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Molecular analysis of the 21-kb bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a.

The complete 21,344-bp DNA sequence of the bacteriocin-encoding plasmid pEF1 from Enterococcus faecium 6T1a was determined. Thirty-four putative open reading frames which could code for proteins longer than 42 amino acids were found. Those included the structural genes encoding for the previously described bacteriocins enterocin I and J (also named as enterocins L50A and L50B). After comparison to sequences in public databases, analysis of the gene organization of pEF1 suggests a modular structure with three different functional domains: the replication region, the bacteriocin region and the mobilization plus UV-resistance region. This genetic mosaic structure most probably evolved through recombination events promoted by transposable elements. The hypothesis that the bacteriocin cluster on pEF1 could act as a functional plasmid stabilization module in E. faecium 6T1a is discussed.

Amino Acid Sequence↗

Impact of specific pbp5 mutations on expression of beta-lactam resistance in Enterococcus faecium.

We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all beta-lactams. Affinity for penicillin generally correlated with beta-lactam MICs for the mutants, but these associations were not strictly proportional.

Ampicillin Resistance↗

Identification of an unusual VanA element in glycopeptide-resistant Enterococcus faecium in Brazil following international transfer of a bone marrow transplant patient.

A vancomycin-resistant Enterococcus (VRE) was isolated from a blood culture of a patient in a Brazilian hospital who had a treatment history of a bone marrow transplant in the USA. The organism was identified as Enterococcus faecium, which exhibited an MIC (minimum inhibitory concentration) >or= 256 microg/mL for vancomycin. This was confirmed by E-test and the vanA gene was detected by PCR. Overlapping PCR revealed a left IR deletion and an additional 1.5 kb fragment between vanSH genes. DdeI digestion of vanRSHAX genes showed the determinant to be a T type variant, and the element was cloned and sequenced. These results revealed an IS1251 downstream of nucleotide 5820 of the VanA element. Insertions like this have not been reported previously in Brazil, but have been detected in the USA. The genotype and association with a patient previously treated in the USA suggest that this VRE was introduced from abroad, probably through inter-hospital strain spread.

Aged↗

Treatment of experimental adjuvant arthritis with the combination of methotrexate and lyophilized Enterococcus faecium enriched with organic selenium.

The efficacy of combination therapy with methotrexate (MTX) and probiotic bacteria Enterococcus faecium enriched with organic selenium (EFSe) in rats with adjuvant arthritis was determined. Rats with adjuvant arthritis were given MTX (0.3 mg/kg 2-times weekly, orally); lyophilized E. faecium enriched with Se (15 mg/kg, 5 d per week, orally); and a combination of MTX plus EFSe for a period of 50 d from the immunization. Levels of serum albumin, serum nitrite/nitrate concentrations, changes in hind paw swelling, arthrogram score, bone erosions, whole body bone mineral density (BMD) and bone mineral content (BMC) were assayed in the rats as variables of inflammation and destructive arthritis-associated changes. Treatment with MTX and with the combination MTX + EFSe significantly inhibited markers of both inflammation and arthritis. Significant differences in favor of combination therapy with MTX + EFSe as compared to MTX alone were seen in serum albumin concentration, hind paw swelling and arthrogram score. Reductions in radiographic scores were also more pronounced in the combination therapy group. Combination therapy, but not MTX alone, inhibited the reduction of BMD and BMC; treatment with lyophilized EFSe alone had no significant effect on adjuvant arthritis in rats. The potent therapeutic effect of low dosage MTX therapy in combination with lyophilized EFSe on adjuvant arthritis in rats was shown.

Animals↗

Persistence of animal and human glycopeptide-resistant enterococci on two Norwegian poultry farms formerly exposed to avoparcin is associated with a widespread plasmid-mediated vanA element within a polyclonal enterococcus faecium population.

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that "plasmid addiction systems" may contribute to the persistence of GREF in nonselective environments.

Animal Husbandry↗

Natural history of colonization with vancomycin-resistant Enterococcus faecium.

