AhrC and Eep are biofilm infection-associated virulence factors in enterococcus faecalis

Enterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species

Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY

This is the published version. Copyright 2007 American Society for MicrobiologyConjugative transfer of the Enterococcus faecalis plasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently

Introducción al muestreo de poblaciones y comunidades microbiológicas

Se identifican y comentan brevemente los principales tópicos de muestreo que pueden interesar a un investigador de poblaciones y comunidades microbiológicas naturales: definición de universo y unidades de estudio, determinación del tamaño de una muestra, definición de variables acerca de las cuales se desea efectuar estimaciones muestrales, factores de que depende la precisión de una estimación, diseños clásicos que otorgan alta “representatividad” a una muestra, importancia del azar en la selección muestral y limitaciones en las estimaciones proyectadas a partir de “miniunidades

Antibiotic resistance in the environment, with particular reference to MRSA

The introduction of β-lactam antibiotics (penicillins and cephalosporins) in the 1940s and 1950s probably represents the most dramatic event in the battle against infection in human medicine. Even before widespread global use of penicillin, resistance was already recorded. E. coli producing a penicillinase was reported in Nature in 1940 (Abraham, 1940) and soon after a similar penicillinase was discovered in Staphylococcus aureus (Kirby, 1944). The appearance of these genes, so quickly after the discovery and before the widespread introduction of penicillin, clearly shows that the resistance genes pre-dated clinical use of the antibiotic itself

Crossed immunoelectrophoresis from sodium dodecyl sulfate-polyacrylamide gels after fixation and staining and without nonionic detergent intermediate layers

A modified method is described for crossed immunoelectrophoresis in which the first-dimension separation has been carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The described method does not require nonionic detergents and is carried out after fixation and staining of the polyacrylamide gel. This permits more precise alignment of immunoprecipitates with polypeptide bands as well as allowing direct testing of an individual polypeptide band for reaction with antibody.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24266/1/0000531.pd

Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions

The prgB gene encodes the surface protein Asc10, which mediates cell aggregation resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis. Previous Tn5 insertional mutagenesis and sequencing analysis of a 12-kb fragment of pCF10 indicated that a region containing prgX, -Q, -R, -S, and -T, located 3 to 6 kb upstream of prgB, is required to activate the expression of prgB. Complementation studies showed that the positive regulatory region functions in cis in an orientation-dependent manner (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). In order to determine the involvement of each gene in the activation of prgB, Tn5 insertional mutagenesis and exonuclease III deletion analyses of the regulatory region were carried out. The results indicate that prgQ and -S are required for the expression of prgB, while prgX, -R, and -T are not required. Western blot (immunoblot) analysis of these mutants shows that prgQ is also essential for the expression of prgA (encoding the surface exclusion protein Sec10), which is located between prgB and the positive-control region. Complementation analysis demonstrates that a cis-acting regulatory element is located in the prgQ region and that pCF10 sequences in an untranslated region 3' from prgQ are an essential component of the positive-control system. Analyses of various Tn5 insertions in pCF10 genes suggest that transcription reading into this transposon is terminated in E. faecalis but that outward-reading transcripts may initiate from within the ends of Tn5 or from the junction sequences.</jats:p

Peptides as quorum sensing molecules : measurement techniques and obtained levels in vitro and in vivo

The expression of certain bacterial genes is regulated in a cell-density dependent way, a phenomenon called quorum sensing. Both Gram-negative and Gram-positive bacteria use this type of communication, though the signal molecules (auto-inducers) used by them differ between both groups: Gram-negative bacteria use predominantly N-acyl homoserine lacton (AHL) molecules (autoinducer-1, AI-1) while Gram-positive bacteria use mainly peptides (autoinducer peptides, AIP or quorum sensing peptides). These quorum sensing molecules are not only involved in the inter-microbial communication, but can also possibly cross-talk directly or indirectly with their host. This review summarizes the currently applied analytical approaches for quorum sensing identification and quantification with additionally summarizing the experimentally found in vivo concentrations of these molecules in humans

Enterococcal sex pheromone precursors are part of signal sequences for surface lipoproteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73811/1/j.1365-2958.2000.01687.x.pd

Acceleration of Enterococcus faecalis Biofilm Formation by Aggregation Substance Expression in an Ex Vivo Model of Cardiac Valve Colonization

Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10) protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10+ and Asc10− E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2–4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis

COMUNIDAD ESTACIONAL DE MICROHONGOS EN LA LITERA DE CONIFERAS DEL N.E. ARGENTINO: ENFASIS EN TAXA DE POTENCIAL INTERES MEDICO

Se analizó la colonización in vitro de microhongos filamentosos de rápido crecimiento en la litera de coníferas (Pinus elliottii) en el N.E argentino, mediante muestras estacionales de acículas senescentes de 4 localidadescon semejanzas climáticas, edáficas y vegetacionales. Veinte muestras (5 de cada lugar) se sembraron en agar papa dextrosa y agar agua (ambos adicionados con dichlorán) durante 21 días a t° ambiente, para enumerarla presencia relativa de su micota. Se contabilizó un total de 63 géneros fúngicos y 117 especies, representadas mayoritariamente por anamorfos de Ascomycota (85%), Ascomycota (9,9%) y Zygomycota (4,1%). Los hongos dematiáceos fueron dominantes (81,9%) y los 5 génerosmas frecuentes y constantes en las 4 estaciones y localidades, presentaron un variado comportamiento estacional, tales como: Cladosporium (31,8%), Alternaria (13,5%), Epicoccum (8,8%), Trichoderma (5,7%) y Fusarium (4,9%), con el 65% de la presencia total. Los géneros con mayor variedad de especies fueron: Curvularia (7), Fusarium (6), Phoma (6), Trichoderma (5) y Cladosporium (4). Las especies de interés médico fueron 52, con una presencia del 73,3% de la micota total. Las frecuencias más altas fueron: A.alternata, A.tenuisssima, Bipolaris cynodontis, C.cladosporiodes, C.oxysporum, Curvularia lunata, E. nigrum, F. semitectum, Nigrospora sphaerica, T. harzianum y T.viride