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Biomedical subjects

B E Murray

Publications and source records attributed to B E Murray.

At least 109 records · Page 6Linked to original sources
PubMed1992

Sequence analysis of the beta-lactamase repressor from Staphylococcus aureus and hybridization studies with two beta-lactamase-producing isolates of Enterococcus faecalis.

The putative beta-lactamase (Bla) repressor gene, blaI, from the staphylococcal plasmid pI524 was isolated, and the DNA sequence was determined. The sequence of blaI was found to be identical to the blaI sequence from pI9789 (blaI blaZ seg-1), a plasmid related to pI524. A blaI probe from pI524 was hybridized with plasmid and genomic DNA from Bla+ Enterococcus faecalis isolates HH22 and PA. The Bla structural gene of HH22 has been previously shown to be of staphylococcal origin, but DNA homologous to the staphylococcal Bla repressor was not found, indicating that the constitutive production of beta-lactamase in these E. faecalis isolates may be the result of a missing repressor protein.

Base Sequence↗
PubMed1992

Comparison of enterococcal and staphylococcal beta-lactamase-encoding fragments.

A restriction map of a 13.5-kb EcoRI fragment encoding beta-lactamase from a plasmid isolated from enterococcal strain PA was prepared and found to differ markedly from the published maps of beta-lactamase-encoding EcoRI fragments of two staphylococcal plasmids and the plasmid from enterococcal strain HH22. This comparison also showed that one of two contiguous EcoRV fragments that encompass the beta-lactamase gene differs in size in the PA strain from that found in the other strains. However, restriction sites in the beta-lactamase structural gene (blaZ) were identical in all four plasmids. Further studies compared the beta-lactamase genes of four clinical enterococcal isolates from various geographic locations with those described above. Isolates from Virginia and Florida generated EcoRV fragments identical to those from the plasmid from strain PA, while isolates from Lebanon and Argentina showed EcoRV fragments analogous to those from HH22 and the staphylococci studied. Although there is evidence suggesting that some of these beta-lactamase-producing enterococcal isolates represent a single strain, this study indicates that there is significant variation in the plasmids that encode beta-lactamase.

Autoradiography↗
PubMed1992

Comparative in vitro activity of PD 127391, a new fluoroquinolone agent, against susceptible and resistant clinical isolates of gram-positive cocci.

We examined the in vitro activity of PD 127391, an investigational fluoroquinolone antibacterial agent, against staphylococci (including methicillin-resistant Staphylococcus aureus), enterococci (including beta-lactamase-producing and highly gentamicin-resistant isolates), and streptococci. The compound was active against all organisms tested and compared favorably with antimicrobial agents routinely used to treat infections with these organisms. On the basis of MICs for 90% of the strains tested, PD 127391 was 32-fold more active against all staphylococci, 16-fold more active against methicillin-resistant S. aureus, 8-fold more active against all streptococci, and 4-fold more active against all enterococci than ciprofloxacin. PD 127391 was shown to be more active than sparfloxacin, which in turn was shown to be more active than ciprofloxacin, against these gram-positive cocci. PD 127391 shows promise for the treatment of infections with gram-positive cocci, including organisms which are resistant to other commonly used antimicrobial agents.

Anti-Infective Agents↗
PubMed1992

Identification by DNA sequence analysis of a new plasmid-encoded trimethoprim resistance gene in fecal Escherichia coli isolates from children in day-care centers.

