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PubMed2006

New bisanthraquinone antibiotics and semi-synthetic derivatives with potent activity against clinical Staphylococcus aureus and Enterococcus faecium isolates.

The escalation of antibiotic resistance among Gram-positive pathogens presents increasing treatment challenges and requires the development of innovative therapeutic agents. Here, we present the antimicrobial properties of structurally unusual bisanthraquinone metabolites produced by a marine streptomycete and four semi-synthetic derivatives. Biological activities were measured against clinically derived isolates of vancomycin-resistant Enterococcus faecium (VRE), and methicillin-susceptible, methicillin-resistant, and tetracycline-resistant Staphylococcus aureus (MSSA, MRSA, and TRSA, respectively). The most potent antibiotic displayed MIC(50) values of 0.11, 0.23, and 0.90microM against a panel (n=25 each) of clinical MSSA, MRSA, and VRE, respectively, and was determined to be bactericidal by time-kill analysis.

Anthraquinones↗

PubMed1993

Triple combination penicillin-vancomycin-gentamicin for experimental endocarditis caused by a highly penicillin-and glycopeptide-resistant isolate of Enterococcus faecium.

A combination of low-dose penicillin (75,000 IU/kg twice daily [b.i.d.]) vancomycin (30 mg/kg b.i.d.) and gentamicin (6 mg/kg b.i.d.) has been shown to be as effective as a combination of high-dose penicillin (500,000 IU/kg b.i.d.) and gentamicin (6 mg/kg b.i.d.) in the treatment of rabbit endocarditis caused by an Enterococcus faecium strain moderately resistant to beta-lactams and highly resistant to glycopeptides. The same regimens were evaluated against an E. faecium strain highly resistant to both penicillin (MIC, 128 micrograms/mL) and vancomycin (MIC, 512 micrograms/mL). High doses of penicillin-gentamicin and vancomycin-gentamicin had no effect in in vitro killing-curve studies or in rabbits after treatment for 5 days. High doses of penicillin-vancomycin were only bacteriostatic in killing curves and provided a small reduction in the bacterial titers of the vegetations. In contrast, high-dose penicillin-vancomycin-gentamicin was bactericidal in vitro and highly effective in treating rabbits. However, the emergence of a bacterial subpopulation resistant to the synergistic effect of penicillin and vancomycin could reduce the clinical utility of this combination.

Animals↗

PubMed2001

Aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im detected together in strains of both Escherichia coli and Enterococcus faecium.

Escherichia coli SCH92111602 expresses an aminoglycoside resistance profile similar to that conferred by the aac(6')-Ie-aph(2")-Ia gene found in gram-positive cocci and was found to contain the aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im (only 44 nucleotides apart). aph(2")-Ib had been reported previously in Enterococcus faecium SF11770. aac(6')-Im had not been detected previously in enterococci and was found to be present also 44 nucleotides downstream from aph(2")-Ib in E. faecium SF11770. aph(2")-Ib and aac(6')-Im are separate open reading frames, each with its own putative ribosome binding site, whereas aac(6')-Ie-aph(2")-Ia appears to be a fusion of two genes with just one start and one stop codon. The deduced AAC(6')-Im protein exhibits 56% identity and 80% similarity to the AAC(6')-Ie domain of the bifunctional enzyme AAC(6')-APH(2"). Our results document the existence of a member of the aph(2") family of genes in gram-negative bacteria and provide evidence suggesting the horizontal transfer of aph(2")-Ib and aac(6')-Im as a unit between gram-positive and gram-negative bacteria.

Aminoglycosides↗

PubMed2003

Axe-Txe, a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant, clinical isolate of Enterococcus faecium.

Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic-based toxin-antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co-expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM-yoeB) present in the E. coli chromosome function as a toxin-antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe-Txe is one of the first functional proteic toxin-antitoxin systems to be accurately described for Gram-positive bacteria.

Amino Acid Sequence↗

PubMed1999

vanA and vanB incorporate into an endemic ampicillin-resistant vancomycin-sensitive Enterococcus faecium strain: effect on interpretation of clonality.

