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PubMed1993

Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Sequence determination of the flanking regions of the vancomycin resistance van gene cluster carried by pIP816 in Enterococcus faecium BM4147 revealed similarity to transposons of the Tn3 family. Imperfect inverted repeats (36 of 38 bp) delineated a 10,851-bp element designated Tn1546. The 4-kb region located upstream from the vanR gene contained two open reading frames (ORF) transcribed in opposite directions. The deduced amino acid sequence of ORF1 (988 residues) displayed, respectively, 56 and 42% identity to those of the transposases of Tn4430 from Bacillus thuringiensis and of Tn917 from Enterococcus faecalis. The product of ORF2 (191 residues) was related to the resolvase of Tn917 (33% amino acid identity) and to the Res protein (48%) of plasmid pIP404 from Clostridium perfringens. Tn1546 transposed consecutively in Escherichia coli from plasmid pUC18 into pOX38 and from pOX38 into various sites of pBR329. Transposition was replicative, led to the formation of cointegrates, and produced a 5-bp duplication at the target site. Southern hybridization and DNA amplification revealed the presence of Tn1546-related elements in enterococci highly resistant to glycopeptides. Analysis of sequences surrounding these elements indicated that transposition plays a role in dissemination of the van gene cluster among replicons of human clinical isolates of E. faecium.

Alanine↗
PubMed2002

Analysis of the conjugal transfer system of the pheromone-independent highly transferable Enterococcus plasmid pMG1: identification of a tra gene (traA) up-regulated during conjugation.

pMG1 (65.1 kbp) is a pheromone-independent Enterococcus faecium conjugative plasmid conferring gentamicin resistance. pMG1 is able to transfer to enterococci at a high frequency in broth mating experiments, and it does not respond to the sex pheromones which are involved in the high-frequency transfer system of some conjugative plasmids in Enterococcus faecalis. To analyze regulation of tra gene expression in pMG1, transcripts of pMG1 were examined during conjugation. RNA samples were prepared from mating mixtures 20, 40, 80, and 160 min after initiation of mating and were subsequently analyzed by Northern hybridization by using a variety of pMG1 DNA fragments as probes. One transcript of gene 71ORF2 increased to the maximal level 20 min after the start of mating. The level of this transcript decreased after 40 min of mating to the same level as the level in the control donor culture. The increase was not observed in cultures of the donor cells or recipient cells. These findings suggested that the increase was a conjugation-specific event. The 71ORF2 gene of pMG1 was disrupted by using the suicide vector pMG226 carrying an erythromycin resistance gene that is expressed in enterococci. The transfer frequencies of mutant plasmid pMG229, which had a disrupted 71ORF2 gene in pMG1, and the parent plasmid pMG1 were 1.6 x 10(-7) and 1.1 x 10(-3) per donor cell, respectively, in broth mating experiments, and the transfer frequencies of pMG229 and pMG1 were 2.7 x 10(-3) and 8.5 x 10(-2) per donor cell, respectively, in filter mating experiments. This indicated that the transfer frequency of plasmid pMG229 was reduced during broth mating and was not altered during filter mating. 71ORF2, which is designated traA, is a gene involved in the tra gene system for conjugation, and the product of this gene is associated with the formation or stabilization of mating aggregates during broth mating.

Base Sequence↗
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↗
PubMed2004

Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis.

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

Bacterial Proteins↗
PubMed2005

Molecular characterization of a widespread, pathogenic, and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island.

Enterococcus faecalis, a common cause of endocarditis and known for its capacity to transfer antibiotic resistance to other pathogens, has recently emerged as an important, multidrug-resistant nosocomial pathogen. However, knowledge of its lineages and the potential of particular clones of this species to disseminate and cause disease is limited. Using a nine-gene multilocus sequence typing (MLST) scheme, we identified an evolving and widespread clonal complex of E. faecalis that has caused outbreaks and life-threatening infections. Moreover, this unusual clonal complex was found to contain isolates of unexpected relatedness, including the first known U.S. vancomycin-resistant enterococcus (E. faecalis strain V583), the first known penicillinase-producing (Bla(+)) E. faecalis isolate, and the previously described widespread clone of penicillinase producers, a trait found in <0.1% of E. faecalis isolates. All members of this clonal cluster (designated as BVE for Bla(+) Van(r) endocarditis) were found to contain a previously described putative pathogenicity island (PAI). Further analysis of this PAI demonstrated its dissemination worldwide, albeit with considerable variability, confirmed its association with clinical isolates, and found a common insertion site in different clonal lineages. PAI deletions, MLST, and the uncommon resistances were used to predict the evolution of the BVE clonal cluster. The finding of a virulent and highly successful clonal complex of E. faecalis with different members resistant to the primary therapies of choice, ampicillin and vancomycin, has important implications for the evolution of virulence and successful lineages and for public health monitoring and control.

