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PubMed2000

Characterization of a divergent vanD-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil.

Enterococcus faecium 10/96A from Brazil was resistant to vancomycin (MIC, 256 microg/ml) but gave no amplification products with primers specific for known van genotypes. A 2,368-bp fragment of a van cluster contained one open reading frame encoding a peptide with 83% amino acid identity to VanH(D), and a second encoding a D-alanine-D-lactate ligase with 83 to 85% identity to VanD. The divergent glycopeptide resistance phenotype was designated VanD4.

Amino Acid Sequence↗
PubMed2002

Isolation and partial characterization of an antibacterial substance produced by Enterococcus faecium.

A strain of Enterococcus faecium isolated from Bulgarian yellow cheese "kashkaval" produced a bacteriocin-like substance named enterococcin A 2000. The antibacterial substance had a low molar mass (< 2 kDa), was relatively stable toward heat but was sensitive to selected proteolytic enzymes. It was active against Gram-positive bacteria including enterococci, such as Listeria, Bacillus and Streptococcus, and also against Gram-negative E. coli. Production of enterococcin A 2000 has a maximum near the end of the exponential phase of producer growth. The peptide was purified by ammonium sulfate precipitation, butanol extraction, followed by cation-exchange chromatography and reversed-phase chromatography. A partial sequence of purified enterococcin A 2000 indicated that this substance does not belong to the class IIa of bacteriocins presenting the consensus anti-Listeria motif YGNGV.

Amino Acid Sequence↗
PubMed2003

VanD-type vancomycin-resistant Enterococcus faecium 10/96A.

VanD type Enterococcus faecium 10/96A is constitutively resistant to vancomycin and to low levels of teicoplanin by nearly exclusive synthesis of peptidoglycan precursors terminating in D-alanyl-D-lactate (L. M. Dalla Costa, P. E. Reynolds, H. A. Souza, D. C. Souza, M. F. Palepou, and N. Woodford, Antimicrob. Agents Chemother. 44:3444-3446, 2000). A G(184)S mutation adjacent to the serine involved in the binding of D-Ala1 in the D-alanine:D-alanine ligase (Ddl) led to production of an impaired Ddl and accounts for the lack of D-alanyl-D-alanine-containing peptidoglycan precursors. The sequence of the vanD gene cluster revealed eight open reading frames. The organization of this operon, assigned to a chromosomal location, was similar to those in other VanD type strains. The distal part encoded the VanH(D) dehydrogenase, the VanD ligase, and the VanX(D) dipeptidase, which were homologous to the corresponding proteins in VanD-type strains. Upstream from the structural genes for these proteins was the vanY(D) gene; a frameshift mutation in this gene resulted in premature termination of the encoded protein and accounted for the lack of penicillin-susceptible D,D-carboxypeptidase activity. Analysis of the translated sequence downstream from the stop codon, but in a different reading frame because of the frameshift mutation, indicated homology with penicillin binding proteins (PBPs) with a high degree of identity with VanY(D) from VanD-type strains. The 5' end of the gene cluster contained the vanR(D)-vanS(D) genes for a putative two-component regulatory system. Insertion of ISEfa4 in the vanS(D) gene led to constitutive expression of vancomycin resistance. This new insertion belonged to the IS605 family and was composed of two open reading frames encoding putative transposases of two unrelated insertion sequence elements, IS200 and IS1341.

Amino Acid Sequence↗
PubMed2005

In vitro inhibition of Helicobacter pylori by Enterococcus faecium GM-1.

A strain of Enterococcus faecium that exhibits antibacterial activity against Helicobacter pylori was isolated from the feces of newborn babies. This strain was selected for its ability to inhibit the growth of H. pylori and to withstand harsh environmental conditions, such as acidic pH and high bile concentration. Biochemical tests and 16S rRNA sequencing specific for Enterococcus faecium GM-1 were used to identify the isolated bacterial strain. In vitro studies were used to investigate the inhibitory effects of E. faecium GM-1 on H. pylori. These results showed that the culture supernatant of E. faecium GM-1 significantly decreased the viability and urease activity of H. pylori. This inhibitory activity remained after adjustment of pH of culture supernatant to neutral. However, treatment with proteolytic enzymes reduced the anti-H. pylori activity of GM-1. Therefore, some substance(s) of E. faecium GM-1 other than pH and lactic acid might be associated with this inhibitory activity. Analysis by electron microscopy also demonstrated that the addition of GM-1 destroyed the cell structure of H. pylori. Additional studies suggested that the binding of H. pylori to human colonial cells decreased in the presence of GM-1.

