Food Control 44 (2014) 22e25

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Food Control

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Short communication Susceptibility of perfringens to antimicrobials produced by lactic acid : 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 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 40 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 in meat products. Lysozyme is mainly added centrifuged (5000 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 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 reuteri INIA P572 (INIA as the MIC of each antimicrobial that caused complete growth in- culture collection) was used as the reuterin-producing strain hibition of the tested C. perfringens in 4 out of 4 replicates. (Rodríguez, Arqués, Rodríguez, Nuñez, & Medina, 2003) and was cultured in MRS broth (de Man, Rogosa, & Sharpe, 1960; Biolife, 3. Results and discussion Milano, Italy) at 37 C for 18 h, in anaerobic jars. Bacteria were stored at 80 C as stock cultures in their corresponding culture The MICs of the antimicrobials against C. perfringens are pre- media supplemented with 5% glycerol, and sub-cultured twice sented in Table 1. Reuterin exhibited antimicrobial activity against before their use in experiments. Culture media for experiments all tested C. perfringens, although its effectiveness depended on the were litmus milk (Biolife), and modified RCM broth (mRCM), which specific isolate or strain and the culture medium used. One mech- does not contain either cysteine hydrochloride or agar, as cysteine anism by which 3-HPA, the bioactive form of reuterin, may inhibit could counteract the effects of reuterin (Schaefer et al., 2010; vegetative bacteria growth is by modifying thiol groups in proteins Vollenweider, Evers, Zurbriggen, & Lacroix, 2010). and small molecules, what eventually leads to a disturbance of the redox status of the cell, ultimately resulting in cell death (Schaefer 2.2. Reuterin, nisin, lysozyme and sodium nitrite solutions et al., 2010; Vollenweider et al., 2010). Vegetative cells of tested C. perfringens were more sensitive to reuterin (MIC range 2.03e A cell-free supernatant of L. reuteri INIA P572 containing 65 mM 4.06 mM) than other clostridia previously reported such as Clos- of reuterin was obtained by modification of the method described tridium clostridioforme (15e30 mM) (Cleusix, Lacroix, Vollenweider, by Talarico, Casas, Chung, and Dobrogosz (1988). Briefly, L. reuteri Duboux, & Le Blay, 2007) and most dairy-related Clostridium spe- INIA P572 was grown overnight at 1% in 1 l of MRS broth at 37 Cin cies (4.06e16.25 mM) (Ávila et al., 2014). However, tested anaerobiosis, harvested by centrifugation (4500 g, 5 min) and C. perfringens showed slightly higher MICs than those described for gently washed in sterile aqueous solution of glycerol (100 mM). In Clostridium difficile (MIC < 1.9 mM) (Cleusix et al., 2007) and order to obtain reuterin from glycerol, the collected cell biomass Clostridium sporogenes (1.1 mM) (Chung, Axelsson, Lindgren, & was suspended into 250 ml sterile aqueous solution of glycerol Dobrogosz, 1989). Mostly, spores were more resistant than vege- (100 mM) and incubated at 37 C for 3 h under anaerobic condi- tative cells. There is no information about the mechanism of action tions. After centrifugation (6600 g, 5 min), the supernatant was of reuterin on spores but given that reuterin may negatively affect a collected, filter-sterilized (0.22 mm) and maintained at 40 C for large number of cellular targets, it might also interact with 24 S. Garde et al. / Food Control 44 (2014) 22e25

Table 1 membrane disruption is essential to inhibit spore development a Minimal inhibitory concentration of reuterin, nisin and sodium nitrite against into vegetative cells. These authors also anticipated that lipid II vegetative cells (106 cells/ml) and spores (105 spores/ml) of Clostridium perfringens, binding may also be involved in nisin inhibition of other spore- in mRCM and milk, after 7 d at 37 C under anaerobic conditions. forming bacteria from the genera Bacillus and Clostridium. The re- m Reuterin (mM) Nisin ( g/ml) Sodium nitrite sults of Udompijitkul et al. (2012) also indicate that nisin does not (mg/ml) inhibit the germination of spores of C. perfringens, suggesting that mRCM Milk mRCM Milk mRCM Milk nisin effectively arrests the outgrowth of germinated spores. Nisin C. perfringens Vegetative 2.03 4.06 3.13 3.13 >300 >300 action against vegetative cells can either be bactericidal or bacte- CECT 486 cells riostatic depending on the nisin concentration, bacteria concen- > > Spores 2.03 