Location: Characterization and Interventions for Foodborne Pathogens
2021 Annual Report
Objectives
1: Molecular characterization of Shiga-toxin producing Escherichia coli (STEC) and extra-intestinal pathogenic E. coli (ExPEC) with specific emphasis elucidating the responses to food-related stresses, and genomic and proteomic studies to assess virulence and to identify genetic markers for detection and typing.
1A: Perform molecular characterization of acid tolerance in STEC.
1B: Perform molecular characterization of ExPEC.
1C: Develop molecular genoserotyping and pathotyping platforms for E. coli.
ID: Characterization of STEC isolates from swine.
1E: Develop and evaluate immunologic-based methods for detection of STEC.
2: Genomic and proteomic analysis of Campylobacter with emphasis on virulence and the molecular characterization of the effects of acidification and other food-processing related stresses on survival Campylobacter in poultry products.
2A: Determine composition and effects that different poultry exudates play in the survival of the contaminating Campylobacter species.
2B: Investigate attachment and formation of biofilms by Campylobacter species on poultry skin in the presence of different poultry exudates.
2C: Investigate practical methods, chemical and microbiological based, for acidification of poultry exudate and their effects on the survival of contaminating Campylobacter spp.
3: Functional and molecular characterization of L. monocytogenes serotypes with emphasis on elucidating responses to food-related stresses through functional genomics; and determining virulence differences among L. monocytogenes strains and serotypes through comparative genomics.
3A: Determine strain variations in growth/survival with exposure to weak organic acids and olive leaf extracts among different L. monocytogenes serotypes.
3B: Determine genes that are essential for the survival and growth of L. monocytogenes under weak organic acid conditions in RTE meat.
3C: Investigate molecular responses of L. monocytogenes exposed to the olive leaf extracts using transcriptomics.
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
Work on the project plan continues to progress well, with the goals of the three sub-objectives for the 60th-month milestones having all been fully or substantially met.
Pathogenic bacteria known as extraintestinal pathogenic E. coli (ExPEC) are important causes of infections, including urinary tract infections, bloodstream infections, and meningitis. Food may be a source of ExPEC strains that cause human infections; therefore, a better understanding of the relationship between human clinical ExPEC strains and strains found in food animals is needed. Initial work on ExPEC focused on characterizing hundreds of strains isolated from humans and poultry in collaboration with another ARS scientist at Wyndmoor, Pennsylvania. Genetic-based PCR methods were used to characterize different ExPEC strains and trace the association between foodborne ExPEC and human diseases. In addition, significant progress was made on determining the prevalence of ExPEC in poultry. In further studies, the genome sequences of several clinical and food isolates of ExPEC were determined. The genome sequence from ExPEC strain FEX669 revealed that this chicken isolate contained numerous virulence factors that could facilitate extraintestinal and even systemic infection and provided useful information to understand how poultry may be an important source for human ExPEC infections. Three clinical ExPEC strains belonging to E. coli Sequence Type 131 (ST131), strains H45, H43ii, and H43iii, recovered from urine samples of patients in Lagos, Nigeria, were also sequenced. The importance of these three ST131 strains is that this group of pathogens has emerged as a leading cause of urinary tract and bloodstream infections in humans, and they are resistant to many antibiotics. The prevalence of E. coli ST131 is possibly attributed to its increased antibiotic resistance, enhanced virulence (disease-causing potential), and greater propensity to transfer genetic materials than non-ST131 E. coli. The genomic information from these strains is useful for understanding the dissemination and pathogenicity of E. coli ST131, as well as for facilitating the development of novel antimicrobial therapies. In addition, the complete genome sequence of ExPEC strain FEX669, isolated from retail chicken meat, was determined. A total of 27 virulence genes were identified on the FEX669 chromosome. They included genes for iron acquisition (chuA, fyuA, iroN, sitA, iucC, irp2, and iutA), attachment (hra, focC, sfaD, and yfcV), and toxin production (hlyF, vat, and usp). The food product origin of strain FEX669 and the presence of virulence genes that could facilitate extraintestinal and even systemic infection is a cause for concern. Genomic sequencing is a critical first step to understand the epidemiology of ExPEC in humans and chicken and their potential to cause illness. The information is important for the development of strategies to control ExPEC and prevent contamination from poultry products.
