Virulence factors and<Emphasis Type="Italic">in Vitro</Emphasis> adherence of<Emphasis Type="Italic">Enterococcus</Emphasis> strains to urinary catheters

The ability to adhere in vitro to urinary catheters and the presence of enterococcal virulence factors was determined in 30 Enterococcus urinary isolates (12 E. faecalis, 12 E. faecium, 3 E. casseliflavus, 3 E. gallinarum). Silicone, siliconized latex and polyvinyl chloride (PVC) were examined by sonication quantitative culture technique and scanning electron microscope. As compared to E. faecalis and E. faecium, E. casseliflavus and E. gallinarum displayed lower adhesion to all synthetic materials. All the tests performed showed higher adherence of all tested strains to siliconized latex and silicone than to PVC. Biofilmforming ability was observed in 5 E. faecalis but in none of the remaining strains. The gene coding enterococcal surface protein (Esp) was detected in 7 E. faecalis and 6 E. faecium strains. Gelatinase was found in 1 E. faecalis, 2 E. faecium and hemolysins were found in 6 E. faecalis and 1 E. faecium strains. All E. casseliflavus and E. gallinarum strainswere negative for these traits. Hydrophobic type of cell surface (measured by its affinity for n-hexadecane) was shown in a few isolates. Bacterial adherence was not significantly associated with the above pathogenic factors.     

The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis

Enterococci have emerged as one of the leading causes of nosocomial bloodstream, surgical site, and urinary tract infections. More recently, enterococci have been associated with biofilms, which are bacterial communities attached to a surface and encased in an extracellular polymeric matrix. The enterococcal cell surface-associated protein, Esp, enhances biofilm formation by Enterococcus faecalis in a glucose-dependent manner. Mature Esp consists of a nonrepeat N-terminal domain and a central region made up of two types of tandem repeats followed by a C-terminal membrane-spanning and anchor domain. This study was undertaken to localize the specific domain(s) of Esp that plays a role in Esp-mediated biofilm enhancement. To achieve this objective, we constructed in-frame deletion mutants expressing truncated forms of Esp in an isogenic background. By comparing strains expressing the mutant forms of Esp to those expressing wild-type Esp, we found that the strain expressing Esp lacking the N-terminal domain formed biofilms that were quantitatively less in biovolume than the strain expressing wild-type Esp. Furthermore, an E. faecalis strain expressing only the N-terminal domain of Esp fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. This suggested that the minimal region contributing to Esp-mediated biofilm enhancement in E. faecalis was confined to the nonrepeat N-terminal domain. Expression of full-length E. faecalis Esp in heterologous host systems of esp-deficient Lactococcus lactis and Enterococcus faecium did not enhance biofilm formation as was observed for E. faecalis. These results suggest that Esp may require interaction with an additional E. faecalis-specific factor(s) to result in biofilm enhancement.     

Specificity of L,D-transpeptidases from gram-positive bacteria producing different peptidoglycan chemotypes

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the L,D-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldt(fm) from Enterococcus faecium, was previously shown to bypass the D,D-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and beta-lactam antibiotics. Ldt(fm) homologues from Bacillus subtilis (Ldt(Bs)) and E. faecalis (Ldt(fs)) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP(3)) and L-Lys(3)-L-Ala-L-Ala at the third position of the stem peptide, respectively, instead of L-Lys(3)-d-iAsn in E. faecium. Ldt(fs) differed from Ldt(fm) and Ldt(Bs) by its capacity to hydrolyze the L-Lys(3)-D-Ala(4) bond of tetrapeptide (L,D-carboxypeptidase activity) and pentapeptide (L,D-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldt(fs), which was also specific for its cognate acyl donor, Ldt(fm) tolerated substitution of L-Lys(3)-D-iAsn by L-Lys(3)-L-Ala-L-Ala. Likewise, Ldt(Bs) tolerated substitution of mesoDAP(3) by L-Lys(3)-D-iAsn and L-Lys(3)-L-Ala-L-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the L,D-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving L,D-transpeptidases.     

Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan

The L,D-transpeptidase Ldt(fm) catalyzes peptidoglycan cross-linking in beta-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldt(fm) homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the L,D-transpeptidation reaction.     

Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem

The beta-lactam antibiotics mimic the D-alanyl(4)-D-alanine(5) extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the dd-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high level resistance to ampicillin. Ldt(fm) is specific for the L-lysyl(3)-D-alanine(4) bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine(5). This specificity was proposed to account for resistance, because the substrate of Ldt(fm) does not mimic beta-lactams in contrast to the D-alanyl(4)-D-alanine(5) extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a beta-lactam of the carbapenem class, totally inhibited Ldt(fm) at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt(fm) is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt(fm) by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt(fm) containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt(fm) is likely to involve rupture of the beta-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of beta-lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.     

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.     

Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase

During the final stages of cell-wall synthesis in bacteria, penicillin-binding proteins (PBPs) catalyse the cross-linking of peptide chains from adjacent glycan strands of nascent peptidoglycan. We have recently shown that this step can be bypassed by an L,D-transpeptidase, which confers high-level beta-lactam-resistance in Enterococcus faecium. The resistance bypass leads to replacement of D-Ala4-->D-Asx-L-Lys3 cross-links generated by the PBPs by L-Lys3-->D-Asx-L-Lys3 cross-links generated by the L,D-transpeptidase. As the first structure of a member of this new transpeptidase family, we have determined the crystal structure of a fragment of the L,D-transpeptidase from E.faecium (Ldt(fm217)) at 2.4A resolution. Ldt(fm217) consists of two domains, the N-terminal domain, a new mixed alpha-beta fold, and the ErfK_YbiS_YhnG C-terminal domain, a representative of the mainly beta class of protein structures. Residue Cys442 of the C-terminal domain has been proposed to be the catalytic residue implicated in the cleavage of the L-Lys-D-Ala peptide bond. Surface analysis of Ldt(fm217) reveals that residue Cys442 is localized in a buried pocket and is accessible by two paths on different sides of the protein. We propose that the two paths to the catalytic residue Cys442 are the binding sites for the acceptor and donor substrates of the L,D-transpeptidase.     

Inactivation kinetics of a new target of beta-lactam antibiotics

Peptidoglycan is predominantly cross-linked by serine DD-transpeptidases in most bacterial species. The enzymes are the essential targets of β-lactam antibiotics. However, unrelated cysteine LD-transpeptidases have been recently recognized as a predominant mode of peptidoglycan cross-linking in Mycobacterium tuberculosis and as a bypass mechanism conferring resistance to all β-lactams, except carbapenems such as imipenem, in Enterococcus faecium. Investigation of the mechanism of inhibition of this new β-lactam target showed that acylation of the E. faecium enzyme (Ldt(fm)) by imipenem is irreversible. Using fluorescence kinetics, an original approach was developed to independently determine the catalytic constants for imipenem binding (k(1) = 0.061 μM(-1) min(-1)) and acylation (k(inact) = 4.5 min(-1)). The binding step was limiting at the minimal drug concentration required for bacterial growth inhibition. The Michaelis complex was committed to acylation because its dissociation was negligible. The emergence of imipenem resistance involved substitutions in Ldt(fm) that reduced the rate of formation of the non-covalent complex but only marginally affected the efficiency of the acylation step. The methods described in this study will facilitate development of new carbapenems active on extensively resistant M. tuberculosis.     

The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.     

