Ann Lab Med.  2023 May;43(3):263-272. 10.3343/alm.2023.43.3.263.

Virulence-associated Genome Sequences of Pasteurella canis and Unique Toxin Gene Prevalence of P. canis and Pasteurella multocida Isolated from Humans and Companion Animals

• Yoshida H
• Kim JM
• Maeda T
• Goto M
• Tsuyuki Y
• Shibata S
• Shizuno K
• Okuzumi K
• Kim JS
• Takahashi T

• KMID: 2551710
• DOI: http://doi.org/10.3343/alm.2023.43.3.263

Abstract

Background
Comparative analysis of virulence factors (VFs) between Pasteurella canis and Pasteurella multocida are lacking, although both cause zoonotic infections. We determined the virulence-associated genome sequence characteristics of P. canis and assessed the toxin gene prevalence unique to P. canis among clinical isolates of P. canis and P. multocida.
Methods
We selected 10 P. canis and 16 P. multocida whole-genome sequences (WGSs) from the National Center for Biotechnology database. The VFanalyzer tool was used to estimate P. canis-characteristic VFs. Amino acid sequences of VFs were compared with multiple-aligned sequences. The genome structure containing P. canis-characteristic and adjacent loci was compared to the corresponding P. multocida genome structure. After designing primer sequences and assessing their accuracy, we examined the gene prevalence of the P. canis-characteristic VFs using PCR among clinical isolates of P. multocida and P. canis.
Results
Using VFanalyzer, we found virulence-associated cytolethal distending toxin (cdt)A–cdtB–cdtC loci common to all P. canis WGSs that were not found in P. multocida WGSs. Similarities in the multiple alignments of CdtA–CdtB–CdtC amino acid sequences were found among the 10 P. canis WGSs. Shared or similar loci around cdtA–cdtB–cdtC were identified between the P. canis and P. multocida genome structures. The PCR-based cdtA–cdtB–cdtC prevalence differed for P. canis and P. multocida clinical isolates.
Conclusions
P. canis-specific cdtA–cdtB–cdtC prevalence was identified among clinical isolates. These three loci may be unique toxin genes and promising targets for the rapid identification of P. canis in clinical settings.

Keyword

Pasteurella canis; Pasteurella multocida; Genome sequence; Unique toxin gene; Cytolethal distending toxin; Japan; Korea; PCR; Zoonoses; Virulence factors

Figure

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  Fig. 1 Multiple amino acid (AA) sequence alignments of cytolethal distending toxin (Cdt)A, B, and C between HL_NV12211 (harboring CdtA–CdtB–CdtC of 251 AAs, 280 AAs, and 180 AAs, respectively) and the remaining nine whole-genome sequences using ClustalW version 2.1. “*” indicates positions having a single, fully conserved residue; “:” indicates that one of the following “strong” groups (STA/NEQK/NHQK/NDEQ/QHRK/MILV/MILF/HY/FYW) is fully conserved; and “.” indicates that one of the following “weaker” groups (CSA/ATV/SAG/STNK/STPA/SGND/SNDEQK/NDEQHK/NEQHRK/FVLIM/HFY) is fully conserved. Gray shading shows AA substitutions compared to the consensus sequence of HL_NV12211.

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  Fig. 2 Genome structure containing the cytolethal distending toxin (cdt)A–cdtB–cdtC loci of Pasteurella canis and adjacent loci from strain HL_NV12211 (GenBank accession no. CP085871.1) (upper) and the comparative structure from Pasteurella multocida subsp. multocida ATCC 43137(T) (GenBank accession no. CP008918.1) (lower). Asterisks show putative Holliday junction resolvase. K7G93_001965, K7G93_001967, and DR93_66 represent loci encoding hypothetical proteins. Abbreviations: relA, GTP diphosphokinase; rlmD and rumA, 23S rRNA (uracil(1939)-C(5))-methyltransferase; recO, DNA repair protein; rsmE, 16S rRNA (uracil(1498)-N(3))-methyltransferase; eno, phosphopyruvate hydratase; pyrG, CTP synthase.

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  Fig. 3 Gel electrophoresis images of amplified cdtA–cdtB–cdtC products using DNA from clinical isolates. Asterisks indicate the positive control isolate (PA42). Abbreviations: M, size marker; NC, negative control.

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