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B E Murray

Publications and source records attributed to B E Murray.

At least 19 recordsLinked to original sources
PubMed2003

Influence of Enterococcus faecalis proteases and the collagen-binding protein, Ace, on adhesion to dentin.

Enterococcus faecalis is a pathogen that persists in medicated root canals. Here, we tested the hypothesis that the E. faecalis proteases, serine protease and gelatinase, and the collagen-binding protein (Ace) contribute to adhesion to the root canal. Scanning electron microscopy was used to examine dentin binding by four E. faecalis strains: OG1RF, the wild type, and three mutant derivatives of OG1RF, TX5128, TX5243 and TX5256 deficient in serine protease and gelatinase, serine protease, and Ace, respectively. For each strain, 20 root halves were exposed to 3 x 10(9) to 5 x 10(9) cells/ml for 6 h, and 50 fields per root half were examined for adherent bacteria. Statistical analysis revealed that adherence of OG1RF was significantly greater than the mutant strains (P < 0.001), while significant differences were not detected between the protease mutants. The data indicate that serine protease and Ace aid E. faecalis binding to dentin, while the role of gelatinase is uncertain.

Analysis of Variance↗
PubMed2001

A simple model host for identifying Gram-positive virulence factors.

We demonstrate the use of the nematode Caenorhabditis elegans as a facile and inexpensive model host for several Gram-positive human bacterial pathogens. Enterococcus faecalis, Streptococcus pneumoniae, and Staphylococcus aureus, but not Bacillus subtilis, Enterococcus faecium, or Streptococcus pyogenes, kill adult C. elegans. Focusing our studies on the enterococcal species, we found that both E. faecalis and E. faecium kill C. elegans eggs and hatchlings, although only E. faecalis kills the adults. In the case of adults, a low inoculum of E. faecalis grows to a high titer in the C. elegans intestine, resulting in a persistent infection that cannot be eradicated by prolonged feeding on E. faecium. Interestingly, a high titer of E. faecium also accumulates in the nematode gut, but does not affect the longevity of the worms. Two E. faecalis virulence-related factors that play an important role in mammalian models of infection, fsr, a putative quorum-sensing system, and cytolysin, are also important for nematode killing. We exploit the apparent parallels between Gram-positive infection in simple and more complex organisms by using the nematode to identify an E. faecalis virulence factor, ScrB, which is relevant to mammalian pathogenesis.

Animals↗
PubMed2001

In vitro activity of 19 antimicrobial agents against enterococci from healthy subjects and hospitalized patients and use of an ace gene probe from Enterococcus faecalis for species identification.

We tested 165 enterococcal isolates, biased toward vancomycin resistant (VR) isolates, collected during recent years from fecal samples of healthy subjects and clinical specimens of hospitalized patients (mostly from United States and some from Europe) for susceptibility to 19 antimicrobials. Nosocomial isolates, whether VR or not, were more often highly resistant to aminoglycosides and clindamycin than fecal isolates from healthy community volunteers and more often resistant to erythromycin, chloramphenicol, trimethoprim, levofloxacin and, for E. faecium, ampicillin (93 vs. 0%). Resistance rates were similar between nosocomial and community-fecal isolates for minocycline, rifampin and quinupristin-dalfopristin (Q-D). None of the 165 enterococci tested hybridized with aph(2'')-Ic and aph(2'')-Id probes for recently described gentamicin resistance genes and 37 of the 39 isolates with high level resistance (HLR) to gentamicin hybridized with an intragenic aac(6')-aph(2'') probe. Of the two newer drugs tested, daptomycin MIC90s were 0.25 microg/mL for E. faecalis and 1 microg/mL for E. faecium, regardless of their vancomycin resistance level or source. For Q-D, none of 28 E. faecium from community based healthy subjects in the USA and 7 of 66 E. faecium from hospitalized patients in the United States were resistant. Among these 7 Q-Dr United States isolates and 7 Q-Dr isolates from Europe (MICs of Q-D of 4-8 microg/mL), none hybridized with vat(D) (formerly satA) and vat(E) (formerly satG) DNA probes, indicating the involvement of other mechanism/s of resistance in these isolates. We also demonstrated that an intragenic probe of the gene ace from E. faecalis showed specific hybridizations to all E. faecalis isolates, suggesting the usefulness of this gene for identification of this species.

