Host-pathogen interactions and their application to biosensors and other novel technologies for the rapid detection of food borne pathogens

Amy E Fleishman Littlejohn, Purdue University

Abstract

The biggest challenge facing rapid detection and identification of foodborne microorganisms is having a high enough cell density for detection. To expedite the process of detection and identification a device has been developed to rapidly concentrate and recover microorganisms from various matrices. The automated Cell Concentration and Recovery (CCR) device developed utilizes cross-flow hollow fiber micro filtration to rapidly (10 - 20 min) reduce the total volume of the sample from as many as 1 L – 10 L to a total volume of 0.5 ml – 1.5 ml while capturing the microorganisms contained in the sample. The ability to concentrate microorganisms away from food matrices eliminates the need for an enrichment step decreasing the time for positive identification of a target microorganism by up to 9 – 12 h. Although this cell concentration step seems to be a very simplistic step physical separation of microorganisms from complex matrices quickly while maintaining cost-effectiveness is a major challenge.^ Physical stresses such as shear forces, pressure shifts, rapidly changing solute concentrations and interactions with food particles can greatly affect microorganisms within a sample in a multitude of ways. For this reason, it is important to have a bacterial strain that is both hardy enough to be utilized as an internal control for the evaluation of physiological stresses experienced during the concentration process, but also as an efficient reporter. Here, a bioluminescent strain, Pseudomonas fluorescents M3A, was utilized to validate the concentration ability of the CCR while monitoring the physiological state of the microorganism in real-time. Light production is an energy demanding and any environment that places stress on the cell will result in decreased light generation. This light signal is very easily detected and has several advantages over other markers, such as fluorescence, that are present in detectable levels even in stressed and dead bacteria. Furthermore, matrices containing plant material or other organic material that auto-fluorescence may produce a false positive signal. Therefore, bioluminescence is a far more powerful tool for the monitoring of physiological stress and cell count when compared to other labeling methods. ^ Once a concentrated microbial sample is obtained, it can be subjected to traditional identification methods such as PCR, biochemical tests and sequencing. Although these methods are the "gold standard" they are too time consuming and often result in the destruction of the microbial sample, making it unavailable for additional testing. Developing simpler but more effective methods for the specific capture and identification of microorganisms without destroying the viability of the microorganism would circumvent these problems. ^ Here, biochips have been evaluated for the specific capture of target microorganisms by exploiting what we know about their host: pathogen interactions as a detection and identification method.^ Additionally, we have evaluated a novel detection tool, BActerial Rapid Detection using Optical light-scattering Technology (BARDOT) sensor for the identification of Vibrio spp. from both live oysters and after resuscitation from a "Viable But Not Culturable State" (VBNC). Furthermore, BARDOT was utilized to determine the efficacy of the reduction of microbial load and diversity on fresh produce following either in-package Atmospheric Cold Plasma or chlorine dioxide ClO₂ treatment.^ This work represents exciting and novel methods for rapid microbial detection and identification in a collaborative effort across six laboratories at two different universities.^

Degree

Ph.D.

Advisors

Bruce Applegate, Purdue University.

Subject Area

Biology, Microbiology

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