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PubMed2001

Distribution of aminoglycoside resistance genes in recent clinical isolates of Enterococcus faecalis, Enterococcus faecium and Enterococcus avium.

Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Distribution of genes encoding seven AMEs was investigated by multiplex PCR for 279 recent clinical isolates of enterococci derived from a university hospital in Japan. The aac(6')-aph(2"), which is related to high level gentamicin resistance, was detected at higher frequency in Enterococcus faecalis (42.5%) than in Enterococcus faecium (4.3%). Almost half of E. faecalis and E. faecium isolates possessed ant(6)-Ia and aph(3')-IIIa. The profile of AME gene(s) detected most frequently in individual strains of E. faecalis was aac(6')aph(2") + ant(6)-Ia + aph(3')-IIIa, and isolates with this profile showed high level resistance to both gentamicin and streptomycin. In contrast, AME gene profiles of aac(6')-Ii+ ant(6)-Ia+aph(3')-IIIa, followed by aac(6')-Ii alone, were predominant in E. faecium. Only one AME gene profile of ant(6)-Ia+aph(3')-IIIa was found in Enterococcus avium. The ant(4')-Ia and ant(9)-Ia, which have been known to be distributed mostly among Staphylococcus aureus strains, were detected in a few enterococcal strains. An AME gene aph(2")-Ic was not detected in any isolates of the three enterococcal species. These findings indicated a variety of distribution profiles of AME genes among enterococci in our study site.

Aminoglycosides↗
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↗
PubMed2004

VanD-type vancomycin-resistant Enterococcus faecium and Enterococcus faecalis.

Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 microg/ml, respectively) and to low levels of teicoplanin (4 microg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 microg/ml) and susceptible to teicoplanin (0.25 microg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with d-alanyl-d-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci. VanX(D) activity, which is not required in the absence of d-Ala-d-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY(D) activity in the membrane fractions was inhibited by penicillin G. The mutations E(13)G in the region of d-alanine:d-alanine ligase (which is implicated in d-Ala1 binding in A902) and S(319)N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of d-Ala-d-Ala-containing peptidoglycan precursors. The same 7-bp insertion in vanS(D) of BM4539 and BM4540 and a 1-bp deletion in vanS(D) of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS(D), had a G(140)E mutation in VanR(D) that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanS(D) or vanR(D) gene that lead to constitutive resistance to vancomycin.

Amino Acid Sequence↗
PubMed1992

Susceptibility testing of clinical isolates of Enterococcus faecium and Enterococcus faecalis.

We collected 103 clinical Enterococcus faecium isolates from across Canada, performed standard broth microdilution susceptibility testing, and compared these results with results from the MicroScan Pos MIC Type 6 panel (Baxter Health Care Corp., West Sacramento, Calif.) and the AMS-Vitek Gram-Positive Susceptibility card (Vitek Inc., St. Louis, Mo.). High-level aminoglycoside resistance to gentamicin and streptomycin was detected by a single-concentration agar method with 1,000 micrograms of each aminoglycoside per ml. In addition, we tested the effect of the lower calcium content in broth media as recommended in National Committee for Clinical Laboratory Standards (NCCLS) guideline M7-A2 on the activity of the highly calcium-dependent agent daptomycin. Of the 103 E. faecium isolates, there were 4 and 30 isolates with high-level gentamicin resistance (HLGR) and high-level streptomycin resistance (HLSR), respectively. An additional 39 (37 with HLGR and 36 with HLSR) E. faecium isolates were tested by both the MicroScan and the AMS-Vitek systems. The AMS-Vitek card demonstrated sensitivities of 95 and 82% for detecting HLGR strains and HLSR strains, respectively. The MicroScan panel demonstrated improved sensitivities for detecting HLGR (42 to 97%) and HLSR (64 to 84%) when readings were performed manually instead of being generated automatically. Ampicillin resistance (MIC, greater than or equal to 16 micrograms/ml) was detected in 23 of the 103 E. faecium isolates. Only 14 and 20 of these were detected by the MicroScan panels and AMS-Vitek cards, respectively. beta-Lactamase activity was not detected in any isolates. The lower calcium content in broth media recommended by NCCLS guideline M7-A2 markedly reduced the in vitro activity of daptomycin against Enterococcus spp.

Calcium↗
PubMed2000

Streptogramin resistance and shared pulsed-field gel electrophoresis patterns in vanA-containing Enterococcus faecium and Enterococcus hirae isolated from humans and animals in Spain.