Bacterial Proteins↗
PubMed2005

The N-terminal domain of enterococcal surface protein, Esp, is sufficient for Esp-mediated biofilm enhancement in Enterococcus faecalis.

Enterococci have emerged as one of the leading causes of nosocomial bloodstream, surgical site, and urinary tract infections. More recently, enterococci have been associated with biofilms, which are bacterial communities attached to a surface and encased in an extracellular polymeric matrix. The enterococcal cell surface-associated protein, Esp, enhances biofilm formation by Enterococcus faecalis in a glucose-dependent manner. Mature Esp consists of a nonrepeat N-terminal domain and a central region made up of two types of tandem repeats followed by a C-terminal membrane-spanning and anchor domain. This study was undertaken to localize the specific domain(s) of Esp that plays a role in Esp-mediated biofilm enhancement. To achieve this objective, we constructed in-frame deletion mutants expressing truncated forms of Esp in an isogenic background. By comparing strains expressing the mutant forms of Esp to those expressing wild-type Esp, we found that the strain expressing Esp lacking the N-terminal domain formed biofilms that were quantitatively less in biovolume than the strain expressing wild-type Esp. Furthermore, an E. faecalis strain expressing only the N-terminal domain of Esp fused to a heterologous protein anchor formed biofilms that were quantitatively similar to those formed by a strain expressing full-length Esp. This suggested that the minimal region contributing to Esp-mediated biofilm enhancement in E. faecalis was confined to the nonrepeat N-terminal domain. Expression of full-length E. faecalis Esp in heterologous host systems of esp-deficient Lactococcus lactis and Enterococcus faecium did not enhance biofilm formation as was observed for E. faecalis. These results suggest that Esp may require interaction with an additional E. faecalis-specific factor(s) to result in biofilm enhancement.

Bacterial Proteins↗
PubMed2006

Putative surface proteins encoded within a novel transferable locus confer a high-biofilm phenotype to Enterococcus faecalis.

Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.

Aminoacyltransferases↗
PubMed2006

Sequence analysis of Enterococcus faecium strain 10/96A (VanD4), the original vancomycin-resistant E. faecium strain in Brazil.

Enterococcus faecium strain 10/96A (VanD4) was the first vancomycin-resistant enterococcus (VRE) isolated in Brazil. Subsequent Brazilian VRE strains have all had the VanA phenotype. Multilocus sequence typing showed that strain 10/96A was isolated sporadically, has a unique sequence type (ST 281), and was not the progenitor of the VRE strains isolated from hospital outbreaks in Brazil.

Brazil↗
PubMed1988

Laboratory detection of high-level aminoglycoside-aminocyclitol resistance in Enterococcus spp.

Methods for detection of high-level resistance to aminoglycoside-aminocyclitol antibiotics were evaluated using 104 blood isolates of enterococci (97 Enterococcus faecalis and 7 Enterococcus faecium). Kanamycin was used to predict resistance to amikacin. Discrepancies between methods were resolved by time-kill studies. Four methods (MicroScan, macrotube, microtiter, and disk diffusion) for detecting resistance to gentamicin and streptomycin were compared, using 51 consecutive strains. There were 13 gentamicin-resistant strains, all of which were detected by macrotube, microtiter, and disk diffusion. MicroScan detected 2 (15%) of the 13. Of the 18 streptomycin-resistant strains, 17 (93%) were detected by disk diffusion, 16 (89%) by microtiter, 9 (50%) by macrotube, and 6 (33%) by MicroScan. An additional 53 consecutive strains were examined only by disk diffusion and microtiter for resistance to gentamicin, streptomycin, and kanamycin. The entire population of 104 strains contained 35 gentamicin-, 22 streptomycin-, and 54 kanamycin-resistant enterococcal isolates. All 35 gentamicin-resistant strains were detected by both methods. Of the 22 streptomycin-resistant strains, 1 was detected only by microtiter, 2 only by disk diffusion, and 19 by both methods. Of the 54 kanamycin-resistant strains, 1 was detected only by microtiter, 2 only by disk diffusion, and 51 by both methods. One additional strain which was resistant only by disk diffusion was susceptible to amikacin plus penicillin by time-kill studies. Disk diffusion is a suitable method for detection of high-level aminoglycoside-aminocyclitol resistance in E. faecalis and is well suited for sporadic testing. Additional data are necessary to determine the suitability of these tests for E. faecium.

Anti-Bacterial Agents↗
PubMed1992

Typing of Enterococcus species by DNA restriction fragment analysis.

