Progress 02/10/16 to 02/09/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Record of Any Impact of Maximized Teleworking Requirement: Though all milestones were at least partially met, the limited access to the lab has had an obvious impact on the pace of research progress. ACCOMPLISHMENTS 02 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.
Impacts
(N/A)
Publications
- 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.
- 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.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Work pertaining to the project plan continues to progress well with the goals of the three sub-objectives for the 48th month milestones having all been substantially met. Specifically, as part of a collaboration with the Food Safety Inspection Service (FSIS) we have investigated methods for the reduction of a cocktail of Campylobacter strains in poultry products. This effort included measuring Campylobacter survival in 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 (Sub- objective 2A, 48 month). Previously, we demonstrated that the short chain fatty acid, butyrate, was able to, in a dose dependent fashion, reduce Campylobacter motility and biofilm formation. 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 on-going 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 began investigating whey, a naturally acidifying agent, in our studies. Whey samples contain lactic acid bacteria (LAB) populations that are likely in a 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 were 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. This past year, progress was made on all three main objectives. The research focuses on using omics technologies and systems biology to understand how foodborne pathogens tolerate stresses encountered in food environments and how food processing conditions may induce resistance to stresses. The research is also focused on identifying food and animal reservoirs for emerging foodborne pathogens, and work will provide information for understanding how these pathogens cause disease in humans and identification of genetic markers for detection and typing. Related to Objective #1 of the project plan, significant progress was made in developing diagnostic method for molecular serotyping of E. coli. Traditionally, serotyping has been used to distinguish > 180 different E. coli O-serogroups (O-antigen) and 53 H-types (H-flagellar antigen) based on cell surface structures). However, this procedure, while laborious and often inaccurate, can only be performed in specialized laboratories. In our research, more than 70 genomes of E. coli reference O-group strains were analyzed to determine the DNA sequence of the cluster of genes involved in production of cell surface polysaccharides that define the different E. coli O-serogroups. This research was intended to develop more rapid and simple methods for detecting, typing, and identifying different serogroups of pathogenic E. coli. Working with CRADA partners, including the E. coli Reference Center at Pennsylvania State University and Life Technologies Corporation, we determined the unique genes that can be targeted to identify the different O- and H-groups. Based on this genetic information, a molecular DNA sequencing-based platform known as AmpliSeq was developed to determine the presence of O- and H-group genes, as well as virulence genes (involved in causing disease) in E. coli strains. The specificity of the method was tested with all the E. coli reference strains and the other field strains isolated from humans, animals, and the environment. The new developed molecular method is inexpensive and will greatly enhance the ability to identify, detect, and type pathogenic E. coli and will eliminate the use of the labor-intensive and inaccurate traditional serotyping procedure. Other work focused on characterizing hundreds of extraintestinal pathogenic E. coli (ExPEC) strains that were isolated from human and poultry in collaboration with another ARS scientist at Wyndmoor, Pennsylvania. ExPEC that are present in produce, poultry and meat can cause illness in humans. Molecular techniques were used to trace the association between foodborne ExPEC and human diseases. Significant progress was made on determining the prevalence of ExPEC in poultry. Genetic-based PCR methods were used to characterize different ExPEC strains. The genome sequence of several ExPEC isolates from human was determined (#8), which 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 development of strategies to control ExPEC and prevent contamination from poultry products. Understanding the nature and behavior of extraintestinal pathogenic E. coli (ExPEC) strains through whole genome sequencing. Pathogenic bacteria known as extraintestinal pathogenic E. coli (ExPEC) are important causes of infections, including urinary tract infections, bloodstream infections, and meningitis. The source of these pathogens is believed to be foodborne. ARS researchers at Wyndmoor, Pennsylvania, determined the complete sequence of the DNA of three ExPEC strains known as E. coli Sequence Type 131 (ST131), strains H45, H43ii, and H43iii, recovered from urine samples of patients in Lagos, Nigeria was determined.The importance of these three ST131 strains is that this group of pathogens has emerged as a leading cause 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 compared to 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. Related to Objective #3 of the project plan, significant progress was made towards understanding the survival mechanisms of L. monocytogenes after exposure to olive leaf extract (OLE). Beneficial to human health, OLE is an herbal supplement with antimicrobial properties. Therefore, OLE was explored as a natural antimicrobial to control foodborne pathogens in food. Lab results showed that OLE inhibited 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 to use for gene expression analyses. Bacterial cells treated with sub-lethal dose of OLE were used to study gene expression profiles. The RNA-Seq method was used to measure the level of gene expression in L. monocytogenes after exposure to OLE. Increased or decreased expression of a number of genes have been identified, which provides the information essential to understanding the specific mechanisms and genes required for growth /survival in food- related stress conditions. This information is also necessary for the design of interventions that will allow complete inactivation of L. monocytogenes. Additional research showed that OLE can be used as an antimicrobial film to inhibit the growth of foodborne pathogens, indicating the potential use of OLE as food packaging material, which will be further explored in the future. Butyrate signaling through lysR reduces Campylobacter mobility and biofilm formation. The application of butyrate in concentrations sufficient to reduce Campylobacter mobility and biofilm formation were determined to also reduce the expression of the lysR gene, a proposed transcription regulator. A Campylobacter strain lacking a functional version of the gene was constructed in order to determine specifically how this gene product contributes to Campylobacter motility and biofilm formation. Progress was also made in search for new antimicrobials for use as food additives and preservatives to inactivate pathogens and to increase the shelf life of foods. Compared to synthetic compounds, plant extracts are generally more likely to be accepted as generally recognized as safe (GRAS), may have a lower cost, and are typically eco-friendlier. In this project cycle, over 1000 plant extracts provided by the Baruch S. Blumberg Institute through a Material Transfer Research Agreement (MTRA) were screened for inhibition of growth of L. monocytogenes. Twelve plant extracts were identified with antimicrobial activity to L. monocytogenes. The Minimal Inhibition of Concentration (MIC) of this compound was comparable to the known antibiotics used in the clinical studies. Accomplishments 01 Natural antimicrobials from plant extracts for use in controlling pathogens. Listeria monocytogenes is a foodborne bacteria that causes a disease known as listeriosis. Consumers prefer the use of natural antimicrobials to inhibit the growth of foodborne pathogens because of safety concerns. ARS researchers at Wyndmoor, Pennsylvania, evaluated 800 different plant extracts from around worldwide for their effectiveness in inhibiting the growth of L. monocytogenes. Twelve of the plant extracts showed notable activity against the pathogen, and the concentrations needed to stop bacteria growth were determined. The extracts caused extensive cell damage to the cell wall and the tails involved in the movement (flagella). These plant extracts can be used as new preservatives by the food industry to reduce the risk of contamination from L. monocytogenes.
