International Journal of Food Microbiology 381 (2022) 109888 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro Removal of Listeria monocytogenes biofilms on stainless steel surfaces through conventional and alternative cleaning solutions T. Mazaheri a, B.R.H. Cervantes-Huamán a, L. Turitich b, C. Ripolles-Avila a, *, J.J. Rodríguez-Jerez a a Area of Human Nutrition and Food Science, Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), CP 08193 Barcelona, Spain b Centre d'Innovació, Recerca i Transferència en Tecnologia dels Aliments (CIRTTA), TECNIO-UAB, XIA, Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), CP 08193 Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Conventional treatments are not effective enough to completely remove Listeria monocytogenes biofilms from Listeria monocytogenes surfaces, thus implying the presence of certain persistent bacterial forms. In this study, eleven treatments (i.e. two Biofilms enzymatic agents applied at two different temperatures and concentrations, two alkaline cleaners and one acid Cell detachment detergent) were used to remove mature L. monocytogenes S2-bac biofilms. A combined treatment was then Biocidal effectivity Food safety selected for its application to four different L. monocytogenes strains (i.e. CECT 5672, CECT 935, S2-bac, EDG-e). Effectivity of the treatments was evaluated quantitatively using TEMPO and qualitatively by direct epifluorescent microscopy (DEM). Bacterial detachment obtained after the application of acid, alkaline and chlorinated alkaline treatments were 6.03, 6.24 and 4.76 Log CFU/cm2, respectively. Enzymatic treatments applied at 50 ◦ C obtained the greatest detachment and biocidal activity. The results derived from the observation of the remaining biofilm structure by DEM proved that conventional treatments were unable to completely remove conformed structures with the potential risk this entails. Last, the application of a combined treatment using a chlorinated alkaline cleaner followed by an enzymatic treatment enhanced the dispersal of the bacterial cells from surfaces, thus consolidating this as a good option to recommend for the 5-step cleaning procedure. 1. Introduction through the food chain is not enough to reduce its presence (EFSA- ECDC, 2021), questions are raised about Listeria spp. lifestyle in the food In the food and beverage industry, equipment surfaces are consid­ processing environment context and new ways to eliminate the path­ ered an important source of microbial contamination, associated over ogen are being sought (Zwirzitz et al., 2021). time with foodborne disease outbreaks and consequently impacting There are thirteen different serotypes of L. monocytogenes (i.e. 1/2a, public health (Faille et al., 2018). Among the pathogens that stand out 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e and 7), although most of due to food contamination as a consequence of cross-contamination the human diseases produced are associated with serotypes 1/2a, 1/2b, from industrial surfaces is Listeria monocytogenes (Churchill et al., 1/2c and 4b (Maćkiw et al., 2020). Of these, serotype 4b is the one 2019). In 2020, this pathogen produced a total of 780 reported cases of considered the most pathogenic and 1/2a the one that is most prevalent invasive listeriosis in Europe, presenting a mortality rate of 13 % (EFSA- in the food industry environment (Laksanalamai et al., 2014; Lee et al., ECDC, 2021), one of the highest rates among the zoonotic agents. Recent 2012). Therefore, it is important to include various L. monocytogenes foodborne outbreaks have been related to L. monocytogenes presence in serotypes in the studies concerned with its control to represent industrial foods from distinct origins (i.e. vegetable and animal) such as the one reality. L. monocytogenes occurrence in the food industry has been linked to the consumption of enoki mushrooms (U.S. FDA/USDA/CDC, related to the pathogen's ability to survive in a wide range of environ­ 2020), hard-boiled eggs (U.S. FDA/USDA/CDC, 2019) and Bologna ments, such as in cold temperatures, with low oxygen, low pH or even a sausages (Allam et al., 2018; Salama et al., 2018). Due to the non- lack of nutrients (Sadekuzzaman et al., 2017). To do so, L. monocytogenes decreasing trend of listeriosis cases and the fact that its control forms biofilms, biological structures which are considered an * Corresponding author. E-mail address: carolina.ripolles@uab.cat (C. Ripolles-Avila). https://doi.org/10.1016/j.ijfoodmicro.2022.109888 Received 31 March 2022; Received in revised form 16 August 2022; Accepted 22 August 2022 Available online 26 August 2022 0168-1605/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). T. Mazaheri et al. International Journal of Food Microbiology 381 (2022) 109888 Table 1 Enzymatic and chemical cleaning detergents used in the treatments for the elimination of L. monocytogenes S2-bac mature biofilms. Type of Cleaner Composition In-use temperature In-use concentartion treatment (◦ C) (%) Ethoxylated sodium lauryl ether glycolate Amines, C12–14 (even numbered)- alkyldimethyl, N-oxides Anionic surfactants (<5 %) Product A 20 and 50 1 and 3 Non-ionic surfactants (<5 %) Proteases (<5 %) Phenoxyethanol Enzymatic Nonylphenoxypoly (ethyleneoxy)ethanol (10 %) Protease (Savinase® <3 %) Product B 20 and 50 1 and 3 α-amylase (Termamyl® <3 %) Thyme oil (0.5 %) Cinnamon oil (0.5 %) Phosphoric acid (25–50 %) Acid 20 1 Amines, C12–14 (even numbered)-alkyldimethyl, N-oxides (1–5 %) Potassium hydroxide (<25 %) Conventional Alkaline 40 1 Sodium hydroxide (2–5 %) Chlorinated Sodium hydroxide (8.5 %) 20 1 alkaline Sodium hypochlorite (6 %) assemblage of microbial cells adhered to surfaces, embedded in a matrix used for mature L. monocytogenes biofilm elimination, modelling S2-bac of extracellular polymeric substances that consists of polysaccharides, strain as a reference according to our previous study (Mazaheri et al., proteins and DNA (González-Rivas et al., 2018). This structure defines 2020). The second specific objective was to evaluate a combined treat­ the main physiological processes in relation to their resistance and ment to remove mature biofilms formed by four more L. monocytogenes persistence (Mosquera-Fernández et al., 2016), which are important strains (i.e. CECT 5672, CECT 935, S2-bac, EDG-e, from distinct sero­ aspects for the development of control strategies for their elimination. types and origins). Biofilm thickness and shape is directly related to antimicrobial diffusion, causing the cells that form the biofilms to increase their capacity to resist 2. Materials and methods disinfectants, thus limiting their efficacy and the further elimination of these microbial communities (Torlak and Sert, 2013). This enables the 2.1. Bacterial strains bacteria to persist in food processing environments on locations that are not easy to clean using conventional cleaning solutions, such as cutting L. monocytogenes strain S2-bac obtained from Ortiz et al. (2014) was machines, smoking areas, and totes and cracks in the floors (Cripe and selected for the