In our ongoing studies of trimethoprim resistance (Tmpr) in day-care centers (DCC), we have shown a high rate of fecal colonization with Tmpr Escherichia coli and, using total plasmid content analysis, have shown that this is due to a diversity of strains. In the present study, we analyzed 367 highly Tmpr (MIC, greater than or equal to 2,000 micrograms/ml) isolates of E. coli from 72 children over a 5-month period and found at least 83 distinct plasmid patterns, indicating that at least 83 strains were involved. Several strains were particularly common in a given DCC, including one found in 61% of children with Tmpr E. coli; these common strains usually persisted within a DCC for several months. Colony lysates were hybridized with gene probes for dihydrofolate reductases (DHFR) types I, II, III, V, and VII; 21% hybridized under stringent conditions, and all of these were with type I (17%) or type V (4%) probes. Tmpr was cloned from a probe-negative Tmpr transconjugant, and an intragenic probe was prepared from this clone. Approximately 21% of the Tmpr E. coli strains (76 isolates) in the DCC were found to have this new gene, 74 of which were in one DCC. The DNA sequence of this gene was determined, and the predicted amino acid sequence was shown to have between 32% and 39% identity with the amino acid sequences for types I, III, V, VI, and VII and the partial sequence of type IV and approximately 26% identity with types IX and X DHFR. This confirms the uniqueness of this gene, which has tentatively been named dhfrxii, and its translation product, DHFR type XII.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of strains of enteroinvasive Escherichia coli O143, including isolates from a large outbreak in Houston, Texas.

A large diarrhea outbreak due to enteroinvasive Escherichia coli (EIEC) serogroup O143 occurring in Houston, Tex., provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. This was done by comparing the plasmid patterns and the chromosomal restriction endonuclease digestion patterns by pulsed-field gel electrophoresis (PFGE) of EIEC from the outbreak, other E. coli from the same serogoup (O143), and EIEC isolated from other patients with diarrhea. Among the isolates studied, there was marked restriction fragment length polymorphism. All 3 non-O143 EIEC isolates had very different restriction endonuclease digestion patterns, as did 5 of 5 O143 non-EIEC isolates and 6 of 15 O143 EIEC isolates. Four Houston outbreak O143 EIEC isolates had the same restriction pattern as an O143 EIEC strain isolated 2 months before in Mexico and was nearly identical to another two O143 EIEC Mexican isolates. These related strains also had the same plasmid pattern; however, the presence of only a few plasmid bands, versus the 21 to 30 chromosomal bands seen with PFGE, suggests that plasmid patterns could be a less specific way to distinguish different strains. These results demonstrate that PFGE can distinguish between different E. coli strains of the same serogroup and phenotype. This technique can also identify relatedness within O143 EIEC, and our data suggest the spread of a strain of EIEC from Mexico to Houston, where it caused a large outbreak. PFGE may be useful to study the epidemiology of EIEC.

DNA, Bacterial↗
PubMed1992

Polymerase chain reaction for diagnosis of enterohemorrhagic Escherichia coli infection and hemolytic-uremic syndrome.

Two pairs of oligonucleotide primers were designed to amplify fragments of the genes for Shiga-like toxin I (SLT-I) and SLT-II in a single reaction. A 370-bp segment and a 283-bp segment were amplified for SLT-I and SLT-II, respectively. The specificities of the polymerase chain reaction (PCR) amplification products were confirmed by using radioactively labeled oligonucleotide probes. SLT sequences were amplified from DNA isolated from 13 previously characterized enterohemorrhagic Escherichia coli (EHEC) strains. No amplification product was produced by using DNA from 20 non-EHEC strains. As little as one bacterial genome was detectable. PCR was then applied to DNA isolated directly from stool samples. We had to remove inhibitors of PCR that were present in lysates prepared from stool samples before amplification was achieved. First, we evaluated the sensitivity of PCR for the detection of known numbers of EHEC added to normal stools. Second, three children with SLT in their stools were shown to have SLT sequences in their stools by PCR. Two of these children had hemolytic-uremic syndrome, and a third child was asymptomatic. Stool specimens collected from another 26 asymptomatic children were negative by PCR for SLT sequences. PCR can be used to diagnose EHEC infections without prior culture of stool specimens.

Bacterial Toxins↗
PubMed1992

Problems and dilemmas of antimicrobial resistance.

