Objective To establish the standard strains of Borrelia burgdorferi of five genotypes (B. bavariensis, B. garinii, B. afzelii, B. burgdorferi sensu stricto and B. yangtzensis) in China and provide evidence for the pathogen identification, diagnostic reagents and vaccine development for Lyme disease prevention and control.

Methods According to Technical Requirements for Evaluation of National Standard Strains of Pathogenic Microorganisms (WS/T 812－2022), B. burgdorferi strains of five genotypes isolated in China (PD91, MD86, FP1, CS4 and QX-S13) were selected. First, the five strains were cultured in BSK II medium and the morphological characteristics of the strains were observed under darkfield microscope. The strain's whole genome DNA was extracted, the 5S–23S rRNA intergenic spacer region was amplified by polymerase chain reaction and sequenced, and the product sequences of different generations of each strain were compared. Multilocus sequence analysis (MLSA) was used to genotype the isolates, meantime the MLSA clustering analysis results of different generations of the strains were compared to verify the genetic stability of the strains. Whole genome sequencing was conducted to analyze genomic features of the 5 strains through the Illumina NovaSeq platform.

Results All the 5 strains had typical morphological characteristics of B. burgdorferi. Through Specific genetic testing and MLSA, strain PD91, MD86, FP1, CS4 and QX-S13 were identified as B. bavariensis, B. garinii, B. afzelii, B. burgdorferi sensu stricto and B. yangtzensis, respectively, and phylogenetic analysis showed that the strains were the same branches genotypes of international reference strains. The stability experiments results showed that the sequence similarity of the 5S–23S rRNA intergenic spacer region of different generations of each strain reached 100.00%, the results of MLSA clustering of the 5 strains in the 10th and 20th cultures were consistent, indicating good genetic stability. The whole genome sequencing results showed that the genome size was 1.26–1.80 Mbp and the G+C content was 27.63%–28.20%. The functional annotation and distribution of metabolic modules of chromoso genome for the five strains were almost same: mainly for cell growth maintaining, reproduction, cell motility, defense and others for cell survival.

Conclusion The five strains have typical biological and morphological characteristics of B. burgdorferi, and their genotypes and molecular characteristics are stable, indicating that they can be used as standard strains for B. burgdorferi of five genotypes in China. This study provided evidence for the establishment of a standardized strain system of B. burgdorferi in China, and relevant resources for the strain identification, diagnostic reagents and vaccine development in Lyme disease prevention and control.