The incidence of virulence factors among 48 Enterococcus faecium and 47 Enterococcus faecalis strains from foods and their antibiotic susceptibility were investigated. No strain was resistant to all antibiotics, and for some strains, multiple resistances were observed. Of E. faecium strains, 10.4% were positive for one or more virulence determinants, compared to 78.7% of E. faecalis strains. Strains exhibiting virulence traits were not necessarily positive for all traits; thus, the incidence of virulence factors may be considered to be strain specific.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...

The aggregation substance (AS) of Enterococcus faecalis, encoded on sex pheromone plasmids, is a surfacebound glycoprotein that mediates aggregation between bacteria thereby facilitating plasmid transfer. Sequencing of the pAD1-encoded Asa1 revealed that this surface protein contains two RGD motifs which are known to ligate integrins. Therefore, we investigated the influence of AS on the interaction of E. faecalis with human monocyte-derived macrophages which constitutively express ␤ 2 integrins (e.g., CD18). AS was found to cause a greater-than-fivefold increase in enterococcal adherence to macrophages and a greater-than-sevenfold increase in phagocytosis. Adherence was mediated by an interaction between the RGD motif and the integrin CD11b/CD18 (complement receptor type 3) as demonstrated by inhibition studies with monoclonal antibodies and RGD peptide. AS-bearing enterococci were significantly more resistant to macrophage killing during the first 3 h postinfection, probably due to inhibition of the respiratory burst as indicated by reduced concentrations of superoxide anion.Enterococci are gram-positive cocci which inhabit the gastrointestinal tract as well as the vagina and the oral cavity. Enterococcus faecalis accounts for 90% of human enterococcal infections, the most common being urinary tract infections, followed by abdominal infections, wound infections, bacteremia, and infective endocarditis (31, 39). Although infections due to E. faecalis have increased substantially during the last 10 years, the understanding of virulence mechanisms is still limited (24). One of the postulated virulence factors is the aggregation substance (AS), a sex pheromone plasmid-encoded surface protein which promotes the conjugative transfer of sex pheromone plasmids by formation of mating aggregates between donor and recipient cells (6,13,52). DNA sequencing of the structural gene for the pAD1-encoded AS revealed the presence of two Arg-Gly-Asp (RGD) sequences (16); RGD is a well-known motif recognized by a family of eukaryotic receptors, the integrins (38). Integrins consist of noncovalently linked ␣ and ␤ chains and are expressed on leukocytes, thrombocytes, endothelium, and various epithelial cells (21, 37, 42). Our group first suggested an interaction of AS with integrins, since we found that AS augmented adherence to porcine renal tubular cells which could be inhibited competitively by an RGD-Ser (RGDS) peptide (26). This hypothesis was corroborated by in vitro experiments with human polymorphonuclear leukocytes (PMN) which demonstrated that AS promotes opsonin-independent binding of E. faecalis via a ␤ 2 integrinmediated mechanism (46). It is assumed that many enterococcal infections are endogenous, originating from the intestinal tract (25, 51). Wells et al. speculated that macrophages may serve as a vehicle facilitating translocation from the intestinum into the lymph system and bloodstream (49, 50). However, this can occur only if enterococci are able to survive within macrophages. Indeed, Gentry-Weeks et al. demons...

In this study, the search for new antibiotics was combined with quantitative ecological studies. The cultured fraction of the associated bacterial communities from ten different Mediterranean sponge species was investigated. To obtain quantitative and qualitative data of sponge-associated bacterial communities and to expand the cultured diversity, different media were used. The largest morphological diversity and highest yield of isolates was obtained by using oligotrophic media, which consisted of natural habitat seawater amended with (1% additional carbon sources. The dominant bacterial morphotypes were determined and bacterial isolates were tested for antimicrobial activity and identified using 16S rDNA sequencing. The sponge-associated most abundant morphotypes were all affiliated to the Alphaproteobacteria and showed antimicrobial activity against at least one of the tested strains. In contrast, the ambient seawater was dominated by Gammaproteobacteria. One single alphaproteobacterium, which was related to Pseudovibrio denitrificans, was shown to dominate the cultured community of at least six of the sponges. This designated MBIC3368-like alphaproteobacterium has been isolated from sponges before and seems to be restricted to associations with members of the phylum Porifera. It displays a weak and unstable antimicrobial activity, which gets easily lost during cultivation. However, this bioactive bacterium was present in the sponges by up to 10(6) cells per gram wet-weight sponge tissue and dominated the cultured fraction with up to 74%. The association of this alphaproteobacterium with sponges is probably evolutionary young and facultative and possibly involves biologically active secondary metabolites. Besides a demonstrated vertical transfer, additional horizontal transfer between the sponges is assumed. Members of the genus Bacillus displaying antimicrobial activity were found regularly, too. However, actinomycetes, which are known for their production of bioactive substances, were present in very low abundance.

