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Mechanisms of clinical resistance to fluoroquinolones in Enterococcus faecalis.

About 10% of 100 clinical isolates of Enterococcus faecalis were resistant to greater than or equal to 25 micrograms of norfloxacin, ofloxacin, ciprofloxacin, and temafloxacin per ml. In this study, the DNA gyrase of E. faecalis was purified from a fluoroquinolone-susceptible strain (ATCC 19433) and two resistant isolates, MS16968 and MS16996. Strains MS16968 and MS16996 were 64- to 128-fold and 16- to 32-fold less susceptible, respectively, to fluoroquinolones than was ATCC 19433; MICs of nonquinolone antibacterial agents for these strains were almost equal. The DNA gyrase from ATCC 19433 had two subunits, designated A and B, with properties similar to those of DNA gyrase from other gram-positive bacteria such as Bacillus subtilis and Micrococcus luteus. Inhibition of the supercoiling activity of the enzyme from ATCC 19433 by the fluoroquinolones correlated with their antibacterial activities. In contrast, preparations of DNA gyrase from MS16968 and MS16996 were at least 30-fold less sensitive to inhibition of supercoiling by the fluoroquinolones than the gyrase from ATCC 19433 was. Experiments that combined heterologous gyrase subunits showed that the A subunit from either of the resistant isolates conferred resistance to fluoroquinolones. These findings indicate that an alteration in the gyrase A subunit is the major contributor to fluoroquinolone resistance in E. faecalis clinical isolates. A difference in drug uptake may also contribute to the level of fluoroquinolone resistance in these isolates.

Adenosine Triphosphate↗

[Adhesion capacity of Enterococcus faecium (SF 68) and Enterococcus faecalis to various substrates].

The author's studied the adherence ability of Enterococcus faecium SF 68 compared to that of Enterococcus faecalis IM 11f, on various substrates: vascular catheters and cardiac valves of rabbit, immunodepressed rats and diabetic rats. The bacterial adherence test was calculated by the number of bacteria adhered for microscopic field enlarged to 2000. The data obtained by scanning electron microscope SEM (Cambridge Stereoscan 150 MK2) revealed a different adherence action of the two germs on the various substrates. All the adherence tests showed a higher and uniform adherence activity of E. faecalis compared to that of E. faecium.

Animals↗

In vitro adhesion to human cells by viable but nonculturable Enterococcus faecalis.

The ability of viable but nonculturable (VBNC) Enterococcus faecalis to adhere to Caco-2 and Girardi heart cultured cells and to urinary tract epithelial cells (ECs) was studied. Enterococci were harvested during the vegetative growth phase (early exponential and stationary), in the VBNC state, and after recovery of the ability to divide. VBNC bacteria maintained their adherence capability but the efficiency of attachment was reduced by about 50 to 70%, depending on the target cell employed. The decrease was transient, since enterococci that regained their culturability showed adherence values similar to those observed for actively growing cells. Analysis of the invasive properties of E. faecalis revealed that the VBNC state caused a decrease in the number of bacteria that entered the cultured HEK cells as a result of the reduction in the number of adhering bacteria. These results highlight the importance of studies of the VBNC phenomenon, with respect to both microbial survival in the environment and the impact on human health.

Bacterial Adhesion↗

Citrate metabolism by Enterococcus faecalis FAIR-E 229.

