strains isolated from cheese in the EU study, our study was concerned only with strains of these two species.

Enterococci were obtained from six partners associated with the EU project and were identified to species level. In this study, safety aspects were studied for 48 E. faecium and 47 E. faecalis strains that were preselected as possible starter cultures for cheese manufacture on the basis of functional properties, such as proteolytic and lipolytic activities, and production of volatile compounds. Information on these strains is available in the catalogue of enterococci of the FAIR-E collection (29). Two of the E. faecium strains used in this study (E 24 and E 25) are commercial probiotic strains. All enterococci were grown in MRS broth (Merck, Darmstadt, Germany) or Todd-Hewitt broth (THB; Difco, Heidelberg, Germany) at 37°C. Escherichia coli strains were grown on a rotary shaker at 250 rpm in Luria-Bertani broth at 37°C with ampicillin (150 µg/ml) or kanamycin (50 µg/ml) added where appropriate.

Production of Gel was tested on Todd-Hewitt agar containing 30 g of gelatin/liter, as described by Coque et al. (6). Production of -hemolysin was indicated by the formation of clear zones surrounding the colonies on blood agar plates. Blood agar plates were prepared using Columbia blood agar base (Merck) with 5% defibrinated human blood (containing all four blood types). Production of AS by enterococci was studied in the clumping assay in the presence of sex pheromone. Sex pheromone was obtained by growing the pheromone producer E. faecalis JH2-2 (Table 1) in THB at 37°C for 18 h. Pheromone-containing supernatant obtained after centrifugation at 10,823 × g was sterilized by autoclaving for 15 min, diluted 1:5 in sterile THB, and used in clumping assays in microtiter plates. Two-hundred-microliter volumes were inoculated (0.5%) with the test Enterococcus strain, incubated at 37°C, and visually examined for cell clumping after 2, 4, 8, and 24 h. The plasmidless strain, E. faecalis OG1X, was used as a negative control, while two variants of E. faecalis OG1X containing either plasmid pAD1 or pAM373 (9, 22) were used as positive controls (Table 1). In order to account for constitutive clumping in the absence of AS, all enterococci were also tested in THB without sex pheromone. Strains that were positive in the clumping assay or that clumped constitutively were tested for the AS genes asa1 and asa373 using gene probes. Total genomic DNA was isolated according to the methods of Pitcher et al. (24), cut with the restriction enzymes HindIII and EcoRI, subjected to electrophoresis on 0.7% agarose gels, and transferred onto nylon membranes (Hybond N+; Amersham Pharmacia Biotech, Freiburg, Germany). Plasmid pQESD3.7 containing the asa1 gene (21) and plasmid pOK12asa373 containing the asa373 gene (22) were cut with PstI and BsrGI/HpaII restriction enzymes, respectively. The fragments corresponding to the AS genes were eluted from the gel, labeled, and used as probes together with the enhanced chemiluminescence direct nucleic acid labeling and detection kit (Amersham Pharmacia), according to the manufacturer's instructions.

PCR amplification was used to detect the enterococcal surface protein gene (esp). Primers esp11 and esp12 were used to amplify the part of the gene encoding the N-terminal region of Esp according to the methods of Shankar et al. (25). DNA from E. faecalis strain MMH594 (Table 1) was used in PCR experiments as a positive control.

E-test (Viva Diagnostika, Cologne, Germany) antibiotic strips were used to determine the susceptibility of the strains to ampicillin, benzylpenicillin, chloramphenicol, tetracycline, erythromycin, ciprofloxacin, streptomycin (high range), gentamicin (high range), and vancomycin (Viva Diagnostika). Commercial Mueller-Hinton II agar plates (Becton Dickinson, Heidelberg, Germany) were seeded with enterococci, and antibiotic strips were placed onto the plates according to the manufacturer's instructions. Plates were incubated at 37°C, and results were read after 24 and 48 h of incubation. The MIC determination was based on the reference agar dilution method that has been previously described (22a).

None of the E. faecium strains and 23 (48.9%) of the E. faecalis strains produced Gel. Kühnen et al. (17) reported a high (63.7%) incidence of Gel production among clinical enterococci. Similarly, Singh et al. (26) reported Gel production by E. faecalis for 54% of endocarditis isolates, 58% of nosocomial clinical isolates, and 62% of nosocomial fecal isolates. The incidence of Gel production among food E. faecalis strains in our study seems to be slightly lower than that reported for clinical strains by Kühnen et al. (17) and Singh et al. (26). This value was also only slightly lower than the 56% incidence among nine E. faecalis strains isolated from food reported by Eaton and Gasson (8). The high incidence of Gel activity among enterococci in this study may be explained by the fact that most enterococci originated from cheese, a protein-rich source. Thus, production of protease may be a selection mechanism for enterococci growing in cheese, as it may enable them to utilize cheese protein as a source for amino acids. However, the incidence of Gel activity of the enterococci in this study may be expected to be even higher, as Eaton and Gasson (8) showed that Gel genes may be silent and that the phenotype may be negative, even though a Gel gene is present. Similar to the results of Eaton and Gasson (8), none of the 48 E. faecium strains in this study showed Gel activity, suggesting that Gel production by this species is not common.

