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PubMed1992

Overproduction of a penicillin-binding protein is not the only mechanism of penicillin resistance in Enterococcus faecium.

In 1989 and 1990, a large number of ampicillin-resistant strains of Enterococcus faecium were isolated from infected patients treated at intensive care units in Berlin, Germany. Twenty-five clinical isolates, including five different biotypes as classified by acid production from various sugars and a wide range of susceptibilities to ampicillin (MICs between 0.5 and 128 micrograms/ml), were selected for a detailed analysis of penicillin-binding proteins (PBPs). All strains contained a slowly reacting PBP with low penicillin affinity known to be present in enterococci. Overproduction of this PBP relative to susceptible isolates was noted, especially in all strains for which the MIC of ampicillin was 8 micrograms/ml, to a lesser degree in the more resistant strains, but not at all in the three highly resistant isolates for which the MIC was 128 micrograms/ml. In these three strains, this PBP appears to have a reduced affinity for beta-lactams. The results suggest that overproduction of PBP 6 correlates only with intermediate resistance levels and that higher resistance is mediated by yet another, still unknown mechanism, probably including reduction of beta-lactam affinity in one or more PBPs.

Bacterial Proteins↗

PubMed1997

Clonal dissemination and colony morphotype variation of vancomycin-resistant Enterococcus faecium isolates in metropolitan Detroit, Michigan.

Thirty-two isolates of vancomycin-resistant Enterococcus faecium (VRE) recovered from 25 patients hospitalized at six hospitals in the metropolitan Detroit, Mich., area over a 32-month period were examined for relatedness by repetitive-sequence PCR (rep-PCR). All isolates were shown to carry the vanA gene by PCR. The rep-PCR patterns generated from each isolate showed that the first three VRE isolates obtained from hospital A between June 1992 and February 1994 were distinct strains. Thereafter, all VRE isolates originating from hospital A and those collected from five other area hospitals had identical rep-PCR patterns. On detailed examination, subcultures of 25 of the 32 VRE isolates produced two distinct colony types characterized phenotypically by a rough and a smooth appearance, respectively. Both colony types retained the vanA locus and the rep-PCR pattern of the primary isolate. These data suggest that a single strain of VRE with the capacity to produce two colonial variants has been disseminated to several Detroit-area hospitals. The clinical significance of the colonial morphotypes is unclear.

Anti-Bacterial Agents↗

PubMed1999

Characterization and heterologous expression of the genes encoding enterocin a production, immunity, and regulation in Enterococcus faecium DPC1146.

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10, 879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis.

Amino Acid Sequence↗

PubMed1998

Comparison of genomic methods for differentiating strains of Enterococcus faecium: assessment using clinical epidemiologic data.

Genomic DNA extracted from 45 vancomycin-resistant Enterococcus faecium (VRE) isolates was cleaved with HindIII and HaeIII and subjected to agarose gel electrophoresis. The ability of this method (restriction endonuclease analysis [REA]) to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). Chart reviews were performed to provide a clinical correlation of possible epidemiologic relatedness. A likely clinical association was found for 29 patients as part of two outbreaks. REA found 21 of 21 isolates were the same type in the first outbreak, with PFGE calling 19 strains the same type. In the second outbreak with eight patient isolates, HindIII found six were the same type and two were unique types. HaeIII found three strains were the same type, two strains were a separate type, and three more strains were unique types, while PFGE found three were the same type and five were unique types. No single "ideal" method can be used without clinical epidemiologic investigation, but any of these techniques is helpful in providing focus to infection control practitioners assessing possible outbreaks of nosocomial infection.

Anti-Bacterial Agents↗

PubMed2005

Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1.

The plasmid pJB01 (GenBank Accession No. AY425961) isolated from the pathogenic bacterium, Enterococcus faecium JC1, is 2235 base pairs in length and consists of a putative double-strand origin (dso), a single-strand origin, a counter-transcribed RNA, and three open reading frames. A comparison of a few replication factors and motifs, bind and nic regions, for replication initiation on the nucleotide sequence level revealed that it belongs to the pMV158 family among RC-replicating plasmids. A runoff DNA synthesis assay demonstrated that nicking occurred between G525 and A526, which is located on the internal loop of a putative secondary structure in the dso. Unlike all the other plasmids of the pMV158 family having two or three direct repeats, pJB01 has three non-tandem direct repeats of 5'-CAACAAA-3' separated by four nucleotides, as the RepB-binding site in the dso. Moreover, the nick site on the internal loop is located at 77 nucleotides upstream from the RepB-binding region. Irrespective of the structural difference of direct repeats from other members of the pMV158 family, we think, it is still a new member of this plasmid family. The introduction of mutations in conserved regions of RepB confirmed that RepB N-moiety is important for nicking/nick-closing activity. Within N-moiety, especially all of the motif R-III, the Y100 in R-IV and Y116 in R-V residues, played particularly critical roles in this activity, however, for its binding, both of the N- and C-moieties of RepB were needed.

