Biomedical subjects

Barbara E Murray

Publications and source records attributed to Barbara E Murray.

17 recordsLinked to original sources

PubMed2004

Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis.

Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).

Bacterial Adhesion↗

PubMed2004

Translocation of Enterococcus faecalis strains across a monolayer of polarized human enterocyte-like T84 cells.

We used a two-chamber system to study transcytosis of Enterococcus faecalis across monolayers of human colon carcinoma-derived T84 cells, which show structural resemblance to the native intestine. Among 16 E. faecalis isolates from different sources, the well-characterized strain OG1RF and 8 other isolates (2 endocarditis isolates, 1 urine isolate, and all 5 fecal isolates) showed translocation in this assay, while 6 clinical isolates (3 endocarditis and 3 urine isolates), the recipient strain JH2-2, and the control, Escherichia coli DH5alpha, had no detectable translocation. Of two OG1RF mutants involving the previously studied epa (enterococcal polysaccharide antigen) gene cluster, known to be needed for virulence and resistance to killing by polymorphonuclear leukocytes, one epa mutant (TX5179) was unable to translocate, while TX5180, with an epa disruption farther downstream, showed a moderate decrease in translocation relative to that of the wild-type strain OG1RF (P < 0.01), indicating that the epa gene cluster is important for translocation across a T84 monolayer. This observation was confirmed by complementation of the epa mutant (TX5179) with epa genes and restoration of its translocation ability. In conclusion, we have demonstrated translocation of at least some strains of E. faecalis across T84 monolayers, although strains differ considerably in this ability, and we have demonstrated that epa mutations can cause marked changes in successful translocation. These results suggest that this model may be a useful in vitro system for studying the process of translocation from the intestinal tract.

Bacterial Translocation↗

PubMed2004

Molecular epidemiology of the fsr locus and of gelatinase production among different subsets of Enterococcus faecalis isolates.

We examined 215 Enterococcus faecalis isolates and found that neither the two-component regulatory locus fsr (E. faecalis regulator) nor gelatinase production was more common in disease-associated isolates than in isolates colonizing healthy individuals (ca. 60 to 65%). The majority of gelatinase-negative isolates, including 14 endocarditis isolates (of 80 isolates tested), contained the previously described 23.9-kb deletion and lacked fsrA and fsrB. While these findings indicate that neither fsr nor gelatinase is required for E. faecalis to cause infection, this study did not address whether fsr or gelatinase affects the severity of disease, as it does in animal models.

Base Sequence↗

PubMed2003

Conventional and molecular epidemiology of trimethoprim-sulfamethoxazole resistance among urinary Escherichia coli isolates.

BACKGROUND: Antibiotic resistance is increasing in Escherichia coli, the most common cause of urinary tract infections, but its epidemiology has not been well described. We evaluated the epidemiology of trimethoprim-sulfamethoxazole-resistant E. coli in a large, public health care system in Denver, Colorado. METHODS: Outpatients with E. coli urinary tract infections during the first 6 months of 1998 were evaluated retrospectively. A prospective study was then performed to confirm the rate of trimethoprim-sulfamethoxazole resistance. We used several strain-typing methods (pulsed-field gel electrophoresis, ribotyping, serotyping) to evaluate the molecular epidemiology of the resistance. RESULTS: The rate of trimethoprim-sulfamethoxazole resistance was similar in the retrospective (24% [161/681]) and prospective (23% [30/130]) phases of the study (P = 0.89). Almost all trimethoprim-sulfamethoxazole-resistant strains (98%) were resistant to at least one other antibiotic. Risk factors for infection with a resistant strain included age < or =3 years, Hispanic ethnicity, recent travel outside the United States, and a prior urinary tract infection. However, rates of resistance were >15% among nearly all of the subgroups. Most strains had high-level resistance (>1000 microg/mL) to trimethoprim-sulfamethoxazole. Of the 23 resistant isolates evaluated, 10 (43%) belonged to the clone A group. There was no correlation between conventional epidemiologic characteristics and the molecular mechanism of resistance or strain type. CONCLUSION: Resistance to trimethoprim-sulfamethoxazole among E. coli isolates among patients in a Denver public health care system is common, with high rates of resistance even among patients without risk factors.

Adolescent↗

PubMed2003

Relapse of type A beta-lactamase-producing Staphylococcus aureus native valve endocarditis during cefazolin therapy: revisiting the issue.

Our experience with a patient with methicillin-susceptible Staphylococcus aureus aortic native valve endocarditis, who had a relapse involving fever and positive blood culture results while receiving cefazolin, led us to evaluate this organism's ability to hydrolyze cefazolin at high inocula, a previously well-documented phenomenon. Analysis of the infecting strain disclosed a high minimum inhibitory concentration of cefazolin when a large inoculum was used, as well as rapid and complete cefazolin degradation, which was associated with regrowth in a time-kill experiment. DNA sequencing of the beta-lactamase gene showed that it was identical to that of the S. aureus type A beta-lactamase, known to efficiently inactivate cefazolin. A word of caution is given regarding the use of this antibiotic for treatment of endocarditis caused by this type of S. aureus isolate.

