title: Use of signature-tagged mutagenesis to identify genes associated with colonization of sheep by E. coli O157:H7.
abstract: Outbreaks of Escherichia coli O157:H7 in the United States due to contaminated foods are a public health issue and a continuing problem. The major reservoir for these organisms is the gastrointestinal tract of ruminants where they are a member of the resident microbiota. Several factors that contribute to the colonization of cattle have been identified, but a systematic screen of genes that might contribute to the colonization and persistence phenotype in mature ruminants has not been reported. Using a sheep model of persistence, signature tagged mutagenesis (STM) was used to screen 1326 mutants for a persistence-negative phenotype of E. coli O157:H7. We identified 9 genes by STM that appeared to be required for colonization and/or survival in sheep. Three of the genes had functions associated with central metabolism (thiK, ftrA and nrdB), one was involved with LPS formation (wbdP), one encodes a non-LEE encoded effector protein (nleB) and one was a methyltransferase encoded on a prophage (Z2389). The remaining three genes did not have homology with any known genes. Six sheep given ΔwbdP and 2 sheep each were given mutants (ΔthiK (Z1745), ΔftrA (Z2164) and Z2389). The ΔwbdP mutant was recovered from the feces of 4/6 sheep at 6 days pi with a mean number of 1.42log10CFU/g feces compared to 4.6log10CFU/g feces for the wild type strain. This difference was significant (P<0.001) over the time course of the experiment (days 6-23). Both ΔthiK and ΔftrA mutants were recovered from 1 of 2 sheep at 9 days PI by enrichment procedures (<50CFU/g feces) whereas mutant Z2389 was not recovered from either animal past 2 days pi. The roles of all of these gene products require further study to determine how the persistence phenotype of a given strain of E. coli O157:H7 interacts with host factors.
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, 50011, United States. Electronic address: ncornick@iastate.edu.
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Cornick
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, 50011, United States.
authors.forename: Josh
authors.initials: J
authors.lastname: Pitzer
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, 50011, United States.
authors.forename: Amy F
authors.initials: AF
authors.lastname: Helgerson
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, 50011, United States.
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Madsen
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, 50011, United States.
authors.forename: Kathy T
authors.initials: KT
authors.lastname: Kurth
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, 50011, United States.
authors.forename: Qianjun
authors.initials: Q
authors.lastname: Xiao
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, 50011, United States.
authors.forename: F Chris
authors.initials: FC
authors.lastname: Minion
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion / Q000235:genetics; D015169:Colony Count, Microbial / Q000662:veterinary; D004927:Escherichia coli Infections / Q000382:microbiology / Q000662:veterinary*; D019453:Escherichia coli O157 / Q000235:genetics* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics*; D005243:Feces / Q000382:microbiology; D041981:Gastrointestinal Tract / Q000382:microbiology; D016254:Mutagenesis, Insertional; D012756:Sheep; D012757:Sheep Diseases / Q000382:microbiology*; D037521:Virulence Factors / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D037521:Virulence Factors
keywords: Colonization; E. coli O157:H7; Sheep