High-level Vancomycin-resistant Enterococci Research Articles

Rapid detection of high-level vancomycin-resistant genes, vanA, vanB, vanD and vanM, in enterococci

Objective To develop a multiple polymerase chain reaction (PCR) technique based assay for rapid detection of vanA, vanB, vanD and vanM in high-level vancomycin-resistant enterococci. Methods After analyzing the uncleotide sequence divergence among D-Ala∶D-Lac ligase genes, an multiplex PCR assay for vanA, vanB, vanD and vanM genes in high-level vancomycin-resistant enterococci were designed. By using recombination plasmids containing vanA, vanB, vanD and vanM genes as positive control, and non-vancomycin resistant enterococci (non-VRE) common pathogenic bacterial DNA as negative control, the sensitivity and specificity of the assay were evaluated. Fifty vancomycin-resistant enterococci (VRE) isolates were detected by the assay. Fifty clinical strains of VRE were isolated from 9 hospitals in Shanghai from January 2006 to December 2014. The results were compared with the conventional PCR and sequencing methods. Results The identity of the D-Ala∶D-Lac ligase genes were 60.8%—71.3% of vanA, vanB, vanD and vanM genes. The multiplex PCR assay could identify the genotypes of the positive control samples accurately. No false positive results were found in negative control samples. Among fifty VRE strains detected by the assay, 18 were vanA genotype and 32 were vanM genotype. Comparison of the multiplex PCR assay and sequencing methods revealed sensitivity and specificity of 100%. The detection limit of the assay was 2×10 copies/PCR reaction. The experiment could be done within 3.5 h. Conclusions A multiplex PCR assay is developed to rapid identify the genotype of the high-level vancomycin-resistant enterococci, which can be used for the molecular epidemiology research and detection of VRE. Key words: Enterococcus; Vancomycin resistance; Genes; Genotype

High-level vancomycin resistant Enterococcus faecium related to humans and pigs found in dust from pig breeding facilities

Environmental dust from animal breeding facilities was never screened for the presence of enterococci, nor of vancomycin-resistant enterococci (VRE), despite the possibility of being a vehicle of transmission of strains and antibiotic resistance genes between food-producing animals and man. Bio-security measures in pig facilities include disinfection with biocides to avoid the dissemination of opportunistic pathogenic bacteria, namely enterococci and in particular VRE. We thus undertook collection of enterococci and VRE in a representative number of breeding pig facilities in Portugal (n=171) and analyzed their susceptibility to benzalkonium chloride (BC) and chlorhexidine (CHX). A prevalence of 15% of VRE was found, with 6% high-level resistance found, and MIC values for CHX and BC were similar to those commonly found among enterococcal isolates from related environments, 8μg/ml and 4μg/ml, respectively. Among the isolated high-level vancomycin resistant Enterococcus faecium carrying the vanA genotype, we found multilocus sequence types closely related to pig and human isolates from European countries and Brazil. These results strongly advise constant surveillance of this environment and its inclusion in future epidemiologic studies on VRE.

Bacteraemia associated with a vancomycin-resistant Enterococcus gallinarum strain harbouring both the vanA and vanC1 genes

A case of a post-surgical patient who developed a fatal bloodstream infection caused by high-level vancomycin-resistant Enterococcus gallinarum is reported. The isolate was found to carry both the vanC1 and vanA genes. This is the first report of an invasive infection associated with a vanA E. gallinarum isolate in Brazil.

Molecular characterization of low-level vancomycin-resistant enterococci found in coastal water of Thermaikos Gulf, Northern Greece

Enterococcus includes species that may pose emerging health risks and has been used as biomarkers for environmental contamination while little is known concerning their occurrence in marine water. Classification of enterococci in environmental samples can be problematic and requires polyphasic taxonomy. In this study, we investigated the presence of vancomycin-resistant enterococci (VRE) in the inner bay of Thermaikos Gulf in Northern Greece. Based on physiological and biochemical criteria, 121 presumptive enterococcal strains were identified. High-level VRE were undetectable in seawater and only 35 vancomycin gene-negative strains possessed low-level vancomycin resistance. Genotyping by pulsed field gel electrophoresis (PFGE) proved to be more reliable for marine enterococcal discrimination and revealed distinguished characteristics of the seawater enterococci, indicating high genetic diversity. Random amplified polymorphic DNA-PCR (RAPD-PCR) was unable to separate distinct species analyzed in this study. This study indicates the need of polyphasic taxonomy for seawater enterococcal species’ identification and provides information for future biomonitoring programs of Thermaikos Gulf.

