Regulation of the pAD1 sex pheromone response of Enterococcus faecalis by direct interaction between the cAD1 peptide mating signal and the negatively regulating, DNA-binding TraA protein

The Enterococcus faecalis conjugative plasmid pAD1 (60 kb) encodes a mating response to the recipient-produced peptide sex pheromone cAD1. The response involves two key plasmid-encoded regulatory proteins: TraE1, which positively regulates all or most structural genes relating to conjugation, and TraA, which binds DNA and negatively regulates expression of traE1. In vitro studies that included development of a DNA-associated protein-tag affinity chromatography technique showed that TraA (37.9 kDa) binds directly to cAD1 near its carboxyl-terminal end and, as a consequence, loses its affinity for DNA. Analyses of genetically modified TraA proteins indicated that truncations within the carboxyl-terminal 9 residues significantly affected the specificity of peptide-directed association/dissociation of DNA. The data support earlier observations that transposon insertions near the 3′ end of traA eliminated the ability of cells to respond to cAD1.

Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (from prgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.

Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation.

Conjugative transfer of the plasmid pCF10 by Enterococcus faecalis donor cells occurs in response to a peptide sex pheromone, cCF10, secreted by recipients. The plasmid-encoded cCF10 binding protein, PrgZ, is similar in sequence to binding proteins (OppAs) encoded by oligopeptide permease (opp) operons. Mutation of prgZ decreased the sensitivity of donor cells to pheromone, whereas inactivation of the chromosomal E. faecalis opp operon abolished response at physiological concentrations of pheromone. Affinity chromatography experiments demonstrated the interaction of the pheromone with several putative intracellular regulatory molecules, including an RNA molecule required for positive regulation of conjugation functions. These data suggest that processing of the pheromone signal involves recruitment of a chromosomal Opp system by PrgZ and that signaling occurs by direct interaction of internalized pheromone with intracellular effectors.

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Detecção de Enterococcus resistentes a vancomicina em criações comerciais de ovinos e caprinos das regiões centro-leste e nordeste do Estado de São Paulo

As exigências das condições higiênico-sanitárias na produção de animais de interesse zootécnico vêm aumentando progressivamente dada à necessidade de aliar-se produtividade a produtos de alta qualidade para atender a mercados consumidores cada vez mais exigentes. Nesse sentido, a utilização de antimicrobianos, tanto na profilaxia como na terapêutica, permanece como estratégia de controle para vários microrganismos patogênicos, de importância não apenas para a produção animal como também para a saúde humana, ainda que restrições ao uso indiscriminado desses produtos têm se intensificado. Não obstante, o uso excessivo desses produtos está associado à seleção de microrganismos resistentes nas áreas de produção. Por outro lado, investigações sobre circulação de cepas resistentes em rebanhos animais, até então restritas a populações humanas, ainda permanecem limitadas no Brasil. Bactérias do gênero Enterococcus, integrantes usuais da microbiota gastrointestinal animal e humana, são indicadoras ambientais de contaminação fecal e tem-se tornado objeto de preocupação em saúde pública e veterinária dada a ocorrência de cepas resistentes à vancomicina (VRE). O presente trabalho teve como objetivo isolar, quantificar e caracterizar VRE presentes em amostras fecais de ovinos oriundos de pequenas propriedades das regiões centro-leste e nordeste do estado de São Paulo. Para tanto, 132 amostras fecais foram coletadas diretamente do reto dos animais ou do piso das instalações. As amostras foram semeadas em ágar m-Enterococcus e subcultivadas em Ágar Bile Esculina acrescido de 6 µg/mL de vancomicina (ABEV), para confirmação de Enterococcus spp e detecção de cepas resistentes. Procedeu-se igualmente a observação da morfologia, características tintoriais, bioquímicas e moleculares. O número máximo de Enterococcus spp. encontrado foi de 2,6 × 105 e 1,70 × 105 UFC/g de fezes do ambiente e dos animais, respectivamente. Na caracterização bioquímica espécies mais prevalentes foram: Enterococcus faecalis e Vagococcus fluvialis. No ABEV, houve crescimento de colônias VRE em 33 das 84 amostras de ovinos-caprinos e em 21 das 48 amostras ambientais, representando, respectivamente 46,7% e 29,3% das amostras analisadas. A análise por multiplex PCR das 54 cepas VRE obtidas indicaram que 23 (43%), 22 (41%), 2 (3,5%) e 2 (3,5%) foram positivas, respectivamente, para os genes vanC2/C3, vanC1, vanA e vanB, sendo que para 5,3% dos isolados nenhum produto foi amplificado, sugerindo a possível ocorrência de genes dos demais grupos van conhecidos entre os isolados. Os resultados obtidos indicam, de forma inédita no país, a circulação de VRE em propriedades produtoras de ovinos e caprinos, sem ocorrência de manifestações clínicas aparentes nos animais, porém com possíveis riscos à saúde dos produtores e profissionais envolvidos, bem como a eventuais consumidores.