Clonal spread and horizontal transfer in the spread of vancomycin resistance genes were investigated. Multiplex PCR, pulsed-field gel electrophoresis (PFGE), hybridization of enterococcal plasmids with the vanA and vanB probes, and sequencing of a fragment of vanB were used in the analysis. Before May 1996, 12 vancomycin-resistant Enterococcus faecium (VRE) isolates were found in Finland. Between May 1996 and October 1997, 156 VRE isolates were found in the Helsinki area. Between December 1997 and April 1998, fecal samples from 359 patients were cultured for VRE. One new case of colonization with VRE was found. During the outbreak period, 88% (137 of 155) of the VRE isolates belonged to two strains (VRE types I and II), as determined by PFGE. Each VRE type I isolate possessed vanB, and five isolates also had vanA. Of the 34 VRE type II isolates, 27 possessed vanA and 7 possessed vanB. Fifteen of 21 (71%) ampicillin-resistant, vancomycin-sensitive E. faecium (VSE) isolates found during and after the outbreak period in one ward were also of type II. Two VSE type II isolates were found in the hospital before the outbreak in 1995. By PFGE, the three groups (vanA, vanB, or no van gene) of type II shared the same band differences with the main type of VRE type II with vanA. None of the differences was specific to or determinative for any of the groups. Our material suggests that vanA and vanB incorporate into an endemic ampicillin-resistant VSE strain.

Ampicillin Resistance↗

PubMed2001

Evaluation of a biochemical test scheme for identifying clinical isolates of Enterococcus faecalis and Enterococcus faecium.

AIMS: To evaluate the full test scheme of Facklam and Sahm (1995) for the identification of clinical enterococcal isolates to genus and species level. METHODS AND RESULTS: Fifty-nine clinical isolates, previously provisionally classed as enterococci on the basis of just four biochemical tests of Facklam and Sahm and one other test, were subjected to genus and species identification using the full identification scheme of Facklam and Sahm; 98% of these strains were confirmed to be enterococci and of these, 69% were identified as Enterococcus faecalis and 31% as Enterococcus faecium. Six tests in the scheme (out of 24) gave anomalous or unreliable results for some strains, and two gave unexpected results for the majority of strains presumptively identified as Ent. faecium. CONCLUSIONS: Nine (out of 12) genus tests and nine (out of 12) species tests from the Facklam and Sahm scheme were reliable. Testing for the presence of the Lancefield antigen D was also useful. The majority of presumptive Ent. faecium strains gave different results for the sorbitol and raffinose tests from that expected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the level of reliability for each of the tests in a current enterococcal identification scheme for differentiating clinical isolates, and showed that two tests gave consistently different test results from those expected for Ent. faecium.

Bacterial Typing Techniques↗

PubMed2001

The relationship of a clonal outbreak of Enterococcus faecium vanA to methicillin-resistant Staphylococcus aureus incidence in an Australian hospital.

Australian isolates of vancomycin-resistant enterococci (VRE) have been widely scattered geographically, predominantly polyclonal and of the VanB phenotype. Forty-nine VRE were isolated from 47 patients in our hospital from October 1996 to December 1999. Forty-four of these VRE were Enterococcus faecium with a vanA glycopeptide resistance genotype. Four isolates were pathogenic. Thirty-five VRE were from an outbreak in the Renal and Infectious Diseases Units over a four-month period. Pulsed-field gel electrophoresis (PFGE) demonstrated that 41 of the 49 VRE were indistinguishable or closely related. Enhanced environmental cleaning, strict contact isolation of colonized patients and reducing inpatient admissions terminated the epidemic. Cohorting of methicillin-resistant Staphylococcus aureus (MRSA)-positive patients was restricted because VRE patients occupied the isolation facilities. This resulted in a statistically significant increase in MRSA infections across the hospital. VRE epidemics have the ability to influence the epidemiology of other nosocomial pathogens when infection control resources are exhausted.

Australia↗

PubMed2001

Cerebrospinal fluid concentrations of quinupristin-dalfopristin in a patient with vancomycin-resistant Enterococcus faecium [correction of faecalis] ventriculitis.

