Location: Characterization and Interventions for Foodborne Pathogens
2024 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 projects address the need for understanding how foodborne bacterial pathogens (Shiga toxin-producing and extraintestinal Escherichia 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.
Studies have begun on the role of the E. coli O157:H7 RpoS and Rcs regulons in the response to environmental stresses. Previously isolated 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 were screened by the polymerase chain reaction (PCR) to reveal that 4 strains carried mutations in the rcs operon and 8 strains carried uncharacterized mutations. In E. coli O157:H7 stress responses and biofilm formation are co-regulated; thus, it is suspected that the RV strains have mutations involved in the stress response pathways that enhance their survival under starvation or other stress conditions. This year, analysis of long-read whole genome-sequences of the 13 E. coli O157:H7 RV were completed and analyzed to identify the potential mutations in stress-related genes. Based on these analyses, more detailed investigations into the stress-responses in these RV strains will be undertaken. To this end, the RpoS-regulated catalase activity was determined in strains RV01-13 and results correlated well with the genotypic data. Additional genotypic and phenotypic stress response analyses are underway.
This year studies of the sanitizer- and stress-induced viable-but-not-culturable (VBNC) state in L. monocytogenes were continued. Previous work on this project involved the use of bleach-treated L. monocytogenes to generate the VBNC status. This allowed for the testing of molecular methodologies to evaluate the VBNC status including (1) viability PMA-qPCR (optimized reagent concentration and crosslinking time, tested DNA extraction kits and qPCR reagents and instruments); and (2) live/dead staining of bacteria using microscopy and flow cytometer sorting. This year, additional stress conditions that may induce VBNC were tested, including a sanitizer (peracetic acid), a biosurfactant (rhamnolipids), and bacteriocins. Furthermore, preliminary transcriptomic experiments and data analyses were conducted under selected conditions. Further investigation will continue to understand the mechanisms and genes/pathway involved in the VBNC phenotype.
Investigations of genome evolution of L. monocytogenes exposed to long-term nutrient-limiting, non-selective stress conditions are being conducted. L monocytogenes strains belonging to different serogroups were collected for this study. The genomes of all the strains selected for these studies have been sequenced. Studies on the growth rates and biofilm-formation of 30 strains of L. monocytogenes were conducted using different media and at different temperatures for an extended period of 10 days. The results were useful for strain selection for long-term evolution studies; establishing basic bacterial growth parameters and culturing conditions. This year a group of 4 of the L. monocytogenes strains with varying biofilm forming abilities were selected from this strain collection and further tested for growth/survival for up to 35 days. In addition, the feasibility of long-read sequencing technologies, established molecular techniques and, bioinformatic analysis workflows 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 rates continues.
Work continues to describe the regulatory control of C. jejuni’s cysteine synthase gene (cysM) a putative virulence factor for both poultry colonization and human disease. The single nucleotide polymorphism (A>G) at position 20 of the upstream untranslated region (UTR) of cysM has been shown to change transcriptional control from an inducible to constitutive form. The transcriptional start site for the promoter contained within the UTR has been determined. The inducible control demonstrated by the A-SNP form of the UTR has been shown to function through blocking transcriptional initiation at the previously identified proper transcription start site. This results in transcription initiation occurring at a multitude of different locations within the downstream gene’s coding region. This produces incomplete mRNA transcripts and likely truncated proteins resulting in blocking of the downstream gene function. Additionally, induction of the cysM gene has been demonstrated through the addition of sodium sulfide to C. jejuni strains growing in a minimal media devoid of sulfur. Current experiments are ongoing to determine if sulfur is the inducible element controlling the A-SNP form of the UTR’s transcriptional control and the exact mechanism of the control.
