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PubMed2006

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Adhesins, Bacterial↗
PubMed1996

Overgrowth of Enterococcus faecium in the feces of patients with hematologic malignancies.

The objective of this study was to characterize the overgrowth and susceptibility of Enterococcus faecium, compared with that of other enterococci, in the feces of patients with hematologic malignancy. During a 52-week period, surveillance samples of feces were cultured weekly. Three hundred thirty-three samples were obtained from 92 patients. E. faecium outnumbered other enterococci in 170 (44%) of the samples, while the opposite was true in 119 (31%) of the samples. Fecal overgrowth of E. faecium (> or = 9.0 log10 cfu/g) was found in 62 samples (16%), while overgrowth of other enterococci was documented in 20 samples (5%) (P < .001). Treatment with third-generation cephalosporins preceded the overgrowth of E. faecium in 93% of the patients. Resistance of E. faecium isolates to ampicillin, high-level gentamicin, and vancomycin was detected in 41%, 4%, and 2% of the patients, respectively. There were 6 patients with enterococcal bacteremia (due to E. faecium in 5 and Enterococcus faecalis in 1) during the study period. The high prevalence of resistance to ampicillin and imipenem rendered few (if any) intravenous antibiotics able to prevent fecal overgrowth of E. faecium.

Aminoglycosides↗
PubMed2001

Possible horizontal transfer of the vanB2 gene among genetically diverse strains of vancomycin-resistant Enterococcus faecium in a Korean hospital.

A total of 25 isolates of vanB-containing Enterococcus faecium were recovered from patients in a single Korean hospital over a 20-month period. There were two distinct vanB2 patterns among the 11 pulsed-field gel electrophoresis types; 17 contained the prototype vanB2 and 8 contained a novel vanB2 with a 177-bp deletion in vanY(B). Both vanB2 genes were transmissible in vitro at a mean frequency of 1.1 x 10(-8) transconjugants/donor. These results suggest the horizontal spread of vanB2 is occurring among genetically diverse strains of E. faecium in Korean hospitals.

Adult↗
PubMed2006

Cometabolism of citrate and glucose by Enterococcus faecium FAIR-E 198 in the absence of cellular growth.

Citrate metabolism by Enterococcus faecium FAIR-E 198, an isolate from Greek Feta cheese, was studied in modified MRS (mMRS) medium under different pH conditions and glucose and citrate concentrations. In the absence of glucose, this strain was able to metabolize citrate in a pH range from constant pH 5.0 to 7.0. At a constant pH 8.0, no citrate was metabolized, although growth took place. The main end products of citrate metabolism were acetate, formate, acetoin, and carbon dioxide, whereas ethanol and diacetyl were present in smaller amounts. In the presence of glucose, citrate was cometabolized, but it did not contribute to growth. Also, more acetate and less acetoin were formed compared to growth in mMRS medium and in the absence of glucose. Most of the citrate was consumed during the stationary phase, indicating that energy generated by citrate metabolism was used for maintenance. Experiments with cell-free fermented mMRS medium indicated that E. faecium FAIR-E 198 was able to metabolize another energy source present in the medium.

Cheese↗
PubMed1995

Vancomycin-resistant Enterococcus faecium bacteremia: risk factors for infection.

We describe an outbreak of vancomycin-resistant Enterococcus faecium (vanA phenotype) bacteremia on the oncology ward of a tertiary care community hospital. In 10 of the 11 cases the patients had leukemia and were neutropenic (median duration of neutropenia, 21 days) at the time of bacteremia. On average, patients received six antibiotic agents for a total of 61 agent-days prior to development of vancomycin-resistant E. faecium bacteremia. The mortality rate was 73%. Molecular typing of 22 isolates revealed that the majority (83%) represented a common strain, indicating nosocomial spread. When the 11 cases were compared to 22 matched control patients, gastrointestinal colonization with vancomycin-resistant E. faecium (odds ratio [denominator, 0] infinity, P = .005) and the use of antimicrobial agents with significant activity against anaerobes (metronidazole, clindamycin, and imipenem; odds ratio infinity, P = .02) were found to be risk factors for the development of vancomycin-resistant E. faecium bacteremia. Since no proven therapy for such infection exists, there is an urgent need to identify effective measures to prevent and control the development of vancomycin-resistant E. faecium bacteremia.

Adult↗
PubMed2006

High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis.