Enterococci are a frequent cause of hospital-acquired infection, being associated with urinary tract infections, wound sepsis, bacteremia, and endocarditis. The source of infection is usually thought to be endogenous, but some evidence points to cross-infection between patients. A better understanding of the epidemiology of enterococci has been limited by the lack of a good discriminatory typing system. This report describes the application of two DNA-based typing methods to Enterococcus faecalis and Enterococcus faecium: comparison of restriction fragments from total DNA by conventional electrophoresis and comparison of restriction fragments hybridizing to an rRNA gene probe (ribotyping). Comparison of restriction fragments (from SstI digestion) by conventional electrophoresis was simple and highly discriminatory. The results of analysis of blood culture isolates and of repeat isolates from individual patients are reported. Ribotyping (with BscI digestion) was more applicable at the level of species discrimination.

Bacteremia↗
PubMed1992

Detection of vancomycin resistance in Enterococcus species.

Enterococcus faecalis and Enterococcus faecium isolates that are resistant to vancomycin have recently been identified in North America and Europe. Of 155 clinical isolates of enterococci (113 E. faecium and 42 E. faecalis), we found that 98 were resistant, 52 were moderately susceptible, and 5 had intermediate susceptibilities to vancomycin by using broth microdilution susceptibility testing according to the National Committee for Clinical Laboratory Standards (NCCLS) (Approved Standard M7-A2). Using NCCLS disk diffusion methodology (Approved Standard M2-A4), we evaluated the NCCLS supplemental M100-S3 revisions for zone diameter interpretive standards and incubation conditions and found 5.8% minor errors. A total of 234 isolates, which included an additional 79 E. faecium isolates that were moderately susceptible to vancomycin, were used to evaluate the Vitek GPS-TA card (bioMerieux, Inc., Hazelwood, Mo.) and the Pos MIC type 6 panel (MicroScan; Baxter Health Care Corp., West Sacramento, Calif.) for the detection of vancomycin resistance. The Vitek card was 100% specific and 72% sensitive, whereas the MicroScan panel with the Walk/Away system was 98% specific, with a sensitivity of 93% which increased to 99% when readings were performed manually. An agar screen plate method was evaluated with vancomycin concentrations of 6, 8, 10, or 12 micrograms/ml; plates were inoculated so as to obtain a final concentration of 10(5) CFU per spot. This method was found to be 100% sensitive and specific at all concentrations.

Drug Resistance, Microbial↗
PubMed1998

Apparent increased prevalence of high-level aminoglycoside-resistant Enterococcus durans resulting from false identification by a semiautomated software system.

Identification of enterococci by using a semiautomated system (PASCO; Difco Laboratories, Detroit, Mich.) in the AHEPA University Hospital, Thessaloniki, Greece, revealed a high proportion of Enterococcus durans, particularly among isolates highly resistant to gentamicin and streptomycin. When 14 isolates were further tested by a conventional biochemical scheme and by PCR, all were reidentified as Enterococcus faecalis. Antibiotic resistance and pulsed-field gel electrophoresis patterns showed that unrelated strains were misidentified.

Anti-Bacterial Agents↗
PubMed1999

Vancomycin-resistant Enterococcus faecium colonization in children.

Nosocomial vancomycin-resistant Enterococcus (VRE) infections have been described in only small numbers of pediatric patients. In none of these studies were multivariate analyses performed to assess which factors were independent risk factors in these patients. In the present cohort study of patients admitted to our hematology/oncology unit, surveillance cultures revealed a colonization rate of 24% and all isolates were identified as Enterococcus faecium. Risk factors associated with colonization with VRE identified by multiple logistic regression analysis included young age and chemotherapy with antineoplastic agents, cefotaxime, vancomycin, and ceftazidime. A molecular epidemiological tool, pulsed-field gel electrophoresis, was used to determine the relatedness of the VRE isolates detected. DNA analysis by this method identified two major clusters of VRE isolates. Young children with gastrointestinal colonization with VRE, without evidence of clinical infection, can serve as a reservoir for the spread of VRE.

Anti-Bacterial Agents↗
PubMed2001

Selection of a teicoplanin-resistant Enterococcus faecium mutant during an outbreak caused by vancomycin-resistant enterococci with the vanB phenotype.

Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant Enterococcus faecalis (VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant Enterococcus faecium (VREM) strains. Two similar restriction fragment length polymorphism types of the vanB gene cluster characterized VRES and VREM isolates, and they both contained the same vanB2 variant of the vanB gene. Two vancomycin-susceptible E. faecium (VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of vanB gene cluster copies. Most probably they reflected the dynamic evolution of an E. faecium strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene cluster, including the regulatory genes vanR(B) and -S(B), and the vancomycin-inducible promoter P(YB). Expression of the resistance genes vanH(B), -B, and -X(B) was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.