In addition to foodborne diarrheal disease some Escherichia coli strains can cause extraintestinal illness. Urinary tract infections are one example of extraintestinal illness caused by a group of uropathogenic E. coli (UPEC). The reservoirs for UPEC are not known, but recent studies suggest that UPEC might be foodborne. To examine the possibility that food might be a reservoir for ExPECs, a total of 79 extra intestinal pathogenic E. coli isolates (44 poultry and 35 human clinical-isolates) were confirmed by PCR, characterized for biofilm formation and antimicrobial resistance profiles, and sequenced. Comparative genome analysis revealed significant relatedness between human and poultry isolates, including virulence gene profiles. These results suggest poultry as a potential reservoir for UPEC. In addition, this work revealed unique characteristics and genes of UPEC that will allow the development of improved tools to screen high risk UPEC in food. Among six poultry UPEC strains genetically related to human clinical isolates, two strains were selected to examine gene expression on chicken meat. Growth modeling and preliminary experiments to determine the appropriate conditions for transcriptomic experiments are complete and transcriptomic experiments will commence within the next few months.
Accomplishments
1. Development of antimicrobial inhibitors through in silico computational research. Fosfomycin, a broad-spectrum antibiotic that has been used to treat deadly foodborne illness caused by the bacterium L. monocytogenes, works by inhibiting the activity of the MurA enzyme that is critical for bacterial cell wall formation. However, L. monocytogenes can develop resistance to fosfomycin, resulting in loss of effectiveness against the pathogen. In this study, ARS researchers in Wyndmoor, Pennsylvania, developed a computational-based approach to identify additional potential inhibitors to the action of MurA. Over 1.4 million compounds were screened computationally, and the 33 top compounds were screened for antibacterial activity. Two compounds that inhibited growth of both Listeria innocua and Escherichia coli were identified. These two compounds have the potential to be developed as new antibiotics. Furthermore, they could have potential in treating infections caused by fosfomycin-resistant bacterial strains. This work provides an innovative approach for identifying novel antibiotics to treat bacterial infections.
2. Characterization of extraintestinal pathogenic Escherichia coli (E. coli) from human clinical and poultry food samples. Worldwide, over 400 million cases of urinary tract infections are reported annually. Extraintestinal pathogenic E coli (ExPEC) is a major cause of urinary tract infections. However, reservoirs of these pathogenic E. coli are not fully understood. To examine the possibility that food might be a reservoir for ExPECs, a total of 79 extra intestinal pathogenic E. coli isolates (44 poultry- and 35 human clinical-isolates) were confirmed by PCR, characterized, and sequenced by ARS researchers in Wyndmoor, Pennsylvania. Forty-eight isolates were determined to be antibiotic resistant, 7 isolates showed reduced sensitivity to commonly used sanitizer, and 5 isolates were able to form an increased level of biofilm. Genome sequencing and analysis identified 65 virulence genes common between poultry and human isolates, suggesting the possibility of poultry as a reservoir for E. coli causing human urinary tract infections.
3. Antimicrobial resistance among meat-borne Escherichia coli (E. coli). While not all E. coli cause human illness, numerous outbreaks of foodborne diarrheal disease occur every year due to pathogenic E. coli. Sanitizers are used during meat processing to reduce the number of bacteria, including human pathogens. It has been shown that resistance to antibiotics may be associated with resistance to common sanitizers. Increased resistance to antibiotics and/or sanitizers confers a selective advantage to pathogenic bacteria such as diarrheagenic E. coli, both inside the animal host and on the meat processing surfaces. This increases the risk of these pathogens reaching consumers. In addition, antimicrobial resistant E. coli in meat may indicate the presence of other antibiotic resistant bacteria in the same environment, as bacteria are known to exchange genetic material. In this study, ARS researchers in Wyndmoor, Pennsylvania, examined the antibiotic and sanitizer resistance among 3,367 E. col isolated from meat and 1,218 antibiotic resistance E. coli were identified. Antibiotic resistance was higher among diarrheagenic E. coli than non-pathogenic E. coli. In this study sanitizer tolerance did not differ between antibiotic resistance and antibiotic susceptible E. coli. In addition, 27 multidrug resistant Shiga toxin-producing E. coli were characterized for resistance to common interventions used in the meat industries (lactic acid and quaternary ammonium compounds) and biofilm formation. These findings contribute to understanding the emerging risk of antimicrobial resistant foodborne E. coli.
