Biomedical subjects

B E Murray

Publications and source records attributed to B E Murray.

At least 91 records · Page 5Linked to original sources

PubMed1994

Analysis by PCR and direct DNA sequencing of gyrA mutations associated with fluoroquinolone resistance in Enterococcus faecalis.

A region of gyrA, the gene encoding subunit A of DNA gyrase, that is known to be associated with resistance was amplified and sequenced from 16 Enterococcus faecalis and Enterococcus faecium isolates. Six ciprofloxacin-resistant clinical isolates (MICs of ciprofloxacin, 32 to 64 micrograms/ml) and one multistep resistant laboratory mutant of E. faecalis (MIC of ciprofloxacin, 128 micrograms/ml) contained a change from serine to arginine or to isoleucine at codon 83 or a change from glutamic acid to lysine or to glycine at codon 87 (Escherichia coli GyrA coordinates); these changes have been associated with fluoroquinolone resistance in other species. No difference in the region studied was found in two ciprofloxacin-resistant E. faecium isolates (MICs, 32 micrograms/ml) or in four laboratory derived, spontaneous ciprofloxacin-resistant mutants of E. faecalis (MICs, 8 to 16 micrograms/ml), suggesting that other mechanisms may be responsible for fluoroquinolone resistance in some enterococci.

Amino Acid Sequence↗

PubMed1994

Resistance of Enterococcus faecium to neutrophil-mediated phagocytosis.

During a previous study of the opsonic requirements for neutrophil (polymorphonuclear leukocyte [PMN])-mediated killing of enterococci, we identified two strains of Enterococcus faecium (TX0015 and TX0016) that were resistant to PMN-mediated killing. To better define the mechanism of this resistance, we examined phagocytosis with a fluorescence assay and found that TX0016 was completely resistant to phagocytosis by PMNs; this finding was confirmed by electron microscopy. Examination of multiple strains of enterococci revealed that all 20 strains of Enterococcus faecalis tested were readily phagocytosed (mean, 18 intracellular organisms per PMN; range, 7 to 28). In contrast, only 13 (50%) of 26 strains of E. faecium tested were susceptible to phagocytosis (> or = 7 organisms per PMN); the other 13 strains showed < or = 3 organisms per PMN. Enterococcus casseliflavus ATCC 25788 and one strain of Enterococcus hirae were also resistant to phagocytosis, while two strains of Enterococcus durans, Enterococcus mundtii ATCC 43186, and one strain each of Enterococcus raffinosus and Enterococcus solitarius were readily phagocytosed. Exposure of E. faecium TX0016 to sodium periodate, but not to the protease trypsin or pronase or to phospholipase C, eliminated resistance to phagocytosis. Sialic acid, a common periodate-sensitive structure used by microorganisms to resist opsonization, could not be demonstrated in E. faecium TX0016 by the thiobarbituric acid method, nor was phagocytosis of TX0016 altered by neuraminidase treatment. This study suggests that there is a difference in susceptibility to phagocytosis by PMNs between different species of enterococci and that a carbohydrate-containing moiety which is not sialic acid may be involved in the resistance of E. faecium TX0016 to phagocytosis.

Bacterial Capsules↗

PubMed1994

Roles of antibodies and complement in phagocytic killing of enterococci.