Antibiosis↗
PubMed1996

Production and characterization of enterocin 900, a bacteriocin produced by Enterococcus faecium BFE 900 from black olives.

Enterococcus faecium BFE 900 isolated from black olives produced a bacteriocin termed enterocin 900, which was antagonistic towards Lactobacillus sake, Clostridium butyricum, enterococci as well as Listeria spp. including Listeria monocytogenes. Enterocin 900 was inactivated by pepsin, alpha-chymotrypsin, proteinase K and trypsin but not by catalase, alpha-amylase, or other non-proteolytic enzymes tested. The bacteriocin was heat stable, retaining activity after heating at 121 degrees C for 15 min. Enterocin 900 was active at pH values ranging from 2.0-10.0, with highest activity at pH 6.0. Bacteriocin production occurred in the late logarithmic growth phase when culture density was ca. log 8.0 CFU ml-1. Enterocin 900 was produced in media with initial pH ranging from 6.0-10.0, but not in media with a pH lower than 6.0. Medium composition, especially the concentrations of peptone and yeast extract influenced bacteriocin production, with no bacteriocin being produced in the absence of either of these compounds. No plasmids could be isolated from Enterococcus faecium BFE 900, indicating that the gene for bacteriocin activity is located on the chromosome.

Bacteriocins↗
PubMed1994

Bactericidal activities of peptide antibiotics against multidrug-resistant Enterococcus faecium.

Multidrug-resistant Enterococcus faecium has emerged as a serious pathogen for which no effective therapy has been established. In this report, we describe the activities of two peptide antibiotics, ramoplanin and daptomycin, against 15 isolates of E. faecium resistant to vancomycin, ampicillin, and aminoglycosides using time-kill experiments. Both antibiotics were rapidly bactericidal when tested in broth; however, the addition of 50% serum resulted in significant regrowth. The combination of ampicillin with either ramoplanin or daptomycin largely prevented this regrowth. These peptide antibiotics showed good activity against these pathogens. While the development of daptomycin has been halted, ramoplanin may hold promise for the therapy of multidrug-resistant E. faecium, especially when combined with ampicillin.

Ampicillin↗
PubMed1995

Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium.

A clinical isolate of Enterococcus faecium that contains a chromosomally encoded vanA gene cluster, Tn1546::IS1251, transferred vancomycin resistance to the plasmid-free strain Enterococcus faecalis JH2-2 during filter matings. Hybridization of a vanHAXY probe to SmaI restriction-digested genomic DNA separated by pulsed-field gel electrophoresis showed that the vanA gene cluster was located on a 40-kb fragment in the original donor strain and on fragments of different sizes (150 to 450 kb) in the transconjugants. No hybridization to vanA gene cluster probes was obtained with plasmid DNA preparations from the donor or transconjugants. These results suggested that in each case, the van genes had integrated into the recipient chromosome. The transconjugants in turn could act as donors of vancomycin resistance, and resistance was transferable to a Rec- recipient. The results of restriction analyses and DNA hybridizations of genomic DNA from the donor and transconjugants were consistent with the transfer of a mobile element that includes the 12.3-kb Tn1546::IS1251 gene cluster and at least 13 kb of additional DNA. This element has been tentatively designated Tn5482. DNA sequence analysis of a fragment predicted to contain the left end of Tn5482 revealed two insertion sequence-like elements: IS1216V and an apparently truncated IS3-like element. Restriction mapping and DNA hybridization patterns of the van gene clusters of three additional clinical isolates from New York City showed an element similar to Tn5482. Transfer of Tn5482 and related elements may be involved in dissemination of vancomycin resistance.

Amino Acid Sequence↗
PubMed2000

Specific antibody promotes opsonization and PMN-mediated killing of phagocytosis-resistant Enterococcus faecium.

