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B E Murray

Publications and source records attributed to B E Murray.

At least 55 records · Page 3Linked to original sources
PubMed1998

A cluster of genes involved in polysaccharide biosynthesis from Enterococcus faecalis OG1RF.

Our previous work identified a cosmid clone containing a 43-kb insert from Enterococcus faecalis OG1RF that produced a nonprotein antigen in Escherichia coli. In the present work, we studied this clone in detail. Periodate treatment of lysates of the clone confirmed that the antigen was carbohydrate in nature. Analysis of DNA sequences and transposon insertion mutants suggested that the insert contained a multicistronic gene cluster. Database comparison showed that the cluster contained genes similar to genes involved in the biosynthesis of dTDP-rhamnose, glycosyltransferases, and ABC transporters involved in the export of sugar polymers from both gram-positive and gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of the clone. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide.

Antigens, Bacterial↗
PubMed1998

Repetitive sequence-based PCR versus pulsed-field gel electrophoresis for typing of Enterococcus faecalis at the subspecies level.

Repetitive sequence-based PCR was compared to pulsed-field gel electrophoresis (PFGE) for the ability to discriminate Enterococcus faecalis isolates at the subspecies level. The BOXA2R primer, derived from repetitive sequences in Streptococcus pneumoniae, was applied to 41 isolates of E. faecalis collected from various sources. The REP1R-Dt and REP2-Dt primers, derived from the gram-negative repetitive extragenic palindromic element, were also applied to 18 selected isolates. Of the 41 isolates examined, 7 were beta-lactamase producing and 8 were vancomycin resistant. By PFGE, 17 isolates had distinct patterns; the other 24 were classified into eight different clonal groups. By PCR using the BOXA2R primer, 16 isolates generated distinct patterns; the other 25 were classified into nine different clonal groups. There were only minor differences in the PCR results obtained by using the BOXA2R primer and the REP1R-Dt and REP2-Dt primers. Two isolates among vancomycin-resistant enterococci from the greater Houston, Tex., area were related by PFGE, distinct by PCR with the BOXA2R primer, and related by PCR with the REP1R-Dt and REP2-Dt primers. Clonal relationships among the remaining 39 isolates were similar by both PFGE and PCR. PCR reliably discriminated all epidemiologically unrelated isolates. Although PCR is less time consuming than PFGE, PCR results were more difficult to interpret than PFGE results, perhaps because fewer bands were generated by PCR than by PFGE and some PCR products were inconsistently seen.

Bacterial Typing Techniques↗
PubMed1998

Excitation-contraction-relaxation cycle: role of Ca2+-regulatory membrane proteins in normal, stimulated and pathological skeletal muscle (review).

Extremely large protein complexes involved in the Ca2+-regulatory system of the excitation-contraction-relaxation cycle have been identified in skeletal muscle, i.e. clusters of the Ca2+-binding protein calsequestrin, apparent tetramers of Ca2+-ATPase pump units and complexes between the transverse-tubular alpha1-dihydropyridine receptor and ryanodine receptor Ca2+-release channel tetramers of the sarcoplasmic reticulum. While receptor interactions appear to be crucial for signal transduction during excitation-contraction coupling, avoidance of passive disintegration of junctional complexes and stabilization of receptor interactions may be mediated by disulfide-bonded clusters of triadin. Oligomerization of Ca2+-release, Ca2+-sequestration and Ca2+-uptake complexes appear to be an intrinsic property of these muscle membrane proteins. During chronic low-frequency stimulation, the expression of triad receptors is decreased while conditioning has only a marginal effect on Ca2+-binding proteins. In contrast, muscle stimulation induces a switch from the fast-twitch Ca2+-ATPase to its slow-twitch/cardiac isoform. These alterations in Ca2+-handling might reflect early functional adaptations to electrical stimulation. Studying Ca2+-homeostasis in transformed muscles is important regarding the evaluation of new clinical applications such as dynamic cardiomyoplasty. Studies of Ca2+-handling in skeletal muscle fibers have not only increased our understanding of muscle regulation, but have given important insights into the molecular pathogenesis of malignant hyperthermia, hypokalemic periodic paralysis and Brody disease.

Animals↗
PubMed1997

Cross-linking analysis of the ryanodine receptor and alpha1-dihydropyridine receptor in rabbit skeletal muscle triads.