evaluation and effectivity comparison of 11 different Losikoff, 2021), thereby considered a critical problem for the food in­ treatments on the basis of it showing the maximum matrix production dustry (McEntire, 2018; Sadekuzzaman et al., 2017). It is also important and greatest resistance (Mazaheri et al., 2020). After comparison of the to detect foodborne pathogens rapidly and efficiently to reduce the 11 treatments, two of the agents were chosen to remove mature biofilms probability of a pathogen remaining on surfaces and so that suitable of different L. monocytogenes strains [i.e. CECT 5672 and CECT 935, action can be undertaken (Ripolles-Avila et al., 2020). A good hygiene obtained from the Spanish Type Culture Collection (CECT, Paterna, program of such actions must be applied in all food sectors (Ben Braïek Spain) and L. monocytogenes EDG-e, which was isolated from an Iberian et al., 2020). Thus, cleaning and disinfection processes are highly pig processing plant (Ortiz et al., 2016)]. All the strains were obtained as important for the food industry to achieve a minimum risk for the safety freeze-dried cultures and recovered by culturing them in Tryptic Soy and quality of food products (Holah et al., 2014; Yang et al., 2016). As Broth (TSB; Oxid, Madrid, Spain) for 48 h at 30 ◦ C. Suspensions were previously suggested by Waghmare and Annapure (2015), sanitation then cultured on Tryptic Soy Agar (TSA; Oxid, Madrid, Spain) and programs in the food industry are commonly based on chlorine, incubated at 37 ◦ C for 24 h. Isolated colonies were used to prepare stock including sodium hypochlorite, since this is an effective oxidizing cultures on TSA, which were stored for up to 1 month at 4 ◦ C. compound for microbial activities and more economical than other chemical products. 2.2. Surfaces The greatest challenge for the cleaning and disinfection procedures applied in the food industry is to find biofilms in their mature state Stainless steel coupons AISI 316 grade 2B (2 cm in diameter and 1 (Ripolles-Avila et al., 2020). After a biofilm is found in this state, the mm thick) were submited to a cleaning and disinfection procedure, ac­ application of antimicrobials is not an effective solution to remove and cording to European standard UNE-EN 13697:2015 (AENOR, 2015). eliminate the structure from the surface (Srey et al., 2013). Mazaheri First, the coupons were cleaned with detergent (ADIS Hygiene, Madrid, et al. (2020) and Ripolles-Avila et al. (2020) recently demonstrated that Spain) for at least 1 h, washed with running tap water, and further aggressive treatments using a combination of enzymes need to be disinfected with 70 % isopropanol (Panreac Química, Castellar del applied to eliminate mature L. monocytogenes biofilms. In this case, Vallès, Spain). The surfaces were susequently dried in a laminar flow enzymatic action causes a disruption of the biofilm extracellular matrix cabinet (PV-30/70, Telstar, Terrasa, Spain). Last, to complete the ster­ and stimulates the release of microbial cells to facilitate their elimina­ ilization stage, the coupons were autoclaved at 121 ◦ C for 15 min before tion by applying a disinfectant product (Pleszczyńska et al., 2017). their use. Nevertheless, it becomes important to extend these types of studies and to compare regular products employed in the food industry. To this end, the general objective of this study was to compare different strategies to 2.3. Biofilm formation on surfaces eliminate mature L. monocytogenes biofilms formed on stainless steel surfaces to find out the best cleaning methodology to apply. The first Several colonies obtained from the incubation of L. monocytogenes specific objective was to assess the effectiveness of eleven treatments strains in TSA at 37 ◦ C for 24 h were inoculated into TSYEB gluc1%+Nacl2% [i.e. TSB enriched with 0.3 % w/v yeast extract (BD, Madrid, Spain), 1 % 2 T. Mazaheri et al. International Journal of Food Microbiology 381 (2022) 109888 Table 2 Comparison between different cleaning treatments for the removal of L. monocytogenes S2-bac mature biofilms. Values correspond to the mean ± standard error (n = 9). Treatment Temperature (◦ C) Concentration (%) Reduction Log (CFU/cm2) Detachment (%) 1 3.23 ± 0.31cd 52.41 ± 4.97cd 20 3 4.08 ± 0.55bc 67.70 ± 8.23bc Product A 1 6.24 ± 0.00a 100 ± 0.00a 50 3 5.96 ± 0.28a 95.73 ± 4.27a 1 2.60 ± 0.39d 42.24 ± 6.13d 20 3 3.42 ± 0.57cd 55.58 ± 8.87cd Product B 1 4.99 ± 0.28b 80.07 ± 4.59b 50 3 6.10 ± 0.09a 97.86 ± 1.42a Acid 20 1 6.03 ± 0.10a 96.57 ± 1.72a Alkaline 40 1 6.24 ± 0.00a 100 ± 0.00a Chlorinated alkaline 20 1 4.76 ± 0.73b 77.45 ± 11.14b a-d Means within a column without a common superscript differ significantly (P < 0.05). w/v glucose (Biolife, Madrid, Spain), and 2 % w/v sodium chloride [1 g of tryptone (BD, Madrid, Spain), 8.5 g of NaCl (Panreac, Castellar (Panreac, Castellar del Vallès, Spain)] until reaching 0.2 McFarland del Vallès, Spain) and 30 g Tween 80 (Scharlab, Barcelona, Spain) for units, equivalent to approximetely 106 CFU/ml (Ripolles-Avila et al., every 1000 ml of sterile distilled water in pH (7.0 ± 0.2)]. In the case of 2018). The resulting microbial suspension was then used for the surface the control surfaces, these were washed twice with 3 ml of sterile inoculation. For this, 30 μl were transferred to the middle of each distilled water and placed directly in a sterile flask for vortexing with stainless steel coupon and placed in sterile Petri dishes, which were glass beads and 10 ml of neutralizer solution without the disinfection inserted in a humidity chamber for biofilm formation (Fuster-Valls et al., treatment. 2008; Ripolles-Avila et al., 2018). The surfaces were incubated statically The TEMPO system (bioMérieux, Marcy l'Etoile, France) was used to for seven days at 30 ◦ C with washing and renewal of nutrients. For this quantify the L. monocytogenes cells within the biofilms. The resulting procedure, the stainless steel coupons were washed twice with 3 ml of suspension after biofilm cell recovery was decimally diluted in Tryptone sterile distilled water to remove non-adhered bacterial cells, and then Saline Solution [TSS; 1 g of tryptone and 8.5 g of NaCl per litre in pH (7.0 30 μl of TSYEBgluc1%+NaCl2% were added to stimulate biofilm growth and ± 0.2)]. Prior to the quantification, the TEMPO vials were hydrated with maturation at 48 h + 24 h + 24 h + 72 h (Ripolles-Avila et al., 2018). 