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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Molecular Characterization of Foodborne Pathogens Research » Research » Research Project #441489

Research Project: Molecular Analysis of Foodborne Pathogen Responses to Stressors

Location: Molecular Characterization of Foodborne Pathogens Research

2022 Annual Report

Objective 1: Characterization of environmental and food-related stress responses of Shiga-toxin producing Escherichia coli (STEC). Sub-objective 1.1: Analysis of STEC O157:H7 responses to acid, heat and biocides with emphasis on the role of RpoS and Rcs regulons. Sub-objective 1.2: Genomic adaptation of environmentally-adapted STEC O157:H7 upon exposure to synthetic gastric fluid. Sub-objective 1.3: Genomic adaptation of environmentally-adapted STEC O157:H7 upon exposure to SOS-inducing stress. Objective 2: Molecular analysis of Campylobacter’s responses to biotic and abiotic stresses encountered in host- and food-environments. Sub-objective 2.1: Consequences of Campylobacter exposure to short-chain fatty acids. The molecular level effects on motility, attachment/invasion of eukaryotic cell lines, and biofilm formation. Sub-objective 2.2: Campylobacter molecular responses during co-incubation with bacteria isolated from poultry environments. The effects the other bacteria have on Campylobacter survival, aggregation (auto-aggregation and co-aggregation), attachment and biofilm development on poultry skin. Objective 3: Molecular analysis of Listeria monocytogenes’ responses to biotic and abiotic stresses encountered in food and food-processing environments. Sub-objective 3.1: Studies of sanitizer- and stress-induced viable-but-nonculturable (VBNC) state in L. monocytogenes. Sub-objective 3.2: Investigation of molecular responses of L. monocytogenes exposed to modiffied atmosphere packaging (MAP) in chicken meats using transcriptomics. Sub-objective 3.3: Investigation of genome evolution of L. monocytogenes exposed to long-term nutrient-limiting, non-selective stress condition. Objective 4: Phenotypic and genetic characterization of extra-intestinal pathogenic Escherichia coli (ExPEC) isolated from poultry and produce. Sub-objective 4.1: Analysis of ExPEC isolated from chickens and humans: biofilm assays, virulence gene profiles, antimicrobial resistance profiles, whole genome comparison of ExPEC strains isolated from chicken and human infections. Sub-objective 4.2: Transcriptomics of ExPEC strains in chicken meat.

The goal of this project is to use omic technologies (proteomic, genomic, and transcriptomics methods) and bioinformatics in a systems approach to understand how pathogens become resistant to food-related stresses, to determine their pathogenicity, and to identify markers for detection and typing. Pathogens that will be investigated include: Shiga toxin-producing Escherichia coli (STEC) and extraintestinal pathogenic E. coli (ExPEC), Campylobacter species, and Listeria monocytogenes. We will use omic technologies to analyze a large variety of strains of each of the pathogens to identify genes and proteins necessary for pathogens to survive stresses encountered in food environments and cause human illness. Research on pathogenic E. coli will focus on examining the association between acid tolerance in STEC and virulence potential, curli expression, biofilm formation, and persistence. This work will provide information to understand the virulence characteristics of STEC and how food environment-related conditions may impact the virulence and persistence in the food environment. We will examine poultry and swine as reservoirs for food-borne infections linked to ExPEC and STEC, respectively, and characterize isolated strains to determine their virulence. The omic data will also reveal genetic markers for identification, molecular typing, and detection of these pathogens. In previous work, we found that the use of certain polyphosphates commonly used during poultry processing increased the survival of Campylobacter by causing subtle changes in pH. Building on our previous research, we will investigate strain diversity and mechanisms of tolerance to stresses, including acid and exposure to antimicrobial compounds, as well as investigate factors affecting attachment and biofilm formation of Campylobacter. In addition, there has been limited effort to identify the microbial makeup of poultry and the processing environment and how these may provide a survival advantage for Campylobacter. Thus, we will investigate environmental stresses that affect the survival and persistence of Campylobacter during poultry processing and the role that the microbial ecology of this environment plays in this process. Finally, we will examine stress responses in L. monocytogenes and explore novel approaches to control this pathogen and determine the genes and proteins that help the pathogen overcome stresses. Genes that are essential for the survival and growth of L. monocytogenes under weak organic acid conditions in RTE meat will be determined. We will also investigate the effect of olive leaf extracts on inactivation of L. monocytogenes, and using transcriptomics, we will determine the molecular responses of this pathogen when exposed to the olive leaf extracts. The research will expand the knowledge on the survival mechanisms of important food-borne pathogens, provide insight into the evolution of pathogens, as well as tools to detect, identify, and type food-borne pathogens, and will assist in the development of practical preservation systems that minimize health risks and assist regulators in making science-based food safety decisions.

