Prevalence of the fsr locus in Enterococcus faecalis infections.

The fsr locus of Enterococcus faecalis confers virulence in animal models. A retrospective analysis of fsr prevalence in diverse E. faecalis clinical isolates demonstrated fsr in all endocarditis isolates versus 53% of stool isolates (P = 0.005). This supports a role for fsr-mediated virulence in the pathogenesis of enterococcal infections in humans.

Prolonged colonization with vancomycin-resistant Enterococcus faecium in long-term care patients and the significance of "clearance".

Little is known about the persistence of colonization with vancomycin-resistant Enterococcus faecium (VRE) in the nononcologic, non-intensive care unit patient. We studied all patients who had VRE isolated on > or =2 occasions of > 1 year apart (Study A) and those who had been "cleared" of VRE colonization after 3 negative stool cultures (Study B). Twelve patients had stored VRE isolates recovered > 1 year apart (Study A), and 58% of paired isolates were genotypically related according to pulsed field gel electrophoresis patterns. In Study B, stool samples were obtained weekly from 21 "cleared" patients for 5 weeks, which revealed that 24% were VRE positive. For these culture-positive patients, 72% of the cultures failed to detect VRE. Recent antibiotic use was significantly more common in the culture-positive patients, as compared with culture-negative patients (P=.003). Colonization with VRE may persist for years, even if the results of intercurrent surveillance stool and index site cultures are negative. Cultures for detection of VRE in stool samples obtained from patients declared "cleared" are insensitive.

Methicillin-resistant Staphylococcus aureus: comparison of susceptibility testing methods and analysis of mecA-positive susceptible strains.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the mecA gene used as the "gold standard" assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecA was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that mecA- and PBP 2a-positive S. aureus isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA.

Linezolid resistance in a clinical isolate of Staphylococcus aureus.

The new oxazolidinone antimicrobial, linezolid, has been approved for the treatment of infections caused by various gram-positive bacteria, including meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Although instances of linezolid resistance in VRE have been reported, resistance has not been encountered among clinical isolates of S aureus. We have characterised an MRSA isolate resistant to linezolid that was recovered from a patient treated with this agent for dialysis-associated peritonitis.

Vancomycin-resistant enterococci: mechanisms and clinical observations.

Enterococci are not generally regarded as highly virulent bacterial pathogens. However, resistance to many antimicrobial drugs complicates treatment of enterococcal infections. Acquired resistance to high concentrations of glycopeptide antibiotics, specifically vancomycin, has exacerbated this problem. This article seeks to concisely review the mechanisms of that resistance and its effects on clinical management of enterococcal infections, as well as clinical microbiology and infection control.

A hospital epidemic of vancomycin-resistant Enterococcus: risk factors and control.

OBJECTIVE: To determine risk factors for vancomycin-resistant Enterococcus (VRE) colonization during a hospital outbreak and to evaluate Centers for Disease Control and Prevention (CDC)-recommended control measures. DESIGN: Epidemiological study involving prospective identification of colonization and a case-control study. SETTING: A university hospital. PARTICIPANTS: Patients on eight wards involved in outbreak from late 1994 through early 1995. METHODS: Cases were matched by ward and culture date with up to two controls. Risk factors were evaluated with four multivariate models using conditional logistic regression. The first evaluated proximity to other VRE patients and isolation status. The second evaluated proximity to unisolated VRE cases and three variables independently predictive after adjustment for proximity. The third evaluated seven significant univariate predictors in addition to proximity to unisolated VRE in backward, stepwise logistic regression. The fourth assessed proximity to VRE with all other variables collected, clustered in a principal components analysis. Pulsed-field gel electrophoresis was performed to assess clonality of two outbreak strains. RESULTS: The incidence of transmission declined significantly after CDC guidelines were implemented. Proximity to unisolated VRE cases during the prior week was a significant predictor of acquisition in each of four multivariate models. Other significant risk factors in multivariate models included a history of major trauma and treatment with metronidazole. Pulsed-field gel electrophoresis confirmed the clonality of two outbreak strains. CONCLUSIONS: VRE was transmitted between patients during a hospital epidemic, with proximity to previously unisolated VRE patients being an important risk factor. Weekly surveillance cultures and contact isolation of colonized patients significantly reduced spread

Restoration of vancomycin susceptibility in Enterococcus faecalis by antiresistance determinant gene transfer.