Anti-Bacterial Agents↗

PubMed2003

A potential virulence gene, hylEfm, predominates in Enterococcus faecium of clinical origin.

An open reading frame (hyl(Efm)) with homologies to previously described hyaluronidase genes has been identified in nonstool isolates of Enterococcus faecium. E. faecium isolates (n=577) from diverse sources were screened for the presence of hyl(Efm) and esp(Efm), a putative virulence gene associated with epidemic E. faecium strains. The presence of esp(Efm) was roughly twice that of hyl(Efm), but both were found primarily in vancomycin-resistant E. faecium isolates in nonstool cultures obtained from patients hospitalized in the United States. These data suggest that specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Differences in the prevalence of these strains may help explain variations in the clinical importance of multiresistant E. faecium across different continents.

Anti-Bacterial Agents↗

PubMed2003

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family.

A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.

Adhesins, Bacterial↗

PubMed2003

Activity of tigecycline (GAR-936), a novel glycylcycline, against Enterococci in the mouse peritonitis model.

A novel glycylcycline agent, tigecycline (GAR-936), was evaluated in vivo in the mouse model of peritonitis against three Enterococcus faecalis and four Enterococcus faecium isolates with different susceptibilities to vancomycin and tetracyclines, all of which were inhibited by </=0.125 micro g of tigecycline/ml. Using a single subcutaneous dose, tigecycline displayed a protective effect (50% protective dose, </=5.7 mg/kg of body weight) against all strains tested, including two with Tn925 (from the Tn916 family), which contains the Tet(M) tetracycline resistance determinant, as well as VanA and VanB strains. As expected, tetracycline and minocycline were ineffective against the isolates carrying Tn925.

Animals↗

PubMed2003

In vivo efficacy of the ketolide ABT-773 (cethromycin) against enterococci in a mouse peritonitis model.

Using six Enterococcus faecalis and five Enterococcus faecium strains, the ketolide ABT-773 (ABT), now known as cethromycin, was found to have in vivo efficacy against both erythromycin (ERY)-susceptible (Ery(s)) and -intermediate (Ery(i)) enterococci (ABT 50% protective doses [PD(50)s], 0.5 to 4.1 and 10.3 to 16.2 mg/kg of body weight, respectively). Against four highly Ery-resistant (Ery(r)) strains for which ABT MICs were low, ABT showed much greater activity (PD(50), 6.3 to 32.5 mg/kg) than ERY (PD(50), >200 mg/kg) but was not protective for strains for which ABT MICs were high. In conclusion, ABT-773 showed in vivo efficacy and considerably greater activity than ERY in a mouse peritonitis model.

Animals↗

PubMed2003

An Enterococcus faecium secreted antigen, SagA, exhibits broad-spectrum binding to extracellular matrix proteins and appears essential for E. faecium growth.

A gene encoding a major secreted antigen, SagA, was identified in Enterococcus faecium by screening an E. faecium genomic expression library with sera from patients with E. faecium-associated endocarditis. Recombinant SagA protein showed broad-spectrum binding to extracellular matrix (ECM) proteins, including fibrinogen, collagen type I, collagen type IV, fibronectin, and laminin. A fibrinogen-binding protein, purified from culture supernatants of an E. faecium clinical isolate, was found to match the N-terminal sequence of the predicted SagA protein and to react with the anti-SagA antibody, confirming that it was the SagA protein; this protein appeared as an 80- to 90-kDa smear on a Western blot that was sensitive to proteinase K and resistant to periodate treatment and glycoprotein staining. When overexpressed in E. faecium and Escherichia coli, the native and recombinant SagA proteins formed stable oligomers, apparently via their C-terminal domains. The SagA protein is composed of three domains: (i) a putative coiled-coil N-terminal domain that shows homology to the N-terminal domain of Streptococcus mutans SagA protein (42% similarity), previously shown to be involved in cell wall integrity and cell shape maintenance, and to the P45 protein of Listeria monocytogenes (41% similarity); (ii) a central domain containing direct repeats; and (iii) a C-terminal domain that is similar to that found in various proteins, including P45 (50% similarity) and P60 (52% similarity) of L. monocytogenes. The P45 and P60 proteins both have cell wall hydrolase activity, and the latter has also been shown to be involved in virulence, whereas cell wall hydrolase activity was not detected for SagA protein. The E. faecium sagA gene, like the S. mutans homologue, is located in a cluster of genes encoding proteins that appear to be involved in cell wall metabolism and could not be disrupted unless it was first transcomplemented, suggesting that the sagA gene is essential for E. faecium growth and may be involved in cell wall metabolism. In conclusion, the extracelluar E. faecium SagA protein is apparently essential for growth, shows broad-spectrum binding to ECM proteins, forms oligomers, and is antigenic during infection.