Impacts
(N/A)
Publications
- Wang, D., Yu, J., Wang, Y., Zhang, M., Li, P., Liu, M., Liu, Y. 2019. Development of a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay for detection of African Swine Fever Virus (ASFV). Journal of Virological Methods. 27.
- Huang, X., Hu, M., Zhou, X., Liu, Y., Shi, C., Shi, X. 2020. The role of yoaE gene regulated by CpxR in the survival ability of Salmonella Enterica serovar Enteritidis in antibacterial egg white. mSphere.
- Yang, Y., Sommers, C.H., Adenipkun, E., Ceruso, M., Jackson, C.R., Woodley, T.A., Barrett, J.B., Hiott, L.M., Liu, Y., Frye, J.G. 2020. Draft genomic sequence of three Escherichia coli Sequence Type 131 isolates (H45, H43ii, and H43iii) from patients in Lagos, Nigeria. Microbiology Resource Announcements. 9:17.
- Zhang, Z., Yang, J., Xu, X., Zhou, X., Shi, C., Zhao, X., Liu, Y., Shi, X. 2020. Co-existence of mphA, oqxAB and blaCTX-M-65 on the IncHI2 plasmid in highly drug-resistant Salmonella enterica serovar Indiana ST17 isolated from retail foods and humans in China. Food Control. doi.org/10.1016/j. foodcontrol.
- Du, R., Qu, Y., Qi, P.X., Sun, X., Liu, Y., Zhao, M. 2020. Natural flagella-templated Au nanowires as a novel adjuvant against Listeria monocytogenes. The Royal Society of Chemistry. Nanoscale. 12:5627-5635.
- Wang, D., Wang, Y., Zhang, M., Liu, Y. 2020. Evaluation of a loop-mediated isothermal amplification (LAMP) method for the detection of Salmonella spp. in terms of sensitivity and applicability. Journal of Nutrition and Metabolism. 3(2):5-5.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. This report documents progress for the parent project 8072-42000-082-00D, Molecular Characterization of Foodborne Pathogen Responses to Stress, which falls under NP108. The project plan was approved (from 2016 to 2021) , and this past year, progress was made on all three main objectives. The research focuses on using omics technologies to understand how food-borne pathogens tolerate stresses encountered in food environments and how food processing conditions may induce resistance to stresses. The research is also focused on identifying food and animal reservoirs for emerging foodborne pathogens, and work will provide information for understanding how these pathogens cause disease in humans and identification of genetic markers for detection and typing. Related to Objective #1 of the project plan, significant progress was made in developing a diagnostic method for molecular serotyping of E. coli. Traditionally, serotyping has been used to distinguish >180 different E. coli O-serogroups (O-antigen) and 53 H-types (H-flagellar antigen) based on cell surface structures). However, this procedure, while laborious and often inaccurate, can only be performed in specialized laboratories. In our research, more than 70 genomes of E. coli reference O-group strains were analyzed to determine the DNA sequence of the cluster of genes involved in production of cell surface polysaccharides that define the different E. coli O-serogroups. This research was intended to develop more rapid and simple methods for detection, typing, and identification of different serogroups of pathogenic E. coli. Working with CRADA partners, we determined the unique genes that can be targeted to identify the different O- and H-groups. Based on this genetic information, a molecular DNA sequencing-based platform known as AmpliSeq was developed to determine the presence of O- and H-group genes, as well as virulence genes (involved in causing disease) in E. coli strains. The specificity of the method was tested with all the E. coli reference strains and the other field strains isolated from humans, animals, and the environment. The new developed molecular method is inexpensive and will greatly enhance the ability to identify, detect, and type pathogenic E. coli and will eliminate the use of the labor-intensive and inaccurate traditional serotyping procedure. Other work focused on characterizing hundreds of extraintestinal pathogenic E. coli (ExPEC) strains that were isolated from human and poultry in collaboration with another ARS scientist at Wyndmoor, Pennsylvania. ExPEC that are present in produce, poultry and meat can cause illness in humans. Molecular techniques were used to trace the association between foodborne ExPEC and human diseases. Significant progress was made on determining the prevalence of ExPEC in poultry. Genetic-based PCR methods were used to characterize different ExPEC strains. The genome sequence of several ExPEC isolates from human and chicken was determined, which 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 development of strategies to control ExPEC and prevent contamination from poultry products. Work pertaining to Objective #2 continues to progress well and all the subobjectives for the 36-month milestones have been at least substantially met. Specifically, the effectiveness of irradiation, high pressure processing and cold storage interventions for reducing Campylobacter numbers in poultry products was determined resulting in two publication. Campylobacter strains with different biofilm forming potential were identified and a direct relationship between the amounts of biofilm a Campylobacter strain can produce and the bacteria⿿s ability for movement using their flagellar organelle was established. Campylobacter strains that were strong swimmers also produced greater amounts of biofilm compared with Campylobacter that had poor swimming movement and produced less biofilm material. Reducing the bacteria⿿s ability to swim using flagella with a specific chemical also reduced the Campylobacter⿿s ability to form biofilms. Finally, we have described the potential use of acidic whey as a wash to reduce Campylobacter numbers on poultry products. Whey is an edible byproduct resulting from the manufacture of cheese. Whey is known to be acidic and is rich in a variety of probiotics including lactic acid bacteria. Initial studies have shown that whey solutions were able to produce large reductions (>5 logs) in Campylobacter numbers, even when applied as a minor component. Related to Objective #3 of the project plan, significant progress was made towards understanding the survival mechanisms of L. monocytogenes after exposure to olive leaf extract (OLE). Beneficial to human health, OLE is an herbal supplement with antimicrobial properties. Therefore, OLE was explored as a natural antimicrobial to control foodborne pathogens in food. Our results showed that OLE inhibited 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 to use for gene expression analyses. The RNA- Seq method was used to measure the level of gene expression in L. monocytogenes after exposure to sub-lethal dose of OLE. Increased or decreased expression of several genes have been identified, which provides the information essential to understanding the specific mechanisms and genes required