Identification of the mechanism responsible for attenuation of rifampicin-passaged Flavobacterium psychrophilum, etiologic agent of bacterial coldwater disease

Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. Presently there are no licensed vaccines for this disease, but earlier work showed that a rifampicin-attenuated strain of F. psychrophilum CSF 259-93B.17 (B17) caused no disease while inducing protection against challenge with the virulent CSF 259-93 strain. Attenuation by passage with rifampicin has been described for several bacterial pathogens, but the mechanism of attenuation is unknown. We hypothesize that passage with rifampicin leads to an accumulation of genomic mutations that, by chance, decrease virulence. Consequently, we examined LPS, proteomic and single nucleotide polymorphism (SNP) differences for two pathogenic F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin. No apparent changes in LPS and only a few proteomic differences were observed among rifampicin-passaged strains relative to the wild-type strains. Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different portions of the gene. One rifampicin-passaged CSF 259-93 strain (CR) demonstrated attenuation (4% mortality) in challenged fish, but only accumulated seven nonsynonymous SNPs compared to its parent strain. A CSF 259-93 strain passaged without rifampicin (CN) was partially attenuated (28% mortality) compared to the parent strain (54.5% mortality). In addition to a Ser492Phe rpoB mutation and a proteomic changes, the CR strain exhibited changed colony morphology and decreased gliding motility, similarly to the B17 strain. The opposite was observed among THC 02-90 wild-type and passaged strains with no significant changes in fish mortalities, despite numerous SNPs leading to nonsynonymous amino acid changes and accumulated by these strains during passage with (n= 174) and without rifampicin (n= 126). While only four SNPs leading to nonsynonymous amino acid substitution significantly attenuated the CR and CN strains, a specific Ser492Phe rpoB mutation may contribute to differential gene regulation and further attenuation of the CR strain.