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Title: Potential mechanisms of attenuation for rifampicin-passaged strains of Flavobacterium psychrophilum

Author
item GLINIEWICZ, KAROL - Washington State University
item WILDUNG, MARK - Washington State University
item ORFE, LISA - Washington State University
item Wiens, Gregory - Greg
item CAIN, KENNETH - University Of Idaho
item LAHMERS, KEVIN - Washington State University
item SNEKVIK, KEVIN - Washington State University
item CALL, DOUGLAS - Washington State University

Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/10/2015
Publication Date: 9/16/2015
Citation: Gliniewicz, K., Wildung, M., Orfe, L.H., Wiens, G.D., Cain, K., Lahmers, K.K., Snekvik, K.R., Call, D.R. 2015. Potential mechanisms of attenuation for rifampicin-passaged strains of Flavobacterium psychrophilum. BMC Microbiology. 15(179):1-15. DOI 10.1186/s12866-015-0518-1.

Interpretive Summary: Pathogenic bacterial strains that have lost virulence are often useful vaccines. Laboratory culture of pathogenic bacteria on media containing the antibiotic rifampicin has long been known to cause a loss of virulence. Previously, an attenuated strain of Flavobacterium psychrophilum was selected following culture on rifampicin and shown to effectively induce protection in rainbow trout and coho salmon against bacterial cold water disease in laboratory trials. While attenuation by passage with rifampicin selection has been described for other pathogens, the mechanism of attenuation is unknown. We hypothesized that rifampicin passage leads to an accumulation of genetic mutations that, by chance, reduce virulence. In this paper, we examined the proteins, the surface components of the bacteria as well as the genome sequences of several F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin. Our findings demonstrated that rifampicin selection does not uniformly lead to strain attenuation. By examining the genetic differences between strains we identified several mutations that may be the cause of attenuation. These studies further our understanding of how strains are attenuated by culture in the presence or absence of rifampicin.

Technical Abstract: Background: Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B 17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent CSF 259–93 strain. We hypothesized that rifampicin passage leads to an accumulation of genomic mutations that, by chance, reduce virulence. To assess the pattern of phenotypic and genotypic changes associated with passage, we examined proteomic, LPS and single-nucleotide polymorphism (SNP) differences for two F. psychrophilum strains (CSF 259–93 and THC 02-90) that were passaged with and without rifampicin selection. Results: Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different DNA bases depending on the strain. One rifampicin-passaged CSF 259–93 strain (CR) was attenuated (4 % mortality) in challenged fish, but only accumulated eight nonsynonymous SNPs compared to the parent strain. A CSF 259–93 strain passaged without rifampicin (CN) accumulated five nonsynonymous SNPs and was partially attenuated (28 % mortality) compared to the parent strain (54.5 % mortality). In contrast, there were no significant change in fish mortalities among THC 02–90 wild-type and passaged strains, despite numerous SNPs accumulated during passage with (n = 174) and without rifampicin (n = 126). While only three missense SNPs were associated with attenuation, a Ser492Phe rpoB mutation in the CR strain may contribute to further attenuation. All strains except CR retained a gliding motility phenotype. Few proteomic differences were observed by 2D SDS-PAGE and there were no apparent changes in LPS between strains. Comparative methylome analysis of two strains (CR and TR) identified no shared methylation motifs for these two strains. Conclusion: Multiple genomic changes arose during passage experiments with rifampicin selection pressure. Consistent with our hypothesis, unique strain-specific mutations were detected for the fully attenuated (CR), partially attenuated (CN) and another fully attenuated strain (B17).