Multilocus variable number tandem repeat analysis as a tool to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A

Aims:  To identify variable number tandem repeat (VNTR)-containing loci, and to use multilocus VNTR (MLVA) to discern genetic relationships among strains of Yersinia enterocolitica biovar 1A isolated from diverse sources.
Methods and Results:  The whole genome sequence of Y. enterocolitica 8081 was analysed and eight VNTR loci with repeat sizes between 4 and 9 bp, and each containing more than four repeat copies were selected for MLVA typing of 88 strains of Y. enterocolitica . Of these, four loci were polymorphic and generated 26 MLVA genotypes among 81 strains of Y. enterocolitica biovar 1A. MLVA was found to be quite discriminatory (DI = 0·87). Cluster analysis and population modelling using minimum spanning tree (MST) clearly clustered Y. enterocolitica biovar 1A into two major groups.
Conclusions:  The MLVA is easy to perform and can be used to discern clonal relationship among strains of Y. enterocolitica . Also the phylogenetic relationships obtained with MLVA genotypes were in good agreement with those established by other typing methods.
Significance and Impact of the Study:  The MLVA method reported is relatively more discriminatory than the other genotyping methods and has the potential to be used as an epidemiological tool for the study of strains of Y. enterocolitica biovar 1A.     

Uptake and persistence of human associated Enterococcus in the mussel Mytilus edulis: relevance for faecal pollution source tracking

Aims:  Micro-organisms and molecular markers for microbial source tracking (MST) in coastal waters are often present at low numbers, and often exhibit significant variability in time and space. In this study, we investigated the uptake, accumulation, and persistence of human associated Enterococcus in the mussel Mytilus edulis .
Methods and Results:  The human associated molecular markers esp in Enterococcus faecium , and M66 in Enterococcus faecalis were targetted by PCR in seawater and mussel samples from coastal sites affected by sewage contamination. Both native mussels and mussels transplanted from pristine to polluted sites were included. The results showed that the esp and M66 markers were often not detectable in seawater whereas mussels were enriched in the markers. Human associated E. faecalis accumulated rapidly in M. edulis , and reached maximum levels after 4–6 h with concentration 30–300 times greater than in the surrounding seawater. Enterococcus faecalis retained in M. edulis showed a survival comparable to planktonic E. faecalis in seawater with half lives of 30 and 22 h, respectively. Human associated markers remained detectable for 120 h in M. edulis after faecal contamination.
Conclusions:  The study demonstrated that native and transplanted M. edulis can accumulate and retain human associated molecular markers relevant for MST.
Significance and Impact of the Study:  Mussels should be considered as additional targets in MST studies in coastal waters.     

Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates

Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants. The genus Enterococcus is of increased significance as a cause of nosocomial infections, and this trend is exacerbated by the development of antibiotic resistance. In this study, we investigated the incidence of known virulence determinants in starter, food, and medical strains of Enterococcus faecalis, E. faecium, and E. durans. PCR and gene probe strategies were used to screen enterococcal isolates from both food and medical sources. Different and distinct patterns of incidence of virulence determinants were found for the E. faecalis and E. faecium strains. Medical E. faecalis strains had more virulence determinants than did food strains, which, in turn, had more than did starter strains. All of the E. faecalis strains tested possessed multiple determinants (between 6 and 11). E. faecium strains were generally free of virulence determinants, with notable exceptions. Significantly, esp and gelE determinants were identified in E. faecium medical strains. These virulence determinants have not previously been identified in E. faecium strains and may result from regional differences or the evolution of pathogenic E. faecium. Phenotypic testing revealed the existence of apparently silent gelE and cyl genes. In E. faecalis, the trend in these silent genes mirrors that of the expressed determinants. The potential for starter strains to acquire virulence determinants by natural conjugation mechanisms was investigated. Transconjugation in which starter strains acquired additional virulence determinants from medical strains was demonstrated. In addition, multiple pheromone-encoding genes were identified in both food and starter strains, indicating their potential to acquire other sex pheromone plasmids. These results suggest that the use of Enterococcus spp. in foods requires careful safety evaluation.     

