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PubMed2001

Evaluation of a biochemical test scheme for identifying clinical isolates of Enterococcus faecalis and Enterococcus faecium.

AIMS: To evaluate the full test scheme of Facklam and Sahm (1995) for the identification of clinical enterococcal isolates to genus and species level. METHODS AND RESULTS: Fifty-nine clinical isolates, previously provisionally classed as enterococci on the basis of just four biochemical tests of Facklam and Sahm and one other test, were subjected to genus and species identification using the full identification scheme of Facklam and Sahm; 98% of these strains were confirmed to be enterococci and of these, 69% were identified as Enterococcus faecalis and 31% as Enterococcus faecium. Six tests in the scheme (out of 24) gave anomalous or unreliable results for some strains, and two gave unexpected results for the majority of strains presumptively identified as Ent. faecium. CONCLUSIONS: Nine (out of 12) genus tests and nine (out of 12) species tests from the Facklam and Sahm scheme were reliable. Testing for the presence of the Lancefield antigen D was also useful. The majority of presumptive Ent. faecium strains gave different results for the sorbitol and raffinose tests from that expected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the level of reliability for each of the tests in a current enterococcal identification scheme for differentiating clinical isolates, and showed that two tests gave consistently different test results from those expected for Ent. faecium.

Bacterial Typing Techniques↗
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↗
PubMed2000

Incomplete cross resistance against ionophores in Enterococcus faecium and Enterococcus faecalis strains from pigs and poultry.

Thirty-two Enterococcus faecium strains and 33 Enterococcus faecalis strains were tested for their susceptibility to the ionophore antibiotics salinomycin, narasin, monensin, and lasalocid. Enterococcal strains originated from poultry in which these products are in use as coccidiostats, and from pigs in which these products are allowed as growth promoters. Resistance against salinomycin and narasin in enterococci was frequent among poultry strains, whereas in pig strains, resistance was less common. No resistance was found against monensin and lasalocid. Full cross resistance between salinomycin and narasin was evident. There was no cross resistance between these two ionophores and monensin and lasalocid.

Animals↗
PubMed2005

Analysis of the prevalence of tetracycline resistance genes in clinical isolates of Enterococcus faecalis and Enterococcus faecium in a Japanese hospital.

Prevalence of seven tetracycline resistance (TC(R)) genes--tet(L), tet(M), tet(K), tet(O), tet(S), tet(T), and tet(U)--which are known to be distributed to gram-positive cocci was analyzed for 224 Enterococcus faecalis and 46 Enterococcus faecium clinical isolates obtained in a Japanese hospital. Any of the TC(R) genes was detected in 75.9% of all the enterococcal strains. The tet(M) was detected at highest rates in both E. faecalis (75.0%) and E. faecium (69.6%), followed by tet(L), which was harbored in 6.7% of E. faecalis isolates and 30.4% of E. faecium isolates. The tet(O), tet(S), and tet(T) were detected in E. faecalis at low frequencies mostly associated with tet(M), while tet(K) and tet(U) were not detected. Nucleotide sequences of tet(S) from E. faecalis strains were identical to that reported in Listeria monocytogenes. Sequences of tet(O) from two E. faecalis strains were almost identical to each other and also to those from Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Campylobacter jejuni, and Campylobacter coli, although minor sequence divergence was observed. The tet(T), which had been reported only in Streptococcus pyogenes, was found in five E. faecalis strains. Sequence of the enterococcal tet(T) differed from that of S. pyogenes by only four nucleotides (four amino acids) and showed high sequence identity (99.8%, amino acid level). Enterococcal strains with any one TC(R) gene or those with two TC(R) genes showed generally similar MICs of tetracyclines, and no evident difference in resistance level was observed.

Amino Acid Sequence↗
PubMed1995

Enterococcus faecium and Enterococcus faecalis bacteremia: acquisition and outcome.

The incidence of enterococcal bacteremia due to Enterococcus faecium is increasing. To understand the clinical significance of E. faecium bacteremia, we compared 16 patients who were bacteremic due to E. faecium to 56 patients who were bacteremic due to Enterococcus faecalis. E. faecium bacteremia developed most frequently in severely ill patients with fever or hypothermia accompanied by CNS, cardiovascular, and/or pulmonary dysfunction, while E. faecalis bacteremia occurred most often in less seriously ill patients. Nosocomial acquisition, cancer, neutropenia, renal insufficiency, current corticosteroid therapy, and previous treatment with broad-spectrum antibiotics were significantly more frequently associated with E. faecium bacteremia. Mortality was significantly higher among patients infected with E. faecium than among those infected with E. faecalis (50% vs. 11%; P = .001); this was true particularly among patients with monomicrobial or nosocomial bacteremia, those who had previously received antibiotic treatment, and those with cancer. Death due to enterococcal bacteremia was observed only among severely ill patients. These findings suggest that E. faecium often infects debilitated patients and that such infection appears to be a significant factor contributing to mortality.

