Comparative polyphasic characterization of Streptococcus phocae strains with different host origin and description of the subspecies Streptococcus phocae subsp. salmonis subsp. nov.

A polyphasic study was undertaken to clarify the taxonomic position of Streptococcus phocae strains isolated from Atlantic salmon (Salmo salar) cage-farmed in Chile. Four salmon and three seal isolates showed minor differences in the SDS-PAGE protein analysis. Thus, a major protein band present in the salmon isolates, of approximately 22.4 kDa, was absent in the pinniped strains, regardless of the growth media employed. In addition, the pinniped strains showed protein bands with molecular masses of 71.5 and 14.2 kDa, when grown on trypticase soy agar supplemented with 1% NaCl, or 25.6 kDa, when grown on Columbia blood agar, not present in the Atlantic salmon strains. A high similarity in the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS spectra of the strains was observed, although some minor peaks were absent in the fish isolates. Fatty acid methyl esters from isolates with different host origin significantly (P<0.05) differed in the content of C16:0, C17:0, C18:1ω9c, C20:4ω6,9,12,15c and summed features 3, 5 and 8. The salmon isolates formed a separate cluster in the phylogenetic analysis of housekeeping genes, separately or as concatenated sequences. Sequence divergences among salmon and seal strains were in the range of inter-subspecies differentiation for groEL (2.5%), gyrB (1.8%), recN (2.1%), rpoB (1.7%) and sodA (2.0%) genes. DNA-DNA hybridization results confirmed those of sequencing, showing reassociation values between seal and salmon strains close to the borderline of species definition. Differences in growth at low temperatures and in the haemolytic capacities were also observed between both groups of isolates. On the basis of all these results, the salmon isolates represent a novel subspecies of S. phocae, for which the name Streptococcus phocae subsp. salmonis subsp. nov. is proposed. The type strain is C-4T (=CECT 7921T=DSM 24768T). The subspecies Streptococcus phocae subsp. phocae subsp. nov. is automatically created. An emended description of S. phocae is also provided.