Food Microbiology 105 (2022) 104026 Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Dual-species biofilms formation between dominant microbiota isolated from a meat processing industry with Listeria monocytogenes and Salmonella enterica: Unraveling their ecological interactions C. Ripolles-Avila, M. Guitan-Santamaria, K. Pizarro-Giménez, T. Mazaheri, J.J. Rodríguez-Jerez * Human Nutrition and Food Science Area, Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona (UAB), Edifici V, Campus de la UAB, 08193, Bellaterra (Cerdanyola del Vallès), Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Alternatives to combat the persistence of pathogens need to consider the microbiota established on industrial Biofilms surfaces as they can influence the protection or replacement (i.e. reduction/inhibition) of pathogens. The Interactions objective of the present study was to determine the ecological interactions established in dual-species biofilms Persistence between Listeria monocytogenes and Salmonella enterica as target pathogens, and isolates recovered from a meat Biocontrol L. monocytogenes processing facility (i.e. Pseudomonas fluorescens, Pseudomonas fragi, Bacillus safensis, Bacillus megaterium, and Salmonella spp. Candida zeylanoides). Results showed different ecological relations in biofilms depending on the species evalu­ ated. Pseudomonas spp. did not influence the growth of either pathogen, although tested species tended to protect the pathogens in the structures generated. B. megaterium and C. zeylanoides affected the two pathogens differ­ ently, demonstrating a reduction of L. monocytogenes adhered cells within the formed biofilm. B. safensis reduced or presented non-influence on S. enterica depending on the incubation conditions. Contrarily, B. safensis was the microorganism that demonstrated the highest replacement capacity for L. monocytogenes, reducing its growth by up to 4 log CFU/cm2. The in vitro study of bispecies biofilms is important for the food industry, helping to un­ derstand how they behave and to find an effective way to eliminate them. 1. Introduction et al., 2011; Fagerlund et al., 2017). Furthermore, tolerance has been related to the resistance of microbial cells when they are exposed to Surfaces in the food industry are regularly exposed to cleaning and sub-lethal concentrations of disinfectants (Capita et al., 2014; Molina-­ disinfection processes since the presence of biofilms, the most wide­ González et al., 2014; Rodríguez-Melcón et al., 2019). Regarding spread microbial form in the environment, is constantly detected on cleaning and disinfection procedures, their aim is to reduce the numbers them (González-Rivas et al., 2018; Mazaheri et al., 2021). Biofilms are of microorganisms to an acceptable level to manage risk (Ripolles-Avila biostructures where microorganisms are embedded in a produced et al., 2019c). However, there are intrinsic and extrinsic factors such as organic matrix essentially composed of polysaccharides, proteins, and temperature, water availability, and the presence of organic matter that nucleic acids (Flemming and Wingender, 2010). This matrix contributes constantly influence the growth of microorganisms (Fagerlund et al., to the attachment of microbial cells to the surface and triggers tolerance 2021). Such factors imply that a whole microbial community could be to environmental stresses (Álvarez-Ordóñez and Briandet, 2016). Bio­ able to survive and remain on different surfaces of the food processing films are difficult to remove completely, becoming a source of facilities conforming an ecosystem named resident microbiota (Bright­ contamination for food products due to the continuous shedding of cells well et al., 2006; Hascoët et al., 2019). It has been indicated that the and spores from food and non-food contact surfaces (González-Rivas resident microbiota directly influences (i.e. modulate the growth of a et al., 2018; Ripolles-Avila et al., 2018b). Biofilms provide protection to microorganism) the presence of certain foodborne pathogens such as cells by presenting a physical barrier, induce stress proteins, permit gene Listeria monocytogenes (Fagerlund et al., 2021). exchange, prevent desiccation and could also produce certain substances A great diversity of resident microbiota, including both microbial for protecting the niche against competitive microorganisms (Bridier families and species capable of adhering to industrial surfaces and * Corresponding author. E-mail address: josejuan.rodriguez@uab.cat (J.J. Rodríguez-Jerez). https://doi.org/10.1016/j.fm.2022.104026 Received 17 November 2021; Received in revised form 8 March 2022; Accepted 11 March 2022 Available online 14 March 2022 0740-0020/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). C. Ripolles-Avila et al. Food Microbiology 105 (2022) 104026 developing biofilms, is found in the food industry (Speranza et al., resident microbiota in the persistence of L. monocytogenes and Salmonella 2017). On industrial surfaces (i.e. contact and non-contact food sur­ spp. is essential for the development of new strategies for their control. faces), species from the Enterobacteriaceae family, Pseudomonas spp., To this end, the present study focuses