for growth /survival in food-related stress conditions. This information is also necessary for the design of interventions that will allow complete inactivation of L. monocytogenes. Additional research showed that OLE can be used as an antimicrobial film to inhibit the growth of foodborne pathogens, indicating the potential use of OLE as food packaging material, which will be further explored in the future. Significant progress was also made in search for new antimicrobials for use as food additives and preservatives to inactivate pathogens and to increase the shelf life of foods. Compared to synthetic compounds, plant extracts are generally more likely to be accepted as generally recognized as safe (GRAS), may have a lower cost, and are typically more eco- friendly. In this project cycle, over 1000 plant extracts provided by the Baruch S. Blumberg Institute through a Material Transfer Research Agreement (MTRA) were screened for inhibition of growth of L. monocytogenes. A plant extract from Stenotus armerioides (Thrift mock golden weed) was identified with antimicrobial activity to L. monocytogenes. This plant extract was further fractionated, and an active compound was purified, identified, and characterized. The Minimal Inhibition of Concentration (MIC) of this compound was comparable to the known antibiotics used in the clinical studies. To further confirm this result, the compound was synthesized in vitro, which showed the same antimicrobial activity as the natural plant extract. In addition, results from Ames test showed that this compound did not possess mutagenic activity. Taken together, our results showed that thrift mock golden weed extract has the potential to be used a sanitizer or packaging material in the food industry to control foodborne pathogens such as L. monocytogenes. Further research will focus on antimicrobial screening for growth inhibition of more foodborne pathogens such as Shiga toxin-producing E. coli (STEC), and Salmonella spp. This research is being conducted in collaboration with Blumberg Institute and Villanova University. Accomplishments 01 Whole genome sequencing for understanding the antibiotic sensitivity of Shiga toxin-producing E. coli (STEC). Shiga toxin-producing E. coli (STEC) can cause serious outbreaks and sporadic cases of food-borne illness. STEC strains belonging to serogroup O111 are harmful pathogens associated with food. The current methods for detection of STEC in food need novobiocin as a selective agent in the enrichment media and selective agars to prevent the growth of background bacteria. However, novobiocin also inhibits the growth of some STEC, particularly STEC O111, which may lead to false-negative detection results. ARS scientists at Wyndmoor, Pennsylvania, first determined the genome sequence (an organism⿿s complete set of DNA) of seven STEC O111 strains with different sensitivities to novobiocin. The genes or alterations in the genome involved in novobiocin sensitivity were identified, and the genome sequences were deposited to GenBank, a public gene sequence database. This information is important for understanding the characteristics of this pathogen at the molecular level for development of enrichment media and selective agars that will improve detection of STEC O111 and prevent the release of contaminated food products to the consumers. 02 Understanding the nature and behavior of L. monocytogenes strains through DNA sequencing. Listeria monocytogenes is an important foodborne pathogen that causes listeriosis associated with high mortality rates. L. monocytogenes is very difficult to control in the food industry since it can survive under very harsh conditions such as high salt, low pH, and low temperature. ARS scientists in Wyndmoor, Pennsylvania determined the genome sequence of seven strains of L. monocytogenes that varied in their ability to cause disease and response to stresses to gain a better understanding about how to control this pathogen. Thus, these sequences were also compared to determine the genes involved in virulence (disease causing ability) and stress responses. The genome sequences were deposited in a GenBank sequence database that can be accessed by the public. The information obtained from this research helps in the design of strategies to control L. monocytogenes in food, and potentially in the development of more effective therapeutic approaches. 03 Olive leaf extract as a natural antimicrobial compound for the food industry. There is a need for novel methods to control pathogenic bacteria in the food industry. Olive leaf extract (OLE) is often used as an herbal supplement and is considered beneficial to human health. ARS researchers in Wyndmoor, Pennsylvania studied the application of OLE as an antimicrobial agent for controlling major foodborne pathogenic bacteria, such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis, and Staphylococcus aureus. The research showed that an antimicrobial film prepared with OLE was able to inhibit growth of foodborne pathogens. Therefore, OLE has the potential to be used as a natural antimicrobial food packing material to control foodborne pathogens in food and the food processing environment. 04 Biofilm formation and flagella for Campylobacter spp. Campylobacter bacteria form biofilms that allow them to persist on foods and in food processing environments increasing their potential to cause human disease. We identified Campylobacter strains that formed either strong or weak biofilms. Next we determined that the ability to form strong biofilms correlated with the ability of Campylobacter to move using hair-like appendages called flagella. A chemical was identified that stopped the bacteria⿿s movement using the flagella. The same chemical was also shown to reduce Campylobacter biofilm formation. Our work had demonstrated that flagella play an important role in Campylobacter biofilm formation and have identified a chemical that can interfere with the functionality of flagella and reduce biofilm formation. Our research has provided a new approach for reducing Campylobacter biofilms which could lead to reductions in the numbers of Campylobacter found on food products and in food production facilities. Fewer Campylobacter will mean fewer sick consumers.
Impacts
(N/A)
Publications
- Smith, J., Gunther, N.W. 2019. Commentary: Campylobacter and hemolytic uremic syndrome. Foodborne Pathogens and Disease. 16(2):90-93.
- Gunther, N.W., Abdul Wakeel, A.Y., Scullen, O.J., Sommers, C.H. 2019. The evaluation of gamma irradiation and cold storage for the reduction of Campylobacter jejuni in chicken livers. Food Microbiology. 82:249-253.
- Accumannola, G.M., Richards, V.A., Gunther, N.W., Lee, J. 2018. Purification and characterization of the thermostable metalloprotease produced by Serratia grimesii isolated from channel catfish. Journal of the Science of Food and Agriculture. 99:2428-2437.
