Integrating Novel Biopolymers into Microbial Enteropathogen-Reduction Studies (ChitoBiOMEs)

Non-Technical Summary:<br/>
Many health related cases due to the presence of foodborne pathogens like Escherichia coli, Salmonella typhimurium and Clostridium difficile are being reported daily and many are acquired during hospitalization. Most recently, highly infective strains of these pathogens have surfaced leaving investigators to speculate that these are associated with the consumption of certain foods, like poultry. Chitosan, a N-deacetylated derivative of chitin, consist of 2-amino-2-deoxy (1-4)-beta-D-glucopyranose residues (or D-glucosamine units) and is derived from the partial alkaline deacetylation of chitin. Chitins, chitosan's precursor, are cellulosic type biopolymers which are widely distributed in nature, such as in the cell wall of fungi and exoskeleton of insects and crustaceans. Recent studies have reported that chitosans and its oligomers have antimicrobial properties. However, little data is available on in vivo microbial studies involving chitosan and its derivatives. Hence, our "ChitoBiOMEs" program addresses the role of chitosan on microbial growth in chicken ceca. In this study, chitosan will be incorporated into the feed mixture for broiler chicken and the growth of three strains of bacteria, Salmonella Typhimurium, Campylobacter jejuni, and Escherichia coli O157:H7 will be observed using molecular biology techniques. Phenotype microarrays, for the first time, will be used as a powerful platform to study the effects of chitosan derivatives on the phenomic activity of the microbiota associated with chicken ceca. With the involvement of USDA and TAMU, this cooperative project will complement current ARS research to develop cost effective means to prevent and/or control foodborne pathogens in poultry that are causative agents of disease in consumers. We will address larger problems, such as the need to use biodegradable products to improve healthy food production. Thus, new intervention strategies, involving the incorporation of chitosan and its derivatives into existing commercial management programs of chicken, will be investigated to reduce foodborne pathogen colonization in poultry. Simultaneously, the "ChitoBiOMEs" project will complement current ARS programs to prepare young scientists for research careers in agricultural research and with ARS. Thus, these students would be trained in areas of research and instruction that would make them attractive candidates for graduate school and research positions in government, academia and industry.
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Approach:<br/>
Chitosan will be modified using chemical and physical procedures and its chemical integrity, thermal stability and surface morphology will be assessed before and after modification. Techniques such as Fourier Transform Infrared Spectroscopy, 1H-NMR and 13C-NMR will be used for structure elucidation. Thermogravimetric Analysis and Differential Scanning Calorimetry will be used to assess the thermal stability of chitosan and its derivatives; while, Atomic Force Microscopy will be used to assess the surface morphology of chitosan derivatives. The molecular weights will be determined using Gel Permeation Chromatography. Subsequently, in vitro studies will be performed to determine the effects of chitosan and its derivatives on the growth of three strains of bacteria, Salmonella Typhimurium, Campylobacter jejuni, and Escherichia coli O157:H7, that occupy the ceca of broiler chickens. In vivo experiments will follow with fifteen day-of-hatch broiler chicks placed on pine litter in floor pens. Contents from the duodenum, ileum, one cecum, and colon of five chicks will be removed and examined by Denaturing Gradient Gel Electrophoresis (DGGE) for modifications in microbial populations.
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It is hypothesized that incorporation of chitosan and its derivatives would selectively enrich the microbiota in chicken ceca. Therefore, phenotype microarray experiments will be performed on ceca samples of chickens that were treated with chitosan derivatives to evaluate the differences in microbial population. Furthermore, students majoring in Agriculture at PVAMU will be fully engaged in performing hands-on laboratory research, thus learning a variety of techniques, ranging from polymer chemistry, microbiology and phenomics. Additionally, the students will participate in activities such as research forums, seminars, fieldtrips, research symposia and scientific meetings to further enhance their learning experience on becoming a scientist. Longitudinal data collection will be utilized to assess programmatic progress. Closed-end surveys will generally be used to assist the PI and Co-PIs in ensuring that the objectives and specific aims are being achieved.