Performing Department
Food Science
Non Technical Summary
The overall aim of this research is to allow for a better understanding of the fundamental importance of commensal microbiota--in a given environment--on hNoV survival and transmission. We hypothesize that hNoV interacts with the leafy green phyllospheres as well as the surrounding production environment through 1) adsorption to the EPS, lipopolysacharride (LPS), and/or peptidoglycan (PG) of bacteria as a whole cell or as individual components and 2) interaction with other microbes found in the leafy green phyllosphere and production environment thus aiding in the persistence and transmission of hNoV.
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Goals / Objectives
ObjectivesTo characterize the interactions of human noroviruses (hNoVs) and hNoV surrogates with microbes identified as standard microbiota in the food production environment.To determine the role microbe-microbe interactions play in enhancing virus persistence and transmission.
Project Methods
Objective 1: To characterize the interactions of hNoVs and hNoV surrogates with microbes identified as standard microbiota in the food production environment.Confirmation of bacteria expressing HBGA-like substances. Pure cultures of the bacteria listed in Table 1 will be evaluated for expression of HBGA-like substances on the cell surface as described previously [30]. In addition to pure, characterized bacterial cultures, commercially available leafy greens will also be screened for candidate bacterial isolates following previously described methods [25]. Up to five candidate bacterial isolates will be identified by sequencing of the 16S rRNA gene using established primers 27F, 530F, 907R, and 1492R [30] and subsequently evaluated for HBGA-like substances.Measurement of binding activity of cultivable surrogates and HuNoV VLPs to bacterial cells. Virus binding to isolated bacterial cells will be investigated by ELISA and transmission electron microscopy (TEM).Isolation of EPS and LPS for binding assays. There is no standard method for the isolation of EPS therefore a previously described method for monoculture EPS isolation will be utilized [30]. Following EPS and LPS extraction, binding assays will be performed by ELISA as described for the whole cell assays with minor differences [30]. Similar to [27], commercially available biotinylated LPS will also be evaluated. Assay controls using enzymatic reactions will be used to confirm specific binding to each cellular component.Peptidoglycan binding assays. Following isolation of EPS from Gram-positive bacteria, the remaining bacterial cells will then be used to extract PG [44]. The isolated PG will be labeled with 0.5% Congo red and washed with saline followed by centrifugation to remove any unbound solution of Congo red. Propidium iodide will be added to the labeled PG that will be evaluated for qualitative and quantitative characteristics by scanning confocal microscopy using the facilities available through the Arkansas P3 Center. Binding assays will be performed as described previously [27]. Commercially available PG from B. subtilis will also be evaluated.Objective 2. To determine the role microbe-microbe interactions play in enhancing virus persistence and transmission.hNoV surrogate stability assays. Viruses alone will serve as a control. pH stability (2, 3, 7, 9, 10), ultraviolet irradiation (254 nm) at various exposure times, and bleach inactivation (200, 1,000, 5,000 ppm) on stainless steel surfaces for various contact times will be evaluated and the effect of bacteria and its cellular components on viral stability will be determined by plaque assay. In general, it is presumed that viral particles associated with environmental materials are physically more stable than freely moving viral particles [20].hNoV surrogate infectivity assays. To determine the effect of whole cell bacteria as well as the individual components (EPS, LPS, PG) on infectivity, traditional plaque assays will continue to be performed using the cultivable virus surrogates--MNV and AiV. These surrogates were selected based on their similarities as well as differences with hNoVs, specifically their receptor binding properties--TuV binds A/B antigens while MNV bind sialic acids-- and genetic relatedness. After exposure to bacterial cells and individual cell components under specific conditions (time, temperature, etc.), plaque assays will be performed as described previously. Impact of whole bacterial cells and their components on infectivity will be determined by an increase or decrease in plaque forming units.hNoV surrogate - microbe interactions: impact on sanitation technologies. We plan to evaluate the use of an ozone washer on inactivation of microbes on fresh produce in retail food service operations (FSOs). A batch wash ozone sanitation system (BWOSS) will be used to evaluate the efficacy of an ozone washer to inactivate bacteria, viruses, and FLP on fresh produce. This research will investigate the impact of microbe-microbe relationships (virus-bacteria and virus-FLP) on the fresh produce and the ability for the BWOSS to inactivate these pathogens when in combination. Here, the focus will be on leafy greens, culinary herbs, and microgreens.Preparation of bacteria for inoculation will be done in accordance with AOAC International Official Method 920.09 as described in [1] while hNoV surrogates (MNV, TuV, AiV) and FLP will be prepared as described previously with modification of final inoculum preparation in buffer equivalent to bacterial organisms [10,17]. Each fresh produce type will be prepared according to standard protocols prior to inoculation with microbes. Each produce sample will be inoculated with ~6 log CFU or PFU by distributing small droplets across the surface of the produce sample. The produce items will be stored overnight at 4°C prior to treatment in BWOSS. For a given experiment, samples will be collected every 10 min over a 40 min period; therefore, 12 units of each produce type will be needed including 2 positive controls and 2 negative, non-inoculated controls for time 0. At each time interval, 2 samples will be pulled and processed for microbial recovery by stomaching in Whirl-pak bags with phosphate buffer at 260 RPM for 1 min. Water samples will also be collected and neutralized with sodium thiosulfate at the end of the 40 min exposure period to determine presence of microbes. Detection of APC and TC will be performed using Petrifilm plates; bacteria will be plated on appropriate selective medium; and virus plaque assays will be employed for detection of viral surrogates and pathogens [10,17]. All microbe--produce type--contact time combinations will be completed in triplicate with duplicate biological replications, and separate experiments will be conducted with bacteria and viruses due to anticipated sample processing differences. For instance, for each microbe inoculated on cut leafy greens there will be 24 samples to analyze for a total of 216 samples.Initially, microbes will be analyzed separately as a control and then the specific interactions will also be evaluated. Bacteria will be incubated in 10ml of nutrient broth at 30°C overnight with shaking at 200 rpm, followed by centrifugation 8000 x g for 10min. The pellet will be adjusted to 109 CFU/ml then suspended in 10ml of PBS. Then 1ml of cell solution will be centrifuged at 3000 x g the supernatant will then be removed. The bacterial pellet will be suspended 400μl of adjusted hNoV surrogate at 105 PFU/ml and allowed to associate at room temperature for 1h. Virus and FLP cultivation and microbe-microbe association will follow [24] method with a few modifications. The experiment will be performed in sterile polystyrene test tubes containing 106 amoebas and 1ml Dulbecco modified Eagle medium (DMEM) and 1ml of virus. This will create a 1:1 ratio of FLP to virus. This will be incubated for 1 hour at 25°C, then centrifuged at 100 x g for 5 minutes. The pellet will be washed three times with PBS to remove the excess virus. The pellet will then be left in PBS at 25°C until used.Two separate scenarios introducing important risk factors will also be considered, and the BWOSS will be evaluated as a preventive control step. The first scenario will consider temperature abuse (7°C) during transportation/storage of fresh produce items. The second scenario will be related to BWOSS treatment of fresh produce with visible "decay juices" with and without removal of the associated decaying produce. Fresh produce will be inoculated as described above and then stored overnight at 4°C prior to treatment in the BWOSS.