3 ml of sterile distilled water and then, 1 ml of the dilution to be After the renewal of nutrients was completed, the surfaces were again quantified added to them. The TEMPO vials were then vortexed to ho­ placed back in the humid chamber to complete the 7-day incubation mogenize their content, transferred into an enumeration card by the period. TEMPO filler and incubated at 30 ◦ C for 48 h. 2.4. Cleaning agents 2.6. Observation of resident L. monocytogenes biofilm communities by direct epifluorescent microscopy (DEM) Two enzymatic products obtained from iTram Higiene (Vic, Spain) and three chemical products obtained from Proquimia (Vic, Spain) were After the surfaces were subjected to the different treatments, the used in this study (Table 1). The in-use concentrations of the agents were coupons were washed twice with 3 ml of sterile distilled water, further prepared by diluting them in hard water following international stan­ stained with 5 μl of Live/Dead BackLight bacterial viability kit (Molec­ dard UNE-EN 13697:2015 (AENOR, 2015). Hard water was obtained by ular Probes, Oregon, USA) and incubated at 20–22 ◦ C for 15 min. To adding 3 ml of solution A [19.84 g of MgCl2 (Sigma, Madrid, Spain) and differentiate between intact and damaged membranes, two fluorescent 46.24 g of CaCl2 (Sigma, Madrid, Spain) per 1000 ml of distilled water], dyes, SYTO9 and propidium iodide (PI), were used. The first dye enters 4 ml of solution B [35.02 g NaHCO3 (PanReac Applichem, Madrid, both live and dead bacterial cells and dyes them green, whereas PI Spain) per 1000 ml of distillate water] and 100 ml of interfering solution penetrates only the cells with damaged membranes and reduced SYTO9 [1.5 g of bovine serum albumin (Sigma, Madrid, Spain) per 100 ml of dye, producing a red color. All the stained surfaces were evaluated with distilled water]. Prior to being mixed to obtain the hard water, all these an Olympus BX51/BX52 epifluorescence microscope (Olympus, Tokyo, solutions were sterilized by a filter membrane (Millex-GP 0.22 μm, Japan) equipped with a 100 W mercury lamp (USH-103OL, Olympus) Merck, Barcelona, Spain) and then mixed with distilled water to obtain a and a dual-pass filter cube (U-M51004 F/Re-V2, Olympus), and coupled final volume of 500 ml. After a week, the inoculated surfaces were rinsed to a digital camera (DP73, Olympus). The biofilms were observed with a twice with 3 ml of sterile distilled water to remove any unattached cells twenty magnification (20×) lens. The obtained images were analysed prior to subjecting them to the cleaning treatment. To evaluate the effect using the analySIS Auto 3.2 software (Soft Imaging System, Münster, of treatments on biofilms, the coupons were placed in a sterile flask with Germany). 3 ml of the treatment solutions covering the surfaces (Mazaheri et al., 2020; Ripolles-Avila et al., 2020). All treatments were applied for 15 2.7. Evaluation of the biocide activity of the cleaning agents washing min but under different conditions (i.e. temperature and concentration), solution as summarised in Table 1, and according to the technical data sheet of the products. The antimicrobial efficacy of the cleaning agents against L. mono­ cytogenes was evaluated after the cleaning treatment was applied to the 2.5. Quantification of microbial cells by TEMPO surfaces. The washing solution (i.e. remaining solution after a surface was treated) was filtered using a sterile membrane (MF-Millipore, Cel­ For biofilm cell recovery and quantification after treatment appli­ lulose Mixed Esters, Hydrophilic; 0.45 μm, 25 mm). The membrane and cation, the surfaces were rinsed with 3 ml of sterile distilled water to filtration equipment was sterilized by autoclave at 121 ◦ C for 15 min remove the non-attached cells and the disinfectant residues. The biofilm before use. The membrane was rinsed twice with 10 ml Buffered Peptone cells were subsequently detached by vortexing the surfaces at 40 Hz for Water (BPW; Oxoid, Ltd., Basingstoke, United Kingdom) to remove any 90 s (Ripolles-Avila et al., 2019a). To do so, each coupon was introduced residue of the cleaning agents, placed in TSA and incubated at 30 ◦ C for in a sterile flask containing glass beads and 10 ml of neutralizer solution 48 h. 3 T. Mazaheri et al. International Journal of Food Microbiology 381 (2022) 109888 (Colagiorgi et al., 2016) and therefore the treatment would be pro­ moting protein denaturation and matrix disruption. On the other hand, acid detergents act as descalers, favoring the elimination of mineral deposits (Fagerlund et al., 2020). In this case, the application of phos­ phoric acid as the acid treatment obtained a high cell detachment (i.e. 6.03 Log CFU/cm2), which could be due to oxidative action of the product, increasing the concentration of hydrogen ions and affecting cell viability (Arias-Moliz et al., 2008; Hashim et al., 2020). Despite the effectiveness having been shown to be high in the present study, its transfer to industrial environments with certain amounts of residues could reduce its action of eliminating the biofilm matrix and structure. In this regard, Parkar et al. (2004) demonstrated differences between the effect of alkaline and acid cleaning agents on the biofilm matrix compared to enzymatic treatments. As a green alternative for industrial surface cleaning, enzymatic detergents have been established as a viable option for the fight against biofilms in the food industry (Delhalle et al., 2020). In the present study, Fig. 1. Epifluorescence digital images of Live/Dead-stained mature enzymatic treatments were applied at two different concentrations and L. monocytogenes S2-bac biofilms. Magnification 20×. temperatures to observe the effect with varying parameters. The results showed that Product A (1 % and 3 %) and Product B (3 %) applied at 50 ◦ C obtained the highest detachment percentage, consolidating them 2.8. Calculations and statistical analysis as the most effective treatments as well as alkaline and acid treatments (P > 0.05). In this case, the substrate specificity of the enzymes can All the experiments were performed in triplicate on three different contribute to a higher efficiency for biofilm removal compared to days (n = 9). The bacterial counts were converted into decimal loga­ alkaline and acidic cleaning agents due to the enzyme's capacity to rithmic values to almost match the assumption of a normal distribution. disrupt and break up biofilm matrix (Fagerlund et al., 2020). Moreover, Cell reduction after treatments was calculated by the differentiation when Product A and Product B were applied at the recommended tem­ between initial biofilm cell count and cell counts remaining on the perature (i.e. 50 ◦ C) with the lowest concentration (i.e. 1 %), their effect surface after the treatments. Biocidal effect (i.e. dead cells) were differed significantly (P < 0.05), with Product A showing higher effec­ calculated from the difference between the cells detached from the tivity, even though this product contained just one type of enzyme. surface after the treatment and the ones present on the washing solution. However, biofilm matrix is completely heterogeneous, and even more so The statistical analysis was performed using One Way ANOVA and the if we take into consideration the fact that in food processing environ­ Fisher LSD Test with the STATISTICA 7.0.61.0 statistical software ments different microbial species coexist within the biofilm structure. To package. Statistical significance was defined as P < 0.05. this effect, enzymatic formulations composed of mixtures of enzymes that attack different substrates to destabilize the matrix, including 3. Results and discussion proteases, cellulases, polysaccharide depolymerases, alginate lyases, dispersing B and DNases, are