Prevalence of virulece-associated genes of Enterococcus faecalis clinical strains isolated from patients and volunteers

Enterococcus faecalis is an important cause of serious hospitals infections. Several E. faecalis putative virulence determinants have been identified. The aim of our study was to determine the prevalence of virulence factors among 180 strains of E. faecalis isolated from humans from different clinical sources in Poland. Tested strains were investigated for the presence of cylA, cylB, cylM, gelE, asal, esp, efaA and ace by using PCR method. Among all strains ace and efaA were most often detected. However, in opposite to strains obtained from faeces of volunteers, most of clinical strains carried esp (64,4% vs. 28,9%) and cylA (44,4% vs. 20%), cylB (41,5% vs. 20%), cylM (45,2% vs. 20%), respectively. Twenty different virulotype were represented by tested strains. Presence of all tested virulence determinants were the most frequently observed among clinical strains. There was no significant association between virulence factors and clinical source of isolation.     

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.     

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.     

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.     

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.     

Occurrence of virulence factors among human intestinal enterococcal isolates

AIMS: The aim of this study was to investigate the frequency of enterococcal virulence factors among human intestinal Enterococcus faecalis strains and to find out whether the pattern differs from that seen in published reports on food and clinical isolates. METHODS AND RESULTS: The E. faecalis isolates were cultured from human faecal samples obtained from five ulcerative colitis patients in remission phase. The species identification was based on API120 strips and species-specific PCR primers. The isolates were further characterized using the pulsed-field gel electrophoresis. The presence of seven different known enterococcal virulence factors among the confirmed E. faecalis isolates were screened using PCR techniques and published primers. CONCLUSIONS: Among the 35 isolates representing nine different pulsotypes the most frequent virulence factors were cpd (33 isolates), agg (25 isolates), gelE (22 isolates) and esp (15 isolates). No complete sets of genes associated for the production of functional cytolysin were encountered indicating that intestinal enterococci may differ in this respect from clinical strains. SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results, the commensal enterococcal strains appear to differ from clinical isolates in their complement of presumed virulence factors.     

Virulence factors in food,clinical and reference Enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannon's index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.     

Genetical conditioning of fluoroquinolone resistance mechanisms of clinical Enterococcus faecalis strains. I. Characterization of tested strains

The first part of the study presents the resistance profiles of 14 selected antibiotic agents for 180 clinical E. faecalis strains. Distribution of virulence factors for tested strains were characterized using PCR method. The results proved that clinical fluoroquinolone resistant E. faecalis strains possess MDR (multidrug-resistant) phenotype and presence of 7-8 tested virulence determinants.     

Vancomycin-resistant Enterococcus spp. in marine environments from the West Coast of the USA

Aims:  The aim of the study was to determine if vancomycin-resistant Enterococcus spp. [VRE] carrying vanA and/or vanB genes were present in public marine beaches and a fishing pier [2001–2003, 2008] from Washington and California [2008].
Methods:  PCR assays for the vanA and/or vanB genes with verification by DNA–DNA hybridization of the PCR products were used. Positive isolates were speciated using the BD BBL Crystal™ Identification and/or by sequencing the 16S ribosomal region.
Results:  Eighteen (8%) of 227 isolates including Enterococcus faecalis , Enterococcus faecium , Enterococcus casseliflavus/gallinarum and a Staphylococcus epidermidis carrying vanA and/or vanB genes, from four of six Washington and one of two California sites, were identified. Selected VRE and the S. epidermidis were able to transfer their van genes to an E. faecalis recipient at frequencies ranging from 1·9 × 10⁻⁶ to 6·7 × 10⁻⁹.
Conclusions:  Vancomycin-resistant Enterococcus spp. was isolated from five of the seven sites suggesting that other North America public beaches could be the reservoirs for VRE and should be assessed.
Significance & Impact of the Study:  This is the first report of isolation and characterization of VRE strains (and a vanB Staphylococcus sp.) from North American environmental sources suggesting that public beaches may be a reservoir for possible transmission of VRE to beach visitors.     

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.     

Enterococcus in wound infections: virulence and antimicrobial resistance

Enterococci, a complex group of facultative pathogens have become increasingly isolated in various hospital settings. They are considerable frequently cultured from traumatic and surgical wounds. We investigated 57 strains of the species E. faecalis, E. faecium and E. casseliflavus isolated from infected wounds. Their ability to produce virulence factors and their sensitivity to antibiotics were evaluated using phenotypic and genotyping methods. In the phenotype studies, significant portion of the isolates produced biofilm (66.7%) and gelatinase (36.8%). Nearly 30% of the strains expressed hemolytic properties. Only a few produced DNAse (15.8%) and lipase (7.0%). The genes esp, gelE, cylA, cylB, cylM and agg were detected in most of the isolates (38.6-87.7%). All the isolated enterococci were susceptible to vancomycin and were characterized by their low resistance to antibiotics, except aminoglycosides (HLR).     

