Rapid and accurate identification of human isolates of Pasteurella and related species by sequencing the sodA gene

Abstract

The identification of Pasteurella and related bacteria remains a challenge. Here, a 449- to 473-bp fragment (sodA(int)) internal to the sodA gene, encoding the manganese-dependent superoxide dismutase, was amplified and sequenced with a single pair of degenerate primers from the type strains of Pasteurella (18 strains), Gallibacterium (1 strain), and Mannheimia (5 strains) species. The sodA(int)-based phylogenetic tree was in general agreement with that inferred from the analysis of the corresponding 16S rRNA gene sequences, with members of the Pasteurella sensu stricto cluster (Pasteurella multocida, Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis) forming a monophyletic group and Gallibacterium and Mannheimia being independent monophyletic genera. However, the sodA(int) sequences showed a markedly higher divergence than the corresponding 16S rRNA genes, confirming that sodA is a potent target to differentiate related species. Thirty-three independent human clinical isolates phenotypically assigned to 13 Pasteurella species by a reference laboratory were successfully identified by comparing their sodA(int) sequences to those of the type species. In the course of this work, we identified the first Gallibacterium anatis isolate ever reported from a human clinical specimen. The sodA(int) sequences of the clinical isolates displayed less than 2.5% divergence from those of the corresponding type strains, except for the Pasteurella pneumotropica isolates, which were closely related to each other (> 98% sodA(int) sequence identity) but shared only 92% sodA(int) identity with the type strain. The method described here provides a rapid and accurate tool for species identification of Pasteurella isolates when access to a sequencing facility is available.

FIG. 1.

Amplification of type strains of Pasteurella and related species with the primers d1 and d2 and separation of the sodA_int amplicons (arrowheads) by 1% agarose gel electrophoresis. Lanes: 1 and 21, 50-bp DNA ladder (Fermentas, Lithuania); 2, P. avium; 3, P. gallinarum; 4, P. volantium; 5, P. skyensis; 6, P. testudinis; 7, A. ureae; 8, Mannheimia sp.; 9, P. trehalosi; 10, P. canis; 11, P. dagmatis; 12, P. multocida; 13, P. stomatis; 14, P. aerogenes; 15, P. mairii; 16, G. anatis; 17, H. parainfluenzae; 18, P. bettyae; 19, P. langaaensis, 20, P. caballi. The size of the amplicons is either 503 bp (lanes 2 to 9), 506 bp (lanes 10 to 17), 515 bp (lane 18), 518 bp (lane 19), or 527 bp (lane 20).

FIG. 2.

Additional amino acids (boxed) in the deduced SodA sequence of Pasteurella bettyae, Pasteurella caballi, and Pasteurella langaaensis are located in the same region. The corresponding codons are absent from sodA sequences of Pasteurella multocida and other bacterial species. The authenticity of these additional codons was confirmed by sequencing the sodA_int fragments of independent P. caballi and P. bettyae isolates. Amino acid numbering refers to the SodA sequence of P. multocida Pm70 (accession no. AAK02085).

FIG. 3.

Phylogenetic trees showing relationships among sodA_int (A) and rrs (16S rRNA gene) (B) sequences from type strains of Pasteurella, Gallibacterium, and Mannheimia species. The trees were established by using the neighbor-joining method from an analysis of the sodA_int sequences obtained in this work (listed in Table 1) (A) and rrs sequences obtained from GenBank (B). The sodA_int and rrs sequences of A. ureae, H. influenzae, and H. parainfluenzae type strains are also included in order to show the relationships between representatives of the main genera of the family Pasteurellaceae. The sodA_int and rrs sequences of E. coli strain K-12 were used as outgroups to root the trees. The value on each branch is the estimated confidence limit (expressed as a percentage) for the position of the branch as determined by bootstrap analysis. Only bootstrap values of greater than 75% are indicated. The scale bar represents 10% differences in nucleotide sequences.