Surface properties of enterococcus faecalis in relation to infective endocarditis

The effect of growth conditions on both the appearance and the antigenic profile of cells of Enterococcus faecalis was investigated using electron micrographs of ruthenium red stained and sectioned cells and SDS-PAGE and blotting techniques respectively. Three specific antigens of molecular weights 73, 40 and 37 kdaltons were of particular interest being expressed most strongly after growth in serum. This medium was deemed to most closely mimic jn vjvo growth conditions reflecting an environment similar to that which the microorganisms would encounter during bacteraemia, preceding the colonisation of the endocardium and the development of infective endocarditis. The 40 and 37 kdalton antigens were shown by immunoqold labelling to be exposed on the surface of the cells although they did not appear to be connected with the fimbriae shown to exist on some of the E. faecalis cells examined by negative staining. The 73, 40 and 37 kdalton antigens were crudely purified using sarkosyl and ammonium sulphate precipitation, and used as the basis of a serodiagnostic test for E. faecalis endocarditis using an ELISA system. This was tested in a blind trial and the success rates were 94% for positives, 90% for negatives with endocarditis caused by other organisms and 80% for E. faecalis infections other than endocarditis. The binding of E.faecalis cells to the serum proteins fibronectin and albumin was investigated using 125I labelled proteins, followed by Scatchard analysis. This showed that· E.faecalis cells do loosely bind large amounts of both of these proteins, thus surely affecting the way in which the host's immune system perceives the cells. The E.faecalis receptor for fibronectin was partially characterised and appeared to involve protein and/or carbohydrate containing components. but did not involve LTA or the 40 and 37 kdalton species specific antigens.

Estudio in vitro de la efectividad de las distintas técnicas de irrigación en la eliminación del enterococcus faecalis

La irrigación es un paso indispensable en la desinfección del conducto radicular Con la que se pretende eliminar la capa residual, compuesta por restos orgánicos e inorgánicos, incluyendo microorganismos que podrían permanecer viables en su interior y ser la causa de reagudizaciones Está demostrado que la instrumentación rotatoria reduce el número de bacterias sólo en un 50%. Como consecuencia, se han empleado numerosos irrigantes a lo largo de los años para la desinfección del conducto radicular A día de hoy, no existe un irrigante que pueda efectuar por sí mismo una irrigación efectiva, por tanto se recurre a la combinación de irrigantes, utilizando uno como desinfectante y otro como quelante. Los más comunes son el hipoclorito sódico y el ácido etilendiaminotetraacético (EDTA). Existen zonas del sistema de conductos inaccesibles a la instrumentación, tanto manual como mecánica, por lo que además de los sistemas de irrigación convencionales, han surgido nuevos mecanismos del irrigación y agitación para que el irrigante alcance esas zonas El conocimiento de la flora bacteriana del interior de los conductos radiculares, ha contribuido a un aumento de la preocupación acerca de la capacidad de descontaminación de los mismos...

Antimicrobial resistance and species composition of Enterococcus spp. isolated from waters and sands of marine recreational beaches in Southeastern Brazil

Density, species composition and antimicrobial resistance in bacteria of the Enterococcus genus were evaluated in seawater and sands from 2 marine recreational beaches with different levels of pollution. The 2 beaches showed predominance of Enterococcus faecalis and Enterococcus faecium, in the water and the sand. Dry sand presented higher densities of Enterococcus sp. and higher frequency of resistant strains than wet sand and seawater. The beach with a higher degree of pollution presented higher percentages of resistant strains (66.7% and 61.5%, in sand and in water, respectively) and resistance to a larger number of antimicrobials compared with the less polluted beach, Ilha Porchat (35.7% and 31.25% of resistant strains in sand and water, respectively). in water samples, the highest frequencies of resistance were obtained against streptomycin (38.5%) and erythromycin (25%), whilst in sand, the highest frequencies were observed in relation to erythromycin and tetracycline (38.1% and 14.3%, respectively). These results show that water and sands from beaches with high indexes of faecal contamination of human origin may be potential sources of contamination by pathogens and contribute to the dissemination of bacterial resistance. (C) 2007 Elsevier Ltd. All rights reserved.

