Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
(N/A)
Non Technical Summary
According to CDC and FDA, foodborne outbreaks from bacterial pathogens are persisting and slightly increasing, depending on the causal pathogen. This trend is, in spite an extensive effort to control and reduce enteric pathogens from food, is consistent and results in the need for new approaches to understand why the pathogens are continuing to persist in the food supply. Non-culturable bacteria are ever present in the food chain. Genetic diversity is associated with non-culturable capabilities that have not be systematically defined as to how this leads to persistance. With the increased use of whole genome sequencing (WGS) it is apparent that genomic diversity has a role in thwarting the control efforts in non-culturable bacteria but in pathogen detection as well. Coupled to expansive genome diversity, the most prevalent organisms adapt quickly to common bacterial control methods that lead to genome evolution. In conjunction with diversity, variable gene expression regulation has been found to alter foodborne pathogen success for persistence and entry to non-culturablility. These three areas have not yet been linked to increases in outbreaks in part because of the complex interplay between these underlying mechanisms that were not previously aligned for easy investigation. However, it is extremely plausible that the organisms persist to cause disease in humans via food vehicles that account for the long-term persistence of food-associated pathogens. One of the metabolic changes that is under-recognized is the ability of these bacteria to become non-culturable (NC). As we see with microbiome studies, NC organisms are dominant in the gut, the environment, and food. Induction of this metabolic state is easily achieved with processing methods (i.e. salt, restricted sugar, temperature changes, redox alteration) that would lead to false negative surveillance tests. Relatively little information is available for this capability for foodborne pathogens. However, by merging the current technologies - genome diversity, gene expression regulation, and induced evolution (niche adaptation) - it is now possible to untangle this possibility that would likely create new targets for pathogen control in the food chain.This shift is leading to more points of control that are difficult to manage or lead to evolution of niche adaptation by emerging genotypes that lead to new outbreaks. Controlling pathogens is a growing challenge that needs new methods and ideas of how to achieve this important goal. We propose to use bacteriophage specifically tuned to NC cells as a method to directly inhibit or kill so that they cannot resuscitate when in contact with the host cells. This approach is being used for biocontrol of food pathogens and has expansive potential as a method to clear the NC pathogen directly within the food.This proposal will define new control options for NC pathogens. The large genomic diversity of pathogens is a new concept but one that is expanding with every new WGS. This coupled to the limitations of enrichment methods to find Salmonella and Listeria, it is imperative that we provide new options for processing to restrict the growth and infectivity of bacteria. Importantly, the enteric pathogens in food also become NC and can remain 'hidden' from detection because they don't grow during enrichment or on plates. This proposal works at the intersection of: 1) population genomics, 2) infectivity and metabolic changes at NC, and 3) mitigation of NC cells with phage. In combination this work will define new targets to control NC pathogen persistence, and infectivity using Salmonella and Listeria as exemplars NC for foodborne pathogens.
Animal Health Component
15%
Research Effort Categories
Basic
65%
Applied
15%
Developmental
20%
Goals / Objectives
Hypothesis: NC pathogens are unwittingly induced using common food processing conditions that enable persistence of these metabolically different and undetectable organisms in food and the food processing environment using common plating methods.ObjectivesAim 1: Induce the NC state in Salmonella and Listeria. We will use methods described here to induce NC in both genera. Verification will be done as described with plate counts, PCR and ATP concentration to define the point of switch to NC from active growth.Aim 2: Determine the induced metabolic changes of NC pathogens. Once the pathways and genes are defined to be important in NC induction, their distribution in the genus will be determined using a population genome comparison. This result will enable identification of new diagnostic genes (or transcripts) to find NC pathogens in the food chain.Aim 3: Determine the ability of NC pathogens to resuscitate, express virulence, and produce adhesion factors using gut epithelial cells (gut association) and immune cells (mesenchymal stem cells) association. Using host association models and gene expression we will examine the exact gene targets for use in food. Specific mammalian-derived products (serum, bone marrow, etc.) will be added to examine resuscitation. We will specifically look for NC initiation, resuscitation, and metabolic routes that can be targets for reducing NC FBP.Aim 4: Determine bacteriophage infection for NC cells of Salmonella and Listeria. It is unknown if phage can be used to control NC cells, even though phage can infect NC cells. This aim seeks to determine the infection dynamics as a point of control for NC pathogens in the food chain.
