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.

Highlights

  • Increased global processing and distribution of food has raised awareness of food safety in regards to accidental or purposeful introduction of a biological contaminates into the food network [1, 2]

  • Various studies have shown that the spent culture medium of pathogenic bacteria, such as Mycobacterium tuberculosis, stimulated enhanced growth of dormant and small inoculum cultures [32,33,34]

  • Studies conducted by Halmann and colleagues have described the presence of a growth-initiation substance (GIS) in the culture filtrate using small inocula of virulent strains of F. tularensis [35, 36]

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Summary

Introduction

Increased global processing and distribution of food has raised awareness of food safety in regards to accidental or purposeful introduction of a biological contaminates into the food network [1, 2]. It is important to note that the gold-standard to validate F. tularensis detection using serology and various PCR platforms remains cultivation of the organism, which requires growth on cysteine or thioglycolate enriched medium and incubation times of 2–4 days at 37 ̊C [5, 23]. Studies utilizing these tools have been widely applied to F. tularensis detection in patients and animal carcasses; few techniques have been reported for identification of F. tularensis in food and environmental matrices [24,25,26,27]. Inasmuch as the potential for biocontamination with F. tularensis and the presence of resident microbes, which may outcompete F. tularensis growth and act as PCR inhibitors, there remains a critical need for improved cultivation and unambiguous detection of F. tularensis in food and environmental matrices

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