Amino Acid Sequence↗

PubMed1994

Bacteriocin production by Enterococcus faecium NA01 from 'wara'--a fermented skimmed cow milk product from west Africa.

An Enterococcus faecium strain from Nigerian fermented skimmed cow milk ('wara') produced bacteriocin inhibitory towards Lactobacillus, Enterococcus and Listeria strains. The bacteriocin (designated enterocin 01) was inactivated by proteases, heat-stable at 100 degrees c and active at pH 2.0-6.0. The Ent. faecium isolate harboured plasmids of ca 36.3 and 23.1 kb. Curing experiments with ethidium bromide resulted in a bacteriocin-negative mutant which had not lost immunity to the bacteriocin. Slight differences in plasmid profiles between wild-type and mutant indicated a possible plasmid-coded bacteriocin production.

Africa↗

PubMed2003

Intra-hospital dissemination of quinupristin/dalfopristin- and vancomycin-resistant Enterococcus faecium in a paediatric ward of a German hospital.

OBJECTIVES: To demonstrate nosocomial transmission of Enterococcus faecium resistant to quinupristin/dalfopristin and vancomycin/teicoplanin among paediatric patients in a German hospital ward. MATERIALS AND METHODS: Multiply-resistant E. faecium were isolated from three female patients aged 9 months, 2 and 15 years during a 10 day time span. Antibiotic susceptibilities were determined by microbroth dilution. Clonal relatedness among the isolates was investigated via SmaI-macrorestriction analysis by PFGE, multilocus sequence typing (MLST), and plasmid profiling. Presence of virulence and resistance determinants was tested by polymerase chain reaction (PCR). Selected resistance genes were localized by Southern hybridizations. RESULTS: A single E. faecium isolate per patient was investigated. All exhibited resistances to quinupristin/dalfopristin, vancomycin/teicoplanin, streptomycin (high-level), penicillin/ampicillin, erythromycin, oxytetracycline, chloramphenicol, rifampicin and fusidic acid. The isolates were susceptible to linezolid only and intermediately resistant to fluoroquinolones including moxifloxacin. PFGE revealed identical patterns for all three isolates. PCRs for virulence determinants hyaluronidase and enterococcal surface protein, esp, were negative, whereas PCR for the enterocin A gene was positive. MLST identified clonal type [8-5-1-1-1-1-1] belonging to a clonal subgroup C1 of hospital- and outbreak-related E. faecium. Southern hybridizations located several resistance genes (erm(B), vat(D), vanA) on a large plasmid, which was transferable in mating experiments with an E. faecium recipient. CONCLUSIONS: These data show routes of dissemination of resistance to multiple antibiotics including streptogramins and glycopeptides in E. faecium via vertical and/or horizontal gene transfer. The isolates spread in the absence of a direct selective pressure, as none of the patients had received earlier streptogramin or glycopeptide therapy.

Anti-Bacterial Agents↗

PubMed1996

Vancomycin-resistant Enterococcus faecium infections in the ICU and quinupristin/dalfopristin.

The incidence of vancomycin resistance among enterococci, and Enterococcus faecium in particular, has increased sharply in the last few years. This shift toward infection with resistant Gram-positive organisms is thought to be the consequence of certain features specific to the intensive care setting: a high concentration of severely compromised patients; continued use of indwelling devices and invasive procedures; and widespread, empiric use of antimicrobial agents directed against Gram-negative bacilli. Measures that can be taken to prevent the development of bacterial resistance in the ICU include strict adherence to infection control policies and asepsis, and rational use of antibiotics. Current antimicrobial regimens for serious enterococcal infections consist of a combination of ampicillin, penicillin G, or vancomycin plus streptomycin or gentamicin. High levels of resistances among some enterococcal isolates, however, may render these strategies ineffective. A new agent, quinupristin/dalfopristin (RP 59500), has demonstrated encouraging in vitro activity against vancomycin-resistant E. faecium. Initial clinical reports, though limited, are similarly promising. Although phase III clinical trials with RP 59500 are not completed, the agent is available through an emergency-use program for patients with severe Gram-positive infections who cannot tolerate or do not respond to all other clinically appropriate antibiotics.