Adhesins, Bacterial↗
PubMed2001

Disruption of an Enterococcus faecium species-specific gene, a homologue of acquired macrolide resistance genes of staphylococci, is associated with an increase in macrolide susceptibility.

The complete sequence (1,479 nucleotides) of msrC, part of which was recently reported by others using a different strain, was determined. This gene was found in 233 of 233 isolates of Enterococcus faecium but in none of 265 other enterococci. Disruption of msrC was associated with a two- to eightfold decrease in MICs of erythromycin azithromycin, tylosin, and quinupristin, suggesting that it may explain in part the apparent greater intrinsic resistance to macrolides of isolates of E. faecium relative to many streptococci. This endogenous, species-specific gene of E. faecium is 53% identical to msr(A), suggesting that it may be a remote progenitor of the acquired macrolide resistance gene found in some isolates of staphylococci.

Amino Acid Sequence↗
PubMed2001

Characterization of emeA, a NorA homolog and multidrug resistance efflux pump, in Enterococcus faecalis.

We hypothesized that multidrug resistance efflux pumps (MDRs) may be contributing to the drug resistance of enterococci. We recently identified potential MDR-encoding genes in the Enterococcus faecalis V583 genome. Among the putative MDRs, we found a gene that encodes a NorA homolog and have characterized this enterococcal MDR in the present study. A mutant from which the enterococcal NorA homolog has been deleted has reduced resistance to several NorA substrates. Complementation of the deletion mutant with the wild-type gene verified the involvement of this enterococcal gene in resistance to ethidium bromide (EtBr) and norfloxacin. Known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibit the efflux of EtBr and norfloxacin in wild-type strain OG1RF. A fluorescence assay with EtBr allowed us to quantitate the efflux capability of the enterococcal NorA pump. On the basis of these results, we have named this enterococcal gene emeA (enterococcal multidrug resistance efflux).

Amino Acid Sequence↗
PubMed2001

In vitro activities of a new ketolide, ABT-773, against multidrug-resistant gram-positive cocci.

The in vitro activities of ABT-773 were evaluated against 324 strains of gram-positive bacteria, including multidrug-resistant Staphylococcus spp. and Enterococcus spp. ABT-773 had lower MIC ranges, MICs at which 50% of isolates are inhibited (MIC(50)s), and MIC(90)s than erythromycin or clindamycin for almost all isolates tested. The MICs of ABT-773 were also lower than those of quinupristin-dalfopristin (Q-D) for methicillin-susceptible Staphylococcus aureus, Rhodococcus spp., and Streptococcus spp., while the MICs of Q-D were lower than those of ABT-773 for methicillin-resistant S. aureus and Enterococcus faecium, including vancomycin-resistant isolates.

Anti-Bacterial Agents↗
PubMed2001

Characterization of fsr, a regulator controlling expression of gelatinase and serine protease in Enterococcus faecalis OG1RF.

We have previously identified a locus, fsr, a homologue of staphylococcal agr loci, which positively regulates the expression of gelatinase and serine protease (encoded by gelE and sprE, respectively) in Enterococcus faecalis OG1RF. The expression of the three genes in the fsr locus, fsrA, fsrB, and fsrC, appears to be autoregulated, and we have shown that mutants with insertion disruptions in each of these three genes were significantly attenuated in a mouse peritonitis model compared to the parent strain. In the present study, we showed that fsrB and fsrC are highly expressed in the postexponential growth phase and that their expression is cell density dependent. Reverse transcriptase PCR using primers covering the intergenic regions in the fsr/gelE loci confirmed that fsrB and fsrC, as well as gelE and sprE, are cotranscribed. We also showed, using a nonpolar fsrB deletion mutant, that fsrB, the homologue of agrB of staphylococci with unknown function, is required for the regulatory function of fsr. Primer extension and analysis of transcriptional fusions indicated the presence of promoters immediately upstream of fsrA, of fsrB, and of gelE and that the fsrB and gelE promoters are fsr dependent, while the fsrA promoter is an fsr-independent weak constitutive promoter. Two conserved 7-bp direct repeats were found immediately upstream of the fsrB and gelE promoters, similar to the repeats found upstream of P2 and P3 promoters of the agr locus; deletions and mutations in the repeated sequences completely abolished the fsrB and gelE promoter activities, suggesting that the repeats are important for the regulatory function in the fsrB and gelE promoter regions.

