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PubMed1999

Competitive polymerase chain reaction for quantification of nonculturable Enterococcus faecalis cells in lake water.

Among the survival strategies developed by bacteria when faced with adverse environmental conditions, the viable but nonculturable (VNC) state has been described. In this state, bacteria are unable to form colonies but are still alive and capable of metabolic activity. The VNC state has been described in numerous Gram-negative species, but recently also in Enterococcus faecalis, a Gram-positive species which can be found in the environment. In this study we describe a competitive PCR (cPCR) protocol to detect and quantify a specific sequence of DNA from culturable and nonculturable E. faecalis cells present in water samples. The protocol was found to be specific and capable of detecting amounts of DNA up to 0.1 pg corresponding to approximately 2 cells ml(-1). Moreover, it allows an internal standard to be used to quantify the amount of specific DNA present in samples from different environments. The application of this cPCR method to water samples from Lake Garda enabled us to demonstrate the presence of nonculturable forms of E. faecalis in lake water and to quantify their DNA and the corresponding concentration of nonculturable cells.

Journal Article↗
PubMed1989

Endocarditis due to streptomycin-susceptible Enterococcus faecalis with high-level gentamicin resistance.

A case of community-acquired endocarditis caused by Enterococcus (Streptococcus) faecalis with high-level resistance to gentamicin sulfate but not to streptomycin sulfate is described. Killing curves performed using achievable serum levels showed synergistic killing when streptomycin but not gentamicin, tobramycin, or amikacin was combined with penicillin G sodium or vancomycin hydrochloride. Combination therapy with vancomycin and streptomycin resulted in cure. Serum bactericidal levels indicated activity of the synergistic, as well as a nonsynergistic (vancomycin plus gentamicin), combination. Routine screening of blood isolates for high-level resistance to streptomycin and gentamicin can provide guidance for selection of therapeutic combinations in serious enterococcal infections, including endocarditis.

Aged↗
PubMed1989

Saliva-mediated aggregation of Enterococcus faecalis transformed with a Streptococcus sanguis gene encoding the SSP-5 surface antigen.

The interaction of a high-molecular-weight salivary glycoprotein (agglutinin) with Streptococcus sanguis M5 leads to the formation of bacterial aggregates. We have previously shown that the SSP-5 surface antigen from S. sanguis M5 binds the salivary agglutinin and therefore may be involved in the aggregation process. Here we report the transformation of a nonaggregating Enterococcus faecalis strain with the SSP-5 gene and show that the protein is expressed on the cell surface and confers an aggregation-positive phenotype. E. faecalis S161 protoplasts were transformed with pAM401 EB-5, a shuttle vector containing the S. sanguis SSP-5 gene, resulting in the isolation of E. faecalis S161EB-5. Crude cell extracts from this transformant and from S. sanguis M5 were analyzed by Western blotting. Extracts from S. sanguis M5 possessed peptides of 190 and 205 kilodaltons that reacted strongly with polyclonal antibodies against the recombinant SSP-5 antigen. E. faecalis S161EB-5 contained only the 190-kilodalton immunoreactive protein, suggesting that the antigen may be processed differently in E. faecalis S161EB-5. The parent strain, E. faecalis S161, did not react with this antibody preparation. Immunogold labeling of intact E. faecalis S161EB-5 and S. sanguis M5 with anti-SSP-5 immunoglobulin G showed that both organisms expressed similar levels of the antigen. Both organisms formed visible aggregates upon incubation with salivary agglutinin. These results suggest that the SSP-5 antigen may mediate both the binding of agglutinin to S. sanguis M5 and the subsequent formation of bacterial aggregates.

Agglutinins↗
PubMed1988

Evaluation of the Cobas Bact automated system for susceptibility testing of Enterobacteriaceae, Pseudomonas aeruginosa, and Enterococcus faecalis to azlocillin, mezlocillin, and ciprofloxacin compared to NCCLS and DIN standards.

The aim of automated susceptibility testing systems like the Cobas Bact is to provide the clinicians with rapid and reliable results for the care of patients and to decrease the work load in microbiological laboratories. Because data about accuracy on mezlocillin, azlocillin and ciprofloxacin were lacking, we investigated 184 bacterial strains and compared the results of the Cobas Bact susceptibility testing to standardized agar dilution and agar diffusion methods. Essential correlations for all methods compared exceeded 90% for the three chemotherapeutics and all species investigated, with the exception of Pseudomonas aeruginosa. On an average only 1.5% very major errors were observed with the several species of Enterobacteriaceae, whereas P. aeruginosa and Enterococcus faecalis were characterized by the complete absence of very major errors when Cobas Bact was correlated to NCCLS agar diffusion.

Autoanalysis↗
PubMed2004

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources.

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.

Anal Canal↗
PubMed1990

Genetic functions and cell-cell interactions in the pheromone-inducible plasmid transfer system of Enterococcus faecalis.

Pheromone-inducible plasmid transfer is a novel form of bacterial conjugation which has, to date, been observed only in Enterococcus (Streptococcus) faecalis. This process includes several important stages of interaction between the donor and recipient cell. The initial interaction is the transmission of a chemical signal from the recipient to the donor cell. Recent evidence has shown that the signal is in the form of a small hydrophobic peptide, which is capable of inducing a complex mating response in the donor cell at concentrations as low as 1-5 molecules per responder cell. Most E. faecalis strains produce multiple pheromones, each of which induces a response only in cells carrying a particular plasmid (or member of a family of related plasmids). Genetic functions ascribed to the pheromone response include: (i) cell-cell aggregation, which promotes initial close contact between mating cells; (ii) surface exclusion, which prevents plasmid transfer between aggregated donor cells; and (iii) highly efficient DNA transfer, which requires other unidentified functions in addition to aggregation. The first two processes appear to be mediated by proteinaceous surface antigens.

Amino Acid Sequence↗
PubMed1996

[Augmented ability of spleen cells to produce interferons and prevention from lethal infection of herpes simplex virus in mice orally treated with Enterococcus faecalis preparation, FK-23].

Effects of the oral or intraperitoneal administration of an Enterococcus preparation, FK-23, to mice on the interferon (IFN) production by their spleen cells and on the host defense against the infection with herpes simplex virus (HSV)-1 were examined. Spleen cells were obtained from the mice intraperitoneally treated with cyclophosphamide (CY) and subsequently orally administered FK-23 preparation, and then cultured with phytohemagglutinin-P or bacterial lipopolysaccharide in vitro. They produced higher titers IFN than those obtained from control mice which were not treated with the FK-23 preparation. The IFN activity was neutralized mainly by antiIFN-beta antibody. Correspondingly, oral (5 mg/mouse) or intraperitoneal (1 mg/mouse) administration of the FK-23 preparation protected some of the CY-pretreated mice from death by HSV-1 infection.

Administration, Oral↗
PubMed1993

Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: implications for their activity in vivo.

The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecalis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.

Aerobiosis↗