Enterocin P (EntP), a sec-dependent bacteriocin from Enterococcus faecium P13, was produced by Lactococcus lactis. The EntP structural gene (entP) with or without the EntP immunity gene (entiP) was cloned in (1), plasmid pMG36c under control of the lactococcal constitutive promoter P32, (2) in plasmid pNG8048e under control of the inducible PnisA promoter, and (3) in the integration vector pINT29. Introduction of the recombinant vectors in L. lactis resulted in production of biologically active EntP in the supernatants of L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000, and the coproduction of nisin A and EntP in L. lactis subsp. lactis DPC5598. The level of production of EntP, detected and quantified by specific anti-EntP antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, by the recombinant L. lactis strains depended on the host strain, the expression vector, and the presence of the entiP gene in the constructs of the recombinant L. lactis strains. The highest amount of EntP was produced with derivatives containing entP and entiP, for both L. lactis IL1403 and L. lactis NZ9000. These derivatives produced up to five- to six-fold more EntP than E. faecium P13. Mass spectrometry analysis revealed that EntP purified from L. lactis IL1403 (pJP214) has a molecular mass identical to that purified from E. faecium P13, suggesting that the synthesis, processing, and secretion of EntP progresses efficiently in recombinant L. lactis hosts.

Bacterial Proteins↗
PubMed2000

Genotypic characterisation of endemic VanA Enterococcus faecium strains isolated in a paediatric hospital.

A total of 36 vancomycin-resistant Enterococcus faecium isolates obtained from 30 patients during a 28-month period in a paediatric university hospital was analysed by pulsed-field gel electrophoresis (PFGE) combined with Southern hybridisation of a vanA-specific DNA probe. All the isolates hybridised with the vanA probe. Seventeen different PFGE patterns and 11 PFGE subtypes were identified among the 36 clinical isolates, and the size of probe-positive bands ranged from c. 30 to 300 kb. These data are consistent with an increase in the overall genomic diversity of vancomycin-resistant E. faecium isolates during the study period. Two periods were distinguished. The prevalence of a single clone in the initial period suggested transmission between patients in three wards. During the following period, multiple genotypes of vancomycin-resistant E. faecium were identified, indicative of multiple introductions or the dissemination of resistance genes by recombinant transposition.

Adolescent↗
PubMed2003

Rifampicin resistance and its fitness cost in Enterococcus faecium.

OBJECTIVES: The genetic basis of rifampicin resistance and the associated fitness cost in Enterococcus faecium were investigated. METHODS: Twelve spontaneous rifampicin-resistant E. faecium mutants were selected from four parent strains recently isolated from porcine faecal material. The DNA sequence of the complete rpoB gene from the parent strains and of nucleotides -189 to +1785 from the mutants was determined from PCR amplicons. The fitness of the mutants was assessed by determining growth rate, by direct growth competition and by the ability of some of the mutants to survive in the pig intestine. RESULTS: The rpoB genes of the parent strains diverged from each other by 1-10% and each encoded proteins that were 1208 amino acids in length. All mutants had a single amino acid substitution in the region implicated in rifampicin resistance in other organisms. Six mutants carried the substitution H489Y/Q, two mutants carried the substitution R492H, one mutant carried the substitution Q480H, two mutants carried the substitutions S494L and V224I, and one mutant carried the substitutions G485D and V224I. Per generation fitness costs of the mutants ranged from a gain of 2.5% to a cost of 10%. Mutants with the substitution H489Y/Q were the most fit, whereas the double mutants were the least fit. The mutant with the substitution H489Q was able to survive in the pig gut for 12 days. There was some correlation between the rifampicin MIC and fitness cost, with higher MICs being associated with higher fitness costs. CONCLUSIONS: Substitutions in RpoB are associated with rifampicin resistance in E. faecium. The fitness cost of resistance is variable and can sometimes be absent.

Amino Acid Substitution↗
PubMed1997

Molecular epidemiology of glycopeptide-resistant Enterococcus faecium on a renal unit.