- Bobokalonov, J., Liu, Y., Shahrin, T., Liu, L.S. 2018. Transcriptomics analysis on the regulation of tomato ripening by the ethylene inhibitor 1- methylcyclopropene. Journal of Plant Studies. 7(2):49-60.
- Liu, Y., Xu, A., Fratamico, P.M., Sommers, C.H., Rotundo, L., Boccia, F., Jiang, Y., Ward, T.J. 2018. Draft whole genome sequence of seven L. monocytogenes strains with variation in virulence and stress responses. Microbiology Resource Announcements. 7(13).
- Rotundo, L., Boccia, F., Fratamico, P.M., Xu, A., Sommers, C.H., Liu, Y., Bono, J.L., Pepe, T. 2018. Draft genome sequences of seven strains of Shiga toxin-producing Escherichia coli O111 with variation in their sensitivity to novobiocin. Microbiology Resource Announcements. 7(10).
- Smith, J., Fratamico, P.M. 2018. Emerging and re-emerging zoonotic food- borne pathogens. Foodborne Pathogens and Disease. 15(12).
- Gunther, N.W., Abdul Wakeel, A.Y., Ramos, R.V., Sheen, S. 2019. The evaluation of hydrostatic high pressure and cold storage parameters for the reduction of Campylobacter jejuni in chicken livers. International Journal of Food Microbiology. 82:249-253.
- Andreozzi, E., Gunther, N.W., Reichenberger, E.R., Cottrell, B.J., Rotundo, L., Nunez, A., Uhlich, G.A. 2018. Pch genes control biofilm and cell adhesion in a clinical serotype O157:H7 isolate. Frontiers in Microbiology. 9(2829).
- Patel, I.R., Gangiredla, J., Lacher, D.W., Mammel, M.K., Bagi, L.K., Baranzoni, G., Fratamico, P.M., Roberts, E.L., Debroy, C., Lindsey, R.L., Stripling, D., Martin, H., Smith, P., Strockbine, N.A., Elkins, C.A., Scheutz, F., Feng, P.C. 2018. Interlaboratory evaluation of the FDA-ECID microarray for profiling Shiga toxin-producing Escherichia coli. Journal of Food Protection. 81:1275-1282.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. This report documents progress for 8072-42000-082-00D, Molecular Characterization of Foodborne Pathogen Responses to Stress, which falls under National Program 108. The research focuses on using omics technologies and systems biology to understand how foodborne pathogens tolerate stresses encountered in food environments and how food processing conditions may induce resistance to stresses. The research also focuses on identifying food and animal reservoirs for emerging foodborne pathogens. The research will provide information for understanding how these pathogens cause disease, as well as for identification of genetic markers for detection and typing. Related to Objective 1, progress was made in gaining a better understanding of the mechanism of acid tolerance in Shiga toxin-producing E. coli (STEC) O157:H7 and non-O157 STEC. The ability to survive low pH conditions results in a low infectious dose and the ability to overcome low pH conditions in food that normally should inactivate the pathogens. To gain a better understanding of genes involved in acid tolerance, the genomes of >25 strains of E. coli O157:H7, consisting of both acid tolerant and acid sensitive strains, were sequenced. Gene expression studies were also performed on a representative set of acid sensitive and acid tolerant strains. Overall, results showed that a complex mechanism regulates acid tolerance in E. coli O157:H7. For example, a comparison of gene expression analyses of acid sensitive and acid resistant strains indicated that a gene called csg (curli adhesive fimbriae) and another called hde (acid induced chaperone) were responsible for the characteristics of the strains when exposed to acid conditions. These genes may serve as suitable targets for development of interventions to control STEC. In addition, E. coli strains that caused a laboratory infection were sequenced using three different DNA sequencing platforms, and the sequence data were combined and analyzed. The strains included those that were used in the laboratory experiments, as well as the same strains isolated from the scientist/patient. It was determined that the antibiotics that were administered to the patient during hospitalization caused an increase in the production of the dangerous Shiga toxins, and this led the patient to acquire hemolytic uremic syndrome and encephalitis. For this work, an enzyme-linked immunoassay developed in collaboration with a CRADA partner was used to quantify the level of Shiga toxins, and with collaborators at the University of Texas, the results were confirmed by using a Shiga toxin gene expression assay. In collaboration with CRADA partners, other research focused on developing a DNA sequence-based platform known as AgriSeq, using PCR primers specific for each E. coli O-group and H-type, as well as several virulence genes (stx, eae, and extra-intestinal E. coli virulence genes) as a method for molecular serotyping and virulotyping (presence of specific set of virulence genes) of E. coli. Related to Objective 2, the research is currently focused on three separate areas: the primary work detailed in the five-year plan, work in collaboration with scientists at the Food Safety Inspection Service (FSIS) addressing outbreaks of Campylobacter infection associated with chicken liver, and work in collaboration with researchers at Villanova University under a Material Transfer Research Agreement, developing new surface disinfecting products. Work pertaining to the 5-year plan continues to progress well, and the sub-objectives for the 24th month milestones have been at least substantially met. Specifically, a method for investigating Campylobacter attachment onto poultry skin under processing and storage conditions has been fully developed and utilized. Intervention techniques for reducing Campylobacter numbers in chicken liver have been investigated and reported in the literature. This has produced specific recommendations for the FSIS to consider for mitigation of disease outbreaks associated with chicken liver. Finally, several novel multi- cationic quaternary ammonium compounds have been identified that produce equal or greater reductions in Campylobacter numbers as compared to commercial products at the same concentrations. Listeria monocytogenes is a major foodborne pathogen that causes a serious human illness known as listeriosis, and the pathogen is relatively resistant to commonly used inactivation treatments. Related to Objective 3, significant progress was made towards understanding the survival mechanisms of L. monocytogenes with exposure to organic acids. The level of gene expression in L. monocytogenes was measured with exposure to organic acids using a technology known as RNA-Seq. Global profiling of gene expression of L. monocytogenes allowed us to to identify highly expressed and repressed genes, which reflect the pathogen�s response to the experimental conditions (exposure to organic acids). This information provides information essential to determine specific mechanisms and genes required for growth or survival in the selected food-related stress conditions. Progress was also made towards understanding the survival mechanisms of L. monocytogenes with treatment with olive leaf extract (OLE). OLE is a natural material that has antimicrobial properties, and it is also used as an herbal supplement due to its health promoting properties. Thus, due to these