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. 2020 Dec 21;15(12):e0244227.
doi: 10.1371/journal.pone.0244227. eCollection 2020.

Whole genome sequencing of Clostridioides difficile PCR ribotype 046 suggests transmission between pigs and humans

Anders Werner  1 , Paula Mölling  2 , Anna Fagerström  2 , Fredrik Dyrkell  3 , Dimitrios Arnellos  3 , Karin Johansson  2 , Martin Sundqvist  2 , Torbjörn Norén  2
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Free PMC article

Whole genome sequencing of Clostridioides difficile PCR ribotype 046 suggests transmission between pigs and humans

Anders Werner et al. PLoS One. .
Free PMC article

Abstract

Background: A zoonotic association has been suggested for several PCR ribotypes (RTs) of Clostridioides difficile. In central parts of Sweden, RT046 was found dominant in neonatal pigs at the same time as a RT046 hospital C. difficile infection (CDI) outbreak occurred in the southern parts of the country.

Objective: To detect possible transmission of RT046 between pig farms and human CDI cases in Sweden and investigate the diversity of RT046 in the pig population using whole genome sequencing (WGS).

Methods: WGS was performed on 47 C. difficile isolates from pigs (n = 22), the farm environment (n = 7) and human cases of CDI (n = 18). Two different core genome multilocus sequencing typing (cgMLST) schemes were used together with a single nucleotide polymorphisms (SNP) analysis and the results were related to time and location of isolation of the isolates.

Results: The pig isolates were closely related (≤6 cgMLST alleles differing in both cgMLST schemes) and conserved over time and were clearly separated from isolates from the human hospital outbreak (≥76 and ≥90 cgMLST alleles differing in the two cgMLST schemes). However, two human isolates were closely related to the pig isolates, suggesting possible transmission. The SNP analysis was not more discriminate than cgMLST.

Conclusion: No general pattern suggesting zoonotic transmission was apparent between pigs and humans, although contrasting results from two isolates still make transmission possible. Our results support the need for high resolution WGS typing when investigating hospital and environmental transmission of C. difficile.

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Conflict of interest statement

D.A. and F.D. are employees of 1928 Diagnostics. This does not alter our adherence to PLOS ONE policies on sharing data and materials. A.W., P.M., A.F., K.J., M.S., and T.N. declare no competing interests.

Figures

Fig 1
Fig 1. Ridom™ SeqSphere+ cgMLST scheme tree.
Neighbour-joining tree of all isolates based on the Ridom™ SeqSphere+ core genome multilocus sequencing typing (cgMLST) scheme version 2 (2147 genes) and MLST scheme (7 genes), ignoring missing alleles, with year of isolation presented after the isolate’s name and coloured according to the source of isolation.
Fig 2
Fig 2. 1928D cgMLST analysis tree.
Unweighted pair group method with arithmetic mean (UPGMA) tree including all isolates with distances based on the 1928D core genome multilocus sequencing typing (cgMLST) scheme (2,631 genes), ignoring missing alleles. Coloured according to the isolation source. Sequence type (MLST), the presence of toxin genes (A = tdcA, B = tdcB, CDT = binary toxin), and percentage of good cgMLST genes (CORE) are presented after each isolate.
Fig 3
Fig 3. SNP analysis tree.
Unweighted pair group method with arithmetic mean (UPGMA) tree including all isolates based on single nucleotide polymorphisms (SNP) differences in the 1928D analysis, coloured according to the isolation source. Sequence type (multilocus sequencing typing (MLST)), the presence of toxin genes (A = tdcA, B = tdcB, CDT = binary toxin), and the proportion of genome aligned (ALN) are presented after each isolate. The SNP analysis confirmed the clustering observed in both core genome (cg) MLST analyses.
Fig 4
Fig 4. Ridom™ SeqSphere+ cgMLST analysis minimum spanning tree.
Minimum spanning tree of all isolates based on the Ridom™ SeqSphere+ core genome multilocus sequencing typing (cgMLST) scheme version 2 (2147 genes) and MLST scheme (7 genes) with numbers of allelic differences shown on connecting lines (distances not to scale), ignoring missing alleles. Year of isolation is presented after the isolate’s name, nodes are coloured according to the isolation source, and genetically closely related isolates (≤6 cgMLST alleles differing) are shaded grey.
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Grants and funding

K.J., P.M., T.N. and M.S. has received funding for this study from the Research Committee of Region Örebro County (grant OLL-674241). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript. D.A. and F.D. employees of 1928 Diagnostics. 1928 Diagnostics provided support in the form of salaries for D.A. and F.D. and made the analysis available for free, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.