OBJECTIVE: To determine the incidence, duration, and genetic diversity of colonization with vancomycin-resistant Enterococcus faecium (VREF). SETTING: Oncology unit of a 650-bed university hospital. METHODS: Surveillance perianal swab cultures were performed on admission and weekly. The molecular relatedness of VREF isolates was determined by pulsed-field gel electrophoresis and by the hybridization pattern of the vanA resistance determinant. RESULTS: During 8 months of surveillance, the VREF colonization rate was 16.6 patients per 1,000 patient-hospital days, which was 10.6 times greater than the VREF infection rate. Eighty-six patients with VREF colonization were identified. Colonization persisted for at least 7 weeks in the majority of patients. Of 36 colonized patients discharged from the hospital and then readmitted, an average of 2 1/2 weeks later, 22 (61%) patients still were colonized with VREF. Of the 14 patients who were VREF-negative at readmission, only three patients remained culture-negative throughout hospitalizations. PFGE demonstrated that colonization with the same VREF isolate may persist for at least 1 year, and patients may be colonized with more than one strain of VREF. CONCLUSION: VREF colonization is at least 10-fold more prevalent than infection among oncology patients. Colonization often persists throughout lengthy hospitalizations and may continue for long periods following hospitalization.

Adult↗

Kinetic mechanism of the GCN5-related chromosomal aminoglycoside acetyltransferase AAC(6')-Ii from Enterococcus faecium: evidence of dimer subunit cooperativity.

The aminoglycoside 6'-N-acetyltransferase AAC(6')-Ii from Enterococcus faecium is an important microbial resistance determinant and a member of the GCN5-related N-acetyltransferase (GNAT) superfamily. We report here the further characterization of this enzyme in terms of the kinetic mechanism of acetyl transfer and identification of rate-contributing step(s) in catalysis, as well as investigations into the binding of both acetyl-CoA and aminoglycoside substrates to the AAC(6')-Ii dimer. Product and dead-end inhibition studies revealed that AAC(6')-Ii follows an ordered bi-bi ternary complex mechanism with acetyl-CoA binding first followed by antibiotic. Solvent viscosity studies demonstrated that aminoglycoside binding and product release govern the rate of acetyl transfer, as evidenced by changes in both the k(cat)/K(b) for aminoglycoside and k(cat), respectively, with increasing solvent viscosity. Solvent isotope effects were consistent with our viscosity studies that diffusion-controlled processes and not the chemical step were rate-limiting in drug modification. The patterns of partial and mixed inhibition observed during our mechanistic studies were followed up by investigating the possibility of subunit cooperativity in the AAC(6')-Ii dimer. Through the use of AAC-Trp(164) --> Ala, an active mutant which exists as a monomer in solution, the partial nature of the competitive inhibition observed in wild-type dead-end inhibition studies was alleviated. Isothermal titration calorimetry studies also indicated two nonequivalent antibiotic binding sites for the AAC(6')-Ii dimer but only one binding site for the Trp(164) --> Ala mutant. Taken together, these results demonstrate subunit cooperativity in the AAC(6')-Ii dimer, with possible relevance to other oligomeric members of the GNAT superfamily.

Acetyltransferases↗

Vancomycin-resistant Enterococcus faecium in hospitalized children.

OBJECTIVE: Determine the epidemiology and risk factors for colonization with vancomycin-resistant Enterococcus faecium. DESIGN: Survey; case-control study. SETTING: Children's hospital. PATIENTS: Pediatric oncology patients. INTERVENTION: Contact isolation, restriction of vancomycin prescribing. RESULTS: There was a high prevalence of colonization with vancomycin-resistant enterococci among pediatric oncology patients. The length of hospitalization and the administration of vancomycin and other intravenous antibiotics was associated with colonization. Prevention of colonization was associated with restriction of vancomycin use and contact isolation. CONCLUSIONS: Vancomycin use may predispose to colonization with vancomycin-resistant E faecium. Vancomycin-resistant E faecium may be nosocomially spread. Contact isolation and restriction of vancomycin use may prevent spread of vancomycin-resistant E faecium.

Bacterial Typing Techniques↗

Physical and genetic map of Enterococcus faecium ATCC19434 and demonstration of intra- and interspecific genomic diversity in enterococci.

A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000-3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons.

Chromosomes, Bacterial↗

Comparative in vitro activity of vancomycin and other antimicrobial agents against methicillin-resistant staphylococcus aureus and enterococcus faecium in the Tohoku district of Japan.

Susceptibility patterns of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium obtained from various hospitals of the Tohoku district were documented. MICs of 6 antimicrobial agents against a total of 480 strains (380 strains were MRSA and 100 were E. faecium) were estimated. All MRSAs were susceptible to vancomycin, teicoplanin and quinupristin/dalfopristin, but all of them were resistant to ampicillin and benzylpenicillin. None of the E. faecium strains were found to be resistant to vancomycin, teicoplanin and quinupristin/dalfopristin. Excluding these, almost all strains of E. faecium were resistant to the remaining drugs. These data suggest that despite the emergence of vancomycin resistance to E. faecium in Europe and in the United States, vancomycin, teicoplanin and quinupristin/dalfopristin will nevertheless provide effective bactericidal activity in the Tohoku area of Japan.