Many clinical isolates of Enterococcus faecium are resistant to neutrophil (PMN)-mediated phagocytosis and killing in the presence of normal human serum. We have now examined the ability of specific polyclonal rabbit antibodies to promote opsonization and killing of phagocytosis-resistant E. faecium. Immune rabbit serum generated against formalin-killed E. faecium TX0016, a phagocytosis-resistant strain, markedly promoted binding of TX0016 organisms to PMNs and PMN-mediated killing. These effects were dramatically reduced by (a) adsorption of immune serum with E. faecium TX0016, but not by adsorption with a strain of E. faecium susceptible to phagocytosis, and (b) incubation of immune serum with carbohydrate purified from TX0016, but not by incubation with a surface protein extract from TX0016. IgG purified from immune serum was unable by itself to promote bacterial binding to PMNs. However, specific IgG was able to promote binding to PMNs and PMN-mediated killing in the presence of normal human serum as a complement source, as were F(ab')(2) and Fab fragments produced from it, and the alternative pathway of complement was sufficient to promote IgG- and F(ab')(2)-mediated opsonization. PMN complement receptor type 3, but not complement receptor type 1, was involved in bacterial binding to PMNs induced by the combination of F(ab')(2) fragments and normal human serum. These results suggest that opsonization by antibodies potentially directed against bacterial carbohydrate, in conjunction with complement activation, has an important role in the host defense against phagocytosis-resistant E. faecium.

Animals↗
PubMed1994

Colonization pattern of vancomycin-resistant Enterococcus faecium.

BACKGROUND: Vancomycin-resistant Enterococcus faecium is increasingly recognized as a serious problem by hospital epidemiologists. Understanding its colonization patterns may help in designing strategies to control its nosocomial spread in the hospital. METHODS: Twenty patients, selected at random, with vancomycin-resistant E. faecium isolated from cultures of various body sites were studied to determine sites of colonization. For 12 of these patients, cultures of environmental surfaces of their rooms and wards were also obtained. RESULTS: Eighteen patients (90%) had vancomycin-resistant E. faecium grown in stool cultures. In five patients (25%), vancomycin-resistant E. faecium was cultured from other sites: groins (four), popliteal fossae (three), mouth (one), and an open wound site (one). Patients with positive cultures from the groins and popliteal fossae also had growth of vancomycin-resistant E. faecium in cultures of diarrhea soiling those sites. No patients had the organism isolated from their nares. Vancomycin-resistant E. faecium grew in cultures obtained from bedside stand tables, over-bed tables, used linen, and bedside rails. CONCLUSIONS: In the 20 patients studied, colonization of vancomycin-resistant E. faecium was limited chiefly to the enteric tract. Absence of colonization of such a secluded area with poor antibiotic penetrability as the nares is encouraging. In our study, vancomycin-resistant E. faecium was isolated from various environmental surfaces from the rooms and wards of patients with vancomycin-resistant E. faecium in their stools.

Administration, Oral↗
PubMed2004

A novel putative enterococcal pathogenicity island linked to the esp virulence gene of Enterococcus faecium and associated with epidemicity.

Enterococcus faecalis harbors a virulence-associated surface protein encoded by the esp gene. This gene has been shown to be part of a 150-kb putative pathogenicity island. A gene similar to esp has recently been found in Enterococcus faecium isolates recovered from hospitalized patients. In the present study we analyzed the polymorphism in the esp gene of E. faecium, and we investigated the association of esp with neighboring chromosomal genes. The esp gene showed considerable sequence heterogeneity in the regions encoding the nonrepeat N- and C-terminal domains of the Esp protein as well as differences in the number of repeats. DNA sequencing of chromosomal regions flanking the esp gene of E. faecium revealed seven open reading frames, representing putative genes implicated in virulence, regulation of transcription, and antibiotic resistance. These flanking regions were invariably associated with the presence or absence of the esp gene in E. faecium, indicating that esp in E. faecium is part of a distinct genetic element. Because of the presence of virulence genes in this gene cluster, the lower G+C content relative to that of the genome, and the presence of esp in E. faecium isolates associated with nosocomial outbreaks and clinically documented infections, we conclude that this genetic element constitutes a putative pathogenicity island, the first one described in E. faecium. Except for the presence of esp and araC, this pathogenicity island is completely different from the esp-containing pathogenicity island previously disclosed in E. faecalis.