on determining the ecological Acinetobacter spp., Bacillus spp., Staphylococcus spp., lactic acid bacteria relationship established in biofilms between L. monocytogenes and Sal­ (LAB), and certain yeasts generally tend to predominate (Fagerlund monella enterica var. Typhimurium, and Pseudomonas fluorescens, Pseu­ et al., 2017; Møretrø and Langsrud, 2017). A previous study from our domonas fragi, Bacillus safensis, Bacillus megaterium, and Candida research group evaluated the resident microbiota on the surfaces of an zeylanoides. The influence of time on the generation of these structures Iberian pig processing plant and correlated the microbial profiles when was also evaluated. L. monocytogenes was present or absent from the surfaces (Hascoët et al., 2019). It was demonstrated that abundance in Pseudomonas spp. influ­ 2. Materials and methods enced positively L. monocytogenes presence and other genera such as Bacillus spp. diminished the presence of the pathogen. On this regard, 2.1. Surfaces Hascoët et al., (2021) demonstrated that mature preformed Bacillus safensis biofilms can inhibit adhesion and the biofilm formation of AISI 316 stainless steel coupons (2 cm in diameter and 1 mm thick) multiple L. monocytogenes strains, eliminating the pathogen by a were used as a highly employed material in the design of food contact currently unidentified mechanism. So, resident microbiota in food pro­ surfaces. Prior to use, the surfaces were subjected to a cleaning and cessing plants can influence the growth of pathogens residing in the food disinfection procedure, as indicated in European standard UNE-EN environment (Fagerlund et al., 2021). This is because of biofilms can be 13697:2015. For that, first, coupons were cleaned with a non- formed by single species or by different species. In that last case, in­ bactericidal detergent (ADIS Higiene, Madrid, Spain) for 1 h, after­ teractions between cohabiting species influence their assembly and wards exposed to 70% iso-propanol (Panreac, Castellar del Vallès, function, as well as lead to outgoing properties that are specific to Spain) for 15 min, and air-dried in a laminar flow cabinet for 30 min. To multi-species biofilms (Røder et al., 2020). For example, mixed biofilms ensure that the coupons were completely sterile, they were autoclaved are more resistant towards distinct compounds such as antimicrobials for 15 min at 121 ◦ C before microbial inoculation. than monospecies biofilms. This is because they generate interspecies interactions for metabolites exchange, production of signalling mole­ 2.2. Microbial cultures used and growth conditions cules and genetic exchange that enhance resistance (Brooks and Flint, 2008; Burmølle et al., 2014; González-Rivas et al., 2018; Kumar and Seven different microorganisms were used to develop dual-species Anand, 1998). Moreover, multi-species biofilms tend to present a more biofilms, among which L. monocytogenes CECT 5672 (Spanish Collec­ complex structure (Røder et al., 2020). This is because specific matrix tion of Type Cultures; University of Valencia, Valencia, Spain) and compounds are produced only by the community, not by the individual S. enterica var. Typhimurium CECT 4594 (University of Valencia, species grown in isolation (Guillonneau et al., 2018). Valencia, Spain) were established as target pathogens. For the dual- Two of the most relevant pathogens for the food industry at a Eu­ species biofilms, P. fluorescens, P. fragi, B. safensis, B. megaterium, and ropean level are Salmonella spp. and L. monocytogenes. The first one is the C. zeylanoides were used, all isolated as part of the dominant microbiota second zoonotic agent most involved in foodborne diseases, represent­ on the surfaces in a meat industry (Hascoët et al., 2019). ing 26.66% of the cases reported in 2019; and the second one is the The strains were obtained as freeze-dried cultures preserved at 4 ◦ C. pathogen causing the highest number of deaths with a mortality rate of First, the cultures were rehydrated in 9 mL of Soy Tryptone Broth (TSB; 17.6% (EFSA-ECDC, 2021). Contamination by these pathogens can Oxoid, Madrid, Spain) and incubated at 30 ◦ C for 48 h. The cultures were occur at any level of the production chain, from farms with few hygienic then transferred to Tryptone Soy Agar plates (TSA; Oxoid, Madrid, measures to the food industry, due to contaminated surfaces, floors, Spain) and incubated at 30 ◦ C for 24 h in the case of all the bacteria and drains, and utensils, among others, and even because of poor handling 48 h for the yeast. From this culture, isolated colonies were transferred by food handlers (Ehuwa et al., 2021; Mazaheri et al., 2021). Both onto TSA plates and incubated again at 30 ◦ C for 24 h to obtain stock pathogens have the ability to form monospecies or mixed species bio­ cultures that were kept refrigerated at 4 ◦ C for a maximum of one month. films in food and on different industrial surfaces (Ripolles-Avila et al., 2018a, 2019d), although mixed biofilms formed with non-pathogenic 2.3. Inoculum preparation, surface inoculation, and incubation species that belong to the resident microbiota of the food industry are conditions most frequently found (Hascoët et al., 2019). It has