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Control of an outbreak of vancomycin-resistant Enterococcus faecium in an oncology ward in South Africa: effective use of limited resources.

An outbreak of vancomycin-resistant enterococci (VRE) occurred in an adult oncology ward of a large teaching hospital in Johannesburg, South Africa. The outbreak strain was identified as an Enterococcus faecium carrying the vanA resistance genotype. Macro-restriction analysis showed that the majority of strains were clonally related. Modified infection control interventions were implemented and control of the outbreak was achieved. Although the epidemiology of VRE is well documented in Europe, North America and Australia, this problem has only recently emerged in South Africa. The epidemiology of the outbreak appears similar to that described for outbreaks elsewhere.

Adult↗

Clinical and molecular epidemiology of hospital Enterococcus faecium isolates in eastern France. Members of Réseau Franc-Comtois de Lutte contr les Infections Nosocomiales.

We carried out a surveillance study of Enterococcus faecium isolates in the Franche-Comtéregion of France over three years. Clinical and epidemiological strains were characterized by antibiotype and genotype (pulsed field gel electrophoresis, PFGE). Three case-control studies were performed to identify risk factors for colonization/infection with three defined resistant phenotypes (amoxycillin, high-level gentamicin and high-level kanamycin). The crude incidence of colonization/infection was 0.156%, and 68.8% of cases were classified as hospital-acquired. Incidence did not differ according to the type of hospitalization (middle term or acute care). The urinary tract was the major site of infection. Resistance rates were: 45.8% (amoxycillin), 18.7% (high-level gentamicin), 61.4% (high-level kanamycin) and 3.1% (vancomycin). No isolate produced b-lactamase and one isolate carried the vanA gene. PFGE revealed two major epidemic patterns each including resistant strains isolated in different hospitals and during different periods in the study. Previous antimicrobial treatment was not identified as a risk factor for colonization/infection with any resistant phenotype. Despite the low frequency of vancomycin-resistant isolates in this study, resistant strains were widely disseminated and had characteristics enabling them to persist and spread. If these strains acquired the vanA gene, the risk of an outbreak would be large. So, the prevalence of vancomycin-resistant E. faecium in hospitals should be carefully monitored in the future.

Adult↗

Nosocomial outbreak of ampicillin resistant Enterococcus faecium: risk factors for infection and fatal outcome.

A nosocomial outbreak caused by ampicillin resistant Enterococcus faecium (ARE) was detected at a Norwegian university hospital in January 1995. Prior to this outbreak, ARE were not common in this hospital or other hospitals in Norway. During 1995 and 1996, a total of 149 cases with clinical ARE infection were detected prospectively. A case control study was performed by allocating controls matched for gender, age and ward of admission. Altogether, 123 case control pairs with mean age 70.1 years were included. Isolates from 89 (72. 4%) of the cases were identical or related to the defined outbreak strain as determined by pulsed-field gel electrophoresis (PFGE). In 75 of the patients (60.9%), ARE caused urinary tract infection, five (4.1%) had bacteraemia, 33 (26.8%) had wound infection and 10 (8.1%) had other infections. In a logistic regression model for 1:1 matched samples, the following factors were identified as significant risk factors for ARE infection: underlying neurological disease (OR=33.5), prescription of antimicrobial agents for more than 10 days (OR=8. 99), prescription of cephalosporins (OR=4.69), underlying gastrointestinal disease (OR=3.36) and length of hospital stay per day (OR=1.04). The intrahospital death rate for the cases was 18.7% compared with 8.9% for the controls, corresponding to an excess mortality attributable to ARE infection of 9.8%. A history of carbapenem prescription was the only independent factor contributing to death (OR=5.64) when comparing ARE patients dying in hospital to those surviving.

Aged↗

Evaluation of chlorhexidine and povidone iodine activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis using a surface test.

Most published studies of the activity of biocides against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) have been based on suspension tests. This study was undertaken to provide information on the effect of chlorhexidine and povidone iodine on bacteria dried on to surfaces, a situation in which biocide activity is known to be reduced. The inactivation of MRSA (10 strains), methicillin-sensitive Staphylococcus aureus (MSSA, 10 strains), VRE (nine strains) and vancomycin-sensitive Enterococcus faecalis (VSE, 10 strains) by 0.5% aqueous chlorhexidine gluconate or 10% povidone iodine was evaluated by applying the European surface test method. Povidone iodine was equally active against resistant and sensitive strains of both species with microbicidal effects (ME), i.e. the log(10)concentration of micro-organisms compared with controls treated with distilled water, after 1.5 min of 3.14 and 3.49 for VRE and VSE respectively, and 3.47 and 3.78 for MRSA and MSSA. Chlorhexidine was equally active against VRE and VSE (ME 3.37 vs. 3. 56 after 7 min, respectively), but was significantly less active against MRSA as opposed to MSSA (ME 3.07 vs. 3.83 after 10 min, P= 0. 017).

