Biotypic and genotypic diversity in Pasteurella canis isolated from host animals and humans: differences in trehalose fermentation and nucleotide sequences encoding trehalose-6-phosphate hydrolase (treC)

Abstract

The biotypic and genotypic features of Pasteurella canis isolated from dogs, cats, and humans were clarified by repetitive sequence-based fingerprinting and nucleotide sequences encoding trehalose-6-phosphate hydrolase (treC). Thirty P. canis and 48 P. multocida isolates were collected from dogs, cats, and humans to perform biotyping. The genotyping of P. canis by fingerprinting was followed by dendrogram construction. The whole-genome sequences (WGSs) were searched for the enzyme-coding nucleotide sequences around the main and adjacent loci constituting the operon. Full-length nucleotide sequences encoding the enzyme were determined using polymerase chain reaction and direct sequencing. Biotypic results were compared to the dendrogram and nucleotide sequence data. We observed a difference in trehalose fermentation with a positivity rate of 46.7%. Two (A-1/A-2) and three (B-1/B-2/B-3) clades were located on the dendrograms generated based on two repetitive sequence-based fingerprinting techniques, showing no association between trehalose fermentation and the clades. Based on the WGSs, two variants of the gene, namely, a 1,641 bp gene treC and a pseudogene (1,335 bp) of treC with its first 306 nucleotides deleted, were observed. Trehalose-positive isolates harbored treC, whereas trehalose-negative isolates lacked treC with or without the pseudogene. Our observations suggest biotypic and genotypic diversity among the P. canis isolates from animal and human hosts, with respect to trehalose fermentation and treC nucleotide sequences. This is the first report on the diversity of treC nucleotide sequences among these isolates.

Keywords: Pasteurella canis; Pasteurella multocida; trehalose fermentation; trehalose-6-phosphate hydrolase.

Fig. 1.

Unweighted Pair Group Method with Arithmetic mean dendrograms based on polymorphic DNA fingerprinting date using repetitive extragenic palindromic sequence-based polymerase chain reaction (PCR) [A] and enterobacterial repetitive intergenic consensus-based PCR [B] procedures. We contained a DNA specimen of Pasteurella multocida subsp. multocida (PA60) isolated from a human patient as a positive control. Biotypic results of trehalose (TRE) fermentation are shown at the right-side of each isolate. Gray shading indicates TRE fermentation-positive/human isolates.

Fig. 2.

Genome structures containing trehalose-6-phosphate hydrolase gene (treC) with adjacent locus from Pasteurella canis HL1500 (GenBank accession No. CP083396.1) (upper-side) and treC pseudogene with adjacent loci from Pasteurella canis HL_NV12211 (CP085871.1) (lower-side). Primer names, primer sequences (5′→3′), and primer positions (arrows with dotted lines) when performing amplification and sequencing are shown under loci and non-coding region. treC encodes trehalose-6-phosphate hydrolase. treC pseudogene showed deletion at 1st-306th nucleotide positions of treC.