RESUMEN
Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra.
Asunto(s)
Bacteriófagos , Enterococcus faecium , Especificidad del Huésped , Enterococos Resistentes a la Vancomicina , Enterococcus faecium/efectos de los fármacos , Bacteriófagos/genética , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Terapia de Fagos/métodos , Infecciones por Bacterias Grampositivas/microbiología , Resistencia a la Vancomicina , Vancomicina/farmacología , Humanos , Antibacterianos/farmacologíaRESUMEN
Enterococcus cecorum, a commensal Gram-positive bacterium of the chicken gut, has emerged as a worldwide cause of lameness in poultry, particularly in fast-growing broilers. It is responsible for osteomyelitis, spondylitis, and femoral head necrosis, causing animal suffering, mortality, and antimicrobial use. Research on the antimicrobial resistance of E. cecorum clinical isolates in France is scarce, and epidemiological cutoff (ECOFF) values are unknown. To determine tentative ECOFF (COWT) values for E. cecorum and to investigate the antimicrobial resistance patterns of isolates from mainly French broilers, we tested the susceptibility of a collection of commensal and clinical isolates (n = 208) to 29 antimicrobials by the disc diffusion (DD) method. We also determined the MICs of 23 antimicrobials by the broth microdilution method. To detect chromosomal mutations conferring antimicrobial resistance, we investigated the genomes of 118 E. cecorum isolates obtained mainly from infectious sites and described previously in the literature. We determined the COWT values for more than 20 antimicrobials and identified two chromosomal mutations explaining fluoroquinolone resistance. The DD method appears better suited for detecting E. cecorum antimicrobial resistance. Although tetracycline and erythromycin resistances were persistent in clinical and nonclinical isolates, we found little or no resistance to medically important antimicrobials.
Asunto(s)
Antiinfecciosos , Enfermedades de las Aves de Corral , Animales , Antibacterianos/farmacología , Enfermedades de las Aves de Corral/microbiología , Pollos/microbiología , Farmacorresistencia Bacteriana , Pruebas de Sensibilidad MicrobianaRESUMEN
Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection.
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Macrófagos/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Simbiosis/fisiología , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismo , Sepsis/inmunología , Sepsis/microbiología , Infecciones Estafilocócicas/inmunología , Pez CebraRESUMEN
Transcription initiation and RNA processing govern gene expression and enable bacterial adaptation by reshaping the RNA landscape. The aim of this study was to simultaneously observe these two fundamental processes in a transcriptome responding to an environmental signal. A controlled σE system in E. coli was coupled to our previously described tagRNA-seq method to yield process kinetics information. Changes in transcription initiation frequencies (TIF) and RNA processing frequencies (PF) were followed using 5' RNA tags. Changes in TIF showed a binary increased/decreased pattern that alternated between transcriptionally activated and repressed promoters, providing the bacterial population with transcriptional oscillation. PF variation fell into three categories of cleavage activity: (i) constant and independent of RNA levels, (ii) increased once RNA has accumulated, and (iii) positively correlated to changes in TIF. This work provides a comprehensive and dynamic view of major events leading to transcriptomic reshaping during bacterial adaptation. It unveils an interplay between transcription initiation and the activity of specific RNA cleavage sites. This study utilized a well-known genetic system to analyze fundamental processes and can serve as a blueprint for comprehensive studies that exploit the RNA metabolism to decipher and understand bacterial gene expression control.
Asunto(s)
Adaptación Fisiológica , ARN Bacteriano/genética , ARN/genética , Iniciación de la Transcripción Genética , Escherichia coli , ARN/metabolismo , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Bacteriano/metabolismoRESUMEN
Enterococcus faecalis is an opportunistic pathogen with an intrinsically high resistance to lysozyme, a key effector of the innate immune system. This high level of resistance requires a complex network of transcriptional regulators and several genes (oatA, pgdA, dltA and sigV) acting synergistically to inhibit both the enzymatic and cationic antimicrobial peptide activities of lysozyme. We sought to identify novel genes modulating E. faecalis resistance to lysozyme. Random transposon mutagenesis carried out in the quadruple oatA/pgdA/dltA/sigV mutant led to the identification of several independent insertions clustered on the chromosome. These mutations were located in a locus referred to as the enterococcal polysaccharide antigen (EPA) variable region located downstream of the highly conserved epaA-epaR genes proposed to encode a core synthetic machinery. The epa variable region was previously proposed to be responsible for EPA decorations, but the role of this locus remains largely unknown. Here, we show that EPA decoration contributes to resistance towards charged antimicrobials and underpins virulence in the zebrafish model of infection by conferring resistance to phagocytosis. Collectively, our results indicate that the production of the EPA rhamnopolysaccharide backbone is not sufficient to promote E. faecalis infections and reveal an essential role of the modification of this surface polymer for enterococcal pathogenesis.
