Molecular characterization of a widespread, pathogenic, and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island

Enterococcus faecalis, a common cause of endocarditis and known for its capacity to transfer antibiotic resistance to other pathogens, has recently emerged as an important, multidrug-resistant nosocomial pathogen. However, knowledge of its lineages and the potential of particular clones of this species to disseminate and cause disease is limited. Using a nine-gene multilocus sequence typing (MLST) scheme, we identified an evolving and widespread clonal complex of E. faecalis that has caused outbreaks and life-threatening infections. Moreover, this unusual clonal complex was found to contain isolates of unexpected relatedness, including the first known U.S. vancomycin-resistant enterococcus (E. faecalis strain V583), the first known penicillinase-producing (Bla(+)) E. faecalis isolate, and the previously described widespread clone of penicillinase producers, a trait found in <0.1% of E. faecalis isolates. All members of this clonal cluster (designated as BVE for Bla(+) Van(r) endocarditis) were found to contain a previously described putative pathogenicity island (PAI). Further analysis of this PAI demonstrated its dissemination worldwide, albeit with considerable variability, confirmed its association with clinical isolates, and found a common insertion site in different clonal lineages. PAI deletions, MLST, and the uncommon resistances were used to predict the evolution of the BVE clonal cluster. The finding of a virulent and highly successful clonal complex of E. faecalis with different members resistant to the primary therapies of choice, ampicillin and vancomycin, has important implications for the evolution of virulence and successful lineages and for public health monitoring and control.     

Intra- and interspecies genomic transfer of the Enterococcus faecalis pathogenicity island

Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transferred. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens.     

Horizontal transfer of virulence genes encoded on the Enterococcus faecalis pathogenicity island

Enterococcus faecalis, a leading cause of nosocomial antibiotic resistant infections, frequently possesses a 150 kb pathogenicity island (PAI) that carries virulence determinants. The presence of excisionase and integrase genes, conjugative functions and multiple insertion sequence elements suggests that the PAI, or segments thereof, might be capable of horizontal transfer. In this report, the transfer of the E. faecalis PAI is demonstrated and a mechanism for transfer elucidated. In filter matings, chloramphenicol resistance was observed to transfer from strain MMH594b, a clinical isolate possessing the PAI tagged with a cat marker, to OG1RF (pCGC) with a frequency of 3.2 x 10(-10) per donor. Secondary transfer from primary transconjugant TCRFB1 to strain JH2SS in filter and broth matings occurred with a frequency of 1 and 2 x 10(-1) per donor respectively. Analysis of the transconjugants demonstrated that a 27,744 bp internal PAI segment was capable of excision and circularization in the donor, and is mobilized as a cointegrate with a pTEF1-like plasmid. High-frequency transfer also occurred from TCRFB1 to JH2SS during transient colonization of the mouse gastrointestinal tract. This is the first demonstration of the horizontal transfer of PAI-encoded virulence determinants in E. faecalis and has implications for genome evolution and diversity.     

Incidence of virulence factors and antibiotic resistance among Enterococci isolated from food

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.     

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.     

Genotypic Diversity and Virulence Characteristics of Clinical and Environmental Vibrio vulnificus Isolates from the Baltic Sea Region

The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region.     

Dogs leaving the ICU carry a very large multi-drug resistant enterococcal population with capacity for biofilm formation and horizontal gene transfer

The enterococcal community from feces of seven dogs treated with antibiotics for 2-9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×10(8) CFU gram(-1) of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks.     

Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.     

Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance

Vancomycin-resistant Enterococcus faecium spread dramatically in hospital settings in the USA in the 1990s and reached endemicity at the turn of the century. Similarly, rising prevalence rates are currently observed in several European countries, with prevalence rates of greater than 10% reported in seven of these. On the basis of multilocus sequence typing (MLST), the population structure of E. faecium was elucidated and the existence of a distinct high-risk enterococcal clonal complex, designated clonal complex-17 (CC17), which is associated with the majority of hospital outbreaks and clinical infections in five continents, was revealed. This complex is correlated with ampicillin and quinolone resistance and with the presence of a putative pathogenicity island. Preliminary MLST data suggest that similar hospital-adapted complexes might also exist in E. faecalis.     

