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Food Microbiology 82 (2019) 30–35 Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Lactococcus lactis subsp. lactis as a natural anti-listerial agent in the mushroom industry T Lionel Kenneth Dygicoa,e, Paula M. O'Connord,g, Maria Hayesc, Cormac G.M. Gahane,f,g, ∗ Helen Groganb, Catherine M. Burgessa, a Food Safety Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland b Horticulture Development Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland c Department of Food BioSciences, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland d Teagasc Food Research Centre, Moorepark, Fermoy, Cork, Ireland e School of Microbiology, University College Cork, Cork, Ireland f School of Pharmacy, University College Cork, Cork, Ireland g APC Microbiome Institute, University College Cork, Cork, Ireland ARTICLE INFO ABSTRACT Keywords: Mushroom growth substrates from different commercial producers of mushrooms (Agaricus bisporus) were Listeria monocytogenes screened for the presence of bacteria with potential for use as biocontrol agents for controlling Listeria mono- Lactococcus lactis cytogenes in the mushroom production environment. Eight anti-listerial strains were isolated from different Mushroom industry sources and all were identified using 16s rRNA gene sequencing as Lactococcus lactis subsp. lactis. Whole-genome Competitive exclusion sequencing of the Lc. lactis isolates indicated that strains from different sites and substrate types were highly similar. Colony MALDI-TOF mass spectrometry found that these strains were Nisin Z producers but inhibitory activity was highly influenced by the incubation conditions and was strain dependant. The biofilm forming ability of these strains was tested using a crystal violet assay and all were found to be strong biofilm formers. Growth of Lc. lactis subsp. lactis using mixed-biofilm conditions with L. monocytogenes on stainless steel resulted in a 4-log reduction of L. monocytogenes cell numbers. Additional sampling of mushroom producers showed that these anti-listerial Lc. lactis strains are commonly present in the mushroom production environment. Lc. lactis has a generally regarded as safe (GRAS) status and therefore has potential for use as an environmentally benign solution to control L. monocytogenes in order to prevent product contamination and to enhance consumer con- fidence in the mushroom industry. 1. Introduction Canada for L. monocytogenes contaminated sliced mushroom products, although there were no incidents of listeriosis reported (Canadian Food Listeria monocytogenes is the causative agent of listeriosis in humans Inspection Agency, 2012; 2014, 2015). Additionally, there have been and a foodborne pathogen of significant public health concern. It is eleven notifications of mushrooms contaminated with L. monocytogenes ubiquitous in nature and it can contaminate food crops and food pro- since 2013 in the European Commission's Rapid Alert System for Food cessing environments (Ferreira et al., 2014). Although there has been (RASFF) database. These recalls resulted in an economic and reputa- an isolated report of L. monocytogenes associated septicaemia in an el- tional loss for the industry. Thus, it is important to take proactive steps derly man linked with the consumption of wild-collected salted mush- to maintain this industry's current reputation for food safety by ex- rooms (Lactarius rufus, Junttila and Brander, 1989), there have been, to ploring novel biocontrol agents to provide enhanced assurance of pro- the authors' knowledge, no reports of listeriosis due to the consumption duct quality and safety. of fresh cultivated mushrooms (Agaricus bisporus). However, there is The primary concern in food production and processing environ- concern in the mushroom industry as studies have shown that L. ments, including the mushroom industry, is that L. monocytogenes bio- monocytogenes can be found in mushroom production facilities which films can form on approved materials (e.g. stainless steel, rubber and therefore pose a risk of product contamination (Murugesan et al., 2015; polymers), including food contact surfaces (Beresford et al., 2001; Viswanath et al., 2013). In recent years, multiple recalls occurred in Bridier et al., 2014; Di Bonaventura et al., 2008). Studies of ∗ Corresponding author. E-mail address: [email protected] (C.M. Burgess). https://doi.org/10.1016/j.fm.2019.01.015 Received 12 January 2018; Received in revised form 25 January 2019; Accepted 25 January 2019 Available online 28 January 2019 0740-0020/ © 2019 Elsevier Ltd. All rights reserved. L.K. Dygico et al. Food Microbiology 82 (2019) 30–35 disinfectants or sanitizing agents that are currently used in the food crop. Batches of fresh mushroom casing were obtained from four dif- industry (i.e. quaternary ammonium compounds, chlorine based com- ferent farms, phase-3 substrate from one farm and SMS from two farms. pounds and oxidising agents) have found that they can reduce biofilm All the farms were located in different regions of Ireland. For the pre- on different