Related to Objective 2, we have investigated methods for the reduction of Campylobacter strains in poultry products. This effort included measuring Campylobacter survival in a competitive growth experiment with lactobacillus in chicken livers and liver exudate. Certain collections of naturally occurring lactobacillus strains derived from cow and goat products were shown to significantly reduced Campylobacter strains during co-incubation in chicken livers. Previously, we demonstrated that the short-chain fatty acid, butyrate, could reduce Campylobacter motility and biofilm formation in a dose-dependent manner. Following up on this work, we used a whole-cell comparative proteomic approach to study Campylobacter attachment/biofilm formation in the presence of butyrate. From this research, we observed that the expression of the lysR regulatory gene is significantly reduced in the presence of butyrate. To fully characterize the effect that lysR has on Campylobacter motility and biofilm formation, we constructed an isogenic “knock out” mutant of lysR. Our ongoing research is attempting to determine the exact phenotypic results that inactivating this specific gene has on Campylobacter biofilm formation. After observing that the primary impact of poultry exudate on Campylobacter survival was the result of the acidity levels of the exudate, we investigated whey, a naturally acidifying agent, in our studies. Whey samples contain lactic acid bacteria (LAB) populations that are likely in large part responsible for the acidity of the whey. The LAB containing acidic whey samples derived from multiple animal sources and at different times during the year were applied to a cocktail of Campylobacter strains to observe their effects on Campylobacter survival. Subsequently, individual LAB strains were isolated from the whey samples and individually tested for their influence on Campylobacter survival. A collection of different LAB species was identified that significantly reduced Campylobacter survival. Several of the strains produced organic acids that were responsible for the observed Campylobacter reductions. However, several of the LAB isolates did not appear to acidify their immediate environment. It currently appears that several of these LAB isolates produce antimicrobial peptides called bacteriocins that have activity against Campylobacter. We are currently in the process of more fully characterizing these potentially unique antimicrobial elements.
Related to Objective 3 of the project plan, progress was made towards understanding the response of L. monocytogenes upon exposure to olive leaf extract (OLE). OLE is an herbal supplement with antimicrobial properties and is considered beneficial to human health. Therefore, OLE was explored as a natural antimicrobial to control foodborne pathogens in food. OLE inhibited the growth of L. monocytogenes in milk. The survival of L. monocytogenes in milk with different concentrations of OLE was conducted to determine the inactivation rates and the optimal dose of OLE for gene expression analyses. L. monocytogenes was treated with a sub-lethal dose of OLE and gene expression was measured by RNA-Seq. Changes in expression were observed for numerous genes. The data is being analyzed to understand the specific genes and genetic mechanisms required for growth and survival in the presence of OLE. This information will be helpful for the design of interventions that will allow the inactivation of L. monocytogenes. Additional research showed that OLE could be used in an antimicrobial film to inhibit the growth of foodborne pathogens, indicating the potential use of OLE as food packaging material.
Accomplishments
2. Pathogen typing methods to track foodborne pathogens in food-processing environments. Listeria monocytogenes and Salmonella species are important foodborne pathogens that cause many cases of human illness. Since these pathogens can cause sporadic or persistent environmental contamination in food processing facilities, multiple environmental sampling tests (ESTs) have been developed to detect contamination with these pathogens. In this study, ARS scientists at Wyndmoor, Pennsylvania, developed strain typing methods for the analysis of presumptive-positive ESTs to track down sources of contamination. Results showed that the strain typing methods for L. monocytogenes and Salmonella are reliable, robust, and highly discriminatory; able to accurately distinguish strains of the two pathogens. These methods allow food processors to rapidly and accurately monitor and track down sources of contamination in processing facilities to prevent contaminated food from reaching the consumer.
Review Publications
Elders, J., Liu, Y., Kanrar, S., Gehring, A.G., Sommers, C.H., Johnston, B.D., Johnson, J.R. 2021. Complete genome sequence of Escherichia coli strain FEX669, a ColV plasmid-containing isolate from retail chicken meat. Microbiology Resource Announcements. 10(8):e01340. https://doi.org/10.1128/MRA.01340-20.
