Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Determine whether Campylobacter resistance to selected individual classes of antimicrobial proteins are or are not consistently manifest across all antimicrobial protein classes tested. Approach (from AD-416): Ferment Lactobacillus salivarius NRRL 5-30514 in specified microbiological broth system. Purify the spent (fermented) medium by ammonium sulfate precipitation, dialysis and column chromatography to obtain purified OR-7. This research relates to inhouse project objective 1. Develop and evaluate current and novel strategies to reduce food-borne pathogens in the poultry production environment. Lactobacillus salivarius strain NRRL B-30514 has demonstrated anti- Campylobacter growth activity and was subsequently shown to produce an antimicrobial peptide typical of class II bacteriocins designated OR-7. The purified protein was encapsulated in polyvinylpyrrolidone, added to chicken feed and during feeding trials consistently reduced the bacterial load of C. jejuni in chicken gastrointestinal tracts. Genomic DNA from L. salivarius B30514 was extracted and subjected to pyrosequencing by current 454 GS FLX sequencer. The 454 sequencing was performed twice, producing approximately 200 Mbp of sequence information to obtain a draft genome sequence of Lactobacillus salivarius strain NRRL B-30514. Properties including potential fermentation activity and bacteriocin production suggest that the bacterium has potential uses as a probiotic lactic acid bacterium and for enhancing bacteriocin production. Sequence analysis of the genome will allow for enhancing these beneficial properties of probiotic lactic acid bacteria.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Determine whether Campylobacter resistance to selected individual classes of antimicrobial proteins are or are not consistently manifest across all antimicrobial protein classes tested. Approach (from AD-416) Ferment Lactobacillus salivarius NRRL 5-30514 in specified microbiological broth system. Purify the spent (fermented) medium by ammonium sulfate precipitation, dialysis and column chromatography to obtain purified OR-7. Bacteriocins (BCNs) are antimicrobial peptides with narrow or broad spectra of antimicrobial activity produced by many lactic-acid bacteria commonly found in animal or human gastrointestinal tracts. Campylobacter jejuni is a commensal bacterium of chickens that is one of the leading causes of human food-borne disease. Recently, several unique anti- Campylobacter BCNs have been identified from lactic acid bacteria isolated from chicken intestines. These BCNs dramatically reduced C. jejuni colonization in poultry and are being directed toward on-farm control of Campylobacters. However, no information concerning prevalence, development and mechanisms of BCN resistance in Campylobacter exists. Susceptibilities to the anti-Campylobacter BCNs OR-7 and E-760 were examined with 137 C. jejuni isolates and 20 C. coli isolates. Only one C. coli strain displayed resistance to the BCNs and this resistance could be transferred at intra- or interspecies levels among Campylobacter strains by natural transformation. Genomic examination of the OR-7-resistant mutants using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to resistance and acquired resistances to the BCNs. This represents a major step forward in understanding BCN resistance in Campylobacters, which will facilitate the development of effective BCN-based strategies to reduce Campylobacter loads in poultry for improving human food safety.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Determine whether Campylobacter resistance to selected individual classes of antimicrobial proteins are or are not consistently manifest across all antimicrobial protein classes tested. Approach (from AD-416) Ferment Lactobacillus salivarius NRRL 5-30514 in specified microbiological broth system. Purify the spent (fermented) medium by ammonium sulfate precipitation, dialysis and column chromatography to obtain purified OR-7. This research relates to inhouse objective 1: Assess the effectiveness and further development of bacteriocins (anti-bacterial peptides) and bacteriophage by in vitro bacterial growth inhibition in culture and in vivo experimentation via challenge in chickens. The project is proceeding toward its goals. We have prepared, purified and delivered a small quantity of bacteriocin E-760 to Professor Lin, University of Tennessee. As the minimum inhibitory concentrations (MIC�s) were in the predicted concentrations of ~0.5 mg/ml we are confident of the product that was delivered. We continue attempting to produce additional purified bacteriocin materials for further testing. The current approach being taken is to produce the materials in heterologous (Saccharomyces cerevisiae) systems. We are attempting to understand the biology of this system so that larger quantities of the materials can be produced. Thus far, susceptibilities of 137 C. jejuni and 20 C. coli isolates for two bacteriocins (BCNs;OR-7 and E-760)were examined. Only one C. coli strain displayed resistance to the BCNs (MIC = 64�g/ml) while others were susceptible with MIC ranging from 0.25 to 1 �g /ml. BCN- resistant (BCNR) C. coli mutant was also obtained by in vitro selection but no high-level BCNR mutants were selected. The BCN resistance in C. coli could be transferred to C. jejuni strains by natural transformation with frequency about 10-7 CFU/�g DNA/recipient cell. Using DNA microarray, we compared the transcriptome of a BCNR C. jejuni mutant with that of parent strain NCTC 11168. Microarray analysis revealed 9 genes were upregulated and 10 genes were downregulated more than two fold in the BCNR mutant. The majority of genes upregulated in the BCNR mutant encoded for zinc metallopeptidase, galactosyltransferase, and three different efflux pumps. Inactivation of these efflux pumps showed that only CmeABC efflux pump contributed Campylobacter resistance to the BCNs. Genomic examination of a 81-176-derived BCNR mutant using in vivo random transposon mutagenesis also indicated that CmeABC contributes C. jejuni to both intrinsic and acquired resistance to the BCNs. Together, this study represents the first report and a major step forward in understanding BCN resistance in Campylobacter.
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