surfaces, but they are not consistently effective in dealing liminary screening for anti-listerial bacteria (ALB), 25 g of substrate was with L. monocytogenes biofilms, as the reduction levels were dependent diluted in 225 ml of maximum recovery diluent (MRD, Oxoid, UK) and on biofilm age, type of surface, and substrate on which the biofilms homogenised in a stomacher for 5 min. Ten-fold serial dilutions of the were formed (Amalaradjou et al., 2009; O’Neill et al., 2015; Pan et al., homogenised samples were then carried out and 100 μl of each dilution 2006; Yang et al., 2009). In addition, several studies suggest that lis- were spread plated on different media: LB agar (Oxoid, UK), TSA sup- terial biofilms that have been exposed to certain antimicrobials, which plemented with 0.6% yeast extract (TSAYE, Oxoid, UK), Brain Heart did not result in complete removal of the pathogen, induced a change in Infusion (BHI) agar (Oxoid, UK), de Man, Rogosa and Sharpe agar biofilm structure with increased antimicrobial resistance (Ibusquiza (MRS, Oxoid, UK), GM17 (M17 supplemented with 0.5% glucose, et al., 2011; Pan et al., 2006; Yun et al., 2012). This shows the need for Oxoid, UK), LM17 (M17 supplemented with 0.5% lactose, Oxoid, UK) novel agents that can effectively control listerial biofilm and ideally are and Mueller-Hinton agar (Oxoid, UK). The plates were then incubated environmentally benign. As an alternative to chemical disinfectants, at a number of temperatures and atmospheres to promote ideal con- biological control (biocontrol) options could be promising alternatives, ditions for antagonistic activity: aerobically for 24 h at 37 °C, anaero- as they are considered natural, have a “green” image and are en- bically for 24 h at 37 °C, aerobically for 24 h at 25 °C and anaerobically vironmentally friendly. for 24 h at 25 °C. After incubation, the plates were overlaid with soft −1 Currently, the mushroom industry uses the commercial product TSAYE (0.75% agar) seeded with approximately log10 6 CFU ml of L. ® Nemasys (BASF, Germany) as a biocontrol agent which utilises a ne- monocytogenes 6179 and then incubated again aerobically for 24 h at matode called Steinernema feltiae, to control sciarid fly larvae during 37 °C. Positive results for inhibition were identified as visually detect- mushroom production cycles. However, such an approach against food- able zones of clearing around a colony or colonies after incubation. borne pathogens that could contaminate the crop has not been con- sidered. One option for this approach during production would be to 2.3. Identification of anti-listerial agents in cell-free supernatants utilise the bacteriocin nisin, which is used in the food industry as a food additive that acts as a natural preservative, mainly as an anti-botulinic Cell-free supernatants (CFS) of isolates with antagonistic activity agent in cheese and liquid eggs, sauces and canned foods (Balciunas from the previous test were analysed for their anti-listerial activity et al., 2013). Nisin is produced by certain strains of Lactococcus lactis using a sensitive well diffusion assay described by Alvarez-Ordóñez subsp. lactis and is the best-studied bacteriocin. Another way of utilising et al. (2013), with minor modifications. Briefly, two types of solid bacteriocin-producing bacteria (BPB) is through competitive exclusion, media were utilised for this assay: an underlay (1% agarose [Sigma- where the BPB could be used to take over the niche occupied by pa- Aldrich, UK], 0.03% TSB [Oxoid, UK] and 0.02% Tween 20 [Sigma- thogenic bacteria, such as L. monocytogenes, to potentially inhibit the Aldrich, UK]) and overlay (1% agarose, 1X TSB). Wells with a diameter growth of the pathogen. Within a food production environment, bac- of 8 mm were made on the underlay that was seeded with approxi- −1 teria that have GRAS status, such as lactic acid bacteria (LAB) are ideal mately log10 6 CFU ml of indicator L. monocytogenes 6179 strain. as they pose low risk to public health. Studies investigating such ap- Then, 50 μl aliquots of neutralised CFS were added in to the wells and proaches have found that competitive exclusion bacteria can control L. allowed to diffuse for 2 h. The overlay was poured over the underlay monocytogenes biofilms formed on surfaces found in food processing media, allowed to solidify and incubated at 37 °C for 24 h. Other L. facilities (García-Almendárez et al., 2008; Zhao et al., 2013). The aim of monocytogenes strains and foodborne contaminants such as Escherichia this study was to isolate and evaluate bacteriocins or bacteriocin-pro- coli (NCTC 9001), Staphylococcus aureus (NCTC 6571) and Salmonella ducing bacteria naturally present in mushroom growth substrates, Typhimurium (SARB 67) were also tested for inhibition (Table 1). This which can prevent or eliminate L. monocytogenes and L. monocytogenes experiment was repeated three times and the activity was interpreted as biofilm formation. the presence or absence of a zone of clearance. The proteinaceous nature of the anti-listerial agents in the CFS was then confirmed using 2.