Evolution and comparative genomics of Campylobacter jejuni ST-677 clonal complex

Abstract

Campylobacter is the most common bacterial cause of gastroenteritis in the European Union with over 200,000 laboratory-confirmed cases reported annually. This is the first study to describe findings related to comparative genomics analyses of the sequence type (ST)-677 clonal complex (CC), a Campylobacter jejuni lineage associated with bacteremia cases in humans. We performed whole-genome sequencing, using Illumina HiSeq sequencing technology, on five related ST-677 CC isolates from two chicken farms to identify microevolution taking place at the farms. Our further aim was to identify novel putative virulence determinants from the ST-677 CC genomes. For this purpose, clinical isolates of the same CC were included in comparative genomic analyses against well-known reference strains of C. jejuni. Overall, the ST-677 CC was recognized as a highly clonal lineage with relatively small differences between the genomes. Among the farm isolates differences were identified mainly in the lengths of the homopolymeric tracts in genes related to the capsule, lipo-oligosaccharide, and flagella. We identified genomic features shared with C. jejuni subsp. doylei, which has also been shown to be associated with bacteremia in humans. These included the degradation of the cytolethal distending toxin operon and similarities between the capsular polysaccharide biosynthesis loci. The phase-variable GDP-mannose 4,6-dehydratase (EC 4.2.1.47) (wcbK, CAMP1649), associated with the capsular polysaccharide biosynthesis locus, may play a central role in ST-677 CC conferring acid and serum resistance during different stages of infection. Homology-based searches revealed several additional novel features and characteristics, including two putative type Vb secretion systems and a novel restriction modification/methyltransferase gene cluster, putatively associated with pathogenesis and niche adaptation.

Keywords: Campylobacter jejuni; bacteremia; capsule; niche adaptation; phase variation; serum resistance.

Fig. 1.—

BRIG image highlighting the CJIEs using strain RM1221 as a reference. Upper and lower identity thresholds of 90% (dark color) and 70% (light color) were used for visualizing the difference between sequences. White areas correspond to sequences with less than 70% similarity (BLASTN) against the reference.

Fig. 2.—

NeighborNet phylogeny of the farm and clinical isolates. Networks were drawn from the shared loci of each set of isolates. (A) ST-677 CC (1,286 shared loci). ST-677 isolates are shown in black and ST-794 in red. (B) ST-677 (1,319 shared loci). Numbers of allelic differences between the strains are indicated by lines and Arabic numbers. Loci and alleles were defined using BIGSdb (http://pubmlst.org/campylobacter/, last accessed September 12, 2014).

Fig. 3.—

ClonalFrame genealogy depicting the evolutionary relationship of the ST-677 CC isolates. In parentheses, the presence of CJIEs is indicated (“d” meaning large deletion in the CJIE1-like element). ST-677 isolates are shown in black and ST-794 in red.

Fig. 4.—

ACT comparison of the CPS gene cluster (from kpsC, cjj5070_05410c to kpsF, cjj5070_05770c) of strain 5070 against ATCC43432 (Penner HS4 type strain) and strain 269.97. Cjd, C. jejuni subspecies doylei; and Cjj, C. jejuni subsp. jejuni.

Fig. 5.—

Evolution of wcbK (CAMP1649) in a representative selection of C. jejuni and C. coli strains. (A) Bayesian phylogenetic tree based on a selection of isolates obtained from Campylobacter PubMLST and GenBank containing the CAMP1649 locus. Branches indicated by * are supported by more than 85%, and the branch indicated by # is supported by more than 90%. Positions of the strains selected for the sequence alignment (shown in panel B) are indicated by strain numbers in the phylogenetic tree. Dark blue circles represent C. coli isolates. (B) Homopolymeric G-tract was identified to be present only in one cluster of isolates (yellow clade). Cjd, C. jejuni subspecies doylei; Cjj, C. jejuni subsp. jejuni; and Cc, C. coli.

Fig. 6.—

BRIG images visualizing genes involved in iron uptake in C. jejuni using NCTC11168 as a reference. (A) Chromosomal location and distribution of selection of genes involved in iron uptake in reference strain NCTC11168. Genes found to be missing in the ST-677 CC are highlighted in red. (B) Detailed analysis of the sequence conservation in selected genes. White areas correspond to sequences with less than 70% similarity (BLASTN) against the reference.