Anti-Infective Agents, Local↗

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↗

Treatment of a vancomycin-resistant Enterococcus faecium ventricular drain infection with quinupristin/dalfopristin and review of the literature.

Central nervous system infections involving vancomycin-resistant Enterococcus faecium (VREF) are infrequently described and pose significant therapeutic difficulties, because these organisms are intrinsically resistant to many antibiotics. We describe the use of intrathecal quinupristin/dalfopristin to treat a VREF-associated infection in a neuro--surgical patient.

Acetamides↗

[Reduction of acute recurrence in patients with chronic recurrent hypertrophic sinusitis by treatment with a bacterial immunostimulant (Enterococcus faecalis Bacteriae of human origin].

A double-blind, placebo-controlled multicenter study in 157 patients with chronic recurrent sinusitis investigated the occurrence of acute relapses during treatment of patients with a bacterial immunostimulant (3 x 30 drops/day), comprised of cells and autolysate of human Enterococcus faecalis bacteria (Symbioflor 1, n = 78) in comparison to placebo (n = 79). The study included a treatment period of 6 months and a follow-up period of 8 months. Under verum the occurrence of relapses (50 incidents) was about half (56%) the number observed under placebo (90 incidents). In the Kaplan-Meier test the verum preparation emerged as significantly superior (p = 0.045, log rank test) compared to placebo. This superiority of verum was found during the treatment period with 17 vs. 33 relapses (p = 0.019) as well as during the follow-up observation with 33 vs. 57 relapses (p = 0.013). The time interval to the first relapse was clearly longer under verum (513 days) than under placebo (311 days). The relative risk for a relapse under the test preparation compared to placebo was 49.0% during the treatment and 55.8% during the follow-up period. Severity of the acute relapses was comparable in both groups. However, antibiotic therapy was only required in 2 patients treated with verum compared to 6 patients in the placebo group. Both preparations were well tolerated and serious side effects did not occur in either group. No changes in laboratory tests--hematology and clinical chemistry--were observed. Potential immunomodifying effects of the test preparation in view of the significant reduction in relapses were discussed.

Adjuvants, Immunologic↗

[The effect of a bacterial immunostimulant (human Enterococcus faecalis bacteria) on the occurrence of relapse in patients with].

The following double-blind, placebo-controlled, multicenter study investigated the influence of a bacterial immunostimulant (Symbioflor 1, cells and autolysate of human Enterococcus faecalis) on the occurrence of relapses in patients with chronic recurrent bronchitis (n = 136; placebo n = 66, verum n = 70) in a 6 months treatment period and a follow-up period of 8 months, compared to placebo. Under verum 39 incidents of relapses were recorded, which was about 60% the number observed among the patients treated with placebo (66 incidents). The verum preparation exhibited superior clinical efficacy compared to placebo (p = 0.001) in the Kaplan-Meier test. This better clinical efficiency of the test preparation was particularly observed during the treatment period, with 12 vs. 27 relapses (p = 0.013), but less during the follow-up observation period, with 27 vs. 39 relapses (p = 0.127). In addition, the time span until occurrence of the first relapse was clearly longer under verum (699 days) than under placebo (334 days) and after the end of the observation period 91% of patients under verum experienced only one relapse compared to 62% in the placebo group (p = 0.01). Severity of relapses under verum was also reduced significantly (chi 2; p = 0.001. Only 4 patients under verum required antibiotic therapy compared to 13 patients under placebo. Verum was equally well tolerated as placebo, with no serious side effects in either group. No changes in laboratory tests--haematology and clinical chemistry--were observed. It can be concluded, that previously demonstrated immunomodifying effects of the test preparation have clinical relevance for the treatment of chronic recurrent bronchitis because not only the number but also the severity of acute relapses could be clearly reduced. This is discussed in view of the current literature.

Adjuvants, Immunologic↗

[Linezolid in two premature babies with necrotizing enterocolitis and infection with vancomycin-resistant enterococcus].

The use of the antibiotic Linezolid in two premature babies with necrotizing enterocolitis and infection with vancomycin-resistant enterococcus is described. Therapy with Linezolid in cause of necrotizing enterocolitis is an exception. Actually there are no proven recommendations for the use in this age group. Despite the successfully results more investigations for the use of linezolid in newborns and prematures are necessary.

Acetamides↗

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↗

Quinupristin-dalfopristin-resistant Enterococcus faecium on chicken and in human stool specimens.

