High-Resolution Melting Genotyping of Enterococcus faecium Based on Multilocus Sequence Typing Derived Single Nucleotide Polymorphisms

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 “melting types” (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure

Environmental waters as a source of antibiotic-resistant Enterococcus species in Belgrade, Serbia

Despite the number of studies on antibiotic-resistant enterococci from Serbian clinical settings, there are no data about environmental contamination with these bacteria. Thus, this study investigated the prevalence of antibiotic-resistant enterococci in Belgrade, Serbia. Enterococcus species collected from ten surface water sites, including a lake, two major river systems, and springs, were tested. Among enterococci, we found single (21.7 %), double (17.4 %), and multiple antibiotic resistance patterns (56.3 %). Vancomycin-resistant strains were not found, indicating that their abundance in Belgrade is tightly linked to clinical settings. The multiple drug-resistant strains Enterococcus faecalis, Enterococcus faecium, and Enterococcus mundtii were frequently detected in the lake during the swimming season and in the rivers near industrial zones. We confirmed the presence of ermB, ermC, ant(6)-Ia, tetM, and tetL and mutations in gyrA genes. The phylogenetic analysis of 16S rRNA gene of E. faecium isolates that harbor esp gene classified them into two groups based on high-bootstraps scores in the tree analysis. Pulsed-field gel electrophoresis analysis of antibiotic-resistant enterococci revealed genomic similarity ranging from 75 to 100 %. This study indicates the importance of anthropogenic impact to the spread of antibiotic-resistant enterococci in environmental waters of Belgrade, Serbia