Asunto(s)
Antígenos de Superficie/inmunología , Enterococcus faecalis/patogenicidad , Infecciones por Bacterias Grampositivas/inmunología , Infecciones por Bacterias Grampositivas/microbiología , Muramidasa/inmunología , Polisacáridos/inmunología , Virulencia , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Enterococcus faecalis/genética , Enterococcus faecalis/inmunología , Infecciones por Bacterias Grampositivas/metabolismo , Muramidasa/metabolismo , Mutagénesis , Mutación , Polisacáridos/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/inmunología , Pez Cebra/microbiologíaRESUMEN
Surface properties like hydrophobicity, aggregation ability, adhesion to mucosal surfaces and epithelial cells and transit time are key features for the characterization of probiotic strains. In this study, we used two Lactobacillus paracasei subsp. paracasei strains (BGNJ1-64 and BGSJ2-8) strains which were previously described with very strong aggregation capacity. The aggregation promoting factor (AggLb) expressed in these strains showed high level of binding to collagen and fibronectin, components of extracellular matrix. The working hypothesis was that strains able to aggregate have an advantage to resist in intestinal tract. So, we assessed whether these strains and their derivatives (without aggLb gene) are able to bind or not to intestinal components and we compared the transit time of each strains in mice. In that purpose parental strains (BGNJ1-64 and BGSJ2-8) and their aggregation negative derivatives (BGNJ1-641 and BGSJ2-83) were marked with double antibiotic resistance in order to be tracked in in vivo experiments in mice. Comparative analysis of binding ability of WT and aggregation negative strains to different human intestinal cell lines and mucin revealed no significant difference among them, excluding involvement of AggLb in interaction with surface of intestinal cells and mucin. In vivo experiments showed that surviving and transit time of marked strains in mice did not drastically depend on the presence of the AggLb aggregation factor.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/microbiología , Intestinos/microbiología , Lacticaseibacillus paracasei/crecimiento & desarrollo , Lacticaseibacillus paracasei/fisiología , Unión Proteica , Animales , Adhesión Bacteriana/fisiología , Células CACO-2 , Moléculas de Adhesión Celular/fisiología , Colágeno/metabolismo , Fibronectinas/metabolismo , Células HT29 , Interacciones Microbiota-Huesped/fisiología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Ratones Endogámicos C57BL , Mucinas/metabolismo , Probióticos , Análisis de la Onda del Pulso , Propiedades de SuperficieRESUMEN
Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine-rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram-positive bacterial genomes have a unique SRR glycoprotein-encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface-exposed proteins - SrpA, SrpB and SrpC - that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases - GtfE/F - encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto-aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Mucosa Intestinal/microbiología , Glicoproteínas de Membrana/metabolismo , Streptococcus salivarius/patogenicidad , Animales , Adhesión Bacteriana/genética , Células Epiteliales/microbiología , Tracto Gastrointestinal/microbiología , Glucosiltransferasas/genética , Glicosilación , Humanos , Masculino , Ratones , Modelos Animales , Streptococcus salivarius/genética , Streptococcus salivarius/metabolismoRESUMEN
Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first snapshot of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5' RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites (TSSs) and 352 processing sites (PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding transcription start sites mapped. We discovered 85 novel putative regulatory RNAs, small- and antisense RNAs, and 72 transcriptional antisense organizations. Presented data constitute a significant insight into bacterial RNA landscapes and a step toward the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.