The clinical and molecular epidemiology of glycopeptide-resistant Enterococcus faecium was investigated during an outbreak on a renal unit. Forty-nine patients were colonized or infected during a 15-month period. Sites of colonization included faeces, urine, intravenous (IV) catheter tips and wound swabs. Ten patients had infections, which included five bacteraemias and three episodes of peritonitis. Pulsed-field gel electrophoresis of 43 patient isolates of glycopeptide-resistant E. faecium identified seven strains during the first 7 months of the outbreak. Three of these strains affected five or more patients. One strain accounted for 17/43 isolates. Isolates that were possibly related to another renal unit strain were cultured from patients at two other Manchester hospitals. These isolates were epidemiologically-related, and may represent a single Manchester epidemic strain. Of five patients who had multiple isolates of glycopeptide-resistant E. faecium, three had isolates representing a single strain and two were colonized or infected by more than one strain.

Adult↗
PubMed1996

Persistence of vancomycin-resistant Enterococcus faecium gastrointestinal tract colonization in antibiotic-treated mice.

Colonization with vancomycin-resistant Enterococcus faecium (VREF) is strongly associated with previous antimicrobial therapy. The gastrointestinal (GI) tract appears to be the major reservoir for this organism. We used antibiotic-treated Swiss Webster mice to study GI tract colonization with a characterized strain of VREF (E. faecium 228). Mice were pretreated with antibiotics in their daily drinking water and inoculated with 10(9) colony-forming units (CFU) of E. faecium 228 by oral gavage. We were able to establish persistent colonization with high concentrations of E. faecium 228 (> 8.0 log10 CFU/g of feces) in animals treated with 5 mg/ml of streptomycin plus 1 mg/ml of cefotetan. RP 59500, a streptogramin antibiotic with good in vitro activity against VREF, was administered orally in mice (n = 8) colonized with E. faecium 228. After 14 days of treatment VREF was undetectable in feces of all treated mice (< 3.0 CFU/g). Seven days after discontinuation of RP 59500, VREF was present in the feces of all animals. VREF isolates recovered after treatment remained susceptible to RP 59500. Attempts to eradicate E. faecium 228 colonization by oral administration of a vancomycin-sensitive E. faecium strain (SF68) or Lactobacillus spp. were unsuccessful as long as animals continued to receive streptomycin and cefotetan. Recovery of E. faecium 228 from cultures of livers and gallbladders in some animals with persistent GI tract colonization suggests that the organisms may also colonize the hepatobiliary system.

Animals↗
PubMed2004

Cloning of the bile salt hydrolase (bsh) gene from Enterococcus faecium FAIR-E 345 and chromosomal location of bsh genes in food enterococci.

Enterococcus faecium strain FAIR-E 345 isolated from food was shown to possess bile salt hydrolase (Bsh) activity in a plate screening assay and by high-performance liquid chromatography analysis. The bsh gene was cloned and sequenced. DNA sequence analysis revealed that it encoded a protein of 324 amino acids, with pI 4.877. A bsh gene probe was prepared from the cloned bsh gene and was used for probing plasmid and total genomic DNA of Bsh-positive enterococci isolated from food to determine the genomic location of their bsh genes. This probe was able to detect the bsh gene among total genomic DNA preparations but not from plasmid preparations of 10 plasmid-bearing Enterococcus strains. However, the probe could detect the bsh gene from total genomic DNA preparations of 12 Enterococcus strains that did not contain detectable plasmid DNA. In no cases did the probe hybridize with plasmid DNA preparations, suggesting that the bsh gene among enterococci is probably generally chromosomally encoded. This presumptive chromosomal location of bsh genes among food enterococci suggests that transfer of this trait by conjugative plasmids is unlikely.

Amidohydrolases↗
PubMed2003

Identification of a novel insertion sequence in vanB2-containing Enterococcus faecium.

AIMS: The characterization of a novel insertion sequence (IS) in vanB2-containing Enterococcus faecium was conducted. METHODS AND RESULTS: Direct PCR amplification of ORFC region of Tn5382 from DNA extracted from vanB2-containing E. faecium, and sequence analysis were performed. A novel IS was identified. It is 1418 bp in length and contains one putative open reading frame that is similar to transposase. There exists inverted terminal repeats of 12 bp, but direct repeats are not present. According to high similarity to putative transposases of IS3 members, such as, IS150, IS861, IS1077 and IS911, we designated it ISEnfa3. SIGNIFICANCE AND IMPACT OF STUDY: Since ISEnfa3 was detected in all vanB2-containing strains examined so far, it could be used as a tool for epidemiological study.

