Development of a novel immunochromatographic assay for rapid detection of VanA ligase-producing vancomycin-resistant enterococci.

We developed a novel immunochromatographic assay (ICA) (EZ-Step VanA rapid kit; Dinona, Korea) for the detection of VanA ligase from vancomycin-resistant enterococci (VRE). Of eight monoclonal antibodies screened by ELISAs, the VanA ligase ICA constructed with 1H9 plus 3G11 showed the greatest reactivity. The detection limit of the kit was 6.3 × 10(6) CFU per test. Of 127 vancomycin-resistant microorganisms, 100 vanA VRE were positive in the VanA ligase ICA, and 27 non-vanA vancomycin-resistant isolates were negative. These results were consistent with those of the PCR analyses. Thus, our ICA is a reliable and easy-to-use immunological assay for detecting VanA-producing VRE in clinical laboratories.

[Environmental contamination during a vancomycin-resistant Enterococci outbreak at a hospital in Argentina]. / Contaminación ambiental durante un brote de enterococo resistente a vancomicina en un hospital de Argentina.

INTRODUCTION: Vancomycin-resistant enterococci isolates (VRE) have caused numerous outbreaks in intensive care units (ICUs). A contaminated hospital environment, the hands of health care workers (HCW), and carrier patients may play important roles in perpetuating the chain of transmission in these outbreaks. The aims of this study were to report the first VRE outbreak in our center and assess the role of environmental contamination and HCW hands in the spread of new cases of enterococcal infection. MATERIAL AND METHOD: Between August and December 2003, surveillance cultures were performed with samples from all patients (n = 113) admitted to the ICU, as well as cultures of samples from the environment (n = 69) and HCW hands (n = 23). RESULTS: Eighteen clinical samples from 8 patients and 7 environmental samples yielded Enterococcus faecium (24 strains) and E. avium (1 strain). VRE was not detected on HCW hands. All the VRE isolates belonged to a single clone and carried the vanA gene. CONCLUSION: Environmental contamination provides an important reservoir for future outbreaks of VRE, perpetuating transmission of the microorganism in the hospital setting.

Evaluation of the AdvanDx VRE EVIGENE assay for detection of vanA in vancomycin-resistant Staphylococcus aureus.

AdvanDx VRE EVIGENE, a commercial vanA/vanB DNA hybridization assay to identify vancomycin-resistant enterococci (VRE), was evaluated for the detection of vanA in Staphylococcus aureus. Performance was assessed using S. aureus, VRE, and vancomycin-intermediate and -susceptible isolates. The assay demonstrated 100% sensitivity and specificity when analyzed visually and by optical density.

[Bacteremia caused by Enterococcus gallinarum with a high level of glycopeptide resistance: 1st documented cases in Argentina]. / Bacteriemia por Enterococcus gallinarum con alto nivel de resistencia a glicopéptidos: primer caso documentado en Argentina.

A case of bacteremia due to high-level-vancomycin- (MIC = 64 micrograms/ml) and high-level-teicoplanin- (MIC = 32 micrograms/ml) resistant Enterococcus gallinarum is described. Both genes, van C1 and van A, respectively conferring natural low-level resistance and acquired high-level resistance to vancomycin, were found in the enterococcal genoma. The present is the first report of an E. gallinarum isolate showing the van A genotype in Argentina.

Evaluation of a novel method based on amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting) for typing strains of vancomycin-resistant Enterococcus faecium.

In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of a novel assay based on the fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites (ADSRRS fingerprinting) was tested. A large number of vancomycin-resistant Enterococcus faecium (VREM) isolates from haematological ward patients of the Clinical Hospital in Gdansk were examined. We found that ADSRRS fingerprinting analysis is a rapid method that offers good discriminatory power. The method demonstrated also excellent reproducibility. The usefulness of the ADSRRS fingerprinting method for molecular typing was compared with pulsed field gel electrophoresis (PFGE) method, which is currently considered the gold standard for molecular typing of isolates recovered from patients and the environment in the course of investigation and control of nosocomial outbreaks. Clustering of ADSRRS fingerprinting data matched pulsed field gel electrophoresis data. The features of ADSRRS fingerprinting technique is discussed in comparison with conventional methods. Data presented here demonstrate the complexity of the epidemiological situation concerning VREM that may occur in a single medical ward.