4. Developed an end-to-end pipeline for high throughput analysis of Escherichia coli (E. coli) genomes. Genomes of important foodborne pathogens like Salmonella and E. coli are routinely sequenced for clinical surveillance and scientific research. Assembly and analysis of whole genomes is complex and time consuming, hence requires efficient software able to assemble and analyze multiple genomes on a larger scale. ARS scientists in Wyndmoor, Pennsylvania,and Clay Center, Nebraska, developed a set of computational tools referred to as the GEA (Gammaproteobacteria Epidemiologic Annotation) pipeline. The pipeline was built, tested, and validated for assembly, analysis, characterization, and annotation of large batches of E. coli and Salmonella genomes using the Center for Genomic Epidemiology command-line tools and high-performance computing. High volume annotations of 14,000 Salmonella genomes and the in-house analysis of 88 genomes of pathogenic E. coli demonstrated robustness of the pipeline.
Review Publications
Armstrong, C.M., He, Y., Chen, C., Counihan, K.L., Lee, J., Reed, S.A., Capobianco Jr, J.A. 2023. Use of a commercial tissue dissociation system to detect Salmonella-contaminated poultry products. Analytical and Bioanalytical Chemistry. https://doi.org/10.1007/s00216-023-04668-w.
Guragain, M., Bagi, L.K., Schmidt, J.W., Paoli, G., Kalchayanand, N., Bosilevac, J.M. 2024. Antibiotic resistance and disinfectant resistance among Escherichia coli isolated during red meat production. Journal of Food Protection. 87(6):100288. https://doi.org/10.1016/j.jfp.2024.100288.
Guragain, M., Kanrar, S., Bagi, L.K., Chen, C. 2023. Complete closed genome sequences of three multidrug resistant uropathogenic Escherichia coli. Microbiology Resource Announcements. 12(10):e00422-23. https://doi.org/10.1128/MRA.00422-23.
Armstrong, C.M., Capobianco Jr, J.A., Nguyen, S.C., Guragain, M., Liu, Y. 2024. High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA. Scientific Reports. 14:7026. https://doi.org/10.1038/s41598-024-56911-8.
Dickey, A.M., Schmidt, J.W., Bono, J.L., Guragain, M. 2024. The GEA pipeline for characterizing Escherichia coli and Salmonella genomes. Scientific Reports. 14. Article 13257. https://doi.org/10.1038/s41598-024-63832-z.
Hanes, R., Liu, Y., Huang, Z. 2024. Druggability analysis of protein targets for drug discovery to combat Listeria monocytogene. Microorganisms. 12(6):1073. https://doi.org/10.3390/microorganisms12061073.
He, Y., Capobianco Jr, J.A., Armstrong, C.M., Chen, C., Counihan, K.L., Lee, J., Reed, S.A., Tilman, S.M. 2024. Detection and isolation of Campylobacter spp. from raw meat. The Journal of Visualized Experiments (JoVE). http://dx.doi.org/10.3791/66462.
Wang, R., Guragain, M., Chitlapilly Dass, S., Palanisamy, V., Bosilevac, J.M. 2024. Impact of intense sanitization on environmental biofilm communities and the survival of Salmonella enterica at a beef processing plant. Frontiers in Microbiology. 15. Article 1338600. https://doi.org/10.3389/fmicb.2024.1338600.
Liu, Y., Kanrar, S., Elder, J., Ream, A.R., Huang, L., Gehring, A.G., Uhlich, G.A., Clabots, C., Johnson, J.R. 2023. Whole genome sequencing and characterization of Escherichia coli human isolate DP033. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00792-23.
Yang, Y., Sommers, C., Liu, Y. 2024. Draft genomic sequence for uropathogenic Staphylococcus saprophyticus ATCC 49453. Microbiology Resource Announcements. https://doi.org/10.1128/mra.01142-23.