Progress Report
The specific goals of this projects address the need to understand how foodborne bacterial pathogens (Shiga toxin-producing and extraintestinal Escherichia coli (E. coli), Campylobacter jejuni, and Listeria monocytogenes) respond to selected food-production/processing related, and other relevant intrinsic and extrinsic stresses. There is a need to develop E. coli serotype O157:H7 nonantibiotic interventions that do not precipitate the stress-induced release and activation of virulence factor-encoded prophage and transferrable genetic elements. One method is to stimulate existing regulatory pathways that repress bacterial persistence and virulence genes. One such regulatory pathway is regulated through the prophage-encoded transcription factor (TF) PchE that inhibits biofilm formation and attachment to cultured epithelial cells by reducing curli fimbriae expression and increasing flagella expression. To identify pchE regulators that might be used in intervention strategies to reduce environmental persistence or host infections, we performed a computational search of the genome of an E. coli O157:H7 strain for pchE promoter sequences for binding sites used by known TFs. A common site shared by MarA/SoxS/Rob TFs was identified and the typical MarA/Rob inducers, salicylate and decanoate, were tested for biofilm and motility effects. Experiments revealed that sodium salicylate, a proven biofilm inhibitor, but not sodium decanoate, strongly reduced O157:H7 biofilms by a pchE-independent mechanism. Both salicylate and decanoate enhanced O157:H7 motility dependent on pchE using media and incubation temperatures optimum for culturing human epithelial cells. However, induction of pchE by salicylate did not activate the SOS response. MarA/SoxS/Rob inducers provide new potential agents for controlling O157:H7 interactions with the host and its persistence in the environment. As a follow-up to these experiments, we initiated a study profiling the changes in gene expression that occur when an environmentally-adapted serotype E. coli O157:H7 is exposed to sodium salicylate. We will also test the effect of sodium salicylate E. coli O157:H7 attachment and infectivity using cultured mammalians cells. We constructed a two-plasmid system for introducing genes into Campylobacter jejuni (C. jejuni) under the control of a constitutive promoter and then integrating the gene expression portion into the chromosome of a target strain. The first plasmid, the construction plasmid, pBlueKan+cysMPro, allows for easy placement of a gene of interest behind a kanamycin resistance marker gene and a constitutive cysM promoter. The second plasmid, the integration plasmid, pCJR01, possesses a highly conserved sequence of the C. jejuni’s 16S/23S rRNA region, allowing for integration of the cassette into the C. jejuni chromosome. We used non-motile C. jejuni strains, where the genes for the flagellar subunits flaA and flaB had been removed, to test our two-plasmid system for gene expression. We were successful in restoring partial motility to these strains using our system for the integration and expression of the flaA gene alone and the flaA and flaB genes in tandem. The development and application of these genetic tools were reported in a peer-reviewed publication, has resulted in two invention disclosures, and the plasmids are being deposited in a public non-profit plasmid repository so that they can be made available to other researchers. Progress has been made on studies to understand the effect of abiotic (short chain fatty acids; SCFAs) and biotic (Lactobacillus) stresses on Campylobacter jejuni. SCFAs are the product of bacterial fermentation within animal digestive tracts. This includes the digestive tracts of both poultry species grown for food and humans. Additionally, the digestive tracts of poultry are a reservoir for the human pathogens Campylobacter jejuni. Previous research on other food safety relevant bacteria has demonstrated that exposure to SCFAs has the potential for reducing the pathogenic abilities of those organisms. Therefore, environmental exposure to SCFAs and the bacteria that produce the SCFAs have the potential for protecting humans from Campylobacter. Our work this year has demonstrated that the three most common SCFAs found in the human digestive system (butyrate, acetate, and propionate) have a negative effect on the motility and to a lesser degree the biofilm formation of Campylobacter jejuni strains. Additionally, SCFA concentrations producing maximum negative effects on C. jejuni motility as well as the minimum concentrations necessary to produce motility effects in C. jejuni strains have been determined. In addition, co-incubation studies with C. jejuni strains and other bacterial strains isolated from poultry have also been completed to investigate the effect of biotic stresses due to the presence of polymicrobial communities. The non-campylobacter strains isolated from poultry have predominately belonged to the genus Lactobacillus. Through co-culture experiments we learned that the influence that the non-Campylobacter strains of Lactobacillus have on C. jejuni appear to be primarily tied to lactic acid production. Strong producers of lactic acid were found to have a negative effect on C. jejuni survival while bacteria that produce low levels of lactic acid seem to have little or no adverse effects on C. jejuni. Studies have begun on the role of the E. coli O157:H7 RpoS and Rcs regulons in the responses to environmental stresses. We have previously isolated several red variant (RV) mutants of E. coli O157:H7 strain 380-94 (strains RV01-RV13) with varying degrees of enhanced capability of Congo red binding and biofilm development. Limited (targeted) characterization of these mutants using PCR showed that four RV strains carried mutations in the rcs operon and 8 RV strains carried uncharacterized mutations. The regulation of biofilm and stress response are interconnected in E. coli O157:H7; thus, these RV strains will likely have mutations involved in the stress response pathways that enhance their survival under starvation or other stress conditions. This year, whole genome-sequencing (WGS) and targeted sequencing of specific stress genes/operons was undertaken. PacBio whole genome sequencing of the 13 E. coli O157:H7 RV derived from the stress-tolerant parent strain 380-94 is in progress and is expected to be completed by the end of this fiscal year. Analysis of the WGSs will focus on, but not be limited to, the presence/absence/mutations in the transmissible Locus of Stress Tolerance genomic island, prophage locations, stx and other virulence genes, as well as rpoS and rcs regulons. Primers were designed to sequence rpoS and rcs operons, PCR amplification of rpoS genes is completed, and Sanger sequencing of the rpoS PCR products will soon be completed for strain 380-94 and the 13 RV stains. The E. coli O157:H7 RV01-13 strains were tested for the catalase activity as a phenotypic indicator of the RpoS activity. Preliminary stress response tests will commence in the coming months. This year studies of the sanitizer- and stress-induced viable-but-not-culturable (VBNC) state in L. monocytogenes were initiated. Using bleach-treated L. monocytogenes as a model, molecular methodologies for determination of the VBNC status were tested including (1) viability PMA-qPCR (optimized reagent concentration and crosslinking time, tested DNA extraction kits and qPCR reagents and instruments); (2) live/dead staining of bacteria using microscopy and flow cytometer sorting. Additional methods examining cell membrane permeability and redox potential will be evaluated during the remainder of this year. In addition, development, and evaluation of a C. elegans bioassay to evaluate virulence potential of VBNC L. monocytogenes will be initiated during the latter part of this year. Investigations of genome evolution of L. monocytogenes exposed to long-term nutrient-limiting; non-selective stress condition have been initiated. L monocytogenes strains belonging to different serogroups have been collected for this study. Most of the strains have been fully sequenced, but stains for which genome sequences are not available were sequenced this year. Studies to determine the growth rates and capacity for biofilm formation of the L. monocytogenes strains in the collection are currently underway. Studies on the growth and long-term survival under nutrient-limiting conditions, as well as design and construction of strains with elevated mutation rate were also begun.

