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Piscirickettsia salmonis, a serious bacterial pathogen of farmed marine fish, previously considered culturable only in eukaryotic cell-culture systems, was grown for the first time on agar and broth containing enhanced levels of cysteine, thus greatly increasing the potential for isolation, in vitro culture and study of this organism. Virulence towards Atlantic salmon following passage on agar media was retained in a controlled laboratory trial. Of the studied temperatures, optimal growth on agar was observed at 22 degrees C.

Bacterial isolates from diseased farmed tilapia (Oreochromis sp.) from Costa Rica (PQ 1104), Atlantic salmon (Salmo salar) from Chile (PQ 1106) and three-line grunt (Parapristipoma trilineatum) from Japan (Ehime-1) were characterized by phenotypic and molecular taxonomic methods. These isolates were Gram-negative, oxidase negative, non-motile, strictly aerobic cocco-bacilli, produced H2S from cysteine supplemented media, which is phenotypically consistent with the genus Francisella. Comparison of 16S rRNA gene sequences and five partial housekeeping gene sequences (groEL, shdA, rpoB, rpoA and pgm) confirmed these isolates to be members of the genus Francisella, with high 16S rRNA similarity (> 99 %) to Francisella philomiragia subsp noatunensis, F. piscicida and Francisella philomiragia subsp philomiragia isolates. Despite the close 16s rRNA relationship with the aforementioned Francisella taxa, isolates PQ 1104 and Ehime-1 form a separate clade on phylogenetic analysis of the 16s rRNA gene and all housekeeping genes investigated, whereas isolate PQ 1106 is highly similar to F. philomiragia subsp noatunensis (NCIMB 14265T) and F. piscicida (DSM 18777T). DNA-DNA hybridization studies revealed mean reassociation values of 60.3 and 72.6 % between isolate PQ 1104 and F. philomiragia subsp noatunensis (NCIMB 14265T) and F. philomiragia subsp philomiragia (ATCC 25015), respectively. Thus, on the basis of molecular genetic evidence, we propose that isolates PQ 1104 and Ehime-1 should be recognised as Francisella asiatica sp. nov. with type strain PQ 1104T (NCIMB and CCUG number not received yet). No separation between F. piscicida and F. philomiragia subsp noatunensis were identified by the same methods and these species constitute heterotypic synonyms for which the epithet noatunensis has priority. However, given the increased evidence of ecological differentiation within the F. philomiragia group and the existence of a specific fish pathogenic clade, we propose that the F. philomiragia subsp noatunensis be elevated to species level as F. noatunensis comb. nov., sp.

Increasing frequencies of community-acquired methicillin-resistant