been highlighted that persistence of certain pathogens such as L. monocytogenes is related A subculture was performed from the stock culture by plating it on to environmental niches shared with different species of the resident TSA plates. The plates were incubated at 30 ◦ C for 24 h, obtaining mi­ microbiota found in food factories such as Actinobacteria, Pseudomo­ crobial cultures in a stationary phase. The inoculum was then prepared nadaceae or Dipodascaceae (Fagerlund et al., 2021). Normally, biofilm by transferring isolated colonies to sterile 9 mL tubes of peptone water communities have different social behaviors which are broadly catego­ (BioMérieux, Marcy l’Etoile, France). The microbial concentration was rized as competitive, cooperative or neutral relations (Røder et al., standardized using a densitometer (Densimat, BioMérieux, Marcy 2020). In this regard, Lee et al. (2014) demonstrated that the composi­ l’Etoile, France) that measures turbidity in McFarland units. Isolated tion and spatial organization of the species within biofilms impact the colonies were transferred to obtain a turbidity of 1.5 McFarland units, survival of the entire community when exposed to stressors. Therefore, equivalent to 108 CFU/mL. Once this suspension was obtained, 100 μL and as discussed by Fagerlund et al. (2021), the focus of future studies were transferred to a 9.9 mL of peptone water, thus obtaining a con­ could change from reductionist approaches (i.e. monospecies biofilms) centration of 106 CFU/mL, which was used for the biofilm formation. to more complex and realistic laboratory models (i.e. mixed biofilms), Dual-species biofilms were formed by preparing a suspension of allowing a further investigation of interactions between species residing 50:50 between L. monocytogenes or S. Typhimurium as target pathogens, in factory environments. and the rest of the microorganisms. Once the mixed species suspension While different strategies have been developed to control biofilms was prepared, 30 μL were inoculated in the center of each coupon, such as chemical (i.e. biocides that inactivate microorganisms), physical resulting in an approximate surface microbial concentration of 4 log (e.g. UV, cold plasma, among others) or biological (e.g. bacteriophages) CFU/cm2. In parallel, a control of L. monocytogenes and S. Typhimurium approaches, there seems to be no clear direction on how to deal with the in monospecies biofilm was also evaluated with the same conditions as risk biofilms pose (Mazaheri et al., 2020; Ripolles-Avila et al., 2020). described previously. Therefore, the evaluation and understanding of the role played by the All the inoculated surfaces were introduced into sterile Petri dishes 2 C. Ripolles-Avila et al. Food Microbiology 105 (2022) 104026 and subsequently placed in a humidified chamber (saturated relative humidity >90%) using pieces of paper towels that were moistened with sterile distilled water, with the objective of promoting microbial growth and the consequent formation of biofilms (Fuster-Valls et al., 2008; Log CFU/cm2 Ripolles-Avila et al., 2018a, 2019a, 2019b). The surfaces were incubated at 30 ◦ C for different periods, which were selected at 72 h, 72 + 24 h, 72 + 48 h, and 72 + 72 h. A washing protocol and renewal of nutrients was established at 72 h with the aim of eliminating non-adhered microor­ ganisms, in addition to renewal of the culture medium to enhance the formation of the biofilm structure (Ripolles-Avila et al., 2018a). This was done by, first, washing the surfaces with 3 mL of sterile distilled water in duplicate to remove loosely attached cells and other impurities, Incubation period (hours) and last, by adding 30 μL of sterile TSB to the coupons to renew the nutrients. Once this procedure was finished, the surfaces were again Fig. 1. Growth evolution of Listeria monocytogenes ( ) and Salmonella enterica placed in the humid chamber and incubated for the corresponding 24, ser. Typhimurium ( ) in the monospecies biofilm during the different incuba­ 48, or 72 h. tion periods. Microbial counts expressed in Log CFU/cm2 (n = 9). Error bars indicate standard error of the mean (SEM). 2.4. Quantification of the microbial load biofilms under these experimental conditions. This fact is in correlation To quantify the microbial cells present in the dual-species biofilms, with the study conducted by Reis-Teixeira et al., (2017), which obtained cells were recovered and inoculated in different culture media. To this no increases in sessile populations between 24 h and 192 h of incubation effect, after the incubation period (i.e. 72 h, 72 + 24 h, 72 + 48 h, and 72 on stainless steel surfaces. Ripolles-Avila et al., (2018a) carried out a + 72 h) the surfaces were washed with 3 mL of sterile distilled water to study for the optimization of an L. monocytogenes biofilm formation eliminate possible non-adhered cells, and further placed in a sterile flask model, concluding that the constitution of a mature biofilm at the containing 3 g of glass beads (2 mm in diameter) and 9 mL of sterile microscopic level was obtained at one week of incubation. Consistent peptone water. The flasks were then vortexed for 90 s at a frequency of with the results obtained