1. A Natural Antimicrobial Against L. Monocytogenes Derived from Olive Leaves. Foodborne pathogenic bacteria, including Listeria monocytogenes are a serious public health concern. Antimicrobial agents can be used to control the proliferation of L. monocytogenes and other foodborne pathogens, and consumers favor more natural antimicrobial agents to control pathogens in food. Olive leaf extract (OLE), derived from leaves of olive trees, has been used in traditional medicine for its health benefits, and it has potential application as a natural antimicrobial agent used as a food additive or incorporated into food packaging materials. However, mechanisms that contribute to the inhibition of growth of L. monocytogenes by OLE are unknown. ARS scientists in Wyndmoor, Pennsylvania studied gene expression of L. monocytogenes when exposed to OLE, revealing several potential antimicrobial targets. Information from this study indicates that OLE has the potential to be used as a natural antimicrobial to control foodborne pathogens in food and the food environment. In addition, the minimal inhibitory concentration (MIC), which is the lowest concentration of an antimicrobial agent that prevents growth of a bacterium, was determined for OLE against L. monocytogenes. Determining the MIC of OLE will reduce the cost of application and avoid undesirable changes in taste with use of high amounts of OLE. Knowledge of an effective and adequate MIC value is critical for the viable and broad applications of OLE by the food industry to control pathogens.