Amino Acid Sequence↗
PubMed2001

Enterococcus faecalis multi-drug resistance transporters: application for antibiotic discovery.

Using bioinformatics approaches, 34 potential multidrug resistance (MDR) transporter sequences representing 4 different transporter families were identified in the unannotated Enterococcus faecalis database (TIGR). A functional genomics campaign generating single-gene insertional disruptions revealed several genes whose absence confers significant hypersensitivities to known antimicrobials. We constructed specific strains, disrupted in a variety of previously unpublished, putative MDR transporter genes, as tools to improve the success of whole-cell antimicrobial screening and discovery. Each of the potential transporters was inactivated at the gene level and then phenotypically characterized, both with single disruption mutants and with 2-gene mutants built upon a delta norA deleted strain background.

ATP-Binding Cassette Transporters↗
PubMed2000

Comparative analysis of the isoform expression pattern of Ca(2+)-regulatory membrane proteins in fast-twitch, slow-twitch, cardiac, neonatal and chronic low-frequency stimulated muscle fibers.

Although all muscle cells generate contractile forces by means of organized filament systems, isoform expression patterns of contractile and regulatory proteins in heart are not identical compared to developing, conditioned or mature skeletal muscles. In order to determine biochemical parameters that may reflect functional variations in the Ca(2+)-regulatory membrane systems of different muscle types, we performed a comparative immunoblot analysis of key membrane proteins involved in ion homeostasis. Cardiac isoforms of the alpha(1)-dihydropyridine receptor, Ca(2+)-ATPase and calsequestrin are also present in skeletal muscle and are up-regulated in chronic low-frequency stimulated fast muscle. In contrast, the cardiac RyR2 isoform of the Ca(2+)-release channel was not found in slow muscle but was detectable in neonatal skeletal muscle. Up-regulation of RyR2 in conditioned muscle was probably due to degeneration-regeneration processes. Fiber type-specific differences were also detected in the abundance of auxiliary subunits of the dihydropyridine receptor, the ryanodine receptor and the Ca(2+)-ATPase, as well as triad markers and various Ca(2+)-binding and ion-regulatory proteins. Hence, the variation in innervation of different types of muscle appears to have a profound influence on the levels and pattern of isoform expression of Ca(2+)-regulatory membrane proteins reflecting differences in the regulation of Ca(2+)-homeostasis. However, independent of the muscle cell type, key Ca(2+)-regulatory proteins exist as oligomeric complexes under native conditions.

Animals↗
PubMed2000

Vancomycin-resistant enterococci isolated from animals and food.

One hundred and one chicken products, boiled ham and turkey cold meat were acquired from 18 different supermarkets in Spain during October 1997 to June 1998 and were analyzed for vancomycin-resistant enterococci (VRE). In the same way, 50 intestinal chicken samples from a slaughterhouse were also studied. VRE were detected in 25 of 92 samples of food of chicken origin (27.2%), but no VRE were found in cooked pork or turkey products. VRE were also detected in 8 of 50 intestinal chicken samples from the slaughterhouse (16%). VRE were identified as Enterococcus durans (n = 11), Enterococcus faecalis (n = 10), Enterococcus faecium (n = 10) and Enterococcus hirae (n = 2). All these strains were characterized as belonging to the vanA genotype by polymerase chain reaction. Ampicillin, quinupristin/dalfopristin and high level aminoglycoside resistance were frequently found among these strains. Heterogeneity was observed in susceptibility patterns among VRE strains, even in those of the same species. The high rate of colonization of chicken products by vanA containing enterococci detected 6 months to 1 year after the banning of avoparcin as a growth promoter, supports other studies suggesting that the food chain could be a source of VRE colonization in humans and thus a source of VRE infections.