more effective when applied at an indus­ 3.1. Effect of eleven treatments on the removal of mature L. trial level (Bridier et al., 2015). In the present study, Product B was monocytogenes S2-bac biofilms composed of different enzymes, such as α-amylase, protease and different essential oils including thyme and cinnamon oils, which can Considering the resistance profile of different strains when con­ cause better outcomes in industrial experiments. Other authors have ducting experimental studies for their elimination is important for also reported the higher dispersal activity of proteases and amylases drawing conclusions that can be extrapolated to the microbial set. In the combined in the formulations of detergent for different food industries present study, L. monocytogenes S2-bac was chosen as a representative and uses (Guerrero-Navarro et al., 2022; Mitidieri et al., 2006). strain because it was the most resistant strain after the application of an Moreover, it has been indicated that the effectiveness of cleaning enzymatic treatment in a previous study (Mazaheri et al., 2020). agents can depend on the structure and matrix produced by the different Comparative scientific studies are important to critically select the L. monocytogenes strains (Ripolles-Avila et al., 2018; Ripolles-Avila et al., appropriate type of cleaning agent for any particular situation (Liikanen 2019b). Moreover, according to Mazaheri et al. (2020), L. monocytogenes et al., 2002), one of them being the removal of mature biofilms. Table 2 S2-bac generated a more robust matrix, and this could have been the shows the results obtained in terms of cell reduction and subsequent reason why enzymatic treatment was more effective than a chlorinated detachment after the different applied cleaning treatments. As can be alkaline product when assessing L. monocytogenes biofilm removal. observed, the treatments with the greatest effectiveness (i.e. highest L. monocytogenes S2-bac belongs to the serotype 1/2a, the serotype most detachment percentage) were the enzymatic treatments applied at 50 ◦ C frequently found in the food industry, as discussed in previous sections. (except Product B at 1 % concentration) and the alkaline and acid Serotype 1/2a could generate more compact and robust structures when treatments, which exerting a similar effect as shown by the non- consolidating biofilms, which could also be the reason why this serotype significant differences (P > 0.05) obtained between them. First, the is more widespread in processing plants (D’Arrigo et al., 2020). conventional detergents used in this study were classified into two Our cleaning tests showed that it is essential to use the correct con­ different groups depending on the pH of application, thus finding the centrations of agents and the recommended temperatures, as also indi­ acid and alkaline detergents. The results derived from the present study cated by Parkar et al. (2004) and Guerrero-Navarro et al. (2022). In this demonstrated that both alkaline and acid treatments are highly effective last study, it was reported that enzymatic cleaning products in food in detaching mature L. monocytogenes biofilms. Alkaline detergents can processing plants are not always used according to recommendations, denature proteins due to the action of hydroxyl ions, can saponify fats for example in cold storage rooms which are not able to reach 50 ◦ C, and, at high concentrations, can have a bactericidal action (Lelieveld, leading to a decrease in the application temperature and concentrations 2013). In this case, cell detachment from the surface after the alkaline failing to remove all surface cells, indicating that effectiveness is directly treatment was applied could be related to the fact that extracellular L. dependent on both parameters. To this effect, when the concentration of monocytogenes biofilm matrices are composed mainly of proteins Product B increased to 3 % and was applied at 50 ◦ C, the treatment was 4 T. Mazaheri et al. International Journal of Food Microbiology 381 (2022) 109888 A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9 A - 10 A - 11 Fig. 2. Epifluorescence digital images of Live/Dead-stained mature L. monocytogenes S2-bac biofilms after the application of tested treatments: Product A 1 % - 20 ◦ C (A-1); Product A 3 % - 20 ◦ C (A-2); Product A 1 % - 50 ◦ C (A.3), Product A 3 % - 50 ◦ C (A-4); Product B 1 % - 20 ◦ C (A-5), Product B 3 % - 20 ◦ C (A-6); Product B 1 % - 50 ◦ C (A-7), Product B 3 % - 50 ◦ C (A-8); Acid (A-9), Alkaline (A-10), Chlorinated alkaline (A-11). Magnification 20×. Table 3 Table 4 Antimicrobial activity of the treatments applied for the elimination of L. mon­ Counts in Log CFU/cm2 of L. monocytogenes cells that conformed the mature ocytogenes S2-bac mature biofilms. Values correspond to the mean ± standard biofilms before and after the application of the combined treatment used for error (n = 9). their elimination. Treatment Temperature Concentration Dead cells (Log L. monocytognes strains Control Combined treatment (◦ C) (%) CFU/cm2) CECT 5672 7.05 ± 0.15a < 0.10 ± 0.00a 1 1.05 ± 0.27d S2-bac 6.87 ± 0.11ab < 0.10 ± 0.00a 20 3 2.81 ± 0.60ef EDG-e 6.64 ± 0.15b < 0.10 ± 0.00a Product A 1 5.00 ± 0.35ab CECT 935 6.63 ± 0.14b 0.17 ± 0.17a 50 3 5.75 ± 0.26b a-b 1 0.00 ± 0.06cd Means within a column without a common superscript differ significantly (P 20 < 0.05). 3 0.00 ± 0.37c Product B 1 2.34 ± 0.17e 50 3 4.64 ± 0.54a 1.04 Log CFU/cm2 more effective in terms of cell detachment. All the Acid 20 1 5.52 ± 0.25ab Alkaline 40 1 5.26 ± 0.25ab enzymatic treatments, when applied at 20 ◦ C, were significantly (P < Chlorinated 0.05) less effective than when applied at the highest temperature. The 20 1 3.41 ± 0.45f alkaline results also demonstrated that when applied at 3 %, all the enzymatic a-f Means within a column without a common superscript differ significantly (P < treatments were significantly (P < 0.05) more effective. 0.05). The application of any of the cleaning treatments proven to be highly 5 T. Mazaheri et al. International Journal of Food Microbiology 381 (2022) 109888 Table 5 the mature biofilms were exposed to the enzymatic treatments, the Microbial dead cell counts (Log CFU/ml) obtained from the treatment washing remaining structure was made up of small, scattered colonies or dis­ solutions. Values correspond to the mean ± standard error (n = 9). aggregated cells, most of which were either damaged or dead (Fig. 2 A-1; L. monocytognes strains Cleaner Dead cells (Log CFU/ ml) A-2; A-3; A-4; A-5; A-6; A-7; A-8). This result has been observed by other CECT 5672 Chlorinated alkaline 4.27 ± 0.58b authors (Mazaheri et al., 2020; Ripolles-Avila et al., 2020). Qualita­ S2-bac 5.41 ± 0.17a tively, the images coincide with the results obtained at a quantitative EDG-e 2.89 ± 0.24c level. In this regard, it was observed that increasing the concentration of CECT 935 3.99 ± 0.46b enzymes from 1 % to 3 % at the same temperature slight reduced the CECT 5672 5.80 ± 0.25a number of remaining biofilm cells on the surfaces (e.g., at 20 ◦ C of S2-bac 5.76 ± 0.14a EDG-e