8.13 3.13 3.13 300 300 tration, physiological state of the bacteria and the prevailing C. perfringens Vegetative 2.03 4.06 3.13 0.78 >300 >300 LBMM 391.1 cells conditions, while nisin action against bacterial spores in the ma- Spores 8.13 16.25 3.13 1.56 300 >300 jority of cases is sporostatic rather than sporicidal (Delves- C. perfringens Vegetative 2.03 4.06 3.13 3.13 >300 >300 Broughton, Blackburn, Evans, & Hugenholtz, 1996). A concentra- LBMM 395.3 cells tion of 3.13 mg/ml of nisin inhibited the growth of vegetative cells > Spores 8.13 8.13 3.13 3.13 300 300 and spores of all tested C. perfringens in mRCM, under our study a Data are expressed as the minimal antimicrobial concentration which caused a conditions. Meghrous, Lacroix, and Simard (1999) reported that complete inhibition of clostridial growth in the 4 replicates. Range of concentration vegetative cells of C. perfringens were inhibited by 0.50e0.75 mg/ml e e m tested for reuterin: 32.5 0.06 mM; for pure nisin: 25 0.03 g/ml; for sodium ni- m trite: 300e0.59 mg/ml. of nisin in RCM at 37 C for 12 h, and also observed that 23 g/ml of nisin prevented the outgrowth of C. perfringens spores up to 10 d. sulfhydryl groups of compounds or molecules present in the spore Udompijitkul et al. (2012) found that 0.8e1.7 mg/ml of nisin structure, thereby causing the inhibition of spores. MIC values inhibited C. perfringens vegetative cells, and 0.1 mg/ml of nisin found for C. perfringens spores (2.03e16.25 mM) are consistent with completely inhibited outgrowth of C. perfringens spores in TGY. the data reported by Ávila et al. (2014) for C. tyrobutyricum, Clos- MICs of 0.05e1.56 mg/ml for nisin were reported for vegetative cells tridium butyricum and Clostridium beijerinckii in mRCM (4.06e of dairy-related clostridia in mRCM (Ávila et al., 2014). Further- 16.25 mM) and for C. beijerinckii and C. sporogenes in milk (8.13e more, lengthening of lag phase was also observed in previous 32.5 mM). studies with C. perfringens vegetative cells (Guerlava et al., 1998; Culture media affected the activity of reuterin against Scannell et al., 2000). In milk, nisin sensitivity of C. perfringens C. perfringens vegetative cells and spores, resulting in higher MIC was strain-dependent (Table 1). Reported MICs of nisin for dairy- values in milk than in mRCM. On the one hand, culture media related C. tyrobutyricum, C. butyricum, C. beijerinckii and enhance or hamper the recovery and growth of microorganisms. C. sporogenes strains were 0.10e6.25 mg/ml for vegetative cells, and On the other hand, the hydroxy group of 3-HPA makes the aldehyde 0.20e25 mg/ml for spores (Ávila et al., 2014). Nisin has been shown function highly reactive to available amino-, hydroxyl-, carboxyl- to be effective in the microbial control of a number of dairy prod- and sulfhydryl-functional groups not only from bacteria but also ucts. Its main commercial application is in processed cheese and from the growth medium (Lüthi-Peng et al., 2002; Vollenweider & cheese products (e.g., block cheese, soft white cheeses, slices, Lacroix, 2004). Lüthi-Peng et al. (2002) described higher instability spreads, sauces, dips) to prevent proliferation of surviving endo- þ of reuterin (3-HPA) in milk and MRS (with 1 g/l sodium thio- spore formers, mainly the gas-producing clostridia and C. botuli- glycolate) than in water, and suggested that sugar or polypeptides num, as reviewed by Delves-Broughton (2005) and Gálvez, López, and amino acids were likely responsible for such instability. They Abriouel, Valdivia, and Omar (2008). These authors also reported observed a higher loss of unbound 3-HPA after its incubation in a the use of nisin in other pasteurized dairy products, such as chilled lactose solution than in a glucose solution. Vollenweider et al. desserts, flavored milk, cream, clotted cream, mascarpone, or can- (2010) reported a decrease of 3-HPA after incubation with ned evaporated milks. An alternative to nisin addition is food cysteine hydrochloride and cysteine, and a small but significant inoculation with a suitable nisin producing strain for in situ binding of 3-HPA to and histidine. Therefore, mRCM production. broth, with glucose and without cysteine hydrochloride, seems to Similar to our results, previous studies have shown that Clos- meet better conditions than milk for the biological activity of reu- tridium sensitivity to reuterin and nisin varies with bacterial strain terin. Nonetheless, reuterin has been successfully added to dairy and, generally, that vegetative cells are more affected than spores products such as milk (Arqués et al. 2004; 2008, 2011; El-Ziney & (Ávila et al., 