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PubMed2004

The emergence of glycopeptide-resistant Enterococcus faecium in a university hospital in southwestern Greece.

BACKGROUND: Enterococci and especially glycopeptide-resistant strains (GRE) are widely distributed in the hospital environment, by acquiring resistance determinants and virulence factors. METHODS: The study included 48 GRE isolated during a 1-year period from different inpatients in a tertiary hospital in southwestern Greece. Antibiotic susceptibility was determined by the Etest, and the presence of resistance and virulence genes was shown by PCR. Clonal types were identified by pulsed-field gel electrophoresis of SmaI DNA digests. RESULTS: All GRE were multi-resistant of the VanA phenotype, verified by the detection of the gene by PCR. Two major clones were distributed in all hospital wards. The majority of the strains (46 of 48) harbored the esp gene, while 27 GRE expressed also the gelE and/or as genes. CONCLUSIONS: The spread of two clones expressing the vanA gene and virulence factors were responsible for the emergence of GRE in the University Hospital of Patras.

Anti-Bacterial Agents↗

PubMed2005

Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2''-phosphotransferase-Ib [APH(2'')-Ib].

Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2'')-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2(1), with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 A, beta = 98.4 degrees, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 A resolution were collected from a native APH(2'')-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.

Bacterial Proteins↗

PubMed2002

Effect of gastrointestinal bleeding and oral medications on acquisition of vancomycin-resistant Enterococcus faecium in hospitalized patients.

There has been minimal investigation of medications that affect gastrointestinal function as potential risk factors for the acquisition of vancomycin-resistant enterococci (VRE). We performed a retrospective case-control study, with control subjects matched to case patients by time and location of hospitalization. Strict exclusion criteria were applied to ensure that only case patients with a known time of acquisition of VRE were included. Control patients were patients with > or =1 culture negative for VRE. The risk factors identified were use of vancomycin (odds ratio [OR], 3.2; 95% confidence interval [CI], 1.7-6.0; P=.0003), presence of central venous lines (OR, 2.2; 95% CI, 1.04-4.6; P=.04), and use of antacids (OR, 2.9; 95% CI, 1.5-5.6; P=.002). Two protective factors included gastrointestinal bleeding (OR, 0.26; 95% CI, 0.08-0.79; P=.02) and use of Vicodin (Knoll Labs; hydrocodone and acetaminophen; OR, 0.93; 95% CI, 0.90-0.97; P=.0003). Changes in gastrointestinal function, whether due to bleeding or to the effects of oral medications, may affect whether patients become colonized with VRE.

Administration, Oral↗

PubMed1996

Limiting vancomycin use to combat vancomycin-resistant Enterococcus faecium.

The implementation, monitoring, and impact of a program to restrict vancomycin use are described. A vancomycin restriction program was implemented in February 1995 at an acute care teaching hospital after guidelines for vancomycin use were established by a multidisciplinary group. Pharmacists reviewed each vancomycin order, suggested alternative treatments when vancomycin use did not comply with the guidelines, and set duration limits for all orders. Orders requiring greater clinical experience for review were referred to a pharmacist or a physician in the division of infectious diseases. The program was monitored by an infectious diseases pharmacist. Data collected after the program was established showed that the volume of vancomycin use decreased substantially. Problems in enforcing the restriction program included administration of vancomycin for surgical prophylaxis before the order reached the pharmacy, continuing use of vancomycin for initial empirical treatment of febrile neutropenic and immunocompromised patients, inadequate tracking of the evaluations, and deficiencies in the evaluations related to a need for continuing education of the pharmacists about the program. Use of vancomycin decreased after a pharmacy-enforced restriction program was implemented.

Anti-Bacterial Agents↗

PubMed2001

Successful treatment of vancomycin-resistant Enterococcus faecium meningitis with linezolid: case report and literature review.

