Food Control 44 (2014) 22e25 Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Short communication Susceptibility of Clostridium perfringens to antimicrobials produced by lactic acid bacteria: Reuterin and nisin Sonia Garde a, Natalia Gómez-Torres a, Marta Hernández b, Marta Ávila a,* a Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña km 7, 28040 Madrid, Spain b Instituto Tecnológico Agrario de Castilla y León (ITACyL), Carretera de Burgos km 119, 47071 Valladolid, Spain article info abstract Article history: The effectiveness as antimicrobials of lactic acid bacteria produced compounds reuterin and nisin was Received 4 February 2014 assessed against vegetative cells and spores of Clostridium perfringens isolates (from ovine milk obtained Received in revised form in farms with diarrheic lambs) and C. perfringens CECT 486 (type A toxin producer). We also tested the 14 March 2014 inhibitory effect of lysozyme and sodium nitrite on Clostridium. Minimal inhibitory concentrations (MIC) Accepted 22 March 2014 of antimicrobials were determined in modified RCM (mRCM) and milk by using a broth microdilution Available online 2 April 2014 method, after 7 d at 37 C under anaerobic conditions. The sensitivity of C. perfringens to the tested antimicrobials was strain and culture medium-dependent. In general, vegetative cells exhibited higher Keywords: e Reuterin sensitivity than spores. Reuterin (MIC values 2.03 16.25 mM) inhibited the growth of vegetative cells Nisin and the outgrowth of spores of all tested C. perfringens, both in mRCM and milk, with higher resistance in Nitrite milk. Nisin (MIC values 0.78e12.5 mg/ml) was also effective against vegetative cells and spores of tested Clostridium perfringens C. perfringens in both culture media. However, lysozyme (up to 400 mg/ml) did not control the growth of Spores any of the tested Clostridium. Sodium nitrite only inhibited the outgrowth of spores of two C. perfringens Vegetative cells isolates at the maximum concentration assayed (300 mg/ml) exclusively in mRCM medium. These results suggest that reuterin and nisin have the potential to control the growth of C. perfringens, and might help to ensure safety at different stages of the food chain. Future studies in food/feed products would be necessary to further corroborate this hypothesis. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction subsequently form spores in the large intestine, producing the enterotoxin at the same time (EFSA, 2005; Sutton & Hobbs, 1971). Spore-forming Clostridium perfringens is widespread in the Antimicrobial compounds obtained from lactic acid bacteria environment and it is frequently involved in food- and nonfood- may help to inhibit C. perfringens throughout the food chain, by borne gastrointestinal illnesses, especially C. perfringens type A themselves or in combination with other treatments. Reuterin is a producing CPE-enterotoxin (Udompijitkul, Paredes-Sabja, & Sarker, broad spectrum antimicrobial compound produced and secreted by 2012). Due to its presence in the intestinal tract of animals, car- the food grade lactic acid bacterium Lactobacillus reuteri (Axelsson, casses may become contaminated with C. perfringens during Chung, Dobrogosz, & Lindgren, 1989). Reuterin is produced as a slaughtering. Inadequate cooling and cooking of contaminated byproduct of glycerol fermentation (Axelsson et al., 1989; Talarico & meat or fish has caused foodborne infections in humans (Allaart, Dobrogosz, 1990) and consists of hydrated, non-hydrated, and van Asten, & Gröne, 2013). C. perfringens can be present in milk dimeric forms of 3-hydroxypropionaldehyde (3-HPA) and dairy products (El-Bassiony, 1980) as a result of contamination (Vollenweider, Grassi, König, & Puhan, 2003). It has high potential with soil or faecal matter, since it widely occurs in the intestinal as a food preservative since it is water soluble, stable and active tract of dairy ruminants. Food poisoning caused by C. perfringens over a wide pH range (Axelsson et al., 1989). Furthermore, the 3- gives rise to abdominal pain, nausea and acute diarrhea after the HPA reuterin form is a metabolite and not sensitive to proteases ingestion of 108 or more enterotoxin-producing vegetative cells, a or lipases, and no natural resistance mechanism has been described proportion of which survive the acid of the stomach and and consequently no resistance genes have been identified (Stevens, Vollenweider, & Lacroix, 2010). The antimicrobial activity * Corresponding author. Tel.: þ34 91 3476884; fax: þ34 91 3572293. of reuterin against a variety of both gram-negative and gram- E-mail address: [email protected] (M. Ávila). positive pathogens has been evaluated by different