Among both E. faecium and E. faecalis, some strains produced -hemolysin (Table 2) with the incidence of this trait being higher for E. faecalis strains (21.3%), than for E. faecium strains (8.3%). Hemolysin plays an important role in enterococcal virulence, as it may increase the severity of the infection (11, 15). Ike et al. (13) showed that 60% of clinical E. faecalis isolates were hemolytic compared to 17% of strains from uninfected sources. The incidence of -hemolysin in our study was much lower than that reported by Ike et al. (13). In addition, this value was much lower than the 44% incidence for E. faecalis strains from foods reported by Eaton and Gasson (8). Interestingly, the hemolysin trait appeared to be linked with AS production (presence of the asa1-related gene) for 7 of the 10 Hly⁺ E. faecalis isolates in our study. Production of hemolysin and AS is known to be often (85% of hemolytic strains) linked on pheromone-responsive plasmids. However, some hemolytic strains did not exhibit a clumping response, and their hemolysin genes probably reside on the chromosome (13). Four E. faecium strains in this study also showed beta-hemolytic activity, but a clumping response was not detected. In contrast, the E. faecium strains from food studied by Eaton and Gasson (8) did not show cytolysin activity. Thus, our results suggest that both E. faecalis and E. faecium strains isolated from foods may produce -hemolysin and that this trait is not exclusive to clinical isolates.

Only one (2.1%) of the E. faecium strains but 17 (36.2%) of the E. faecalis strains produced Esp (Table 2). Shankar et al. (25) showed that 29% of their blood isolates and 42% of endocarditis E. faecalis isolates were positive for the esp gene, while only 3% of isolates from stool showed this trait. The reason for the high incidence of Esp production among E. faecalis strains in this study is not known. Our result correlated well with that of Eaton and Gasson (8), who showed an incidence of 33% for Esp production among E. faecalis isolates from food. Similar to the results of Eaton and Gasson (8), who showed that none of the E. faecium strains from food produced Esp, a low incidence among E. faecium strains was also found in our study. In addition to its role in adhesion, Esp is also thought to play a role in evasion of the immune response (25). Thus, enterococci possessing this trait would clearly be undesirable for use in foods as a starter culture.

None of the E. faecium strains showed a clumping phenotype in the clumping assay, while 18 (38.3%) E. faecalis strains exhibited a clumping reaction (Table 2). Because an additional six E. faecalis strains clumped constitutively, i.e., also in the absence of pheromone, it was unclear whether clumping was as a result of AS. By probing for the asa1 and asa373 genes, it was established that, of the six strains clumping constitutively, five (E 237, E 238, E 260, E 342, and E 372) were positive for the AS gene(s) (Table 2). Thus, 23 (48.9%) and 15 (31.9%) of the E. faecalis strains in total were positive for the asa1 and asa373 genes, respectively (Table 2). Interestingly, while some strains were positive for only the asa1 gene, none of the E. faecalis strains was positive only for the asa373 gene, which always occurred when the asa1 gene was present.

AS is an important virulence factor of enterococci, and over one-third of clinical isolates of E. faecalis are known to carry pheromone response plasmids (3). In our study, production of AS was common among E. faecalis strains isolated from foods. In contrast, E. faecium strains did not appear to produce AS, which was not surprising, as pheromone response plasmids are generally associated with E. faecalis (7). Eaton and Gasson (8) showed that 67% of the E. faecalis strains isolated from food produced asa1-related AS, while none of the E. faecium strains were positive for such AS genes. The much higher incidence reported by Eaton and Gasson (8) may be explained by the lower number (nine E. faecalis strains isolated from food) which they investigated.

Ten (20.8%) E. faecium strains and six (12.8%) E. faecalis strains were susceptible to all antibiotics tested. All E. faecalis strains and all but one E. faecium strain were susceptible to vancomycin (Table 3). The E. faecium strains were mostly resistant to ciprofloxacin (56.3%), followed by penicillin (45.8%), erythromycin (27.1%), chloramphenicol (10.4%), tetracycline (6.3%), streptomycin (4.2%), gentamicin (2.1%), and vancomycin (2.1%). None were resistant to ampicillin (Table 3). In contrast, the E. faecalis strains were mostly resistant to chloramphenicol (63.8%), followed by streptomycin (46.8%), tetracycline (44.7%), erythromycin (31.9%), ciprofloxacin (27.7%), gentamicin (25.5%), penicillin (12.8%), and ampicillin (2.1%), while none were resistant to vancomycin (Table 3). No strains tested were resistant to all the antibiotics used in this study, while multiple resistance to five or more antibiotics was observed (Table 3).

