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PubMed2006

Complete sequence of the enterocin Q-encoding plasmid pCIZ2 from the multiple bacteriocin producer Enterococcus faecium L50 and genetic characterization of enterocin Q production and immunity.

The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.

Amino Acid Sequence↗

PubMed1998

Enterococcus faecium bacteremia: does vancomycin resistance make a difference?

BACKGROUND: Enterococcus faecium has received increased attention, primarily due to the emergence of vancomycin resistance. The purpose of this investigation was to study the epidemiological characteristics of vancomycin-resistant E faecium (VRE) bacteremia and to determine the clinical impact of vancomycin resistance on the outcome of patients with this infection. METHODS: We retrospectively analyzed the clinical features and outcome of 53 patients with E faecium bacteremia. RESULTS: From January 1992 until December 1995, there were 32 episodes of bacteremia caused by vancomycin-susceptible E faecium (VSE) and 21 caused by VRE. An intra-abdominal site was the most common source of bacteremia in both groups. All of the VRE and 78% of VSE bacteremia cases were nosocomially acquired. Previous administration of vancomycin was associated with VRE bacteremia (P<.001), as were indwelling bladder catheters (P=.01). Fifty-nine percent of the patients with VSE bacteremia survived vs 24% with VRE (P=.009), despite similar severity-of-illness scores. In 62% of the patients with VRE sepsis, death was related to the bacteremia (P=.01). Patients infected with VRE had longer hospitalizations than those with VSE (34.8 vs 16.7 days, respectively) (P=.004), were more likely to be on the medical service (P=.03), and on the average, had hospitalization costs of more than $27,000 per episode than did patients with VSE bloodstream infection ($83,897 vs $56,707, respectively) (P=.04). CONCLUSIONS: Vancomycin-resistant E faecium bacteremia is a complication of prolonged hospitalization in debilitated patients. Vancomycin resistance has a negative impact on survival in patients with E faecium bacteremia and leads to higher health care costs.

Anti-Bacterial Agents↗

PubMed1996

Risk factors associated with vancomycin-resistant Enterococcus faecium infection or colonization in 145 matched case patients and control patients.

Risk factors and mortality associated with vancomycin-resistant Enterococcus faecium (VREF) infection or colonization were examined at a tertiary care hospital by comparing 145 patients who had VREF isolates (cases) to 145 patients with vancomycin-susceptible Enterococcus faecium (VSEF) isolates (controls). The number of deaths per 100 person-days of hospitalization after diagnosis did not differ significantly between VREF patients (1.2) and VSEF patients (0.8). Multivariate analyses found that the duration of hospitalization ( > or = 7 days), intrahospital transfer between floors, use of antimicrobials (i.e., vancomycin and third-generation cephalosporins), and duration of vancomycin use ( > or = 7 days) was independently associated with VREF infection or colonization. This study, which has a large sample size, confirms some earlier observations regarding risks for VREF infection or colonization and identifies factors that may be potentially exploited to develop interventional strategies for the control of this emerging nosocomial problem.

Adolescent↗

PubMed1999

Isolation and molecular characterization of vancomycin-resistant Enterococcus faecium in Malaysia.

Nineteen strains of vancomycin-resistant Enterococcus faecium isolated from 10 of 75 (13.3%) tenderloin beef samples were examined for resistance to selected antibiotics, presence of plasmids, and genetic diversity by random amplification of polymorphic DNA analysis. All strains showed multiple resistant to the antibiotics tested. Multiple antibiotic indexing of the vancomycin-resistant E. faecium strains showed that all (100%) originated from high risk contamination environments where antibiotics were often used. Plasmids ranging in size from 1.5 to 36 megadalton were detected in 15 of 19 (79%) strains. Thus, three plasmid profiles and eight antibiotypes were observed among the E. faecium strains. A high degree of polymorphism was obtained by combining the results of the two primers used; with the 19 E. faecium strains being differentiated into 19 RAPD-types. These preliminary results suggest that RAPD-PCR has application for epidemiologic studies and that resistance patterns and plasmid profiling could be used as an adjunct to RAPD for the typing of E. faecium in the study area.

Animals↗

PubMed2003

Analysis of daptomycin efficacy and breakpoint standards in a murine model of Enterococcus faecalis and Enterococcus faecium renal infection.

