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.     

Role of pathogenicity island-associated integrases in the genome plasticity of uropathogenic Escherichia coli strain 536

The genome of uropathogenic Escherichia coli isolate 536 contains five well-characterized pathogenicity islands (PAIs) encoding key virulence factors of this strain. Except PAI IV(536), the four other PAIs of strain 536 are flanked by direct repeats (DRs), carry intact integrase genes and are able to excise site-specifically from the chromosome. Genome screening of strain 536 identified a sixth putative asnW-associated PAI. Despite the presence of DRs and an intact integrase gene, excision of this island was not detected. To investigate the role of PAI-encoded integrases for the recombination process the int genes of each unstable island of strain 536 were inactivated. For PAI I(536) and PAI II(536), their respective P4-like integrase was required for their excision. PAI III(536) carries two integrase genes, intA, encoding an SfX-like integrase, and intB, coding for an integrase with weak similarity to P4-like integrases. Only intB was required for site-specific excision of this island. For PAI V(536), excision could not be abolished after deleting its P4-like integrase gene but additional deletion of the PAI II(536)-specific integrase gene was required. Therefore, although all mediated by P4-like integrases, the activity of the PAI excision machinery is most often restricted to its cognate island. This work also demonstrates for the first time the existence of a cross-talk between integrases of different PAIs and shows that this cross-talk is unidirectional.     

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.     

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.     

Molecular characterization of Enterococcus faecium isolated from hospitalized patients in Korea

AIMS: Multilocus sequence typing (MLST) was performed for vancomycin-resistant Enterococcus faecium (VREF) from diverse geographical areas in Korea to obtain insights into the genetic relationships with other molecular profiles. To understand the diversity of lineages, vancomycin-susceptible E. faecium (VSEF) were included. METHODS AND RESULTS: A total of 60 E. faecium isolates were analysed by MLST and esp profile. Molecular typing of Tn1546 of 30 VREF strains was evaluated by overlapping PCR of Tn1546 and DNA sequencing. Seven sequence types (ST) were found among 30 VSEF isolates, and four STs were found among 30 VREF isolates. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Of the 60 E. faecium isolates, 35 isolates were positive for the esp gene. On molecular typing of Tn1546, all VREF isolates were divided into four main types. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type represented different STs. CONCLUSIONS: An association between Tn1546 typing and MLST was not found. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the horizontal spread of Tn1546 between strains plays a major role in the dissemination of vancomycin resistance in Korea.     

Molecular analysis of the 21-kb bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a

The complete 21,344-bp DNA sequence of the bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a was determined. Thirty-four putative open reading frames which could code for proteins longer than 42 amino acids were found. Those included the structural genes encoding for the previously described bacteriocins enterocin I and J (also named as enterocins L50A and L50B). After comparison to sequences in public databases, analysis of the gene organization of pEF1 suggests a modular structure with three different functional domains: the replication region, the bacteriocin region and the mobilization plus UV-resistance region. This genetic mosaic structure most probably evolved through recombination events promoted by transposable elements. The hypothesis that the bacteriocin cluster on pEF1 could act as a functional plasmid stabilization module in E. faecium 6T1a is discussed.     

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.     

Relationship between biofilm formation, the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium

One-hundred and twenty-eight enterococcal isolates were examined for their ability to form biofilm in relation to the presence of the gene encoding the enterococcal surface protein (esp), production of gelatinase and to the source of isolation. Neither esp nor gelatinase seemed to be required for biofilm formation: both Enterococcus faecalis and Enterococcus faecium did not show a correlation between the presence of either esp or the production of gelatinase and biofilm formation. However, in E. faecium while esp was found in isolates from either source, the presence of both esp and biofilm together was only found in strains from clinical settings, suggesting that there exists a synergy between these factors which serves as an advantage for the process of infection.     

Describing ancient horizontal gene transfers at the nucleotide and gene levels by comparative pathogenicity island genometrics

MOTIVATION: Lateral gene transfer is a major mechanism contributing to bacterial genome dynamics and pathovar emergence via pathogenicity island (PAI) spreading. However, since few of these genomic exchanges are experimentally reproducible, it is difficult to establish evolutionary scenarios for the successive PAI transmissions between bacterial genera. Methods initially developed at the gene and/or nucleotide level for genomics, i.e. comparisons of concatenated sequences, ortholog frequency, gene order or dinucleotide usage, were combined and applied here to homologous PAIs: we call this approach comparative PAI genometrics. RESULTS: YAPI, a Yersinia PAI, and related islands were compared with measure evolutionary relationships between related modules. Through use of our genometric approach designed for tracking codon usage adaptation and gene phylogeny, an ancient inter-genus PAI transfer was oriented for the first time by characterizing the genomic environment in which the ancestral island emerged and its subsequent transfers to other bacterial genera.     

Contribution of the enterococcal surface protein Esp to pathogenesis of Enterococcus faecium endocarditis

The enterococcal surface protein Esp, specifically linked to nosocomial Enterococcus faecium, is involved in biofilm formation. To assess the role of Esp in endocarditis, a biofilm-associated infection, an Esp-expressing E. faecium strain (E1162) or its Esp-deficient mutant (E1162Δesp) were inoculated through a catheter into the left ventricle of rats. After 24 h, less E1162Δesp than E1162 were recovered from heart valve vegetations. In addition, anti-Esp antibodies were detected in Esp-positive E. faecium bacteremia and endocarditis patient sera. In conclusion, Esp contributes to colonization of E. faecium at the heart valves. Furthermore, systemic infection elicits an Esp-specific antibody response in humans.     