Amino Acid Sequence↗
PubMed1997

Synergic activity of vancomycin-quinupristin/dalfopristin combination against Enterococcus faecium.

Clinical specimens were cultured, and the strains identified by the Vitek system as Enterococcus faecium were characterized by their DNA. The MIC vancomycin, quinupristin/dalfopristin and teicoplanin for each isolate was determined. Ten vancomycin-sensitive and ten vancomycin-resistant strains of E. faecium were tested. Quinupristin/dalfopristin at 0.25 x MIC and vancomycin at 0.5 x MIC separately as well as in combination were added to Trypticase Soy Broth tubes inoculated with a 24 h culture. The results obtained by determining cfu at 2, 4, 8, 12 and 24 h indicated that the combination of subinhibitory concentrations of quinupristin/dalfopristin plus vancomycin produced after 24 h, in vancomycin-resistant strains, a consistent degree of synergy. Synergy was observed up to only 12 h when similar combinations were employed for vancomycin-sensitive strains. Vancomycin-sensitive strains tended to be slightly less susceptible to quinupristin/dalfopristin than vancomycin-resistant strains.

Colony Count, Microbial↗
PubMed2003

The stimulating effect of a harsh environment on the bacteriocin activity by Enterococcus faecium RZS C5 and dependency on the environmental stress factor used.

Bacteriocin production by Enterococcus faecium RZS C5 occurs in a growth-associated way but is generally switched off in the very early growth phase. The influence of environmental stress on the bacteriocin production kinetics by E. faecium RZS C5 was analysed at a controlled temperature of 35 degrees C and constant pH 6.5. The effect of environmental stress on bacteriocin production was depending on the type of stress applied. Oxidative stress did not interfere with cell growth or bacteriocin activity. In contrast, salt stress decreased both the cell growth and the specific bacteriocin production. Nevertheless, moderate levels of sodium chloride improved bacteriocin activity because they increased the biomass concentration at which bacteriocin production was switched off. Environmental stress due to limitations in sugar or complex nutrients did not affect the early shut-off mechanism or the specific bacteriocin production. However, bacteriocin stability decreased or increased at low levels of sugar or complex nutrients, respectively.

Bacteriocins↗
PubMed1992

A truncated Tn916-like element in a clinical isolate of Enterococcus faecium.

A 58.7-kb nonconjugative plasmid (pKQ1) previously reported in a clinical isolate of Enterococcus faecium was found to contain both a tetM and an erythromycin resistance (erm) determinant. The plasmid contained a region homologous to the A, F, H, and G HincII fragments of Tn916. However, the 4.8-kb B fragment of Tn916 which contained the tetM determinant was replaced by a 7.3-kb fragment, and the 3.6-kb HincII C fragment of Tn916 was missing. An element homologous to Tn917 was juxtaposed to the truncated Tn916-like element. The Tn917-like element was similar in size to the erm transposon Tn917 as determined by a ClaI restriction digest which spanned approximately 99% of the transposon. When Bacillus subtilis or Streptococcus sanguis were transformed with pKQ1, no zygotically induced transposition of the tetM element was detected. Similarly no transposition of the Tn917-like element was detected.

Bacillus subtilis↗
PubMed2000

Anti-Listeria effect of enterocin A, produced by cheese-isolated Enterococcus faecium EFM01, relative to other bacteriocins from lactic acid bacteria.

Enterocin A produced by Enterococcus faecium EFM01 displayed a narrow antimicrobial spectrum, mainly directed against Listeria spp. In particular, the bacteriocin was extremely active against 13 Listeria monocytogenes strains. This high specificity of action of enterocin A for Listeria spp. relative to lactic acid bacteria, together with its broad range of activity from pH 4.0 to pH 9.0, are factors which may be of great interest with respect to the potential antilisterial use of this bacteriocin in fermented foods. Assessment of the effect of enterocin A concentration on the extent and kinetics of bactericidal activity on L. monocytogenes Lm 6 (107 cfu ml-1 in culture broth), suggested that viability losses of higher than 5 log10, and time intervals necessary for maximum loss of viability of less than 2 h, could not be obtained. Moreover, it was shown that both parameters are closely dependent on the Listeria strain used. On the other hand, at concentrations inducing destruction of approximately 2 log10 cycles, maximum loss of viability was achieved within time intervals which varied widely from one lactic acid bacteria bacteriocin to another.

Bacteriocins↗