Comparison of genotypic and phenotypic cluster analyses of virulence determinants and possible role of CRISPR elements towards their incidence in Enterococcus faecalis and Enterococcus faecium

Enterococcus faecalis and Enterococcus faecium are human commensals frequently found in fermented foods or used as probiotics, but also recognized as opportunistic pathogens. We investigated 62 Enterococcus strains isolated from clinical, food and environmental origins towards a rationale for safety evaluation of strains in food or probiotic applications. All isolates were characterised with respect to the presence of the virulence determinants fsrB, sprE, gelE, ace, efaAfs/fm, as, esp, cob and the cytolysin operon. In addition RAPD-PCR was used to obtain genomic fingerprints that were clustered and compared to phenotypic profiles generated by MALDI-TOF-MS. The gelatinase phenotype (GelE) and the haemolytic activity (β-haemolysis) were analysed. E. faecium strains contained esp and efaAfm only, and none of them contained any CRISPR elements. The amenability of E. faecalis strains to acquisition of virulence factors was investigated along the occurrence of CRISPR associated (cas) genes. While distribution of most virulence factors, and RAPD versus MALDI-TOF-MS typing patterns were unrelated, 2 out of 5 RAPD clusters almost exclusively contained clinical E. faecalis isolates, and an occurrence of CRISPR elements versus reduced number of virulence factors was observed. The presence of the cytolysin operon, cob and as encoding pheromone and aggregation substance, respectively, significantly corresponded to absence of cas. As their production promote genetic exchange, their absence limits further gene acquisition and distribution. Thus, absence of the cytolysin operon, cob and as in a cas positive environment suggests itself as promising candidate for E. faecalis evaluation towards their occurrence in food fermentation or use as probiotics.     

A novel peptidoglycan cross-linking enzyme for a beta-lactam-resistant transpeptidation pathway

The beta-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the beta-lactam ring and the d-alanyl(4)-d-alanine(5) extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in abeta-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldt(fm), catalyzes in vitro the cross-linking of peptidoglycan subunits in a beta-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldt(fm) for the l-lysyl(3)-d-alanine(4) peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic beta-lactams in contrast to d-alanyl(4)-d-alanine(5) in the pentapeptide donors required for dd-transpeptidation. Ldt(fm) homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens.     

Adhesion to bile drain materials and physicochemical surface properties of Enterococcus faecalis strains grown in the presence of bile

The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of Enterococcus faecalis strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for E. faecalis not expressing Agg or Esp and E. faecalis with Esp on its surface. E. faecalis expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of E. faecalis.     

Effects of Enterococcus faecium and dried whey on broiler performance, gut histomorphology and intestinal microbiota

The experiment was conducted to study the effects of supplementing a broiler starter diet with the probiotic Enterococcus faecium NCIMB 10415 and dried whey (80% lactose) on chick performance, gut histomorphology and intestinal microbiota. One-day-old male Ross 308 strain broiler chickens were fed diets containing: (i) control feed, (ii) control + 3.5% dried whey, (iii) control + 0.2% E. faecium, and (iv) control + 3.5% dried whey + 0.2% E. faecium. Birds were maintained in battery brooders confined in an environmentally controlled experimental room. The experiment lasted for 21 days. Birds fed E. faecium or E. faecium + dried whey exhibited significantly improved weight gain and feed conversion rate (FCR). Weight gain and FCR of treatment groups 1-4 were 628.7, 657.8, 690.9, 689.3 and 1.218, 1.193, 1.107, 1.116, respectively. Lactic acid bacteria counts in both the ileal content and excreta were significantly affected by dietary treatment. Supplementation of the E. faecium and dried whey separately and in combination increased lactic acid bacteria colonization in the ileal content from 4.2 to 5.0, 7.8 and to 5.1 log cfu/g, respectively (treatments 1-4). Similarly, supplementation of dried whey and E. faecium separately and in combination increased lactic acid bacteria in the excreta from 5.3 to 5.5, 8.0 and to 7.2 log cfu/g, respectively. Addition of the probiotic E. faecium increased villus height in the ileum (p < 0.05). Thus, supplementation of E. faecium enhanced broiler chick performance with respect to weight gain and FCR. No additive effect of E. faecium and dried whey was detected. Further studies are needed to investigate the relationship between E. faecium and dried whey with respect to gut histomorphology.     