Animals↗
PubMed2000

Streptogramin resistance and shared pulsed-field gel electrophoresis patterns in vanA-containing Enterococcus faecium and Enterococcus hirae isolated from humans and animals in Spain.

The present study was performed to determine if any of the 45 vanA-containing Enterococcus faecium or 18 vanA-containing E. hirae strains were shared by chickens (32 E. faecium/l7 E. hirae) and humans (13 E. faecium/1 E. hirae) using pulsed field gel electrophoresis (PFGE) and to study quinupristin-dalfopristin (Q-D) resistance. Seven of the 45 E. faecium isolates (from 2 outpatients and from 5 poultry products) were resistant to Q-D (MIC > or = 16 microg/ml); one strain was shown to have satA by PCR and sequencing and, in the other six isolates, the recently described satG gene was demonstrated. Six different PFGE patterns were detected among the 7 Q-D E. faecium-resistant isolates. None of the E. hirae isolates showed Q-D resistance. Among the 45 vanA -containing E. faecium strains, 25 unrelated clones were found by PFGE with highly diverse patterns and an indistinguishable PFGE pattern was observed in vanA-containing E. faecium strains from two humans and two poultry products. A single PFGE pattern was detected in 17 of 18 vanA-containing E. hirae isolates, obtained from one human and 16 chicken samples. Based on the presence of indistinguishable PFGE patterns among VR E. faecium and E. hirae from humans and chickens, we conclude that horizontal transfer of these strains could occur between both groups.

Acetyltransferases↗
PubMed2000

Specific antibody promotes opsonization and PMN-mediated killing of phagocytosis-resistant Enterococcus faecium.

Many clinical isolates of Enterococcus faecium are resistant to neutrophil (PMN)-mediated phagocytosis and killing in the presence of normal human serum. We have now examined the ability of specific polyclonal rabbit antibodies to promote opsonization and killing of phagocytosis-resistant E. faecium. Immune rabbit serum generated against formalin-killed E. faecium TX0016, a phagocytosis-resistant strain, markedly promoted binding of TX0016 organisms to PMNs and PMN-mediated killing. These effects were dramatically reduced by (a) adsorption of immune serum with E. faecium TX0016, but not by adsorption with a strain of E. faecium susceptible to phagocytosis, and (b) incubation of immune serum with carbohydrate purified from TX0016, but not by incubation with a surface protein extract from TX0016. IgG purified from immune serum was unable by itself to promote bacterial binding to PMNs. However, specific IgG was able to promote binding to PMNs and PMN-mediated killing in the presence of normal human serum as a complement source, as were F(ab')(2) and Fab fragments produced from it, and the alternative pathway of complement was sufficient to promote IgG- and F(ab')(2)-mediated opsonization. PMN complement receptor type 3, but not complement receptor type 1, was involved in bacterial binding to PMNs induced by the combination of F(ab')(2) fragments and normal human serum. These results suggest that opsonization by antibodies potentially directed against bacterial carbohydrate, in conjunction with complement activation, has an important role in the host defense against phagocytosis-resistant E. faecium.

Animals↗
PubMed2000

Efficacy of telithromycin (HMR 3647) against enterococci in a mouse peritonitis model.