Daptomycin efficacy against clinical isolates of Enterococcus faecalis, Enterococcus faecium, and a lab-derived daptomycin-resistant isolate of E. faecalis was investigated in a mouse model of renal infection. The daptomycin MICs against these enterococci ranged from 0.5 to 50 micro g/ml. The objective of this study was to determine the relationship between the MICs of drugs against E. faecalis and E. faecium and the level of daptomycin exposure needed to evaluate the drug's efficacy. Correlating the required therapeutic exposures of mice with the exposures achieved clinically allowed us to project enterococcal breakpoint values. Mice pretreated with carrageenan were infected intravenously with 3 x 10(8) to 4 x 10(8) CFU of E. faecalis or E. faecium. Daptomycin (5 to 50 mg of drug/kg of body weight) or saline control was administered 4 h postinfection and continued once daily for 2 days (three total doses). On day 4, infected kidneys were harvested, homogenized, and dilution plated. Efficacy was defined as a > or = 2-log(10) (99%) reduction in bacterial burden in infected kidneys. At clinically relevant dosages and exposures (area under the curve, 400 to 600 microg.hr/ml), daptomycin demonstrated similar and marked efficacy against all clinical enterococcal isolates tested. Daptomycin achieved efficacy with comparable doses against both vancomycin-sensitive (MIC, < or = 4 microg/ml) and -resistant enterococcal strains tested. Efficacy was also established against the lab-derived daptomycin-resistant E. faecalis isolate. In this murine renal infection model, clinically relevant exposures of daptomycin were effective against E. faecalis and E. faecium strains for which MICs were < or = 8 microg/ml. These murine efficacy data for daptomycin, along with surveillance data and human pharmacokinetic exposures achieved, suggest a breakpoint concentration value of < or = 8 microg/ml (susceptible) and > or = 16 microg/ml (resistant) for daptomycin against E. faecium and E. faecalis.

Animals↗

PubMed1993

Comparison of high-level gentamicin-resistant Enterococcus faecium isolates from different continents.

Eight clinical isolates of Enterococcus faecium highly resistant to gentamicin (MIC, > 1,000 mg/liter) from patients in six hospitals on three continents were investigated for evidence of spread of either a clone of high-level gentamicin-resistant (HLGR) E. faecium or wide dissemination of a gentamicin resistance (Gmr) plasmid. A combination of ribotypes, plasmid profiles, and extended antimicrobial susceptibilities enabled us to distinguish all but two of the isolates and did not suggest clonal dissemination of a single strain. Two isolates from hospitals situated close together appeared identical by these methods. All of the isolates carried Gmr plasmids which appeared to be closely related following digestion with restriction endonucleases. Cross-hybridization studies confirmed extensive DNA homology between these plasmids. The fragments of these plasmids which hybridized with a probe specific for the aac6'aph2" resistance gene did not resemble those seen in the Gmr transposon Tn5281, which was characterized previously in E. faecalis HH22. This study suggests that there has been widespread dissemination of a single Gmr plasmid and its derivatives amongst isolates of HLGR E. faecium, although a Gmr plasmid from an HLGR E. faecium isolated in the United States showed little homology with the other Gmr plasmids studied.

Conjugation, Genetic↗

PubMed1999

Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339.

VanD-type resistance to glycopeptides in Enterococcus faecium BM4339 is due to constitutive synthesis of D-alanyl-D-lactate-terminating peptidoglycan precursors (B. Périchon, P. Reynolds, and P. Courvalin, Antimicrob. Agents Chemother. 41:2016-2018, 1997). The sequence of a 5,780-bp fragment was determined and revealed six open reading frames. The 3' distal part encoded the VanHD dehydrogenase, the VanD ligase, and the VanXD DD-dipeptidase, which were highly similar to the corresponding proteins in VanA and VanB types of resistance. The deduced VanYD protein was homologous to penicillin-binding proteins that display DD-carboxypeptidase activity. The 5' end coded for the putative VanRD-VanSD two-component regulatory system. Due to a frameshift mutation in the chromosomal ddl gene, BM4339 produced an impaired D-alanine:D-alanine ligase. However, since expression of the resistance genes is constitutive, growth of E. faecium BM4339 was not dependent on the presence of glycopeptides in the culture medium.

Amino Acid Sequence↗

PubMed2002

[A critical pneumonia by multidrug-resistant Enterococcus faecium in a chronic hemodialysis patient. A case report].

