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United States Department of Agriculture

Agricultural Research Service

Title: Isolation of a Lactobacillus Salivarius: Its Bacteriocin Is Inhibitory to Campylobacter Jejuni in Chickens

Authors
item Stern, Norman
item Svetoch, E - RUSSIAN ST DEPT
item Eruslanov, B - RUSSIAN ST DEPT
item Perelygin, V - RUSSIAN ST DEPT
item Mitsevich, E - RUSSIAN ST DEPT
item Mitsevich, I - RUSSIAN ST DEPT
item Pokhilenko, V - RUSSIAN ST DEPT
item Levchuk, V - RUSSIAN ST DEPT
item Svetoch, O - RUSSIAN ST DEPT
item Seal, Bruce

Submitted to: Antimicrobial Agents and Chemotherapy
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 7, 2006
Publication Date: September 1, 2006
Citation: Stern, N.J., Svetoch, E.A., Eruslanov, B.V., Perelygin, V.V., Mitsevich, E.V., Mitsevich, I.P., Pokhilenko, V.D., Levchuk, V.P., Svetoch, O.E., Seal, B.S. 2006. Isolation of a lactobacillus salivarius: its bacteriocin is inhibitory to campylobacter jejuni in chickens. Antimicrobial Agents and Chemotherapy. Vol 50:3111-3116.

Interpretive Summary: Campylobacter jejuni is a poultry-borne disease causing bacteria. High numbers of this organism on processed poultry carcasses leads to unacceptably high rates of human disease. We screened >1,200 competing Lactobacilli bacteria from chicken guts for Campylobacter-killing ability and identified one (Lactobacillus salivarius- NRRL B-30514) as the best in this capacity. We then isolated a particular protein (bacteriocin) from this Lactobacillus strain that served to kill Campylobacter. This bacteriocin was then produced and purified in sufficient quantities and added to chicken feed. The chicken feed was provided for 3 days to groups of Campylobacter-colonized chickens. The birds were sacrificed and the numbers of Campylobacter in bacteriocin treated birds were compared to numbers found in Campylobacter-colonized, but untreated birds. The results indicated at least one-million fold reduction in Campylobacter in the treated birds. This treatment appears useful in reducing human exposure of Campylobacter and corresponding reductions in human disease.

Technical Abstract: We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of lactic acid bacteria. One, Lactobacillus salivarius (NRRL B-30514), was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation (CAP). Ten-µl of the CAP created a zone of C. jejuni growth inhibition; growth within the zone resumed when the CAP was pre-incubated with protease enzymes. The bacteriocin (OR-7) was further purified using molecular sieving and ion exchange chromatography. SDS-PAGE electrophoresis estimated the bacteriocin molecular weight as ~6,000 Da and, MALDI-TOF analysis at 5,123 Da. The isoelectric point of the antimicrobial peptide was determined at pH ~9.0. The amino acid sequence determined was consistent with Class IIa bacteriocins. Bacteriocin activity was stable following exposure to 90oC for 15 minutes. The purified protein was encapsulated in polyvinylpyrrolidone (PVP), and added to feed at 250 mg/Kg feed. In duplicate experiments, 10 day-of-hatch chicks were placed in each of 9 isolation units; 2 groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit); at 7 days-of-age, one group of birds were treated with bacteriocin emended feed for three days and one group were left untreated. At 10 days-of-age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization by at least one-million fold as compared with levels found in the untreated groups. Non-challenged birds were never colonized. Bacteriocin from L. salivarius NRRL B-30514 appears useful to control C. jejuni in poultry.

Last Modified: 12/20/2014
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