Asunto(s)
Regiones no Traducidas 5'/genética , Mapeo Cromosómico/métodos , Enterococcus faecalis/genética , ARN Bacteriano/genética , Análisis de Secuencia de ARN/métodos , Lugares Marcados de Secuencia , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Desnaturalización de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Procesamiento Postranscripcional del ARN , Sitio de Iniciación de la Transcripción , TranscriptomaRESUMEN
Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.
Asunto(s)
Enterococcus faecalis/genética , Interacciones Huésped-Patógeno/genética , Profagos/genética , Factores de Virulencia/genética , Activación Viral/genética , Cromosomas Bacterianos/genética , Infección Hospitalaria/genética , Enterococcus faecalis/patogenicidad , Genoma Bacteriano , Humanos , Profagos/metabolismo , Profagos/patogenicidad , Factores de Virulencia/metabolismoRESUMEN
Enterococcus faecalis is a commensal bacterium of the human intestine and a major opportunistic pathogen in immunocompromised and elderly patients. The pathogenesis of E. faecalis infection relies in part on its capacity to colonize the gut. Following disruption of intestinal homeostasis, E. faecalis can overgrow, cross the intestinal barrier, and enter the lymph and bloodstream. To identify and characterize E. faecalis genes that are key to intestinal colonization, our strategy consisted in screening mutants for the following phenotypes related to intestinal lifestyle: antibiotic resistance, overgrowth, and competition against microbiota. From the identified colonization genes, epaX encodes a glycosyltransferase located in a variable region of the enterococcal polysaccharide antigen (epa) locus. We demonstrated that EpaX acts on sugar composition, promoting resistance to bile salts and cell wall integrity. Given that EpaX is enriched in hospital-adapted isolates, this study points to the importance of the epa variability as a key determinant for enterococcal intestinal colonization.
Asunto(s)
Antígenos de Superficie/metabolismo , Enterococcus faecalis/crecimiento & desarrollo , Enterococcus faecalis/metabolismo , Intestinos/microbiología , Polisacáridos/metabolismo , Animales , Antígenos de Superficie/genética , Ácidos y Sales Biliares/genética , Ácidos y Sales Biliares/metabolismo , Pared Celular/genética , Pared Celular/metabolismo , Farmacorresistencia Microbiana , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidad , Genes Bacterianos , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Masculino , Ratones , Microbiota/genética , Polisacáridos/genética , Ramnosa/metabolismoRESUMEN
BACKGROUND: Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function. RESULTS: In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model. CONCLUSIONS: Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.
Asunto(s)
Proteínas Bacterianas/genética , Enterococcus faecalis/fisiología , Operón , Peritonitis/microbiología , Fagocitosis , Animales , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidad , Regulación Bacteriana de la Expresión Génica , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Macrófagos/metabolismo , Ratones , VirulenciaRESUMEN
We discovered a chromosomal locus containing 2 toxin-antitoxin modules (TAs) with an antisense transcriptional organization in the E. faecalis clinical isolate V583. These TAs are homologous to the type I txpA-ratA system and the type II mazEF, respectively. We have shown that the putative MazF is toxic for E. coli and triggers RNA degradation, and its cognate antitoxin MazE counteracts toxicity. The second module, adjacent to mazEF, expresses a toxin predicted to belong to the TxpA type I family found in Firmicutes, and the antisense RNA antidote, RatA. Genomic analysis indicates that the cis-association of mazEF and txpA-ratA modules has been favored during evolution, suggesting a selective advantage for this TA organization in the E. faecalis species. We showed regulatory interplays between the 2 modules, involving transcription control and RNA stability. Remarkably, our data reveal that MazE and MazEF have a dual transcriptional activity: they act as autorepressors and activate ratA transcription, most likely in a direct manner. RatA controls txpA RNA levels through stability. Our data suggest a pivotal role of MazEF in the coordinated expression of mazEF and txpA-ratA modules in V583. To our knowledge, this is the first report describing a crosstalk between type I and II TAs.
