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PubMed1996

Gentamicin resistance gene transfer from Enterococcus faecalis and E. faecium to Staphylococcus aureus, S. intermedius and S. hyicus.

Transfer of gentamicin resistance from Enterococcus faecalis and E. faecium to Staphylococcus aureus, S. hyicus and S. intermedius was accomplished by filter mating. Transfer from E. faecalis was plasmid mediated and a probe to the gentamicin resistance gene aac(6')-aph(2") hybridized to HindIII fragments of various sizes (7.5, 6.2 and 4.7 kbp) of these plasmids. Transfer from E. faecium was apparently to the chromosome in 9 of 10 instances with HindIII fragments of 3.3 or 3.0 kbp hybridizing to the probe. These results are consistent with transposon mediated resistance. Plasmid transfers to S. intermedius were at much lower frequencies than to S. aureus or to S. hyicus but presumed transposon transfer direct to the chromosome was equal in all three species.

Burns↗
PubMed2005

Vancomycin for multi-drug resistant Enterococcus faecium cholangiohepatitis in a cat.

A 12-year-old, neutered male domestic shorthair cat was evaluated with a life-long history of intermittent, predominantly small bowel diarrhea and a 3 day history of hematochezia. At presentation, the cat had increased liver enzyme activities and an inflammatory leukogram. Histopathology demonstrated inflammatory bowel disease (IBD), cholangiohepatitis and pancreatitis. The cholangiohepatitis was associated with a multi-drug resistant Enterococcus faecium. Gallbladder agenesis was also documented. Treatment with vancomycin was safely instituted for 10 days. Clinical signs resolved, however, cure of the bacterial cholangiohepatitis was not achieved. The risk of vancomycin resistant enterococci (VRE) in human and veterinary medicine is discussed.

Animals↗
PubMed2005

Organization of heat shock dnaK and groE operons of the nosocomial pathogen Enterococcus faecium.

Enterococcus faecium is a frequently antibiotic-resistant opportunistic pathogen that is commonly recovered from hospitalized patients. The genetic organization of the dnaK operon was analyzed and was shown to consist of at least four heat shock genes, hrcA-grpE-dnaK-dnaJ. The dnaK/J intergenic region was 140 bp shorter than in E. faecalis. The dnaK operon was expressed from a putative sigma(A)-type promoter (PhrcA) upstream of the hrcA start codon and was preceded by two conserved CIRCE sequences. Northern hybridization revealed the presence of multiple mRNAs in the dnaK operon. Conversely, the groE operon was transcribed as a single mRNA. Induction of dnaK and groEL genes occurred in response to either heat shock or exposure to other stress agents.

Bacterial Proteins↗
PubMed2005

Detection of the esp gene in high-level gentamicin resistant Enterococcus faecalis strains from pet animals in Japan.

We investigated the prevalence of the esp gene and the susceptibility to gentamicin in Enterococcus faecalis and E. faecium strains obtained from pet animals. Nine of 30 E. faecalis and 2 of 38 E. faecium strains from the pet animals had the esp gene. Three esp-positive E. faecalis strains, which were isolated from two dogs and a cat, showed gentamicin MICs of > or =256 microg/ml and harbored the high-level gentamicin resistance (HLGR) gene, aac(6')-Ie-aph(2'')-Ia. Of the nine esp-positive E. faecalis strains, five, including the three strains with the HLGR gene, were closely related by numerical analysis of PFGE patterns. Longitudinal investigation needs to elucidate whether the HLGR gene was incorporated into a subpopulation of the esp-positive E. faecalis.

Animals↗
PubMed1997

Glucose starvation response in Enterococcus faecalis JH2-2: survival and protein analysis.

