title: Potential mechanisms of attenuation for rifampicin-passaged strains of Flavobacterium psychrophilum.
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.
  
  
  CONCLUSIONS
  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).
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA. karol@uidaho.edu.|Present address: Department of Biological Sciences, University of Idaho, Moscow, Idaho, USA. karol@uidaho.edu.
authors.forename: Karol
authors.initials: K
authors.lastname: Gliniewicz
authors.affiliation: Molecular Biology and Genomics Core, Washington State University, Pullman, WA, USA. wildung@wsu.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: Wildung
authors.affiliation: Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA, USA. lorfe@vetmed.wsu.edu.
authors.forename: Lisa H
authors.initials: LH
authors.lastname: Orfe
authors.affiliation: USDA-ARS-National Center for Cool and Cold Water Aquaculture, Leetown, WV, USA. greg.wiens@ars.usda.gov.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Wiens
authors.affiliation: Department of Fish and Wildlife Resources, University of Idaho, Moscow, ID, USA. kcain@uidaho.edu.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Cain
authors.affiliation: Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA. klahmers@vt.edu.|Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA, USA. klahmers@vt.edu.
authors.forename: Kevin K
authors.initials: KK
authors.lastname: Lahmers
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA. ksnek@wsu.edu.|Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, WA, USA. ksnek@wsu.edu.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Snekvik
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA. drcall@wsu.edu.|Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA, USA. drcall@wsu.edu.
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Call
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005393:Fish Diseases / Q000382:microbiology; D045826:Flavobacteriaceae Infections / Q000382:microbiology / Q000662:veterinary*; D005417:Flavobacterium / Q000187:drug effects* / Q000254:growth & development* / Q000472:pathogenicity; D008070:Lipopolysaccharides / Q000032:analysis; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D020641:Polymorphism, Single Nucleotide; D020543:Proteome / Q000032:analysis; D012293:Rifampin / Q000378:metabolism*; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D012692:Serial Passage; D016019:Survival Analysis; D014774:Virulence
chemical_list: D000900:Anti-Bacterial Agents; D008070:Lipopolysaccharides; D020543:Proteome; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin