Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system

Abstract

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 degrees 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.

FIG. 1.

Direct detection of bacteriocin OR-7 from the crude antimicrobial preparation of Lactobacillus salivarius NRRL B-30514 culture after SDS-PAGE. Following SDS-PAGE, the gel was laid onto a plate seeded with Campylobacter jejuni to determine which band(s) corresponded to the antimicrobial activity and its specific molecular mass. Lane 1 contains molecular mass markers (in kilodaltons). The bands and zones of inhibition in lanes 2 and 4 are pure bacteriocin and a crude preparation demonstrating antimicrobial activity. Lane 3 is the crude antimicrobial preparation after adsorption with C. jejuni. The zone of growth inhibition (arrow) had a mass (M) of ∼6 kDa.

FIG. 2.

Direct detection of OR-7 from the crude antimicrobial preparation of Lactobacillus salivarius NRRL B-30514 culture after isoelectric focusing. The focusing gel was laid over a plate seeded with Campylobacter jejuni to demonstrate antimicrobial activity at the specified isoelectric point. Lane 1 contains pI standards (pI values for Serva marker proteins were 10.25, 10.04, 9.75, 9.30, 8.65, 8.45, and 8.15). The zones of clearance surrounding the highlighted band (arrow) in lanes 2 and 3 are crude antimicrobial preparations and pure OR-7 bacteriocin demonstrating antimicrobial activity relating to the peptide migrating at pI ∼9.3.