Submitted to: Campylobacter Helicobacter and Related Organisms International Workshop
Publication Type: Abstract only
Publication Acceptance Date: 6/30/2005
Publication Date: 9/6/2005
Citation: Stern, N.J., Svetoch, E., Eruslanov, B.V., Kovalev, Y.N., Volodina, L.I., Perelygin, V.V., Mitsevich, E.V., Mitsevich, I.P., Pokhilenko, V.D., Borzenkov, V.N. 2005. Isolation of lactobacillus salivarius inhibitory to campylobacter jejuni characterization of associated bacteriocin, and poultry treatment. Campylobacter Helicobacter and Related Organisms International Workshop. I-39. Interpretive Summary:
Technical Abstract: We evaluated anti-Campylobacter activity among 11,790 isolates of lactic acid bacteria from poultry production environments. We measured zones of C. jejuni inhibition surrounding the candidate strains and observed 279 isolates exhibiting antagonism. One Lactobacillus salivarius strain, PVD32, was identified and deposited under provisions of the Budapest Treaty (NRRL B-30514). The cell-free, ammonium sulfate precipitate from the culture was termed the crude antimicrobial preparation (CAP). A zone of C. jejuni growth inhibition surrounding 10 ul of the CAP was observed. C. jejuni growth resumed when the CAP was pre-incubated with protease enzymes, thus demonstrating the peptide characteristic consistent with bacteriocin definition. The bacteriocin was further purified using a combination of ammonium sulfate precipitation, CM-Sepharose, Superose, and ion exchange chromatography. SDS-PAGE electrophoresis provided an estimated molecular weight of ~6 KDa. MALDI-TOF analysis refined the molecular weight as 5,123 Da. The isoelectric point of the active fraction was determined at a pH of ~9.0. The amino acid sequence of the bacteriocin was determined. The bacteriocin activity was stable following exposure to 90oC for 15 minutes. The protein was purified and micro-encapsulated in polyvinylpyrrolidone (PVP), and added to feed at levels of 250 mg/Kg feed. 10-day of hatch chicks were placed in 9 isolation units; 2 units of birds were challenged with each of 4 C. jejuni isolates (one isolate per unit); the one additional unit served as an untreated, unchallenged group; at 7 days of age, birds in one unit per isolate were provided bacteriocin emended feed for 3 days while a corresponding unit was not given emended feed. The trials were duplicated. At day 10, all birds were sacrificed and the cecal content was plated directly onto Campy-Cefex agar (detection limit 100 cfu/gm) to enumerate the challenge strains. Bacteriocin treatment consistently reduced colonization by at least a one-million fold as compared with levels found in the untreated control groups. None of the non challenged birds were colonized. The bacteriocin from this Lactobacillus salivarius appears useful to control C. jejuni in poultry.