Complete Genome Sequences of Historic Clostridioides difficile Food-Dwelling Ribotype 078 Strains in Canada Identical to That of the Historic Human Clinical Strain M120 in the United Kingdom.

Clostridioides (Clostridium) difficile is a spore-forming anaerobic bacterium that causes severe intestinal diseases in humans. Here, we report the complete genome sequence of the first C. difficile foodborne type strain (PCR ribotype 078) isolated from food animals in Canada in 2004, which has 100% similarity to the genome sequence of the historic human clinical strain M120.

ANNOUNCEMENT

The isolation of multidrug-resistant hypervirulent Clostridioides difficile PCR ribotype 027 and 078 strains from food animals and retail foods in Canada in 2004 (1–3) coincided with a major outbreak of severe C. difficile infections in humans in Canada and the United Kingdom in the same year (4, 5). Further epidemiological evidence has indicated that C. difficile could be a foodborne pathogen, thereby explaining a major fraction of infections acquired in hospitals and the community (6–8). Here, we applied a genome-based strategy to further test such a hypothesis and to determine the genetic features that allow highly virulent strains to move between humans and animals. We report the complete genome sequences of three historical Canadian C. difficile PCR ribotype 078 food strains (3) using Pacific Biosciences RS II sequencing.

In brief, pure bacterial isolates (cultured in tryptone soy agar–5% sheep blood under anaerobic conditions at 37°C; Thermo Fisher Scientific) were used for DNA extraction (QIAamp DNA blood minikit, Gram-positive bacterial protocol; Qiagen). Determinations of DNA concentration and size were performed using a Qubit fluorometer (Thermo Fisher Scientific). Genomic DNA was sheared in Covaris g-Tubes with the help of a fix-angled rotor centrifuge (Eppendorf) using the following parameters: shear for 1 min at 3,500 rpm, flip the tube, and shear for 1 min at 3,500 rpm. Sheared DNA was collected and purified using AMPure PB beads according to the PacBio protocol. Target 20-kb SMRTbell templates were prepared following an exonuclease VII reaction, DNA damage repair reaction, end repair reaction, overnight ligation, heat kill, and exonuclease III/VII digestion of misligated products. SMRTbell template size selection was performed using BluePippin (Sage Science) protocols. Sequencing primer annealing to the SMRTbell templates was followed by DNA polymerase P6 binding. SMRTbell templates bound to MagBeads were sequenced using single-molecule real-time (SMRT) version 3 cells (6Pac cells; 2 cells/isolate). The genomes were assembled and polished using the HGAP3 and Quiver software packages, respectively, within the SMRTPortal (version 2.3.0) with default settings. The genomes were additionally polished with Arrow (the resequencing pipeline run with default parameters), which is within the latest SMRT Analysis software bundle (SMRTLink, version 5.1.0) and checked for misassemblies, but none were identified. Annotations of the genomes were completed using Prokka within EDGE bioinformatics (9, 10).

Interestingly, whole-genome alignment and single-nucleotide polymorphism (SNP) analyses, with the help of the PhaME software (M. Shakya/P. S. G. Chain et al., unpublished data), revealed nearly 100% nucleotide sequence identity with the genome sequence of C. difficile strain M120 (BioProject number PRJNA42467), a PCR ribotype 078 isolate obtained from a UK patient in 2007 (11). Our whole-genome sequence analysis (Table 1) indicates that, regardless of geographic distances, these historic C. difficile strains concurrently had similar genomic elements to explain disease in both animals and humans.

TABLE 1

Genome insights and GenBank accession numbers for three Clostridioides difficile strains isolated from the Canadian food production system

C. difficile strain	GenBank accession no.	Genome size (bp)	No. of genes	No. of virulence genes	No. of antibiotic resistance genes	Fold coverage (×)
R1	CP026613	4,093,143	3,579	11	16	362
R2	CP026614	4,093,145	3,577	11	16	371
R3	CP026615	4,093,148	3,580	11	16	339

Data availability.

The complete sequences and annotations of these C. difficile genomes are deposited in the GenBank database with accession numbers CP026613 (C. difficile R1), CP026614 (C. difficile R2), and CP026615 (C. difficile R3).