AUTHOR=Ong Chian Teng , Blackall Patrick. J. , Boe-Hansen Gry B. , deWet Sharon , Hayes Ben J. , Indjein Lea , Korolik Victoria , Minchin Catherine , Nguyen Loan To , Nordin Yusralimuna , Siddle Hannah , Turni Conny , Venus Bronwyn , Westman Mark E. , Zhang Zhetao , Tabor Ala E. 

TITLE=Whole-genome comparison using complete genomes from Campylobacter fetus strains revealed single nucleotide polymorphisms on non-genomic islands for subspecies differentiation

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1452564

DOI=10.3389/fmicb.2024.1452564

ISSN=1664-302X

ABSTRACT=<sec><title>Introduction</title><p>Bovine Genital Campylobacteriosis (BGC), caused by <italic>Campylobacter fetus</italic> subsp. venerealis, is a sexually transmitted bacterium that significantly impacts cattle reproductive performance. However, current detection methods lack consistency and reliability due to the close genetic similarity between <italic>C. fetus</italic> subsp. venerealis and <italic>C. fetus</italic> subsp. fetus. Therefore, this study aimed to utilize complete genome analysis to distinguish genetic features between <italic>C. fetus</italic> subsp. venerealis and other subspecies, thereby enhancing BGC detection for routine screening and epidemiological studies.</p></sec><sec><title>Methods and results</title><p>This study reported the complete genomes of four <italic>C. fetus</italic> subsp. fetus and five <italic>C. fetus</italic> subsp. venerealis, sequenced using long-read sequencing technologies. Comparative whole-genome analyses (<italic>n</italic> = 25) were conducted, incorporating an additional 16 complete <italic>C. fetus</italic> genomes from the NCBI database, to investigate the genomic differences between these two closely related <italic>C. fetus</italic> subspecies. Pan-genomic analyses revealed a core genome consisting of 1,561 genes and an accessory pangenome of 1,064 genes between the two <italic>C. fetus</italic> subspecies. However, no unique predicted genes were identified in either subspecies. Nonetheless, whole-genome single nucleotide polymorphisms (SNPs) analysis identified 289 SNPs unique to one or the <italic>C. fetus</italic> subspecies. After the removal of SNPs located on putative genomic islands, recombination sites, and those causing synonymous amino acid changes, the remaining 184 SNPs were functionally annotated. Candidate SNPs that were annotated with the KEGG “Peptidoglycan Biosynthesis” pathway were recruited for further analysis due to their potential association with the glycine intolerance characteristic of <italic>C. fetus</italic> subsp. venerealis and its biovar variant. Verification with 58 annotated <italic>C. fetus</italic> genomes, both complete and incomplete, from RefSeq, successfully classified these seven SNPs into two groups, aligning with their phenotypic identification as CFF (<italic>Campylobacter fetus</italic> subsp. fetus) or CFV/CFVi (<italic>Campylobacter fetus</italic> subsp. venerealis and its biovar variant). Furthermore, we demonstrated the application of mraY SNPs for detecting <italic>C. fetus</italic> subspecies using a quantitative PCR assay.</p></sec><sec><title>Discussion</title><p>Our results highlighted the high genetic stability of <italic>C. fetus</italic> subspecies. Nevertheless, <italic>Campylobacter fetus</italic> subsp. venerealis and its biovar variants encoded common SNPs in genes related to glycine intolerance, which differentiates them from <italic>C. fetus</italic> subsp. fetus. This discovery highlights the potential of employing a multiple-SNP assay for the precise differentiation of <italic>C. fetus</italic> subspecies.</p></sec>