Drug Resistance, Microbial↗

PubMed2006

Emergence of linezolid resistance in the vancomycin-resistant Enterococcus faecium multilocus sequence typing C1 epidemic lineage.

A relatively high rate of vancomycin-resistant Enterococcus faecium not susceptible to linezolid was observed in intensive care unit patients. Linezolid-resistant isolates carried the G2576T mutation in the 23S rRNA gene, belonged to different clones, and shared the same allelic profile, which clusters in the C1 multilocus sequence typing epidemic lineage.

Acetamides↗

PubMed1998

Transferable, plasmid-mediated vanB-type glycopeptide resistance in Enterococcus faecium.

An approximately 60-kb transferable, vanB-carrying plasmid has been identified in a clinical Enterococcus faecium strain. A similar plasmid has been observed in an unrelated E. faecium strain, suggesting that plasmid transfer of vanB operons occurs in nature and plays a role in the dissemination of VanB-type resistance among strains of E. faecium.

Anti-Bacterial Agents↗

PubMed1997

Growth and bacteriocin production by Enterococcus faecium DPC1146 in batch and continuous culture.

Production of the bacteriocin enterocin 1146 (E1146) by Enterococcus faecium DPC1146 was studied in batch and continuous fermentation. Growth was strongly inhibited by lactic acid. In batch fermentations maximum E1146 activity (2.8 MBU L-1) was obtained in 9 h with 20 g L-1 glucose. Increase in initial glucose concentration did not lead to a proportional increase in E1146 activity. A simple linear model was found to be adequate to explain the relationship between specific bacteriocin production rate and specific growth rate in batch fermentations with initial glucose concentration higher than 20 g L-1. Maximum bacteriocin activity (2.9-3.2 MBU L-1) was obtained in continuous fermentations at dilution rates between 0.12 and 0.17 h-1 and specific bacteriocin production rate increased linearly with dilution rate.

Bacteriocins↗

PubMed1994

Endocarditis due to high level gentamicin resistant Enterococcus faecium.

We describe the first reported case in the literature of bacterial endocarditis caused by Enterococcus faecium that was highly resistant to gentamicin. The case is unusual in that it followed a successfully treated Streptococcus sanguis endocarditis. The micro-organism was susceptible only to the glycopeptide antibiotics, vancomycin and teicoplanin and to agents such as pristinamycin and daptomycin which are not routinely available for treatment. It illustrates the therapeutic dilemma posed by serious infections with such micro-organisms and supports previous observations that early heart valve replacement surgery may be necessary to achieve cure of endocarditis due to enterococci which are highly resistant to gentamicin. It further emphasises the importance of screening for high-level aminoglycoside resistance in enterococci in all life threatening enterococcal infections, including endocarditis, septicaemia and meningitis when aminoglycoside-penicillin synergy is required for successful treatment.

Aged↗

PubMedDate not supplied

[The growth characteristics of Enterococcus faecium under batch cultivation conditions].

Growth peculiarities of the strain of homoenzymic lactic acid bacteria Enterococcus faecium under periodic cultivation have been studied. The effect of carbohydrates source on the level of biomass accumulation and efficiency of its use by the strain is shown. Glucose concentration in the medium being increased from 1.0 to 3.5%, the economic effect decreases from 16.0 to 10.6%. It is established that only at the beginning of cultivation the specific growth rate of the strain is determined by the concentration of lactic acid. This dependence can be described by Ierusalimsky's equation. The obtained values of micronm and Kp are 1.5 h(-1) and 6.5 g/l, respectively. Biomass concentration being above 0.3 g ACB/l, the strain growth depends on two factors: inhibition of lactic acid and limitation of the sources of nitrogen nutrition. Interaction of these two factors results in considerable decrease of specific rate of bacterial growth.

Culture Media↗

PubMed2005

Production of enterocin P, an antilisterial pediocin-like bacteriocin from Enterococcus faecium P13, in Pichia pastoris.

The gene encoding mature enterocin P (EntP), an antimicrobial peptide from Enterococcus faecium P13, was cloned into the pPICZalphaA expression vector to generate plasmid pJC31. This plasmid was integrated into the genome of P. pastoris X-33, and EntP was heterologously secreted from the recombinant P. pastoris X-33t1 derivative at a higher production and antagonistic activity than from E. faecium P13.