Copper resistance in Enterococcus faecium, mediated by the tcrB gene, is selected by supplementation of pig feed with copper sulfate

The tcr gene cluster mediates in vitro copper resistance in Enterococcus faecium. Here we describe the selection of tcr-mediated copper resistance in E. faecium in an animal feeding experiment with young pigs fed 175 mg copper/kg feed (ppm), which is the concentration commonly used for piglets in European pig production. tcr-mediated copper resistance was not selected for in a control group fed low levels of copper (6 ppm). We also show coselection of macrolide- and glycopeptide-resistant E. faecium in the animal group fed the high level of copper. Finally, we identify the tcr genes in the enterococcal species E. mundtii, E. casseliflavus, and E. gallinarum for the first time.     

Enterococcal surface protein Esp is not essential for cell adhesion and intestinal colonization of Enterococcus faecium in mice

Background  

Enterococcus faecium has globally emerged as a cause of hospital-acquired infections with high colonization rates in hospitalized patients. The enterococcal surface protein Esp, identified as a potential virulence factor, is specifically linked to nosocomial clonal lineages that are genetically distinct from indigenous E. faecium strains. To investigate whether Esp facilitates bacterial adherence and intestinal colonization of E. faecium, we used human colorectal adenocarcinoma cells (Caco-2 cells) and an experimental colonization model in mice.     

粪肠、屎肠球菌及相近种部分持家基因的系统发育分析

【目的】利用16S rRNA、clpX和recA基因分子标记研究Enterococcus faecalis、Enterococcus faecium及相近种间的种系发育关系,并比较这些基因序列对E.faecalis、E.faecium及相近种的区分能力。【方法】以分离自传统乳制品中的9株E.faecium和1株E.durans分离株为研究对象,以clpX和recA基因片段为标记,通过PCR扩增、测序,结合已公布的近缘种相应序列构建系统发育树并与16S rRNA基因进行比较。【结果】在基于clpX和recA基因的进化树中,10株试验菌株与E.faecalis始终处于同一分支。与该物种这两个基因的平均相似性为99.6%和98.6%,与另一分支的Faecium-group(E.durans和E.faecium)的平均相似性仅为61.5%和33.5%。相近种E.durans和E.hirae间这两个基因的差异性为20.3%和39.0%;在基于16S rRNA基因的进化树中,试验菌株与Faecium-group(E.lactis、E.faecium、E.durans、E.hirae)处于同一分支。与这些成员间该基因的相似性大于99.6%,与E.faecalis基因的平均相似性可达98.4%。相近种间该基因相似性无明显差异。【结论】按照10株试验菌株clpX和recA基因的分析结果可将由传统生理生化和16S rRNA基因序列鉴定的9株E.faecium和1株E.durans归类为E.faecalis,clpX和recA基因可用于部分相近种的分类鉴定。     

Adhesion to Bile Drain Materials and Physicochemical Surface Properties of Enterococcus faecalis Strains Grown in the Presence of Bile

The aim of this study is to determine whether growth in the presence of bile influences the surface properties and adhesion to hydrophobic bile drain materials of Enterococcus faecalis strains expressing aggregation substance (Agg) or enterococcal surface protein (Esp), two surface proteins that are associated with infections. After growth in the presence of bile, the strains were generally more hydrophobic by water contact angles and the zeta potentials were more negative than when the strains were grown in the absence of bile. Nitrogen was found in lower surface concentrations upon growth in the presence of bile, whereas higher surface concentrations of oxygen were measured by X-ray photoelectron spectroscopy. Moreover, an up to twofold-higher number of bacteria adhered after growth in bile for E. faecalis not expressing Agg or Esp and E. faecalis with Esp on its surface. E. faecalis expressing Agg did not adhere in higher numbers after growth in bile, possibly because they mainly adhere through positive cooperativity and less through direct interactions with a substratum surface. Since adhesion of bacteria is the first step in biomaterial-centered infection, it can be concluded that growth in bile increases the virulence of E. faecalis.     

Genome-wide identification of ampicillin resistance determinants in Enterococcus faecium

Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.