IN SILICO DEVELOPMENT OF HIGH-RESOLUTION MLVA TYPING SCHEME FOR ENTEROCOCCUS FAECIUM

Abstract

Emergence of enterococci as nosocomial pathogens is frequently associated with hospital outbreaks. Vancomycin resistance is especially perturbing as it limits the possible therapeutic options. Based on vast in silico analysis, we introduce a new multiple-locus variable-number tandem repeat analysis (MLVA) scheme for genotyping of Enterococcus faecium isolates, parameters of which are comparable to these of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The scheme was tested in silico on all available at this time E. faecium genomes in NCBI Genbank. Searching for suitable variable number of tandem repeats (VNTR) loci was conducted with a set of free access applications. Ten VNTR loci were selected according to their polymorphic structure and stability using the Primer-BLAST utility of NCBI. Primers were designed to be compatible in a multiplex reaction and the method was adapted for high resolution separation techniques. As a result, a total of 60 MLVA profiles and 35 MLST profiles were generated from the analysis of 114 sequenced genomes. Minimum spanning trees were created for both MLVA and MLST in order to analyze the genetic relatedness between isolates. Hunter Gaston discriminatory index was measured for both MLVA (0,959) and MLST (0,926). Typeability was also measured for both methods (MLVA – 85.9%; MLST – 89.4%). These results suggest that the new MLVA scheme is suitable for epidemiological studies of hospital-adapted E. faecium isolates.