in the present study, neither were any signif­ 40 Hz, with the objective of detaching the cells conforming the dual- icant differences (P > 0.05) observed between the lowest incubation species biofilms (Ripolles-Avila et al., 2019a). periods. In the case of S. Typhimurium, the 72-h period was the one with For the cell count, the resulting suspension was decimally diluted in the greatest growth compared to the rest of the incubation periods (P < Tryptone Saline Solution (TSS; 1 g of tryptone [BD, Madrid, Spain] and 0.05). This observation may be because a washing was carried out and 8.5 g of sodium chloride per liter; pH 7.0 ± 0.2). Next, 1 mL of different the culture medium renewed after the first 72 h, causing the loss of dilutions was inoculated onto a Petri dish, subsequently adding the non-adhered cells and other impurities. If the biofilm formation was not corresponding culture medium, TSA for the total count and ALOA very compact, the washing could have caused the detachment of a large (BioMérieux, Marcy l’Etoile, France) or SS (Salmonella-Shigella agar; number of cells through passive desorption processes or by grazing and Condalab, Madrid, Spain) for the specific counts of L. monocytogenes and erosion (Krsmanovic et al., 2021), generating therefore decrease in mi­ S. Typhimurium, respectively. The plates were then incubated at 37 ◦ C crobial counts. This fact was also observed by Ripolles-Avila et al., for 24 h for the counts on the selective agar and 30 ◦ C for 48 h for the (2018a) in which it was determined that reaching biofilm maturity total counts. This process was carried out for each of the mixtures and could be compromised if L. monocytogenes did not have a minimum of the different incubation times. 48 h of incubation as a period for initial attachment. Likewise, Marsh et al., (2003) observed that when the microbial concentration on a 2.5. Statistical analysis surface is low due to short incubation times, the washing and nutrient renewal process could modify the structural pattern of cell adhesion The experiments were performed in triplicate on three separate days described as “honeycomb”, generating a lower adhered microbial load. (n = 9). CFU values for L. monocytogenes and S. Typhimurium were used Nevertheless, when there was a high microbial density, these same steps to calculate by difference the CFU numbers present of the microor­ did not influence the pattern of cell adhesion and as a result a higher ganism faced with the pathogen. Once the microbial counts were ob­ adherent load was obtained. In the evaluated incubation periods, it was tained, they were subjected to a decimal logarithmic transformation to observed that S. Typhimuirum counts decreased non-significantly (P > reduce the variability in the microbiological analyses. To verify the 0.05). This could be because as incubation time passes, a degradation of conditions prior to performing an analysis of variance, the Shapiro-Wilk nutrients occurs in biofilms due to their consumption, thus generating a test for normality was carried out on the studied populations, and the decline in the number of microorganisms (Donlan, 2002; Ripolles-Avila Levene test was performed to verify the equality between variances. et al., 2018a). To contrast the results obtained with their maturity level, Once the test conditions were met, the results were evaluated by analysis it would be interesting to evaluate L. monocytogenes and S. Typhimuirum of variance (ANOVA), with a posteriori contrast using the Tukey test. The formed biofilms by direct epifluorescence microscopy (DEM) under the statistical software package R v 3.6.3 was used for all the tests. A value of experimental conditions tested. P < 0.05 was established as the level of significance. 3.2. Impact of dominant species in the meat industry on L. monocytogenes 3. Results and discussion and S. Typhimurium in dual-species biofilms 3.1. Monospecies biofilms of L. monocytogenes and S. Typhimurium 3.2.1. Pseudomonas spp. as predominant genera Studies on the type of interaction exerted by the residential micro­ Microbial counts obtained for the controls (i.e. monospecies bio­ biota of industrial surfaces against pathogens of the food industry are films) over the different incubation periods is shown in Fig. 1. Regarding nowadays highly interesting. This is because these could be used for the the counts obtained from L. monocytogenes in the monospecies biofilms, development of new control strategies such as biocontrol alternatives in no significant differences (P < 0.05) were observed between any of the the future (Ripolles-Avila et al., 2019c). In this regard, it has been shown evaluated incubation periods. For that, it can be presumed that neither that among Gram-negative microbiota the predominance of Pseudo­ the washing nor time affected L. monocytogenes counts when producing monas spp. stands out (Røder et al., 2016; Stellato et al., 2016), and 3 C. Ripolles-Avila et al. Food Microbiology 105 (2022) 104026 especially P. fluorescens and P. fragi (Hascoët et al., 2019). A predomi­ Typhimurium cell abundance. Similarly, Ripolles-Avila et al. (2019b) nance of P. fluorescens over L. monocytogenes can be observed in all the observed that P. fragi does