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

Abstract

Introduction: Bovine Genital Campylobacteriosis (BGC), caused by Campylobacter fetus 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 C. fetus subsp. venerealis and C. fetus subsp. fetus. Therefore, this study aimed to utilize complete genome analysis to distinguish genetic features between C. fetus subsp. venerealis and other subspecies, thereby enhancing BGC detection for routine screening and epidemiological studies.

Methods and results: This study reported the complete genomes of four C. fetus subsp. fetus and five C. fetus subsp. venerealis, sequenced using long-read sequencing technologies. Comparative whole-genome analyses (n = 25) were conducted, incorporating an additional 16 complete C. fetus genomes from the NCBI database, to investigate the genomic differences between these two closely related C. fetus subspecies. Pan-genomic analyses revealed a core genome consisting of 1,561 genes and an accessory pangenome of 1,064 genes between the two C. fetus 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 C. fetus 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 C. fetus subsp. venerealis and its biovar variant. Verification with 58 annotated C. fetus genomes, both complete and incomplete, from RefSeq, successfully classified these seven SNPs into two groups, aligning with their phenotypic identification as CFF (Campylobacter fetus subsp. fetus) or CFV/CFVi (Campylobacter fetus subsp. venerealis and its biovar variant). Furthermore, we demonstrated the application of mraY SNPs for detecting C. fetus subspecies using a quantitative PCR assay.

Discussion: Our results highlighted the high genetic stability of C. fetus subspecies. Nevertheless, Campylobacter fetus subsp. venerealis and its biovar variants encoded common SNPs in genes related to glycine intolerance, which differentiates them from C. fetus subsp. fetus. This discovery highlights the potential of employing a multiple-SNP assay for the precise differentiation of C. fetus subspecies.

Keywords: Campylobacter fetus; SNPs; complete genomes; genome comparison; glycine; subspecies; veterinary science.

Figure 1

The pangenome of the 25 Campylobacter fetus strains. Dark purple indicates the presence of the gene ortholog, while light purple represents the absence of the gene ortholog. International Organization for Standardization (ISO) country code: Canada (CA), United States (US), France (FR), Uruguay (UY), Argentina (AR), United Kingdom (UK), Australia (AU), and New Zealand (NZ).

Figure 2

The Campylobacter-specific virulence factors among the 25 Campylobacter fetus strains. The color key indicates the percentage of identity between the genomic regions identified in each isolate and the Campylobacter-specific virulence factors, in which yellow represents 100% identity and purple represents 0% identity. International Organization for Standardization (ISO) country code: Canada (CA), United States (US), France (FR), Uruguay (UY), Argentina (AR), United Kingdom (UK), Australia (AU), and New Zealand (NZ).

Figure 3

Correlation between the 25 Campylobacter fetus strains based on their average nucleotide identity. The color key indicates the degree of correlation between the strains, in which red represents the highest correlation while blue represents the lowest correlation. The dendrogram demonstrates the hierarchical relationship between the 25 Campylobacter fetus strains based on their average nucleotide identity. International Organization for Standardization (ISO) country code: Canada (CA), United States (US), France (FR), Uruguay (UY), Argentina (AR), United Kingdom (UK), Australia (AU), and New Zealand (NZ).

Figure 4

Whole-genome alignment of 25 Campylobacter fetus subspecies. Reference: published C. fetus subsp. venerealis ATCC 19438^T [GCA_008271385.1]. Query were complete genome sequences of C. fetus subsp. Fetus (n = 11) and C. fetus subsp. venerealis (n = 14) subspecies. Strains labeled in red are C. fetus subsp. fetus, isolated and labeled in blue, are C. fetus subsp. venerealis and strains labeled in green are C. fetus subsp. venerealis bv. intermedius. Black arcs represent the putative genomic islands. Orange arcs represent the genes that were used in PCR C. fetus subspecies identification. Purple lines represent candidate SNPs identified in the 25 C. fetus subspecies.

Figure 5

Phylogenetic tree based on single nucleotide polymorphisms (SNPs) between the 25 Campylobacter fetus strains. Strains labeled in red are C. fetus subsp. fetus, strains labeled in blue are C. fetus subsp. venerealis, and strains labeled in green are C. fetus subsp. venerealis bv. intermedius. All C. fetus subsp. fetus isolates clustered into a separate branch from all C. fetus subsp. venerealis and C. fetus subsp. venerealis bv. intermedius isolates, indicating that there were distinctive SNPs. C. fetus subsp. venerealis and C. fetus subsp. venerealis bv. intermedius isolates from Australia clustered separately from non-Australian isolates. International Organization for Standardization (ISO) country code: Canada (CA), United States (US), France (FR), Uruguay (UY), Argentina (AR), United Kingdom (UK), Australia (AU), and New Zealand (NZ).

Figure 6

(A) Screening for single nucleotide polymorphisms (SNPs) suitable for reliable differentiation between Campylobacter fetus subsp. fetus and Campylobacter fetus subsp. venerealis. (B) Candidate single nucleotide polymorphisms (SNPs) are categorized into Clusters of Orthologous Groups (COGs). The blue section represents coding sequences (CDS) responsible for “cellular processes and signaling” (n = 45). The orange section represents the CDS responsible for “information storage and processing” (n = 21). The gray section represents CDS responsible for “metabolism” (n = 44). The yellow section represents CDS belonging to multiple groups of COGs (n = 4). The light blue section represents CDS, which was poorly categorized (n = 21). The green section represents CDS with no match return from querying the COG database (n = 10).

Figure 7

STRING network of SNP-coding CDS, which had more than eight degrees of association. The thickness of the network edges indicates the confidence level of the functional interactions.

Figure 8

(A) Standard curve for the mraY SNP assay plotting the quantification cycle (Cq) value against the starting quantity of C. fetus venerealis DNA. Data points are from 1 ng to 0.1 pg, with 1 pg indicated by an arrow. The R² of the standard curve is 0.994. (B) Allelic discrimination plot for mraY Taqman SNP assay using Relative Fluorescent Units (RFU) for VIC and FAM. Red—CFF control (ATCC 27374), brown—CFF isolate (strain BT376/03), green—CFV control (ATCC 19438), blue—CFV isolates (strains A8, 957, 76223, 924, and 926), and light blue—other Campylobacter species (C. sputorum, C. ureolyticus, C. hyointestinalis, and A. cryoaerophilus).