Asunto(s)
Antitoxinas/genética , Toxinas Bacterianas/genética , Enterococcus faecalis/genética , ARN sin Sentido/genética , Secuencia de Aminoácidos , Proteínas de Unión al ADN/genética , Endorribonucleasas/genética , Enterococcus faecalis/patogenicidad , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Estabilidad del ARN/genéticaRESUMEN
The Enterococcus faecalis leucine-rich protein ElrA promotes virulence by stimulating bacterial persistence in macrophages and production of the interleukin-6 (IL-6) cytokine. The ElrA protein is encoded within an operon that is poorly expressed under laboratory conditions but induced in vivo. In this study, we identify ef2687 (renamed elrR), which encodes a member of the Rgg (regulator gene for glucosyltransferase) family of putative regulatory proteins. Using quantitative reverse transcription-PCR, translational lacZ fusions, and electrophoretic mobility shift assays, we demonstrate that ElrR positively regulates expression of elrA. These results correlate with the attenuated virulence of the ΔelrR strain in a mouse peritonitis model. Virulence of simple and double elrR and elrA deletion mutants also suggests a remaining ElrR-independent expression of elrA in vivo and additional virulence-related genes controlled by ElrR.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecalis/metabolismo , Enterococcus faecalis/patogenicidad , Regulación Bacteriana de la Expresión Génica/fisiología , Operón/fisiología , Animales , Proteínas Bacterianas/genética , Enterococcus faecalis/genética , Regulación Bacteriana de la Expresión Génica/genética , Ratones , Operón/genética , Virulencia/genética , Virulencia/fisiologíaRESUMEN
Enterococcus faecalis is an opportunistic pathogen responsible for a wide range of life-threatening nosocomial infections, such as septicemia, peritonitis, and endocarditis. E. faecalis infections are associated with a high mortality and substantial health care costs and cause therapeutic problems due to the intrinsic resistance of this bacterium to antibiotics. Several factors contributing to E. faecalis virulence have been identified. Due to the variety of infections caused by this organism, numerous animal models have been used to mimic E. faecalis infections, but none of them is considered ideal for monitoring pathogenesis. Here, we studied for the first time E. faecalis pathogenesis in zebrafish larvae. Using model strains, chosen isogenic mutants, and fluorescent derivatives expressing green fluorescent protein (GFP), we analyzed both lethality and bacterial dissemination in infected larvae. Genetically engineered immunocompromised zebrafish allowed the identification of two critical steps for successful establishment of disease: (i) host phagocytosis evasion mediated by the Epa rhamnopolysaccharide and (ii) tissue damage mediated by the quorum-sensing Fsr regulon. Our results reveal that the zebrafish is a novel, powerful model for studying E. faecalis pathogenesis, enabling us to dissect the mechanism of enterococcal virulence.
Asunto(s)
Modelos Animales de Enfermedad , Enterococcus faecalis/patogenicidad , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/patología , Pez Cebra/microbiología , Animales , Animales Modificados Genéticamente , Evasión Inmune , Huésped Inmunocomprometido , Infecciones Oportunistas/microbiología , Infecciones Oportunistas/patología , Análisis de Supervivencia , Virulencia , Imagen de Cuerpo EnteroRESUMEN
Enterococcus faecalis is a commensal bacterium and a major opportunistic human pathogen. In this study, we combined in silico predictions with a novel 5'RACE-derivative method coined '5'tagRACE', to perform the first search for non-coding RNAs (ncRNAs) encoded on the E. faecalis chromosome. We used the 5'tagRACE to simultaneously probe and characterize primary transcripts, and demonstrate here the simplicity, the reliability and the sensitivity of the method. The 5'tagRACE is complementary to tiling arrays or RNA-sequencing methods, and is also directly applicable to deep RNA sequencing and should significantly improve functional studies of bacterial RNA landscapes. From 45 selected loci of the E. faecalis chromosome, we discovered and mapped 29 novel ncRNAs, 10 putative novel mRNAs and 16 antisense transcriptional organizations. We describe in more detail the oxygen-dependent expression of one ncRNA located in an E. faecalis pathogenicity island, the existence of an ncRNA that is antisense to the ncRNA modulator of the RNA polymerase, SsrS and provide evidences for the functional interplay between two distinct toxin-antitoxin modules.
