title: A combined enrichment and aptamer pulldown assay for Francisella tularensis detection in food and environmental matrices.
abstract: Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1-106 CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology.
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, St. Paul, Minnesota, United States of America.
authors.forename: Elise A
authors.initials: EA
authors.lastname: Lamont
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, St. Paul, Minnesota, United States of America.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, Utah, United States of America.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Enomoto
authors.affiliation: Molecular Ecology Group, Wageningen University, Dreijenplen 10, 6703HB, Wageningen, Netherlands.
authors.forename: Klaudyna
authors.initials: K
authors.lastname: Borewicz
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, St. Paul, Minnesota, United States of America.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Abdallah
authors.affiliation: Department of Veterinary Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, United States of America.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Isaacson
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, St. Paul, Minnesota, United States of America; Department of Veterinary Biomedical Sciences, University of Minnesota, St. Paul, Minnesota, United States of America.
authors.forename: Srinand
authors.initials: S
authors.lastname: Sreevatsan
mesh_terms: D052157:Aptamers, Nucleotide; D001483:Base Sequence; D002336:Carnosine / Q000494:pharmacology; D004783:Environmental Microbiology; D005516:Food Microbiology / Q000379:methods*; D005604:Francisella tularensis / Q000187:drug effects / Q000254:growth & development / Q000302:isolation & purification*; D008969:Molecular Sequence Data
chemical_list: D052157:Aptamers, Nucleotide; D002336:Carnosine