not adhere with the necessary force to the experimental conditions studied in the present research (Fig. 2-A1), surface, implying that part of the biofilm structure can be eliminated coinciding with findings reported by Haddad et al., (2021) regarding during washing, reducing population numbers. Furthermore, an in­ dominance of P. fluorescens over L. monocytogenes although without crease of S. Typhimurium presence, mixed with P. fluorescens when the influencing pathogen counts when compared to the monospecies bio­ abundance was slightly higher, was observed 24 h after washing and films. According to the results of Pang and Yuk (2019), the presence of renewal of the culture medium. This fact could mean that Pseudomonas L. monocytogenes does not affect P. fluorescens cell density and it can spp. develops colonization and biofilm formation in the upper parts of constitute up to 90% of the population of these structures. This shows a the structure, something that has also been proven by other authors competitive advantage under the applied conditions of Pseudomonas (Puga et al., 2018; Ripolles-Avila et al., 2019b). All this could imply that spp., which can be attributed to its growth capacity and rapid utilization if L. monocytogenes or S. Typhimurium are found in the deepest parts of of nutrients over L. monocytogenes in dual-species biofilms. This same the biofilm under industrial conditions and a cleaning procedure is dominance of Pseudomonas spp. was demonstrated by Heir et al. (2018), applied to industrial surfaces, both pathogens could be released and obtaining significantly lower counts of L. monocytogenes in the presence mobilized outside, potentially causing subsequent recontamination of P. fluorescens and P. fragi. Furthermore, the results of the confronta­ problems in the food industry. tion between L. monocytogenes and P. fragi in dual-species biofilms Table 1 shows the derived cell counts within the formed biofilms showed a clear predominance of P. fragi over the pathogen in all con­ after the different incubation periods. In general, no significant differ­ ditions (Fig. 2-A.2). Sasahara and Zottola (1993) reported that a mixed ences (P > 0.05) were observed between the controls (i.e. monospecies culture with P. fragi improves the production of exopolymers and the L. monocytogenes biofilms) and the L. monocytogenes counts obtained binding and formation of colonies of L. monocytogenes. In the same from the study groups (i.e. dual-species P. fluorescens and P. fragi bio­ study, it was hypothesized that P. fragi produced a substance that films). This indicates that L. monocytogenes presence and counts were not attracted L. monocytogenes cells, demonstrating a high consolidation of influenced by either Pseudomonas species employed in the present study. mixed biofilms. However, in the present study, no significant differences Of note is that L. monocytogenes cell counts increased after washing and were obtained in the abundance of the pathogen in the presence of renewal of the nutrients, although without significant differences (P > P. fragi among any of the times studied (P = 0.34; P = 0.91; P = 0.82). In 0.05). In this context, a reduction of P. fluorescens was observed after the contrast, Norwood and Gilmour (2001) supported the decrease in application of a wash and renewal of nutrients. This could be due to a competitiveness of L. monocytogenes in the presence of P. fragi due to greater L. monocytogenes adhesion to stainless steel surfaces or to an competition for nutrients. Furthermore, they also suggest the production exterior placement of P. fluorescens within the biofilm, as observed by of antagonist compounds together with Staphylococcus xylosus. Puga et al. (2018). These authors pointed out that P. fluorescens is found In the case of S. Typhimurium, the results showed that for both in the outermost layer of the biofilm, generating an overproduction of P. fluorescens and P. fragi the percentage of the microbial population was matrix for its protection (Puga et al., 2016, 2018). By applying the wash, significantly higher than that of the pathogen itself (P < 0.05), pre­ some Pseudomonas spp. cells could then be detached from the top layers, senting consequently a greater abundance (Fig. 2-B.1 and B.2). How­ but L. monocytogenes could not as the pathogen would be protected in the ever, S. Typhimurium counts when compared with its control (i.e. generated structure. This is an interesting finding since it could indicate monospecie biofilm) did not present significant differences (P > 0.05) that when Pseudomonas spp. is present in mixed biofilms in food facil­ with pseudomonads dual-biofilms. This could also be because Pseudo­ ities, L. monocytogenes may not disappear, thus becoming an initial hy­ monas spp. presents a fast-growing pattern (Thirup et al., 2000), which pothesis for an industrial study as can be translated to two potential could lead to a greater predominance within the structure. From the problems: (i) the detection of the pathogen on surfaces can be difficult beginning, a greater predominance of both Pseudomonas spp. was with the usual sampling techniques, what could cause contaminated observed. Nevertheless, at 72 h, after the washing and nutrients renewal, points to be underestimated and give therefore a false sense of safety; there was a decrease in Pseudomonas spp. and an increase in S. and (ii), after applying a regular sanitization process there could be a Incubation period Incubation period L. monocytogenes P. fluorescens L. monocytogenes P. fragi Incubation period Incubation period S. Typhimurium P. fluorescens S. Typhimurium P. fragi Fig. 2. Comparison of the abundance percentage (%) of L. monocytogenes (A) and S. Typhimurium (B) when forming dual-species biofilms with P. fluorescens (1) and P. fragi (2) in the 4 different incubation periods evaluated. 