Citrate metabolism by Enterococcus faecalis FAIR-E 229 was studied in various growth media containing citrate either in the presence of glucose or lactose or as the sole carbon source. In skim milk (130 mM lactose, 8 mM citrate), cometabolism of citrate and lactose was observed from the first stages of the growth phase. Lactose was stoichiometrically converted into lactate, while citrate was converted into acetate, formate, and ethanol. When de Man-Rogosa-Sharpe (MRS) broth containing lactose (28 mM) instead of glucose was used, E. faecalis FAIR-E 229 catabolized only the carbohydrate. Lactate was the major end product, and small amounts of ethanol were also detected. Increasing concentrations of citrate (10, 40, 70, and 100 mM) added to MRS broth enhanced both the maximum growth rate of E. faecalis FAIR-E 229 and glucose catabolism, although citrate itself was not catabolized. Glucose was converted stoichiometrically into lactate, while small amounts of ethanol were produced as well. Finally, when increasing initial concentrations of citrate (10, 40, 70, and 100 mM) were used as the sole carbon sources in MRS broth without glucose, the main end products were acetate and formate. Small amounts of lactate, ethanol, and acetoin were also detected. This work strongly supports the suggestion that enterococcal strains have the metabolic potential to metabolize citrate and therefore to actively contribute to the flavor development of fermented dairy products.

Cheese↗

Catabolite repression in Enterococcus faecalis.

Metabolism of citrate, pyruvate and sugars by Enterococcus faecalis E-239 and JH2-2 and an isogenic, catabolite derepressed mutant of JH2-2, strain CL4, was investigated. The growth rates of E. faecalis E-239 on citrate and pyruvate were 0.58 and 0.63 h(-1), respectively, indicating that both acids were used as energy sources. Fructose and glucose prevented the metabolism of citrate until all the glucose or fructose had been metabolised. Diauxie growth was not observed but growth on glucose and fructose was much faster than on citrate. In contrast, citrate was co-metabolized with galactose or sucrose and pyruvate with glucose. When glucose was added to cells growing on citrate, glucose metabolism began immediately but inhibition of citrate utilisation did not begin for approximately 1.5 h. Growth rates of E. faecalis JH2-2 and its isogenic, catabolite derepressed mutant, strain CL4, on citrate, were 0.41 and 0.36 h(-1), respectively. The catabolite derepressed mutant was able to co-metabolise citrate and glucose at all concentrations of glucose tested (3-25 mM), while its parent, could only metabolise citrate once all the glucose had been consumed. In strains JH2-2 and E-239, the growth rate on citrate decreased as the glucose concentration increased and, in 25 mM glucose, consumption of citrate was inhibited for several hours after glucose had been consumed. These results indicate that catabolite repression by glucose and fructose occurs in enterococci.

Carbohydrate Metabolism↗

Relationship between stress response toward bile salts, acid and heat treatment in Enterococcus faecalis.

Stress tolerance and cross-protection in Enterococcus faecalis ATCC19433 were examined after exposure to bile salts, acid or heat shock. Bile salts and heat adapted cells demonstrated induced homologous tolerance and cross-resistance. No cross-protection of heat adapted cells against acid stress is observed and pretreatment with bile salts even sensitized the cells to this challenge. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins. Some of these polypeptides are induced by more than one treatment. The greatest overlap is observed between bile salts and heat treatments. Eighteen stress proteins, including DnaK and GroEL, are common between these stresses.

Bile Acids and Salts↗

Selection of glycopeptide-resistant mutants of VanB-type Enterococcus faecalis BM4281 in vitro and in experimental endocarditis.

Enterococcus faecalis BM4281 is resistant to vancomycin, susceptible to teicoplanin (VanB phenotype), and intrinsically resistant to low levels of gentamicin. The efficacy of glycopeptides against BM4281 was investigated in a rabbit model of experimental endocarditis for reduction of bacterial counts in cardiac vegetations and selection of mutants with increased resistance to glycopeptides. Teicoplanin led to a 100-fold reduction of bacteria in the vegetations, whereas vancomycin had no effect. Monotherapy with either antibiotic selected mutants with homogeneous or heterogeneous resistance to high levels of both glycopeptides. Vancomycin also selected mutants that required the antibiotic for growth. The combination of gentamicin plus teicoplanin was bactericidal, prevented the emergence of mutants, and allowed sterilization of the vegetations in 25% of the rabbits, indicating that the combination may be an alternative if penicillin cannot be used against VanB-type enterococci.