Vancomycin-resistant enterococci (VRE) are a rare cause of meningitis, occurring primarily in patients who have undergone neurosurgical procedures. We describe the first reported case of VRE meningitis successfully treated with linezolid. A 56-y-old female with subarachnoid hemorrhage underwent ventriculostomy and embolization of cerebral aneurysms. Her postoperative course was complicated by multiple infections needing repeated antibiotic courses, culminating in the development of VRE meningitis. She was treated with 600 mg of i.v. linezolid (MIC < 0.75 microg/ml) every 12 h for 6 weeks. After the fourth dose, peak and trough linezolid concentrations were 11.45 and 0.14 microg/ml in serum and 3.19 and 2.39 microg/ml in cerebral spinal fluid (CSF). On Day 19 of linezolid therapy, serum and CSF trough concentrations were 1.53 and 2.98 microg/ml, respectively. Linezolid achieved sufficient CSF concentrations to bring about clinical and bacteriological cure. We conclude that i.v. linezolid may be a useful option for treating VRE meningitis. We also present findings of a literature review, which identified 11 cases of VRE meningitis treated with other pharmacologic agents with mixed success.

Acetamides↗

PubMed2005

Diversity and stability of plasmids from glycopeptide-resistant Enterococcus faecium (GRE) isolated from pigs in Denmark.

In this paper, we examine the plasmid variation between a subset of unrelated GRE isolated from pigs in Denmark between 1995 and 2001 (five from each of the years). The isolates were tested with PFGE, plasmid RFLP, and subsequently Southern blotting with an IS1216V probe. Of the 35 isolates, 31 belonged to the same PFGE type (type I), and the last four belonged to a completely different PFGE type (type II). All 35 isolates contained the same type of large plasmid (approximate size of 150-200 kb), which could be divided into eight different variant types (V0 to V7). Most variance among the plasmid was seen at the end of the 7-year period, coinciding with the ban in 1998 of the macrolide tylosin as growth promoter for pig production. The stability of the plasmid in its original host was compared with stability of the same plasmid in BM4105RF, when both strains were maintained in liquid cultures without the presence of antibiotics for 1,100 generations. Here, the plasmid proved far more stable in its original host environment than in the new host.

Animals↗

PubMed2000

Evaluation of fluorescence-based amplified fragment length polymorphism analysis for molecular typing in hospital epidemiology: comparison with pulsed-field gel electrophoresis for typing strains of vancomycin-resistant Enterococcus faecium.

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the "gold standard" for molecular typing in epidemiology.

Bacterial Typing Techniques↗

PubMed1993

Purification and characterization of VanR and the cytosolic domain of VanS: a two-component regulatory system required for vancomycin resistance in Enterococcus faecium BM4147.

Resistance to the glycopeptide antibiotic vancomycin requires five genes. Two of these, vanR and vanS, have sequence homology to cytoplasmic response regulatory (VanR) and transmembrane sensory (VanS) proteins of two-component regulatory systems used to sense and transduce environmental signals. We report the overproduction and purification to homogeneity of VanR (27 kDa) and of a fusion protein of VanS (residues 95-374, the cytosolic domain) to the maltose binding protein (MBP), yielding a MBP-VanS protein of 76 kDa. The MBP-VanS fusion protein displayed an ATP-dependent autophosphorylation on a histidine residue with a rate of 0.17 min-1 and a phosphorylation stoichiometry of 10-15%. 32P-PhosphoMBP-VanS transferred the phosphoryl group to VanR. 32P-Phospho VanR showed chemical stability anticipated for an aspartyl phosphate and was relatively stable to hydrolysis (t1/2 = 10-12 h). Thus, the vancomycin resistance operon appears to have collected and specifically tailored the His kinase and Asp phosphoryl receptor of two-component signal transduction logic for sensing extracellular vancomycin and turning on structural genes, vanA and vanH, to make altered peptidoglycan structures such that vancomycin does not bind.

ATP-Binding Cassette Transporters↗

PubMed2000

[Epidemiologic increase of various genotypes of vancomycin-resistant Enterococcus faecium in a university hospital].

After a report of a possible relationship between an outbreak of vancomycin-resistant enterococci (VRE) in a nearby hospital and earlier admission of two of the patients with this VRE in the University Medical Centre of Utrecht (UMCU), the Netherlands, an extensive search for VRE carriers was started in the UMCU. In the study period of two months, VRE carriership was diagnosed in 51 patients in nine of the 11 wards investigated. Twenty-six patients in eight wards were colonized with the same VRE genotype as in the nearby hospital; spread was demonstrated in three wards. In addition, six patients of one ward were colonized with a second genotype and seven other patients with a third genotype, while 12 patients were carriers of a unique genotype. Most carriers were found in the internal medicine/nephrology and dialysis ward. Far-reaching measures (such as cohort nursing, admission stops, use of gowns and gloves, disinfection and restriction of use of vancomycin) taken in the four wards where spread was demonstrated, appeared effective but in three wards, spread was again demonstrated later. Frequent readmissions and transfers of patients appear to play an important part in this matter. None of the 51 colonized patients developed a serious VRE infection.

