Efficiency of the Cepheid Xpert vanA/vanB assay for screening of colonization with vancomycin-resistant enterococci during hospital outbreak

This study aimed to assess the efficiency of the Cepheid Xpert vanA/vanB test for detecting vancomycin-resistant enterococci (VRE) colonization during a VanA Enterococcus faecium outbreak and to compare the Cepheid Xpert vanA/vanB (Cepheid, Sunnyvale, USA) test to a culture method with chromogenic medium chromID VRE agar (bioMérieux). The Cepheid Xpert vanA/vanB assay showed sensitivity 61.5%, specificity 79.2%, positive predictive value 61.5% and negative predictive value 79.2%. The results obtained in this study demonstrate that a positive result in the Cepheid Xpert vanA/vanB test for vanA enables the rapid (less than 1 h) presumptive, prior to culture, recognition of patients colonized with VRE. However, the Cepheid Xpert vanA/vanB assay cannot be the only test used to screen patients during an ongoing VRE outbreak, because additional culturing of all samples negative for both vanA and vanB or positive for vanB should be performed in order to confirm the carrier status of the patient.     

Evaluation of chromogenic media for the isolation of vancomycin-resistant enterococci from stool samples

Aims:  This study sought to evaluate the performance of two chromogenic media designed for the isolation of vancomycin-resistant enterococci (VRE) and compare them with a traditional bile-esculin medium for the isolation of VRE from stool samples.
Methods and Results:  A total of 285 stool samples were inoculated onto Chromogenic VRE Agar (AES VRE agar; AES Chemunex), chromID VRE (bioMérieux) and VRE Agar (Oxoid) both directly and also following broth enrichment. In total 18 strains of vancomycin-resistant Enterococcus faecium were recovered, including 17 harbouring the vanA gene and one with vanB . On direct culture, the sensitivity of the three media was 66·7%, 77·8% and 44·4% and after broth enrichment 66·7%, 83·3% and 77·8% using AES VRE Agar, chromID VRE and Oxoid VRE Agar respectively.
Conclusions:  All three media are useful tools for the isolation of VRE from stool samples. AES VRE Agar and bioMérieux chromID VRE are easier to use than Oxoid VRE Agar due to diffusion of black coloration from the latter.
Significance and Impact of the Study:  This is the first study to evaluate the performance of AES VRE Agar and the first to compare two media containing synthetic chromogens for the isolation of VRE.     

High prevalence of vancomycin-resistant enterococci in Swedish sewage

In Europe the use of the growth promoter avoparcin is considered to have selected for vancomycin-resistant enterococci (VRE). Sweden ceased using avoparcin in 1986, and only occasional cases of VRE from hospitals have been reported since 1995. Within the framework of a European study, samples from urban raw sewage, treated sewage, surface water, and hospital sewage in Sweden (n = 118) were screened for VRE. Surprisingly, VRE were isolated from 21 of 35 untreated sewage samples (60%), from 5 of 14 hospital sewage samples (36%), from 6 of 32 treated sewage samples (19%), and from 1 of 37 surface water samples. Thirty-five isolates from 33 samples were further characterized by geno- and phenotyping, MIC determination, and PCR analysis. Most isolates (30 of 35) carried the vanA gene, and the majority (24 of 35) of the isolates were Enterococcus faecium. Most of the VRE were multiresistant. The typing revealed high diversity of the isolates. However, one major cluster with seven identical or similar isolates was found. These isolates came from three different sewage treatment plants and were collected at different occasions during 1 year. All VRE from hospital sewage originated from one of the two hospitals studied. That hospital also had vancomycin consumption that was 10-fold that of the other. We conclude that VRE were commonly found in sewage samples in Sweden. The origin might be both healthy individuals and individuals in hospitals. Possibly, antimicrobial drugs or chemicals released into the sewage system may sustain VRE in the system.     

