Molecular characterization and mode of action of a bacteriocin produced by Lactococcus lactis subsp. lactis CRL 1584

PASTERIS S.E.; ALE C.E.; VERA PINGITORE E.; NADER-MACIAS M.E.

Tucumán

Simposio; IV SIMPOSIO INTERNACIONAL DE BACTERIAS LÁCTICAS (SIBAL).; 2013

Cerela-Conicet

Lactococcus lactis CRL 1584 isolated from a Lithobates catesbeianus (bullfrog) hatchery inhibits the growth of Citrobacter freundii (a red-leg syndrome related pathogen in raniculture) and Listeria monocytogenes (a food-borne bacteria) by a synergic effect of lactic acid, hydrogen peroxide and a bacteriocin. The bacteriocin could be used to control pathogenic microorganisms in raniculture and food-spoilage bacteria. The aim of this work was to advance in the characterization of the bacteriocin through the determination of the molecular localization of the encoding genes and its mode of action on L. monocytogenes, looking at the cell damage at the ultrastructural level and its viability. To identify the bacteriocin genes in L. lactis CRL 1584, degenerate primers were designed. The DNA was isolated and amplified by PCR, and agarose electrophoresis gels were applied to identify the amplicons. The amplified fragments were purified, sequenced and analyzed. The presence of extrachromosomal elements was also studied. Plasmid curing experiments by using novobiocin were performed to obtain a plasmid-free strain. The PCR amplification by using the nisin family oligonucleotides showed a DNA fragment of 450 bp. The sequence revealed a bacteriocin gene encoding peptides with a high degree of similarity to nisin Z. L. lactis showed only one plasmid and the plasmid-free variant have maintained the antimicrobial activity against L. monocytogenes similarly than the wild-type strain, indicating that bacteriocin genes would be encoded in the chromosome. On the other hand, supernatants of L. lactis CRL 1584 cultures were collected and concentrated to 4,200 AU/mL of bacteriocin. Then, fractions of supernatants were treated as follows: a-neutralized supernatant+catalase (N+C), b- neutralized supernatant+catalase+chymotrypsin (N+C+Chym). Chymotrypsin+heating (Chym+h) was used as control. These fractions were supplemented with BHI broth and inoculated with 5 x 105 cfu/ml L. monocytogenes Scott A. Samples were taken for viability and ultrastructural evaluation of the pathogenic cells. Bacteriocin showed a bactericidal effect on L. monocytogenes. After 60 min of incubation, L. monocytogenes viability decreased in 0.7 log units and no viable cells were detected after 150 min in N+C fractions. In both, N+C+Chym and Chym+h fractions there were no differences in the bacterial growth that showed 3 log units higher than the initial cell number at the end of the assay (300 min). The ultrastructural studies on L. monocytogenes cells revealed a clumping of the cytoplasmic material with an increased periplasmic space and an altered pattern of electron density in the cell wall at 60 min. These changes were more evident at 120 min of incubation. These results, described for the first time in L. lactis from a L. catesbeianus hatchery, help on the understanding of the mode of action of the bacteriocin. This antimicrobial substance could be applied as a biopreservative for bullfrog carcasses and also included in beneficial products for raniculture.