EFFECT OF LATOCOCCIN ON THE GROWTH AND VIABILITY OF Citrobacter freundii, A RED-LEG ASSOCIATED PATHOGEN IN RANICULTURE

PASTERIS, S.E.; GUIDOLI, M.; OTERO M. C.; BÜHLER, M.I.; NADER MACÍAS M. E.

SAINT MALO. FRANCIA

Simposio; SECOND INTERNATIONAL SYMPOSIUMN ON ANTIMICROBIAL PEPTIDE: FOOD, VETERINARY, MEDICAL, AND NOVEL APPLICATIONS.; 2009

Raniculture is the activity that includes the reproduction,breeding and fattening of frogs to obtain meat and by-products. Rana catesbeiana (R.c.) is the selected specie for this purpose, being the production an intensive process. In these conditions, the animals are more susceptible to infectious diseases, such as red leg syndrome (RLS). Sometimes, bullfrog farmers use antibiotics to control bacterial diseases that modify both intestinal and skin microbiota and contribute to the spread of antibiotic resistance [1]. Thus, an alternative way to prevent diseases is the application of probiotics[2], which are defined as “microbial cells that are administered to reach the gastrointestinal tract and maintain their viability, with the aim of improving host health” [3]. Lactic acid bacteria (LAB) are used as probiotics in aquaculture and were described as autochthonous microorganisms in raniculture [1]. Lactococcus lactis subsp lactis CRL 1584 was isolated from a R.c. hatchery [1], and is able to produce lactic acid, H2O2 and a bacteriocin (lactococcin) identified previously as Nisin Z. The purpose of this work was to evaluate the effect of lactococcin on the growth and viability of Citrobacter freundii, as a frog’s pathogen.  Lactococcin production was determined by the agar well diffusion method by using Listeria monocytogenes as indicator strain. The bacteriocin was synthesized in LAPTg medium at 37°C following the microbial growth, with a maximum of 1,760 AU/mL at the end of the exponential growth phase (5 h). In these conditions, LAB strain also produced the highest lactic acid amounts (4.85 g/L). To evaluate the effect of the antimicrobial compounds on the pathogen growth and viability, cocultures between L. lactis and C. freundii were performed in LAPTg broth at 37°C. The results showed that the number of CFU/mL of the pathogenic bacteria decreased from the beginning of the assay to 0 values at 8 h. These results indicate that the inhibition was produced by lactic acid, H2O2 and/or lactococcin or either by a synergic effect between all of them. Later, C. freundii cells obtained during the exponential growth were resuspended and maintained for 7 h in untreated or treated (neutralized, added with catalase or chymotrypsin) cell-free LAB strain supernatants. When suspended into untreated supernatants, the pathogen viability decreased 6 log U, while into neutralized supernatant cells viability remains constant. However, when resuspended in catalase or chymotrypsin treated supernatants, the number of CFU/mL decreased, but not to the low levels of the crude supernatant (1-2 log U), These data indicate that the inhibitory activity is the result of a synergic effect between all the antimicrobial compounds, as was demonstrated when the pathogen was treated individually with lactic acid, H2O2 or supernatant containing lactococcin. These results contribute to the knowledge of the chemical nature of the inhibitory effect of a potential probiotic bacterium for raniculture.