Product A 5.42 ± 0.22a application; Fig. 2 A-1 and A-5 vs A-2 and A-6). However, when the CECT 935 5.28 ± 0.26a temperature was increased to 50 ◦ C, a complete disintegration of the a-c mature biofilm structure and a distortion of the proportion of dead cells Means within a column without a common superscript differ significantly (P < 0.05). were observed (e.g., at 3 %; Fig. 2 A-2 and A-6 vs A-4 and A-8). Each enzyme needs an optimal temperature to exhibit its maximum activity and, in the event that combinations of enzymes are used, it is important effective must be complemented with the application of a disinfection to establish the temperature closest to the optimal activity of each procedure since cleaning treatments remove a high percentage of mi­ enzyme used. Fagerlund et al. (2020) reported that the optimum tem­ croorganisms but cannot eliminate them completely (González-Rivas perature for the application of enzymatic detergents is between et al., 2018). Therefore, the effectiveness of the treatments applied in 45 ◦ C–55 ◦ C. The most suitable temperature of the enzymatic treatments this study would increase with the application of the disinfection process applied to exhibit the best cell detachment activity and a complete (Ripolles-Avila et al., 2020). The only treatment that would not improve elimination of the biofilm structure was 50 ◦ C, coinciding with Ripolles- in terms of effectivity is the applied chlorinated alkaline detergent since Avila et al. (2019b), who applied a similar treatment for the elimination it is a single-step cleaning and disinfection treatment. The results of the of Salmonella enterica and Cronobacter sakazakii biofilms. present study demonstrated that 22.55 % of L. monocytogenes S2-bac Differently from what was obtained following the application of the cells remained adhered on the surface after the application of the enzymatic treatments, the rest of the applied treatments (i.e. chlorinated chlorinated alkaline treatment. The findings presented are in agreement alkaline, alkaline and acid) did not disperse the structure of the biofilm, with what has been reported by other authors such as Kim et al. (2018) consequently leaving both microbial population and biofilm structure and Ripolles-Avila et al. (2020), who demonstrated that chlorinated on the surface. Although a reddish color is observed in the whole alkaline detergents can detach a certain number of cells that conform structure on the images, the cells can be damaged rather than these structures, but that the treatments are not completely effective as completely dead. In the case of the alkaline and acid treatments, a they do not completely disperse the structure. By scanning electronic subsequent disinfection would be applied, so there may be a greater microscopy, Mendonca et al. (1994) showed that L. monocytogenes cells reduction in the microbial load. Moreover, in the food industry, all exposed to pH 9.00, 10.00, 11.00 and 12.00 did not leak constituents evaluated treatments would have also been accompanied with physical and did not change their cell structure, thus generating lower biocidal removal so this could also influence by potentiating the detachment effect than other pHs. Chlorinated alkaline treatment is recommended in effect. On the other hand, the application of the chlorinated alkaline the 5 cleaning steps for areas where risk assessment concludes that the treatment would not entail no other subsequent treatment, implying that zone does not pose a potential risk. However, the microbial population the endured structure would remain on the surface. This can aggravate that will resist treatment must be considered, consolidate again the the state of hygiene, since by not completely eliminating the structure, structures, their acquired resistance and their capacity to migrate to L. monocytogenes cells could regenerate and re-start the formation of other places of the food industry, thus posing a risk of re-contamination. biofilms (Thomas and Sathian, 2014). In this regard, Mnif et al. (2020) demonstrated that after treating biofilms with alkaline and acid agents, 3.2. Impact on the structure and matrix of the treated L. monocytogenes the remaining adhered biofilm cells re-consolidated the structure and S2-bac biofilms increased their resistance to chemical agents. The structure and viability of L. monocytogenes S2-bac biofilms were 3.3. Biocidal effect of the eleven treatments on the detached also investigated by direct epifluorescence microscopy (DEM) before L. monocytogenes S2-bac cells that conformed the biofilm and after the biofilms were subjected to cleaning treatments. Fig. 1 shows mature L. monocytogenes S2-bac biofilms obtained after a one- Table 3 shows the biocidal activity of each product, calculated from week incubation period at 30 ◦ C. In this regard, the formation of bio­ the solution where the surfaces were treated. The formulation used for films can be determined from the organization of the cells from which the design of the detergents could also have biocidal effects when they are formed, observed by DEM (Ripolles-Avila et al., 2018). As applied as treatments. For this reason, this was considered important to observed in the results, the biofilm had reached its maturity as cell evaluate. The maximum antimicrobial activity found was for the treat­ distribution on the surface was in geometric shapes and covered a large ment with Product A applied at 3 % and 50 ◦ C, obtaining a microbial part the surface, while leaving interstitial spaces that can be assumed to reduction of 5.75 log CFU/cm2. This low microbial load observed in the be water channels. The results are in concordance with what was re­ treatment solution (i.e. implying greater effectiveness) could have two ported by Mazaheri et al. (2020) and Ripolles-Avila et al. (2018), who explanations: (i) the low count in the washing solution is derived from demonstrated that L. monocytogenes conformed mature biofilms at one the fact that L. monocytogenes S2-bac remained on the surface because week of incubation. This also concurs with the findings of Centorame the treatment was not effective; (ii) could be related to the fact that the et al. (2017), who affirm that biofilms achieve a more complex organi­ cells released from the surface and passed into the washing liquid were zation and a higher density of attached cells after this minimum period in a non-viable state (i.e. antimicrobial effect). It was demonstrated that of incubation. Moreover, conditioning in a humidity-saturated chamber it was the second case since both the quantification and DEM studies on led to the formation of mature biofilms, as also stated by Mai et al. the cells that remained after treatment on the surface showed that (2006), since this is a primary determinant in the adhesion of microor­ Product A was completely disintegrated and effective, indicating that ganisms and a way to help them to distribute on the surface. this enzymatic product applied under these conditions has antimicrobial L. monocytogenes S2-bac cells and structure remaining on the surface