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Title: Isolation of Lactobacillus salivarius 1077 (NRRL B-50053) and characterization of its bacteriocin and spectra of antimicrobial activity

Author
item SVETOCH, EDWARD - State Research Center For Applied Microbiology And Biotechnology
item ERUSLANOV, BORIS - State Research Center For Applied Microbiology And Biotechnology
item LEVCHUK, VLADIMIR - State Research Center For Applied Microbiology And Biotechnology
item PERELYGIN, VLADIMIR - State Research Center For Applied Microbiology And Biotechnology
item MITSEVICH, EVGENY - State Research Center For Applied Microbiology And Biotechnology
item MITSEVICH, IRINA - State Research Center For Applied Microbiology And Biotechnology
item STEPANSHIN, JURI - State Research Center For Applied Microbiology And Biotechnology
item DYATLOV, IVAN - State Research Center For Applied Microbiology And Biotechnology
item Seal, Bruce
item Stern, Norman

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/17/2011
Publication Date: 4/1/2011
Citation: Svetoch, E.A., Eruslanov, B.V., Levchuk, V.P., Perelygin, V.V., Mitsevich, E.V., Mitsevich, I.P., Stepanshin, J., Dyatlov, I., Seal, B.S., Stern, N.J. 2011. Isolation of Lactobacillus salivarius 1077 (NRRL B-50053) and characterization of its bacteriocin and spectra of antimicrobial activity. Applied and Environmental Microbiology. 77(8):2749-2754.

Interpretive Summary: Pathogenic bacteria continue to contaminate raw poultry and result if disease. We have been finding antagonistic bacteria that produce antimicrobial proteins which kill pathogens. One of these antagonists, Lactobacillus salivarius L-1077, was isolated from poultry and kills the pathogen Campylobacter jejuni. The isolate was then used to produce bacteriocin (BCN). Using a variety of biochemical techniques we purified and characterized the BCN. The BCN had a molecular weight of 3,454 Da and the iso-electric point was 9.1. Minimum inhibitory concentrations (MIC) of BCN L-1077 against 33 pathogen isolates were quite excellent. Subsequently, the therapeutic benefit of BCN L-1077 was demonstrated in market-age (40 to 43 day old) broiler chickens colonized with both C. jejuni and Salmonella enteritidis. As compared with untreated control birds, both C. jejuni and S. enteritidis in colonized ceca were diminished by ten-thousand fold and, S. enteritidis in both the liver and spleen of treated birds were reduced by 6 to 8 log10/gm over the non-treated control birds. BCN L-1077 appears to hold promise in controlling C. jejuni/S. enteritidis in commercial broiler chickens.

Technical Abstract: Lactobacillus salivarius 1077 (NRRL B-50053) was isolated from poultry intestinal materials after demonstrating in-vitro anti-Campylobacter jejuni activity. The isolate was then used for in-vitro fermentation. The protein content of the cell-free supernatant from the spent medium was precipitated by ammonium sulfate and dialyzed to produce the crude antimicrobial preparation. The bacteriocin (BCN)-like sensitivity to proteolytic enzymes and resistance to lysozyme, lipase and 100o C was observed with this preparation. The polypeptide was further purified by gel-filtration, ion-exchange and hydrophobic-interaction chromatography. MALDI TOF MS, Edman degradation and iso-electro focusing were used to characterize its 3,454 Da molecular weight, the amino acid sequence of its 37 residue components and the 9.1 iso-electric point of the BCN. BCN L-1077 contained the Class II BCN signature N-terminus sequence of YGNGV. Minimum inhibitory concentrations (MIC) of BCN L-1077 against 33 bacterial isolates (both Gram-negative and Gram-positive) ranged from 0.09 to 1.5 mg/ml. Subsequently, the therapeutic benefit of BCN L-1077 was demonstrated in market-age (40 to 43 day old) broiler chickens colonized with both C. jejuni and Salmonella enteritidis. As compared with untreated control birds, both C. jejuni and S. enteritidis in colonized ceca were diminished by > 4 log10 and, S. enteritidis in both the liver and spleen of treated birds were reduced by 6 to 8 log10/gm over the non-treated control birds. BCN L-1077 appears to hold promise in controlling C. jejuni/S. enteritidis in commercial broiler chickens.