In mature skeletal muscle, excitation-contraction (EC) coupling is thought to be mediated by direct physical interactions between the transverse tubular, voltage-sensing dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR) Ca2+ release channel of the sarcoplasmic reticulum (SR). Although previous attempts at demonstrating interactions between purified RyR and alpha1-DHPR have failed, the cross-linking analysis shown here indicates low-level complex formation between the SR RyR and the junctional alpha1-DHPR. After cross-linking of membranes highly enriched in triads with dithiobis-succinimidyl propionate, distinct complexes of more than 3000 kDa were detected. This agrees with numerous physiological and electron-microscopic findings, as well as co-immunoprecipitation experiments with triad receptors and receptor domain-binding studies. However, a distinct overlap of immunoreactivity between receptors was not observed in crude microsomal preparations, indicating that the triad complex is probably of low abundance. Disulphide-bonded, high-molecular-mass clusters of triadin, the junctional protein proposed to mediate interactions in triads, were confirmed to be linked to the RyR. Calsequestrin and the SR Ca2+-ATPase were not found in cross-linked complexes of the RyR and alpha1-DHPR. Thus, whereas recent studies indicate that the two receptors exhibit temporal differences in their developmental inductions and that receptor interactions are not essential for the formation and maintenance of triads, this study supports the signal transduction hypothesis of direct physical interactions between triad receptors in adult skeletal muscle.

Animals↗
PubMed1997

Vancomycin-resistant enterococci.

Enterococci have been recognized as an important cause of nosocomial infections for almost 20 years and as a cause of endocarditis for almost a century. While long known for their capacity of displaying multiple antibiotic resistant traits, the extent to which this could occur was not fully appreciated until the emergence of enterococci with acquired resistance to vancomycin; this resistance has been particularly problematic because it often occurs in the uncommon subset of enterococci that are also highly resistant to ampicillin-a combination with devastating therapeutic consequences. The observation that vancomycin resistance can be transferred to and expressed in other gram-positive organisms, for which vancomycin is often considered the primary therapeutic alternative, is a chilling reminder of just how close we may be to a wide array of potentially untreatable "killer" microbes.

Anti-Bacterial Agents↗
PubMed1997

The effect of antibiotic exposure on adherence to neutrophils of Enterococcus faecium resistant to phagocytosis.

Many clinical isolates of Enterococcus faecium are resistant to neutrophil-mediated phagocytosis and killing. As antibiotic exposure may alter bacterial surface properties and promote phagocytosis, we used a fluorescence microscopy assay to examine the effect of antibiotic pretreatment on the resistance to phagocytosis of six strains of E. faecium. Using two antimicrobial agents with good in-vitro activity against E. faecium, namely quinupristin/ dalfopristin and sparfloxacin, we found that exposure to quinupristin/dalfopristin at concentrations both below and above the MIC promoted bacterial adherence to neutrophils (PMNs) for all of three strains of vancomycin-susceptible E. faecium, while sparfloxacin was similarly effective in two of these three strains. In contrast, neither antibiotic was effective in promoting PMN adherence for three vancomycin-resistant strains of E. faecium. The variability amongst strains in response to antibiotic exposure suggests that either the mechanisms of resistance to phagocytosis, or its regulation, may be different amongst different strains of E. faecium.

Anti-Bacterial Agents↗
PubMed1997

Enterococcus faecalis antigens in human infections.

Genomic libraries of two Enterococcus faecalis strains, OG1RF and TX52 (an isolate from an endocarditis patient), were constructed in cosmid vectors pBeloBAC11 and pLAFRx, and screened with a serum from a rabbit immunized with surface proteins of an E. faecalis endocarditis isolate and sera from four patients with enterococcal endocarditis. Seventy-five cosmid clones reacted with at least two of the sera. Thirty-eight of the 75 immunopositive clones were considered to contain distinct inserts based on their DNA restriction patterns and were chosen for further subcloning into a pBluescript vector. Each sublibrary was screened with one of the five sera, and the DNA sequence of the immunopositive subclones was determined. Analysis of these sequences revealed similarities to a range of proteins, including bacterial virulence factors, transporters, two-component regulators, metabolic enzymes, and membrane or cell surface proteins. Fourteen subclones did not show significant similarity to any sequence in the databases and may contain novel genes. Thirteen of the immunopositive cosmid clones did not yield immunopositive subclones, and one such cosmid clone produced a nonprotein antigen in Escherichia coli.