Amino Acid Sequence↗
PubMed2006

Control of Listeria monocytogenes in goat's milk and goat's jben by the bacteriocinogenic Enterococcus faecium F58 strain.

The bacteriocinogenic Enterococcus faecium F58 strain, a natural goat's jben cheese isolate, lacks decarboxylase activity involved in most biogenic amine formation. It was also sensitive to 13 antibiotics assayed and free of virulence and vancomycin resistance genes. The F58 strain reached the stationary phase after 12 h of growth in sterile goat's milk, and the production of enterocin F-58 (Ent L50) was first detected after 48 h (400 AU/ml), thereafter remaining stable up to 5 days. The effectiveness of the F58 strain in controlling Listeria monocytogenes serovar 4b in reduced fat and whole goat's milk, and in goat's jben has been examined. Coculture experiments of F58-L. monocytogenes in both types of milk demonstrated that listeriae were not eliminated, although reductions by 1 to 4 log units were found. Nevertheless, when the F58 strain was previously inoculated in whole milk and left to grow for 12 h before contamination, the pathogen was completely eliminated after 130 h of coculture. Production of jben cheese contaminated with L. monocytogenes prior to packaging, using preparations of F58-producer strain, caused a significant decrease in the number of viable listeriae, which were undetectable after 1 week of cheese storage at 22 degrees C. Altogether, results from this study suggest that E. faecium F58 strain may be used as an adjunct culture in cheese to control contamination and growth of L. monocytogenes by in situ enterocin production, thus providing an additional hurdle to enhance control of this pathogen.

Animals↗
PubMed1995

Detection of aminoglycoside-penicillin synergy against Enterococcus faecium using high-content aminoglycoside disks.

Thirty-seven Enterococcus faecium strains were screened for high-level aminoglycoside resistance with an agar diffusion test using high-content aminoglycoside disks (300 micrograms of streptomycin and 120 micrograms of gentamicin, tobramycin, kanamycin or amikacin). The inhibition zones obtained were correlated with results of time-kill penicillin-aminoglycoside synergy studies. An 11 mm breakpoint differentiated strains susceptible or resistant to the synergy of streptomycin plus penicillin. Irrespective of the inhibition zones obtained with tobramycin and kanamycin disks, Enterococcus faecium strains never showed synergy with penicillin in combination with these aminoglycosides. Penicillin-amikacin synergy cannot be predicted by the amikacin disks. Nevertheless, even though kanamycin disks do not predict penicillin-kanamycin synergy, they can be used to predict penicillin-amikacin synergy. In summary, high-content streptomycin, gentamicin and kanamycin disks can be used to predict the susceptibility of Enterococcus faecium strains to the synergistic combination of penicillin plus one of the aminoglycosides (streptomycin, gentamicin or amikacin, respectively).

Aminoglycosides↗
PubMed1992

The VanS-VanR two-component regulatory system controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Plasmid pIP816 of Enterococcus faecium BM4147 confers inducible resistance to vancomycin and encodes the VanH dehydrogenase and the VanA ligase for synthesis of depsipeptide-containing peptidoglycan precursors which bind the antibiotic with reduced affinity. We have characterized a cluster of five genes of pIP816 sufficient for peptidoglycan synthesis in the presence of vancomycin. The distal part of the van cluster encodes VanH, VanA, and a third enzyme, VanX, all of which are necessary for resistance. Synthesis of these enzymes was regulated at the transcriptional level by the VanS-VanR two-component regulatory system encoded by the proximal part of the cluster. VanR was a transcriptional activator related to response regulators of the OmpR subclass. VanS stimulated VanR-dependent transcription and was related to membrane-associated histidine protein kinases which control the level of phosphorylation of response regulators. Analysis of transcriptional fusions with a reporter gene and RNA mapping indicated that the VanR-VanS two-component regulatory system activates a promoter used for cotranscription of the vanH, vanA, and vanX resistance genes.

Amino Acid Sequence↗
PubMed2002

Automated ribotyping of vancomycin-resistant Enterococcus faecium isolates.

Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.

Alleles↗
PubMed1995

Overexpression, purification, and characterization of VanX, a D-, D-dipeptidase which is essential for vancomycin resistance in Enterococcus faecium BM4147.