天津地区气单胞菌分离株的鉴定与多位点序列分型

[目的]研究气单胞菌菌株分类情况,并分析其致病性.[方法]采集环境样品和鱼类标本,分离并鉴定气单胞菌菌株,并运用多位点序列分型(Multilocus sequence typing,MLST)方法进行分类研究,利用PCR和测序方法分析毒力基因Aera、Hly、Aha1、GCAT和Nuc的分布.[结果]通过对分离菌株的16S rRNA基因进行分析,确认属于4种不同气单胞菌的7个分离株.发现所有菌株至少有1种毒力基因阳性,其中3株具有4种毒力基因.药物敏感实验显示,6株分离株对3种或3种以上抗菌素具有多重耐药性.最后,对看家基因gyrB、groL、gltA、metG、ppsA和recA进行分析,与MLST数据库中的等位基因序列比对,发现7株分离株均为新的不同的序列型(Sequence type,ST).[结论]气单胞菌具有较高的遗传多样性.     

Population structure and evolution of pathogenicity of Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is an enteric human pathogen but is widespread in the environment. Pathogenicity is determined by a number of virulence factors, including the virulence plasmid pYV, the high-pathogenicity island (HPI), and the Y. pseudotuberculosis-derived mitogen (YPM), a superantigen. The presence of the 3 virulence factors varies among Y. pseudotuberculosis isolates. We developed a multilocus sequence typing (MLST) scheme to address the population structure of Y. pseudotuberculosis and the evolution of its pathogenicity. The seven housekeeping genes selected for MLST were mdh, recA, sucA, fumC, aroC, pgi, and gyrB. An MLST analysis of 83 isolates of Y. pseudotuberculosis, representing 19 different serotypes and six different genetic groups, identified 61 sequence types (STs) and 12 clonal complexes. Out of 26 allelic changes that occurred in the 12 clonal complexes, 13 were mutational events while 13 were recombinational events, indicating that recombination and mutation contributed equally to the diversification of the clonal complexes. The isolates were separated into 2 distinctive clusters, A and B. Cluster A is the major cluster, with 53 STs (including Y. pestis strains), and is distributed worldwide, while cluster B is restricted to the Far East. The YPM gene is widely distributed on the phylogenetic tree, with ypmA in cluster A and ypmB in cluster B. pYV is present in cluster A only but is sporadically absent in some cluster A isolates. In contrast, an HPI is present only in a limited number of lineages and must be gained by lateral transfer. Three STs carry all 3 virulence factors and can be regarded as high-pathogenicity clones. Isolates from the same ST may not carry all 3 virulence factors, indicating frequent gain or loss of these factors. The differences in pathogenicity among Y. pseudotuberculosis strains are likely due to the variable presence and instability of the virulence factors.     

Genetic diversity and safety aspects of enterococci from slightly fermented sausages

AIMS: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR and partial sequencing of 16S rRNA and sodA genes were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All the E. faecalis strains carried virulence genes (efaAfs, esp, agg and gelE) and all E. faecium isolates carried efaAfm gene. Enterococcus faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides) and E. gallinarum and E. casseliflavus/flavescens isolates showed vanC1 and vanC2/C3 genotypes (low-level resistance only to vancomycin) respectively. CONCLUSIONS: E. faecalis has been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed.     

Frequency of virulence genes and antibiotic resistances in Enterococcus spp. isolates from wastewater and feces of domesticated mammals and birds, and wildlife

Enterococci are gastrointestinal tract residents and also an important cause of nosocomial infections. To understand which species, virulence determinants, and antibiotic resistances are prevalent in enterococci shed by various hosts groups, a total of 1460 strains isolated from 144 fecal samples obtained from wastewater, domesticated mammals and birds, and wildlife were characterized. Identification of isolates to the species level showed that Enterococcus faecalis was dominant in domesticated mammals and birds and wildlife feces, whereas Enterococcus faecium was dominant among wastewater isolates, and that no single Enterococcus species could be associated with a specific host group. The frequency of 12 virulence determinants was evaluated among isolates, but no single virulence determinant could be associated with a specific host group. Resistance to 12 antibiotics was evaluated among isolates, and it was shown that the highest frequency of resistance at breakpoint concentration was found in domesticated mammals and birds (P?≤ 0.05 for 4 antibiotics). Our results suggests that (1) species identification and virulence typing of Enterococcus spp. isolates are not useful for the identification of the host groups responsible for fecal contamination of water by microbial source tracking and that (2) antibiotic use for clinical, veterinary, or animal husbandry practices is promoting resistance.     

