1a. 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.
1b. 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.
3. Progress Report:
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.
1. 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.
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