4 C. Ripolles-Avila et al. Food Microbiology 105 (2022) 104026 Table 1 Growth evolution of L. monocytogenes and S. Typhimurium in dual-species biofilms with both Pseudomonas species during the different incubation periods and their comparison in mono-species biofilms. Counts expressed in Log CFU/cm2 (n = 9) ± standard error of the mean (SEM). Pathogen’s cell count in mono-species and dual-species biofilms (log CFU/cm2) Incubation period L. monocytogenes L. monocytogenes + P. L. monocytogenes + P. S. Typhimurium S. Typhimurium + P. S. Typhimurium + P. a (hours) fluorescens a fragi a b fluorescens b fragi b 72 5.42 ± 0.13Aa 5.79 ± 0.20Aa 5.08 ± 0.31Aa 6.25 ± 0.12Aa 6.25 ± 0.11Aa 6.18 ± 0.11Aa 72 þ 24 4.84 ± 0.27Aa 5.52 ± 0.22Aa 4.68 ± 0.22Aa 5.62 ± 0.23Ba 5.65 ± 0.16Ba 5.66 ± 0.17Ba 72 þ 48 5.34 ± 0.24Aa 5.34 ± 0.24Aa 5.12 ± 0.35Aa 5.61 ± 0.14Ba 5.72 ± 0.13Ba 5.55 ± 0.16Ba 72 þ 72 5.36 ± 0a34Aa 4.99 ± 0.33Ba 5.17 ± 0.27Aa 5.50 ± 0.21Ba 5.47 ± 0.14Ba 5.72 ± 0.14Ba A− B Values of a column lacking a common uppercase superscript differ significantly (P < 0.05). a Values of a row by study group (i.e. L. monocytogenes or S. Typhimurium) lacking a common lowercase superscript differ significantly (P < 0.05). a For L. monocytogenes specific count ALOA medium was used. b For S. Typhimurium specific count SS medium was used. reduction in indicators such as total aerobic microbiota, which could fact agrees with results reported by Pang et al. (2020), in which facilitate the mobilization of microorganisms within the bottom layers P. fluorescens presence did not contribute to the development of Salmo­ of the biofilm, leading to an increased risk of L. monocytogenes growth. nella spp. biofilms. However, Pang et al. (2017) observed that the Therefore, anti-biofilm products would need to be developed and more presence of Pseudomonas aeruginosa had an antagonistic effect against sensitive methods applied that allow the pathogen to be detected quickly Salmonella spp., reducing its population level. Further studies will be and efficiently, as well as to be eliminated. required to evaluate the specific mechanisms that biofilm communities Despite the observed trend, non-significant differences were found use for their social behaviors to transfer the in vitro results to an indus­ between 72 h, 72 h + 24 h, and 72 h + 48 h (P = 0.47; P = 0.23, trial level. respectively). However, in the period 72 h + 72 h, L. monocytogenes growth in the presence of P. fluorescens was significantly lower (P < 3.2.2. Bacillus spp. as predominant genera 0.05) compared to the rest of the conditions, albeit presenting an Regarding the interaction with L. monocytogenes, the results showed elevated cell count. One of the possible reasons could be that a clear predominance of B. safensis and B. megaterium in all the condi­ P. fluorescens produces a bioactive substance in a late growth stage that tions studied (Fig. 3-A.1 and A.2). These results coincide with those could affect the pathogen under study. In this regard, Sharma et al. reported by Hascoët et al., (2021), although in their study a preim­ (2020) identified different compounds when combined in co-culture plantation of Bacillus spp. was performed, differing from the present with L. monocytogenes, concluding that bioinoculants could potentially study where the effect was evaluated in biofilms formed at the same time be implemented in agricultural amendments. Nevertheless, with the by direct interaction. Moreover, in the B. safensis case, and differently obtained results, this is an approach that need not be considered. from the results obtained for P. fluorescens, the washing did not influence Moreover, regarding the cell counts obtained from the dual-species L. monocytogenes abundance between the 4 times studied (P = 0.98; P = biofilms of L. monocytogenes and P. fragi, it was observed that in all 0.91, and P = 1.00, respectively, and compared with 72 h as the first conditions L. monocytogenes growth was reduced by P. fragi presence, incubation period). In terms of bacterial counts, Table 2 shows that after although no significant differences were observed in any of the incu­ washing and nutrient renewal, a significant decrease (P < 0.05) in the bation periods (P = 0.41; P = 0.68; P = 0.61; P = 0.64, respectively). L. monocytogenes counts was observed, although at 72 h + 48 h and 72 h The same tendency was observed in the case of S. Typhimurium + 72 h the pathogen counts increased until reaching pre-wash values (P when mixed in dual-species biofilms with both Pseudomonas species (i.e. = 0.71 and P = 