characteristics, OLE is a novel material that can be utilized to control pathogenic bacteria in food. It was demonstrated that OLE inhibited growth of foodborne pathogens, as well as formation of biofilms (aggregates of bacteria attached to a surface), and therefore, OLE has the potential to be used as a natural antimicrobial to control foodborne pathogens in food and the food environment. Bacterial cells treated with sub-lethal doses of olive leaf extract were used to study the gene expression profile of L. monocytogenes. In addition, through a Material Transfer Research Agreement with the Baruch S. Blumberg Institute, over 1,500 plant extracts were screened for their ability to inactivate L. monocytogenes. Several extracts showed antimicrobial activity, and the active compound in one extract was purified and characterized. This compound and others that are being examined can be used in formulations of antimicrobial products/disinfectants for the inactivation of L. monocytogenes in food environments. Accomplishments 01 A method to identify E. coli strains based on specific genetic sequences. Traditionally, a procedure called serotyping has been used to distinguish among the >180 different E. coli O-serogroups and 53 H- types (O-polysaccharide antigen and H-flagellar antigen are E. coli cell surface structures); however, this procedure can only be performed in specialized laboratories, and it is laborious and often inaccurate. To develop more rapid and simple methods for detection, typing, and identification of E. coli belonging to all of the different types and to identify the specific virulence genes (genes associated with causing disease) the strains carry, ARS researchers at Wyndmoor, Pennsylvania sequenced the genomes of E. coli reference O-group strains, determined the DNA sequence of the cluster of genes involved in production of cell surface polysaccharides that define the different E. coli O-serogroups, and the sequences were deposited in the GenBank DNA sequence public database. Working with CRADA partners, unique genetic regions that can be targeted in methods to identify the different O- and H-groups have been determined. Based on this genetic information, a molecular DNA sequencing-based platform known as AgriSeq was developed to test for the presence of O- and H-group genes, as well as virulence genes in E. coli strains. The accuracy of the method was tested with all of the reference strains, as well as with other strains isolated from humans, animals, and the environment. This new molecular method is inexpensive, will greatly enhance the ability to identify, detect, and type E. coli, and will eliminate the use of the labor-intensive and inaccurate traditional serotyping procedure. 02 Understanding the survival mechanism of Listeria monocytogenes with exposure to organic acids. L. monocytogenes is an important foodborne pathogen that causes listeriosis associated with high mortality rates, and furthermore, this pathogen can survive antimicrobial treatments that are normally used during food processing. Organic acids such as lactic acid and diacetic acid have been applied to control L. monocytogenes in ready-to-eat (RTE) meat; however, the mechanism used by L. monocytogenes to adapt to exposure to organic acids remains unclear. ARS researchers in Wyndmoor, Pennsylvania used a procedure known as RNA sequencing (RNA-Seq) to determine the genes that are affected in L. monocytogenes with exposure to levels of sodium lactate normally used during food processing. Genes involved in transport of nutrients into the cell and bacterial movement and attachment, as well as a number of other genes were affected. These results reflect the pathogen�s response to the environmental conditions and provide information essential to determine specific mechanisms required for growth or survival in the selected food-related stress conditions. This study provides insight on the adaptation mechanism of L. monocytogenes with treatment of sodium lactate and will aid in developing more effective strategies to control L. monocytogenes in RTE meat. 03 The effectiveness of gamma irradiation to inactivate Campylobacter jejuni in chicken liver. Recent outbreaks linked to undercooked chicken liver contaminated with C. jejuni necessitate the development of a safer liver product. Intervention methods to reduce the numbers of C. jejuni present on an uncooked chicken liver would reduce the number of disease cases resulting from a contaminated product that may be consumed undercooked. ARS researchers in Wyndmoor, Pennsylvania showed that application of gamma irradiation was successful in reducing Campylobacter numbers on or in the liver without any visual or undesirable changes to the liver product. When irradiation was followed by cold storage, the level of irradiation needed to decrease C. jejuni numbers to undetectable levels was significantly reduced. This work represents the first time that irradiation has been used to inactivate C. jejuni present in chicken liver and therefore has produced novel information with regards to treatment dose and bacterial reductions, as well as the interaction this treatment has with typical food storage conditions. This work is of considerable interest and value to poultry producers, as well as consumers. Since the current methods for processing chicken liver is likely to continue to result in disease outbreaks, this new inactivation method is essential for producing a liver product that is safe for consumers. 04 The effectiveness of high hydrostatic pressure to inactivate Campylobacter jejuni in chicken liver. Recent outbreaks of disease caused by C. jejuni contamination in undercooked chicken liver has necessitated the development of methods to reduce C. jejuni numbers in processed chicken liver. High hydrostatic pressure processing has been used successfully for reducing bacterial counts in a variety of different food products. ARS researchers in Wyndmoor, Pennsylvania showed that a range of high pressure treatments of chicken liver produced only modest reductions in C. jejuni levels. Additionally, the pressure treatments produced undesired changes in the appearance of the liver at the highest pressures tested; therefore, high pressure treatments were also performed in conjunction with reduced temperature storage. With this combination treatment, the reductions in C. jejuni numbers increased; however, they still did not reach the desired levels. This work demonstrated that high pressure treatment in conjunction with cold storage is more effective than high pressure treatment alone; however, this combination treatment will not be sufficient to significantly reduce the number of outbreaks. This work is of considerable interest and value to poultry producers and food safety researchers who will need to include additional technologies in conjunction with high pressure/cold storage treatment if this method is to be utilized as a primary intervention for increasing the safety of chicken liver.
Impacts
(N/A)
Publications
- Baranzoni, G., Fratamico, P.M., Kim, G., Reichenberger, E.R., Funk, J., Manning, S. 2017. Genome sequences of 34 Shiga toxin-producing E. coli isolated from swine and other sources. Genome Announcements.