Amino Acid Sequence↗

PubMed2003

Role of the Enterococcus faecalis GelE protease in determination of cellular chain length, supernatant pheromone levels, and degradation of fibrin and misfolded surface proteins.

Gelatinase (GelE), a secreted Zn-metalloprotease of Enterococcus faecalis, has been implicated as a virulence factor by both epidemiological data and animal model studies. Expression of gelE is induced at a high cell density by the fsr quorum-sensing system. In the present study, GelE was shown to be responsible for the instability of a number of Asc10 (aggregation substance) mutant proteins, implying that GelE functions to clear the bacterial cell surface of misfolded proteins. Disruption of GelE production led to increased cell chain length of E. faecalis, from a typical diplococcus morphology to chains of 5 to 10 cells. This function of GelE was also exhibited when the protein was expressed in Streptococcus pyogenes. GelE-expressing E. faecalis strains were more autolytic, suggesting that GelE affects chain length through activation of an autolysin. GelE was also essential for degradation of polymerized fibrin. GelE expression reduced the titer of cCF10, the peptide pheromone that induces conjugation of pCF10, and pCF10 had increased conjugation into non-GelE-expressing strains. These new functions attributed to GelE suggest that it acts to increase the dissemination of E. faecalis in high-density environments.

Bacterial Proteins↗

PubMed2002

An Enterococcus faecalis ABC homologue (Lsa) is required for the resistance of this species to clindamycin and quinupristin-dalfopristin.

Enterococcus faecalis isolates are resistant to clindamycin (CLI) and quinupristin-dalfopristin (Q-D), and this is thought to be a species characteristic. Disruption of a gene (abc-23, now designated lsa, for "lincosamide and streptogramin A resistance") of E. faecalis was associated with a > or =40-fold decrease in MICs of Q-D (to 0.75 microg/ml), CLI (to 0.12 to 0.5 microg/ml), and dalfopristin (DAL) (to 4 to 8 microg/ml) for the wild-type E. faecalis parental strain (Q-D MIC, 32 microg/ml; CLI MIC, 32 to 48 microg/ml; DAL MIC, 512 microg/ml). Complementation of the disruption mutant with lsa on a shuttle plasmid resulted in restoration of the MICs of CLI, Q-D, and DAL to wild-type levels. Under high-stringency conditions, lsa was found in 180 of 180 isolates of E. faecalis but in none of 189 other enterococci. Among 19 erm(B)-lacking Enterococcus faecium strains, 9 (47%) were highly susceptible to CLI (MIC, 0.06 to 0.25 microg/ml) and had DAL MICs of 4 to 16 microg/ml; for the remaining erm(B)-lacking E. faecium strains, the CLI and DAL MICs were 4 to > 256 and 2 to > 128 microg/ml, respectively. In contrast, none of 32 erm(B)-lacking E. faecalis strains were susceptible (CLI MIC range, 16 to 32 microg/ml; DAL MIC range, > or =32 microg/ml). When lsa was introduced into an E. faecium strain initially susceptible to CLI, the MICs of CLI and DAL increased > or =60-fold and that of Q-D increased 6-fold (to 3 to 6 microg/ml). Introduction of lsa into two DAL-resistant (MICs, > 128 microg/ml), Q-D-susceptible (MICs, 0.5 and 1.5 microg/ml) E. faecium strains (CLI MICs, 12 and >256 microg/ml) resulted in an increase in the Q-D MICs from 3- to 10-fold (to 8 and >32 microg/ml), respectively. Although efflux was not studied, the similarity (41 to 64%) of the predicted Lsa protein to ABC proteins such as Vga(A), Vga(B), and Msr(A) of Staphylococcus aureus and YjcA of Lactococcus lactis and the presence of Walker A and B ATP-binding motifs suggest that this resistance may be related to efflux of these antibiotics. In conclusion, lsa appears to be an intrinsic gene of E. faecalis that explains the characteristic resistance of this species to CLI and Q-D.

Amino Acid Sequence↗

PubMed2002

Virulence effect of Enterococcus faecalis protease genes and the quorum-sensing locus fsr in Caenorhabditis elegans and mice.