The present study was performed to determine if any of the 45 vanA-containing Enterococcus faecium or 18 vanA-containing E. hirae strains were shared by chickens (32 E. faecium/l7 E. hirae) and humans (13 E. faecium/1 E. hirae) using pulsed field gel electrophoresis (PFGE) and to study quinupristin-dalfopristin (Q-D) resistance. Seven of the 45 E. faecium isolates (from 2 outpatients and from 5 poultry products) were resistant to Q-D (MIC > or = 16 microg/ml); one strain was shown to have satA by PCR and sequencing and, in the other six isolates, the recently described satG gene was demonstrated. Six different PFGE patterns were detected among the 7 Q-D E. faecium-resistant isolates. None of the E. hirae isolates showed Q-D resistance. Among the 45 vanA -containing E. faecium strains, 25 unrelated clones were found by PFGE with highly diverse patterns and an indistinguishable PFGE pattern was observed in vanA-containing E. faecium strains from two humans and two poultry products. A single PFGE pattern was detected in 17 of 18 vanA-containing E. hirae isolates, obtained from one human and 16 chicken samples. Based on the presence of indistinguishable PFGE patterns among VR E. faecium and E. hirae from humans and chickens, we conclude that horizontal transfer of these strains could occur between both groups.

Acetyltransferases↗
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↗
PubMed2000

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces.

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.

Animals↗
PubMed2006

Comparison of testing methods for detection of decreased linezolid susceptibility due to G2576T mutation of the 23S rRNA gene in Enterococcus faecium and Enterococcus faecalis.

E-test, Vitek 2, MicroScan, agar dilution, and disk diffusion were compared for detection of decreased linezolid susceptibility due to 23S rRNA gene G2576T mutation among 32 clinical Enterococcus strains initially reported as intermediate or resistant by E-test alone or Vitek 2 confirmed by E-test. Agar and broth dilution methods were in concordance with PCR detection of the mutation, and disk diffusion was somewhat less sensitive but equally specific.

Acetamides↗
PubMed1991

Detection of penicillin-binding proteins immunologically related to penicillin-binding protein 5 of Enterococcus hirae ATCC 9790 in Enterococcus faecium and Enterococcus faecalis.

Penicillin-binding protein (PBP) 5 of Enterococcus hirae ATCC 9790 belongs to the class of the high-molecular mass, low-affinity PBPs which have been correlated with penicillin resistance in most Enterococcus species. Polyclonal antibodies were raised against PBP 5 and used to detect immunologically related membrane proteins in E. faecium and E. faecalis strains. Several strains of both species were found to have a membrane protein of similar molecular mass to E. hirae PBP 5 which reacted with the antibodies. Some E. faecium strains did not react with antibodies but their derivatives with increased penicillin minimal inhibitory concentrations did. In some E. faecalis strains the lack of a PBP 5-related protein was associated with failure to select stable penicillin-resistant derivatives.

Bacterial Proteins↗
PubMed1990

Mechanism of resistance to vancomycin in Enterococcus faecium D366 and Enterococcus faecalis A256.

The role of the glycopeptide-inducible proteins of Enterococcus faecium D366 (39.5 kilodaltons) and Enterococcus faecalis A256 (39 kilodaltons) in the mechanism of resistance to vancomycin and teicoplanin was examined. Crude cell walls from noninduced cells or from induced cells treated with sodium dodecyl sulfate to remove the inducible proteins were shown to bind vancomycin, in contrast to cell walls containing the cytoplasmic membrane-associated induced proteins, which did not bind vancomycin. Cytoplasmic membranes from vancomycin-induced cells did not inactivate (bind) vancomycin or teicoplanin, but they could protect the glycopeptides from being bound to the synthetic pentapeptide. This protection could be competitively abolished by D-alanyl-D-alanine. A decrease in glycopeptide binding to the pentapeptide was observed in a time-dependent fashion after treatment of the pentapeptide with the cytoplasmic membranes from induced cells. We hypothesize that the inducible proteins are responsible for glycopeptide resistance due to the binding to, and subsequent enzymatic modification of, the pentapeptide precursor of peptidoglycan, which is considered to be the natural target of glycopeptides.

Amino Acid Sequence↗
PubMed2000

In vitro activity of quinupristin/dalfopristin and other antibiotics against ampicillin-resistant enterococcus faecium.

BACKGROUND: Enterococcus faecium constitutes approximately 10% of clinical isolates of enterococci and is noted for its antimicrobial resistance. In particular, E faecium is commonly resistant to ampicillin. The optimal treatment for severe infections caused by these multi-resistant organisms has yet to be determined. METHODS: Enterococci tested were isolated from blood, pleural fluid and cerebrospinal fluid. Ampicillin-resistant Enterococcus faecium (AREF) was identified using the API Rapid Strep Kit system. A total of 58 isolates of AREF were enrolled in this study. Ten different antibiotics were tested, including Synercid (quinupristin/dalfopristin), teicoplanin, vancomycin, ampicillin, trimethoprim/sulfamethoxazole (TMP/SMX), ciprofloxacin, gentamicin, chloramphenicol, rifampicin and tetracycline. The agar dilution method described by the National Committee for Clinical Laboratory Standards was used to determine the minimum inhibitory concentrations (MICs) of the antibiotics tested. RESULTS: Teicoplanin showed the best in vitro activity. Its MIC ranged from 0.25 to 2 micrograms/ml with an MIC90 of 1 microgram/ml. The MIC of vancomycin was 0.5-128 micrograms/ml with an MIC90 of 2 micrograms/ml. Three strains were vancomycin resistant, and they were the VanB phenotype. The MIC of quinupristin/dalfopristin was 0.5 to 8 micrograms/ml with an MIC90 of 2 micrograms/ml. Chloramphenicol and tetracycline showed moderate susceptibility. AREF showed high resistance to other antibiotics tested, including ciprofloxacin, gentamicin, TMP/SMX and rifampicin. High-level gentamicin resistance (MIC > 1,000 micrograms/ml) was found in 78% of AREF tested. CONCLUSIONS: Teicoplanin showed the best in vitro activity against AREF. Clinical studies are necessary to confirm the efficacy of quinupristin/dalfopristin in vivo.

