The prevalence and molecular epidemiology of vancomycin-resistant Enterococcus (VRE) carriage in patients admitted to intensive care units in Beijing, China.

BACKGROUND: Vancomycin-resistant Enterococcus (VRE) can be carried in the gut for a long period and its carriage status is associated with subsequent infections. This study aimed to investigate the frequency of intestinal VRE carriage in intensive care patients in Beijing. METHODS: A multicenter, retrospective cross-sectional study was conducted at six hospitals in Beijing, China. All patients admitted to intensive care units (ICUs) between April 2 and May 1, 2017, were enrolled, and their clinical data were gathered by reviewing electronic medical records. Rectal swabs collected from patients were stored at -80 °C in the Institute of Clinical Pharmacology, Peking University First Hospital, and they were selectively cultured for VRE, then the identified strains were analyzed by polymerase chain reaction (PCR) to detect the glycopeptide resistance gene and were characterized by multilocus sequence typing (MLST). RESULTS: Of 148 patients recruited, 46 (31.1%) carried VRE, with the majority (n = 42) being Enterococcus faecium. In total, 78.3% of the VRE were vanA positive and 15.2% vanM positive, while 6.5% undetected glycopeptide resistance gene. The predominant ST was ST78 (47.6%) followed by ST192 (14.3%), ST555 (9.5%), and ST789 (9.5%). Multivariate analysis showed that factors associated VRE carriage were patients aged >65 years (odds ratio [OR], 3.786; 95% confidence interval [CI], 1.402-10.222) and recent third-generation cephalosporins use (OR, 6.360; 95% CI, 1.873-21.601). CONCLUSIONS: The overall proportion of VRE carriage in patients admitted to ICUs was markedly high in Beijing, China. The vanM gene has been spread widely but vanA gene was the dominant resistance determinant in VRE in Beijing.

Evaluation of Quadruple Real-Time PCR Method to Detect Enterococci Carrying Vancomycin-Resistant Genes vanA, vanB, vanM in Rectal Swabs.

Purpose: To evaluate the sensitivity and accuracy of the quadruple real-time PCR method for the detection of enterococci carrying vancomycin-resistant genes vanA, vanB, and vanM in rectal swabs. Methods: Choosing PCR-sequenced DNA extracted directly from rectal swabs as the gold standard, the results of the quadruple real-time PCR method and the traditional method (screening culture combined PCR-sequencing method whose DNA extracted from single colony) were compared with the gold standard. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the quadruple real-time PCR method and the traditional method were obtained. The time required for the three methods was calculated. Results: The results between gold standard and the quadruple real-time PCR method were similar. Compared to the traditional method, the quadruple real-time PCR method had a much higher sensitivity, specificity, PPV, NPV, and consistency. Our study found that the quadruple real-time PCR method is beneficial for detection of enterococci carrying vanM with vancomycin heteroresistance. The traditional method had high specificity and NPV, but its sensitivity and PPV were not ideal. The time needed for gold standard is a minimum of 28 h; the quadruple real-time PCR method takes 2-3 h while the traditional method consumes a minimum of 72 h. Conclusion: The quadruple real-time PCR method can provide a rapid and reliable result for the diagnosis of patients with colonized vancomycin-resistant enterococci. This new method is beneficial for the active screening, timely clinical treatment measures, epidemiological research, and hospital monitoring of the enterococci carrying vancomycin-resistant gene, especially for the enterococci with vancomycin heteroresistance carrying vanM.

Real-time PCR for the rapid detection of vanA, vanB and vanM genes.

BACKGROUND/PURPOSE: To evaluate the ability of quadruple Taqman probe real-time PCR to the detection of vanA, vanB and vanM in enterococcal isolates. METHODS: A total of 343 strains, including 253 vancomycin-resistant enterococcus (VRE) strains and 90 non-VRE strains, were tested by both quadruple Taqman probe real-time PCR and gel-based PCR assay. RESULTS: When differentiating among three genotypes of vanA, vanB and vanM in VRE strains, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic accuracy and consistency of the quadruple Taqman probe real-time PCR were all 100%. Minimum. Inhibitory concentration (MIC) results showed that there was a wide MIC range of vancomycin and teicoplanin for the strains that harboring vanA/vanM gene respectively or harboring vanA and vanM genes simultaneously. However, the VRE strains with vanB genotype all were sensitive to teicoplanin. CONCLUSION: Considering the excellent PPV and low NPV, real-time PCR would be useful to monitor VRE-colonized or infected patients. However, further evaluation of the assay's performance in the clinical specimens is required, especially when considering that high level of PCR inhibitors present in these samples.

Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate.

Vancomycin-resistant enterococci are troublesome pathogens in clinical settings because of few treatment options. A VanA/VanM-type vancomycin-resistant Enterococcus faecium clinical isolate was identified in Japan. This strain, named AA708, harbored five plasmids, one of which migrated during agarose gel electrophoresis without S1 nuclease treatment, which is indicative of a linear topology. We named this plasmid pELF1. Whole genome sequencing (WGS) analysis of the AA708 strain revealed that the complete sequence of pELF1 was 143,316 bp long and harbored both the vanA and vanM gene clusters. Furthermore, mfold analysis and WGS data show that the left end of pELF1 presumably forms a hairpin structure, unlike its right end. The pELF1 plasmid was not digested by lambda exonuclease, indicating that terminal proteins were bound to the 5' end of the plasmid, similar to the Streptomyces linear plasmids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results were also consistent with the exonuclease assay results. In retardation assays, DNAs containing the right end of proteinase K-untreated pELF1 did not appear to move as well as the proteinase K-treated pELF1, suggesting that terminal proteins might be attached to the right end of pELF1. Palindromic sequences formed hairpin structures at the right terminal sequence of pELF1; however, sequence similarity with the well-known linear plasmids of Streptomyces spp. was not high. pELF1 was unique as it possessed two different terminal structures. Conjugation experiments revealed that pELF1 could be transferred to E. faecalis, E. faecium, E. casseliflavus, and E. hirae. These transconjugants exhibited not only high resistance levels to vancomycin, but also resistance to streptomycin, kanamycin, and erythromycin. These results indicate that pELF1 has the ability to confer multidrug resistance to Enterococcus spp. simultaneously, which might lead to clinical hazards.

New colony multiplex PCR assays for the detection and discrimination of vancomycin-resistant enterococcal species.

New colony multiplex PCR assays for detection of seven types of vancomycin-resistance determinants and eight types of Enterococcus species were developed. For 135 enterococcal isolates examined in this study, these assays showed high sensitivity and specificity, and could provide the rapid and accurate detection of vancomycin-resistant determinants and Enterococcus spp.