activity. However, and parallel to this, a subsequent disinfection should after the application of the eleven treatments is shown in Fig. 2. When be applied to further improve the effectiveness of Product A. The factor 6 T. Mazaheri et al. International Journal of Food Microbiology 381 (2022) 109888 A-1 A-2 B-1 B-2 C-1 C-2 D-1 D-2 Fig. 3. Epifluorescence digital images of Live/Dead-stained mature L. monocytogenes biofilms before (1) and after (2) the application of the combined treatments (i.e. chlorinated alkaline 1 % + Product A 1 %) on four different strains: CECT 5672 (A); S2-bac (B); EDG-e (C); CECT 935 (D). Magnification 20×. that reduced the antimicrobial action of Product A was the decrease in obtained for the acidic cleaning agent, similar to what has been obtained the treatment temperature (i.e. 20 ◦ C), the importance of the optimal by other authors (Fagerlund et al., 2020). temperature application having been discussed in the previous two Surprisingly, one treatment that exerted one of the lowest biocidal sections. activities was the chlorinated alkaline detergent (i.e. logarithmic In addition, Product A treatment (i.e. 3 % at 50 ◦ C) did not present reduction of 3.41 Log CFU/cm2). In this case, biofilm removal from the significant differences (P > 0.05) on the exerted biocidal activity with surface was lower, which could also be the reason why the biocidal the alkaline and acid treatments (Table 3). Other authors have reported effectivity was lower (i.e. less microbial load was being released to the similar logarithmic reductions when these treatments were applied to solution). It is also important to point out that the biocidal activity of eliminate biofilms. For example, Taormina and Beuchat (2002) reported alkaline and chlorinated alkaline, although not evaluated in the present antibacterial effectiveness of an alkaline detergent of 5 or 6 Log CFU/ml study, can be influenced by the interaction with organic matter, subse­ reduction. Moreover, approximately 5 logs of dead cells were also quently adversely affecting the efficacy of many biocides such as sodium 7 T. Mazaheri et al. International Journal of Food Microbiology 381 (2022) 109888 hypochlorite, included in the formulation of the chlorinated alkaline product as a second treatment step resulted in the complete dispersion of detergent (Ramos et al., 2013). the structure. 3.4. Effectivity, disaggregation capacity and biocidal effect of the 4. Conclusion combined treatment on biofilms of different L. monocytogenes strains The application of conventional treatments such as alkaline and acid The selected strains to conduct this study were L. monocytogenes detergents for biofilm removal greatly reduce the cells conforming L. CECT 5672, CECT 935, EDG-e and S2-bac, chosen based on assessing monocytogenes mature biofilms, with chlorinated alkaline detergent different serotypes (i.e. 4b and 1/2a). In this case, a comparison between being more effective and therefore an option in terms of conventional an effective treatment and one that demonstrated lower removal ca­ treatments. However, the structure formed on the surfaces was not pacity was included as part of the study with the objective of subjecting dispersed, with cells remaining on the stainless-steel coupons with the treatments to more strains. Hence, chlorinated alkaline (1 % at 20 ◦ C) potential consequences in terms of cell reparation to be viable again and and Product A (1 % at 50 ◦ C) were selected as treatments. Table 4 shows potentially re-contaminate other industrial surfaces. In contrast, not only the effectivity of the applied treatments but also the biofilm applying enzymatic treatments had the two effects, high detachment formation capacity of each strain. Starting from this last point, capacity and high dispersion of the structure, thus being consolidated as L. monocytogenes CECT 5672 was the largest biofilm producer in the most effective treatment. Potentiating chemical-based cleaning de­ conjunction with S2-bac. Strain CECT 5672 has already been reported as tergents effectivity with enzymatic treatments could be an option to a high biofilm producer in comparative studies employing 17 different L. optimize treatments, so combined treatment could therefore be a good monocytogenes strains (Mazaheri et al., 2020; Ripolles-Avila et al., option to recommend when applying 5-step cleaning protocols. 2019a). The results obtained after the application of combined treatments (i. Funding e. chlorinated alkaline and Product A) for 30 min on each L. mono­ cytogenes strain are also presented in Table 4. As described by Fagerlund This study was supported by Research Project grants RTI2018- et al. (2020), the application of chlorinated alkaline helps to remove 098267-R-C32 from the Ministerio de Ciencia, Innovación y organic matter from industrial surfaces. The posterior application of the Universidades. enzymatic product increased the detachment activity of the chlorinated alkaline treatment alone from 77 % to 100 % in the majority of the Declaration of competing interest strains (i.e. > 6 log reduction). This combination can therefore ensure an adequate level of cleaning and elimination of the cells detached from the The authors declare no conflict of interest. biofilms. In this regard, a two-step cleaning with chlorinated alkaline and an enzymatic product produced the largest microbial cells reduction Data availability and could be a recommended treatment to substitute cleaning and disinfection in the same procedure (i.e. 5-step cleaning protocol). Data will be made available on request. Fagerlund et al. (2020) evaluated the same combined treatment (i.e. chlorinated alkaline followed by an enzymatic based cleaner as the Acknowledgements second step), giving a >3 log reduction in L. monocytogenes biofilms formed on stainless steel coupons. The difference in the effectivity in The authors acknowledge Ms. Sarah Davies for the English grammar comparison with the results obtained could be related to the increase in review and Dolors Busquets for her technical support. The authors alone the treatment temperature (i.e. 50 ◦ C). are responsible for the content and writing of the paper. The antimicrobial efficacy of the combined treatment against the mature L. monocytogenes biofilms is shown in Table 5. In this case, the References results are separated from each treatment because although applied in combination (i.e. first the chemical treatment and then the biological AENOR, 2015. UNE-EN 13697 - Chemical disinfectants and antiseptics - quantitative one), the remaining cleaning solution was independent (i.e. two non-porous surface test for the evaluation of bactericidal and/or fungicidal activity different washing solutions rather than a mixed washing solution). of chemical disinfectants used in food, industrial, domestic and institutional areas - Test method a. Again, lower antimicrobial activity was found in the chlorinated alka­ Allam, M., Tau, N., Smouse, S.L., Mtshali, P.S., Mnyameni, F., Khumalo, Z.T.H., line treatment, which can be attributed to the fact that less microbial Ismail, A., Govender, N., Thomas, J., Smith, A.M., 2018. Whole-genome sequences of load was being released. With the application of the subsequent enzy­ Listeria monocytogenes sequence type 6 isolates associated with a large foodborne outbreak in South Africa, 2017 to 2018. Genome Announc. 