The contributions of complement and antibodies to polymorphonuclear leukocyte (PMN)-mediated killing of enterococci were investigated with pooled normal human serum (PNHS) or immune human sera (IHS) from patients with serious enterococcal infections. Each IHS containing antienterococcal antibodies demonstrated by enzyme-linked immunosorbent assay and Western blotting (immunoblotting) was examined with the enterococcus strain isolated from the same patient. PNHS promoted PMN-mediated killing of enterococci similar to that for IHS. PMN-mediated killing was consistently abrogated after preopsonization with heat-inactivated PNHS, but some heat-inactivated IHS supported neutrophil bactericidal activity. Inhibition of the classical pathway of complement by chelation of either PNHS or IHS with Mg-EGTA [Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not alter PMN-mediated killing, suggesting that activation of the alternative pathway of complement is sufficient to promote killing of enterococci by PMNs. PMN-mediated killing assays were also performed with normal rabbit serum and immune rabbit serum against enterococci. Preopsonization with heat-inactivated immune rabbit serum resulted in PMN-mediated killing of enterococci, which was ablated after adsorption of the serum with the same isolate used for immunization. The influence of different phenotypic enterococcal traits on neutrophil-mediated killing was also investigated. Similar kinetics of killing were observed for derivatives of Enterococcus faecalis strains regardless of resistance to antimicrobial agents or production of beta-lactamase, hemolysin, gelatinase, or surface proteins involved in the aggregative response to pheromones. In summary, PMN-mediated killing of enterococci appears to depend primarily on complement activation by either the classical or the alternative pathway. Human antienterococcal antibodies generated during infection variably promoted neutrophil bactericidal activity, while antibody raised in a rabbit supported PMN-mediated killing of the organism examined. Finally, the different phenotypic properties of E. faecalis examined did not influence the neutrophil-mediated killing of these organisms.

Adult↗

PubMed1993

Susceptibility of beta-lactamase-producing enterococci to piperacillin with tazobactam.

The in vitro activity of piperacillin with and without tazobactam was evaluated against different inocula of 12 clinical isolates of beta-lactamase-producing Enterococcus faecalis obtained from different geographic areas. Minimum inhibitory concentrations (MICs) of piperacillin alone at approximately 10(3) colony-forming units (CFU)/spot ranged from 4 to 8 and from 4 to 8 micrograms/ml with piperacillin plus tazobactam. When approximately 10(7) CFU/spot was used, MICs increased to a range of 128-1024 micrograms/ml piperacillin. This inoculum effect was reversed by the addition of tazobactam to piperacillin at a fixed concentration of 1 microgram/ml or at a ratio of 8 : 1 (piperacillin relative to tazobactam) with an MIC90 of 16/2 micrograms/ml for the combination drug. In time-kill studies, four beta-lactamase-producing (Bla+) isolates were tested and demonstrated a decrease of > or = 2 log10 with 8 or 16 micrograms/ml of piperacillin in combination with 4 micrograms of tazobactam, but not with piperacillin alone. A non-beta-lactamase-producing isolate was equally inhibited by piperacillin alone and piperacillin plus tazobactam. Against a Bla+ isolate, the combination of piperacillin with tazobactam with streptomycin resulted in a synergistic effect relative to that of piperacillin with tazobactam; piperacillin plus streptomycin did not show synergism. Piperacillin in combination with tazobactam is active against enterococci that produce beta-lactamase and, in combination with an appropriate aminoglycoside, could be a viable choice for therapy of enterococci that do not have high-level resistance to all aminoglycosides.

Culture Media↗

PubMed1993

Nosocomial outbreak due to Enterococcus faecium highly resistant to vancomycin, penicillin, and gentamicin.

In October 1990, Enterococcus faecium that was highly resistant to glycopeptides, penicillins, and aminoglycosides was isolated from the peritoneal dialysis fluid from a patient in an intensive care unit. Over the following 6 months, multiresistant E. faecium organisms were isolated from cultures of blood, urine, or surgical wound specimens from eight additional patients. Surveillance cultures of groin and/or rectal swabs were positive for eight of 37 patients and four of 62 employees at risk. Restriction endonuclease digestion of chromosomal DNA from outbreak isolates was consistent with dissemination of a single strain throughout the intensive care unit. Strict infection control interventions contained the outbreak after several weeks. Review of patient charts suggested that renal insufficiency, length of hospital stay, duration of antibiotic treatment, and prior treatment with vancomycin were risks for infection due to multiresistant E. faecium. The emergence of multiple-drug-resistant enterococci presents serious infection control and therapeutic dilemmas.

Aged↗

PubMed1993

In vitro activity of azithromycin against bacterial enteric pathogens.