Anti-Bacterial Agents↗
PubMed2002

Native valve endocarditis due to Enterococcus hirae.

Enterococcus hirae is a rare isolate in clinical specimens. We describe a case of native aortic-valve endocarditis that was caused by Enterococcus hirae in a 72-year-old man. This is the first reported case of endocarditis due to this organism.

Aged↗
PubMed2002

Possible connection between a widely disseminated conjugative gentamicin resistance (pMG1-like) plasmid and the emergence of vancomycin resistance in Enterococcus faecium.

A total of 640 vancomycin-resistant Enterococcus faecium (VRE) isolates, which were obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were used in this study. Of the 640 strains, 611 and 29 were VanA and VanB VRE, respectively, based on PCR analysis. Four hundred ninety-two (77%) of the strains exhibited resistance to concentrations of gentamicin from 64 micro g/ml (MIC) to more than 1,024 micro g/ml (MIC). The gentamicin resistance of each of 261 (53%) of the 492 gentamicin-resistant strains was transferred to E. faecium at a frequency of about 10(-5) to 10(-6) per donor cell in broth mating. More than 90% of vancomycin resistances of the 261 strains cotransferred with the gentamicin resistances to E. faecium strains by filter mating. The conjugative gentamicin resistance plasmids were identified and were classified into five types (A through E) with respect to their EcoRI restriction profiles. The transfer frequencies of each type of plasmid between E. faecium strains or Enterococcus faecalis strains were around 10(-3) to 10(-5) per donor cell or around 10(-6) to 10(-7) per donor cell, respectively, in broth mating. Type A and type B were the most frequently isolated, at an isolation frequency of about 40% per VRE isolate harboring the gentamicin resistance conjugative plasmid. The plasmids did not show any homology in Southern hybridization with the pheromone-responsive plasmids and broad-host-range plasmids pAMbeta1 and pIP501. The EcoRI or NdeI restriction fragments of each type of plasmids hybridized to the conjugative gentamicin resistance plasmid pMG1 (65.1 kb), which was originally isolated from an E. faecium clinical isolate, and transfer efficiently in broth mating.

Anti-Bacterial Agents↗
PubMed2003

Influence of transferable genetic determinants on the outcome of typing methods commonly used for Enterococcus faecium.

A variety of methods is used for a molecular typing of Enterococcus spp. and related gram-positive bacteria including macrorestriction analysis using pulsed-field gel electrophoresis (PFGE), ribotyping, rapid amplification of polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). To test the influence of transferable determinants on the outcome of different typing methods commonly used for enterococci, we established a homogenous strain collection of 24 transconjugants resulting from filter matings with antibiotic-resistant Enterococcus faecium. As expected, AFLP, RAPD, and PFGE all identified our model bacteria as strongly related. However, distinct differences in the resolving and discriminatory power of the tested methods could be clearly addressed. In PFGE, 22 of 24 transconjugants possessed less than a three-band difference to the recipient pattern and would be regarded as strongly related. Three different RAPD PCRs were tested; in two reactions, identical patterns for all transconjugants and the recipient were produced. One RAPD PCR produced an identical pattern for 18 transconjugants and the recipient and a clearly different pattern for the remaining 6 transconjugants due to a newly appearing fragment resulting from acquisition of the tetL gene. AFLP clusters all transconjugants into a group of major relatedness. Percent similarities were highly dependent on the method used for calculating the similarity coefficient (curve-based versus band-based similarity coefficient). Fragment patterns of digested plasmids showed the possession of nonidentical plasmids in most transconjugants. PFGE still could be recommended as the method of choice. Nevertheless, the more-modern AFLP approach produces patterns of comparable discriminatory power while possessing some advantages over PFGE (less-time-consuming internal standards). Plasmid fingerprints can be included to subdifferentiate enterococcal isolates possessing identical macrorestriction and PCR typing patterns.

Anti-Bacterial Agents↗
PubMed2004

Enterococcus cecorum empyema thoracis successfully treated with cefotaxime.

We report the first case of Enterococcus cecorum empyema thoracis and spontaneous bacterial peritonitis in a 44-year-old man with underlying cirrhosis. The patient responded to cefotaxime (MIC, 0.25 microg/ml) treatment and drainage of the empyema. Susceptibility of E. cecorum to expanded-spectrum cephalosporins could be due to its production of types of penicillin-binding proteins similar to those produced by Streptococcus species rather than to those produced by Enterococcus species (as predicted by phylogenetic analysis of the 16S rRNA gene sequences).

Adult↗