A 44-year-old man was treated successfully for vancomycin-resistant Enterococcus faecium (VREF) ventriculitis with intrathecal quinupristin-dalfopristin 1 mg, 2 mg, and 4 mg, and other intravenous antibiotics. Cerebrospinal fluid samples were collected before and after the 1-mg and 2-mg doses to determine the concentrations of quinupristin-dalfopristin and its active metabolites. Concentrations were above the minimum inhibitory concentration for VREF immediately after unclamping the extraventricular drain and were quantifiable for at least 7 hours.

Adult↗

PubMed2001

Transient intestinal carriage after ingestion of antibiotic-resistant Enterococcus faecium from chicken and pork.

BACKGROUND: Antibiotic-resistant enterococci are often present in retail meats, but it is unclear whether the ingestion of these contaminants leads to sustained intestinal carriage. METHODS: We conducted a randomized, double-blind study in 18 healthy volunteers. Six ingested a mixture of 10(7) colony-forming units (CFU) of two glycopeptide-resistant strains of Enterococcus faecium obtained from chicken purchased at a grocery store, six ingested 10(7) CFU of a streptogramin-resistant strain of E. faecium obtained from a pig at slaughter, and six ingested 10(7) CFU of a glycopeptide-susceptible and streptogramin-susceptible strain of E. faecium from chicken purchased at a grocery store. Suspensions of enterococci were prepared in 250 ml of whole milk and were well within the amounts deemed acceptable by Danish food regulations. Stool samples were collected before exposure, daily for 1 week after ingestion, and at 14 and 35 days. Resistant enterococci in stools were identified by selective culture techniques; further molecular characterization of the organisms was also conducted. RESULTS: At the outset, none of the subjects were colonized with glycopeptide-resistant or streptogramin-resistant E. faecium. After ingestion of the study strains, these same strains were isolated from the stools of all subjects, in various concentrations. The test strain was isolated in stool from 8 of 12 subjects on day 6, and from 1 of 12 on day 14. All stool samples were negative at 35 days. CONCLUSIONS: The ingestion of resistant E. faecium of animal origin leads to detectable concentrations of the resistant strain in stools for up to 14 days after ingestion. The organisms survive gastric passage and multiply.

Adult↗

PubMed2000

Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a German university pediatric hospital.

Nosocomial Infections caused by vancomycin-resistant enterococci (VRE) are an emerging threat to critically ill patients. At the University Hospital Eppendorf, VRE were isolated from 38 patients between August 1993 and April 1997, of whom 32 were hospitalized at the Department of Pediatrics. Pulsed-field gel electrophoresis revealed that 26 Enterococcus faecium isolates from patients of the Department of Pediatrics were identical or closely related, and that isolates from three additional patients of the same department were possibly related. All of these isolates were of vanA genotype. They were resistant to glycopeptides, ampicillin, ciprofloxacin, clindamycin, and erythromycin. Most isolates displayed high-level resistance to gentamicin, but all remained susceptible to quinupristin/dalfopristin. Implementation of stringent hand disinfection and environmental disinfection policies, as well as measures for patient isolation contained this first outbreak of VRE at a German Children's hospital, which emphasizes the importance of hygienic measures for the control of nosocomial spread of these organisms.

Child, Preschool↗

PubMed2005

Prevention of febrile neutropenia in cancer patients by probiotic strain Enterococcus faecium M-74. Phase II study.

Febrile neutropenia (FN) remains a potentially life-threatening complication of anticancer chemotherapy. Bacterial translocation via intestinal mucosa is a significant mechanism of FN development. Competitive inhibition of bowel colonization by pathogenic microorganisms by lactic acid bacteria could be a useful prevention of FN. The aim of the study was the prevention of FN by probiotic strain Enterococcus faecium M-74 enriched with selenium in leukemic patients. Fourteen (six males/eight females) patients with myelogenous leukemia treated by induction or consolidation chemotherapy were included in the study. Patients received prophylaxis with E. faecium M-74 during one cycle of chemotherapy. The daily dose was 36 x 10(9) CFU tid. Prophylaxis started between day -2 and day +2 of chemotherapy and continued until the absolute neutrophile count (ANC) was >1,000/microl. All patients experienced febrile neutropenia. During 231 days of severe neutropenia, 30 febrile episodes occurred. No any febrile episode or infection provoked by the strain tested was noticed. Tolerance of therapy was excellent without significant adverse effects. Our results demonstrate the safety of the probiotic strain E. faecium M-74 enriched with selenium in leukemic patients with severe neutropenia. However, its administration was not effective in the prevention of febrile neutropenia, but this does not preclude the protective effect of other probiotic strains.