The in vitro activity of azithromycin against enteric bacterial pathogens was determined by agar dilution. Azithromycin was highly active against Campylobacter spp. (MIC for 90% of strains tested [MIC90] = 0.125 micrograms/ml) and against enterotoxigenic, enterohemorrhagic, enteroinvasive, and enteropathogenic Escherichia coli (MIC90 = 2 micrograms/ml), Shigella spp. (MIC90 = 1 micrograms/ml), and Salmonella spp. (MIC90 = 4 micrograms/ml), including Salmonella typhi (MIC90 = 1 microgram/ml). On the basis of the in vitro activity of the drug against these organisms, clinical studies of azithromycin in enteric diseases should be considered; the high intracellular concentrations achieved by azithromycin may be particularly relevant for organisms like S. typhi, Campylobacter spp., and Shigella spp. which typically invade cells as part of their infectious process.

Azithromycin↗

PubMed1993

Genes involved in the regulation of beta-lactamase production in enterococci and staphylococci.

In enterococci, the structural gene for beta-lactamase (blaZ) is identical to blaZ from Staphylococcus aureus. However, in the enterococci studied to date, beta-lactamase is produced constitutively, whereas in staphylococci it is often inducible. Recent reports have revealed the presence of two adjacent genes upstream of the staphylococcal blaZ thought to be the antirepressor (blaR1) and repressor (blaI) genes. In the present study, beta-lactamase expression mutants of the staphylococcal beta-lactamase plasmid pI524 were generated by transposon mutagenesis with the transposon Tn917. Tn917 insertions upstream of blaZ in either blaR1 or blaI resulted in constitutive beta-lactamase production, indicating that the repressor function is lost with insertion of Tn917 into either gene. This finding supports the concept that the staphylococcal beta-lactamase regulatory genes are encoded on a polycistronic mRNA. The corresponding region upstream of the enterococcal blaZ from Enterococcus faecalis HH22 was sequenced and compared with the staphylococcal blaR1 sequence. The two sequences were identical for 893 nucleotides, and then the sequences diverged completely. Therefore, in strain HH22, only 51% of the putative antirepressor gene is present and the repressor gene is also absent. In conclusion, constitutive beta-lactamase production in HH22 appears to be due to a lack of the regulatory genes blaR1 and blaI which regulate expression of blaZ in staphylococci.

Base Sequence↗

PubMed1993

Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function.

A defined synthetic medium was used to determine the amino acid requirements of Enterococcus faecalis OG1 and to demonstrate the absence of a requirement for exogenous purines or pyrimidines. Genomic libraries prepared from strain OG1 were transduced into Escherichia coli auxotrophic mutants, and cloned DNAs which complemented pyrC, pyrD, purF, purL, and guaAB mutations were identified. These and other cloned DNAs with known functions were localized on a restriction map of OG1 which was generated with SfiI (5 fragments), AscI (9 fragments), and NotI (15 fragments); the size of the OG1 chromosome was revised from a previous estimate of approximately 2,750 kb to 2,825 kb. The synthetic medium and the restriction map should be useful for studying enterococcal metabolic functions and the relationships between chromosomally encoded genes.

Amino Acids↗

PubMed1993

Typing of group B streptococci: comparison of pulsed-field gel electrophoresis and conventional electrophoresis.

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 35 group B streptococci were analyzed by pulsed-field gel electrophoresis (PFGE). Nineteen different patterns and four possible variants were identified. Twenty-four isolates were previously analyzed by conventional electrophoresis of HindIII-digested and/or BglII plus EcoRI double-digested chromosomal DNA. Although interpretations by both methods were essentially the same, PFGE identified as variants two isolates that were previously classified as the same isolate. More importantly, PFGE of the chromosomal DNA of group B streptococci digested with SmaI generated more easily defined patterns, since fewer and better separated bands were obtained, whereas digestion with HindIII or EcoRI plus BglII typically generated 100 or more bands. SalI digestion also yielded easily evaluable results, although the SalI fragments were somewhat smaller than those generated by SmaI. In our hands, PFGE patterns were more easily discerned and interpreted than were patterns previously generated by conventional electrophoresis.

Adult↗

PubMed1993

Comparison of ribotyping and pulsed-field gel electrophoresis for subspecies differentiation of strains of Enterococcus faecalis.