Phenotypes and genotypes of vancomycin-resistant enterococci isolated during long-term follow-up in a patient with recurrent bacteremia and colonization.

Twenty-seven isolates of vancomycin-resistant enterococci were obtained at monthly intervals from a bed-ridden man with hypoxic encephalopathy. During the 28-month period of the patient's hospitalization, 3 episodes of bacteremia and one episode of catheter-related infection caused by vancomycin-resistant enterococci occurred. Rectal swabs showed colonization of vancomycin-resistant enterococci for more than 2 years. Three months after termination of antimicrobial therapy, the rectal colonization for vancomycin-resistant enterococci was eradicated. Four species (Enterococcus faecium, Enterococcus gallinarum, Enterococcus faecalis, and Enterococcus casseliflavus) were identified among the 27 vancomycin-resistant enterococcus isolates. Three non-clonal related patterns were found among 17 strains of E. faecium by pulsed-field gel electrophoresis. All of the 3 E. faecalis isolates were of the VanB phenotype, but of the vanA genotype. Linezolid had the most potent in vitro activity against these vancomycin-resistant enterococcus isolates, with minimum inhibitory concentrations >2 microg/mL. Eighty-five percent of these vancomycin-resistant enterococcus isolates were susceptible to tetracycline and 66% were susceptible to quinupristin-dalfopristin. Although a high genetic correlation of E. faecium was identified in the patient with prolonged hospitalization, the isolation of 3 genetically unrelated colonized isolates suggested a lack of correlation between infection and colonization. Precautions against resistant organisms, adapted antibiotic policies, and elimination of patient carriage are useful for controlling the spread of vancomycin-resistant enterococci.

Diversity of Tn1546 elements in clinical isolates of glycopeptide-resistant enterococci from Scottish hospitals.

The Tn1546-related elements of 48 Van glycopetide-resistant enterococci were compared. Ten distinct Tn1546 types were identified with variation primarily due to IS1542 and IS1216V-like insertions. Clonal isolates frequently differed in their Tn1546 type, indicating instability of Tn1546-related elements. A putative hybrid promoter was identified, generated upstream of vanR by the insertion of IS1542. The presence of this hybrid promoter was associated with constitutive expression of the van genes and elevated teicoplanin resistance.

Epidemiological, microbiological, clinical, and prognostic factors of bacteremia caused by high-level vancomycin-resistant Enterococcus species.

A case-control study was performed between 1994 and 1996 in order to study the epidemiological, microbiological, clinical, and prognostic features of high-level vancomycin-resistant enterococcal bacteremia. Seventeen consecutive patients who had clinically significant bacteremia due to vancomycin-resistant enterococci (vanA genotype: 16 Enterococcus faecalis, 1 Enterococcus faecium) were compared with 169 who had vancomycin-susceptible enterococcal bacteremia. The following were selected by multivariate analysis as independent risk factors that influenced the development of high-level vancomycin-resistant enterococcal bacteremia: prior glycopeptide therapy (P=0.049); inclusion in a hemodialysis program (P=0.046); prior therapy with corticosteroids or antineoplastic agents (P=0.029); and prior surgical treatment (P=0.022). The following other factors were selected by univariate analysis: tracheostomy (P=0.002); prolonged hospitalization (P=0.01); and any kind of puncture (P=0.02). The crude associated-mortality rate was 13.4%. Gene amplification of vanA was positive for 17 strains of enterococci. Pulsed-field gel electrophoresis of genomic DNA after SmaI digestion of vanA isolates revealed that one strain predominated (10 isolates), though at least four similar banding patterns were identified (6 isolates). The 16 strains closely related to the outbreak were investigated further. The surgical intensive care unit was the first and most involved service. The hospital outbreak of vanA vancomycin-resistant enterococcal bacteremia occurred between 1994 and 1995 and was caused by Enterococcus faecalis. This is believed to be the first and only such outbreak described in a Spanish hospital thus far.