The aggregation substance of Enterococcus faecalis increased bacterial adherence to and internalization by epithelial cells originating from the colon and duodenum but not by cells derived from the ileum. However, enterococcal translocation through monolayers of intestinal epithelium was not observed.Enterococcus faecalis, a gram-positive facultative anaerobic bacterium, belongs to the normal flora of the intestinal tract and is also found in the vaginal vault and the oral cavity. Enterococci have increasingly gained attention as pathogens, since they have become the fourth leading cause of nosocomial infections in the United States (8). E. faecalis frequently causes local or systemic infections, such as urinary tract and abdominal infections, wound infections, bacteremia, and endocarditis (15). It is assumed that many enterococcal infections are endogenous, resulting from bacterial translocation from the intestinal lumen to extraintestinal sites (16,31).Surprisingly little is known about the virulence factors of E. faecalis. One of the potential virulence factors is the aggregation substance (AS), an adhesin encoded by inducible sex pheromone plasmids (for a review, see reference 5). The AS, a signal peptide-containing protein, appears as a hair-like structure on the cell surface and is incorporated primarily into the ''old'' parts of the cell wall (30). It has been demonstrated that this adhesin is responsible for bacterium-bacterium contact during conjugative transfer of sex pheromone plasmids (5).The AS contains two Arg-Gly-Asp motifs which are known to be recognized by integrins, a family of eukaryotic cell surface receptors (24). Since integrins have been reported to be expressed on intestinal epithelial cells (2), we hypothesized that the AS promotes adherence and possibly invasion into these cells, thereby enabling enterococci to translocate through the intestinal epithelial barrier.(This work was presented in part at the 98th General Meeting of the American Society for Microbiology, Atlanta, Ga., 17 to 21 May 1998.)Three different E. faecalis strains (Table 1), kindly provided by R. Wirth (Institute of Microbiology, University of Regensburg, Regensburg, Germany), were used to study the effect of the AS on adherence, internalization, and translocation through intestinal epithelial cells. The bacteria were cultured in Todd-Hewitt medium (Difco, Augsburg, Germany) supplemented with erythromycin (20 g/ml) for growth of the plasmid-containing strains (OG1X/pAM721 and OG1X/pAM944). Since E. faecalis strains expressing the AS spontaneously clump, all inocula were sonicated (Branson W-450 Sonifier; 80 W, 20 s, continuously) to obtain single-cell suspensions. Salmonella enterica serovar Typhimurium ATCC 14028 and Escherichia coli HB101 grown in Luria-Bertani medium were used as positive and negative controls, respectively. For the experiments described below, the bacteria were harvested in mid-log phase and suspended in tissue culture medium without supplements, which served as the infection medium. Appropriate ...

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Enterococcus faecalis is a leading cause of infections in liver transplant patients. This study reviewed the incidence of virulence factors such as hemolysin, gelatinase, aggregation substances (asa1 and asa373), or the enterococcal surface protein (Esp) in isolates from liver transplant patients. In total, 133 isolates from liver transplant patients were compared with 47 isolates from feces of healthy volunteers and 66 isolates from blood cultures. Amplified fragment length polymorphism (AFLP) analysis indicates that the isolates from different clinical subgroups can be divided into genogroups with an AFLP similarity of >80% and different virulence factors. Hemolysin and asa1 might be associated with infection, as they are more frequent in isolates from blood cultures and transplant patients. Esp might be associated with colonization and spread, because it is more frequent in isolates from feces of healthy volunteers and transplant patients. An epidemic esp gene-positive strain among liver transplant patients supports this hypothesis.

The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under laboratory conditions, at least 105-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no case was transfer from E. faecalisto another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of E. faecalisin the municipal sewage water treatment plant of the city of Regensburg ranged from 105 to 108 events per 4 h, indicating that gene transfer should take place under natural conditions.