Hybridization of EcoRI- and HindIII-digested chromosomal DNAs from 41 isolates of Enterococcus faecalis with probes for rRNA genes was performed (ribotyping). The ability of ribotyping to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). With EcoRI, seven ribopatterns (usually differing by only one band) were found, while PFGE had previously shown 25 clearly different patterns plus six related variants. Digestion with HindIII generated a few additional patterns but still failed to differentiate some strains that had very different PFGE patterns. Ribotyping with BscI has also been reported to be inadequate for subspecies strain differentiation (L. M. Hall, B. Duke, M. Guiney, and R. Williams, J. Clin. Microbiol. 30:915-919, 1992). Although ribotyping with other restriction endonucleases may perform better in distinguishing different strains, at present PFGE appears to be superior for strain differentiation.

DNA, Bacterial↗

PubMed1993

Evaluation of the molecular epidemiology of an outbreak of multiply resistant Shigella sonnei in a day-care center by using pulsed-field gel electrophoresis and plasmid DNA analysis.

Outbreaks of diarrhea in child day-care centers (DCC) are common. This study was undertaken to evaluate the molecular epidemiology of an outbreak of diarrhea due to Shigella sonnei. This outbreak involved 25 of 52 (48%) DCC children and 14 of 132 (11%) teachers and household contacts. S. sonnei isolates from nine children and five contacts were characterized by antimicrobial susceptibility, plasmid content, plasmid DNA restriction fragment pattern, and pulsed-field gel electrophoresis (PFGE) of total genomic DNA; 33 isolates from Houston, Tex., Chicago, Ill., and Mexico City, Mexico, also were studied. All outbreak isolates were resistant to ampicillin and trimethoprim-sulfamethoxazole and shared five to six plasmids ranging from 3.3 to 70 MDa. A total of 8 of 12 temporally associated nonoutbreak Houston isolates had plasmid profiles and restriction fragment patterns similar to those of the outbreak strain, despite possessing different antibiotic susceptibility patterns. PFGE demonstrated identical DNA patterns among outbreak isolates and similar or identical patterns among temporally associated sporadic Houston isolates with plasmid profiles similar to that of the outbreak strain. All other nonoutbreak strains from Houston, Chicago, and Mexico had plasmid profiles, restriction fragment patterns, and PFGE patterns different from those of the outbreak strain. DCC outbreak isolates could be distinguished from most sporadic isolates by antimicrobial susceptibility testing, but plasmid analysis and PFGE could not differentiate common-source isolates from sporadic isolates in the same location during the same time period, indicating that isolates present in the community were genetically similar to those producing outbreaks in the DCC.

Child Day Care Centers↗

PubMed1993

Impact of the fluoroquinolones on gastrointestinal flora.

A number of studies have been performed to evaluate the effect of the fluoroquinolones on gastrointestinal flora. The fluoroquinolones have only slight or no effect on the oropharyngeal flora, except when Neisseria, Haemophilus or Branhamella spp. are present. Studies have consistently shown that Gram-negative facultative bacteria of the lower intestinal flora are strongly suppressed during administration of these agents. Total faecal anaerobes are generally unchanged. The effect of the fluoroquinolones on Gram-positive bacteria is more variable with mild to moderate suppression reported with some agents. In view of the high faecal concentrations of the fluoroquinolones, the general lack of effect on anaerobes is surprising; it may be attributable to the large number of microorganisms found in faeces and faecal binding of the fluoroquinolones. Several recent studies suggest that the effects of some fluoroquinolones on faecal anaerobes and Gram-positive cocci may be more profound in certain patient populations such as bone marrow transplant recipients and patients undergoing gastrointestinal surgery. Colonisation with yeasts and the emergence of resistant bacterial strains have been reported during or after fluoroquinolone administration in some studies. Future studies will need to investigate the effect of the newer agents with greater activity against anaerobes and Gram-positive cocci on the gastrointestinal flora and to continue surveillance for resistant organisms.

Anti-Infective Agents↗

PubMed1992

Infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis.