Distribution and diversity of the enterococcal surface protein (esp) gene in animal hosts and the Pacific coast environment

Aims:  This study sought to evaluate the distribution of the enterococcal surface protein ( esp ) gene in Enterococcus faecium in the Pacific coast environment as well as the distribution and diversity of the gene in Northern California animal hosts.
Methods and Results:  Over 150 environmental samples from the Pacific coast environment (sand, surf zone, fresh/estuarine, groundwater, and storm drain) were screened for the esp gene marker in E. faecium , and the marker was found in 37% of the environmental samples. We examined the host specificity of the gene by screening various avian and mammalian faecal samples, and found the esp gene to be widespread in nonhuman animal faeces. DNA sequence analysis performed on esp polymerase chain reaction amplicons revealed that esp gene sequences were not divergent between hosts.
Conclusions:  Our data confirm recent findings that the E. faecium variant of the esp gene is not human-specific.
Significance and Impact of the Study:  Our results suggest that the use of the esp gene for microbial source tracking applications may not be appropriate at all recreational beaches.     

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization

Aims: To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1–3 and 8). Methods and Results: We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over‐representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic‐uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2_EDL933 or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993–2008. Conclusion: We observed that the tir‐255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. Significance and Impact of the Study: The detection of virulence clade‐specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations.     

Erythromycin resistance and virulence genes in Enterococcus faecalis from swine in China

This study aims to describe the erythromycin resistance phenotypes and genotypes, and the prevalence of virulence genes of Enterococcus faecalis isolated from swine in China. A total of 117 nonreplicate E. faecalis isolates, obtained from 502 clinical samples taken from different pig farms between 2007 and 2009 were included in the study. Minimum inhibitory concentrations were determined using the broth microdilution method. All of the isolates were screened for the presence of seven virulence genes (ace, asa1, cylA, efaA, esp, gelE, and hyl). In addition, the DNA from rythromycin-resistant isolates were amplified with primers specific for erythromycin resistance erm(A), erm(B), erm(C), mef(A/E), and msr(C) genes. Results show that erythromycin, tylosin, and ciprofloxacin resistance rates in E. faecalis were 66.67% (n=78), 66.67% (n=78), and 64.10% (n=75), respectively. About 69.23% of isolates (n=81) were positive for gelE, 48.72% (n=57) for ace, 15.38% (n=18) for efa, 7.69% (n=9) for asa1, and 6.84% (n=8) for esp. Among the erythromycin-resistant isolates, erm(B) (n=54) was the most prevalent resistance gene, followed by erm(A) (n=37). A significant correlation was found between the presence of the gelE virulence gene and erythromycin resistance (P<0.05). These findings suggest that enterococci from swine should be regarded with caution because they can be reservoirs for antimicrobial resistance and virulence genes.     

Detection of virulence factors in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolates from a Tunisian hospital

Phenotypic and genotypic determination of virulence factors were carried out in 46 high-level gentamicin-resistant (HLGR) clinical Enterococcus faecalis (n=34) and Enterococcus faecium (n=12) isolates recovered from different patients in La Rabta Hospital in Tunis, Tunisia, between 2000 and 2003 (all these isolates harboured the aac(6')-aph(2") gene). The genes encoding virulence factors (agg, gelE, ace, cylLLS, esp, cpd, and fsrB) were analysed by PCR and sequencing. The production of gelatinase and hemolysin, the adherence to caco-2 and hep-2 cells, and the capacity for biofilm formation were investigated in all 46 HLGR enterococci. The percentages of E. faecalis isolates harbouring virulence genes were as follows: gelE, cpd, and ace (100%); fsrB (62%); agg (56%); cylLLS (41.2%); and esp (26.5%). The only virulence gene detected among the 12 HLGR E. faecium isolates was esp (58%). Gelatinase activity was detected in 22 of the 34 E. faecalis isolates (65%, most of them with the gelE+-fsrB+ genotype); the remaining 12 isolates were gelatinase-negative (with the gelE+-fsrB- genotype and the deletion of a 23.9 kb fragment of the fsr locus). Overall, 64% of the cylLLS-containing E. faecalis isolates showed beta-hemolysis. A high proportion of our HLGR E. faecalis isolates, in contrast to E. faecium, showed moderate or strong biofilm formation or adherence to caco-2 and hep-2 cells.