We used a mouse peritonitis model to evaluate the in vivo efficacy of telithromycin (HMR 3647) (TEL) and erythromycin (ERY) against four strains of Enterococcus faecalis and three strains of Enterococcus faecium with differing susceptibilities to TEL. TEL was highly active in vivo against Ery-susceptible (Ery(s)) and -intermediate (Ery(i)) strains (MIC of TEL = 0.015 to 0.062 microg/ml) and showed less efficacy against Ery-resistant (Ery(r)) isolates (MIC of TEL = 4 to 16 microg/ml), although this was overcome in part by a second subcutaneous dose. Quinupristin-dalfopristin was also noted to have less efficacy against Ery(r) versus Ery(s) or Ery(i) E. faecium strains, but this difference was reduced by intravenous administration. In conclusion, TEL was more potent in vivo against enterococci than was ERY; its activity was lowered by the presence of erm(B)-mediated Ery(r).

Animals↗
PubMed2000

Analysis of a gene cluster of Enterococcus faecalis involved in polysaccharide biosynthesis.

Previously, we described a gene cluster of Enterococcus faecalis OG1RF that produced an antigenic polysaccharide when cloned in Escherichia coli. The polysaccharide antigen was not detectable in E. faecalis strains, however. Here, we show by reverse transcriptase-PCR that the 16 genes in this region are transcribed in OG1RF. Gene disruption of orfde4, encoding a putative glycosyl transferase, and orfde6, a putative dTDP-rhamnose biosynthesis gene, generated two OG1RF mutants. The mutants showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. In addition, two mucoid E. faecalis isolates from patients with chronic urinary tract infections were found to produce the polysaccharide antigen.

Animals↗
PubMed2000

Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence.

Three agr-like genes (fsrA, fsrB, and fsrC, for Enterococcus faecalis regulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) of Enterococcus faecalis OG1RF. The deduced amino acid sequence of fsrA shows 26% identity and 38% similarity to Staphylococcus aureus AgrA (the response regulator of the accessory gene regulator system in the agr locus), FsrB shows 23% identity and 41% similarity to S. aureus AgrB, and FsrC shows 23% identity and 36% similarity to S. aureus AgrC (the sensor transducer of Agr system). Northern blot analysis suggested that gelE and sprE are cotranscribed and that fsrB and fsrC are also cotranscribed in OG1RF. Northern blot analysis of fsrA, fsrB, fsrC, gelE, and sprE insertion mutants showed that fsrB, fsrC, gelE, and sprE are not expressed in fsrA, fsrB, and fsrC mutants, while insertion in an open reading frame further upstream of fsrA did not effect the expression of these genes, suggesting that agr-like genes may be autoregulated and that they regulate gelE and sprE expression, as further confirmed by complementation of fsr gene mutations with a 6-kb fragment which contains all three fsr genes in the shuttle vector, pAT18. Testing of 95 other isolates of E. faecalis showed that 62% produced gelatinase (Gel(+)), while 91% (including all Gel(+) strains) hybridized to a gelE probe; 71% (including all Gel(+) strains) hybridized to an fsr probe, corroborating the conclusion that both gelE and fsr are necessary for gelatinase production. Testing of fsrA, fsrB, and sprE mutants in a mouse peritonitis model showed that sprE and agr-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for a gelE mutant. These results suggest that sprE and agr-like genes contribute to the virulence of E. faecalis OG1RF in this model.

Amino Acid Sequence↗
PubMed2000

Diversity of ace, a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of ace during human infections.

Our previous work reported that most Enterococcus faecalis strains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46 degrees C, but not 37 degrees C, and we subsequently identified an E. faecalis sequence, ace, that encodes a bacterial adhesin similar to the collagen binding protein Cna of Staphylococcus aureus. In this study, we examined the diversity of E. faecalis-specific ace gene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine E. faecalis strains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5' region of ace that encodes the A domain was sequenced, and these sequences showed > or =97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of S. aureus, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived from E. faecalis strain OG1RF detected Ace in mutanolysin extracts of seven of nine E. faecalis strains after growth at 46 degrees C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients with E. faecalis endocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other E. faecalis antigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an E. faecalis endocarditis patient serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to collagen types I and IV and laminin. In conclusion, these results show that ace is highly conserved among isolates of E. faecalis, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.

Amino Acid Sequence↗