0.60, respectively). Nevertheless, L. monocytogenes no significant differences were found between experimental groups). counts were significantly (P < 0.05) lower when confirming dual-species However, a significant reduction in S. Typhimurium cell numbers (P < biofilms with B. safensis and B. megaterium in comparison with 0.05) was obtained after the washing and the nutrients renewal (i.e. 72 L. monocytogenes monospecies biofilms. In this regard, and for the case of h) in all the cases studied (i.e. in monospecies and Pseudomonas spp. B. safensis, a reduction of between 3.5 and 4 log CFU/cm2 in the dual-species biofilms) (Table 2). In this line, in the last incubation period L. monocytogenes counts were obtained. This effect could be explained by (i.e. 72 h + 72 h), the final S. Typhimurium count in the biofilm formed antibacterial substance production by these type of microorganisms, by P. fragi did not present significant differences (P > 0.05) in com­ including gramicidin (Yang and Yousef, 2018). In this regard, Hascoët parison with the monospecies biofilms. Therefore, the S. Typhimurium et al., (2021) generated an extract derived from the B. safensis growth, population level was not influenced by any of the Pseudomonas species demonstrating that the higher the concentration of the extract when used, with practically the same cellular count obtained (P > 0.05). This testing it against L. monocytogenes the greater the delay in the pathogen’s Table 2 Growth evolution of L. monocytogenes and S. Typhimurium in dual-species biofilms with both Bacillus species during the different incubation periods and their comparison in mono-species biofilms. Counts expressed in Log CFU/cm2 (n = 9) ± standard error of the mean (SEM). Pathogen’s cell count in mono-species and dual-species biofilms (log CFU/cm2) Incubation period L. monocytogenes L. monocytogenes + B. L. monocytogenes + B. S. Typhimurium S. Typhimurium + B. S. Typhimurium + B. a (hours) safensis a megaterium a b safensis b megaterium b 72 5.42 ± 0.13Aa 1.69 ± 0.38Ac 3.5 ± 0.27Ab 6.25 ± 0.12Aa 5.57 ± 0.14Ab 5.28 ± 0.19Ab 72 þ 24 4.84 ± 0.27Aa 0.63 ± 0.17Bc 3.49 ± 0.3Aba 5.62 ± 0.23Ba 5.63 ± 0.20Aa 5.10 ± 0.23Aaa 72 þ 48 5.34 ± 0.24Aa 1.5 ± 0.24Ac 3.55 ± 0.33Aba 5.61 ± 0.14Ba 5.12 ± 0.16Aa 5.47 ± 0.39Aaa 72 þ 72 5.36 ± 0.34Aa 1.95 ± 0.52Ac 3.5 ± 0.39Ab 5.50 ± 0.21Ba 4.23 ± 0.28Bb 5.17 ± 0.47Aba A− B Values of a column lacking a common uppercase superscript differ significantly (P < 0.05). a Values of a row by study group (i.e. L. monocytogenes or S. Typhimurium) lacking a common lowercase superscript differ significantly (P < 0.05). a For L. monocytogenes specific count ALOA medium was used. b For S. Typhimurium specific count SS medium was used. 5 C. Ripolles-Avila et al. Food Microbiology 105 (2022) 104026 Incubation period Incubation period L. monocytogenes B. safensis L. monocytogenes B. megaterium Incubation period Incubation period S. Typhimurium B. safensis S. Typhimurium B. megaterium Fig. 3. Comparison of the abundance percentage (%) of L. monocytogenes (A) and S. Typhimurium (B) when forming dual-species biofilms with B. safensis (1) and B. megaterium (2) in the 4 different incubation periods evaluated. growth, highlighting the fact that the microorganism can be producing S. Typhimurium both B. safensis and B. megaterium significantly (P < an antibacterial substance. Moreover, Hascoët et al., (2021) demon­ 0.05) reduced the pathogen counts in the 72 h and 72 h + 72 h incu­ strated that L. monocytogenes counts decreased significantly in the bation periods (Table 2), although under no test conditions was the presence of preformed B. safensis biofilms, while these preformed bio­ observed reduction as high as for L. monocytogenes. This result could be films were not affected and stable over time. This fact was again explained by this species of Bacillus having a faster growth than S. corroborated by Cervantes-Huaman (2019) who demonstrated that Typhimurium, thus producing a conflict over nutrients and hindering L. monocytogenes did not adhere adequately to the surface and produce a the development of the pathogen. Moreover, when Bacillus spp. reaches robust biofilm when B. safensis was present on the surface as a preformed the stationary phase (i.e. a phase that could be happening at 72 h or at biofilm. All these findings in conjunction with the ones reported in the 72 h + 72 h) phenomena such as nutrient competition, the decrease in present study suggest that B. safensis could be an interesting species for essential factors for respiration, or even variation at a more acidic pH L. monocytogenes replacement on industrial surfaces and a potential can occur (Castañeda-Alvarez and Consuelo-Sánchez, 2016). The spatial species for the biocontrol of this pathogen. When L. monocytogenes distribution of the microbial species within the biofilm could also affect counts in monospecies biofilms were compared with the ones obtained the ecological relation and subsequent treatment choice for their elim­ in dual-species biofilms with B. megaterium, a significant decrease in the ination (Rodríguez-Melcón et al., 2021). There could be a social conflict counts of the pathogen of between 1.5 and 2 log CFU/cm2 were ob­ between the bacteria that are