6, 1–2. https://doi.org/ matic treatment, the antimicrobial activity significantly increased. 10.1128/genomeA.00538-18. Copyright. Arias-Moliz, M.T., Ferrer-Luque, C.M., Espigares-Rodríguez, E., Liébana-Ureña, J., 3.5. Qualitative evaluation of the combined effect of chlorinated alkaline Espigares-García, M., 2008. Bactericidal activity of phosphoric acid, citric acid, and EDTA solutions against Enterococcus faecalis. Oral Surg. Oral Med.Oral Pathol. Oral and enzymatic solutions for L. monocytogenes biofilm removal Radiol. Endodontol. 106, 84–89. https://doi.org/10.1016/j.tripleo.2008.04.002. Ben Braïek, O., Smaoui, S., Ennouri, K., Ben Ayed, R., Hani, K., Mastouri, M., Ghrairi, T., Microscopic images showed how the two treatments combined 2020. In situ Listeria monocytogenes biocontrol and sensory attributes enhancement in raw beef meat by Enterococcus lactis. J. Food Process. Preserv. 1–8 https://doi.org/ heightened the biofilm removal effectivity (Fig. 3). The use of chlori­ 10.1111/jfpp.14633. nated alkaline and enzymatic treatments over fixed periods of 30 min Bridier, A., Sanchez-Vizuete, P., Guilbaud, M., Piard, J.C., Naïtali, M., Briandet, R., 2015. showed almost complete removed the biofilm structures. As previously Biofilm-associated persistence of food-borne pathogens. Food Microbiol. 45, 167–178. https://doi.org/10.1016/j.fm.2014.04.015. described by Mnif et al. (2020) and Ripolles-Avila et al. (2020), and also Centorame, P., D’Angelo, A.R., Di Simone, F., Salini, R., Cornacchia, A., Marrone, R., observed in the present study, chlorinated alkaline treatment was unable Anastasio, A., Pomilio, F., 2017. Listeria monocytogenes biofilm production on food to completely disintegrate the biofilms structure, remaining on the packaging materials submitted to physical treatment. Ital. J. Food Saf. 6, 106–109. https://doi.org/10.4081/ijfs.2017.6654. surface with the potential consequences. One of the direct consequences Churchill, K.J., Sargeant, J.M., Farber, J.M., O’connor, A.M., 2019. Prevalence of Listeria if cells are not completely dead is that they can repair themselves from monocytogenes in select ready-to-eat foods—deli meat, soft cheese, and packaged damages derived from treatments and consolidate again biofilms salad: a systematic review and meta-analysis. J. Food Prot. 82, 344–357. https://doi. structures. Therefore, conventional cleaning and disinfection treatments org/10.4315/0362-028X.JFP-18-158. Colagiorgi, A., Di Ciccio, P., Zanardi, E., Ghidini, S., Ianieri, A., 2016. A look inside the are not considered a good weapon to remove and eliminate bacterial Listeria monocytogenes biofilms extracellular matrix. Microorganisms 4, 1–12. cells from the surfaces. Nevertheless, the application of the enzymatic https://doi.org/10.3390/microorganisms4030022. 8 T. Mazaheri et al. International Journal of Food Microbiology 381 (2022) 109888 Cripe, J., Losikoff, M., 2021. Presence of Listeria monocytogenes and sanitation controls in Mnif, S., Jardak, M., Yaich, A., Aifa, S., 2020. Enzyme-based strategy to eradicate cold-smoked salmon facilities during FDA inspections. Food Prot. Trends 41, monospecies Macrococcus caseolyticus biofilm contamination in dairy industries. Int. 184–194. https://doi.org/10.4315/1541-9576-41.2.184. Dairy J. 100, 104560 https://doi.org/10.1016/j.idairyj.2019.104560. D’Arrigo, M., Mateo-Vivaracho, L., Guillamón, E., Fernández-León, M.F., Bravo, D., Mosquera-Fernández, M., Sanchez-Vizuete, P., Briandet, R., Cabo, M.L., Balsa-Canto, E., Peirotén, Á., Medina, M., García-Lafuente, A., 2020. Characterization of persistent 2016. Quantitative image analysis to characterize the dynamics of Listeria Listeria monocytogenes strains from ten dry-cured ham processing facilities. Food monocytogenes biofilms. Int. J. Food Microbiol. 236, 130–137. https://doi.org/ Microbiol. 92, 1–6. https://doi.org/10.1016/j.fm.2020.103581. 10.1016/j.ijfoodmicro.2016.07.015. Delhalle, L., Taminiau, B., Fastrez, S., Fall, A., Ballesteros, M., Burteau, S., Daube, G., Ortiz, S., López, V., Martínez-Suárez, J.V., 2014. The influence of subminimal inhibitory 2020. Evaluation of enzymatic cleaning on food processing installations andfood concentrations of benzalkonium chloride on biofilm formation by Listeria products bacterial microflora. Front. Microbiol. 11, 1–16. https://doi.org/10.3389/ monocytogenes. Int. J. Food Microbiol. 189, 106–112. https://doi.org/10.1016/j. fmicb.2020.01827. ijfoodmicro.2014.08.007. EFSA-ECDC, 2021. The European union one health 2020 zoonoses report. EFSA J. 19, Ortiz, S., López-Alonso, V., Rodríguez, P., Martínez-Suárez, J.V., 2016. The connection 1–324. https://doi.org/10.2903/j.efsa.2021.6971. between persistent, disinfectant-resistant Listeria monocytogenes strains from two Fagerlund, A., Heir, E., Møretrø, T., Langsrud, S., 2020. Listeria monocytogenes biofilm geographically separate Iberian pork processing plants: evidence from comparative removal using different commercial cleaning agents. Molecules. https://doi.org/ genome analysis. Appl. Environ. Microbiol. 82, 308–317. https://doi.org/10.1128/ 10.3390/molecules25040792. AEM.02824-15. Faille, C., Cunault, C., Dubois, T., Bénézech, T., 2018. Hygienic design of food processing Parkar, S.G., Flint, S.H., Brooks, J.D., 2004. Evaluation of the effect of cleaning regimes lines to mitigate the risk of bacterial food contamination with respect to on biofilms of thermophilic bacilli on stainless steel. J. Appl. Microbiol. 96, 110–116. environmental concerns. Innov. Food Sci. Emerg. Technol. 46, 65–73. https://doi. https://doi.org/10.1046/j.1365-2672.2003.02136.x. org/10.1016/j.ifset.2017.10.002. Pleszczyńska, M., Wiater, A., Bachanek, T., Szczodrak, J., 2017. Enzymes in therapy of Fuster-Valls, N., Hernández-Herrero, M., Marín-de-Mateo, M., Rodríguez-Jerez, J.J., biofilm-related oral diseases. Biotechnol. Appl. Biochem. 64, 337–346. https://doi. 2008. Effect of different environmental conditions on the bacteria survival on org/10.1002/bab.1490. stainless steel surfaces. Food Control 19, 308–314. https://doi.org/10.1016/j. Ramos, B., Miller, F.A., Brandão, T.R.S., Teixeira, P., Silva, C.L.M., 2013. Fresh fruits and foodcont.2007.04.013. vegetables - an overview on applied methodologies to improve its quality and safety. González-Rivas, F., Ripolles-Avila, C., Fontecha-Umaña, F., Ríos-Castillo, A.G., Innov. Food Sci. Emerg. Technol. 20, 1–15. https://doi.org/10.1016/j. Rodríguez-Jerez, J.J., 2018. Biofilms in the spotlight: detection, quantification, and ifset.2013.07.002. removal methods. Compr. Rev. Food Sci. Food Saf. 17, 1261–1276. https://doi.org/ Ripolles-Avila, C., Hascoët, A.S., Guerrero-Navarro, A.E., Rodríguez-Jerez, J.J., 2018. 