- Gurtler, J., Doyle, M.P., Kornacki, J.L., Fratamico, P.M., Gehring, A.G., Paoli, G. 2017. Advantages of virulotyping foodborne pathogens over traditional identification and characterization methods. Foodborne Pathogens Virulence Factors and Host Susceptability. New York, NY: Springer Publishing. p. 3-40.
- Uhlich, G.A., Reichenberger, E.R., Cottrell, B.J., Fratamico, P.M., Andreozzi, E. 2017. Whole-genome sequence of Escherichia coli serotype O157:H7 strain B6914-ARS. Genome Announcements.
- Smith, J., Fratamico, P.M. 2018. Escherichia coli pathotypes. Pathogenic E. coli: Evolution, Omics, Detection, and Control � Caister Academic Press, United KingdomBook Chapter.
- Gehring, A.G., Fratamico, P.M., Lee, J., Ruth, L., He, X., He, Y., Paoli, G., Stanker, L.H., Rubio, F.M. 2017. Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples. Food Control. 77:145- 149.
- Rotundo, L., Fratamico, P.M., Amagliani, G., Carloni, E., Omiccioli, E., Magnani, M. 2018. Comparison of the Diatheva STEC FLUO with BAX system kits for detection of O157:H7 and Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) in ground beef and bean sprout samples using different enrichment protocols. Journal of Food Analytical Methods.
- Amagliani, G., Rotundo, L., Carloni, E., Omiccioli, E., Magnani, M., Brandi, G., Fratamico, P.M. 2018. Detection of Shiga toxin-producing Escherichia coli (STEC) in food: evaluation of culture enrichment conditions. Food Research International. 103:398-405.
- Zhang, X., Ashby, R.D., Solaiman, D., Liu, Y., Fan, X. 2017. Antimicrobial activity and inactivation mechanism of lactonic and free acid sophorolipids against Escherichia coli O157:H7. Biocatalysis and Agricultural Biotechnology. 11(C):176-182. doi: 10.1016/j.bcab.2017.07. 002.
- Liu, Y., Yoo, B., Hwang, C., Suo, Y., Sheen, S., Khosravi, P., Huang, L. 2017. LMOf2365_0442 encoding for a fructose specific PTS permease IIA may be required for virulence in L. monocytogenes Strain F2365. Frontiers in Microbiology. 8:01611.
- Suo, Y., Gao, S., Xie, Y., Liu, Y., Qu, Y., Shen, Y., Zhou, C. 2017. A multi-pathogen selective enrichment broth for simultaneous growth of Salmonella enteria, Escherichia coli O157:H7 and Shigella flexneri. Journal of Food Safety. doi:10.1111/jfs.12388.
- Gunther, N.W., Reichenberger, E.R. 2017. Complete genome sequence of Campylobacter jejuni RM1246-ERRC that exhibits resistance to Quaternary Ammonium Compounds. Genome Announcements.
- Suo, Y., Gao, S., Baranzoni, G., Xie, Y., Liu, Y. 2018. Comparative transcriptome RNA-Seq analysis of Listeria monocytogenes with sodium lactate adaptation. Food Control. 91:193-201.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. This report documents progress for the parent project 8072-42000-082-00D, Molecular Characterization of Foodborne Pathogen Responses to Stress, which falls under NP108. A portion of this work continues research from project 8072-42000-070-00D, Genomic and Proteomic Analysis of Foodborne Pathogens. The project plan was approved in February 2016, and this past year, progress was made on all three main objectives. The research focuses on using omics technologies and systems biology to understand how food-borne pathogens tolerate stresses encountered in food environments and how food processing conditions may induce resistance to stresses. The research is also focused on identifying food and animal reservoirs for emerging foodborne pathogens, and work will provide information for understanding how these pathogens cause disease, as well as for identification of genetic markers for detection and typing. Related to objective #1 of the project plan, progress was made in gaining a better understanding of the mechanism of acid tolerance in Shiga toxin- producing E. coli (STEC) O157:H7. The ability to survive low pH conditions results in a low infectious dose and the ability to overcome low pH conditions in food that normally should inactivate the pathogens. To gain a better understanding of genes involved in acid tolerance, the genomes of >25 strains of E. coli O157:H7, consisting of both acid tolerant and acid sensitive strains, were sequenced and compared. Gene expression studies were also performed on a representative set of acid sensitive and acid tolerant strains. Overall, results showed that a complex mechanism regulates acid tolerance. For example, to survive under acid stress conditions, acid sensitive strains express curli fimbriae, which allows the cells to form biofilms, providing protection and facilitating survival. Gene expression analyses of acid-sensitive and acid-resistant strains indicated that csg (curli adhesive fimbriae) and hde (acid induced chaperone) genes positively correlated with the phenotypic difference between sensitive and resistant strains. Related work showed that 7 other types of STEC could also tolerate acidic conditions in food as well or better than O157:H7, thus, non-O157:H7 STEC can be as dangerous as O157:H7, and control strategies should have the ability to inactivate all highly acid tolerant STEC. This research enhances the understanding of the disease-causing potential of STEC and the mechanism of acid tolerance and provides information to develop strategies to control these harmful pathogens in food. In addition, E. coli strains that caused a laboratory infection were sequenced and analyzed (the two mutant strains used in the laboratory, the parent strains, and the same strains [the mutants] isolated from the patient). It was determined that the antibiotics that were administered to the patient during hospitalization caused an increase in the production of the dangerous Shiga toxins, and this led the patient to acquire hemolytic uremic syndrome and encephalitis. For this work, an enzyme-linked immunoassay developed in collaboration with a CRADA partner was used to quantify the level of Shiga toxins, and with collaborators at a university in Texas, the results were confirmed by using a Shiga toxin gene expression assay. Continuing with the research on DNA sequencing of the E. coli O-group reference strains, the O-antigen gene cluster sequences of all of the E. coli reference strains were identified from the genome sequences, and O-group-specific primers were designed in collaboration with CRADA partners. A molecular sequence-based O- and H- antigen typing kit was designed, and this method can also determine the presence of specific virulence genes in the E. coli strain. Other work focused on characterizing hundreds of STEC strains that were isolated from pigs through funded grants and a collaboration with scientists at Michigan State University. The strains were characterized using genetic- based methods, and the genome sequence of almost 100 swine, cattle, and human STEC isolates was determined. The results showed that some STEC carried by pigs were similar to STEC associated with human illness and some strains from cattle. Among the different E. coli serogroups isolated from swine, human pathogens STEC O157:H7 and O26:H11, carrying genes that are involved in human illness were also isolated from swine. This work represents the largest comprehensive study on swine STEC in the U.S., and the data provide a critical first step to enhance the understanding of the epidemiology of STEC in swine and of the potential of swine STEC to cause illness. The information is important for development of strategies to control STEC shedding on farms and prevent contamination of pork products. Finally, the performance of a commercial kit for detection of STEC was compared to methods used by the Food Safety and Inspection Service (FSIS) for regulatory testing. The ability of the new commercial kit to detect STEC in food was similar to that of