Animals↗

Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis.

Biosynthesis of gelatinase, a virulence factor of Enterococcus faecalis, was found to be regulated in a cell density-dependent fashion in which its production is active in late log to early stationary phase. Addition of early stationary phase culture filtrate to medium shifted the onset of gelatinase production to that of mid-log phase, suggesting that E. faecalis secretes a gelatinase biosynthesis-activating pheromone (GBAP). GBAP was isolated from culture supernatant of E. faecalis OG1S-P. Structural analysis suggested GBAP to be an 11-residue cyclic peptide containing a lactone structure, in which the alpha-carboxyl group of the C-terminal amino acid is linked to a hydroxyl group of the serine of the third residue. A synthetic peptide possessing the deduced structure showed GBAP activity at nanomolar concentrations as did natural GBAP. Database searches revealed that GBAP corresponds to a C-terminal part of a 242-residue FsrB protein. Northern analysis showed that GBAP slowly induces the transcription of two operons, fsrB-fsrC encoding FsrB and a putative histidine kinase FsrC and gelE-sprE encoding gelatinase GelE and serine protease SprE. Strains with an insertion mutation in either fsrC or a putative response regulator gene fsrA failed to respond to GBAP, suggesting that the GBAP signal is transduced by a two-component regulatory system.

Amino Acid Sequence↗

Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic.

Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLS, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.

Amino Acid Sequence↗

Survey for virulence determinants among Enterococcus faecalis isolated from different sources.

A collection of Enterococcus faecalis strains from clinical isolates, healthy individuals and the environment was screened for the presence of virulence factor genes, such as those for collagen-binding protein (ace), endocarditis antigen (efaA), haemolysin activator (cylA), gelatinase (gelE), aggregation substances (asa1 and asa373), a surface protein (esp) and two novel putative surface antigens (EF0591 and EF3314). Apart from some genes that were present in all strains (ace, efaA and EF3314), the gelE gene was the most common factor, although its presence did not correlate with its expression. The genes that encode Esp and CylA were never detected in endocarditis isolates, whereas an association was noted between the esp gene and isolates from urinary tract infection (UTI) and bacteraemia. An aggregation substance gene was always present in commensal strains. As for gelatinase, the presence of the cylA and asa genes did not correlate completely with their phenotypic expression. Generally, isolates from endocarditis, biliary stents and the environment were equipped with fewer virulence factors than isolates from other sources. UTI strains possessed the highest number of factors.

Adhesins, Bacterial↗

Cloning and molecular analysis of genes affecting expression of binding substance, the recipient-encoded receptor(s) mediating mating aggregate formation in Enterococcus faecalis.

Transfer of the conjugative plasmid pCF10 in Enterococcus faecalis strains involves production of a plasmid-encoded aggregation substance on the surface of donor cells in response to stimulation by a pheromone secreted by recipient cells. Aggregation substance then facilitates attachment to recipient cells via a chromosomally encoded receptor, termed binding substance (BS). A BS mutant, strain INY3000, generated by random Tn916 insertions, was previously found to carry copies of the transposon at four unique sites (K. M. Trotter and G. M. Dunny, Plasmid 24:57-67, 1990). In the present study, DNA flanking the Tn916 insertions was used to complement the BS mutation of INY3000 following Tn916 excision from cloned chromosomal fragments. Complementation results showed that three of the four regions mutated in INY3000 play some role in BS expression. Tn5 mutagenesis and DNA sequence analysis of the complementing fragment from one of these regions indicated the presence of three genes (ebsA, ebsB, and ebsC) that affect BS expression. The ebsA and ebsB genes encode peptides likely to function in cell wall metabolism, whereas ebsC may encode a product that suppresses the function or expression of EbsB.

Amino Acid Sequence↗

Comparative genomic hybridization analysis of Enterococcus faecalis: identification of genes absent from food strains.