Gunther, N.W., Abdul Wakeel, A.Y., Reichenberger, E.R., Al-Khalifa, S., Minbiole, K.P. 2018. Quaternary ammonium compounds with multiple cationic moieties (multiQACs) provide antimicrobial activity against Campylobacter jejuni. Food Control. https://doi.org/10.1016/j.foodcont.2018.06.038.
Baranzoni, G., Fratamico, P.M., Boccia, F., Bagi, L.K., Kim, G., Anastasio, A., Pepe, T. 2017. Evaluation of the performance of the IQ-check kits and the USDA microbiology laboratory guidebook methods for detection of Shiga Toxin-Producing E. coli (STEC) and STEC and Salmonella simultaneously in ground beef. Journal of Applied Microbiology. 122:809-816.
Cha, W., Fratamico, P.M., Ruth, L., Bowman, A., Nolting, J., Manning, S., Funk, J. 2018. Prevalence and characteristics of Shiga toxin-producing Escherichia coli in finishing pigs: implications on public health. International Journal of Food Microbiology. 264:8-15.
Debroy, C., Fratamico, P.M., Roberts, E. 2018. Molecular genotyping of Escherichia coli O-serogroups. Animal Health Research Reviews. https://doi.org/10.1017/S1466252317000093.
He, Q., Liu, Y., Liu, D., Guo, M. 2021. Integration of transcriptomic and proteomic approaches unveils the molecular mechanism of membrane disintegration in Escherichia coli O157:H7 with ultrasonic field treatment. Science of the Total Environment. https://doi.org/10.1016/j.scitotenv.2021.148366.
Elders, J., Fratamico, P., Liu, Y., Needleman, D., Bagi, L.K., Tebbs, R., Allred, A., Siddavatam, P., Suren, H., Gujjula, K., Debroy, G., Dudley, E.G., Yan, X. 2021. A targeted sequencing assay for serotyping Escherichia coli using AgriSeq technology. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.627997.
Zhang, F., Zhai, T., Haider, S., Liu, Y., Huang, Z. 2020. Synergistic effect of chlorogenic acid and caffeic acid with Fosfomycin in growth inhibition of a resistant Listeria monocytogenes strain. ACS Omega. 5(13):7537-7544.
Zhang, Z., He, S., Yang, B., Shi, C., Liu, Y., Shi, X. 2021. Genomic characterization of an extensively drug-resistant chicken-borne Salmonella Indiana isolate carrying an IncHI2-IncHI2A plasmid. Food Control. https://doi.org/10.1016/j.foodcont.2020.107761.
Wang, Y., Zhang, M., Wang, D., Zhang, Y., Jiao, X., Liu, Y. 2020. Development of a real-time LAMP assay for monofloral honey authentication using rape honey. CyTA - Journal of Food. https://doi.org/10.1080/19476337.2020.1749135.
Zhang, Z., Chang, J., Xu, X., Zhou, M., Shi, C., Liu, Y., Shi, X. 2021. Dissemination of IncFII plasmids carrying fosA3 and blaCTX-M-55 in clinical isolates of Salmonella Enteritidis. Zoonoses and Public Health. 2021;00:1–9. DOI: 10.1111/zph.12825.
Ceruso, M., Clement, J., Zhang, F., Huang, Z., Pepe, T., Anastasio, A., Liu, Y. 2020. The inhibitory effect of plant extracts on growth of the foodborne pathogen, Listeria monocytogenes. Antibiotics. 9(6) https://doi.org/10.3390/antibiotics9060319.
Li, C., Zhang, Z., Xu, X., He, S., Zhao, X., Yan, C., Xiujuan, Z., Shi, C., Liu, Y., Zhou, M., Shi, X. 2021. Molecular characterization of Cephalosporin-resistant Salmonella Enteritidis ST11 isolates carrying blaCTX-M from children with diarrhea. Foodborne Pathogens and Disease. https://doi.org/10.1089/fpd.2020.2878.