Asunto(s)
Enterococcus faecalis/genética , ARN sin Sentido/genética , ARN no Traducido/genética , Análisis de Secuencia de ARN , Toxinas Bacterianas/genética , Secuencia de Bases , Secuencia Conservada , Enterococcus faecalis/metabolismo , Sitios Genéticos , Estrés Oxidativo , Péptidos/genética , ARN sin Sentido/análisis , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN no Traducido/análisis , ARN no Traducido/metabolismo , Lugares Marcados de SecuenciaRESUMEN
Enterococcus faecalis is an established nosocomial pathogen, yet the pathogenesis of enterococcal infections, particularly of urinary tract infections (UTIs), remains to be fully elucidated. Fibronectin-binding proteins have been identified as potent adhesins in pathogenic Gram-positive cocci. Here, we characterized EfbA, which is encoded by the enterococcal orthologue of Streptococcus pneumoniae pavA. Similar to PavA, the anchorless EfbA protein was localized to the enterococcal cell outer surface and bound to immobilized human fibronectin. In addition to abrogated EfbA expression, deletion of the efbA gene eliminated EfbA from the cell surface and drastically reduced the enterococcal cell binding to immobilized fibronectin. The ΔefbA deletion mutant was highly attenuated vs wild-type in a murine ascending UTI model, consistent with an increased tropism for the kidney relative to the bladder. These results provide the first evidence that EfbA of E. faecalis plays a role in UTIs, probably contributing to the pathogenesis in this site.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecalis/patogenicidad , Infecciones por Bacterias Grampositivas/microbiología , Infecciones Urinarias/microbiología , Animales , Adhesión Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Enterococcus faecalis/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/fisiología , Immunoblotting , Proteínas de la Membrana/metabolismo , Ratones , Microscopía Inmunoelectrónica , Unión Proteica , Proteínas Recombinantes , VirulenciaRESUMEN
Enterococcus cecorum is a member of the normal poultry gut microbiota and an emerging poultry pathogen. Some strains are resistant to key antibiotics and coccidiostats. We evaluated the impact on chicken excretion and persistence of a multidrug-resistant E. cecorum of administering narasin or antibiotics. E. cecorum CIRMBP-1294 (Ec1294) is non-wild-type to many antimicrobials, including narasin, levofloxacin, oxytetracycline and glycopeptides, it has a low susceptibility to amoxicillin, and carries a chromosomal vanA operon. Six groups of 15 chicks each were orally inoculated with Ec1294 and two groups were left untreated. Amoxicillin, oxytetracycline or narasin were administered orally to one group each, either at the recommended dose for five days (amoxicillin, oxytetracycline) or continuously (narasin). Faecal samples were collected weekly and caecal samples were obtained from sacrificed birds on day 28. Ec1294 titres were evaluated by culture on vancomycin- and levofloxacin-supplemented media in 5 % CO2. For inoculated birds given narasin, oxytetracycline or no antimicrobials, vancomycin-resistant enterococci were searched by culture on vancomycin-supplemented media incubated in air, and a PCR was used to detect the vanA gene. Ec1294 persisted in inoculated chicks up to day 28. Compared to the control group, the Ec1294 titre was significantly lower in the amoxicillin- and narasin-receiving groups on days 21 and 28, but was unexpectedly higher in the oxytetracycline-receiving group before and after oxytetracycline administration, preventing a conclusion for this group. No transfer of the vanA gene to other enterococci was detected. Other trials in various experimental conditions should now be conducted to confirm this apparent absence of co-selection of the multi-drug-resistant E. cecorum by narasin or amoxicillin administration.