BACKGROUND: The combination of the streptogramins quinupristin and dalfopristin was approved in the United States in late 1999 for the treatment of vancomycin-resistant Enterococcus faecium infections. Since 1974, another streptogramin, virginiamycin, has been used at subtherapeutic concentrations to promote the growth of farm animals, including chickens. METHODS: To determine the frequency of quinupristin-dalfopristin-resistant E. faecium, we used selective medium to culture samples from chickens purchased in supermarkets in Georgia, Maryland, Minnesota, and Oregon and stool samples from outpatients. RESULTS: Between July 1998 and June 1999, samples from 407 chickens from 26 stores in four states were cultured, as were 334 stool samples from outpatients. Quinupristin-dalfopristin-resistant E. faecium was isolated from 237 chicken carcasses and 3 stool specimens. The resistant isolates from stool had low-level resistance (minimal inhibitory concentration [MIC], 4 microg per milliliter; resistance was defined as a MIC of at least 4 microg per milliliter). The resistant isolates from chickens in general had higher levels of resistance (MICs ranging from 4 to 32 microg per milliliter; MIC required to inhibit 50 percent of isolates, 8 microg per milliliter). CONCLUSIONS: Quinupristin-dalfopristin-resistant E. faecium contaminates a large proportion of chickens sold in U.S. supermarkets. However, the low prevalence and low level of resistance of these strains in human stool specimens suggest that the use of virginiamycin in animals has not yet had a substantial influence. Foodborne dissemination of resistance may increase, however, as the clinical use of quinupristin-dalfopristin increases.

Animal Feed↗

Epidemiology of vancomycin-resistant Enterococcus faecium under a selective isolation policy at an urban county hospital.

BACKGROUND: We report our experience in a county hospital with the use of selective contact isolation for patients with vancomycin-resistant Enterococcus faecium (VREF). About 12% of patients with VREF are isolated for reasons such as draining wounds and uncontrolled diarrhea. METHODS: Passive surveillance identified all inpatients (181) from 1995 to 1999 with cultures positive for VREF. Data were collected via electronic databases and from prospectively maintained infection control records. Isolates were typed with use of pulsed-field gel electrophoresis. RESULTS: Nearly all patients (175/181) with VREF had been admitted at least 48 hours or had a history of previous hospitalization. Most patients (69%) had urine cultures positive for VREF without blood cultures positive for the organism. Only 12 of 127 (9.%) patients with complete data had VREF infection on the basis of receiving treatment and/or having more than 1 blood culture positive for VREF. After VREF became endemic, statistically significant increased prevalence was not detected via surveillance of clinical cultures nor sequential point-prevalence studies. Two major genotypes carrying vanB resistance genes were identified and persisted throughout the period studied. VREF persisted in individual patients up to 46 months. CONCLUSIONS: The number of VREF infections in this facility has been low, despite appreciable colonization, for an extended period during which selective isolation was used.

Adult↗

The capsular polysaccharide of Enterococcus faecalis and its relationship to other polysaccharides in the cell wall.

With the goal of identifying and characterizing traits of Enterococcus faecalis that play key roles in human disease, we identified an operon specifying synthesis of a capsular carbohydrate of the type most commonly expressed by clinical isolates. This surface-exposed carbohydrate consists of glycerol phosphate, glucose, and galactose residues, and its biosynthesis is encoded by a determinant that includes 11 ORFs. Insertional inactivation of genes in this pathway yielded mutants with enhanced susceptibility to phagocytic killing in vitro and compromised in the ability to persist in regional lymph nodes in vivo.

Animals↗

Structure of peptide sex pheromone receptor PrgX and PrgX/pheromone complexes and regulation of conjugation in Enterococcus faecalis.

Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone binding destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.

Base Sequence↗

Characterization of a class of nonformylated Enterococcus faecalis-derived neutrophil chemotactic peptides: the sex pheromones.

Bacteria produce a heterogeneous mixture of neutrophil chemotactic agents in culture filtrates. Formylmethionyl peptides have been shown to comprise a significant portion of the chemotactic activity in bacterial culture filtrates; however, not all of the chemotactic agents in bacterial culture filtrates are formylated peptides. To examine whether nonformylated peptides derived from bacteria could act as chemotactic agents, we studied several nonformylated hepta- and octapeptide Enterococcus faecalis-derived sex pheromones, their modified derivatives, and their competitive inhibitors for activation of rat peritoneal neutrophils. Several of these peptides, in particular cAM373 and cPD1, proved to be potent chemotactic agents in submicromolar concentrations as well as inducers of lysosomal granule enzyme secretion. Moreover, the more biologically active peptides were able to compete with fMet-Leu-[3H]Phe for binding to the formyl peptide receptor. These studies demonstrate that the formylmethionyl moiety may be an absolute requirement only for the binding of di- and tripeptides to the formyl peptide receptor. Larger peptides that may have or that may allow for additional contact points between the peptide and receptor may require N-formylation only relatively. Indeed, by removing this structural restraint, the formyl peptide receptor may interact with an unlimited number of peptide fragments of both infectious and host origins to then modulate neutrophil responses to infection and inflammation.

