A combined enrichment and aptamer pulldown assay for Francisella tularensis detection in food and environmental matrices

• Published in: PloS one Vol. 9; no. 12; p. e114622
• Main Authors: Lamont, Elise A, Wang, Ping, Enomoto, Shinichiro, Borewicz, Klaudyna, Abdallah, Ahmed, Isaacson, Richard E, Sreevatsan, Srinand
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 23.12.2014; Public Library of Science (PLoS)
• Subjects: Analysis; Antibody response; Aptamers; Aptamers, Nucleotide; Bacteria; Base Sequence; Biology and Life Sciences; Carnosine; Carnosine - pharmacology; Cell culture; Chromatography; Deoxyribonucleic acid; Disease control; DNA; Ecology; Enrichment; Environmental Microbiology; Filtrate; Food contamination; Food contamination & poisoning; Food Microbiology - methods; Francisella tularensis; Francisella tularensis - drug effects; Francisella tularensis - growth & development; Francisella tularensis - isolation & purification; Ingestion; Laboratorium voor Microbiologie; Lettuce; Liquid chromatography; Medical research; Medicine; Medicine and Health Sciences; Microbiological Laboratory; Microbiologie; Microbiology; Molecular Sequence Data; Mycobacterium tuberculosis; Pasteurella tularensis; Polymerase chain reaction; Population; Proteins; Public health; Soil contamination; Studies; Tuberculosis; Tularemia; Water pollution
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0114622

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.