Establishment of a Multilocus Sequence Typing Scheme for Pasteurella canis Using Isolates from Infected Humans and Diseased Companion Animals

Abstract

Background: Multilocus sequence typing (MLST) is well-established for Pasteurella multocida but remains undeveloped for Pasteurella canis. We established MLST for P. canis using isolates from humans and companion animals in Japan and Korea to gain insights into its population biology.

Methods: We analyzed 39 and 22 isolates from companion animals and humans, respectively. We selected seven housekeeping genes-adk, aroA, deoD, gdhA, g6pd, mdh, and pgi-used in P. multocida MLST. Primer pairs for PCR amplification and sequencing were designed based on conserved sites in 10 whole-genome sequences. We determined fragment sequences, variable sites, allelic profiles, and sequence types (STs) of each isolate. A phylogenetic tree of concatenated sequences was constructed using the goeBURST algorithm to identify STs and clonal complexes (CCs). ompA, encoding outer membrane protein A, was genotyped for molecular characterization.

Results: The sequenced fragment lengths and allele numbers of the seven genes were 424, 451, 483, 439, 429, 419, and 440 bp and 16, 13, 15, 18, 22, 19, and 18, respectively. ST1-ST47, including CC2, CC10, CC18, CC31, and CC33, were diversely distributed among the isolates from different hosts/countries. In the seven-gene phylogenetic tree, apart from P. multocida, all isolates clustered together. goeBURST diagrams revealed diverse ST distributions among different hosts (animal/human) and countries (Japan/Korea/ others). We found clusters 1-4 in ompA genotyping, indicating that MLST discrimination is higher than ompA typing discrimination.

Conclusions: We established MLST for P. canis isolates from humans and companion animals in Japan and Korea, thereby providing a robust tool for population biology studies.

Keywords: Companion animals; Humans; Japan; Korea; Multilocus sequence typing; One Health; Pasteurella canis; Whole-genome sequences.

Fig. 1. Phylogenetic tree of the concatenated sequences of the seven housekeeping genes in Pasteurella canis isolates constructed using the neighbor-joining method with 1,000 bootstrap replicates (shown below the branches). Evolutionary analysis was conducted using MEGA11. All horizontal branch lengths are drawn to scale. The corresponding STs and CCs are indicated in parentheses. Pasteurella multocida subsp. multocida ATCC 43137(T) (accession No. NZ_CP008918.1) and P. multocida subsp. septica NCTC 11995(T) (NZ_UGSV00000000.1) were used as outgroups.

Abbreviations: ST, sequence type; CC, clonal complex.

Fig. 2. goeBURST diagrams indicating relations among STs and CCs in the 61 Pasteurella canis isolates. The numbers in the circles represent STs, and the numbers near the lines indicate the numbers of alleles differing between two connected STs. Putative CCs are identified by an outer dotted frame and correspond to STs with higher numbers of single-locus variants. (A) Distribution of STs among different hosts. Red and blue indicate isolates in animals and humans, respectively. (B) Distribution of STs among different countries. Dark blue, pink, and green indicate isolates from Japan, Korea, and others. Black arrows indicate STs that are identical between animal and human populations (A) or between Japanese and Korean populations (B).

Abbreviations: ST, sequence type; CC, clonal complex.

Fig. 3. Phylogenetic tree of ompA amino-acid sequences constructed using the neighbor-joining method and maximum-likelihood analysis with the Whelan and Goldman model. Evolutionary analyses were conducted using MEGA11. STs are shown on the right.

Abbreviation:ST, sequence type.