An important obstacle to the long-term efficacy of an antimicrobial agent is the appearance and spread of resistance to the agent. The fact that many antimicrobials are produced by microorganisms in nature may provide long-term selective pressure for the emergence of resistance in antibiotic-producing as well as -nonproducing organisms. Indeed, the rapidity with which many resistances have appeared after the introduction of a new antibiotic suggests that these resistance genes were already present somewhere in nature prior to clinical use. In the hospital setting, the most recent worrisome resistance traits to emerge include plasmid-mediated resistance to imipenem and to third-generation cephalosporins among nosocomial gram-negative bacteria, and the acquisition of resistance to vancomycin by enterococci. Methicillin-resistant staphylococci continue to be a problem and are increasingly resistant to numerous other agents such as rifampin and the newer fluoroquinolones. The most important resistances seen in community-acquired organisms include beta-lactam resistance in pneumococci and combined ampicillin and chloramphenicol resistance in Haemophilus influenzae. Shigellae resistant to essentially all commonly used oral agents are also a problem, particularly in developing countries. No end is in sight to the problem of antimicrobial resistance, and thus new strategies to prevent infections and control resistant organisms continue to be necessary.

Anti-Bacterial Agents↗
PubMed1992

Ceftibuten and trimethoprim-sulfamethoxazole for treatment of Shigella and enteroinvasive Escherichia coli disease.

In a prospective randomized study at two clinical sites, ceftibuten was compared with trimethoprim-sulfamethoxazole (TMP-SMX), both given orally for a period of 5 days, for the treatment of dysentery. Twenty-two children were found to have bacillary dysentery caused by Shigella and/or enteroinvasive Escherichia coli. All organisms isolated were susceptible to ceftibuten; 6 of 20 Shigella strains and 4 of 5 enteroinvasive E. coli were resistant to TMP-SMX. The diarrhea persisted for a mean (+/- SD) period of 2.4 +/- 1.4 days in the ceftibuten-treated patients vs. 3.4 +/- 1.7 days in the TMP-SMX-treated patients. The duration of fever was similar for both treatment groups. Patients treated with ceftibuten or TMP-SMX had equivalent clinical responses unless the pathogen was found to be TMP-SMX-resistant. Those who were randomized to receive TMP-SMX but who were eventually found to have TMP-SMX-resistant organisms had significantly more stools at days 3, 4 and 5 (P less than 0.02 to less than 0.00006) with more watery consistency for these days (P less than 0.02 to less than 0.005) compared to patients treated with ceftibuten. No clinical relapses were reported and no drug-related side effects were observed. We conclude that ceftibuten is at least as effective as TMP-SMX in the treatment of diarrhea caused by Shigella and enteroinvasive E. coli in children.

Adolescent↗
PubMed1991

Evidence for clonal spread of a single strain of beta-lactamase-producing Enterococcus (Streptococcus) faecalis to six hospitals in five states.

Beta-lactamase-producing (Bla+) enterococci have been reported in three state and two countries. Pulsed-field gel electrophoresis was used to compare 14 Bla+ Enterococcus (Streptococcus) faecalis isolated from hospitalized patients in seven states and three continents. The restriction endonuclease digestion patterns of isolates from Connecticut, Massachusetts, Lebanon, and Argentina were all markedly different, indicating that these were different strains. However, isolates from Delaware, Texas, Pennsylvania (Philadelphia and Pittsburgh), Florida, and Virginia were similar, indicating that these isolates were derivatives of a single strain. This conclusion was supported by hybridization using individual fragments as probes. Spread of Bla+ enterococci within the hospital setting was also demonstrated. These findings illustrate the value of pulsed-field gel electrophoresis for epidemiologic analyses and support the importance of identifying and containing organisms with new resistance properties in an effort to decrease their transmission to and from, as well as within, hospitals.

Argentina↗
PubMed1991

Evidence of infection with organisms producing Shiga-like toxins in household contacts of children with the hemolytic uremic syndrome.

