Planktonic Interference and Biofilm Alliance between Aggregation Substance and Endocarditis- and Biofilm-Associated Pili in Enterococcus faecalis.

Like many bacteria, Enterococcus faecalis encodes a number of adhesins involved in colonization or infection of different niches. Two well-studied E. faecalis adhesins, aggregation substance (AS) and endocarditis- and biofilm-associated pili (Ebp), both contribute to biofilm formation on abiotic surfaces and in endocarditis, suggesting that they may be expressed at the same time. Because different regulatory pathways have been reported for AS and Ebp, here, we examined if they are coexpressed on the same cells and what is the functional impact of coexpression on individual cells and within a population. We found that while Ebp are only expressed on a subset of cells, when Ebp and AS are expressed on the same cells, pili interfere with AS-mediated clumping and impede AS-mediated conjugative plasmid transfer during planktonic growth. However, when the population density increases, horizontal gene transfer rates normalize and are no longer affected by pilus expression. Instead, at higher cell densities during biofilm formation, Ebp and AS differentially contribute to biofilm development and structure, synergizing to promote maximal biofilm formation.IMPORTANCE Most bacteria express multiple adhesins that contribute to surface attachment and colonization. However, the network and relationships between the various adhesins of a single bacterial species are less well understood. Here, we examined two well-characterized adhesins in Enterococcus faecalis, aggregation substance and endocarditis- and biofilm-associated pili, and found that they exhibit distinct functional contributions depending on the growth stage of the bacterial community. Pili interfere with aggregation substance-mediated clumping and plasmid transfer under planktonic conditions, whereas the two adhesins structurally complement one another during biofilm development. This study advances our understanding of how E. faecalis, a ubiquitous member of the human gut microbiome and an opportunistic pathogen, uses multiple surface structures to evolve and thrive.

Molecular Characterization of Virulence Genes in Vancomycin-Resistant and Vancomycin-Sensitive Enterococci.

BACKGROUND: The aim of this study was to find out the correlation between presence of virulence (gelatinase [gel E], enterococcal surface protein [esp], cytolysin A [cyl A], hyaluronidase [hyl], and aggregation substance [asa1]) and vancomycin-resistant genes (van A and van B) in enterococci, with their phenotypic expression. MATERIALS AND METHODS: A total of 500 isolates (250 each clinical and fecal) were processed. Enterococci were isolated from various clinical samples and from fecal specimens of colonized patients. Various virulence determinants namely asa1, esp, hyl, gel E, and cyl were detected by phenotypic methods. Minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of virulence and van genes. RESULTS: Out of all the samples processed, 12.0% (60/500) isolates carried van A or van B genes as confirmed by MIC test and PCR methods. Genes responsible for virulence were detected by multiplex PCR and at least one of the five was detected in all the clinical vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE). gel E, esp, and hyl genes were found to be significantly higher in clinical VRE. Of the fecal isolates, presence of gel E, esp, and asa1 was significantly higher in VRE as compared to VSE. The presence of hyl gene in the clinical VRE was found to be statistically significant (P = 0.043) as against the fecal VRE. Correlation between the presence of virulence genes and their expression as detected by phenotypic tests showed that while biofilm production was seen in 61.1% (22/36) of clinical VRE, the corresponding genes, i.e., asa1 and esp were detected in 30.5% (11/36) and 27.8% (10/36) of strains only. CONCLUSION: Enterococcus faecium isolates were found to carry esp gene, a phenomenon that has been described previously only for Enterococcus faecalis, but we were unable to correlate the presence of esp with their capacity to form biofilms.

Virulence factors in vancomycin-resistant and vancomycin - susceptible Enterococcus faecalis from Brazil / Fatores de virulência em Enterococcus faecalis resistentes e suscetível à vancomicina isolados no Brasil

Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.

Enterococci são membros da microbiota comensal de animais e insetos, mas também são importantes patógenos oportunistas. Nosso objetivo foi observar se há qualquer diferença na virulência nos diversos grupos de Enterococcus faecalis, principalmente nos E. faecalis resistente à vancomicina (VREFS) isolados de colonização e infecção. VREFS e E. faecalis sensíveis à vancomicina (VSEFS) do Brasil foram pesquisadas quanto a presença de fatores de virulência. Ensaios fenotípicos foram usados para obter a expressão in vivo, entender o potencial patogênico destas amostras e determinar se existe correlação entre virulência e resistência a antibióticos. Diferentes perfis de virulência foram encontrados sugerindo que o clone que está se disseminado pode ter gerado diversas variações. No entanto, nosso estudo mostrou que um conjunto de fatores parece ser mais comum entre as amostras: gelatinase, substância de agregação e esp (GEA). Estes fatores tem sido correlacionados com a agregação de células e formação de biofilmes. A formação de biofilme pode promover a conjugação de plasmídeos contendo genes de resistência entre as espécies. Curiosamente, o perfil GAE não foi exclusivo para VREFS, foi o segundo mais observado em amostras VSEFS provenientes de colonização e infecção em pacientes hospitalizados e também de swabs retais de voluntários saudáveis. Tais linhagens pacerem representar a "porta de entrada" para novos genes de resistência em E. faecalis e podem contribuir para a disseminação de E. faecalis principalmente nos hospitais.

Virulence factors in vancomycin-resistant and vancomycin- susceptible Enterococcus faecalis from Brazil.

Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.