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods.

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.

Continuing high prevalence of VanA-type vancomycin-resistant enterococci on Norwegian poultry farms three years after avoparcin was banned.

Avoparcin was used as a feed additive in Norwegian broiler and turkey production from 1986 until 1995. It was banned due to the selection of VanA-type vancomycin-resistant enterococci (VRE) in animal husbandry and to reduce the potential for human exposure to VRE. The aim of the present study was to investigate the prevalence of VRE carriage in Norwegian poultry farmers and their poultry three years after avoparcin was banned. Corresponding faecal samples from poultry and humans on farms where avoparcin had previously been used (exposed farms, n = 73) and farms where avoparcin had never been used (unexposed farms, n = 74) were analysed for the presence of VRE. For each farm, one sample from the poultry house and one sample from the farmer were obtained. VRE were isolated from 72 of 73 (99%) and eight of 74 (11%) poultry samples from exposed and unexposed farms, respectively. VRE were isolated from 13 of 73 (18%) and one of 74 (1%) farmer samples from exposed and unexposed farms, respectively. All VRE isolates were highly resistant to vancomycin and possessed the vanA gene, as shown by PCR. The high prevalence of VRE is in accordance with previous Norwegian studies, and shows a remarkable stability of the VanA resistance determinant in an apparently non-selective environment.

Vancomycin-resistant enterococci isolated from animals and food.

One hundred and one chicken products, boiled ham and turkey cold meat were acquired from 18 different supermarkets in Spain during October 1997 to June 1998 and were analyzed for vancomycin-resistant enterococci (VRE). In the same way, 50 intestinal chicken samples from a slaughterhouse were also studied. VRE were detected in 25 of 92 samples of food of chicken origin (27.2%), but no VRE were found in cooked pork or turkey products. VRE were also detected in 8 of 50 intestinal chicken samples from the slaughterhouse (16%). VRE were identified as Enterococcus durans (n = 11), Enterococcus faecalis (n = 10), Enterococcus faecium (n = 10) and Enterococcus hirae (n = 2). All these strains were characterized as belonging to the vanA genotype by polymerase chain reaction. Ampicillin, quinupristin/dalfopristin and high level aminoglycoside resistance were frequently found among these strains. Heterogeneity was observed in susceptibility patterns among VRE strains, even in those of the same species. The high rate of colonization of chicken products by vanA containing enterococci detected 6 months to 1 year after the banning of avoparcin as a growth promoter, supports other studies suggesting that the food chain could be a source of VRE colonization in humans and thus a source of VRE infections.

Carrier rate of resistant enterococci in a tertiary care hospital in Norway.

The prevalence of resistant enterococci varies geographically. In the present study we looked at the carrier rate of resistant enterococci in the hematology and gastrointestinal surgery units of a tertiary care hospital in Norway. Anal swabs were taken from all 82 hospitalized patients on 4 different dates, at least 4 weeks apart, in 1995. 51% had positive cultures for enterococci. 6% of all patients carried enterococci resistant to ampicillin. 7% carried enterococci with high-level gentamicin resistance. Two strains resistant to vancomycin were found, including the first vanA Enterococcus faecium isolated in a Norwegian hospital. There was a correlation between use of antibiotics and being a carrier of enterococci per se, but the correlation with resistant enterococci did not reach statistical significance owing to the small number of isolates. The carrier rates both for presence of enterococci and for resistant enterococci were generally lower than those found in other studies.