Evaluation of Escherichia coli O157:H7 internationalization into romaine lettuce tissues and detection of metabolic changes of internalized bacteria in response to bioavailable carbon using developed bioluminescent assay

Leigh A Farris, Purdue University

Abstract

E. coli O157:H7 has been identified as the causative agent of food-borne illnesses associated with bagged lettuces and spinach. It is hypothesized that the acts of processing, the environment within bagged leafy greens, and exposure to temperatures above 4°C can work in concert to promote the persistence and growth of lettuce-associated E. coli O157:H7. To test this hypothesis, a bioluminescent-based assay to monitor changes in E. coli O157:H7 activity in response to bioavailable carbon was developed, and was adapted to specifically examine the in situ utilization of lettuce-derived carbon. Using a bioluminescent derivative of E. coli O157:H7 in in vitro assays, metabolic variations occurring as a result of the catabolism of simple and complex carbon sources, and manifested as changes in bioluminescence, were observed. In vitro carbon utilization assays using lettuce extracts demonstrated that incubation at higher temperatures (25° and 37°C) results in rapid increases in E. coli O157:H7 activity. The usefulness of this assay in in situ formats was demonstrated by photographic evidence of the metabolism of alfalfa root exudate by E. coli O157:H7. Bioluminescence also allowed the visual confirmation of the internalization of E. coli O157:H7 into the vascular tissues of romaine lettuce leaves. In situ assays performed at 37°C demonstrated the real-time utilization of lettuce-derived carbon sources by internalized E. coli O157:H7. The increased metabolic activity of lettuce-internalized or -associated bacteria was confirmed by imaging using a photon counting camera. Persistence assays demonstrated that lettuce-associated E. coli O157:H7 survive at least two weeks if held at 4°C and quickly become metabolically active when exposed to a temperature of 37°C. Finally, the environment within bagged lettuces was shown to be conducive to increases in E. coli O157:H7 metabolism. Bags of romaine lettuce were inoculated with carbon-limited E. coli O157:H7, and following a brief incubation at 37°C increases in bioluminescence from previously dormant E. coli O157:H7 was observed. It is hypothesized that the chopping of lettuce leaves allows the leaching of organic compounds into the surrounding milieu for use by contaminating E. coli O157:H7. The exposure of E. coli O157:H7 to lettuce-derived carbon sources, combined with exposure to a temperature of 37°C could serve as a metabolic priming mechanism, and may lead to the rapid onset and contribute to the severity of pathogen-associated diseases in human hosts.

Degree

Ph.D.

Advisors

Applegate, Purdue University.

Subject Area

Agronomy|Microbiology

COinS