Comparison of vancomycin-resistant enterococci isolates from human, poultry and pigs in Korea

Vancomycin-resistant enterococci (VRE) have emerged as an important nosocomial pathogen. Since 1989, a rapid increase in the incidence of enterococcal bacteremia and endocarditis by VRE has been reported. The use of avoparcin in animal husbandry is reportedly associated with the appearance of VRE. In this study, a multiplex polymerase chain reaction (PCR) method was established to detect and differentiate resistant types of enterococci, which specifically amplify the four van genes that encode vancomycin resistance elements. Using this method, we investigated the incidence rates and types of VRE from two types of farms: those that had used avoparcin and those that had not used avoparcin. A total of 1091 animal fecal samples were collected from 70 pig farms and 32 poultry farms. A total of 425 enterococci were isolated from the fecal samples. Among the 425 isolates, six showed a pattern of high-level vancomycin resistance (Minimal Inhibitory Concentration, MIC: 64–256 μg/ml). Out of six high-level VRE, three were isolated from poultry farms that had used avoparcin and three were not. The six high-level VRE harbored the vanA gene. Sixty-seven of 425 isolates that showed a pattern of low-level vancomycin resistance (MIC: 4–8 μg/ml) were associated with the presence of vanC-1 or vanC-2/3 gene. We also performed a repetitive extragenic palindromic PCR (rep-PCR) method to compare the genetic relatedness between the high-level VRE of six animal isolates and 31 human isolates. None of the animal isolates had a similar rep-PCR pattern as the human isolates but similarities between human VRE isolates were observed.

Determination of antibiotic resistance and resistance plasmids of clinical Enterococcus species

To determine the antibiotic resistance pattern and resistance plasmids, we studied 23 antibiotic-resistant clinical isolates of Enterococcus spp. which caused infection in Bayindir-Ankara Hospital, Turkey. Biochemical and physiological identification tests were applied by the Vitek system and compared with the results of protein profiles by SDS-PAGE. From 23 isolates, 20 were identified as E. faecalis, 2 as E. faecium and 1 as E. gallinarum. Twenty four antibiotics belong to 10 different groups were used in susceptibility tests. Multiple antibiotic resistance was determined in 10 of 23 Enterococcus spp. Overall resistance to the used antibiotics was 47.3% and low level resistance was 16.6%. Among the isolates tested, 8.7% demonstrated high level gentamicin resistance, 17.4% demonstrated high level streptomycin resistance, and 43.5% demonstrated penicillin resistance. High level vancomycin resistant Enterococcus spp. rate was 34.8%, and 60.9% exhibited low level resistance to vancomycin and teicoplanin. They contain plasmids which varied in numbers between 1 and 11 and the plasmid sizes ranged from 2.08 to 56.15 kb. In curing experiments with acriflavine, two different plasmids were shown in different molecular sizes of 33.49 and 13.6 kb while the first determined glycopeptide and penicillin resistance, the second one determined either glycopeptide or penicillin resistance in two different E. faecalis strains. On the other hand, a 22.58 kb plasmid, determining kanamycin resistance, was detected in an E. faecium strain. After the curing experiments, an elimination of 37.17 and 44.47 kDa protein bands was shown in E. faecium EFA1 and E. faecalis EFA13 in SDS-PAGE, respectively. This survey indicates the increase of antibiotic-resistant enterococci, especially to vancomycin in our hospital isolates.

Vancomycin-resistantEnterococcus faecalisin Serbia

To the Editor: First isolated in France (1), vancomycin-resistant enterococci (VRE) have become pathogens of major importance, particularly in the United States (2). Infections due to VRE are still uncommon in most European countries (3). We report the first isolation of high-level vancomycin-resistant Enterococcus faecalis in Serbia. A 55-year-old woman was admitted to the Clinic for Cardiovascular Diseases, Belgrade, on April 1, 2002, for aortobifemoral bypass surgery. Three weeks after she was admitted to the hospital, an infection developed in the surgical wound and treatment with sulfamethoxazole-trimethoprim (160/800 mg q 12 h) was empirically introduced. Bacteriologic analysis of the wound swab sample showed a methicillin-resistant strain of Staphylococcus aureus, a multiresistant strain of Acinetobacter sp., a commonly susceptible strain of Enterococcus sp., and a VRE strain. According to the results of susceptibility testing, imipenem (1 g q 6 h) was added to the patient’s treatment protocol. VRE were not isolated from subsequent wound samples or any other sample submitted for microbiologic analysis. The patient was discharged at the end of the 14-day treatment period. The isolate was identified as E. faecalis by biochemical characterization, as recommended by Facklam and Collins (4) and confirmed by API 20 Strep (bioMerieux, Marcy-l’Etoile, France). Susceptibility testing, performed by the disk diffusion method, showed that the isolate was resistant to vancomycin, teicoplanin, gentamicin, streptomycin, tetracycline, and ciprofloxacin, while susceptible to ampicillin, amoxicillin, amoxicillin and clavulanic acid, and imipenem. Resistance to vancomycin, teicoplanin, gentamicin, and streptomycin was confirmed by the broth dilution method, according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations (5). The obtained MICs were 256 μg/mL for vancomycin, 64 μg/mL for teicoplanin, >4,000 μg/mL for gentamicin, and >2,000 μg/mL for streptomycin. This phenotype, with high-level resistance to vancomycin and teicoplanin, is typical for the vanA genotype (2). The strain was subsequently genotyped by pulsed-field gel electrophoresis, using previously described methods (6). The presence of the vanA gene was confirmed by polymerase chain reaction assay, according to a previously described procedure (7). E. faecium EF228 was used as the positive control. The enterococci are among the most frequent causes of nosocomial infections, particularly in intensive care units, and present a major therapeutic challenge (2). While the emergence of VRE strains in the United States is probably associated with extensive use of vancomycin, the occurrence of VRE in Europe is possibly due to application of avoparcin (glycopeptide analog) as a growth promoter in animal husbandry (3). However, avoparcin has not been used in Serbia, and vancomycin application has been restricted to hospitalized patients and quite limited due to its high cost. Thus, emergence of VRE strains in Serbia has not been likely. The origin of this VRE isolate is unknown: the strain may have been imported or may have originated from the hospital environment. The first prospective pan-European VRE surveillance study (January–April 1997) showed VanA-VRE strains in only eight European countries, with isolates numbering from one to four per country (3). No epidemiologic relations were established among the VanA isolates, and only 2 out of 18 isolates (11%) were identified as E. faecalis (3). Since our patient-case had no history of travel outside Serbia, we assumed that the VRE isolate originated from the hospital environment. However, a study investigating the occurrence of VRE strains in Belgrade, the capital of Serbia, detected no such isolates in five different hospitals (8). Although the study did not analyze samples from the Clinic for Cardiovascular Diseases, it did include samples from the Clinic for General Surgery, which is located within the same building. The susceptibility of 191 isolates of enterococci to vancomycin was tested by agar dilution method according to NCCLS recommendations. Of the 191 isolates, 159 were classified as susceptible and 32 as intermediately susceptible. This report of the first isolation of VRE in Serbia, as well as the previously shown presence of enterococci displaying intermediary susceptibility to vancomycin, provides the rationale for future active screening for VRE in hospital environments in the region.