Our results showed that a larger number of E. faecium strains than E. faecalis strains were resistant to penicillin, which may be explained by E. faecium being generally more resistant to penicillin than E. faecalis (20). The incidence of ampicillin and vancomycin resistance for both E. faecium and E. faecalis was low, indicating that most of the strains tested did not acquire resistance determinants for these antibiotics. In a study of European cheeses, Teuber et al. (27) also reported a low (4%) incidence of vancomycin-resistant enterococci. While the incidence of aminoglycoside-resistant enterococci was low among E. faecium isolates, it was considerably higher for E. faecalis. These results indicate that especially E. faecalis strains with acquired, high-level aminoglycoside resistance can occur in traditional cheeses. Teuber et al. (27) reported a much higher incidence of gentamicin-resistant enterococci from cheeses, as 80% of isolates were resistant to this compound. The incidence of tetracycline and erythromycin resistance among E. faecalis strains was relatively high at 44.7 and 31.9% of the strains, respectively, and this incidence was greater than for E. faecium strains. Similarly high incidences of erythromycin and tetracycline resistance among cheese enterococcal isolates were described by Teuber et al. (27). This may be explained by the fact that erythromycin resistance plasmids and transposons are commonly found among enterococci (20). The incidence of chloramphenicol resistance was higher for E. faecalis strains (63.8%) than for E. faecium strains (10.4%) in this study, and a high (32%) incidence of chloramphenicol resistance among cheese enterococcal isolates was also reported by Teuber et al. (27).

It is difficult to assess the impact of antibiotic-resistant enterococci from foods on potential human pathogenicity. It is clear that in the hospital environment, antibiotics may influence selection of pathogenic enterococci, which may lead to infections or superinfections (20). Most problematic are strains that have acquired multiple antibiotic resistance, especially resistance to vancomycin and to the synergistic action of -lactams and aminoglycosides (20), which leaves few therapeutic options. Although the enterococci in this study showed acquired resistance traits to a number of antibiotics, they did generally not show resistance to the clinically relevant antibiotic ampicillin or vancomycin, and a low incidence of resistance towards gentamicin was observed especially among the E. faecium strains. These results indicated that these food enterococcal strains were mostly still susceptible to clinically relevant antibiotics.

Antibiotic resistance alone cannot explain the virulence of enterococci. In order to become pathogenic, they need to express virulence traits associated with adhesion, translocation, and evasion of immune responses and cause pathological changes (16). Our results have shown that virulence factors such as hemolysin, AS, Gel, and Esp also occur among food enterococcal isolates. In general, the incidence of these virulence traits was lower among E. faecium strains than among E. faecalis strains and E. faecium harbored fewer virulence traits than E. faecalis. These results correlate well with those of Eaton and Gasson (8). The reason for this may be that the AS and hemolysin virulence factors, for example, are located on pheromone response plasmids, which generally occur in E. faecalis strains only. Only a few (10.4%) E. faecium strains but a higher number (78.8%) of E. faecalis strains in our study were positive for one of the virulence factors tested. However, enterococci exhibiting virulence traits were not necessarily positive for all traits tested. Thus, it appears that the incidence of such virulence factors is strain specific. Should Enterococcus strains be selected as starter cultures, for safety considerations each strain should be tested for the different virulence traits as well as for antibiotic resistance. Yet, the question whether enterococci are safe for use as starter cultures remains difficult to answer. Eaton and Gasson (8) showed that the incidence of virulence factors was highest among clinical enterococcal isolates, followed in decreasing order by food strains and starter strains, suggesting that the food and starter strains have a lower potential for pathogenicity. Clearly, based on the lower incidence of virulence traits among E. faecium strains in this study and that by Eaton and Gasson (8), it would be advisable that this species would be used rather than E. faecalis. One problem that remains, however, is the possibility of transfer of virulence determinants from a strain which harbors these to a starter strain. Eaton and Gasson (8) showed that such transfer is a possibility, especially when pheromone-responsive plasmids are involved. However, these authors were also unable to transfer a plasmid containing virulence determinants into a food E. faecium strain by filter mating (8). The possibility of gene transfer under in vitro and in vivo conditions clearly requires further investigation. Although much progress has been made in determinations of enterococcal virulence factors, some virulence traits may presently remain undiscovered.