Asunto(s)
Antibacterianos , Oxitetraciclina , Animales , Antibacterianos/farmacología , Vancomicina , Pollos , Oxitetraciclina/farmacología , Levofloxacino , Amoxicilina/farmacologíaRESUMEN
Enterococcus cecorum is an emerging pathogen responsible for osteomyelitis, spondylitis, and femoral head necrosis causing animal suffering and mortality and requiring antimicrobial use in poultry. Paradoxically, E. cecorum is a common inhabitant of the intestinal microbiota of adult chickens. Despite evidence suggesting the existence of clones with pathogenic potential, the genetic and phenotypic relatedness of disease-associated isolates remains little investigated. Here, we sequenced and analyzed the genomes and characterized the phenotypes of more than 100 isolates, the majority of which were collected over the last 10 years from 16 French broiler farms. Comparative genomics, genome-wide association studies, and the measured susceptibility to serum, biofilm-forming capacity, and adhesion to chicken type II collagen were used to identify features associated with clinical isolates. We found that none of the tested phenotypes could discriminate the origin of the isolates or the phylogenetic group. Instead, we found that most clinical isolates are grouped phylogenetically, and our analyses selected six genes that discriminate 94% of isolates associated with disease from those that are not. Analysis of the resistome and the mobilome revealed that multidrug-resistant clones of E. cecorum cluster into a few clades and that integrative conjugative elements and genomic islands are the main carriers of antimicrobial resistance. This comprehensive genomic analysis shows that disease-associated clones of E. cecorum belong mainly to one phylogenetic clade. IMPORTANCE Enterococcus cecorum is an important pathogen of poultry worldwide. It causes a number of locomotor disorders and septicemia, particularly in fast-growing broilers. Animal suffering, antimicrobial use, and associated economic losses require a better understanding of disease-associated E. cecorum isolates. To address this need, we performed whole-genome sequencing and analysis of a large collection of isolates responsible for outbreaks in France. By providing the first data set on the genetic diversity and resistome of E. cecorum strains circulating in France, we pinpoint an epidemic lineage that is probably also circulating elsewhere that should be targeted preferentially by preventive strategies in order to reduce the burden of E. cecorum-related diseases.
Asunto(s)
Antiinfecciosos , Enfermedades de las Aves de Corral , Animales , Aves de Corral , Pollos , Estudio de Asociación del Genoma Completo , FilogeniaRESUMEN
The concomitant presence of a complete fsr quorum-sensing system and gelE-sprE operons in Enterococcus faecalis is known to be essential for the detection of gelatinase activity. However, there are reports of the absence of gelatinase activity despite the presence of complete fsr and gelE loci. In order to understand this incongruence between genotype and phenotype we sequenced fsr and gelE loci of the E. faecalis LN68 strain, which was previously found to carry both operons but to lack gelatinase activity. Of the 59 nucleotide differences detected compared with the gelatinase-positive V583 strain, we found a nonsense mutation (a premature STOP codon) predicted to truncate the ATPase sensor domain of the FsrC protein, responsible for sensing and transducing the signal from the quorum-sensing molecule. Strain LN68 was highly affected in the expression of the gelE and sprE genes, further supporting the lack of Fsr-dependent gelE induction. When we constructed a V583 mutant with the same premature stop mutation in the fsrC gene the resulting strain was no longer able to degrade gelatin. We conclude that the reduced ability to transduce the quorum-sensing signal of the prematurely truncated FsrC protein is sufficient to explain the negative gelatinase phenotype. As the incongruent genotype and phenotype is detected in natural isolates, we believe that the silencing of the quorum-sensing system Fsr may be beneficial for some E. faecalis strains.
Asunto(s)
Proteínas Bacterianas/metabolismo , Enterococcus faecalis/enzimología , Gelatinasas/metabolismo , Lactonas/metabolismo , Péptidos Cíclicos/metabolismo , Percepción de Quorum , Transducción de Señal , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Enterococcus faecalis/genética , Enterococcus faecalis/fisiología , Gelatinasas/genética , Regulación Bacteriana de la Expresión Génica , Genotipo , Datos de Secuencia Molecular , FenotipoRESUMEN
Enterococcus faecalis is an opportunistic pathogen responsible for nosocomial infections. Lipoproteins in Gram-positive bacteria are translocated across the plasma membrane and anchored by the fatty acid group. They perform critical roles, with some described as virulence determinants. The aim of this study was to explore the roles of E. faecalis lipoproteins in the stress response and virulence. We constructed a mutant affected in the predicted prolipoprotein diacylglyceryl transferase gene lgt, and examined the role of Lgt in membrane anchoring, growth, the stress response and virulence. Inactivation of lgt enhanced growth in a high concentration of Mn(2+) or under oxidative stress in vitro, and significantly decreased virulence.