Adult↗

PubMed2006

Multilocus sequence typing and analysis of putative virulence factors in vancomycin-resistant and vancomycin-sensitive Enterococcus faecium isolates from Brazil.

Enterococci are leading causes of hospital-acquired infections that are often difficult to treat because of high-level aminoglycoside and glycopeptide resistance. Vancomycin-resistant enterococci are a global problem, and have been isolated with increasing frequency in hospitals in Brazil. The objective of this study was to determine the genetic relatedness of vancomycin-resistant Enterococcus faecium (VREFM) and vancomycin-sensitive E. faecium (VSEFM) isolated from human infections and faecal sources in Brazil, and to compare these isolates with those from domesticated animals. Isolates (n = 56) were classified by multilocus sequence typing (MLST) and assessed for putative virulence traits. The acm gene was detected in 98% of all isolates. The 56 isolates studied comprised 26 different MLST types. VSEFM isolates from the faeces of pigs were found to be distinct from all human isolates characterised previously by MLST, and were assigned new sequence type (ST) numbers. VREFM isolates were represented by four different STs (ST-114, ST-17, ST-281, ST-50). Among the 26 STs identified in this study, eBURST detected three groups of STs with related allelic profiles, and 19 unrelated STs. Among E. faecium isolates from Brazil, the esp gene was restricted to vancomycin-resistant isolates. Furthermore, isolates classified as ST-17 by MLST, an epidemic strain type isolated internationally with the purK-1 gene, were found among VREFM isolates from Brazil that also harboured the esp and hyl genes.

Adhesins, Bacterial↗

PubMed2003

Mutations in the DNA mismatch repair proteins MutS and MutL of oxazolidinone-resistant or -susceptible Enterococcus faecium.

Mutations in mutS and mutL, which encode DNA mismatch repair (MMR) proteins, can confer hypermutator phenotypes and may facilitate the emergence of mutational antibiotic resistance in bacteria. Linezolid-resistant enterococci (LRE) rarely emerge during therapy and contain mutations in 23S rRNA genes. As enterococci with defective MMR could be prone to the development of oxazolidinone resistance mutations, we investigated 13 clinical isolates of Enterococcus faecium, including 2 LRE, for mutations in mutSL. A 4,944-bp fragment spanning mutSL was sequenced from two pairs of linezolid-resistant (MICs, 64 micro g/ml) and linezolid-susceptible (MICs, 2 micro g/ml) E. faecium isolates (one pair from Austria and one pair from the United Kingdom) identical by pulsed-field gel electrophoresis. The pairs represented distinct strains in which linezolid resistance had emerged during therapy. The MutSL peptides of all four isolates had amino acid substitutions compared with the sequence of E. faecium strain DO (used for genome sequencing). These were Val352Ile (one pair of isolates only) and Met628Leu in MutS and Leu387Pro, Tyr406Phe, Thr415Ser, Phe427Leu, and Phe565Ile in MutL. The significance of these changes remains unknown; these isolates did not show a demonstrable hypermutator phenotype. The same substitutions were found in two of nine geographically diverse linezolid-susceptible enterococcal isolates; the other seven isolates had MutSL sequences identical to that of strain DO. Multilocus sequence typing revealed that all isolates with alternate MutSL peptides belonged to a distinct lineage of a prevalent E. faecium clonal complex, designated CC17. Further studies are needed to investigate the prevalence of these MutSL mutations and their possible roles in the emergence of E. faecium strains resistant to oxazolidinones and other antibiotic classes.

Acetamides↗

PubMed2002

Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium.

