Multilocus sequence-typing scheme for Borrelia miyamotoi — the erythema-free ixodid tick-borne borreliosis pathogens

Introduction. Borrelia miyamotoi is a pathogen of erythema-free ixodid tick-borne borreliosis (ITBB), a disease widespread in Russia. To date, there are no generally accepted methods for B. miyamotoi genotyping. The multilocus sequencing typing (MLST) scheme of Borrelia was originally developed for B. burgdorferi, and does not have the required discrimination power for monitoring the ITBB pathogens. The objective of this study is to develop the MLST scheme for B. miyamotoi. Materials and Methods. The whole genome sequences of 10 reference strains (GenBank) were analyzed for the selection of the house-keeping loci. The MLST scheme development was based on principles published by the authors of the method. For this experiment, 81 B. miyamotoi strains and positive clinical samples were used to test the MLST scheme. Results. After analyzing the genomic data, 8 house-keeping loci were chosen for MLST, for which the PCR and sequencing primers were designed. Each MLST loci was represented by several alleles (from 4 to 7) which form 15 sequence types. The genetic diversity of pathogens isolated from ITBB patients and ticks were characterized. Discussion. Based on pairwise distances between allelic profiles, the sequence types can be classified into four groups. The first two groups are clonal complexes; the other two groups are formed by once identified sequence types. The first clonal complex unites 11 sequence types (80 or 88% of the characterized B. miyamotoi), the second consists of 2 sequence types (9 or 9.8%). The genetic differences between B. miyamotoi are associated with the sources of strains and biological isolates. The MLST based classification confirms the previously described genetic heterogeneity of B. miyamotoi populations associated with ecologically unrelated vectors of ITBB pathogens. Conclusion. The proposed MLST scheme is an appropriate tool for ITBB pathogen classification and evolutionary change characterization within clonal complexes.