We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90°C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two 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 was treated with bacteriocin-emended feed for 3 days, and one group was 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 at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.
We evaluated anti-Campylobacter activity among 365 Bacillus and Paenibacillus isolates from poultry production environments. One novel antagonistic Bacillus circulans and three Paenibacillus polymyxa strains were identified and further studied. Cell-free ammonium sulfate precipitate (crude antimicrobial preparation) was obtained from each candidate culture. Zones of Campylobacter growth inhibition surrounding 10 microl of this crude antimicrobial preparation were quantified using a spot test. Campylobacter growth resumed when the preparation was preincubated with selected protease enzymes, demonstrating peptide characteristics consistent with a bacteriocin. These peptides were further purified using combinations of molecular mass resolution and ion exchange chromatography. Molecular masses of the peptides were estimated at approximately 3,500 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing was used to determine the pI values of the peptides. Amino acid sequences of the bacteriocins and more precise molecular masses were obtained by matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF) analysis. The bacteriocin from P. polymyxa NRRL B-30507 had a pI of 4.8, that from P. polymyxa NRRL B-30509 had a pI of 7.2, that from P. polymyxa NRRL B-30508 had a pI of 4.8, and that from B. circulans NRRL B-30644 had a pI of 7.8. The amino acid sequences were consistent with those of class IIa bacteriocins. These antagonists and the corresponding bacteriocins may be useful in the control of Campylobacter infection in poultry.
Campylobacter is a leading cause of food-borne illness in the United States. Recent evidence has demonstrated that bacteriocins produced by Bacillus circulans and Paenibacillus polymyxa reduce cecal Campylobacter colonization in broiler chickens infected with Campylobacter jejuni. As Campylobacter coli is the most prevalent Campylobacter isolate recovered in turkeys, the objectives of the present study were to evaluate the efficacy of these bacteriocins against C. coli colonization and their influence on the gastrointestinal architecture of young turkeys. In 3 separate trials, a total of 135 day-of-hatch poults (n = 45/trial) were orally challenged on d 3 with approximately 10(6) cfu of a mixture of 3 C. coli isolates. Immediately before bacteriocin treatment (d 10), cecal Campylobacter concentrations averaged 1.1 x 10(7) cfu/ g of cecal contents (n = 15/trial). On d 10 to 12 posthatch, 2 bacteriocin treatment groups were given free access to feed supplemented with purified, microencapsulated bacteriocins, whereas the positive control treatment group had access to untreated feed (n = 10/treatment group per trial). At the end of the 3-d dosing period, ceca and duodenal loops were collected for analysis. In each of the 3 separate trials, treatment with bacteriocin eliminated detectable ceca Campylobacter concentrations (detection limit, 1 x 10(2) cfu/g of cecal contents) vs. controls (1.0 x 106 cfu of Campylobacter/g of cecal contents). Duodenum crypt depth and goblet cell numbers were also reduced in turkeys treated with either bacteriocin vs. controls (P < 0.05). The dynamic reduction in crypt depth and goblet cell density in turkeys dosed with bacteriocin may provide clues to how bacteriocins inhibit enteric Campylobacter.
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