We investigated the survival of Enterococcus faecalis following starvation provoked by energy source glucose exhaustion. Inhibition of protein synthesis by chloramphenicol before 3 h of starvation resulted in a dramatic decrease in viable bacteria. Antibiotic treatment of cells after 3 or 6 h of starvation had a progressively lesser influence on bacterial survival. During the first 24 h of deprivation, a total of 42 proteins were identified as glucose-starvation-inducible; 4 temporal classes of proteins (A, B, C and D) were defined in relation to their enhanced synthesis after glucose exhaustion. Our results show that proteins from the two early classes (A and B) seem to be the most important for long-term survival in E. faecalis. One protein of each of these classes was analysed at the molecular level. The N-terminal sequence of one of them, belonging to class A, showed strong homology with the N-terminal sequence of carbamate kinase from Streptococcus faecium. This enzyme could be implicated in the development of alternative metabolic pathways of energy production and could be compared to the Cst proteins of Escherichia coli.

Bacterial Proteins↗
PubMed2001

A hospital epidemic of vancomycin-resistant Enterococcus: risk factors and control.

OBJECTIVE: To determine risk factors for vancomycin-resistant Enterococcus (VRE) colonization during a hospital outbreak and to evaluate Centers for Disease Control and Prevention (CDC)-recommended control measures. DESIGN: Epidemiological study involving prospective identification of colonization and a case-control study. SETTING: A university hospital. PARTICIPANTS: Patients on eight wards involved in outbreak from late 1994 through early 1995. METHODS: Cases were matched by ward and culture date with up to two controls. Risk factors were evaluated with four multivariate models using conditional logistic regression. The first evaluated proximity to other VRE patients and isolation status. The second evaluated proximity to unisolated VRE cases and three variables independently predictive after adjustment for proximity. The third evaluated seven significant univariate predictors in addition to proximity to unisolated VRE in backward, stepwise logistic regression. The fourth assessed proximity to VRE with all other variables collected, clustered in a principal components analysis. Pulsed-field gel electrophoresis was performed to assess clonality of two outbreak strains. RESULTS: The incidence of transmission declined significantly after CDC guidelines were implemented. Proximity to unisolated VRE cases during the prior week was a significant predictor of acquisition in each of four multivariate models. Other significant risk factors in multivariate models included a history of major trauma and treatment with metronidazole. Pulsed-field gel electrophoresis confirmed the clonality of two outbreak strains. CONCLUSIONS: VRE was transmitted between patients during a hospital epidemic, with proximity to previously unisolated VRE patients being an important risk factor. Weekly surveillance cultures and contact isolation of colonized patients significantly reduced spread

Adolescent↗
PubMed1999

Photobactericidal activity of methylene blue derivatives against vancomycin-resistant Enterococcus spp.

The toxicities and phototoxicities of methylene blue and its two methylated derivatives were measured against one standard and three vancomycin-resistant pathogenic strains of Enterococcus spp. Each of the compounds was bactericidal and the derivatives exhibited photobactericidal activity on illumination at a 'light' dose of 6.3 J/cm(2) against one or more of the strains. Increased bactericidal and photobactericidal activity in the methylated derivatives is thought to be due to their higher hydrophobicities allowing greater interaction with the bacterial cell wall. In addition, the derivatives exhibited higher inherent photosensitizing efficacies.

Enterococcus faecalis↗
PubMed2006

Cell preparation of Enterococcus faecalis strain EC-12 prevents vancomycin-resistant enterococci colonization in the cecum of newly hatched chicks.

The use of antimicrobials in broilers is considered to be a cause of the appearance of vancomycin-resistant enterococci (VRE). Once VRE penetration occurs, whatever its origin, it is difficult to expel the enterococci from the intestine because of their multiple resistance, whether natural or acquired. In this study, we evaluated the prevention of VRE colonization by the dietary supplementation of a cell-wall preparation of Enterococcus faecalis strain EC-12 (EC-12) in newly hatched broilers that were challenged by experimental infection with VRE. The chicks were fed a basal diet supplemented with 0.05% (wt/wt) EC-12 powder for 15 d. The control group and that administered Lactobacillus sp. were fed the basal diet. The VRE challenge was administered orally when the chicks were 2 d old (d 0). Dietary EC-12 reduced VRE colonization in the intestine from d 3 to 14. Total IgA in the cecal digesta and total IgG in the serum were higher on d 14 in the EC-12 treatment group. However, VRE-specific and EC-12-specific antibodies were not affected in serum. Hence, it appeared that dietary EC-12 stimulated the gut immune system and reinforced the immune reaction against the VRE challenge to accelerate its defecation from the chick intestine.