We conducted a prospective study in 87 household contacts of 51 children with hemolytic uremic syndrome to determine the frequency of infection with Shiga-like toxin-producing bacteria. Gastrointestinal tract symptoms occurred in only 1 of 87 contacts. Free fecal toxin was detected in 25 of 64 (39%) of the household members. Neutralization with specific antisera to Shiga-like toxins I and II (SLT-I, SLT-II) revealed that in 6 of these household contacts only SLT-I was present in stool, in 10 only SLT-II was present and in 9 both toxins were found. Thirty-three percent of the hemolytic uremic syndrome families in which 2 or more members were studied had more than 1 household member with free fecal toxin in stool. None of the household contacts was found to have E. coli O157:H7 in feces. Serum samples were available in 77 household contacts; 75% (58 of 77) had serum neutralizing titers of greater than or equal to 1:4 to 1 or both toxins. In those contacts for whom paired sera were available, seroconversion was found in 10 of 24 (42%). These data show that household contacts of children with hemolytic uremic syndrome are commonly colonized with Shiga-like toxin-producing E. coli and seroconversion to Shiga-like toxins occurs frequently in family members of children with hemolytic uremic syndrome.

Adult↗
PubMed1991

Characterization of the gentamicin resistance transposon Tn5281 from Enterococcus faecalis and comparison to staphylococcal transposons Tn4001 and Tn4031.

In Enterococcus faecalis, the genetic determinant encoding gentamicin resistance (Gmr) on the conjugative plasmid pBEM10 previously has been shown to be on a mobile element. In the current study, this element, termed Tn5281, was shown to relocate in the absence of homologous recombination in E. faecalis UV202. On the basis of restriction endonuclease analysis and DNA-DNA hybridization studies, Tn5281 was shown to be similar, if not identical, to the Gmr transposons Tn4001 found in Australian isolates of Staphylococcus aureus and Tn4031 found in U.S. isolates of Staphylococcus epidermidis, since all three of these transposons have symmetrically located HindIII (2.5 kb apart), ClaI (slightly more than 2.5 kb apart), and HaeIII (3.9 kb apart) sites. Restriction endonuclease digestion patterns of Tn5281 generated with HincII, ScaI, and AluI were also consistent with Tn4001 and Tn4031. By using a probe specific for the external portion of the terminal inverted repeat of Tn4031, it was determined that each terminus of Tn5281 contained a 0.35-kb HaeIII fragment and a 0.7-kb HindIII-HaeIII fragment. The sizes of these fragments are identical to those found in the staphylococcal transposons, which is a further indication that inverted repeats like IS256 are present in Tn5281. A 1-kb HaeIII fragment in pBEM10 also hybridized with this probe, which indicates that Tn5281 in pBEM10 contains a double copy of the inverted repeat at one end.

Conjugation, Genetic↗
PubMed1991

Nucleotide sequence of the beta-lactamase gene from Enterococcus faecalis HH22 and its similarity to staphylococcal beta-lactamase genes.

The nucleotide sequence of the constitutively produced beta-lactamase (Bla) gene from Enterococcus faecalis HH22 was shown to be identical to the published sequences of three of four staphylococcal type A beta-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred forty nucleotides upstream of the beta-lactamase start codon were determined for an inducible staphylococcal beta-lactamase and were identical to those of the constitutively expressed enterococcal gene, indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The genes for the enterococcal Bla and an inducible staphylococcal Bla were each cloned into a shuttle vector and transformed into enterococcal and staphylococcal recipients. The major difference between the backgrounds of the two hosts was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene. The location of the enzyme was found to be host dependent, since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell-bound enzyme in the enterococcus. On the basis of the identities of the enterococcal Bla and several staphylococcal Bla sequences, these data suggest the recent spread of beta-lactamase to enterococci and also suggest the loss of a functional repressor.

Base Sequence↗
PubMed1991

Lack of homology of enterococci which have high-level resistance to trimethoprim with the dfrA gene of Staphylococcus aureus.