In vitro activity of salicylamide derivatives against vancomycin-resistant enterococci

A series of 13 salicylamide derivatives was assessed for antibacterial activity against three isolates of vancomycin-resistant Enterococcus faecalis (VRE) and Enterococcus faecalis ATCC 29212 as a quality standard. The minimum inhibitory concentration was determined by the broth microdilution method with subsequent subcultivation of aliquots to assess minimum bactericidal concentration. The growth kinetics was established by the time-kill assay. Ampicillin, ciprofloxacin, tetracycline and vancomycin were used as the reference antibacterial drugs. Three of the investigated compounds showed strong bacteriostatic activity against VRE (0.199–25?µM) comparable to or more potent than ampicillin and ciprofloxacin. In addition, these compounds were tested for synergistic effect with vancomycin, ciprofloxacin and tetracycline, while 5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide showed the highest potency as well as synergistic activity with vancomycin against VRE 368. Screening of the cytotoxicity of the most effective compounds was performed using human monocytic leukemia THP-1 cells, and based on LD₅₀ values, it can be stated that the compounds have insignificant toxicity against human cells.     

Evaluation of clonality in enterococci isolated in Brazil carrying Tn1546-like elements associated with vanA plasmids

Fifty-one vancomycin-resistant enterococci samples isolated from different geographic regions in Brazil were studied. All the isolates harboured the vanA gene as demonstrated by PCR analysis, and in a majority of strains the gene was associated with a transferable plasmid of 70 kb. A single variant of the prototype Tn1546 associated with common transferable vanA-containing plasmids has spread among the enterococcal strains circulating in Brazil. The VanA element integrity in these enterococci strains and the different pulsed-field gel electrophoresis patterns suggest horizontal transmission of the vancomycin resistance transposon in Brazilian strains.     

Emergence and characterisation of vanB vancomycin-resistant Enterococcus faecalis in Rio de Janeiro, Brazil

Here we describe the detection and characterisation of three isolates of vancomycin-resistant VanB-type Enterococcus faecalis. Sequence analysis suggested that these isolates harboured the vanB1 gene. The isolates were susceptible to the majority of antimicrobial agents tested, with the exception of chloramphenicol, erythromycin and vancomycin, and showed distinct profiles of high-level resistance to aminoglycosides. Analysis of the clonal relatedness of the vanB E. faecalis isolates showed similar pulsed-field gel electrophoresis profiles. To our knowledge, this is the first report of the occurrence of enterococcal strains carrying vanB genes in Brazil.     

Environmental contamination with vancomycin-resistant enterococci from hospital sewage in Portugal

Vancomycin-resistant enterococci (VRE) were detected in samples of sewage obtained downstream of hospitals of the Porto area in Portugal, and in samples from the Douro Estuary. Clonal analysis, Tn1546 typing, and presence of putative virulence traits indicate the clinical origin of these isolates. This observation highlights the importance of hospital sewage in the VRE contamination of the environment.     

Risk factors for vancomycin-resistant enterococci colonisation in critically ill patients

Vancomycin-resistant enterococci (VRE) are important hospital pathogens and have become increasingly common in patients admitted to the intensive care unit (ICU). To determine the incidence and the risk factors associated with VRE colonisation among ICU patients, active surveillance cultures for VRE faecal carriages were carried out in patients admitted to the ICU of the University Hospital of Uberlandia, Minas Gerais, Brazil. Risk factors were assessed using a case-control study. Seventy-seven patients (23.1%) were found to be colonised with vanC VRE and only one patient (0.3%) was colonised with vanA VRE. Independent risk factors for VRE colonisation included nephropathy [odds ratio (OR) = 13.6, p < 0.001], prior antibiotic use (OR = 5.5, p < 0.03) and carbapenem use (OR = 17.3, p < 0.001). Our results showed a higher frequency (23.1%) of Enterococcus gallinarum and Enterococcus casseliflavus, species that are intrinsically resistant to low levels of vancomycin (vanC), without an associated infection, associated with prior antibiotic use, carbapenem use and nephropathy as comorbidity. This study is the first to demonstrate the risk factors associated with vanC VRE colonisation in ICU hospitalised patients. Although vanA and vanB enterococci are of great importance, the epidemiology of vanC VRE needs to be better understood. Even though the clinical relevance of vanC VRE is uncertain, these species are opportunistic pathogens and vanC VRE-colonised patients are a potential epidemiologic reservoir of resistance genes.     