Enterococcus faecalis, a member of the natural microbiota of animal and human intestinal tracts, is also present as a natural contaminant in a variety of fermented foods. Over the last decade, E. faecalis has emerged as a major cause of nosocomial infections. We investigated the genetic diversity in 30 clinical and food isolates, including strains V583 and MMH594, in order to determine whether clinical and food isolates could be distinguished. Data were obtained using comparative genomic hybridization and specific PCR with a total of 202 probes of E. faecalis, selected using the available V583 genome sequence and part of the MMH594 pathogenicity island. The cognate genes encoded mainly exported proteins. Hybridization data were analyzed by a two-component mixture model that estimates the probability of any given gene to be either present or absent in the strains. A total of 78 genes were found to be variable, as they were absent in at least one isolate. Most of the variable genes were clustered in regions that, in the published V583 sequence, related to prophages or mobile genetic elements. The variable genes were distributed in three main groups: (i) genes equally distributed between clinical and dairy food isolates, (ii) genes absent from dairy food-related isolates, and (iii) genes present in MMH594 and V583 strains only. Further analysis of the distribution of the last gene group in 70 other isolates confirmed that six of the probed genes were always absent in dairy food-related isolates, whereas they were detected in clinical and/or commensal isolates. Two of them corresponded to prophages that were not detected in the cognate isolates, thus possibly extending the number of genes absent from dairy food isolates. Genes specifically detected in clinical isolates may prove valuable for the development of new risk assessment markers for food safety studies and for identification of new factors that may contribute to host colonization or infection.

DNA, Bacterial↗

Growth at high pH increases Enterococcus faecalis adhesion to collagen.

AIM: To evaluate the effect of growth at pH levels from 7.1 to 9.5 on the adherence of Enterococcus faecalis to bovine serum albumin (BSA) and collagen type I. METHODOLOGY: Enterococcus faecalis strain A197A was grown in broth of adjusted pHs varying between 7.1 and 9.5. Aliquots of bacterial suspensions were added to wells coated either with BSA or with collagen type I. Bacteria adhering to the surfaces were stained with crystal violet. Spectrophotometric measurements of the dissolved stain were used to assess the number of bacteria adhering to the surfaces. The data obtained were analysed using the Kolmogorov-Smirnov test, Levene's test and Student's t-test, with alpha = 0.05 as the level for statistical significance. RESULTS: The adhesion of E. faecalis to BSA-coated surfaces decreased inversely with alkalinity of the growth medium. The pH 7.1-grown bacteria bound to BSA significantly more than the other BSA groups. On the contrary, the adhesion to collagen type I-coated surfaces of bacteria grown at pH 8.0 and 8.5 was significantly greater than for those grown at pH 7.1. CONCLUSIONS: A minor increase in pH up to 8.5, which may be a consequence of insufficient treatment with alkaline medicaments such as calcium hydroxide, increases the collagen-binding ability of E. faecalis, in vitro. This can be a critical mechanism by which E. faecalis predominates in persistent endodontic infections.

Animals↗

Enterococcus faecium and Enterococcus faecalis bacteremia: acquisition and outcome.

The incidence of enterococcal bacteremia due to Enterococcus faecium is increasing. To understand the clinical significance of E. faecium bacteremia, we compared 16 patients who were bacteremic due to E. faecium to 56 patients who were bacteremic due to Enterococcus faecalis. E. faecium bacteremia developed most frequently in severely ill patients with fever or hypothermia accompanied by CNS, cardiovascular, and/or pulmonary dysfunction, while E. faecalis bacteremia occurred most often in less seriously ill patients. Nosocomial acquisition, cancer, neutropenia, renal insufficiency, current corticosteroid therapy, and previous treatment with broad-spectrum antibiotics were significantly more frequently associated with E. faecium bacteremia. Mortality was significantly higher among patients infected with E. faecium than among those infected with E. faecalis (50% vs. 11%; P = .001); this was true particularly among patients with monomicrobial or nosocomial bacteremia, those who had previously received antibiotic treatment, and those with cancer. Death due to enterococcal bacteremia was observed only among severely ill patients. These findings suggest that E. faecium often infects debilitated patients and that such infection appears to be a significant factor contributing to mortality.