Antibodies, Bacterial↗
PubMed1996

Serological investigation of enterococcal infections using western blot.

To assess the usefulness of Western blot in the diagnosis of enterococcal infections, a pilot study was conducted with a newly developed Western blot using sera from patients with confirmed enterococcal infections. Sera from 17 of 19 patients with enterococcal endocarditis reacted strongly to enterococcal antigens on the Western blot, and most produced specific bands at molecular weights 98 kDa and 54 kDa. Sera from patients with bacteremic cholangitis and pyelonephritis reacted frequently as well, but the pattern of bands was different from that observed with endocarditis. Eighty-five percent of 26 sera tested from patients with bacteremia and associated deep-seated infections (endocarditis, cholangitis, and pyelonephritis) were positive on Western blot, compared to 30% of sera from bacteremic patients with no clinically determined deep focus of infection (p < 0.001).

Antibodies, Bacterial↗
PubMed1996

Bactericidal activity of the fluoroquinolone DU-6859a alone and in combination with other antimicrobial agents against multiresistant enterococci.

The in vitro activity of DU-6859a (DU) alone and in combination with various antimicrobials was evaluated against multiresistant enterococci including some isolates with defined gyrA mutations. DU produced rapid in vitro killing against most enterococci that lacked resistance to ciprofloxacin, but it was not bactericidal against strains with MICs of ciprofloxacin of > or = 8 micrograms/ml, or against one of four strains with an MIC of ciprofloxacin of 4 micrograms/ml. The combination of DU with rifampin was antagonistic against two of two isolates tested. Combinations of DU and novobiocin, gentamicin, or a beta-lactam (amoxicillin, ampicillin-sulbactam, or amoxicillin-clavulanate) were generally indifferent. When different beta-lactams were used together, with or without DU, bactericidal activity was observed against some isolates. Despite the absence of synergistic interactions with other agents, DU is a promising fluoroquinolone for use against enterococci, although prior development of resistance to currently available fluoroquinolones diminishes some of its effect.

Anti-Bacterial Agents↗
PubMed1996

Influence of oral glycopeptides on the fecal flora of human volunteers: selection of highly glycopeptide-resistant enterococci.

Changes in fecal flora were evaluated in 22 healthy volunteers administered oral vancomycin or teicoplanin in 1989-1991 in Belgium. Evaluation of 5 colonies per subject revealed no glycopeptide-resistant enterococci in the predominant flora before glycopeptide administration; however, large numbers (mostly Enterococcus faecium) emerged by the end of the study in 14 (64%) of the subjects. Pediococci and lactobacilli also increased in number. In 1992, 40 healthy volunteers and 33 cancer patients were evaluated by plating stool samples directly onto selective media containing vancomycin; low numbers of vancomycin-resistant enterococci (< 50 cfu/g) were found in 11 (28%) of the 40 and 4 (12%) of the 33 samples, respectively. DNA restriction fragment length polymorphism analysis showed that most isolates were different, but all contained vanA in Tn1546-like elements. These results indicate that vanA and Tn1546-like elements were common in Belgium as early as 1989 and that community-based individuals in that location likely form a major reservoir for glycopeptide-resistant enterococci.

Adult↗
PubMed1996

Comparative in-vitro activity of the new fluoroquinolone trovafloxacin (CP-99,219) against gram-positive cocci.

The in-vitro activities of the new fluoroquinolone trovafloxacin (CP-99,219) and ciprofloxacin were determined against 225 Gram-positive cocci. Trovafloxacin was 4-32 fold more active than ciprofloxacin against staphylococci and streptococci and also showed greater activity against enterococci including vancomycin resistant Enterococcus faecium and Enterococcus faecalis that were highly resistant to aminoglycosides and/or produced beta-lactamase. Trovafloxacin was also bactericidal at 3 mg/L against enterococci except for isolates for which the MICs were > or = 2 mg/L.

Anti-Infective Agents↗
PubMed1996

Vancomycin-resistant enterococci from nosocomial, community, and animal sources in the United States.