Project Methods
Bacterial Strains and Growth Conditions: Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 (ATCC 700720; Salmonella WT) and 14028, Heidelberg, Schwarzengrund, Agona, Saint Paul and hypervirulent Typhimurium60 will be used in this study. All isolates will be grown in LB (Difco, BD) at 37°C with shaking at 220 rpm for 14-16 hours before use, harvested by centrifugation, washed twice with sterile saline. Strains will be inoculated into DMEM containing 0.45% glucose (pH 7.2) and 0.19 M MOPS buffer18,29. Samples will be taken daily to determine plate counts and ATP concentration (according to manufacturer's instructions - Promega). Samples will be collected daily for metabolomic analysis18,19,21,65,69. L. monocytogenes Scott A along with other serotypes from the 100K Pathogen Genome Project culture collection that were derived from outbreak strains will be used. At least five isolates of each serotype will be used to determine strain variation in NC induction.Bacterial Association Measurements: Bacterial association was determined using a modified gentamycin protection assay73,77,78 after adding each treatment in a 96-well plate containing an MOI of 1:100 to 1000. This will be done using colonic epithelial cells (Caco2)52,67 and stem cells.Metabolite extraction using Oasis SPE cartridge: Oasis HLB Cartridges will be conditioned with 5 mL of 100% methanol. Flow through was discarded. Cartridges will be equilibrated with 5 mL of LC grade water and flow through was discarded. One mL of sample was loaded into each cartridge and flow through was discarded. Cartridges will be washed with 10 mL of 5% methanol in LC grade water. Flow through was discarded. Cartridges containing the samples will be eluted with 2 mL of 100% methanol. Flow through was collected in 10 mL glass vials and samples will be lyophilized in speedvac with no heat. Dried metabolites will be suspended in 70 μL of 9:1 ACN:H2O. Glass vials will be put into 50 mL falcon tube and centrifuged at 500 rpm for 30 s. The samples (~70 μL) will be transferred into spring inserts in autoclaved 2 mL tubes. Samples will be sonicated for 10 min at 4?C, centrifuged at 5000 x g for 5 min at 4?C. Half of the sample (35 μL) was transferred to glass insert in Aliquot B tube (LC-1, HILIC) and the remaining amount (35 μL) was left in Aliquot A (LC-2, C18). Samples will be stored at -20?C prior to analysis.Instrumental analysis LC-MS to detect metabolic content. Liquid chromatography coupled to mass spectrometry (LC-MS) was carried out using two different analytical platforms to cover a wider number of non-volatile metabolites. The non-polar molecules will be determined by reversed-phase chromatography (RP) and the more polar molecules by hydrophilic interaction liquid chromatography (HILIC). Both procedures will be applied on an Agilent 1290 high-performance liquid chromatography system coupled to an Agilent 6230 time-of-flight (TOF) accurate mass spectrometer (Agilent Technologies, Santa Clara, CA). An electrospray source with an Agilent Jet Stream (AJS) nebulizer was used working in positive mode and acquiring a mass range between 50 and 1700 Thomson (m/z) at 4 spectra/sec and high-resolution mode. Fragmentor voltage was set at 120 V, sheath gas flow was 11 L/min, and sheath gas temperature was 350ºC. The standard of TFANH4 and purine was infused along with the sample. All the analyses will be carried out injecting a 5µL aliquot with samples housed in an autosampler maintained at 4°C. Method blanks prepared with water, acetonitrile and the resuspension mix (10% water in acetonitrile) will be analyzed along with the samples.RP separation was performed by using a Poroshell 120 EC-C18 column (2.7µm, 3.0 mm x 50 mm) from Agilent (Wilmington, DE) thermostated at 30°C. The initial mobile phase was a mixture of 1% phase A (60% acetonitrile in water) and 99% phase B (10% acetonitrile in isopropanol), both containing 10 mM of ammonium formate and formic acid. After injection, a phase B gradient was applied reaching 30% B in 4 min and then rising to 48% B in 1 min, 82% B in 17min and 99% B in 1 min. The total analysis time was 24 min with a flow rate at 0.3 mL/min. A standard solution Waters 6963 RP QC (Waters, Milford, MA) was used as RP quality control and injected along with the samples.HILIC analyses will be carried out by using an Acquity UPLC BEH Amide column (130A, 1.7µm, 2.1 x 100 mm) from Waters (Milford, MA) held at 30°C. Mobile phases consisted of water (A) and 90% acetonitrile in water (B), both at pH 5 with an ammonium acetate and acetic acid buffer.After injection, a linear gradient was applied from 0% A to 10% A in 20 min with a flow rate of 0.3 mL/min. Then, phase A was increased to 95% in 10 min while flow rate was reduced to 0.2 mL/min, to get back to the initial conditions for a total analysis time of 41 min. A Waters 1806006963 HILIC QC (Waters, Milford, MA) and a custom-made QC will be selected as HILIC quality controls. The custom QC include an aqueous mixture at 5 µM of carnitine, lysine, adenylputricine, aminocapricoic acid, ornithine, tigonelline, alaninol, acetylcarnitine, 1-(2 pyramidyl)piperazine, methoxychalcone, cholecalciferol, 13-docosenamide and oleamide.Bacteriophage infection. We will use a collection of phage that are infective to the strains used in both genera. Infection will be monitored using plaque assays with dividing cells to verify infection before using the phage treatment in NC cells. Cell suspensions of NC cells will be used with phage at various MOI so that turbidity change can be used to assess NC cell lysis. The appropriate MOI of phage will be used for cell culture assays to determine the impact of phage infection to inhibit host cell association and infection as described above.An overnight culture of the isolates was used to inoculate a fresh culture and incubated at 37°C for 3 hours. OD will be adjusted to 0.3 - 0.4 at 600 nm before addition of phage. The 5 ml cultures will be collected at 1000g for 20 min at room temperature and re-suspended in Butterfield's Buffer before transferring to sterile 96-well plates and exposed to phage or endolysin with gentle rocking. Automated OD600 will be every 30 minutes for 24 hours at 37°C.