Sources and pathways of spread of vancomycin-resistant enterococci in hemato-oncological patients

The presented study aims at analyzing an increasing prevalence of vancomycin-resistant enterococci (VRE) isolated from various kinds of clinical material obtained from patients in the Department of Hemato-oncology (DHO), University Hospital in Olomouc, Czech Republic. Between January 1 and March 31, 2005, enterococci were isolated by standard microbiological procedures using both clinical material obtained from hospitalized patients and samples from the department environment. Resistance to vancomycin and teicoplanin was determined by a standardized microdilution method. Phenotype determination of resistance to vancomycin was verified by PCR detection of vanA and vanB genes. In VanA Enterococcus faecium, macrorestriction analysis was performed by pulsed-field gel electrophoresis. During the monitored period, a total of 128 Enterococcus sp. strains were isolated, of which 38 (30 %) isolates from 22 different patients were determined as VRE. Dominating were Enterococcus faecium VanA (63 %) and Enterococcus casseliflavus VanC (16 %) strains. At the same time, one Enterococcus faecium VanA strain was acquired from a bed-side table used by a patient in whom a similar strain had been isolated repeatedly from various clinical materials including a rectal swab taken in 2004. Based on the macrorestriction analysis of genome DNA in 24 vancomycin-resistant Enterococcus faecium VanA strains isolated from the patients' clinical material, one strain from the bed-side table surface and one strain isolated from stools in 2004, 8 unique restriction profiles with similarity ranging from 90 % to 100 % were identified, which could be classified into 3 clonal types. Thus, we can assume not only the endogenous origin of the VRE in hemato-oncological patients and their potential selection caused by therapy with broad-spectrum antibiotics but also the ability of the strains to survive in a hospital setting and, subsequently, to be spread clonally by various vectors.     

Predominance of vanA genotype among vancomycin-resistant Enterococcus isolates from poultry and swine in Costa Rica

The use of avoparcin as a growth promoter is considered to have selected for vancomycin-resistant enterococci (VRE). In Costa Rica, the use of avoparcin for poultry and swine was intensive until the product was withdrawn from the market in 2000. We evaluated the presence of VRE in poultry, swine, and cattle fecal samples obtained during 1998 and 1999. A total of 185 VRE isolates were recovered from 116 out of 893 samples. Enterococcus faecium was the most frequently isolated species (50.8%), being predominant among poultry (71.6%) and swine (37.7%) isolates, but it was not recovered from the bovine samples. The second-most-frequently-isolated species from poultry and swine, respectively, were E. durans (23.2%) and E. faecalis (21.7%). E. casseliflavus was the only species obtained from bovine samples, but it was not found among the avian isolates. An evident predominance of the vanA determinant among vancomycin-resistant enterococcal species from poultry and swine, but not from cattle, was observed and was similar to the situation in European countries before avoparcin was forbidden. The diversity of the vanA determinant in the isolates was assessed by detection of the IS1251 insertion in the vanSH intergenic region and of the IS1476 insertion in the vanXY intergenic region. However, in none of the 154 vanA+ isolates recovered in this study were those insertions detected.     

