In vitro inhibition of Citrobacter freundii, a red-leg syndrome associated pathogen in raniculture, by indigenous Lactococcus lactis CRL 1584 Sergio E. Pasteris a , Marcos G. Guidoli a , Marı ´a C. Otero a,b , Marta I. Bu ¨ hler a , Marı ´a E. Nader-Macı ´as b, * a Instituto Superior de Investigaciones Biolo ´gicas (INSIBIO-CONICET) - Instituto de Biologı´a ‘‘Dr. Francisco D. Barbieri’’, Facultad de Bioquı´mica, Quı´mica y Farmacia - Universidad Nacional de Tucuma ´n, Chacabuco 461, CP: T4000ILI, San Miguel de Tucuma ´n, Argentina b Centro de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145, CP: T4000ILC, San Miguel de Tucuma ´n, Argentina 1. Introduction Raniculture represents a growing commercial aqua- culture activity widely distributed throughout the world, Lithobates catesbeianus (Frost et al., 2006) being the selected species. The Food and Agriculture Organization (2001) has indicated that bullfrog meat has a high proportion of essential amino acids, a large degree of absorbable proteins and low contents of sodium chloride and lipids (mainly cholesterol). These commercial require- ments have resulted in an intensive L. catesbeianus production process that increases the risk of bacterial disease outbreaks (Mauel et al., 2002). Red-leg syndrome (RLS) or bacterial dermatosepticemia is one of the main infectious diseases that affect captive bullfrog, causing high economic losses (Texeira et al., 2002; Densmore and Earl Green, 2007). The etiological agents involve Citrobacter freundii, and the widespread therapies include the use of antibiotics (Glorioso et al., 1974; Mauel et al., 2002, Schadich and Cole, 2009; Zhen-Yu et al., 2009). Thus, the Veterinary Microbiology 151 (2011) 336–344 A R T I C L E I N F O Article history: Received 13 November 2010 Received in revised form 18 March 2011 Accepted 23 March 2011 Keywords: Lithobates catesbeianus Raniculture Infectious diseases Lactic acid bacteria Probiotics A B S T R A C T Red-leg syndrome (RLS) is one of the main infectious diseases that cause economic losses in Lithobates catesbeianus hatcheries, Citrobacter freundii being an etiological agent. Treatment or prevention with therapeutics or chemicals results in modifications of the indigenous microbiota, development of antibiotic resistance, presence of their residues in food and enhancement of production costs. Thus, probiotics could be used as an alternative therapy. Lactic acid bacteria are part of the indigenous microbiota of healthy frogs and can prevent pathogen colonization by different mechanisms, including the production of antagonistic substances. In this work, the evaluation and characterization of the inhibition of C. freundii CFb by Lactococcus lactis subsp. lactis CRL 1584, a potentially probiotic candidate, were carried out. This strain produced lactic acid, H 2 O 2 and bacteriocin in static and shaken conditions and inhibited pathogen growth in associative cultures, with an earlier inhibition under agitated conditions. The elimination of each of the antimicrobial metabolites partially abolished the inhibition of the pathogen, suggesting that the inhibitory effect could be attributed to a combined action of the three antagonistic molecules. Electron microphotographs revealed the damage caused by L. lactis CRL 1584 supernatants to C. freundii cells. The addition of pure lactic acid, H 2 O 2 and bacteriocin to the culture media showed that each metabolite caused different morphological modifications in C. freundii, in agreement with the effect on viable cell counts. The results support the possibility that L. lactis CRL 1584 might be considered as a probiotic to be used in the prevention of RLS in raniculture. ß 2011 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +54 381 4310465; fax: +54 381 4311720. E-mail address: fnader@cerela.org.ar (M.E. Nader-Macı ´as). Contents lists available at ScienceDirect Veterinary Microbiology jou r nal h o mep ag e: w ww .els evier .co m/lo c ate/vetm ic 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.03.025