the kits used as part of the FSIS method. Related to Objective #2, the only 12-month milestone was to complete the collection of poultry exudates at the production and retail levels. Two exudate samples of significant volume were collected from production sources and two were collected from retail sources. This collection of exudates should be sufficient for experimental use to meet the subsequent milestones in sub-objective 2A. Work was continued to expand and characterize the laboratory collection of Campylobacter jejuni, and Campylobacter coli strains isolated from environmental and clinical sources. For ten of these Campylobacter strains, whole genome sequencing and characterization was completed. A collaboration with researchers at Villanova University produced a material transfer research agreement (MTRA) focused on investigating the effectiveness on a new class of antimicrobial compounds against Campylobacter strains. The research succeeded in identifying a trait specific to a sub-group of the antimicrobial compounds that increased the compounds� effectiveness in inactivating Campylobacter strains. Related to Objective #3 of the project plan, significant progress was made towards understanding the survival mechanisms of L. monocytogenes with exposure to organic acids. Organic acids such as lactic acid and diacetic acid have been applied to control L. monocytogenes in ready-to- eat (RTE) meat; however, at concentrations used, they are not fully effective in inactivating all of the Listeria. A growth study of L. monocytogenes in RTE meat with 4% sodium lactate was conducted to determine inactivation rates and also the optimal dose of lactic acid to use for gene expression analyses. Bacterial cells treated with 4% sodium lactate for two days were used to study changes in L. monocytogenes gene expression in RTE meat. Increased or decreased expression of a number of genes occurred, and this information is being used in systems biology analyses to determine how L. monocytogenes may survive in food with exposure to lactic acid. Additional work being planned will further allow the identification of highly expressed and repressed genes, which reflect the pathogen�s response to organic acids and will provide the information essential to determine specific mechanisms required for growth or survival in the selected food-related stress conditions. This information is necessary for the design of interventions that will allow complete inactivation of L. monocytogenes. Additional research showed that olive leaf extract and oleuropein, a major compound found in olive leaf extract had antimicrobial properties against L. monocytogenes and other foodborne pathogens. Further research will explore whether exposure to both organic acids and olive leaf extract will result in a synergistic effect for inactivating L. monocytogenes. Accomplishments 01 Swine as a reservoir for Shiga toxin-producing E. coli (STEC) that cause human illness. Although cattle are considered an important reservoir for STEC, food products from other animal species, including swine have been linked to foodborne illness. However, little is known of the prevalence and fecal shedding of STEC in clinically healthy swine over time. In collaboration with scientists in academia, ARS researchers at Wyndmoor, Pennsylvania conducted longitudinal studies to fill this gap by investigating fecal shedding of STEC in swine raised on conventional farms during the finishing period. A majority of the fecal samples tested were positive for the presence of the STEC Shiga toxin genes, and STEC strains of various types were recovered from a large portion of the pigs. The virulence (disease-causing potential) genes carried by the strains differed, some of the STEC carried by the pigs were similar to those associated with human illness and strains from cattle. 02 Method to identify E. coli strains based on differences in genes involved in production of cell surface structures. Traditionally, a procedure called serotyping has been used to distinguish among the > 180 different E. coli O-serogroups and 53 H-types (O-antigen and H- flagellar antigen are E. coli cell surface structures); however, this procedure can only be performed in specialized laboratories, and it is laborious and often inaccurate. To develop more rapid and simple methods for detection, typing, and identification of E. coli belonging to all of the different serogroups, ARS researchers at Wyndmoor, Pennsylvania sequenced the genomes of E. coli reference O-group strains, determined the DNA sequence of the cluster of genes involved in production of cell surface polysaccharides that define the different E. coli O-serogroups, and the sequences were deposited in the GenBank DNA sequence public database. Working with CRADA partners, unique genes that can be targeted in genetic-based methods to identify the different O- and H-groups have been determined. Based on this genetic information, a molecular DNA sequencing-based platform known as AmpliSeq was developed to test for the presence of O- and H-group genes, as well as virulence genes (involved in causing disease) in E. coli strains. This new molecular method is inexpensive and will greatly enhance the ability to identify, detect, and type E. coli and will eliminate the use of the labor-intensive and inaccurate traditional serotyping procedure. 03 Olive leaf extract is a natural compound that has antimicrobial properties. There is a need for novel methods to control pathogenic bacteria in the food supply. Olive leaf extract (OLE) is an herbal supplement that is beneficial to human health. It has antioxidant, as well as antimicrobial properties. Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis, and Staphylococcus aureus are major foodborne pathogens that cause serious human illness. ARS researchers at Wyndmoor, Pennsylvania showed that OLE inhibited growth of these foodborne pathogens, as well as the formation of biofilms (aggregates of bacteria attached to a surface), and therefore, OLE has the potential to be used as a natural antimicrobial to control foodborne pathogens in food and the food environment. Oleuropein is the key compound in OLE that has antimicrobial activity. 04 Antimicrobials known as biscationic quaternary ammonium compounds (QACs) are effective at inactivating Campylobacter. Monocationic QACs have been commercially used as disinfectants for many years and represent a significant portion of the cleaning products market. However, resistance to the QACs has steadily developed over the years of usage. In an effort to address the developing resistance, new QACs were constructed with unique structural changes from currently commercially available QACs. Together with collaborators, ARS researchers at Wyndmoor, Pennsylvania showed that a subgroup of these new QACs proved to be more successful at inactivating a major human pathogen, Campylobacter jejuni, compared to currently commercially available QACs. The chemical structure of the commercially available QACs has only one negative charge, and they are classified as monocationic; however, the more successful sub-group of the experimental QACs have two negative charges, and these are known as biscationic. Additionally, the remaining experimental QACs, a mix of tricationic (three negative charges) and tetracationic (four negative charges) structures, were less successful at inactivating C. jejuni cells compared to the monocationic and biscationic QACs. This research has resulted in the development of a new class of disinfectants with effectiveness higher than commercially available QACs, which will help to reduce contamination by Campylobacter and potentially other foodborne pathogens.