Virulence characteristic and MLST-agr genetic background of high-level mupirocin-resistant, MRSA isolates from Shanghai and Wenzhou, China

The emergence and prevalence of high-level mupirocin-resistant, methicillin-resistant Staphylococcus aureus (MuH MRSA) is challenging the eradication of MRSA nasal carriage and the treatment of skin and soft tissue infections. To understand the potentially pathogenetic capacity and the genetic basis of MuH MRSA, it is important to have a detailed knowledge of the molecular traits of this organism. Fifty three MuH MRSA isolates were gathered from Shanghai (28 isolates) and Wenzhou (25 isolates) in China. These isolates, consisting of 27 different PFGE-SCCmec-spa patterns, were examined by PCR for 35 virulence genes and further typed using agr (accessory gene regulator) typing and MLST (multilocus sequence typing). All 53 strains were positive for the genes hlg/hlg variant and icaD, and negative for seb, sed, see, seh, eta, etb, hld, cap-5, and ACME-arcA. Compared with Wenzhou isolates, Shanghai isolates were more likely to carry seg (P?=?0.002) and several other genes which were not found in Wenzhou strains such as sec, sei, tst (P<0.001 each), and pvl (P?=?0.012), and less likely to contain sea (P<0.001), cna (P?=?0.031), and efb (P?=?0.045). MLST and agr typing showed that ST239-agr1, ST5-agr1, and ST239-agr2 were the common lineages in MuH MRSA isolates from these two different regions. Our results indicated that MuH MRSA strains from two different geographic regions of China have differences in distribution of some virulence genes, while their major MLST-agr genetic backgrounds were accordant.     

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species-specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans , Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti-Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.     

Comparative genomics of Escherichia coli strains causing urinary tract infections

The virulence determinants of uropathogenic Escherichia coli have been studied extensively over the years, but relatively little is known about what differentiates isolates causing various types of urinary tract infections. In this study, we compared the genomic profiles of 45 strains from a range of different clinical backgrounds, i.e., urosepsis, pyelonephritis, cystitis, and asymptomatic bacteriuria (ABU), using comparative genomic hybridization analysis. A microarray based on 31 complete E. coli sequences was used. It emerged that there is little correlation between the genotypes of the strains and their disease categories but strong correlation between the genotype and the phylogenetic group association. Also, very few genetic differences may exist between isolates causing symptomatic and asymptomatic infections. Only relatively few genes that could potentially differentiate between the individual disease categories were identified. Among these were two genomic islands, namely, pathogenicity island (PAI)-CFT073-serU and PAI-CFT073-pheU, which were significantly more associated with the pyelonephritis and urosepsis isolates than with the ABU and cystitis isolates. These two islands harbor genes encoding virulence factors, such as P fimbriae (pyelonephritis-associated fimbriae) and an important immunomodulatory protein, TcpC. It seems that both urovirulence and growth fitness can be attributed to an assortment of genes rather than to a specific gene set. Taken together, urovirulence and fitness are the results of the interplay of a mixture of factors taken from a rich menu of genes.     

Five genes encoding surface-exposed LPXTG proteins are enriched in hospital-adapted Enterococcus faecium clonal complex 17 isolates

Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.     

Antibiotic resistance and virulence factors among clinical and food enterococci isolated in Slovakia

The resistance to antibiotics and the distribution of virulence factors in enterococci isolated from traditional Slovak sheep cheese bryndza was compared with strains from human infections. The occurrence of 4 enterococcal species was observed in 117 bryndza-cheese isolates. The majority of strains were identified as E. faecium (76 %) and E. faecalis (23 %). Several strains of E. durans and 1 strain of E. hirae were also present. More than 90 % of strains isolated from 109 clinical enterococci were E. faecalis, the rest belonged to E. faecium. The resistance to 6 antimicrobial substances (ampicillin, ciprofloxacin, higher concentration of gentamicin, nitrofurantoin, tetracycline and vancomycin) was tested in clinical and food enterococci. A higher level of resistance was found in clinical than in food strains and E. faecium had a higher resistance than E. faecalis; no resistance to vancomycin was detected. The occurrence of 3 virulence-associated genes, cylA (coding for hemolysin), gelE (coding for gelatinase) and esp (coding for surface protein) was monitored. Differences were found in the distribution of cylA gene between clinical and bryndza-cheese E. faecalis strains; in contrast to clinical strains (45 %), cylA gene was detected in 22 % of food isolates. The distribution of 2 other virulence factors, gelE and esp, was not significantly different in the two groups of E. faecalis strains. cylA and gelE genes were not detected in E. faecium but more than 70 % of clinical E. faecium were positive for esp, even thought none of the 79 E. faecium cheese isolates contained this gene.