2. New Genetic Tools for Gene Expression in Campylobacter. According to the CDC the foodborne bacterium Campylobacter is the most common bacterial cause of diarrheal illness in the United States, leading to over 1.5 million illness each year. Despite its prominence and years of research, certain genetic tools needed to study this important pathogen are still lacking. ARS scientist in Wyndmoor, Pennsylvania developed two new genetic tools to accomplish the cloning and expression of exogenous genes in Campylobacter jejuni. The first tool makes the genes active and links them with a marker that is easily followed within C. jejuni. The second tool moves the activated gene and marker into the bacteria’s chromosome. The effectiveness of these tools was demonstrated by restoring C. jejuni’s motility to a strain of the bacterium in which necessary genes were deleted. The development and application of these genetic tools was reported in a peer reveiwed journal, has resulted in two invention disclosures, and they will be deposited in a public non-profit plasmid repository so that they can be made available to other researchers. The tools will help identify genes essential for Campylobacter virulence and persistence in foods, allowing us and others to develop interventions against this human pathogen.

Review Publications
Ceruso, M., Liu, Y., Gunther, N.W., Pepe, T., Anastasio, A., Qi, P.X., Tomasula, M.M., Renye Jr, J.A. 2021. Anti-listerial activity of thermophilin 110 and pediocin in fermented milk and whey. Food Control.
Huang, L., Hwang, C., Liu, Y., Renye Jr, J.A., Jia, Z. 2022. Growth competition between lactic acid bacteria and Listeria monocytogenes during meat fermentation – A Mathematical Modeling. Food Control. 158(2022):111553.
Du, R., Qu, Y., Zhao, M., Liu, Y., Qi, P.X., Sun, X. 2022. Logistic modeling to predict minimum inhibitory concentration of olive leaf extract against Listeria monocytogenes. PLoS ONE.
Wang, D., Wang, Y., Zhu, K., Shi, L., Zhang, M., Yu, J., Liu, Y. 2019. Detection of cassava component in sweet potato noodles by real-time loop-mediated isothermal amplification (Real-time LAMP) method. Molecules. 24(11), 2043.
Qin, X., Dong, R., He, S., Zhou, X., Zhang, Z., Cui, Y., Shi, C., Liu, Y., Shi, X. 2019. Characterization of the role of ybgC in lysozyme resistance of Salmonella Enteritidis. Food Control. 109:106732.
Huang, X., Zhou, X., Jia, B., Li, N., Jia, J., He, M., He, Y., Qin, X., Cui, Y., Shi, C., Liu, Y., Shi, X. 2019. Transcriptional equencing uncovers survival mechanisms of Salmonella enterica serovar enteritidis in antibacterial egg white. mSphere. 4(1). pdf.
Uhlich, G.A., Paoli, G., Kanrar, S. 2020. Escherichia coli serotype O157:H7 PA20R2R complete genome sequence. Microbiology Resource Announcements. 9(50): e01143-20.
Gunther, N.W., Kanrar, S., Uhlich, G.A. 2021. Complete genomic sequences of Campylobacter coli strains isolated from poultry sold in Pennsylvania farmers’ markets. Microbiology Resource Announcements. Available online: Microbiol Resource Announcements 10:e00015-21.