Ampicillin↗

One or two low affinity penicillin-binding proteins may be responsible for the range of susceptibility of Enterococcus faecium to benzylpenicillin.

Three benzylpenicillin-resistant, clinical isolates of Enterococcus faecium (MIC values 16-64 micrograms ml-1) contained six penicillin-binding proteins (PBPs), of which PBP5 was the most abundant and had the lowest affinity for the antibiotic. Four benzylpenicillin-susceptible strains (MIC values 0.031-0.5 microgram ml-1) were obtained as spontaneous derivatives from these above organisms. There were significant decreases in the amounts of PBP5 in each of the derivatives, with the concomitant appearance of a new, higher affinity PBP (5*) in three strains. Increased amounts of PBP5, with no changes in PBP5*, were found in several mutants with intermediate-level benzylpenicillin-resistance (MIC values 1-8 micrograms ml-1) selected from two of the susceptible strains. Examination of 18 other clinical isolates, with a wide range of susceptibilities to benzylpenicillin (MIC values 0.062-128 micrograms ml-1), showed that PBP5* was present in 13 strains, and PBP5 in all of them, but in differing amounts. The results concerning the relative amounts and relative affinities of PBPs 5* and 5 allowed the categorization of the various strains into six groups, within which organisms had somewhat similar susceptibilities to benzylpenicillin.

Bacterial Proteins↗

Characterization of Tn1546 in vancomycin-resistant Enterococcus faecium isolated from canine urinary tract infections: evidence of gene exchange between human and animal enterococci.

Thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a 2-year period (1996 to 1998). Isolated species included Enterococcus faecium (n = 13), Enterococcus faecalis (n = 7), Enterococcus gallinarum (n = 11), and Enterococcus casseliflavus (n = 4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to three or more antibiotics. One E. faecium isolate, CVM1869, displayed high-level resistance to vancomycin (MIC > 32 micro g/ml) and gentamicin (MIC > 2,048 micro g/ml). Molecular analysis of this isolate revealed the presence of Tn1546 (vanA), responsible for high-level vancomycin resistance, and Tn5281 carrying aac6'-aph2", conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine E. faecium clone that had acquired Tn1546, perhaps from a human vancomycin-resistant E. faecium. Transposons Tn5281 and Tn1546 were located on two different conjugative plasmids. Sequence analysis revealed that in Tn1546, ORF1 had an 889-bp deletion and an IS1216V insertion at the 5' end and an IS1251 insertion between vanS and vanH. To date, this particular form of Tn1546 has only been described in human clinical vancomycin-resistant enterococcus isolates unique to the United States. Additionally, this is the first report of a vancomycin-resistant E. faecium isolated from a companion animal in the United States.

Animals↗

Increasing resistance to beta-lactam antibiotics among clinical isolates of Enterococcus faecium: a 22-year review at one institution.

To identify any change in the antibiotic resistance of Enterococcus faecium, we examined the antibiotic susceptibilities of clinical strains (n = 84) isolated at one institution during the 22 years since 1968. A significant increase in resistance to penicillin was observed during the study period: the MICs of penicillin for 50 and 90% of isolates tested were 16 and 64 micrograms/ml, respectively, from 1969 to 1988 (n = 48; geometric mean MIC, 14 micrograms/ml) , whereas they were 256 and 512 micrograms/ml, respectively, from 1989 to 1990 (n = 36; geometric mean MIC, 123 micrograms/ml) (P less than 0.001). A comparable increase in resistance to ampicillin was also noted (P less than 0.001). No strains produced detectable beta-lactamase. In contrast, susceptibilities to vancomycin, teicoplanin, and ciprofloxacin remained stable. High-level resistance to gentamicin was observed in none of 48 isolates from 1969 to 1988, but was present in 22 of 36 strains (61%) from 1989 to 1990 (P less than 0.001) and was significantly associated with resistance (MIC, greater than or equal to 128 micrograms/ml) to penicillin (P less than 0.001). To assess the potential evolution of antibiotic resistance in this species, clinical isolates (n = 24) were compared with strains isolated in 1968 from a human population in the Solomon Islands that was never exposed to antibiotics. Solomon Island isolates were significantly more susceptible than all clinical strains to penicillin, ampicillin, and vancomycin (P less than 0.001 for each), but they exhibited no differences in susceptibility to teicoplanin or ciprofloxacin. The penicillin-binding affinity of penicillin-binding protein 5 (PBP 5) in penicillin-resistant clinical strains (MIC, 512 micrograms/ml) was notably lower than that in strains with more typical susceptibilities, suggesting an alteration in this PBP as a possible mechanism for increased penicillin resistance. Solomon Island strains most susceptible to penicillin demonstrated a prominent PBP 5* and the absence of PBP 5. These changes in the antibiotic resistance of E. faecium emphasize the importance of identifying this species in patients with serious enterococcal infections and the necessity of assessing its susceptibility to both beta-lactams and aminoglycosides if effective therapy is to be identified.

