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Current approach to identify sources of human pathogens is largely dependent on the cultivation and isolation of target bacteria. For rapid pathogen source identification, culture-independent strain typing method is necessary. In this study, we designed new primer set that broadly covers  flaA  short variable region (SVR) of various  Campylobacter  species, and applied the  flaA  SVR sequencing method to examine the diversity of  Campylobacter  spp. in geese fecal samples ( n  = 16) with and without bacteria cultivation. Twenty-three  Campylobacter  strains isolated from the 16 geese fecal samples were grouped similarly by conventional  flaA  restriction fragment length polymorphism (RFLP) method and by the  flaA  SVR sequencing method, but higher discriminant power was observed in the  flaA  SVR sequencing approach. For culture-independent  flaA  SVR sequencing analysis, we developed and optimized the sequence data analysis pipeline to identify as many genotypes as possible, while minimizing the detection of genotypes generated by sequencing errors. By using this pipeline, 51,629 high-quality  flaA  sequence reads were clustered into 16 operational taxonomic units (=genotypes) by using 98% sequence similarity and >50 sequence duplicates. Almost all  flaA  genotypes obtained by culture-dependent method were also identified by culture-independent  flaA  SVR MiSeq sequencing method. In addition, more  flaA  genotypes were identified probably due to high throughput nature of the MiSeq sequencing. These results suggest that the  flaA  SVR sequencing could be used to analyze the diversity of  Campylobacter  spp. without bacteria isolation. This method is promising to rapidly identify potential sources of  Campylobacter  pathogens.

High-Throughput flaA Short Variable Region Sequencing to Assess Campylobacter Diversity in Fecal Samples From Birds