Carrier State↗

PubMed2005

Enterocin AS-48RJ: a variant of enterocin AS-48 chromosomally encoded by Enterococcus faecium RJ16 isolated from food.

The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcusfaecium by species-specific PCR, DNA-rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125 Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly less active on strains of Listeria monocytogenes and Bacillus cereus. Producer strains show cross-immunity. PCR amplification of total DNA from strain RJ16 with primers for the AS-48 structural gene and sequencing of the amplified fragment revealed an almost identical sequence (99.5%), except for a single mutation that predicts the change of Glu residue at position 20 of AS-48 to Val. Therefore, bacteriocin produced by E. faecium RJ16 should be considered a variant of AS-48, which we call AS-48RJ. PCR amplification revealed that strain RJ16 contains the complete as-48. gene cluster. Hybridization with probes for as-48 gene cluster revealed a chromosomal location of as-48 genes in strain RJ16, being the first example of a chromosomal location of this bacteriocin trait. Strain RJ16 produced enzymes of interest in food processing (esterase, esterase lipase and phytase activities), and did not decarboxylate amino acids precursors for biogenic amines. Strain RJ16 did not exhibit haemolytic or gelatinase activities, and PCR amplification revealed the lack of genes encoding for known virulence determinants (aggregation substance, collagen adhesin, enterococcal surface protein, endocarditis antigens, as well as haemolysin and gelatinase production). Strain RJ16 was resistant to ciprofloxacin (MIC > 2 mgl(-1)) and levofloxacin (MIC > 4 mgl(-1)) and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, tetracycline, quinupristin/dalfopristin, vancomycin and teicoplanin. Altogether, results from this study suggest that this broad-spectrum bacteriocin-producing strain may have a potential use in food preservation.

Amino Acid Sequence↗

PubMed2004

Two episodes of vancomycin-resistant Enterococcus faecium outbreaks caused by two genetically different clones in a newborn intensive care unit.

In 2001 two outbreak episodes (January-March and June-July) caused by vancomycin-resistant E. faecium (VRE) of the VanA-type were observed at a neonatal intensive care unit (NICU) of a university hospital in south-west Germany. To identify the initial source and the route of transmission environmental samples were examined as well as stool samples from patients and the staff. VRE was not found in environmental samples. However, stool samples from 24 hospitalised children tested positive and bacterial clonality was assessed by Sma1-based macro restriction analysis. Furthermore, esp gene and vancomycin resistance gene carriage were examined as well as bacteriocin production. PCR analysis showed that all 24 isolates carried vanA gene cluster, encoding resistance to vancomycin and teicoplanin. However, five of the vanA-positive isolates were resistant to vancomycin but not to teicoplanin. Only these five isolates produced bacteriocin, but in none of the isolates esp gene was detected. PFGE revealed that both outbreaks were caused by two different clones. The patient initiating the first episode, was identified whereas the origin of the second episode remained unknown. From one of the 40 staff stool samples VRE was isolated. This strain was related to the clone of the summer outbreak. In conclusion there were two independent episodes of self limiting VRE outbreaks and transmission on the ward is highly probable.

Bacterial Proteins↗

PubMed1998

Infection and colonization with vancomycin-resistant Enterococcus faecium in an acute care Veterans Affairs Medical Center: a 2-year survey.