The d,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel beta-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting from l,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated with no modification of the level of production of the PBPs or of their affinity for beta-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A beta-lactam-insensitive l,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a beta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys(3) --> d-Asx-Lys(3) cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the l,d-transpeptidase.

Alanine↗

PubMed2004

Comparative analysis of amplified fragment length polymorphism and pulsed field gel electrophoresis in a hospital outbreak and subsequent endemicity of ampicillin-resistant Enterococcus faecium.

Reliable molecular methods for determination of relatedness between bacterial isolates have become increasingly important to evaluate outbreaks and endemic situations with nosocomial pathogens. In the present study Simpson's index of diversity with calculated confidence intervals was used to compare amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) analysis of a hospital outbreak of ampicillin-resistant Enterococcus faecium and subsequent endemicity. The outbreak, in a Norwegian tertiary hospital, of infections caused by these enterococci started in 1995 and increased in 1996 after which the situation turned endemic. The purpose of this study was to compare the two methods in this setting and to determine the length of time during an outbreak that these methods are sufficiently valid to be of value for hospital infection control efforts. One hundred and sixty clinical isolates from urine specimens collected during the period 1995-1999 were included. The findings indicate that PFGE and AFLP are equally discriminative and could in this setting be used for typing purposes over the whole 5-year period.

Ampicillin↗

PubMed2001

Bactericidal activities of two daptomycin regimens against clinical strains of glycopeptide intermediate-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and methicillin-resistant Staphylococcus aureus isolates in an in vitro pharmacodynamic model with simulated endocardial vegetations.

Daptomycin is an investigational lipopeptide antibiotic active against gram-positive organisms. The mechanism of action is unique, resulting in interference with cell membrane transport. The bactericidal activity of daptomycin was evaluated against glycopeptide-intermediate susceptible Staphylococcus aureus (GISA), vancomycin-resistant Enterococcus faecium (VREF), and methicillin-resistant S. aureus (MRSA) in an in vitro infection model with simulated endocardial vegetations. Simulated regimens of daptomycin at 6 mg/kg/day (D6) and 10 mg/kg/day (D10) were utilized. MICs and MBCs for daptomycin were determined in the absence and in the presence of albumin with the following results (MIC/MBC): for GISA-992, 0.5/1.0 and 16/16; for VREF-590, 2.0/2.0 and 32/32; and for MRSA-494, 0.25/0.25 and 1.0/4.0 microg/ml, respectively. During the first 8 h daptomycin significantly reduced the inoculum for all organisms. Daptomycin at 6 mg/kg/day and 10 mg/kg/day had log(10) CFU/g reductions of 5 and 6, 3.4 and 5, and 6.4 and 6.5 by 8 h for GISA-992, VREF-590, and MRSA-494, respectively. Against both GISA-992 and VREF-590, the D10 regimen achieved the limit of detection at 72 h, with D6 regimens showing slight regrowth. A concentration-dependent killing effect was noted to occur, with daptomycin demonstrating a more rapid and greater kill from the D10 versus the D6 regimen. The results of this study suggest that daptomycin demonstrates significant (P < 0.05) activity against gram-positive organisms in a simulated sequestered infection site.

Anti-Bacterial Agents↗

PubMed2000

Paradoxical effect of inserting, in Enterococcus faecalis penicillin-binding protein 5, an amino acid box responsible for low affinity for penicillin in Enterococcus faecium.

Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and immunologically related proteins that are characterized by their low affinity for penicillin. For this reason, they are mainly involved in penicillin resistance, due essentially to their ability to take over the function of all other PBPs already bound and inhibited by the beta-lactam. It has been demonstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 or by the presence of specific amino acid sequences in the protein that further decrease the affinity for penicillin. In particular, a specific amino acid box (ANNGA) previously identified in Enterococcus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box ANNGA in Enterococcus faecalis, an enterococcal species usually more sensitive to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-introduced into a pbp5 mutant of E. faecalis obtained by insertion of transposon Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradoxically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is concerned, differences exist in their three-dimensional structure that affect penicillin affinity.

Amino Acid Sequence↗

PubMed1986

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.

Anaerobiosis↗