Amino Acid Sequence↗

Cis-acting, orientation-dependent, positive control system activates pheromone-inducible conjugation functions at distances greater than 10 kilobases upstream from its target in Enterococcus faecalis.

The prgB gene encodes the surface protein, Asc10, which mediates cell aggregation, resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline-resistance plasmid pCF10 in Enterococcus faecalis. Messenger RNA analysis by Northern blot hybridization and primer extension indicates that prgB transcription is pheromone-inducible and monocistronic. Previous transposon mutagenesis and sequencing analysis of a 12-kilobase (kb) region of pCF10 indicated that several genes including prgR and prgS are required to activate expression of prgB. The distance (3-4 kb) between these regulatory genes and prgB suggested that the activation might function in trans. To test this, a promoterless lacZ gene fusion to prgB was constructed and cloned without some or all of the regulatory genes. Several restriction fragments of the regulatory region were cloned in a higher copy-number plasmid, and numerous complementation studies were carried out in E. faecalis. Complementation in trans was not observed in any of these experiments. However, when the regulatory region and target genes were cloned in different sites of the same plasmid, separated by as much as 12 kb, activation of prgB was observed. Interestingly, this activation occurred only when the regions were cloned in the same relative orientation in which they exist on wild-type pCF10. These results suggest that one or more regulatory molecules may bind to an upstream cis-acting site and track along the DNA to reach a target site to activate prgB transcription.

Bacterial Proteins↗

Phosphinate analogs of D-, D-dipeptides: slow-binding inhibition and proteolysis protection of VanX, a D-, D-dipeptidase required for vancomycin resistance in Enterococcus faecium.

VanX is a D-Ala-D-Ala dipeptidase that is essential for vancomycin resistance in Enterococcus faecium. Contrary to most proteases and peptidases, it prefers to hydrolyze the amino substrate but not the related kinetically and thermodynamically more favorable ester substrate D-Ala-D-lactate. The enzymatic activity of VanX was previously found to be inhibited by the phosphinate analogs of the proposed tetrahedral intermediate for hydrolysis of D-Ala-D-Ala. Here we report that such phosphinates are slow-binding inhibitors. D-3-[(1-Aminoethyl)phosphinyl]-D-2-methylpropionic acid I showed a time-dependent onset of inhibition of VanX and a time-dependent return to uninhibited steady-state rates upon dilution of the enzyme/inhibitor mixture. The initial inhibition constant Ki after immediate addition of VanX to phosphinate I to form the E-I complex is 1.5 microM but is then lowered by a relatively slow isomerization step to a second complex, E-I*, with a final K*i of 0.47 microM. This slow-binding inhibition reflects a Km/K*i ratio of 2900:1. The rate constant for the slow dissociation of complex E-I* is 0.24 min-1. A phosphinate analog with an ethyl group replacing what would be the side chain of the second D-alanyl residue in the normal tetrahedral adduct gives a K*i value of 90 nM. Partial proteolysis of VanX reveals two protease-sensitive loop regions that are protected by the intermediate analog phosphinate, indicating that they may be part of the VanX active site.

Bacterial Proteins↗

Enterococcus faecalis pheromone binding protein, PrgZ, recruits a chromosomal oligopeptide permease system to import sex pheromone cCF10 for induction of conjugation.

Conjugative transfer of the plasmid pCF10 by Enterococcus faecalis donor cells occurs in response to a peptide sex pheromone, cCF10, secreted by recipients. The plasmid-encoded cCF10 binding protein, PrgZ, is similar in sequence to binding proteins (OppAs) encoded by oligopeptide permease (opp) operons. Mutation of prgZ decreased the sensitivity of donor cells to pheromone, whereas inactivation of the chromosomal E. faecalis opp operon abolished response at physiological concentrations of pheromone. Affinity chromatography experiments demonstrated the interaction of the pheromone with several putative intracellular regulatory molecules, including an RNA molecule required for positive regulation of conjugation functions. These data suggest that processing of the pheromone signal involves recruitment of a chromosomal Opp system by PrgZ and that signaling occurs by direct interaction of internalized pheromone with intracellular effectors.

Amino Acid Sequence↗