We assessed the ability of gene transfer to reverse vancomycin resistance in class A (VanA) glycopeptide-resistant Enterococcus faecalis. Recombinant shuttle vectors containing a vanH promoter-vanA antisense gene cassette fully restored vancomycin susceptibility through a combined transcriptional activator binding domain decoy and inducible vanA antisense RNA effect.

Diversity of domain V of 23S rRNA gene sequence in different Enterococcus species.

The highly conserved central loop of domain V of 23S RNA (nucleotides 2042 to 2628; Escherichia coli numbering) is implicated in peptidyltransferase activity and represents one of the target sites for macrolide, lincosamide, and streptogramin B antibiotics. DNA encoding domain V (590 bp) of several species of Enterococcus was amplified by PCR. Twenty enterococcal isolates were tested, including Enterococcus faecium (six isolates), Enterococcus faecalis, Enterococcus avium, Enterococcus durans, Enterococcus gallinarum, Enterococcus casseliflavus (two isolates of each), and Enterococcus raffinosus, Enterococcus mundtii, Enterococcus malodoratus, and Enterococcus hirae (one isolate of each). For all isolates, species identification by biochemical testing was corroborated by 16S rRNA gene sequencing. The sequence of domain V of the 23S rRNA gene from E. faecium and E. faecalis differed from those of all other enterococci. The domain V sequences of E. durans and E. hirae were identical. This was also true for E. gallinarum and E. casseliflavus. E. avium differed from E. casseliflavus by 23 bases, from E. durans by 16 bases, and from E. malodoratus by 2 bases. E. avium differed from E. raffinosus by one base. Despite the fact that domain V is considered to be highly conserved, substantial differences were identified between several enterococcal species.

SME-type carbapenem-hydrolyzing class A beta-lactamases from geographically diverse Serratia marcescens strains.

Three sets of carbapenem-resistant Serratia marcescens isolates have been identified in the United States: 1 isolate in Minnesota in 1985 (before approval of carbapenems for clinical use), 5 isolates in Los Angeles (University of California at Los Angeles [UCLA]) in 1992, and 19 isolates in Boston from 1994 to 1999. All isolates tested produced two beta-lactamases, an AmpC-type enzyme with pI values of 8.6 to 9.0 and one with a pI value of approximately 9.5. The enzyme with the higher pI in each strain hydrolyzed carbapenems and was not inhibited by EDTA, similar to the chromosomal class A SME-1 beta-lactamase isolated from the 1982 London strain S. marcescens S6. The genes encoding the carbapenem-hydrolyzing enzymes were cloned in Escherichia coli and sequenced. The enzyme from the Minnesota isolate had an amino acid sequence identical to that of SME-1. The isolates from Boston and UCLA produced SME-2, an enzyme with a single amino acid change relative to SME-1, a substitution from valine to glutamine at position 207. Purified SME enzymes from the U. S. isolates had beta-lactam hydrolysis profiles similar to that of the London SME-1 enzyme. Pulsed-field gel electrophoresis analysis revealed that the isolates showed some similarity but differed by at least three genetic events. In conclusion, a family of rare class A carbapenem-hydrolyzing beta-lactamases first described in London has now been identified in S. marcescens isolates across the United States.

A cluster of VanD vancomycin-resistant Enterococcus faecium: molecular characterization and clinical epidemiology.