Vancomycin resistance in Enterococcus faecium requires five genes: vanR, vanS, vanH, vanA, and vanX. The functions and mechanism of four gene products have been known, with VanR/S for signal transduction and transcriptional regulation and VanH/A to synthesize D-Ala-D-lactate. But the function of the fifth gene product, VanX, has been unknown until very recently, when Reynolds and colleagues discovered D-, D-dipeptidase activity in crude extracts of a VanX overproducer [Reynolds, P. E., et al. (1994) Mol. Microbiol. 13, 1065-1070]. We report here the expression of VanX in Escherichia coli and its purification to homogeneity. VanX has been characterized as a metal-activated D-, D-dipeptidase with an optimal pH range of 7-9. The kcat and Km of D-Ala-D-Ala in the absence of divalent metal are determined to be 4.7 s-1 and 1 mM, respectively. However, in the presence of metal cations, kcat can be as high as 788 s-1. VanX is unable to hydrolyze D-Ala-D-lactate, the substituted moiety in the peptidoglycan that leads to vancomycin resistance, not only because of low binding affinity (Ki estimated at 242 mM) but also due to a kcat less than 0.005 s-1. The more than 10(5)-fold differential in catalytic efficiency of VanX for hydrolysis of D-Ala-D-Ala vs D-Ala-D-lactate leaves D-Ala-D-lactate intact for subsequent incorporation into peptidoglycan.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Enterococcus faecium in hospitals.

Most of the characteristics that have ensured the success of enterococci as nosocomial pathogens were described early in this century. Enterococcus faecium and Enterococcus faecalis, the enterococci most frequently isolated from clinical material, differ fundamentally. The intrinsic antimicrobial resistance of Enterococcus faecium, supplemented by acquired resistance mechanisms, can generate a glycopeptide-multiply-resistant nosocomial pathogen that survives on hands and in the environment, and has the potential for intra-hospital and inter-hospital spread. The use of terms such as 'an enterococcus', 'faecal streptococci' and 'group D streptococci' have hindered, and still hinder, our understanding of a species rapidly emerging as the most problematic of nosocomial pathogens.

Anti-Bacterial Agents↗
PubMed2003

[Integrative therapy for postoperative intractable enterococcus faecium infections].

OBJECTIVE: To assess the treatment of patients with infection of enterococcus faecium after surgery who failed to respond to antibiotics. METHODS: Five patients after surgery were proved to have Enterococcus faecium infection by bacterial culture. They were treated by sensitive antibiotics but failed. Comprehensive treatment prescribed included immunoenhancements, enteral nutrition, and traditional Chinese medicines. RESULTS: Four patients were discharged from the hospital after recovery, and was cared else where after 1 month treatment. CONCLUSION: Comprehensive treatment is a better way to treat patients with refractory enterococcus faecium infection after surgery.

Aged↗
PubMed1994

Overproduction of a low-affinity penicillin-binding protein and high-level ampicillin resistance in Enterococcus faecium.

Five ampicillin-resistant clinical isolates of Enterococcus faecium were analyzed for a correlation between overproduction of the low-affinity penicillin-binding protein (PBP 5) and the level of ampicillin resistance. Comparison was made with one susceptible clinical isolate and its ampicillin-resistant derivative obtained in the laboratory by selection with increasing concentrations of penicillin. Overproduction of the low-affinity PBP relative to the susceptible isolate was noted in moderately resistant strains (MIC, 32 micrograms/ml) but not in highly resistant strains (MIC, 128 micrograms/ml). Polyclonal antibodies specifically reacting with the low-affinity PBP of Enterococcus hirae, Enterococcus faecalis, and Enterococcus faecium (M. Ligozzi, M. Aldegheri, S. C. Predari, and R. Fontana, FEMS Microbiol. Lett. 83:335-340, 1991) were used to determine the amount of this PBP in the E. faecium isolates. In all strains, the antibody preparation reacted with a membrane protein of the same molecular mass as PBP 5. The amount of this protein was very small in the susceptible strain but large in all of the resistant strains. These results suggest that the highly resistant strains also overproduced the low-affinity PBP, which, compared with PBP 5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability.

Ampicillin Resistance↗