Estudio del efecto antimicrobiano del catdex sobre el desarrollo de Enterococcus faecalis

Las bacterias en infecciones endodónticas juegan un papel crucial para el éxito del tratamiento, a pesar de los avances en instrumentos y el gran efecto antimicrobiano del NaOCl existe evidencia científica que no se logra erradicar al 100% la carga bacteriana del sistema de conductos, debido a las condiciones anatómicas e irregularidades del conducto en ocasiones resulta imposible acceder a dichas zonas aunado a la patogenicidad de las bacterias. Actualmente en endodoncia se tiene la necesidad de investigar nuevos materiales irrigantes que se puedan utilizar para la irrigación del sistema de conductos, que sean capaces de brindarnos un mejor efecto antimicrobiano. El NaOCl es el irrigante por excelencia utilizado en endodoncia, desde los años XIX que se introdujo para su uso en la terapia pulpar. A pesar de su efecto antimicrobiano eliminando bacterias, hongos, esporas, virus y que además tiene la capacidad de disolver tejido orgánico y necrótico, sin embargo, también se caracteriza por ser una sustancia químicamente inestable, irritante y además caustico para las células. Una desventaja que tiene su uso durante el tratamiento de endodoncia, es el riesgo que se extruya a los tejidos periapicales y genere una reacción inflamatoria severa. El Cat Dex es un potente agente antimicrobiano para las bacterias patógenas que regularmente encontramos dentro de los conductos y con la ventaja que no resulta ser citotóxico ya que ha sido empleado anteriormente contra células tumorales.

Enterococcus faecalis prophage dynamics and contributions to pathogenic traits

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.

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

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

VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin

Three Enterococcus faecium strains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P(175)L, which suppressed the activity of the host Ddl D-Ala:D-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRS(B) operon that lowered the free energy of pairing from -13.08 to -6.65 kcal/mol, leading to low-level constitutive expression of the resistance genes from the P(RB) promoter, as indicated by analysis of peptidoglycan precursors and of VanX(B) D,D-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription, initiated from the P(YB) resistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the P(RB) regulatory promoter in strain BM4661, where it was superinducible. Strain BM4661 provides the first example of reversion to vancomycin resistance of a VanB-type dependent strain not due to a compensatory mutation in the ddl or vanS(B) gene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough of the resistance genes from the P(RB) promoter in the absence of vancomycin.

A Microleakage Study Of Gutta-percha/ah Plus And Resilon/real Self-etch Systems After Different Irrigation Protocols.

The development and maintenance of the sealing of the root canal system is the key to the success of root canal treatment. The resin-based adhesive material has the potential to reduce the microleakage of the root canal because of its adhesive properties and penetration into dentinal walls. Moreover, the irrigation protocols may have an influence on the adhesiveness of resin-based sealers to root dentin. The objective of the present study was to evaluate the effect of different irrigant protocols on coronal bacterial microleakage of gutta-percha/AH Plus and Resilon/Real Seal Self-etch systems. One hundred ninety pre-molars were used. The teeth were divided into 18 experimental groups according to the irrigation protocols and filling materials used. The protocols used were: distilled water; sodium hypochlorite (NaOCl)+eDTA; NaOCl+H3PO4; NaOCl+eDTA+chlorhexidine (CHX); NaOCl+H3PO4+CHX; CHX+eDTA; CHX+ H3PO4; CHX+eDTA+CHX and CHX+H3PO4+CHX. Gutta-percha/AH Plus or Resilon/Real Seal Se were used as root-filling materials. The coronal microleakage was evaluated for 90 days against Enterococcus faecalis. Data were statistically analyzed using Kaplan-Meier survival test, Kruskal-Wallis and Mann-Whitney tests. No significant difference was verified in the groups using chlorhexidine or sodium hypochlorite during the chemo-mechanical preparation followed by eDTA or phosphoric acid for smear layer removal. The same results were found for filling materials. However, the statistical analyses revealed that a final flush with 2% chlorhexidine reduced significantly the coronal microleakage. A final flush with 2% chlorhexidine after smear layer removal reduces coronal microleakage of teeth filled with gutta-percha/AH Plus or Resilon/Real Seal SE.