VanD-mediated glycopeptide resistance has been reported for an isolate of Enterococcus faecium, BM4339. Three clinical isolates of vancomycin-resistant E. faecium collected from 3 patients during a 6-week period in 1993 had agar dilution MICs of vancomycin and teicoplanin of 128 and 4 microg/mL, respectively. Polymerase chain reaction (PCR) using degenerate primers complementary to genes encoding d-Ala-d-X ligases yielded a 630-bp product that was similar to the published partial sequence of vanD. By use of inverse PCR, vanD, vanHD, and two partial flanking open-reading frames were sequenced. The deduced amino acid sequence of VanD showed 67% identity with VanA and VanB. vanD appeared to be located on the chromosome and was not transferable to other enterococci. The 3 isolates were indistinguishable by pulsed-field gel electrophoresis and differed from BM4339. No other isolates carrying vanD were found in a subset of 875 recent US isolates of vancomycin-resistant enterococci.

Vancomycin-resistant enterococci in intensive care units: high frequency of stool carriage during a non-outbreak period.

BACKGROUND: We aimed to define the epidemiological associations of vancomycin-resistant enterococci (VRE) in intensive care units (ICUs) during a non-outbreak period by examining prevalence, risk factors for colonization, frequency of acquisition, and molecular strain types. DESIGN: A prospective cohort design was followed. Consecutive patient admissions to 2 surgical ICUs at a tertiary care hospital were enrolled. The main outcome measures were results of serial surveillance cultures screened for VRE. RESULTS: Of 290 patients enrolled, 35 (12%) had colonization with VRE on admission. The VRE colonization or infection had been previously detected by clinical cultures in only 4 of these patients. Using logistic regression, VRE colonization at the time of ICU admission was associated with second- and third-generation cephalosporins (odds ratio [OR] = 6.0, P<.0001), length of stay prior to surgical ICU admission (OR = 1.06, P = .001) greater than 1 prior ICU stay (OR = 9.6, P = .002), and a history of solid-organ transplantation (OR = 3.8, P = .021). Eleven (12.8%) of 78 patients with follow-up cultures acquired VRE. By pulsed-field gel electrophoresis, 2 strains predominated, one of which was associated with an overt outbreak on a non-ICU ward near the end of the study period. CONCLUSIONS: Colonization was common and usually not recognized by clinical culture. Most patients who had colonization with VRE and were on the surgical ICU acquired VRE prior to surgical ICU entry. Exposure to second- and third-generation cephalosporins, but not vancomycin, was an independent risk factor for colonization. Prospective surveillance of hospitalized patients may yield useful insights about the dissemination of nosocomial VRE beyond what is appreciated by clinical cultures alone.

Characterization of vancomycin-resistant Enterococcus faecium isolates from the United States and their susceptibility in vitro to dalfopristin-quinupristin.

In the course of clinical studies with the investigational streptogramin antimicrobial dalfopristin-quinupristin, isolates of vancomycin-resistant Enterococcus faecium were referred to our laboratory from across the United States. Seventy-two percent of the strains were of the VanA type, phenotypically and genotypically, while 28% were of the VanB type. High-level resistance to streptomycin or gentamicin was observed in 86 and 81%, respectively, of the VanA strains but in only 69 and 66%, respectively, of the VanB strains. These enterococci were resistant to ampicillin (MIC for 50% of the isolates tested [MIC50] and MIC90, 128 and 256 microg/ml, respectively) and to the other approved agents tested, with the exception of chloramphenicol (MIC90, 8 microg/ml) and novobiocin (MIC90, 1 microg/ml). Considering all of the isolates submitted, dalfopristin-quinupristin inhibited 86.4% of them at concentrations of < or = 1 microg/ml and 95.1% of them at < or = 2 microg/ml. However, for the data set comprised of only the first isolate submitted for each patient, 94.3% of the strains were inhibited at concentrations of < or = 1 microg/ml and 98.9% were inhibited at concentrations of < or = 2 microg/ml. Multiple drug resistance was very common among these isolates of vancomycin-resistant E. faecium, while dalfopristin-quinupristin inhibited the majority at concentrations that are likely to be clinically relevant.

Influence of erythromycin resistance, inoculum growth phase, and incubation time on assessment of the bactericidal activity of RP 59500 (quinupristin-dalfopristin) against vancomycin-resistant Enterococcus faecium.

