Improvement and Validation of a Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA8+) for , , and .

The genotyping of the multidrug-resistant species complex is essential to identify outbreaks and to track their source and spread. The aim of this study was to improve and extend the typeability, availability, cost and time efficiency of an existing multi-locus VNTR analysis (MLVA). A modified scheme (MLVA8+) was adopted and validated for strain-level differentiation of the three species involved in human pathology. A diverse set of 465 clinical isolates from 22 hospitals and 3 outpatient laboratories in Bulgaria were studied, where 315 were carbapenem-resistant. The MLVA8+ typeability was significantly improved and the typing data were validated against 158 isolates which were previously typed by WGS. The MLVA8+ results were highly concordant with the classic 7-locus MLST and the novel MLST, but had greater congruency coefficients (adjusted Wallace). A major advantage was the differentiation of the hybrid cluster ST258 into its corresponding clades. Furthermore, the applicability of MLVA8+ was demonstrated by conducting a retrospective investigation of the intra-hospital spread of KPC-, NDM- and OXA-48-like producers. The MLVA8+ has improved utility and extended typing scope to and , while its cost and time-to-result were reduced.