Animals↗
PubMed2000

Characterization of transposon Tn1549, conferring VanB-type resistance in Enterococcus spp.

Transfer of VanB-type resistance to glycopeptides among enterococci has been reported to be associated with the movement of large chromosomal genetic elements or of plasmids. The authors report the characterization of the 34 kb transposon Tn1549 borne by a plasmid related to pAD1 and conferring vancomycin resistance in clinical isolates of Enterococcus spp. Tn1549 contained 30 ORFs and appeared to be organized like the Tn916 family of conjugative transposons into three functional regions: (i) the right end, implicated in the excision-integration process; (ii) the central part, in which the vanB2 operon replaces the tet(M) gene; and (iii) the left extremity, in which eight of the 18 ORFs could be implicated in the conjugative transfer.

Bacterial Proteins↗
PubMed1993

Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H(+)-ATPase by cloned genes from the same and different species.

We isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which 'G' at position 301 of the alpha-subunit gene of the F1F0 type of H(+)-ATPase was deleted. MS117 had low H(+)-ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH 6.0. When the alpha-subunit gene of E. hirae H(+)-ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH 6.0. We next transformed MS117 with pHY300PLK containing the alpha-subunit gene of Bacillus megaterium F1F0-ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H(+)-ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F1F0 type of H(+)-ATPase.

Bacillus megaterium↗
PubMed2004

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources.

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.

Anal Canal↗
PubMed2002

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↗
PubMed2006

Genetic analysis of bacteriocin 43 of vancomycin-resistant Enterococcus faecium.

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan were tested for bacteriocin production. Of the 277 (44%) bacteriocinogenic strains, 21 were active against E. faecalis, E. faecium, E. hirae, E. durans, and Listeria monocytogenes. Of those 21 strains, a representative bacteriocin of strain VRE82, designated bacteriocin 43, was found to be encoded on mobilizable plasmid pDT1 (6.2 kbp). Nine open reading frames (ORFs), ORF1 to ORF9, were presented on pDT1 and were oriented in the same direction. The bacteriocin 43 locus (bac43) consists of the bacteriocin gene bacA (ORF1) and the immunity gene bacB (ORF2). The deduced bacA product is 74 amino acids in length with a putative signal peptide of 30 amino acids at the N terminus. The bacB gene encodes a deduced 95-amino-acid protein without a signal sequence. The predicted mature BacA protein (44 amino acids) showed sequence homology with the membrane-active class IIa bacteriocins of lactic acid bacteria and showed 86% homology with bacteriocin 31 from E. faecalis YI717 and 98% homology with bacteriocin RC714. Southern analysis with a bac43 probe of each plasmid DNA from the 21 strains showed hybridization to a specific fragment corresponding to the 6.2-kbp EcoRI fragment, suggesting that the strains harbored the pDT1-like plasmid (6.2 kb) which encoded the bacteriocin 43-type bacteriocin. The bac43 determinant was not identified among non-VRE clinical isolates.

Amino Acid Sequence↗
PubMed1992

Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.

Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCl extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage lambda gt11. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The nucleotide sequence of each subclone was determined. The sequence data indicated an open reading frame encoding a polypeptide of 666 amino acid residues, with a calculated molecular mass of 70,678 Da. The first 24 N-terminal amino acids of purified extracellular muramidase-2 were in very good agreement with the deduced amino acid sequence after a 49-amino-acid putative signal sequence. Analysis of the deduced amino acid sequence showed the presence at the C-terminal region of the protein of six highly homologous repeat units separated by nonhomologous intervening sequences that are highly enriched in serine and threonine. The overall sequence showed a high degree of homology with a recently cloned Streptococcus faecalis autolysin.