OBJECTIVE: To investigate the risk factors, clinical features, molecular epidemiology, and treatment outcomes associated with an outbreak of infections due to beta-lactamase-producing, high-level gentamicin-resistant Enterococcus faecalis. DESIGN: Case-control and molecular genetics study. SETTING: Tertiary care Veterans Affairs hospital. PATIENTS: Sixty-five patients infected or colonized with beta-lactamase-producing high-level gentamicin-resistant E. faecalis (case patients) were matched and compared with 65 randomly selected patients infected or colonized with beta-lactamase-negative, gentamicin-susceptible E. faecalis. MEASUREMENTS AND MAIN RESULTS: During the 20-month study period, 124 of 1506 isolates (8.2%) of E. faecalis from 70 patients were found to produce beta-lactamase. Univariate analysis showed older age, higher APACHE II score, nosocomial acquisition, recent surgical procedure, total parenteral nutrition, and antibiotic treatment to be significantly associated with the acquisition of beta-lactamase-producing, high-level gentamicin-resistant E. faecalis. A multivariate analysis, done using stepwise multiple logistic regressions, showed that only two variables remained significant: an APACHE II score greater than 6 (odds ratio, 9.5; 95% CI, 4.4 to 20.3) and antibiotic treatment (odds ratio, 10.2; CI, 4.5 to 23.2). The mortality rate for case patients was 7.7% (5 of 65 patients; CI, 1.2% to 14.2%); no control patient died. Of 23 infections occurring in case patients, 13 were treated with "inappropriate" antibiotics (regimens that included a beta-lactamase-unstable antibiotic); 2 patients improved and 11 had complete resolution of disease. "Appropriate" treatments (regimens that included a beta-lactamase-stable antibiotic) were used in 10 patients; 5 of 10 infections were fatal. Restriction enzyme digests of total chromosomal DNA showed nearly identical patterns for selected isolates of beta-lactamase-producing, high-level gentamicin-resistant E. faecalis, suggesting dissemination through the hospital of a single strain of E. faecalis. CONCLUSIONS: Fatal infections were observed despite treatment with beta-lactamase-stable antibiotics. The risk for infection or colonization with beta-lactamase-producing, high-level gentamicin-resistant E. faecalis was strongly associated with severe underlying disease (acute physiology and chronic health evaluation [APACHE] II score, greater than 6) and previous antibiotic treatment. These results may be useful in targeting high-risk patients for infection-control interventions.

Case-Control Studies↗

PubMed1992

Determination of the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium.

Pulsed-field gel electrophoresis was used to determine the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium. The size determinations of OG1X, a strain of E. faecalis widely used in many laboratories for genetic studies, using Sma I, Not I, and Sfi I alone or in combination, ranged from 2,750 to 2,761 kb. Using the same enzymes as with OG1X, the size of HH-67, a plasmid-free clinical isolate of E. faecalis, was determined to be 2,170-2,288 kb and the size of JH2-2, an E. faecalis recipient strain, ranged from 2,008 to 2,135 kb. The size range generated for GE-1, a plasmid-free E. faecium strain, with the use of Sma I, Not I, and Apa I was 2,045-2,155 kb. Although OG1X differed in size from the other three enterococci, each individual enterococcal strain generated reproducible results in different experiments. However, for both E. faecalis OG1X and E. faecium GE-1, one of the enzymes used generated a considerably smaller molecular size than that generated by the other two enzymes. The discrepancy was due to visually undiscernible comigrating fragments, and serves to point out a potential source of error if fewer than two enzymes are used to size a genome. The size discrepancies were resolved by digesting individual fragments with a second enzyme. The molecular sizes of these enterococcal strains are larger than that recently reported for Campylobacter, smaller than that of Escherichia coli and Pseudomonas aeruginosa, and similar (OG1X) or smaller (JH2-2, HH67, and GE-1) than the 2,819-kb reported for Streptococcus mutans.

Chromosomes, Bacterial↗

PubMed1992

Trimethoprim-resistant Escherichia coli in households of children attending day care centers.