research http://dx.doi.org/10.1016/j.foodcont.2014.03.034 0956-7135/Ó 2014 Elsevier Ltd. All rights reserved. S. Garde et al. / Food Control 44 (2014) 22e25 23 groups (Arqués et al., 2004; Arqués, Rodríguez, Nuñez, & Medina, experiments. The concentration of reuterin (3-HPA) in the cell-free 2008; Arqués, Rodríguez, Nuñez, & Medina, 2011; Axelsson et al., supernatant was determined following the method described by 1989; Bian, Molan, Maddox, & Shu, 2011; El-Ziney & Debevere, Lüthi-Peng, Schärer, and Puhan (2002). Commercial nisin (Sigma, 1998; Spinler et al., 2008). However, we noticed that there is no St. Louis, Mo., USA; 2.5% pure nisin, potency of 106 IU/g), lysozyme information on the antimicrobial activity of reuterin against (Fluka Biochemica, Buchs, Switzerland; w70,000 U/mg) and so- C. perfringens. The bacteriocin nisin, a polypeptide produced by dium nitrite (Sigma) were used. Stock solutions were prepared in Lactococcus lactis, exerts its antimicrobial properties against several distilled water (or in 0.02 N HCl for assays of nisin in mRCM), filter- gram-positive organisms. Nisin is approved as a food preservative sterilized (0.22 mm) and stored at À40 C until use. by the EU (E234), as well as by the World Health Organisation (WHO) and the US Food and Drug Administration (FDA). However, 2.3. Preparation of spore and vegetative cell suspensions few studies have been carried out to test the antimicrobial activity of nisin against C. perfringens (Banerjeee & Sarkar, 2004; Eastoe & For obtaining spores, C. perfringens LBMM 391.1 was grown for Long, 1959; Guerlava, Nolf, & Tholozan, 1998; Scannell, Ross, Hill, 24 h on Ellner’s broth; C. perfringens LBMM 395.3 was grown for & Arendt, 2000; Udompijitkul et al., 2012). 24 h on modified DuncaneStrong sporulation medium (1.5% pro- In addition to lactic acid bacteria antimicrobials, food and/or teose peptone, 1.33% sodium phosphate dibasic, 0.4% yeast extract, feed additives may control C. perfringens growth. Thus, nitrite is 0.4% raffinose, 0.05% cysteine-HCl); C. perfringens CECT 486 was used as an antimicrobial agent primarily to prevent the growth of grown for 4 d on mRCM (all at 37 C, in anaerobiosis). Spores were Clostridium botulinum in meat products. Lysozyme is mainly added centrifuged (5000 Â g, 15 min), washed twice, resuspended into to prevent cheese late blowing caused by Clostridium tyrobutyr- sterile water (108 spores/ml), and stored at e 40 C. Before exper- icum, but it has also been tested as a feed additive to control iments, spores were heat-shocked (80 C, 15 min). For obtaining C. perfringens-associated disease in poultry (Allaart et al., 2013). vegetative cells, C. perfringens isolates were grown in mRCM over- Recent studies carried out with nisin and reuterin at our laboratory night at 37 C in anaerobiosis (108 cells/ml). The concentration of evidenced their broad spectrum of activity against spoilage Clos- vegetative cells and spores was determined by a Thoma counting tridium spp. isolated from late blowing cheeses (Ávila, Gómez-Torres, chamber under phase-contrast microscopy. Hernández, & Garde, 2014). In this study, we perform a preliminary evaluation of the susceptibility of vegetative cells and spores of 2.4. Minimal inhibitory concentration of antimicrobial compounds C. perfringens to nisin and reuterin, as well as to nitrite and lysozyme. Minimal inhibitory concentrations (MICs) of reuterin, nisin, 2. Material and methods lysozyme and sodium nitrite against vegetative cells and spores of C. perfringens were determined in litmus milk and mRCM using a 2.1. Bacterial strains and growth conditions broth microdilution method (Ávila et al., 2014). For each antimi- crobial compound, assays were carried out in duplicate wells in the C. perfringens CECT 486 (type A toxin producer) is a type strain microplates, in duplicate experiments. Final spore concentration in from the Spanish Type Culture Collection (Colección Española de wells was 105 spores/ml, and vegetative cell concentration in wells Cultivos Tipo, Valencia, Spain). C. perfringens isolates LBMM 391.1 was 106 vegetative cells/ml. Positive and negative controls of and LBMM 395.3 (ITACyL culture collection) were obtained from clostridia growth were included. In litmus milk, the growth of the ovine milk in farms with diarrheic lambs. Clostridia were grown in tested C. perfringens was distinguished because they produced acid Reinforced Clostridial Medium (RCM, Difco, Detroit, USA) at 37 C, coagulation of the milk. The absence of growth was confirmed 48 h, in anaerobic jars with an H2 plus CO2 generating kit (Anaer- visually after 7 d at 37 C in anaerobiosis. The results are expressed oGen, Oxoid, Basingstoke, UK). Lactobacillus