The occurrence of vancomycin-resistant enterococci in hematological patients in relation to antibiotic use

Very important bacterial pathogens found in hematological patients at present are vancomycin-resistant enterococci (VRE). The main goal of this retrospective study was to assess their occurrence in relation to antibiotic use. We isolated 1918 Enterococcus strains, in toto, 138 (7.2%) of which proved to be VRE. The VRE most frequently identified were Enterococcus faecium VanA (77%) and Enterococcusfaecalis VanB (12%), mostly isolated from stools (57%). Comparing the development of the selection pressure of antibiotics and percentage of VRE in each period of observation, an effect of the administration of each antibiotic group on the occurrence of VRE can be presumed. A reduction in the administration of third generation cephalosporins, glycopeptides and fluoroquinolones and its replacement by penicillin antibiotics combined with inhibitors of bacterial beta-lactamases, contributed to the cessation of VRE incidence and succeeding reduced occurrence from 15.1% in the second half of 1998 to 6.1% in the first half of 2000.     

Detection of bacteriocin production and virulence traits in vancomycin-resistant enterococci of different sources

Aim: Three hundred and two enterococci were isolated from food, animal and clinical samples in order to evaluate the incidence of vancomycin‐resistant enterococci (VRE) and bacteriocin, cytolysin, haemolysin, gelatinase production. Methods and Results: Among the isolates, 27 (8·9%) were VRE, and 17 (63%) of these showed, by the deferred antagonism method, bacteriocin production against Gram‐positive and some Gram‐negative indicators. Eight bacteriocin producers displayed by polymerase chain reaction an enterocin structural gene: six Enterococcus faecium the Enterocin A, two Enterococcus faecalis the Enterocin P genes. The enterocins AS‐48, 31, L50 and 1071A/B genes were not found. Regarding the virulence factors, two VRE produced gelatinase and seven were haemolytic. Gelatinase gelE gene was found in 19 strains and cytolysin cylL_L gene in eight. Among the strains showing the cylL_L gene, only two E. faecalis expressed a β‐haemolysis. Conclusions: Our results showed the persistence of VRE in food, animal and clinical samples. Many of these strains displayed antibacterial activity and sometimes different components of virulence, which could emphasize their pathogenicity. Significance and Impact of the Study: This work indicates the need of a constant monitoring of enterococci in order to assess their possible pathogenic properties. The strains of interest in the food industry or used as probiotics should be tested for antibiotic resistance and virulence traits.     

Persistence of vancomycin-resistant enterococci in New Zealand broilers after discontinuation of avoparcin use

Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production. Avoparcin was also used from 1977 to 2000. A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials. These isolates were then examined to determine the prevalence of antimicrobial resistance. Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin. The bacitracin MIC was > or =256 microg/ml for 98.7% of isolates, and the avilamycin MIC was > or =8 microg/ml for 14.9% of isolates. No resistance to ampicillin or gentamicin was detected. Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans. However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E. faecium and the remainder were E. faecalis. Vancomycin-resistant E. faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E. faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand. These data demonstrate that vancomycin-resistant E. faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin.     

Prevalence of vancomycin-resistant enterococci in hospitalized patients and those living in the community in the Czech Republic

Between July 1, 2002 and December 31, 2003, rectal swabs from both hospitalized patients and community subjects in the Czech Republic were taken to ascertain the prevalence of vancomycin-resistant enterococci (VRE). The swabs were used for isolating and identifying enterococci and their susceptibility to antibiotics. Vancomycin resistance phenotypes were verified by PCR detection of vanA, vanB, vanC1 and vanC2 genes. A molecular biology analysis was performed in Enterococcus faecium VanA strains. During the observed period, 2691 rectal swabs from the hospitalized patients and 6529 rectal swabs from the subjects in community setting were examined. In total, 31 VRE of hospital origin and 13 community-population strains were isolated. The prevalence of VRE in the gastrointestinal tract was 1.9% in the hospitalized patients and 0.4% in the community subjects. The prevailing strains were Enterococcus faecium VanA (61.3%) in the VRE of hospital origin and Enterococcus gallinarum VanC (46.2%) in the community VRE. Mutual comparison between the hospital and community Enterococcus faecium VanA strains showed no similarity.