Multiresistant enterococci were tested for susceptibility to trimethoprim (TMP). Although most enterococci are inhibited by less than or equal to 1.0 microgram/ml, the MICs for 7 of 29 selected multiresistant isolates were greater than or equal to 8 micrograms/ml, including for two beta-lactamase positive (Bla+) strains, for which the MICs of TMP were greater than 1,000 micrograms/ml, and for another Bla+ strain, for which the MIC was 128 micrograms/ml. None of five isolates tested transferred TMP resistance and none of the resistant isolates hybridized to the dfrA gene of Staphylococcus aureus. Whether TMP resistance in enterococci is due to a mutation(s) or to acquisition of a new gene is not known. Acquisition of resistance to TMP is another example of the multiple antimicrobial resistance typically displayed by enterococci.

Enterococcus faecalis↗
PubMed1991

Epidemiology of an endemic strain of beta-lactamase-producing Enterococcus faecalis.

Three previously reported beta-lactamase-producing (Bla+) enterococci with distinct but related antibiotic resistance phenotypes, plasmid profiles, and plasmid restriction endonuclease digestion patterns were isolated at the West Haven Veterans Administration Medical Center in Connecticut (WH245, WH257, WH571) in July 1986 and March 1987. In this study, we analyzed the whole-cell DNA of these isolates by using pulsed-field gel electrophoresis of large chromosomal fragments generated by SmaI digestion. The three West Haven isolates showed very similar chromosomal restriction endonuclease digestion patterns; these patterns were distinct from those of Bla+ and Bla- enterococci from other geographic areas and from those of other West Haven enterococci, suggesting an endemic Bla+ strain at this institution. Clinical information regarding these isolates suggests that exposure to the genitourinary clinic, obstructive urinary tract disease, and frequent antibiotic therapy may have been common risk factors for acquisition of this endemic strain.

Aged↗
PubMed1991

DNA fingerprinting of Enterococcus faecium by pulsed-field gel electrophoresis may be a useful epidemiologic tool.

Pulsed-field gel electrophoresis was used to compare 34 isolates of Enterococcus faecium from six different geographic locations. This procedure generated an average of 13 discernible fragment bands per isolate (range, 10 to 19 fragment bands) of 34 to 485 kb. The resulting restriction endonuclease digestion patterns were quite heterogeneous and were able to differentiate 27 of 34 isolates from each other, as defined by one or more mismatched fragment bands. Five patterns were shared by two or more isolates, and each set of isolates with matching patterns (shared pattern) originated in the same medical center, suggesting a common epidemiologic background, including highly penicillin resistant isolates in Richmond and Philadelphia. We conclude that pulsed-field gel electrophoresis of DNA digested with low-frequency-cleavage restriction enzymes offers a relatively simple method of comparing E. faecium for the purpose of epidemiologic study.

Bacterial Typing Techniques↗
PubMed1991

New aspects of antimicrobial resistance and the resulting therapeutic dilemmas.

Emergence of resistance to antimicrobial agents among previously susceptible organisms continues to be an important obstacle to the successful treatment of bacterial infections. In hospitals, plasmid-mediated resistance to third-generation cephalosporins and monobactams has recently appeared in gram-negative bacilli, due primarily to mutations in TEM- and SHV-type enzymes. Among nosocomial enterococci, vancomycin resistance, beta-lactamase production, and high-level resistance to all aminoglycosides have recently been added to this organism's already formidable armamentarium of resistance properties. Also, resistance to fluoroquinolones and rifampin has been emerging in methicillin-resistant Staphylococcus aureus. In the community, organisms in which resistance plays a particularly important role are shigellae, Haemophilus influenzae, gonococci, and pneumococci, particularly in developing countries. beta-lactamase-producing meningococci have been reported for the first time. The selective pressure generated by the use of antimicrobial agents, together with the ability of bacteria to acquire and spread resistance and the capacity of humans to transmit bacteria, suggest that antimicrobial resistance will continue to be a problem for the foreseeable future.

Bacterial Infections↗