Amino Acid Sequence↗
PubMed2000

Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q.

Enterococcus faecium L50 grown at 16 to 32 degrees C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47 degrees C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Hâvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47 degrees C and maximally detected at 47 and 37 to 47 degrees C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25 degrees C and are not detected at 37 degrees C or above.

Amino Acid Sequence↗
PubMed1996

Pseudo-outbreak of Enterococcus durans infections and colonization associated with introduction of an automated identification system software update.

Enterococci are an important cause of hospital-acquired infections. Since 1989, there has been an increase in the number of nosocomial enterococcal infections caused by strains resistant to vancomycin in the United States. Although many enterococcal species can colonize humans, only Enterococcus faecalis, E. faecium, E. raffinosus, and E. casseliflavus have been implicated in clusters of infection. In January 1996, the Centers for Disease Control and Prevention received a report of an outbreak of vancomycin-resistant enterococci in which 31 of 84 (36.9%) isolates were identified as E. durans. Twenty-nine isolates identified as E. durans were identified to the species level after the introduction of an automated identification system software update (Vitek gram-positive identification card, version R09.1) for the identification of species of gram-positive organisms. When seven isolates initially reported as E. durans were identified to the species level by alternate methods, they were found to be E. faecium. Subsequently, isolates identified as E. durans by the automated system were reidentified by using a rapid streptococcus test, and no further enterococcal isolate has been confirmed as E. durans. Automated microbial analysis is a potential source of error that is not easily recognized. When laboratory findings are discordant with expected clinical or epidemiologic patterns, confirmatory testing by alternate methods should be performed.

Anti-Bacterial Agents↗
PubMed1997

Emergence and dissemination of a highly vancomycin-resistant vanA strain of Enterococcus faecium at a large teaching hospital.

We prospectively identified patients at the Massachusetts General Hospital from whom vancomycin-resistant enterococci (VRE) were isolated from a clinical specimen from 1 January 1991 through 31 December 1995. VRE strains were available from 139 (82%) of the 169 patients with clinical cases. Of these, 39 (28%) were identical or closely related by pulsed-field gel electrophoresis (i.e., VRE type A strain), including 38 (43%) of 89 VRE strains in 1995. By multivariate analysis, acquisition of the VRE type A strain was associated with receipt of clindamycin (odds ratio [OR] = 10.5), 15 or more days of hospitalization before the first isolation of VRE (OR = 2.9), and residence on one of the general medical floors (OR = 7.8). The VRE type A strain was a vanA strain of Enterococcus faecium and was highly resistant to all antimicrobial agents tested except chloramphenicol. These findings document the rapid dissemination of a highly resistant strain of E. faecium among patients and among other extant VRE strains at the Massachusetts General Hospital in 1995.

Adolescent↗
PubMed2005

Emerging incidence of Enterococcus faecium among hospital isolates (1993 to 2002).

Historically, most clinical microbiology laboratories report that 80 to 90% of enterococci are Enterococcus faecalis, whereas E. faecium accounts for 5 to 10% of isolates. At our medical center from 1993 to 2002, we evaluated the percentages of E. faecium among all enterococcal isolates and the percentages of E. faecium isolates that were vancomycin resistant. Over this 10-year period, the percentage of enterococci that were identified as E. faecium increased from 12.7 to 22.2% (P < 0.001) and the proportion of E. faecium that was vancomycin resistant increased from 28.9 to 72.4% (P < 0.001). Both the percentage of E. faecium among the enterococci and the proportion of vancomycin-resistant E. faecium increased significantly over this 10-year period.

Chicago↗