2014; Hofstetter, Gebhardt, Ho, Gänzle, & Debevere, 1998) and cottage cheese (El-Ziney & Debevere, 1998)as McMullen,2013; Mazzotta, Crandall, & Montville, 1997). One a biopreservative to control the growth of gram-positive and gram- possible explanation might be that, in the experiments performed negative food-borne pathogens. It caused a bacteriostatic or with spores, the added antimicrobials would first react with the bactericidal effect depending on the tested strain and reuterin targeted spores, thus depleting unbound antimicrobials in the concentration. Langa et al. (2013) have achieved in situ production medium down to a non-inhibitory concentration for newly of reuterin in dairy products made from milk with glycerol and emerged vegetative cells. Another possibility is that due to the L. reuteri. Furthermore, it has been recently reported that the reu- length of the incubations in our study, the inhibitory activity of terin produced in cheese by a L. reuteri adjunct inhibited the growth sporostatic antimicrobials could decay as a result of time- and of C. tyrobutyricum and prevented the development of cheese late temperature-dependent loss of stability, permitting C. perfringens blowing (Gómez-Torres, Ávila, Gaya, & Garde, 2014). spores to undergo outgrowth and become vegetative cells at Cells and spores of the tested C. perfringens were sensitive to apparently higher concentration of antimicrobials. nisin. Nisin inhibits growth of vegetative gram-positive bacteria by Nitrite only inhibited the outgrowth of spores of the two forming pores in the cytoplasmic membrane and disrupting cell C. perfringens isolates in mRCM, at the maximum concentration wall biosynthesis by binding to lipid II (Bierbaum & Sahl, 2009). assayed (300 mg/ml). Nitrite concentrations ranging from 200 to Nisin also inhibits the outgrowth of germinated bacterial spores. 400 mg/ml, and from 3000 to 4000 mg/ml were required for inhibi- Recent studies of Gut, Blanke, and van der Donk (2011) with Bacillus tion of spore outgrowth of C. perfringens at pH 6 and 7, respectively anthracis suggest that nisin utilizes lipid II as the germinated spore (Labbe & Duncan, 1970). Other authors reported that the addition of target during outgrowth inhibition, and that the nisin-mediated nitrite to the growth medium at 400 mg/ml delayed the growth of S. Garde et al. / Food Control 44 (2014) 22e25 25 vegetative cells of C. perfringens at 35 C for 5 weeks (Gibson & Gálvez, A., López, R. L., Abriouel, H., Valdivia, E., & Omar, N. B. (2008). Application of Roberts, 1986). Li and McClane (2006) also observed that vegeta- bacteriocins in the control of foodborne pathogenic and spoilage bacteria. Critical Reviews in Biotechnology, 28,125e152. tive cells and spores of C. perfringens type A isolates carrying a Gibson, A. M., & Roberts, T. A. (1986). The effect of pH, sodium chloride, sodium chromosomal cpe gene were particularly nitrite resistant. In regard nitrite and storage temperature on the growth of Clostridium perfringens and to lysozyme, the maximum assayed concentration (400 mg/ml) did faecal streptococci in laboratory media. International Journal of Food Microbi- ology, 3,195e210. not inhibit the growth of any tested C. perfringens in mRCM or milk Gómez-Torres, N., Ávila, M., Gaya, P., & Garde, S. (2014). Prevention of late blowing (data not shown). The lack of antimicrobial activity of lysozyme defect by reuterin produced in cheese by a Lactobacillus reuteri adjunct. Food against two C. perfringens strains derived from a food poisoning Microbiology, 42,82e88. Guerlava, P., Nolf, S., & Tholozan, J. L. (1998). Rapid cooling, moderate heat treat- outbreak was previously described by Hughey and Johnson (1987). ment and nisin addition influence cell homeostasis of Clostridium perfringens On the contrary, Zhang, Darius, Smith, and Ritchie (2006) reported a type A. International Journal of Food Microbiology, 39,195e139, 203. MIC for lysozyme of 156 mg/ml for three avian isolates of Gut, I. M., Blanke, S. R., & van der Donk, W. A. (2011). Mechanism of inhibition of Bacillus anthracis spore outgrowth by the lantibiotic nisin. ACS Chemical Biology, C. perfringens type A in Luria broth after 24 h at 41 C. The differences 6,744e752. between these studies may be attributed to different sensitivities of Hofstetter, S., Gebhardt, D., Ho, L., Gänzle, M., & McMullen, L. M. (2013). Effects of Clostridium strains to lysozyme and/or to assay conditions. nisin and reutericyclin on resistance of of Clostridium spp. to heat e In conclusion, reuterin and nisin, antimicrobial compounds from and high pressure. 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