Impacts
(N/A)
Publications
- Kim, G., Fratamico, P.M., Breidt, F., Oh, D. 2016. Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid. Journal of Applied Microbiology. doi: 10.1111/jam.13223.
- Li, X., Liu, Y., Jia, Q., Lamacchia, V., O'Donoghue, K., Huang, Z. 2016. A systems biology approach to investigate the antimicrobial activity of oleuropein. Journal of Industrial Microbiology and Biotechnology. 43(12) :1705-1717.
- Yu, Q., Niu, M., Yu, M., Liu, Y., Wang, D., Shi, X. 2016. Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shellfish in Shanghai. Food Control. 60:263-268.
- Liu, Y., McKeever, L., Malik, N.S. 2017. Assessment of the antimobial activity of olive leaf extract against foodborne bacterial pathogens. Frontiers in Microbiology. doi: 10.3389/fmicb.2017.00113.
- Kong, Q., Patfield, S.A., Skinner, C.B., Stanker, L.H., Gehring, A.G., Fratamico, P.M., Rubio, F., Qi, W., He, X. 2016. Validation of two new immunoassays for sensative detection of a broad range of shiga toxins. Austin Immunology. 1(2):1007.
- Yoo, B.K., Liu, Y., Juneja, V.K., Huang, L., Hwang, C. 2017. Effect of environmental stresses on the survival and cytotoxicity of Shiga toxin- producing Escherichia coli. Food Quality and Safety. 1(2):139-146. doi: 10.1093/fqsafe/fyx010.
- Yoo, B.K., Liu, Y., Juneja, V.K., Huang, L., Hwang, C. 2016. Effects of stresses on the growth and Cytotoxicity of Shiga-Toxin producing Escherichia coli in ground beef and spinach. Journal of Food Science and Technology. 1:1-7.
- Yan, R., Liu, Y., Gurtler, J., Fan, X. 2017. Sensitivity of pathogenic and attenuated E. coli O157:H7 strains to ultraviolet-C light as assessed by conventional plating methods and ethidium monoazide-PCR. Journal of Food Safety. doi: 10.1111/jfs.12346.
- Edlind, T., Brewster, J.D., Paoli, G. 2017. Enrichment, amplification, and sequence-based typing of Salmonella enterica and other foodborne pathogens. Journal of Food Protection. 80(1):15-24.
- Uhlich, G.A., Chen, C., Cottrell, B.J., Andreozzi, E., Irwin, P.L., Nguyen, L.T. 2017. Gene duplication and promoter mutation expand the range csgD- dependent biofilm responses in a STEC population. Microbiology. 163:611- 621.
- Uhlich, G.A., Paoli, G., Zhang, X., Dudley, E.G., Figler, H.M., Cottrell, B.J., Androzzi, E. 2017. Whole-genome sequence of Escherichia coli serotype O157:H7 strain PA20. Genome Announcements. doi: 10.1128/genomeA. 01460-16.
- Fratamico, P.M., Bosilevac, J.M., Schmidt, J.W. 2017. Methods for detecting pathogens in the beef food chain: an overview. In: Acuff, G., Dickson, J. Ensuring safety and quality in the production of beef. Volume 1: Safety. Cambridge, UK: Burleigh Dobbs Science. p.35-51.
- Fratamico, P.M., Bosilevac, J.M., Schmidt, J.W. 2017. Methods for detecting pathogens in the beef food chain: detecting particular pathogens. In: Acuff, G., Dickson, J. Ensuring safety and quality in the production of beef. Volume 1: Safety. Cambridge, UK: Burleigh Dobbs Science. p.59-72.
- Beier, R.C., Franz, E., Bono, J.L., Mandrell, R.E., Fratamico, P.M., Callaway, T.R., Andrews, K., Poole, T.L., Crippen, T.L., Sheffield, C.L., Anderson, R.C., Nisbet, D.J. 2016. Disinfectant and antimicrobial susceptibility profiles of the big six non-O157 Shiga toxin-producing Escherichia coli strains from food animals and humans. Journal of Food Protection. 79(8):1355-1370.
- Smith, J.L., Fratamico, P.M. 2016. Escherichia coli as other Enterobacteriaceae: food poisoning and health effects. In: Caballero, B., Finglas, P., and Toldra, F. (eds.) Encyclopedia of Food and Health. Oxford: Acad. p. 539-544.
- Baranzoni, G., Fratamico, P.M., Reichenberger, E.R., Kim, G., Breidt, F., Kay, K., Oh, D. 2016. Complete genome sequences of Escherichia coli O157:H7 strains SRCC 1675 and 28RC that vary in acid resistance. Genome Announcements. 4:4. doi: 10.1128/genomeA.00743-16.
- Gunther, N.W., Reichenberger, E.R., Bono, J.L. 2016. Complete genome sequence of UV-resistant Campylobacter jejuni RM3194, including an 81.08- kilobase plasmid. Genome Announcements. 4(2):e00305-16.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. This new Project Plan was recently certified through ARS Office of Scientific Quality Review (OSQR). For further details on current work see the 2016 annual report for project 8072-42000-070-00D.
Impacts
(N/A)
Publications
- Baranzoni, G., Fratamico, P.M., Gangiredla, J., Patel, I., Bagi, L.K., Delannoy, S., Fach, P., Boccia, F., Anastasio, A., Pepe, T. 2016. Characterization of shiga toxin subtypes and virulence genes in Porcine shiga toxin-producing Escherichia coli. Frontiers in Microbiology. 7:574.
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