Staphylococcus pseudintermedius isolated from atopic dogs with pyoderma induces mast cell degranulation.

Abstract

Aims: First, to determine via whole genome sequencing the sequence of the hld gene that encodes δ-toxin and elements of the accessory gene regulator (agr) locus that encode quorum sensing in four Staphylococcus pseudintermedius isolates from atopic dogs; second, to assess degranulation of mast cells by synthetic δ-toxin in vitro, and by culture filtrate containing δ-toxin from the S. pseudintermedius isolates in canine skin in vivo; and third, to determine whether the genetic region (RNAIII) encoding the δ-toxin gene is upregulated in response to increasing bacterial density (quorum sensing) in the isolates.

Methods: Four isolates of S. pseudintermedius were obtained from four dogs with pyoderma and canine atopic dermatitis (cAD). All four isolates were sequenced to compare their genomes and the sequences of the agr and hld elements. Synthetic S. pseudintermedius δ-toxin was applied to a mast cell culture from murine fetal liver cells in vitro. Degranulation was assessed using a β-hexosaminidase assay. Filtered supernatants from cultures of the four S. pseudintermedius isolates were tested by mass spectrometry to detect δ-toxin. These filtrates were then injected into the skin of five normal dogs. The injection sites were biopsied 15 minutes later. Degranulation of canine mast cells was assessed and quantified histologically. To assess up-regulation of the genetic region encoding the δ-toxin gene in response to increasing bacterial density in the four S. pseudintermedius isolates, relative expression of RNAIII was assayed using quantitative PCR after 1, 2, 4, 7 and 8 hours of culture.

Results: Synthetic S. pseudintermedius δ-toxin caused comparable degranulation of MC/9 cells to δ-toxin of Staphylococcus aureus. Mast cell degranulation was demonstrated in the skin of all five normal dogs following intradermal injection of a purified supernatant that contained S. pseudintermedius δ-toxin. The genetic elements of the δ-toxins were described. As the cell density of cultures of the S. pseudintermedius isolates from atopic dogs increased, RNAIII expression increased relative to the reference gene (gyrB), suggesting that RNAIII expression may be controlled by a quorum-sensing mechanism.

Conclusions and clinical relevance: S. pseudintermedius isolates from atopic dogs carry genes encoding δ-toxin, a staphylococcal exotoxin that can degranulate murine mast cells in vitro. An agent in filtered S. pseudintermedius culture known to contain δ-toxin causes degranulation of dermal mast cells in vivo and may play a role in the initiation and/or exacerbation of cAD.