RP 59500, a mixture of two semisynthetic streptogramin antibiotics (quinupristin and dalfopristin), is one of a few investigational agents currently in clinical trials with inhibitory activity against multiple-drug-resistant strains of Enterococcus faecium. We evaluated the bactericidal activity of this antimicrobial against 30 recent clinical isolates of vancomycin-resistant E. faecium, including 23 erythromycin-resistant (MIC, >256 microg/ml) and 7 erythromycin-intermediate (MIC, 2 to 4 microg/ml) strains. All isolates were inhibited by RP 59500 at 0.25 to 1.0 microg/ml. The bactericidal activity of RP 59500 was markedly influenced by the erythromycin susceptibility of the strains and by several technical factors, such as inoculum growth phase and time of incubation of counting plates. As determined by time-kill methods, RP 59500 at a concentration of 2 or 8 microg/ml failed to kill erythromycin-resistant organisms under any conditions. Bactericidal activity was observed against all seven erythromycin-intermediate isolates when log-phase inocula were used and the cells were counted after 48 h of incubation (mean reductions in viable bacteria for RP 59500 at concentrations of 2 and 8 microg/ml, 3.45 and 3.50 log10 CFU/ml, respectively), but killing was diminished when the plates were examined at 72 h (mean killing, 3.06 and 2.95 log10, CFU/ml, respectively). No bactericidal activity was observed when stationary-phase cultures were used. On the basis of these data, we expect that bactericidal activity of RP 59500 against the multiple-drug-resistant E. faecium strains currently encountered would be distinctly uncommon.

Catheter-associated Staphylococcus aureus bacteremia.

The majority of cases of Staphylococcus aureus bacteremia are hospital-acquired, and most are associated with infected intravenous catheters. Preventive measures, early detection of infections, and strategies for effective treatment have become matters of increasing urgency.

In vitro activities in new oxazolidinone antimicrobial agents against enterococci.

The comparative in vitro activities of two new oxazolidinone antimicrobial agents, U-100592 and U-100766, against 180 isolates of enterococci representing several resistance profiles were examined by using an agar dilution technique. The two oxazolidinones inhibited all isolates, including strains resistant to vancomycin, ampicillin, and minocycline, at concentrations between 1 and 4 micrograms/ml.

A gene conferring resistance to vancomycin but not teicoplanin in isolates of Enterococcus faecalis and Enterococcus faecium demonstrates homology with vanB, vanA, and vanC genes of enterococci.

We report the sequence of a 630-bp fragment of a gene associated with resistance to high levels of vancomycin in a clinical isolate of Enterococcus faecalis which retained susceptibility to teicoplanin. This gene was similar to the recently sequenced vanB and partially homologous with vanA, but it showed less-marked similarity to vanC. A DNA probe, derived from this polymerase chain reaction-amplified gene fragment, hybridized specifically with genomic DNA from Enterococcus faecium and E. faecalis isolates which were vancomycin resistant (MICs ranged from 8 to 512 micrograms/ml) but susceptible to teicoplanin. Curing of vancomycin resistance was associated with loss of DNA hybridization with the gene probe. Transfer of DNA which hybridized with the probe accompanied transfer of vancomycin resistance to a susceptible recipient strain. Neither curing nor transfer of vancomycin resistance was consistently related to loss or acquisition, respectively, of plasmid DNA.

Formation constant and stoichiometry of the acetonitrile:18-crown-6 complex by nuclear magnetic resonance, raman and infrared spectroscopy.

Crown ethers are increasingly used in a variety of chemical applications. While crown ether complexes with alkali-metal cations have been extensively studied, relatively little is known about their complexes with neutral molecules to form so-called host: guest complexes. The use of NMR is reported for the determination of the formation constant (2.1 +/- 0.1) for the acetonitrile: 18-crown-6 complex. The suitability of Raman spectroscopy for studies of the solid-phase complex is demonstrated and limitations in the use of infrared spectroscopy are discussed.