BACKGROUND: After our first known patient with vancomycin-resistant enterococci (VRE) infection was admitted in 1993, we observed a gradual increase in infections and colonization caused by this organism. Thus we initiated a prospective study to quantitate the incidence of VRE infection versus colonization, to identify risk factors for VRE, and to define the natural history of VRE colonization among our patients. METHODS: Stool/rectal cultures were performed for patients admitted to the intensive care units at the time of admission and weekly thereafter. Patients found to be carrying VRE were followed with cultures every 2 weeks, and this protocol was continued after transfer to the medical-surgical wards. A surveillance form was initiated on each VRE patient and included demographics, underlying diseases, and risk factors. Environmental cultures in the intensive care units were randomly performed. Patients with positive cultures were isolated. RESULTS: During a 27-month period, 210 patients were found to be colonized or infected with VRE. Ages ranged from 35 to 97 years; the mean age was 65 years. Fourteen percent (29 of 210) of the patients were VRE positive on admission. Nosocomial colonization or infection occurred at an average of 28 days after admission. Seventeen percent (25 of 216) of patients cleared VRE during their hospital stay; 19% (40 of 210) developed 47 infections. One third of infections involved the urinary tract. Liver transplantation, chemotherapy, and total parenteral nutrition were each associated with infection. CONCLUSION: Routine measures as advocated by the Centers for Disease Control and Prevention were not effective in controlling VRE in our patient population.

Adult↗

PubMed2003

[Infections with Van B phenotype Enterococcus faecium and E. faecalis in patients with immune deficiency during the course of hematologic neoplasms].

In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. The aim of this study was to characteristics of the types of resistance to glycopeptide antibiotic and to check the resistance patterns of these pathogens. A trial of monitoring the risk factors for colonization or infection with VRE as well as epidemiological investigation were undertaken. Genus identification of the isolates was performed according to the method of Facklam and Collins, and species were identified using the API 20 Strep test. MICs of different antimicrobial agents were determined by the E-test method. The isolates collected during the investigation demonstrated resistance to multiple antimicrobials, which is a common characteristic of VRE. Isolates were found to be uniformly resistant to penicillin, fluoroquinolones, tetracycline and to high concentrations of aminoglycosides. The only drugs with in vitro activity against the isolates were ampicillin (VRES), linezolid (VRES, VREM) and quinupristin-dalfopristin (VREM). Except for a single VREM isolate, they all revealed the typical VanB phenotype with resistance to vancomycin and susceptibility to teicoplanin. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene claster, including the regulatory genes vanRB and -SB, and the vancomycin--inducible promoter PYB. Our studies have found an association between colonization or infection with VRE and the mean duration of hospital stay, previously administration of glycopeptide, cephalosporins and imipenem. These organisms were a common cause of monoethiological bloodstream infections.

Adult↗

PubMed2001

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on "bryndza", a traditional Slovak dairy product from sheep milk.

"Bryndza" is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in "bryndza" was investigated with the aim to reduce the contaminant agents. "Bryndza" was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, "bryndza" was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 degrees C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in "bryndza" before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10(3) cfu/ml/g, Staphylococcus aureus (10(2) cfu/ml/g) and enterococci (10(4) cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10(2) cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10(3) cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in "bryndza". However, bacteriocin activity was not determined by laboratory analyses.

Animals↗

PubMedDate not supplied

Treatment of vancomycin-resistant Enterococcus faecium infections.

OBJECTIVE: To define the clinical characteristics of patients infected with vancomycin-resistant enterococci (VRE) and the outcome of the infections without the availability of effective antimicrobial therapy. METHODS: Charts of 28 patients with VRE infections were reviewed for demographics, clinical findings at the time of isolation of VRE, underlying medical problems, surgical procedures, invasive devices, treatment with antimicrobial agents, microbiological data, and patients' responses and outcomes. RESULTS: The infections included 6 cases of bacteremia, 9 surgical site infections (SSIs), 4 cases of peritonitis, 2 pelvic abscesses, 7 urinary tract infections (UTIs), and 2 soft tissue infections (STIs). Four of the 6 bacteremia cases were central-line related and resolved with line removal alone; 1 was treated with a combination product of quinupristin and dalfopristin (Synercid) and 1 had persistent bacteremia in the presence of a ventriculoperitoneal shunt. Seven of 9 SSIs resolved with surgical debridement and 2 of the 9 patients received antibiotics for organisms other than VRE. Similarly, 2 patients with STIs were treated with local debridement and antibiotics directed at organisms other than VRE and 2 patients with pelvic abscesses were treated with drainage and surgical debridement with antibiotics directed at other organisms; the infections resolved completely. Patients with peritonitis were treated with removal of their Tenckhoff catheters, drainage, and irrigation and 1 patient was treated with quinupristin-dalfopristin; 3 of 4 patients were cured. Two of 7 patients with UTIs were treated with nitrofurantoin and their urine cultures showed no growth after treatment; however, most patients with UTIs experienced resolution despite a lack of specific antimicrobial therapy. CONCLUSIONS: Although no antimicrobial agents are currently available for VRE infections, VRE line-related bacteremias could be treated by line removal alone. Surgical site infections, STIs, and abscesses could be managed by surgical debridement and drainage without specific antimicrobial agents against VRE and UTIs could be resolved with nitrofurantoin or removal of Foley catheters. Removal of foreign devices, debridement, and surgical drainage seemed to be important in the resolution of VRE infections.