The presence of vancomycin-resistant enterococci (VRE) was looked for in fecal samples from 104 healthy volunteers (3 with hospital exposure), 100 selected hospitalized patients, and various environmental sources (44 commercial chickens, 5 farm-raised chickens, 3 turkeys, and 2 chicken farm lagoon slurries). Five probiotic preparations were also studied. No VRE with vanA or vanB genes were isolated from the healthy volunteers without hospital exposure, environmental sources, or probiotic preparations. VRE with vanB were found in the stools of 16% of the high-risk hospitalized patients and in one volunteer with hospital contact. All VRE examined could be classified into one of two clones by pulsed-field gel electrophoresis. VRE from 11 of the colonized patients were quantified and ranged from 10(3) to 10(6) CFU/g of stool. This study, in contrast to findings in Europe, failed to find evidence of VanA- or VanB-type VRE in the community or environmental sources in Houston, Texas, and suggests that these settings are not a likely source of VRE in hospitals in this geographic area.

Adolescent↗
PubMed1996

Intracellular activities of RP 59500 (quinupristin-dalfopristin) and sparfloxacin against Enterococcus faecium.

RP 59500, a combination of the streptogramins quinupristin and dalfopristin, and sparfloxacin are new antibiotics with good in vitro activities against Enterococcus faecium, which is an increasingly important nosocomial pathogen with resistance to multiple antimicrobials. Since fluoroquinolones and related macrolides have displayed high intracellular concentrations inside host cells, we evaluated the intracellular activities of these agents inside neutrophils against three strains each of vancomycin-susceptible E. faecium (VSEF) and vancomycin-resistant E. faecium (VREF). At concentrations equal to four times the MIC, RP 59500 and sparfloxacin decreased the number of intracellular VSEF organisms, while both antibiotics were at best bacteriostatic against intracellular VREF strains. At concentrations equal to one-fourth of the MIC, both antibiotics were bacteriostatic against intracellular VSEF strains but were ineffective in inhibiting the growth of VREF strains. Despite their anticipated markedly higher intracellular human neutrophil (PMN) concentrations, RP 59500 and sparfloxacin activities in medium alone were equal to or greater than those inside PMNs against almost all strains. We conclude that the intracellular PMN concentrations of these antibiotics may not be directly related to their intracellular activities in our assay. The reason for the differences in their activities against VSEF versus VREF remains undefined.

Ampicillin↗
PubMed1996

Comparison of the beta-lactamase gene cluster in clonally distinct strains of Enterococcus faecalis.

Ten beta-lactamase-producing Enterococcus faecalis isolates were examined for the presence of the staphylococcal beta-lactamase repressor and antirepressor genes. Four isolates, previously shown to be unrelated to each other by pulsed-field gel electrophoresis analysis, were positive for both genes by PCR, although beta-lactamase production was not induced with methicillin. Six isolates, previously shown to be clonally related, were negative for both genes by PCR. The blaZ sequences of eight beta-lactamase-producing E. faecalis isolates were determined. Seven isolates from five distinct clones had sequences identical to that previously reported for E. faecalis HH22, regardless of whether the repressor or antirepressor was demonstrated by PCR. However, blaZ from one isolate differed from those of the other enterococci by 11 nucleotides; this isolate is part of the large clone, as defined by pulsed-field gel electrophoresis and multilocus enzyme analysis, that includes HH22. These findings suggest either that enterococci have acquired the bla gene cluster from more than one source or that the gene cluster has undergone considerable change since acquisition by this clone.

Amino Acid Sequence↗
PubMed1996

In vitro activity of the trinem sanfetrinem (GV104326) against gram-positive organisms.

The in vitro activity of the trinem sanfetrinem (formerly GV104326) (GV) was compared with that of vancomycin, ampicillin, and/or nafcillin against 287 gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and multiresistant enterococci, by the agar and microbroth dilution methods. GV demonstrated 2 to 16 times more activity than ampicillin and nafcillin against the majority of these organisms. The MIC range of GV was 16 to 64 micrograms/ml for 19 Enterococcus faecium strains that were highly resistant to ampicillin (ampicillin MIC range, 64 to 512 micrograms/ml) and vancomycin resistant and 0.25 to 32 micrograms/ml for resistant Rhodococcus spp. Similar activities (+/-1 dilution) were observed by either the agar or the broth microdilution method. GV demonstrated bactericidal activity against a beta-lactamase-producing Enterococcus faecalis strain and against two methicillin-susceptible Staphylococcus aureus strains in 10(5)-CFU/ml inocula. Synergy between GV and gentamicin was observed against an E. faecalis strain that lacked high-level gentamicin resistance. The activity of GV suggests this compound warrants further study.

Ampicillin↗