Performing Department
Center for Food Safety
Non Technical Summary
Contamination of ready-to-eat (RTE) foods with pathogenic microorganisms continue to pose a serious risk to public health. For years, multiple cases of large-scale outbreaks and recalls due to environmental contamination with Salmonella enterica and Listeria monocytogenes have been reported. Reinforcement of current Good Manufacturing Practices, implementation of new FSMA regulations and deployment of new intervention strategies have yet to result in a marked reduction in the incidence of recalls and outbreaks; it is then critical to explore novel technological strategies. The environmental contamination with L. monocytogenes represents a serious risk for several commodities such as dairy foods, fresh produce and frozen foods as this bacterium is capable of widespread contamination of equipment and facilities for packing and processing. S. enterica is a foodborne pathogen of concern in low moisture foods, poultry, meat and produce and is capable of persisting on food contact surfaces.The use of a dynamic and harmless light technology during down time and close of operation could serve as a useful tool in preventing biofilm formation and persistence. Antimicrobial blue light (aBL) technology has been explored for hospital disinfection with very promising results, but its application to control foodborne pathogens remains relatively limited.The goal of this project is to investigate the application of aBL for reducing the surface viability of Listeria monocytogenes and Salmonella enterica biofilms, and of foodborne associated viruses. Specifically, this project will: 1) Determine optimal aBL emission doses to inactivate L. monocytogenes and Salmonella biofilms as single species on inert solid surfaces; 2) Assess the impact of mixed culture biofilms on aBL effectiveness to kill pathogenic bacteira; 3) Determine optimal conditions to inactivate norovirus, Hepatitis A virus and SARS-CoV-2; and 4) Characterize the possible interaction of aBL with sanitizers to inactivate pathogen biofilms. This project will include a series of laboratory experiments in which different surfaces will be inoculated with pathogenic bacteria biofilms and viruses, aBL-treated using different conditions and the effectiveness will be measured by a combination of viability determination and microscopy observations.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Goals / Objectives
Long-term goal: The goal of this project is to investigate the application of aBL for reducing the surface viability of Listeria monocytogenes and Salmonella enterica biofilms, and of foodborne associated viruses.Specific objectives and approach:Determine optimal aBL emission doses to inactivate biofilms of L. monocytogenes and Salmonella as single species on different inert solid surfaces.Assess the impact of mixed culture biofilms on aBL inactivation effectiveness.Determine optimal conditions to inactivate norovirus surrogates, Hepatitis A virus and SARS-CoV-2.Characterize the possible interaction of aBL with sanitizers to inactivate pathogen biofilms.
Project Methods
3.1.2.1 Bacterial strains: A 5-strain mixture of L. monocytogenes selected by their diverse biofilm-forming ability will include strains: 19115, 19117, Coleslaw, G1091, and 2011L-2626. The Salmonella cocktail will include a mixture of five serovars that will be selected for their ability to form biofilms. The strains that will be screened include the following serovars (strain ID): Typhimurium (ATCC 14028, UK1, E2009005811), Enteritidis (PT-30, M4639), Tennessee (E2007000502), Agona (F5567), Newport (AMO076, 11509-K, SN78), Oranienburg (1839) and Heidelberg (6316-J). Each strain will be activated from stock cultures by transfers in tryptic soy broth (TSB) supplemented with 0.6% yeast extract (TSBYE) and incubated at 35 °C for 24 h.3.1.2.2 Biofilm growth: Biofilms will be grown in a Drop Flow Biofilm Reactor (DFBR 600, Biosurface Technologies Corp.) which allows bacterial biofilms to be established on one side of surface plates and continuously submerged in inoculated media. Biofilms will be grown using an inoculum of 5 Log CFU/ml of the cocktail in 1:10 TSB to promote biofilm density for a duration of 48 h and 7 days. The plates will be washed with PBS to remove planktonic cells. The solid surfaces coated with biofilm will be used for the evaluation of the inhibitory effects of blue light.3.1.2.3 Blue light treatments: Biofilm-containing surface plates with an initial count of approximately 6 Log CFU/per plate will be removed from flow cells in the reactor and will be either allowed to dry for 48 h or treated within 2 h after removal (wet biofilms). The bottom face of surface plates and their edges will be disinfected by wiping with a 70% ethanol solution for 5 min. Surface plates will be placed under aBL lamps at distances from 20 to 100 cm depending on the dose and lamp power. Combinations of commercially available LED arrays at 405, 420 and 460 nm currently in use in our lab will be used. We currently have six 405 nm blue light LED array lamps , two 405 nm blue light LED array lamps , one 405 nm blue light LED array lamp , six 420 nm LED array lamps , three 460 nm LED array lamps , two radiometers to measure light intensity and a Gassen spectrometer to measure wavelength.3.1.2.5 Evaluation of biofilm disruption: Biofilm structure changes will be visualized using