Bacteremia↗

In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification.

Disruption of the previously described efaA (from Enterococcus faecalis antigen A) gene was generated in E. faecalis strain OG1RF and loss of an 37-kDa immunoreactive band from the mutant was demonstrated in Western blots. In a mouse peritonitis model, mice infected with the efaAfs (fs=from Enterococcus faecalis) mutant showed more prolonged survival than mice infected with the parent strain OG1RF. These results suggest that efaAfs encodes a function important for infection of mice by enterococci. An efaA-like gene was also identified in E. faecium DNA libraries and its deduced amino acid sequence showed 73% similarity to EfaA of E. faecalis and 42-63% similarities to a group of streptococcal virulence and adhesion associated proteins that are components of ATP-binding cassette transport systems. Intragenic probes representing efaAfs, recAfs, efaAfm (fm=from E. faecium) and gyrAfm were tested for their ability to identify E. faecalis and E. faecium using colony lysates of 133 enterococci and one Streptococcus sp. Probes of E. faecium and E. faecalis origin hybridized to all isolates of E. faecium and E. faecalis, respectively, regardless of their clinical source but not to any of 29 other enterococci. These results suggest that the use of gene probes may prove helpful in identification of isolates of E. faecium and E. faecalis.

Amino Acid Sequence↗

Glucose starvation response in Enterococcus faecalis JH2-2: survival and protein analysis.

We investigated the survival of Enterococcus faecalis following starvation provoked by energy source glucose exhaustion. Inhibition of protein synthesis by chloramphenicol before 3 h of starvation resulted in a dramatic decrease in viable bacteria. Antibiotic treatment of cells after 3 or 6 h of starvation had a progressively lesser influence on bacterial survival. During the first 24 h of deprivation, a total of 42 proteins were identified as glucose-starvation-inducible; 4 temporal classes of proteins (A, B, C and D) were defined in relation to their enhanced synthesis after glucose exhaustion. Our results show that proteins from the two early classes (A and B) seem to be the most important for long-term survival in E. faecalis. One protein of each of these classes was analysed at the molecular level. The N-terminal sequence of one of them, belonging to class A, showed strong homology with the N-terminal sequence of carbamate kinase from Streptococcus faecium. This enzyme could be implicated in the development of alternative metabolic pathways of energy production and could be compared to the Cst proteins of Escherichia coli.

Bacterial Proteins↗

Heterogeneric conjugal transfer of the pheromone-responsive plasmid pIP964 (IncHlyI) of Enterococcus faecalis in the apparent absence of pheromone induction.

Erythromycin-resistant derivatives of the pheromone-responsive plasmid pIP964 from Enterococcus faecalis were constructed to study its host range. This was done by inserting the integrative vector pAT112 and the related replicon pTCR1 harboring oriR of the broad host range plasmid pAM beta 1 into the hemolysin-bacteriocin operon of pIP964, to give pTCR2 and pTCR3, respectively. Plasmid pTCR2 was transferred by filter matings from E. faecalis to Enterococcus faecium and Listeria monocytogenes at frequencies of 2 x 10(-7) and 5 x 10(-7) per donor, respectively, in the apparent absence of pheromone induction and cellular aggregation. In these hosts, pTCR2 remained intact as a self-replicating element and maintained its transfer capabilities. Plasmid pTCR3, but not pTCR2, was transferred at similar frequencies from E. faecalis to Lactococcus lactis and Streptococcus agalactiae. Thus, the transfer system of pIP964 possesses a broader host-range than its replication system.

Cell Aggregation↗