Enterococcus faecium is usually known as a pathogen of nosocomial infections in compromised patients. Recently it is a problem that some of the E. faecium become resistant to many antibiotics. It is not usual that this pathogen causes pneumonia. We report a community-acquired pneumonia by multidrug-resistant E. faecium in an outpatient who had been receiving hemodialysis for the past year. The patient recovered from pneumonia with vancomycin, but had nosocomial pneumonia caused by MRSA later but recovered.

Aged↗

PubMed1996

Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium.

Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in beta-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 micrograms/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k+2/K] values of 17 to 25 M(-1) s(-1)) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 micrograms/ml) and H80721 (MIC, 512 micrograms/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm variants (k+2/K < or = 1.5 M(-1) s(-1)) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.

Amino Acid Sequence↗

PubMed1992

Biosynthesis of modified peptidoglycan precursors by vancomycin-resistant Enterococcus faecium.

In the presence of bacitracin, vancomycin-resistant Enterococcus faecium (vanA phenotype) accumulate UDP-N-acetylmuramyl(UDP-Mur-NAc)-tetrapeptide and a UDP-MurNAc-depsipentapeptide containing lactate substituted for the carboxy-terminal-D-alanine residue. In an in vitro peptidoglycan polymerization assay, the modified precursors function and confer resistance to vancomycin.

Amino Acid Sequence↗

PubMed1994

Evaluation and characterization of multiresistant Enterococcus faecium from 12 U.S. medical centers.

Forty-two Enterococcus faecium isolates resistant to ampicillin, penicillin, gentamicin, streptomycin, vancomycin, and teicoplanin (VanA phenotype) from 12 U.S. medical centers were analyzed by pulsed-field gel electrophoresis of chromosomal DNA. The isolates were tested for susceptibility to 12 alternative drugs. The results indicated both intrahospital and interhospital diversity among multiresistant vanA enterococcal isolates. Furthermore, the finding of isolates with identical pulsed-field gel electrophoresis patterns in different centers strongly suggests some interhospital clonal transmission.

DNA, Bacterial↗

PubMed1993

In vitro activity of novobiocin against multiresistant strains of Enterococcus faecium.

Sixty strains of vancomycin- and ampicillin-resistant Enterococcus faecium were evaluated for their susceptibilities to novobiocin in vitro. In Mueller-Hinton broth, novobiocin inhibited all strains when it was used at a concentration of < or = 2 microgram/ml and 40 of 60 strains when it was used at a concentration of < or = 0.5 micrograms/ml. MICs were 8- to 16-fold higher in 50% serum. Novobiocin alone resulted in 2-log-unit killing at 24 h. Combinations of novobiocin and a fluoroquinolone (either ciprofloxacin or ofloxacin) were additive and bactericidal for quinolone-susceptible strains in either broth or 50% serum. Gentamicin did not affect novobiocin activity, and rifampin and doxycycline were antagonistic.

Anti-Infective Agents↗

PubMed2000

Expression, purification and crystallization of enterococcus faecium streptogramin A acetyltransferase.

The streptogramin A acetyltransferase from Enterococcus faecium (SWISS-PROT P50870) has been overexpressed in Escherichia coli, purified and crystallized. Crystallization conditions were screened using the hanging-drop vapor-diffusion method and resulted in two distinct crystal forms. Form I crystals diffract to 2.5 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 68.6, b = 102.6, c = 107.5 A. Form II crystals diffract to 2.7 A and belong to space group F222, with unit-cell parameters a = 185.8, b = 185.8, c = 186.5 A. Rotation-function and packing analyses for both crystal forms indicate that the asymmetric unit may contain one and two copies of the trimeric enzyme for crystal forms I and II, respectively.

Acetyltransferases↗

PubMed2003

Homemade traditional cheeses for the isolation of probiotic Enterococcus faecium strains.

One hundred twenty-two strains of Enterococcus faecium isolated from Tafí Cheese, a homemade traditional cheese of the highlands in the province of Tucumán, Argentina, were evaluated for their potential application as starter cultures in the manufacture of this traditional cheese. Eleven of the 122 strains showing limited delays in growth in oxgall were selected for the study of bile salts hydrolase activity (BSH), cholesterol reduction, antimicrobial activity, and virulence determinants. Nine strains were able to remove cholesterol in in vitro assays, a property that was closely related to the bile salt hydrolase activity. Only two strains produced active bacteriocins against Listeria strains although genetic evidence for the bacteriocin structural gene was found in six other enterococci strains. No virulence factors were detected in any of the 11 selected strains of enterococci.