CSLM with a Zeiss LSM confocal laser scanning microscope (Carl Zeiss Microscopy). Serial images will be captured and processed by Zeiss Zen 2.3 software following staining with cell fluorescent markers: SYTO®9/propidium iodide, and fluorescent markers targeting extracellular matrix polysaccharides, proteins and DNA.3.2.2.1 Bacteria strains: The same strain mixtures of Listeria and Salmonella strains will be used in this series of experiments. For binary biofilms, the following Lactobacillus strains that we have used previously will be used: Lactobacillus (Lb) fermentum ATCC 14931, Lb. bavaricus LB5, Lb. plantarum CaTC2, Lb. sakei LB706, and Lb. buchneri NCDO110. Pseudomonas strains to be included in this project include: P. flourescens NCTC 10038, P. aeruginosa strains PAO1, NCTC 10332, ATCC 27853, and ATCC 15442.3.2.2.3 Blue light treatments: Chamber slides will be placed under aBL lamps at distances from 20 to 100 cm depending on the conditions determined in Objective 1. LED lamps at the wavelengths selected in Objective 1 will be used to treat coupons at the optimal temperature.3.2.2.4 Microbiological analysis: Chamber slides carrying biofilms will be sealed and transferred to ultrasonic bath for 2 min sonication to recover biofilm cells, and the contents will be transferred into tubes and vortexed. The viable cells in the PBS suspension released from the biofilms will be serially diluted and spread in triplicate onto TSAYE Petri plates. These plates will be incubated for 2 hours at 35 °C to allow recovery of injured cells and double strength molten MOX agar for Listeria and on XLD agar for Salmonella will be overlaid on top of the spread TSAYE. Petri plates will be incubated for an additional 24 h and typical dark colonies for each of these bacteria will be counted and CFU/surface plate. For quantification of Lactobacillus, MRS agar will be directly plated after serial dilution and incubated at 30 °C for 24 h. The Pseudomonas population will be estimated similarly by plating directly onto Pseudomonas isolation agar and incubating at 35 °C for 24 h.3.2.2.5 Fluorescence in-situ hybridization: Before FISH, biofilms will be subjected to the fixation and permeabilization steps depending on the biofilm composition comprising gram-positive and Gram-negative bacteria. Then, the procedure will be conducted as described by (Thurnheer et al., 2004) using 16S rRNA-targeted oligonucleotide probes with conjugates, resulting in different fluorescence of target cells. The microscopic examination will be performed with a Zeiss LSM confocal laser scanning microscope (Carl Zeiss Microscopy). Serial images will be captured and processed by Zeiss Zen 2.3 software (Carl Zeiss). Individual strains will be subjected to FISH to verify corresponding binding to probes and to identify any cross-reactivity.3.2.2.6 Evaluation of biofilm disruption: In parallel chambers, the aBL effect on 48 h and 7 days-biofilms will be determined following staining with cell fluorescent markers: SYTO®9/propidium iodide, and fluorescent markers targeting extracellular matrix polysaccharides, proteins and DNA: FITC-WGA/calcofluor white M2R, SYPRO® Ruby, and deoxyribonuclease 1, respectively. Confocal laser scanning microscopy with image analysis program (COMSTAT2) will be used to obtain qualitative and quantitative data on thickness, loss of structural features, changes in EPS composition and viability of biofilms.Because surfaces can be contaminated via both hand-contact fecal (unhygienic workers) and respiratory (coughing) droplets all viral inoculations on surfaces will be conducted with the viruses re-suspended in two human matrices: feces (human norovirus and hepatitis A virus) and nasal secretions (SARS-CoV-2) to mimic fecal and respiratory transmissions routes. In addition, for all viruses, water or soil as a viral matrix will be included to determine any reduction effects for interference substances on aBL. In order to be able to detect 3-4 log reduction, a high viral titer levels (8 log TCID50/ml) will be used. All research involving SARS-CoV-2 will be done at our CFS BSL3 laboratory according to approved protocols.Virus strains/propagation: The US reference strain SARS-CoV-2 USA-WA 1/2020 (provided by Dr. Jeff Hogan at UGA) will be propagated in Vero E6 cells, Hepatitis A virus (ATCC) will be propagated in FRhk-4 cells, and Tulane virus (provided by Dr. Qiuhong Wang at OSU) in LLC-MK2 as routinely done in our lab. SARS-CoV-2 and HAV will be generated in Dulbecco's Modified Eagle Medium (DMEM), while Tulane virus in Medium 199. All viruses will be generated in media supplemented with 2% Fetal Bovine Serum (FBS) and 1% antimycotic-antibacterial cocktail. Infected cells will be collected from the flasks, centrifuged at low speed (300 xgfor 10 minutes at 4 °C) to pellet the cell debris, while supernatants containing the viruses, will be aliquoted and stored at -80 °C. An aliquot of the viruses will be immediately titrated as described below. Because impurities in the cell culture media and intrinsic organic load from FBS can act as interfering agents reducing the viricidal effect of aBL, all generated viral stocks will be purified and further concentrated to 108 TCID50/ml using Amicon® 100K Ultra-15 (Millipore). The latter is also done to exchange the virus cell culture media with another relevant matrix (water, or 10% filter-sterilized soil, fecal or respiratory solution i.e. saliva).?