Bacteremia↗
PubMed1998

In-vitro selection of resistance to vancomycin and teicoplanin in Enterococcus faecium and Enterococcus faecalis compared with Staphylococcus epidermidis.

Clinical isolates of Enterococcus faecium, Enterococcus faecalis and Staphylococcus epidermidis were studied for their ability to develop resistance by two selection methods, one in broth and one on agar. The MICs for enterococci after exposure to vancomycin ranged from 0.5 mg/L to 4 mg/L, and those after exposure to teicoplanin ranged from 0.25 mg/L to 1 mg/L. No significant increases occurred for E. faecalis, whilst two isolates of E. faecium showed increases of up to eight-fold (from 0.125 mg/L to 1 mg/L). Vancomycin MICs ranged from 1 mg/L to 4 mg/L and teicoplanin MICs reached 32 mg/L for S. epidermidis. Pulsed-field gel electrophoresis analysis of SmaI-digested whole chromosomal DNA was performed to compare the genomic DNA of glycopeptide-exposed and wild-type strains. In-vitro exposure to vancomycin did not alter MICs significantly except in one S. epidermidis isolate for which the MIC reached 4 mg/L, whereas the teicoplanin-exposed cultures of S. epidermidis showed increases of up to 64 times the original MIC. Comparable results were achieved with the two selection methods.

Anti-Bacterial Agents↗
PubMed2003

Prevalence of resistance to ampicillin, gentamicin and vancomycin in Enterococcus faecalis and Enterococcus faecium isolates from clinical specimens and use of antimicrobials in five Nordic hospitals.

We determined the species distribution and prevalence of ampicillin resistance, high-level gentamicin resistance (HLGR) and vancomycin resistance among clinical enterococcal isolates from five Nordic laboratories (Bergen, Tromsø, Uppsala, Aarhus and Reykjavik). Isolates represented three different groups: (i) all blood culture isolates from 1999; (ii) consecutive in-patient isolates (maximum 40); and (iii) consecutive outpatient isolates (maximum 40) collected during March to May 2000. Antimicrobial use data were collected at the national and hospital level. A high proportion (31.4%) of Enterococcus faecium was detected among blood culture isolates, in contrast to only 4.2% among isolates from outpatients. Ampicillin resistance was not found in Enterococcus faecalis, in contrast to 48.8% in E. faecium isolates. HLGR rates varied considerably between laboratories (1.1-27.6%). Acquired vancomycin resistance was not detected. There were no significant differences in the prevalences of HLGR between in-patient and outpatient isolates at individual hospitals. A cluster of clonally related ampicillin-resistant and HLGR E. faecium isolates was demonstrated in one of the hospitals. The lowest level of hospital antimicrobial use, the lowest proportion of E. faecium and the lowest prevalence of resistance were observed in Reykjavik. The study showed a relatively low level of resistance in enterococci, as compared with most European countries and the USA. However, there were large differences between hospitals with regard to the relative proportion of E. faecium isolates, their susceptibility to ampicillin and gentamicin, as well as the prevalence of HLGR in E. faecalis isolates. This indicates a potential for further improvement of antibiotic policies, and possibly hospital infection control, to maintain the low resistance levels observed in these countries.

Ampicillin Resistance↗
PubMed2004

Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis.

OBJECTIVES: The enterococcal surface protein gene, esp, is a major putative pathogenicity marker in clinical isolates of Enterococcus faecium and Enterococcus faecalis. This study demonstrates in vitro conjugative transfer of the esp gene among E. faecium and E. faecalis. MATERIALS AND METHODS: Enterococcal isolates from clinical samples, positive for esp, were mated on filters with enterococcal recipients. Transconjugants were checked for transfer of antibiotic resistance determinants and co-mobilization of the esp gene. They were also characterized by PCR and plasmid profiling/PFGE typing including Southern hybridizations with labelled esp probes. Transfer as triggered by excision was tested using Taqman PCR. RESULTS: Two of five E. faecalis and five of nine E. faecium transferred antibiotic resistance determinants into a recipient. Of the transconjugants analysed by PCR for acquisition of esp, only isolates from two E. faecalis and a single E. faecium mating were positive. In the donor strains, the esp gene was located on the chromosome. Molecular analysis revealed a plasmid localization of esp in the E. faecium transconjugant and chromosome-to-chromosome transfer in E. faecalis. CONCLUSION: The esp gene is transferable by conjugation among enterococcal isolates.

Conjugation, Genetic↗
PubMed2003

Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy).

