Understanding bacterial survival in protective microenvironments for improved response strategies for food defense

Bonnie M Wang, Purdue University

Abstract

Cells in solution are in their least protective state from antimicrobial agents because interaction occurs from all directions. Attaching themselves to surfaces increases their protection from cleaning and disinfection by decreasing the volume of exposure to treatment, while forming protective states such as spores and biofilms enhances resistance and ability to survive. Cells obtain even greater protection in microenvironments which further limits accessibility of antimicrobial agents to those cells deep within the microenvironment. The inability to effectively treat pathogens in protective microenvironments can lead to recontamination of the area outside the microenvironment. Thus, removing and/or eliminating pathogens in microenvironments are crucial in responding to a microbial contamination event. This study evaluated the effect of protective microenvironments (e.g. groove) on food contact surfaces (e.g. stainless steel) on the efficacy of antimicrobial agents (e.g. hypochlorite) for treating biofilms (e.g. Escherichia coli O157:H7) and spores (e.g. Bacillus subtilis). Eight hour Escherichia coli O157:H7 biofilms formed on stainless steel test surfaces with and without fabricated grooves (0.5 in long, 100 or 500 µm wide, and 100, 400, 600, or 750 µm deep) using a flow-through system were treated with hypochlorite solution (20, 200, and 2000 ppm) and gaseous chlorine dioxide (0.7, 1.7, and 2.2 mg l- 1) for 5, 10, and 30 min. A surface isolation system was developed to sample only regions of interest on the test surfaces, as well as ascertain regrowth of treated cells in rich medium post treatment. Results from hypochlorite studies showed that while 20 ppm hypochlorite for 5 min was effective in treating cells in solution and cells attached to flat ungrooved surfaces, treatment was ineffective for disinfecting 8 hr biofilms on test surfaces. Although initial plate count data from 200 ppm hypochlorite for 5 min treatment did not detect viable cells suggesting treatment was effective, the regrowth assay indicated the presence of E. coli O157:H7 post treatment concluding treatment was not successful. When treatment time was increased to 10 min, biofilm on flat ungrooved surfaces were successfully disinfected, but one out of the sixteen 750 µm deep grooved samples still survived. In evaluating narrower grooves 100 µm wide, increased concentration and time (2000 ppm for 30 min) were needed to disinfect the 8 hr biofilm in the groove. Gaseous chlorine dioxide treatment studies on 8 hr biofilms showed while 1.7 mg l-1 for 10 min was sufficient to disinfect the biofilm on the ungrooved surfaces, 2.2 mg l-1 for 10 min was needed to disinfect the biofilm on the deep (750 µm) and wide (500 µm) grooved surfaces. As found with the hypochlorite studies, a longer treatment time of 30 min was needed to successfully disinfect the biofilm on deep and narrow (100 µm) grooved surfaces. Bacillus subtilis spores spotted on the test surfaces and treated with chlorine dioxide confirmed results and suggested that the presence of water in the groove protects the spores from treatment. Drying and dehydration experiments showed that grooves decrease the evaporation rate of water which in turn enhances survival. In summary, deep and narrow grooves decrease treatment efficacy and increase the risk of recontamination by protecting cells in the biofilm or spores from treatment. Grooves with a depth to width ratio greater than one were found to exhibit greater protection against antimicrobial agents. Efficacy of cleaning and disinfection should therefore be validated on these protective surfaces.

Degree

Ph.D.

Advisors

Nivens, Purdue University.

Subject Area

Food Science

COinS