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United States Department of Agriculture

Agricultural Research Service

Title: Dereplication by Automated Ribotyping of a Probiotic Competitive Exclusion Culture Bacterial Isolate Library

Authors
item Sheffield, Cynthia
item Andrews, Kathleen
item Harvey, Roger
item Crippen, Tawni
item Nisbet, David

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 4, 2005
Publication Date: January 20, 2006
Citation: Sheffield, C.L., Andrews, K., Harvey, R.B., Crippen, T.L., Nisbet, D.J. 2006. Dereplication by automated ribotyping of a competitive exclusion culture bacterial isolate library. Journal of Food Protection. 69:228-232.

Interpretive Summary: The treatment of animals with a mix of harmless bacteria in an attempt to prevent infection from disease-causing bacteria is a strategy called competitive exclusion. This strategy is used in place of chemical antibiotics to control disease-causing bacteria in food animals. During competitive exclusion treatment, a mix of many different bacterial species is given to the animal. This mix is originally harvested from healthy animals and its bacterial make-up must be determined prior to its use in competitive exclusion treatment. The process of defining the bacterial make-up of this mix is a complicated and time consuming process when done by selective culturing methods. In the present study, a technique called automated ribotyping was compared to selective culturing techniques for identifying a complicated mix of bacteria. In addition to being more time consuming than ribotyping, selective culturing methods lack the precision of ribotyping and, therefore, often result in the duplication of species. Ribotyping, on the other hand, can identify the individual species of bacteria contained within the competitive exclusion treatment more rapidly and more precisely. A total of 157 isolates from the competitive exclusion mix used in this study were screened and sorted into 23 ribogroups. This is an 85% reduction from the size of the original library. A total of 134 duplicate bacterial species could be discarded from the library by using the ribotyping technique. This is a significant reduction in the time and effort necessary to determine the bacterial make-up in this mix compared to standard selective culture techniques.

Technical Abstract: Concerns over the development of antibiotic resistant bacteria within the food animal industry have intensified the search for more natural approaches to the prevention and treatment of bacterial diseases in food animals. Competitive exclusion cultures are the foundation of a disease-management strategy based on the use of benign bacterial strains to prevent the establishment of pathogenic bacteria within a specific host. Differentiation of phenotypically ambiguous isolates is a critical step in establishing a manageable library of bacteria for use in the development of defined competitive exclusion cultures. Automated ribotyping was employed to dereplicate a large collection of phenotypically indistinguishable isolates from a continuous-flow competitive exclusion culture. A total of 157 isolates were screened by automated ribotyping following an EcoRI restriction enzyme digestion. The isolates were resolved into 23 ribogroups, which represents an 85% reduction in the number of isolates in the bacterial isolate library. Seventy-six percent of the isolates fit into one of 5 ribogroups. This work demonstrated that automated ribotyping is an effective and efficient tool for dereplication of bacterial libraries.

Last Modified: 10/1/2014
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