Enterococci are widely distributed in the environment; within the human body, they are normal commensals of the oral cavity, gastrointestinal tract and vagina. In recent years, enterococci have become one of the most frequent causes of acquired nosocomial infections worldwide. The molecular mechanism of virulence of these bacteria is still not completely understood. The aims of this work were to characterize phenotypically 47 isolates of Enterococcus faecalis and Enterococcus faecium collected in Sardinia (Italy) by their abilities to adhere to different epithelial cell lines (Vero and Caco-2 cells) and to associate their phenotypes with the presence of known virulence genes detected within their genomes by PCR. The following genes were amplified: AS (aggregation substance), esp (surface protein gene), ace (accessory colonization factor), efaA (E. faecalis endocarditis antigen) and gelE (gelatinase). The virulence genes were detected in E. faecalis isolates only, with the exception of esp, which was found in both species. The phenotypic and genotypic results were also compared with the susceptibility of isolates to various antibiotics.

Antigens, Bacterial↗
PubMed2003

Molecular characterization of clinical and environmental isolates of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis from a teaching hospital in Wales.

The present study describes the first molecular characterization of environmental and clinical isolates of vancomycin-resistant enterococci (VRE) in Wales. Over a 3-month period (May-July 2000), 134 isolates of VRE (89 Enterococcus faecium and 45 Enterococcus faecalis) were isolated from the patient environment of the University Hospital of Wales (UHW) in Cardiff, Wales, UK. In addition, over the same time-period, 24 clinical isolates of VRE (20 isolates of E. faecium and four isolates of E. faecalis) were obtained from 14 patients. All study isolates were subjected to PFGE typing and their van genotypes were determined by using multiplex PCR. The vanA PCR product (231 bp) was evident in 146 (92 %) of 158 VRE isolates; the remaining 12 isolates (8 %) were positive for the vanB gene. All isolates of E. faecalis were found to be vanA-positive. In total, 16 PFGE banding profiles (pulsotypes) were observed for environmental isolates of E. faecium, whilst eight pulsotypes were found for isolates of E. faecalis. Some of these pulsotypes were isolated from multiple sites, whereas others were more restricted in their distribution. Eleven pulsotypes were evident for clinical isolates and eight of these (representing 11 isolates) were also encountered in environmental isolates. Eleven clinical isolates of E. faecium (55 %) shared an identical pulsotype that was not detected in environmental isolates. These results demonstrate a heterogeneous environmental population of VRE and an association of certain strains with clinical isolates. Predominance of a single pulsotype (not detected in the environment) amongst clinical isolates suggests non-environmental transmission between patients.

Bacterial Proteins↗
PubMed2005

The tcrB gene is part of the tcrYAZB operon conferring copper resistance in Enterococcus faecium and Enterococcus faecalis.

The plasmid-localized tcrB (transferable copper-resistance gene B) gene from Enterococcus faecium was identified to be part of an operon called the tcrYAZB operon, which has a genetic organization similar to the copYZAB copper-homeostasis gene cluster from Enterococcus hirae. Putative promoter (P(tcr))- and repressor-binding sites highly similar to the E. hirae cop-promoter region were identified upstream of the tcrYAZB genes. The P(tcr) promoter was cloned in both the absence and the presence of the proximal repressor-encoding tcrY gene into a promoter-probe vector. Induction of the promoter was shown in liquid growth medium containing increasing concentrations of copper sulphate. To determine the growth advantage conferred by the tcrYAZB genes in a copper environment, a tcr-deletion mutant was isolated, and its growth was compared with that of its copper-resistant ancestor (strain A17sv1) in sublethal concentrations of copper sulphate. A competition assay using these two isogenic strains showed that copper sulphate concentrations of 3 mmol l(-1) and above are sufficient to select for copper resistance.

Bacterial Proteins↗
PubMed1998

Characterization of new insertion-like sequences of Enterococcus hirae and their dissemination among clinical Enterococcus faecium isolates.

Sequence analysis of different fragments that hybridized with a 4.5-kb EcoRI fragment originally cloned from Enterococcus hirae ATCC 9790 showed 66% homology to IS-like sequences found in staphylococci and lactococci. We tested several enterococcal ATCC strains and found that only E. hirae ATCC 9790 and Enterococcus faecium ATCC 19434 hybridized with the IS-like sequence. Moreover, we wanted to investigate the dissemination of this new IS among E. faecium strains. We analyzed 131 clinical E. faecium isolated in Italy and the USA for the presence of the IS and we found its presence in more than 63% of the isolates. The hybridization patterns obtained vary considerably between unrelated strains and allow further classification among ribotype-grouped species.

Base Sequence↗
PubMed2006

Relationship between biofilm formation, the enterococcal surface protein (Esp) and gelatinase in clinical isolates of Enterococcus faecalis and Enterococcus faecium.