Emergence of CC17 Enterococcus faecium: from commensal to hospital-adapted pathogen

For many years, Enterococcus faecium was considered to be a commensal of the digestive tract, which only sporadically caused opportunistic infections in severely ill patients. Over the last two decades, vancomycin-resistant E. faecium (VREF) has emerged worldwide as an important cause of nosocomial infections, especially in immunocompromised patients. The global Vancomycin-resistant enterococci (VRE) epidemic was preceded by the emergence of ampicillin-resistant E. faecium (AREfm) in the United States in the early 1980s, followed by the rapid emergence of VRE in the 1990s. A similar increase of VRE may occur in countries with still low levels of VRE in hospitals (such as The Netherlands), but increasing incidence of AREfm infections. Molecular epidemiological studies of both human- and animal-derived E. faecium isolates using multilocus sequence typing revealed the existence of host-specific genogroups, including a specific genetic lineage designated CC17, associated with hospital-related isolates. These strains were characterized by ampicillin and quinolone resistance. In addition, the majority of these CC17 isolates contain over hundred hospital-clade-specific genes, including mobile elements, phage genes and plasmid sequences, hypothetical and membrane proteins and antibiotic and regulatory genes and a putative pathogenicity island including the esp gene.     

Enterococcal surface protein Esp does not facilitate intestinal colonization or translocation of Enterococcus faecalis in clindamycin-treated mice

Enterococcal surface protein (Esp) is a cell wall-associated protein of Enterococcus faecalis that has been identified as a potential virulence factor. We used a mouse model to examine whether Esp facilitates intestinal colonization or translocation of E. faecalis to mesenteric lymph nodes. After clindamycin treatment, similar levels of high-density colonization were established after orogastric inoculation of an E. faecalis isolate containing the esp gene within a large pathogenicity island and an isogenic mutant created by allelic replacement of the esp gene with a chloramphenicol resistance cassette (P=0.7); translocation to mesenteric lymph nodes was detected in 3 of 12 (25%) mice in both groups. Isogenic mutants of FA2-2 (a plasmid-free derivative of E. faecalis strain JH2) with or without the esp gene failed to establish colonization of clindamycin-treated mice. These results suggest that Esp does not facilitate intestinal colonization or translocation of E. faecalis.     

Determination of prevalence and economic impact of ovine footrot in central Kashmir India with isolation and molecular characterization of Dichelobacter nodosus

The present study determines the prevalence, economic impact of virulent footrot in central Kashmir, India, along with isolation and molecular characterization of Dichelobacter nodosus (D. nodosus) where so far no such work has been carried out. Over all 12.54% prevalence of footrot was recorded in central Kashmir with highest (15.84%) in district Srinagar, and least (10.89%) in district Budgam, while it was 13.28% in district Ganderbal. Overall economic impact of footrot was estimated to the tune of Rs 15.82 million annually to the sheep farming in central Kashmir. Out of 370 samples collected from footrot lesions of naturally infected sheep, 200 (54.05%) detected D. nodosus positive by polymerase chain reaction (PCR). Out of these, 132 (66.00%) samples carried serogroup B of D. nodosus, five (2.50%) serogroup E, one (0.50%) serogroup I, while, 53 (26.50%) had mixed infection of serogroups B and E, four (2.00%) of serogroups B and I, two (1.00%) of serogroups B and G and the remaining three (1.50%) samples harboured the mixed infection of serogroups B, E and I. Serogroup G was detected for the first time in India. Over all serogroup B was most frequent (97.0%) followed by E (30.5%), while serogoups I (4.0%) and G (1.0%) were least prevalent. A total of 265 D.nodosus strains were isolated out of which 194 (73.20%) were typed as serogroup B, 61 (23.01%) as serogroup E, eight (3.01%) as serogroup I and remaining two (0.75%) belonged to serogroup G. Out of 265 D. nodosus isolates, 164 (61.88%) possessed intA (integrase) gene, thus were considered as virulent strains. Serogroup wise intA gene was found in 121(62.37%) isolates of serogroup B, 36 (59.01%) of E, two (100%) of G and five (62.50%) of I. Out of 20 randomly selected isolates subjected to gelatin gel test, 16 isolates with intA gene produced thermostable protease while four isolates without intA gene revealed the production of thermolabile protease. This indicated a good co-relation between presence of intA gene and gelatin gel test in determination of the D. nodosus virulence. Thus the present investigation suggests the incorporation of serogroups B and E, based on their predominant prevalence, in the formulation of an effective bivalent vaccine to combat footrot in central Kashmir.     

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.     

Detection of human-derived fecal pollution in environmental waters by use of a PCR-based human polyomavirus assay

Regulatory agencies mandate the use of fecal coliforms, Escherichia coli or Enterococcus spp., as microbial indicators of recreational water quality. These indicators of fecal pollution do not identify the specific sources of pollution and at times underestimate health risks associated with recreational water use. This study proposes the use of human polyomaviruses (HPyVs), which are widespread among human populations, as indicators of human fecal pollution. A method was developed to concentrate and extract HPyV DNA from environmental water samples and then to amplify it by nested PCR. HPyVs were detected in as little as 1 microl of sewage and were not amplified from dairy cow or pig wastes. Environmental water samples were screened for the presence of HPyVs and two additional markers of human fecal pollution: the Enterococcus faecium esp gene and the 16S rRNA gene of human-associated Bacteroides. The presence of human-specific indicators of fecal pollution was compared to fecal coliform and Enterococcus concentrations. HPyVs were detected in 19 of 20 (95%) samples containing the E. faecium esp gene and Bacteroides human markers. Weak or no correlation was observed between the presence/absence of human-associated indicators and counts of indicator bacteria. The sensitivity, specificity, and correlation with other human-associated markers suggest that the HPyV assay could be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking "toolbox."     

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.     

Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates

The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.     

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm,a new member of the MSCRAMM family

A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.