Abdominal Abscess↗

PubMed1998

Epidemiology of a dominant clonal strain of vancomycin-resistant Enterococcus faecium at separate hospitals in Boston, Massachusetts.

In 1996, the dominant (43%) strain of vancomycin-resistant enterococci (VRE; type A) at Massachusetts General Hospital was identified at Brigham and Women's Hospital (BWH). To characterize the epidemiology of infection with type A isolates of VRE at BWH, we collected demographic and clinical data for all patients from whom VRE were isolated from a clinical specimen through September 1996. The first clinical isolates from all BWH patients from whom VRE were isolated were typed by pulsed-field gel electrophoresis of SmaI digests of chromosomal DNA. Among patients hospitalized after the first patient at BWH infected with a type A isolate of VRE was identified, exposures were compared between patients who acquired type A isolates of VRE and those who acquired other types of VRE. Isolates from 99 patients identified to have acquired VRE were most commonly from blood (n = 27), urine (n = 19), or wounds (n = 19). Three months after the index patient arrived at BWH and at a time when > or =12 types of strains of VRE were present, type A isolates of VRE became dominant; 39 of 75 (52%) of the study cohort had acquired type A isolates of VRE. We found no association between the acquisition of type A isolates of VRE and transfer from another institution or temporal overlap by service, ward, or floor with patients known to have acquired type A isolates of VRE. By multivariate analysis, only residence in the medical intensive care unit (adjusted odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4 to 107) and the receipt of two or more antibiotics per patient-day (adjusted OR, 12.2; 95% CI, 1.2 to 9.0) were associated with the acquisition of strain A. This strain of VRE, dominant at two Boston hospitals, was associated with intensity of antibiotic exposures (i.e., two or more antibiotics per patient-day). We hypothesize that this strain may have unidentified properties providing a mechanism favoring its spread and dominance over other extant isolates, and further studies are needed to define these properties.

Anti-Bacterial Agents↗

PubMed2002

A variant enterococcal surface protein Esp(fm) in Enterococcus faecium; distribution among food, commensal, medical, and environmental isolates.

Enterococci are increasingly important causes of nosocomial disease. Also, they are associated with food and have a history of use as dairy starter and probiotic cultures. An enterococcal surface protein Esp(fs) is involved the virulence and biofilm-forming capacity of Enterococcus faecalis and recently we demonstrated the presence of a homologue Esp(fm) in E. faecium. Here we describe the complete structure of Esp(fm) and demonstrate that its distribution in E. faecium correlates with disease associated strains from a range of pathological sites.

Amino Acid Sequence↗

PubMed1999

First confirmed case of a vancomycin-resistant Enterococcus faecium with vanA phenotype from Brazil: isolation from a meningitis case in São Paulo.

The importance of enterococci as a nosocomial etiologic agent is well documented; however, enterococci are also capable of causing a variety of community-acquired infections. Vancomycin resistance in a clinical Enterococcus isolate was first reported in 1986, and since then vancomycin-resistant enterococci (VRE) have been reported world-wide. This report describes a case of E. faecium with the VanA phenotype, isolated from meningitis in Sao Paulo, Brazil. Two E. faecium strains were isolated. One strain showed VanA phenotype, and the molecular characterization of the VanA gene was confirmed by polymerase chain reaction. The other strain was susceptible to vancomycin and teicoplanin. The authors would like to call the attention of the scientific community to this first identification of a VRE case in Sao Paulo, Brazil.

Anti-Bacterial Agents↗