10.1111/1541-4337.12378. Establishment of incubation conditions to optimize the in vitro formation of mature Guerrero-Navarro, A.E., Ríos-Castillo, A.G., Ripolles-Avila, C., Zamora, A., Hascoët, A.S., Listeria monocytogenes biofilms on food-contact surfaces. Food Control 92, 240–248. Felipe, X., Castillo, M., Rodríguez-Jerez, J.J., 2022. Effectiveness of enzymatic https://doi.org/10.1016/j.foodcont.2018.04.054. treatment for reducing dairy fouling at pilot-plant scale under real cleaning Ripolles-Avila, C., Cervantes-Huaman, B.H., Hascoët, A.S., Yuste, J., Rodríguez-Jerez, J. conditions. LWT 154. https://doi.org/10.1016/j.lwt.2021.112634. J., 2019a. Quantification of mature Listeria monocytogenes biofilm cells formed by an Hashim, S.T., Fakhry, S.S., Alrubaye, H.H., 2020. Evaluation of the effectiveness of in vitro model: a comparison of different methods. Int. J. Food Microbiol. 289, treatments for sanitizing agents for removal of Listeria monocytogenes biofilm. Plant 209–214. https://doi.org/10.1016/j.ijfoodmicro.2018.10.020. Arch. 20, 249–255. Ripolles-Avila, C., Ríos-Castillo, A.G., Fontecha-Umaña, F., Rodríguez-Jerez, J.J., 2019b. Holah, J.T., BRI UK, C., 2014. Cleaning and disinfection practices in Removal of Salmonella enterica serovar Typhimurium and Cronobacter sakazakii food processing. In: Hygiene in Food Processing. Woodhead Publishing Limited. biofilms from food contact surfaces through enzymatic catalysis. J. Food Saf. 40 https://doi.org/10.1533/9780857098634.3.259. https://doi.org/10.1111/jfs.12755. Kim, C.Y., Zhu, X., Herzberg, M., Walker, S., Jassby, D., 2018. Impact of physical and Ripolles-Avila, C., Ramos-Rubio, M., Hascoët, A.S., Castillo, M., Rodríguez-Jerez, J.J., chemical cleaning agents on specific biofilm components and the implications for 2020. New approach for the removal of mature biofilms formed by wild strains of membrane biofouling management. Ind. Eng. Chem. Res. 57, 3359–3370. https:// Listeria monocytogenes isolated from food contact surfaces in an Iberian pig doi.org/10.1021/acs.iecr.7b05156. processing plant. Int. J. Food Microbiol. 323, 1–9. https://doi.org/10.1016/j. Laksanalamai, P., Huang, B., Sabo, J., Burall, L.S., Zhao, S., Bates, J., Datta, A.R., 2014. ijfoodmicro.2020.108595. Genomic characterization of novel Listeria monocytogenes serotype 4b variant strains. Sadekuzzaman, M., Yang, S., Mizan, M.F.R., Kim, H.S., Ha, S.Do, 2017. Effectiveness of a PLoS One 9. https://doi.org/10.1371/journal.pone.0089024. phage cocktail as a biocontrol agent against L. monocytogenes biofilms. Food Control Lee, S., Ward, T.J., Graves, L.M., Wolf, L.A., Sperry, K., Siletzky, R.M., Kathariou, S., 78, 256–263. https://doi.org/10.1016/j.foodcont.2016.10.056. 2012. Atypical Listeria monocytogenes serotype 4b strains harboring a lineage II- Salama, P.J., Embarek, P.K.B., Bagaria, J., Fall, I.S., 2018. Learning from Listeria: safer specific gene cassette. Appl. Environ. Microbiol. 78, 660–667. https://doi.org/ food for all. Lancet 391, 2305–2306. https://doi.org/10.1016/S0140-6736(18) 10.1128/AEM.06378-11. 31206-6. Lelieveld, H.L.M., 2013. Introduction. In: Hyg. Food Process. Princ. Pract, Second ed. Srey, S., Jahid, I.K., Do Ha, S., 2013. Biofilm formation in food industries: a food safety https://doi.org/10.1016/B978-0-85709-429-2.50022-X concern. Food Control. https://doi.org/10.1016/j.foodcont.2012.12.001. Liikanen, R., Yli-Kuivila, J., Laukkanen, R., 2002. Efficiency of various chemical Taormina, P.J., Beuchat, L.R., 2002. Survival of Listeria monocytogenes in commercial cleanings for nanofiltration membrane fouled by conventionally-treated surface food-processing equipment cleaning solutions and subsequent sensitivity to water. J. Memb. Sci. 195, 265–276. https://doi.org/10.1016/S0376-7388(01) sanitizers and heat. J. Appl. Microbiol. 92, 71–80. https://doi.org/10.1046/j.1365- 00569-5. 2672.2002.01488.x. Maćkiw, E., Stasiak, M., Kowalska, J., Kucharek, K., Korsak, D., Postupolski, J., 2020. Thomas, Mitha, Sathian, C.T., 2014. Cleaning-in-place (CIP) system in dairy plant- Occurrence and characteristics of Listeria monocytogenes in ready-to-eat meat review. IOSR J. Environ. Sci. Toxicol. Food Technol. 8, 41–44. https://doi.org/ products in Poland. J. Food Prot. 83, 1002–1009. https://doi.org/10.4315/JFP-19- 10.9790/2402-08634144. 525. Torlak, E., Sert, D., 2013. Combined effect of benzalkonium chloride and ultrasound Mai, T.L., Sofyan, N.I., Fergus, J.W., Gale, W.F., Conner, D.E., 2006. Attachment of against Listeria monocytogenes biofilm on plastic surface. Lett. Appl. Microbiol. 57, Listeria monocytogenes to an austenitic stainless steel after welding and accelerated 220–226. https://doi.org/10.1111/lam.12100. corrosion treatments. J. Food Prot. 69, 1527–1532. https://doi.org/10.4315/0362- U.S. FDA/USDA/CDC, 2019. Outbreak Investigation of Listeria monocytogenes: hard- 028X-69.7.1527. boiled eggs. https://www.fda.gov/food/outbreaks-foodborne-illness/outbreak- Mazaheri, T., Ripolles-Avila, C., Hascoët, A.S., Rodríguez-Jerez, J.J., 2020. Effect of an investigation-Listeria-monocytogenes-hard-boiled-eggs-december-2019#Recall. enzymatic treatment on the removal of mature Listeria monocytogenes biofilms: a U.S. FDA/USDA/CDC, 2020. Outbreak investigation of Listeria monocytogenes: Enoki quantitative and qualitative study. Food Control 114, 1–8. https://doi.org/10.1016/ Mushrooms (March 2020). https://www.fda.gov/food/outbreaks-foodborne-illne j.foodcont.2020.107266. ss/outbreak-investigation-Listeria-monocytogenes-enoki-mushrooms-march-2020. McEntire, J., 2018. Guidance on environmental monitoring and control of Listeria for the Waghmare, R.B., Annapure, U.S., 2015. Integrated effect of sodium hypochlorite and fresh produce industry. URL. United Fresh Prod. Assoc. (accessed 1.12.21). https:// modified atmosphere packaging on quality and shelf life of fresh-cut cilantro. Food www.unitedfresh.org/guidance-on-environmental-monitoring-and-control-of-Li Packag. Shelf Life 3, 62–69. https://doi.org/10.1016/j.fpsl.2014.11.001. steria-for-the-fresh-produce-industry-2nd-ed/ Yang, Y., Mikš-Krajnik, M., Zheng, Q., Lee, S.B., Lee, S.C., Yuk, H.G., 2016. Biofilm Mendonca, A.F., Amoroso, T.L., Knabel, S.J., 1994. Destruction of Gram-negative food- formation of Salmonella Enteritidis under food-related environmental stress borne pathogens by high pH involves disruption of the cytoplasmic membrane. Appl. conditions and its subsequent resistance to chlorine treatment. Food Microbiol. 54, Environ. Microbiol. 60, 4009–4014. https://doi.org/10.1128/aem.60.11.4009- 98–105. https://doi.org/10.1016/j.fm.2015.10.010. 4014.1994. Zwirzitz, B., Wetzels, S.U., Dixon, E.D., Fleischmann, S., Selberherr, E., Thalguter, S., Mitidieri, S., Souza Martinelli, A.H., Schrank, A., Vainstein, M.H., 2006. Enzymatic Quijada, N.M., Dzieciol, M., Wagner, M., Stessl, B., 2021. Co-occurrence of Listeria detergent formulation containing amylase from Aspergillus niger: a comparative study spp. and spoilage associated microbiota during meat processing due to cross- with commercial detergent formulations. Bioresour. Technol. 97, 1217–1224. contamination events. Front. Microbiol. 12, 1–14. https://doi.org/10.3389/ https://doi.org/10.1016/j.biortech.2005.05.022. fmicb.2021.632935. 9