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.

Effect of parenteral antibiotic administration on the establishment of colonization with vancomycin-resistant Enterococcus faecium in the mouse gastrointestinal tract.

A mouse model of intestinal colonization with vancomycin-resistant enterococci (VRE) was used to study the effect of different beta-lactam antibiotics on establishment of VRE colonization. A clinical VanB VRE isolate, Enterococcus faecium C68 (102 or 104 cfu), was inoculated by gastric gavage in conjunction with subcutaneous administration of antibiotics. The MIC of ceftriaxone and ticarcillin against VRE strain C68 is >10,000 microg/mL, and the MIC of piperacillin is 1250 microg/mL. Ceftriaxone and ticarcillin-clavulanate treatment groups developed persistently high levels of stool VRE compared with both the saline and the piperacillin-tazobactam (Pip-Taz) groups (P<.008). The level of stool VRE in the Pip-Taz group did not differ from that for the saline group. Thus, in this mouse model, beta-lactam antibiotics with minimal anti-enterococcal activity promoted establishment of high-level VRE colonization, but Pip-Taz (a beta-lactam antibiotic with more potent activity against VRE) did not.

VanA-mediated vancomycin-resistant Enterococcus spp. in sewage.

Three high-level vancomycin-resistant Enterococcus strains (two Enterococcus faecium and one Enterococcus durans) were recovered from three of eight sewage samples taken from the general sewage collector at Logroño (Northern Spain). The strains were present in the sewage samples at estimated concentrations of ten resistant bacteria/mL, corresponding to about 0.4% of the enterococcal population. The VanA protein was detected in each strain by immunoblotting of membrane extracts of the vancomycin-induced cells, and the vanA gene was demonstrated in the wild strains and their transconjugants by DNA-DNA hybridization. This is the first, confirmed report of vanA mediated vancomycin resistance in E. durans.

Vancomycin binding to cell walls of non-streptococcal vancomycin-resistant bacteria.

The availability of peptidoglycan for binding to vancomycin was investigated in intrinsically vancomycin-resistant clinical isolates of Leuconostoc mesenteroides, Lactobacillus viridescens and Pediococcus pentosaceus. Intrinsic vancomycin resistance expressed by L. mesenteroides and P. pentosaceus was the result of poor binding of vancomycin to native or SDS-treated cell walls. Pre-exposure of L. viridescens to vancomycin decreased the subsequent binding of vancomycin to the cell walls, suggesting an alternative mechanism to that found in high-level vancomycin-resistant enterococci and intrinsically-resistant L. mesenteroides and P. pentosaceus. Vancomycin binding was sensitive to substitution of larger side chains at the C-terminus. Variations of peptidoglycan peptide types found in L. mesenteroides, L. viridescens and P. pentosaceus could account for their intrinsic vancomycin resistance.

High-level vancomycin-resistant enterococci causing hospital infections

Nosocomial infection or colonization due to enterococci with high-level resistance to vancomycin (minimal inhibitory concentrations [MICs] between 64 and greater than 2000 mg/L) has occurred in 41 patients with renal disease. These vancomycin-resistant enterococci were cultured from many sources including blood. All but one strain contained one or more plasmids ranging in molecular weight from 1.0 to 40 Megadaltons (MDa). Vancomycin resistance was transferable by conjugation to a susceptible recipient strain of Enterococcus faecalis but this was not always associated with plasmid DNA. The emergence of transferable high-level vancomycin resistance in enterococci causing significant clinical infections is of particular importance since vancomycin is widely regarded as a reserve drug for the management of infections with multi-resistant Gram-positive organisms.