Anti-Bacterial Agents↗

Ampicillin plus ciprofloxacin therapy of experimental endocarditis caused by multidrug-resistant Enterococcus faecium.

The combination of ampicillin and ciprofloxacin displayed bactericidal in-vitro activity against two strains of Enterococcus faecium which were highly resistant to ampicillin, vancomycin, and aminoglycosides. This antibiotic combination was used to treat rabbits with experimental endocarditis caused by these strains. Although a significant decrease in vegetation bacterial counts occurred with one strain, the degree of killing did not approach that of penicillin with an aminoglycoside against susceptible enterococci.

Ampicillin↗

A cluster of vancomycin-resistant Enterococcus faecium in an intensive care unit.

OBJECTIVE: To describe the epidemiology of a cluster of vancomycin-resistant Enterococcus faecium (VAREC) in a cardiothoracic surgery intensive care unit. DESIGN: A case series of patients identified through review of surveillance data on nosocomial infections, review of microbiologic records, and culture survey of patients in the unit. RESULTS: Six patients in the cardiothoracic surgery intensive care unit had VAREC with identical antimicrobic susceptibility patterns over a 6-month period. Four patients were identified with VAREC through prospective surveillance and 2 through retrospective review. Prior vancomycin use was seen more commonly in patients with VAREC (6/6, 100%) than in those without VAREC (3/12, 25%) (Fisher's exact test, p = .01). Six of the 7 patients with prior infection developed VAREC (85.7%). A prior nosocomial infection and prior exposure to vancomycin were found to be important variables in a logistic regression analysis. VAREC also was isolated from the environment. A combination of cohorting of patients and staff, and modifications of standard contact isolation practices eliminated the presence of VAREC from the cardiothoracic surgery intensive care unit. CONCLUSIONS: The results suggest that prior administration of vancomycin, especially in the patient who develops nosocomial infection, can influence the acquisition of vancomycin-resistant enterococci and that VAREC may be transmitted from patient to patient. Using a modification of the standard infection control practice of isolation, we were able to control the spread of this resistant strain of E faecium.

Cross Infection↗

First outbreak of colonization and infection with vancomycin-resistant Enterococcus faecium in a tertiary care hospital in Singapore.

We report the first outbreak of vancomycin-resistant Enterococcus faecium colonization and infection among inpatients in the hematology ward of an acute tertiary care public hospital in Singapore. Two cases of bacteremia and 4 cases of gastrointestinal carriage were uncovered before implementation of strict infection control measures resulted in control of the outbreak.

Adult↗

Successful treatment of meningitis due to multiply resistant Enterococcus faecium with a combination of intrathecal teicoplanin and intravenous antimicrobial agents.

Following neurosurgery necessitated by intractable seizures, Enterococcus faecium meningitis that was resistant to ampicillin, a high-level aminoglycoside (MIC, > 2,000 micrograms/mL), and vancomycin developed in a 6-year-old boy. Treatment with intrathecal teicoplanin in combination with intravenous clindamycin, rifampin, and ampicillin was successful. The role of intravenous and intrathecal antibiotics in treatment of this infection is discussed. This case is illustrative of the safety and potential usefulness of intrathecally administered teicoplanin.

Child↗

Dissemination of clonally unrelated erythromycin- and glycopeptide-resistant Enterococcus faecium isolates in a tertiary Greek hospital.

Between September 1999 to February 2001, 25 glycopeptide-resistant Enterococcus faecium (GRE) isolates were recovered from a Greek hospital. The isolates exhibited 13 distinct chromosomal macrorestriction types by pulsed-field gel electrophoresis, and all were erythromycin and vancomycin resistant, carrying the genes vanA and ermB. Vancomycin resistance, always linked with erythromycin resistance, was transferable from 17 isolates. The dissemination of erythromycin-resistant GRE strains may, at least in part, reflect the extensive use of macrolides in husbandry in Greece.

Anti-Bacterial Agents↗