Argentina↗

PubMed1997

Isolation of vancomycin resistant Enterococcus faecium from food.

In a survey of vancomycin resistant Enterococcus faecium (VREF) in Danish meat products, VREF could be detected in 16% of 160 samples of broilers collected at slaughterhouses and in 15% of 26 samples of pork collected from the retail trade. VREF were isolated by enrichment for 24 h in nutrient broth supplemented with vancomycin (50 micrograms/ml) prior to plating on Slanetz and Bartley agar. Using direct plating on Slanetz and Bartley agar, VREF could be isolated from only 1.7% of 540 samples of broilers from slaughterhouses and 2.2% of 90 samples of broilers from retail outlets. VREF was not detected in 124 samples of pork and 128 samples of beef from retail outlets by the direct plating method. An additional enrichment step in nutrient broth supplemented with vancomycin enhanced the detection rate of VREF by approximately three times compared to the direct plating method when investigating the same 160 samples of broilers by the two methods. The implications and public health aspects of VREF in food is discussed.

Animals↗

PubMed2005

Combination treatment of rat adjuvant-induced arthritis with methotrexate, probiotic bacteria Enterococcus faecium, and selenium.

The effects of the probiotic bacteria Enterococcus faecium (EF) and selenium were studied on methotrexate (MTX) treatment in rats with adjuvant arthritis. Arthritic rats were treated orally with the following substances: lyophilized EF (15 mg/kg/day, 5 days a week), sodium selenite pentahydrate (SSe; 0.050 mg/kg containing 0.015 mg/kg selenium, 5 days a week), MTX (0.6 mg/kg/week), and their combinations for the period of 50 days from adjuvant application. Levels of serum albumin, serum nitrite/nitrate concentrations, hind paw swelling, arthrogram scores, whole-body bone mineral density (BMD), and bone erosions were evaluated as markers of inflammation and destructive changes associated with arthritis. Long-term preventive treatment with low-dose MTX significantly inhibited the markers of both inflammation and arthritis. EF or SSe when administered singly or in combination had no significant effect on the given parameters in arthritic rats. EF, but not SSe, potentiated the beneficial effects of MTX, which resulted in a more significant reduction of hind paw swelling, arthrogram scores, and whole-body BMD decrease. EF also had a tendency to improve the effect of MTX on serum albumin and nitrite/nitrate concentrations. Our results indicate that EF may increase the preventive effect of MTX treatment in rat adjuvant arthritis by improving its anti-inflammatory and antiarthritic effects.

Animals↗

PubMed1990

Microbiological study of Enterococcus faecium SF68: post-antibiotic effect and growth curves.

We studied the microbiological characteristics of Enterococcus faecium SF 68 (an oral vaccine) assessing post-antibiotic effect of ampicillin and growth curves in different media. Results showed a good resistance of the microorganism tested which has important implications in clinical practice. The growth ability of Enterococcus faecium SF 68 was similar on all media tested.

Ampicillin↗

PubMed2001

Differences in antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium strains isolated from farm and pet animals.

The prevalence of acquired resistance in 146 Enterococcus faecium and 166 Enterococcus faecalis strains from farm and pet animals, isolated in 1998 and 1999 in Belgium, against antibiotics used for growth promotion and for therapy was determined. Acquired resistance against flavomycin and monensin, two antibiotics used solely for growth promotion, was not detected. Avoparcin (glycopeptide) resistance was found sporadically in E. faecium only. Avilamycin resistance was almost exclusively seen in strains from farm animals. Resistance rates were higher in E. faecium strains from broiler chickens than in strains from other animal groups with tylosin and virginiamycin and in E. faecalis as well as in E. faecium strains with narasin and bacitracin. Resistance against ampicillin was mainly found among E. faecium strains from pets and was absent in E. faecalis. Tetracycline resistance occurred most often in strains from farm animals, while enrofloxacin resistance, only found in E. faecalis, occurred equally among strains from all origins. Resistance against gentamicin was very rare in broiler strains, whereas resistance rates were high in strains from other origins. It can be concluded that resistance against antibiotics used solely for growth promotion was more prevalent in E. faecium strains than in E. faecalis strains. With few exceptions, resistance against the different categories of antibiotics was more prevalent in strains from farm animals than in those from pets.

Animals↗