One-hundred and twenty-eight enterococcal isolates were examined for their ability to form biofilm in relation to the presence of the gene encoding the enterococcal surface protein (esp), production of gelatinase and to the source of isolation. Neither esp nor gelatinase seemed to be required for biofilm formation: both Enterococcus faecalis and Enterococcus faecium did not show a correlation between the presence of either esp or the production of gelatinase and biofilm formation. However, in E. faecium while esp was found in isolates from either source, the presence of both esp and biofilm together was only found in strains from clinical settings, suggesting that there exists a synergy between these factors which serves as an advantage for the process of infection.

Bacterial Proteins↗
PubMed2005

In vitro activity of phenothiazine derivatives in Enterococcus faecalis and Enterococcus faecium.

The antimicrobial activity of the phenothiazine derivatives thioridazine and prochlorperazine have been evaluated with 11 Enterococcus faecalis strains and 9 Enterococcus faecium strains, originating from human infections and animal faecal flora. We found that all E. faecalis and E. faecium strains, regardless of their susceptibility to commonly used antibiotics, were inhibited by thioridazine at a concentration of 16-32 microg/ml and by prochlorperazine at a concentration of 32-64 microg/ml. Combinations of the antibiotics vancomycin or ampicillin and thioridazine and prochlorperazine at subinhibitory concentrations, could render vancomycin- or ampicillin-resistant bacteria sensitive to each of the antibiotics. Verapamil and reserpine, inhibitors of P-glycoprotein-mediated multidrug resistance, did not reduce resistance. Our results outline modification of resistance in enterococci induced by phenothiazine derivatives unrelated to P-glycoprotein-mediated multidrug resistance.

ATP-Binding Cassette Transporters↗
PubMed1992

Identical genes confer high-level resistance to gentamicin upon Enterococcus faecalis, Enterococcus faecium, and Streptococcus agalactiae.

The structural gene coding for the bifunctional aminoglycoside-modifying 6'-acetyltransferase-2''-phosphotransferase (6'AAC-2''APH) enzyme was specifically amplified by the polymerase chain reaction using template DNA of clinical isolates of enterococci (both Enterococcus faecalis and Enterococcus faecium) and the single high-level gentamicin-resistant Streptococcus agalactiae strain identified thus far. The results of the present study demonstrated that the genes encoding this antibiotic resistance trait are highly homologous in these species. In dot blot hybridization assays using nonradioactively labeled oligonucleotide probes, strains with and without high-level gentamicin resistance could be discerned unequivocally. The gene segment encoding 6'-acetylating activity and the gene segment encoding 2''-phosphorylating activity were simultaneously present in all isolates exhibiting high-level gentamicin resistance.

Acetyltransferases↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1996

Glycopeptide susceptibility among Danish Enterococcus faecium and Enterococcus faecalis isolates of animal and human origin and PCR identification of genes within the VanA cluster.

The MICs of vancomycin and avoparcin were determined for isolates of Enterococcus faecium and isolates of Enterococcus faecalis recovered from the feces of humans and animals in Denmark. Two hundred twenty-one of 376 (59%) isolates of E. faecium and 2 of 133 (1.5%) isolates of E. faecalis were resistant to vancomycin (MICs, 128 to > or = 256 micrograms/ml), and all vancomycin-resistant isolates were resistant to avoparcin (MICs, 64 to > or = 256 micrograms/ml). All vancomycin-resistant isolates examined carried the vanA, vanX, and vanR genes, suggesting that a gene cluster similar to that of the transposon Tn1546 was responsible for the resistance.

Animals↗
PubMed1998

Comparative in vitro activities and postantibiotic effects of the oxazolidinone compounds eperezolid (PNU-100592) and linezolid (PNU-100766) versus vancomycin against Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium.

The activities of the oxazolidinone antibacterial agents eperezolid (PNU-100592) and linezolid (PNU-100766) were compared with that of vancomycin against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus (n = 200), coagulase-negative staphylococci (n = 100), and vancomycin-susceptible and -resistant Enterococcus faecalis and Enterococcus faecium (n = 50). Eperezolid and linezolid demonstrated good in vitro inhibitory activity, regardless of methicillin susceptibility for staphylococci (MIC at which 90% of the isolates are inhibited [MIC90] range, 1 to 4 microg/ml) or vancomycin susceptibility for enterococci (MIC90 range, 1 to 4 microg/ml). In time-kill studies, eperezolid and linezolid were bacteriostatic in action. A postantibiotic effect of 0.8+/-0.5 h was demonstrated for both eperezolid and linezolid against S. aureus, S. epidermidis, E. faecalis, and E. faecium.

Acetamides↗