The expression of two Enterococcus faecalis extracellular virulence-related proteins, gelatinase (GelE) and serine protease (SprE), has been shown to be positively regulated by the fsr quorum-sensing system. We recently developed a novel system for studying E. faecalis pathogenicity that involves killing of the nematode worm Caenorhabditis elegans and showed that an E. faecalis fsrB mutant (strain TX5266) exhibited attenuated killing. We explore here the role of the fsr/gelE-sprE locus in pathogenicity by comparing results obtained in the nematode system with a mouse peritonitis model of E. faecalis infection. Insertion mutants of fsrA (TX5240) and fsrC (TX5242), like fsrB (TX5266), were attenuated in their ability to kill C. elegans. A deletion mutant of gelE (TX5264) and an insertion mutant of sprE (TX5243) were also attenuated in C. elegans killing, although to a lesser extent than the fsr mutants. Complementation of fsrB (TX5266) with a 6-kb fragment containing the entire fsr locus restored virulence in both the nematode and the mouse peritonitis models. The fsr mutants were not impaired in their ability to colonize the nematode intestine. These data show that extracellular proteases and the quorum-sensing fsr system are important for E. faecalis virulence in two highly divergent hosts: nematodes and mice.

Animals↗

PubMed2002

Involvement of PhoP-PhoS homologs in Enterococcus faecalis virulence.

Eleven PhoP-PhoS homolog pairs were identified by searching the Enterococcus faecalis V583 genome sequence database at The Institute for Genomic Research with the Bacillus subtilis PhoP-PhoS sequences. Each pair appears to be a potential two-component system composed of a response regulator and a sensor kinase. Seven of the homologs were disrupted in E. faecalis strain OG1RF. TX10293, a mutant disrupted in one of these genes (etaR, the first gene of the gene pair designated etaRS), showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. The predicted EtaR protein sequence showed greatest similarity to LisR of Listeria monocytogenes (77%) and CsrR of Streptococcus pyogenes (70%); EtaS is 53% similar to LisK and 54% similar to CsrS. When grown in vitro, the TX10293 mutant was more sensitive to low pH (pH 3.4) and more resistant to high temperature (55 degrees C) than wild-type OG1RF. In conclusion, many potential two-component systems are identified for E. faecalis, one of which, EtaRS, was shown to be involved in stress response and virulence.

Amino Acid Sequence↗

PubMed2002

Evidence that the enterococcal polysaccharide antigen gene (epa) cluster is widespread in Enterococcus faecalis and influences resistance to phagocytic killing of E. faecalis.

In previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis (epa) from Enterococcus faecalis strain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in Escherichia coli. Disruption of two open reading frames in the epa gene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with E. faecalis endocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecular-weight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF (P < or = 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF (P < or = 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the epa gene cluster of OG1RF (which is also present in the sequenced E. faecalis strain V583) was detected in 12 of 12 other clonally distinct E. faecalis strains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an E. faecalis endocarditis patient serum, and sera from four other patients with E. faecalis endocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the epa gene cluster is widespread among E. faecalis and confers some protection against human host defenses.

Antigens, Bacterial↗

PubMed2002

The Enterococcus faecalis fsrB gene, a key component of the fsr quorum-sensing system, is associated with virulence in the rabbit endophthalmitis model.

We used a rabbit endophthalmitis model to explore the role of fsrB, a gene required for the function of the fsr quorum-sensing system of Enterococcus faecalis, in pathogenicity. A nonpolar deletion mutant of fsrB had significantly reduced virulence compared to wild type. Complementation of mutation restored virulence. These data corroborate the role of fsrB in E. faecalis pathogenesis and suggest that the rabbit endophthalmitis model can be used to study the in vivo role of quorum sensing.

Animals↗

PubMed2002

Molecular typing of selected Enterococcus faecalis isolates: pilot study using multilocus sequence typing and pulsed-field gel electrophoresis.

The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differentiation of Enterococcus faecalis isolates. We sequenced intragenic regions of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen) and one housekeeping gene (pyrC) of 22 E. faecalis isolates chosen largely for their temporal and geographical diversity, but also including some outbreak isolates. MLST analysis of polymorphic regions of these four genes identified 13 distinct sequence types (STs) with different allelic profiles; the composite sequences generated from the four sequenced gene fragments of individual isolates showed 98.3 to 100% identity among the 22 isolates. We also found that the allelic profiles from two sequences, ace and salA, were sufficient to distinguish all 13 STs of this study. The 13 STs corresponded to 12 different PFGE types, with one previously designated PFGE clone (a widespread U.S. clone of beta-lactamase-producing isolates) being classified into two highly related STs which differed at 2 of 2,894 bases, both in the same allele. MLST also confirmed the clonal relationships among the isolates of two other PFGE clonal groups, including vancomycin resistant isolates. Thus, this pilot study with representative E. faecalis isolates suggests that, similar to PFGE, the sequence-based typing method may be useful for differentiating isolates of E. faecalis to the subspecies level in addition to identifying outbreak isolates.

Base Sequence↗