Anti-Bacterial Agents↗

PubMed2001

Clinical and molecular characterization of vancomycin-resistant Enterococcus faecium strains during establishment of endemicity.

To characterize the molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREF) in Tennessee, VREF isolates that were recovered from patients during a 3-year period at a tertiary care center and throughout the state were typed by means of pulsed-field gel electrophoresis. Clinical characteristics and outcomes of patients colonized or infected with different strain types were also examined. A total of 34 different strain types were identified. A single VREF strain (type O) predominated (63 [61%] of 103 single-patient isolates (i.e., 1 isolate per patient) obtained from 7 different health care institutions). There were no significant differences between patients harboring type O strains and those harboring non-type O strains (P> or =.05). The rate of recovery of type O subtypes and strains other than type O increased over the 3-year study period. Establishment of VREF endemicity was initially characterized by clonal spread of type O strains. Subsequently, polyclonal dissemination may have been due to microevolutionary changes among type O strains.

Anti-Bacterial Agents↗

PubMed1997

Overexpression and characterization of the chromosomal aminoglycoside 6'-N-acetyltransferase from Enterococcus faecium.

The chromosomal gene aac(6')-Ii, encoding an aminoglycoside 6'-N-acetyltransferase in Enterococcus faecium, renders this organism resistant to moderate levels of many aminoglycoside antibiotics. The ubiquitous presence of aac(6')-Ii in E. faecium complicates the selection of antibiotics for treatment of infections caused by this organism. In view of the importance of this enzyme, we have initiated studies to gain an understanding of its molecular mechanism of acetyl transfer. The AAC(6')-Ii enzyme was overexpressed in Escherichia coli and purified in a simple three-step procedure which yields 55 mg of pure dimeric protein per liter of cell culture. Steady-state kinetic analyses revealed a broad substrate specificity and demonstrated that acetylation occurs exclusively at position N-6'. k(cat)/Km values were on the order of 10(4) M(-1) s(-1), which is relatively low compared to other aminoglycoside-modifying enzymes. In addition, MIC values were positively correlated with k(cat), the rate when the enzyme is saturated with the aminoglycoside substrate, and not with k(cat)/Km, the rate at low aminoglycoside (sub-Km) concentrations. These results describe an enzyme which is not optimally evolved for aminoglycoside inactivation and suggest that this chromosomally encoded enzyme may have an alternate physiological function.

Acetylation↗

PubMed2000

[Vancomycin-resistant Enterococcus faecium outbreak in a nephrology ward].

In April 2000, an outbreak of vancomycin-resistant Enterococcus faecium (VRE) was discovered in an internal medicine/nephrology and dialysis ward of the Eemland Hospital, Amersfoort, the Netherlands. Although enterococci are considered relatively non-virulent, VRE are resistant to almost all commercially available antibiotics. Surveillance cultures were obtained from all patients at the ward, all patients visiting the dialysis ward and the environment of patients. VRE were determined and clustering of strains was analysed using molecular genotyping. In all, 12 patients were colonized with the outbreak strain. Transmission of VRE usually occurs via the hands of health care workers. The ward was closed for new admissions, patients were divided in cohorts of colonized and non-colonized patients, and rooms were disinfected after patient discharge. Infection control measures (such as handwashing and use of gloves and gowns) were enforced and prescriptions of vancomycin and cephalosporins were reduced. With these measures the outbreak could be controlled. Epidemiological analysis demonstrated that earlier admission and previous use of ciprofloxacin, amoxicillin and amoxicillin-clavulanic acid were risk factors for colonization. A nearby hospital was a possible source of this outbreak.

Aged↗

PubMed1993

Environmental strains of Enterococcus faecium with inducible high-level resistance to glycopeptides.

High-level resistance to glycopeptides in Enterococcus faecium is associated with an inducible 39-kDa cytoplasmic membrane protein. The present paper shows that such glycopeptide-resistant E. faecium strains can not only be isolated in a definite clinical setting but also from waste water of sewage treatment plants. Nearer characterization of these and of clinical isolates by resistance pattern, biotyping, and genotyping (DNA-fingerprinting with pulsed-field gel electrophoresis) has shown that different glycopeptide-resistant E. faecium strains have been isolated from clinical sources and from waste water.

DNA Fingerprinting↗