Graduate Student Exit Seminar: Phuong Le, June 12, 11-12, Bainer 2033

BAE Graduate Student Seminar 

Thursday, June 12, 2014 
11 am 12 noon, 2033 BAINER HALL

Topic: Rapid assessment of oxidative stress response in Escherichia coli and Listeria monocytogenes to evaluate efficacy of sanitation 

Phuong Le 
Biological and Agricultural Engineering 
University of California, Davis 

Sanitation is a critical step to reduce microbial load in many process industries. Particularly in the fresh produce industry, sanitation is used to reduce the risk of cross-contamination in wash water, minimizing foodborne illness. To assess the efficacy of sanitation processes, there is a need to rapidly measure microbial response to sanitizers and determine the critical concentration of sanitizers above which significant reduction in microbial load can be achieved. The goal of this study is to develop a label-free magnetic resonance (MR) imaging and relaxometry method, and an optical spectroscopy method to examine the oxidative stress response of Escherichia coli and Listeria monocytogenes to hydrogen peroxide (H2O2) and sodium hypochlorite (NaOCl). The central hypothesis is that cellular stress response of bacteria is a sensitive indicator of sanitation efficacy, which is measured by traditional plate counting method. 

The results show that oxidative stress response of Escherichia coli and Listeria monocytogenes can be measured using label-free (MR) imaging and relaxometry, based on the activity of catalase enzymes. The oxidative stress response of Escherichia coli and Listeria monocytogenes to H2O2 (0-1% v/v) was correlated with the bacterial reduction, quantified with traditional plate counting method. The comparison between the two methods shows the MR method developed in this study can determine the critical concentration of H2O2 above which a significant reduction in bacterial culturability (at least 4 logs) was achieved even in high organic load environment. In addition, MR method can assess sanitation efficacy of H2O2 for both gram-positive and gram-negative strains. 

Optical spectroscopy together with fluorescent probes was used to assess changes in intracellular reactive oxygen species, membrane permeability, and glucose uptake of Escherichia coli O157:H7 when treated with NaOCl and H2O2. The relative amount of intracellular reactive oxygen species was measured using Aminophenyl fluorescein (APF) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for cells treated with NaOCl and H2O2 respectively. Membrane permeability was measured using Propodium Iodide (PI). Glucose uptake was measured using 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG). Measurements of physiological changes in Escherichia coli O157:H7 using these diverse fluorescent probes were correlated with decrease in microbial viability, measured by plate counting. It was shown that 2-NBDG, but not PI, APF, or H2DCFDA, is a good surrogate measurement to assess reduction in bacterial culturability due to NaOCl and H2O2 treatment respectively. 
Overall, the study demonstrated two approaches to rapidly assess efficacy of sanitation using NaOCl and H2O2. These approaches have the potential to serve as surrogate methods for the traditional plate counting, which is the current gold standard used by the Food and Drugs Administrative, to provide a faster analysis of sanitation processes. 

Coffee and cookies will be served.