Ampicillin Resistance↗
PubMed2006

First genetic characterization of a bacterial beta-phenylethylamine biosynthetic enzyme in Enterococcus faecium RM58.

Enterococcus faecium RM58 produces beta-phenylethylamine and tyramine. A gene from Ent. faecium RM58 coding for a 625 amino-acid residues protein that shows 85% identity to Enterococcus faecalis tyrosine decarboxylase has been expressed in Escherichia coli, resulting in L-phenylalanine and L-tyrosine decarboxylase activities. Both activities were lost when a truncated protein lacking 84 amino acids at its C-terminus was expressed in E. coli. This study constitutes the first genetic characterization of a bacterial protein having L-phenylalanine decarboxylase activity and solves a long-standing question regarding the specificity of tyrosine decarboxylases in enterococci.

Carboxy-Lyases↗
PubMed1998

Growth and adherence on stainless steel by Enterococcus faecium cells.

Enterococcus faecium isolated from Brazilian raw milk was used in this study. For growth studies, E. faecium was inoculated into 10% RSM (reconstituted skim milk) and MRS both, incubated at 6.5 and 9 degrees C for 10 days and at 30, 42, and 45 degrees C for 48 h. Cells were enumerated after spread-plating onto MRS agar and incubating at 30 degrees C for 48 h. The ability of E. faecium cells to adhere to stainless-steel chips (6 by 6 by 1 mm, AISI 304, finish #4) was investigated. MRS broth containing stainless steel chips was inoculated to an initial concentration of 10(3) or 10(6) CFU/ml of E. faecium. Adherent cells were stained with acridine orange and enumerated by epifluorescence microscopy. E. faecium grew between 6.5 and 42 degrees C in MRS and between 9 and 40 degrees C in RSM. In MRS broth with 10(6) or 10(3) CFU/ml, the g (generation time) values were 0.62 and 0.42 h and R (growth rate) values were 1.6 and 2.4 h-1. Values of R = 2.3 h-1 and g = 0.43 h were determined for E. faecium growing in RSM with 10(3) CFU/ml. In MRS broth, for samples with a starting concentration of 10(6) cells per ml, adherence to stainless-steel chips was first observed at 2 h. However, adherence was first observed at 4 h in samples with an initial concentration of 10(3) cells per ml. After 10 h of exposure the number of adherent cells was similar for all samples regardless of initial inoculum. These results indicate that E. faecium readily adheres to stainless steel. It also underscores the need to control E. faecium by using appropriate low storage temperatures and adequate sanitizing practices in the dairy industry.

Animals↗
PubMed1993

Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145.

Enterococcus faecium BM4145, a clinical isolate from urine, was resistant to streptogramin group A antibiotics by inactivation. The strain harbored a plasmid containing a gene, satA, responsible for this resistance; this gene was cloned and sequenced. It encoded SatA, a protein deduced to be 23,634 Da in mass and homologous with a new family of chloramphenicol acetyltransferases described in Agrobacterium tumefaciens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The similarity of SatA to other acetyltransferases, LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) from E. coli, and to two putative acetyltransferases, NodL from Rhizobium leguminosarum and Urf1 from E. coli, was also observed in a region considered to be the enzyme's active site. Acetylation experiments indicated that acetyl coenzyme A was necessary for SatA activity and that a single acetylated derivative of pristinamycin IIA was produced. Other members of the streptogramin A group such as virginiamycin M and RP54476 were also substrates for the enzyme. We conclude that resistance to the streptogramin A group of antibiotics in E. faecium BM4145 is due to acetylation by an enzyme related to the novel chloramphenicol acetyltransferase family.

Acetylation↗
PubMed1999

In situ inversion of the conjugative transposon Tn916 in Enterococcus faecium DPC3675.

Enterococcus faecium DPC3675 is a derivative of E. faecium DPC1146 which contains a single copy of the conjugative transposon Tn916. Although the transposon is observed to be oriented in one direction in individual colonies, DNA extracted from cultures grown from these colonies contains the transposon in both orientations, as determined by PCR analysis and sequencing of the transposon/chromosome junctions. Therefore, Tn916 possesses a hitherto unreported ability to invert within a particular insertion site during growth in broth.

Base Sequence↗