Children in day care centers are frequently colonized with trimethoprim-resistant Escherichia coli. The frequency of transmission to family members was studied. Colonization with trimethoprim-resistant E. coli was detected in 13 (57%) of 23 day care center children. Among the 50 (98%) of 51 household members tested, colonization was detected in 13 (26%), representing 12 (52%) of 23 households tested. Households of colonized center children had significantly more colonized members than did households of noncolonized children (odds ratio, 13.3; 95% confidence interval, 1.3-172.6; P = .01). There was a trend toward higher prevalence of colonization in mothers (35%) and siblings (30%) than in fathers (12%). The same plasmid profiles of trimethoprim-resistant E. coli from center children were found among 8 (67%) of 12 colonized households. There was no association between antibiotic use and fecal colonization with trimethoprim-resistant E. coli. Thus, transmission of trimethoprim-resistant E. coli among children within day care centers and from center children to household members is common.

Carrier State↗

PubMed1992

The adherence of verocytotoxin-producing Escherichia coli to rabbit intestinal cells.

Verocytotoxin-producing Escherichia coli (VTEC) have been recognised recently as an important cause of human disease. The adherence of VTEC to rabbit intestinal tract and the relationship between adherence and other virulence traits were studied. Twenty clinical isolates of VTEC (O157:H7 and other serotypes) and a control, commensal E. coli strain, were examined. Bacteria were evaluated for the presence of surface fimbriae, plasmid profile and hybridisation with a 3.4 kb DNA probe derived from the 60-MDa plasmid of such strains. Adherence was determined by electronmicroscopy and quantitatively with radio-labelled bacteria. Of the VTEC strains, 12 (60%) had surface fimbriae; all O157:H7 and 10 (70%) of 14 of the non-O157:H7 strains hybridised with the probe. No isolate was negative for both of these virulence traits and there was no correlation between their presence. The plasmid profiles varied among the strains, with no correlation to virulence traits. The adherence of VTEC strains differed significantly, ranging from 0.3 to 34.0 bacteria/intestinal cell. The mean adherence of fimbriate strains was greater than that of non-fimbriate strains (3.9 versus 2.7 bacteria/cell), although marked variability was noted in both groups. This study showed that VTEC strains differed markedly in their adherence capability and that neither the presence of fimbriae nor hybridisation with the 3.4-kb probe was essential for adherence. Several distinct mechanisms probably play a role in VTEC adherence.

Animals↗

PubMed1992

Intrahospital spread of a single gentamicin-resistant, beta-lactamase-producing strain of Enterococcus faecalis in Argentina.

Six beta-lactamase-producing (Bla+) isolates of Enterococcus faecalis recovered over a 17-month period from an Argentinian pediatric hospital were found to have identical or almost identical chromosomal restriction patterns by pulsed-field gel electrophoresis, although the plasmid patterns were different. These isolates, like Bla+ enterococci in the United States, hybridized to a staphylococcal Bla gene probe. The presence of a single strain was somewhat surprising, since all isolates transferred Bla by conjugation.

Argentina↗

PubMed1992

Comparison of the gentamicin resistance transposon Tn5281 with regions encoding gentamicin resistance in Enterococcus faecalis isolates from diverse geographic locations.

The genetic determinant encoding gentamicin resistance (Gmr) on the beta-lactamase encoding plasmid pBEM10 of Enterococcus faecalis HH22 is carried on a transposon, termed Tn5281, that is highly related to the staphylococcal Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in United States isolates of Staphylococcus epidermidis. We have now studied plasmid DNA from Gmr strains of E. faecalis isolated from diverse geographical locations (Houston, Pennsylvania, Thailand, and Chile) by using restriction endonuclease analysis and DNA-DNA hybridization to determine whether other Gmr E. faecalis carry Tn5281 or a similar type of element. We also compared these enterococci to several United States isolates of Staphylococcus aureus with nonmobile Gmr determinants. Three E. faecalis isolates (from Houston and Chile) carried Tn5281-like elements, whereas two isolates (from Houston and Pennsylvania) had restriction endonuclease and DNA-DNA hybridization patterns more similar to those of the Tn4001-IS257 hybrid found in the nonmobile Gmr determinants in United States isolates of S. aureus. A strain from Thailand had a third pattern unrelated to either Tn5281 or the nonmobile Gmr determinants present in United States isolates of S. aureus. Our results demonstrate that there is both similarity and diversity between the Gmr determinant of strains of E. faecalis isolated in diverse geographic locations.

Anti-Bacterial Agents↗