1a. Objectives (from AD-416):
1. Utilize genomic, proteomic, and phenotype approaches to improve characterization of foodborne pathogens. 1a. Apply several molecular technologies to further characterize genomic variability of C. jejuni isolates. 1b. Optimize phenotype microarray technology for Campylobacter spp. 1c. Perform proteomic comparisons on the 5 genetically diverse C. jejuni isolates employed in aforementioned genomic characterization investigations (Objective 1: Sub-Objective 1a: C) so as to increase our basic knowledge of differential protein expression. 1d. Perform metagenomic/microbial ecology analyses on chicken gastrointestinal material to increase our basic knowledge of the microbial ecology of the broiler chicken gut. 1e. Develop a database on Campylobacter spp., containing molecular, phylogenetic, proteomic, metabalomic, epidemiologic, and metadata information. 2. Develop, refine, and implement improved methods for the cultural recovery of Campylobacter spp, and potential emerging foodborne pathogens, specifically as it meets regulatory and public health needs. 2a. Utilize genomic, phenotypic, and in silico metabolic reconstruction technologies to develop improved media for isolation of C. jejuni. 2b. Compare existing and newly refined media for recovery of C. jejuni and emerging Campylobacter spp. from the poultry farm environment as well as recovery of isolates most likely involved in public exposure. 3. Using a systems approach, compare Campylobacter spp. with varied colonization abilities for the identification of potential genes or proteins involved in colonization, virulence, and gene expression. 3a. Compare C. jejuni isolates that vary in their ability to colonize broiler chickens, for the identification of genes, proteins, and phenotypes involved in colonization. 3b. Compare C. jejuni isolates that vary in their levels of eukaryotic cell (Caco-2) adherence and invasion for the identification of genes, proteins, and phenotypes involved in virulence.
1b. Approach (from AD-416):
Improved understanding of mechanisms used during poultry colonization as well as defining the impact of applied interventions on Campylobacter spp. and associated chicken microflora is a high priority. The approaches of this project are to 1) further define novel regions in the Campylobacter spp. genome; 2) define colonization mechanisms by comparing Campylobacter spp. isolates exhibiting varying degrees of colonization in chickens; and 3) refine media components and methods for culture based recovery of Campylobacter spp. Our approach follows a coordinate driven model incorporating a comprehensive and multidisciplinary (systems biology) strategy that combines pathogen genomics, proteomics, metabolomics, and metagenomics. The overall impact of this research is to obtain information so as to provide scientifically sound guidance to assist the development of intervention strategies against Campylobacter spp. during broiler production. This should positively impact public health, with a concurrent understanding of the mechanisms and the long-term consequences of intervention application.
3. Progress Report:
Objective 1: Molecular characterization of poultry associated pathogens, coupled with next-generation sequencing, led to the observation that distinct Campylobacter subtypes are associated with specific niches during poultry production, processing, and on the final product. This information allowed for the refined selection of isolates for further characterization (phenotype microarrays and genome sequencing). This data will facilitate the development of specific interventions against Campylobacter along the “farm to fork” continuum, and the development of novel detection tools. Knowledge in these areas should result in reduced carriage of foodborne pathogens in poultry. Objective 2: In an effort to determine if the use of varied media and recovery conditions introduces a bias on the recovery of Campylobacter subtypes during epidemiologic investigations, three integrated broiler flocks were sampled (production, processing, and final product). Samples were cultured for Campylobacter using four different media coupled with four different atmosphere/temperature combinations. Quantitative analyses demonstrated that recovery of Campylobacter was similar among the four media tested, independent of recovery conditions. Subtype analyses of isolates revealed a bias in subtype recovery relative to sample type, media, incubation temperature, and atmosphere. Next-generation sequencing revealed that Campylobacter represented a small proportion (<0.04%) of nucleotide sequences present in feces. Furthermore, next-generation sequencing revealed that more non-Campylobacter sequences were present upon incubation at 42C relative to 37C. Further characterization of distinct niche-dependent subtypes will facilitate the development of specifically targeted interventions at each integrated stage to reduce and eliminate Campylobacter from poultry. Objective 3: Dose-response/transmission parameters were monitored for Campylobacter challenge in the chicken. Individual housing was determined as the most efficient experimental design for ascertaining dose-response relationships/estimates of transmission. The individual housing model was employed to determine the infectious dose of C. jejuni isolates previously determined to differentially invade Caco-2 eukaryotic cell lines. Preliminary data revealed that highly invasive isolates were stronger colonizers relative to the less invasive isolates. Further characterization of isolates with different invasiveness will allow for the delineation of colonization factors.
Oakley, B., Morales, C., Hiett, K.L., Line, J.E., Seal, B.S. 2012. Application of high throughout sequencing to measure performance of commonly used selective cultivation methods for the food-borne pathogen campylobacter. FEMS Microbiology Ecology. 79(2):327-336.