Establishment of a Multilocus Sequence Typing Scheme for Pasteurella canis Using Isolates from Infected Humans and Diseased Companion Animals
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Background: Multilocus sequence typing (MLST) is well-established for Pasteurella multo- cida but remains undeveloped for Pasteurella canis. We established MLST for P. canis us- ing 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, respec- tively. 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 frag- ment sequences, variable sites, allelic profiles, and sequence types (STs) of each isolate. A phylogenetic tree of concatenated sequences was constructed using the goeBURST algo- rithm 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, respec- tively. 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 di- verse ST distributions among different hosts (animal/human) and countries (Japan/Ko- rea/others). We found clusters 1–4 in ompA genotyping, indicating that MLST discrimina- tion is higher than ompA typing discrimination. Conclusions: We established MLST for P. canis isolates from humans and companion ani- mals in Japan and Korea, thereby providing a robust tool for population biology studies.
Abstract
Background: Multilocus sequence typing (MLST) is well-established for Pasteurella multo- cida but remains undeveloped for Pasteurella canis. We established MLST for P. canis us- ing 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, respec- tively. 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 frag- ment sequences, variable sites, allelic profiles, and sequence types (STs) of each isolate. A phylogenetic tree of concatenated sequences was constructed using the goeBURST algo- rithm 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, respec- tively. 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 di- verse ST distributions among different hosts (animal/human) and countries (Japan/Ko- rea/others). We found clusters 1–4 in ompA genotyping, indicating that MLST discrimina- tion is higher than ompA typing discrimination. Conclusions: We established MLST for P. canis isolates from humans and companion ani- mals in Japan and Korea, thereby providing a robust tool for population biology studies.