Location: Characterization and Interventions for Foodborne Pathogens
2023 Annual Report
Objectives
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.
Approach
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 project address the need to understand how foodborne bacterial pathogens (Shiga toxin-producing and extraintestinal Escherichia coli (E. coli), Campylobacter jejuni (C. jejuni), and Listeria monocytogenes) respond physiologically and genetically 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. This year, changes in gene expression that occur when an environmentally adapted serotype O157:H7 E. coli is exposed to sodium salicylate were profiled. The results of these experiments are currently being analyzed. Project scientists also tested the effect of salicylate on E. coli O157:H7 attachment and infectivity using cultured mammalians cells, and initiated a set of dual-RNASeq experiments to determine the effect of E. coli attachment on gene expression in both the bacterial and mammalian cells.
Project scientists previously reported the construction of a two-plasmid system for introducing genes into Campylobacter jejuni under the control of a constitutive promoter and then integrating the gene expression portion into the chromosome of a target strain. 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. This year, further studies on the constitutive promoter used in this gene expression system was found to have interesting properties that can be leveraged to construct a tunable promoter system with further patent potential.
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 pathogen 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.
We previously 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. This year we completed and published a study showing that butyrate decreases C. jejuni motility and biofilm formation by influencing the expression of a LysR-type transcriptional regulator. We investigated the effect that physiologically relevant concentrations of butyrate have on C. jejuni under in vitro conditions. Butyrate at concentrations of 5 and 20 mM negatively impacted C. jejuni motility and biofilm formation. These two traits are believed important for C. jejuni's ability to infect the lower intestines of humans. Proteomic analysis revealed differences in the amounts of several proteins in the presence of butyrate; the level 31 proteins were significantly reduced while the levels of 11 proteins were significantly increased. A LysR-type transcriptional regulator was one of the proteins that was in lower amounts in cells grown in the presence of butyrate. Additional studies showed that overexpression of the LysR protein in C. jejuni reduced butyrate’s effect on motility.
Previous 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. Through co-culture experiments we learned that the influence of Lactobacillus on C. jejuni appears to be primarily due to production of lactic acid. 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. Lactic acid can exist in two different forms (enantiomers D- or L-lactic acid). This year, studies revealed the degree to which the Lactobacillus makes either D-lactic acid or L-lactic acid influences its effect on C. jejuni. Additional, in vivo and in vitro studies will be necessary to understand the role of D- and L-lactic acid on C. jejuni survival.
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 was completed and the genomes, were assembled, annotated, and submitted to NCBI GenBank. 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. 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 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. In addition, development and evaluation of a greater wax moth Galleria mellonella bioassay to evaluate the virulence potential of VBNC L. monocytogenes was initiated. Growth and methods for injecting the moth larvae were established. Testing of L. monocytogenes virulence will be conducted later 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. This year studies of the growth rates and capacity for biofilm formation of the L. monocytogenes strains in the collection (30 strains) were conducted using different media and for 10 days. The results will be useful for strain selection for future long-term evolution studies; established basic bacterial growth parameters and culturing conditions. In addition, the feasibility of long-read sequencing technologies, established molecular techniques and, bioinformatic analysis workflow were evaluated. Studies on the growth and long-term survival under nutrient-limiting conditions, as well as design and construction of strains with elevated mutation rate continues.
Accomplishments
1. A chemical intervention to reduce infectivity of pathogenic E. coli. Enterohemorrhagic Escherichia coli (EHEC) cause severe hemorrhagic diarrhea that can progress to life-threating systemic syndromes. Traditional antibiotic treatments induce virulence gene expression in EHEC leading to more severe illness, sometimes resulting in death. There is a need to identify or develop non-antibiotic interventions that can interfere with EHEC infection, but do not activate EHEC virulence. In the present study EHEC genome sequences were analyzed to identify regulators and chemical inducers that can do just that. ARS scientists in Wyndmoor, Pennsylvania, discovered three potential regulators and cognate chemical inducers. Two were tested, and one, salicylate, was found to increase EHEC motility; reduce biofilm formation and reduce attachment of EHEC to cultured human colonic cells all without inducing EHEC virulence. Salicylate and other potential chemical inducers. Thus, salicylate and the identified cellular regulators represent factors upon which interventions could be developed to reduce infectivity of this deadly foodborne pathogen.
2. A naturally occurring compound may reduce infectivity of an important foodborne pathogen. Among cases of bacterial foodborne gastrointestinal diseases of humans, the bacterium Campylobacter jejuni stands out as causing the largest number of infections annually in the developed world. Infection by Campylobacter is most often associated with the consumption or preparation of undercooked poultry. ARS scientists in Wyndmoor, Pennsylvania, have shown that a compound (the short chain fatty acid, butyrate) naturally present in both poultry and human intestines reduced both motility and biofilm formation by Campylobacter jejuni at physiologically relevant concentrations. Furthermore, a specific cellular regulator associated with this response was identified. Motility and biofilm formation are important for Campylobacter infectivity; thus, both butyrate and the cellular regulator represent factors upon which interventions could be developed to reduce the number of human infections by this highly prevalent foodborne pathogen.
Review Publications
Yang, Y., Sommers, C.H., Sheen, S., Gehring, A.G., Liu, Y. 2022. Draft genomic sequence for a multidrug resistant Klebsiella pneumoniae 060517CS3-g isolated from retail ground chicken meat. Microbiology Resource Announcements. 12(1). https://doi.org/10.1128/mra.00949-22.
Chen, C., Kanrar, S., Paoli, G. 2022. Genome sequences of a stress resistant outbreak-associated Shiga Toxin-Producing Escherichia coli O157:H7 strain and a variant with enhanced congo red-binding capability. Microbiology Resource Announcements. 12(1). https://doi.org/10.1128/mra.00974-22.
Guragain, M., Schmidt, J.W., Kalchayanand, N., Dickey, A.M., Bosilevac, J.M. 2022. Characterization of Escherichia coli harboring colibactin genes (clb) isolated from beef production and processing systems. Nature Scientific Reports. 12. Article 5305. https://doi.org/10.1038/s41598-022-09274-x.
Gunther, N.W., Nunez, A., Bagi, L.K., Abdul Wakeel, A.Y., Ream, A.R., Liu, Y., Uhlich, G.A. 2023. Butyrate decreases Campylobacter jejuni motility and biofilm partially through influence on LysR expression. Food Microbiology. https://doi.org/10.1016/j.fm.2023.104310.
He, Y., Reed, S.A., Gunther, N.W., Armstrong, C.M., Capobianco Jr, J.A. 2023. Complete genome sequence of Campylobacter jejuni BSD5, a multidrug-resistant isolate from a poultry processing facility in the United States. Microbiology Resource Announcements. 12(6). https://doi.org/10.1128/mra.00284-23.
Guragain, M., Schmidt, J.W., Dickey, A.M., Bosilevac, J.M. 2023. Distribution of extremely heat-resistant Escherichia coli in the beef production and processing continuum. Journal of Food Protection. 86(1). Article 100031. https://doi.org/10.1016/j.jfp.2022.100031.
Msanne, J.N., Shao, J.Y., Ashby, R.D., Campos, P., Liu, Y., Solaiman, D. 2022. Draft genome sequences of the sophorolipid-producing yeast pseudohyphozyma bogoriensis ATCC 18809. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00566-22.