located on the periphery and those that are tained. However, the global effect was not as noticeable as with inside the biofilm, since those that are outside consume most of the B. safensis. The obtained results were in concordance with Barbosa et al., resources, notoriously limiting and depriving the nutrients that reach (2005), who reported similar antimicrobial activities by B. megaterium the interior. against L. monocytogenes. Sharma et al., (2020) did not find significant differences between 3.2.3. Candida spp. as predominant genera B. megaterium populations and L. monocytogenes presence or absence, Within the fungal diversity established in the different food in­ implying that the pathogen does not influence B. megaterium growth. In dustries, there is a predominance of the genus Candida spp. (Gounadaki this last study, an extraction was carried out and different bioactive et al., 2008). In the study carried out by Hascoët et al. (2019) different compounds were identified, which could also explain the anti-Listeria species of Candida spp. were isolated, among which C. krusei, C. ciferry, effect and the results obtained in this study. C. zeylanoides were reported, the latter predominating at a percentage of In the case of S. Typhimurium, and in terms of abundance, the pre­ 61%. In the results obtained in the present study, a clear predominance dominance of B. safensis and B. megaterium was way superior to that of C. zeylanoides with respect to L. monocytogenes was observed at all presented by the tested pathogen (Fig. 3-B.1 and B.2), coinciding with incubation times studied (Fig. 4-A). Specifically, when comparing the results obtained for L. monocytogenes. This higher abundance could L. monocytogenes cell counts on the monospecies biofilms and when be explained by the report that Bacillus spp. is a bacterial genera with a conforming dual-species biofilms with C. zeylanoides (Table 3), differ­ strong expansion capacity when conforming biofilms, even able to ences of up to more than 1 log (P < 0.05) were observed, indicating that double in size in just four days (Gingichashvili et al., 2017). As the in­ C. zeylanoides slows L. monocytogenes growth. No studies have been done cubation periods elapsed, the tendency of B. safensis and B. megaterium to understand the relation with C. zeylanoides and L. monocytogenes in and their interaction with S. Typhimurium was the opposite. The pre­ dual-species biofilms. However, notable is the study carried out by disposition of B. safensis was to increase the percentage of its population Agustín and Brugnoni (2018), in which dual-species biofilms with and therefore to decrease the abundance of the pathogen in the L. monocytogenes and Listeria innocua with Candida tropicalis, Candida dual-species biofilms, while in the case of B. megaterium its abundance kefyr, and C. krusei were generated. They concluded that was reduced to be displaced by S. Typhimurium, which increased its L. monocytogenes increases its presence with C. tropicalis and L. innocua in population. Despite this, both species of Bacillus spp. dominated over S. the presence of C. tropicalis and C. krusei. However, L. innocua decreases Typhimurium. its presence with C. kefyr. Between these results and those obtained in In terms of cell counts, when conforming dual-species biofilms with the present study, antagonistic and synergistic interactions between 6 C. Ripolles-Avila et al. Food Microbiology 105 (2022) 104026 Incubation period Incubation period L. monocytogenes C. zeylanoides S. Typhimurium C. zeylanoides Fig. 4. Comparison of the abundance percentage (%) of L. monocytogenes (A) and S. Typhimurium (B) when forming dual-species biofilms with C. zeylanoides in the 4 different incubation periods evaluated. Table 3 Growth evolution of L. monocytogenes and S. Typhimurium in dual-species biofilms with C. zeylanoides during the different incubation periods and their comparison in mono-species biofilms. Counts expressed in Log CFU/cm2 (n = 9) ± standard error of the mean (SEM). Pathogen’s cell count in mono-species and dual-species biofilms (log CFU/cm2) Incubation period (hours) L. monocytogenes a L. monocytogenes + C. zeylanoides a S. Typhimurium b S. Typhimurium + C. zeylanoides b Aa Ab Aa Ab 72 5.42 ± 0.13 4.01 ± 0.33 6.25 ± 0.12 5.64 ± 0.23 72 þ 24 4.84 ± 0.27Aa 2.97 ± 0.35Bb 5.62 ± 0.23Ba 5.37 ± 0.16Aa 72 þ 48 5.34 ± 0.24Aa 3.69 ± 0.34Ab 5.61 ± 0.14Ba 5.76 ± 0.32Aa 72 þ 72 5.36 ± 0.34Aa 2.82 ± 0.56Bb 5.50 ± 0.21Ba 5.53 ± 0.34Aa A− B Values of a column lacking a common uppercase superscript differ significantly (P < 0.05). a Values of a row by study group (i.e. L. monocytogenes or S. Typhimurium) lacking a common lowercase superscript differ significantly (P < 0.05). a For L. monocytogenes specific count ALOA medium was used. b For S. Typhimurium specific count SS medium was used. species of the same genus are revealed. Therefore, more studies are differences (P > 0.05). Viljoen (2001) stated that certain yeasts can needed to understand L. monocytogenes behavior. The results obtained stimulate or inhibit bacterial growth, although this depends on the de­ from the interaction between C. zeylanoides and the pathogen indicate gree of interrelation between them, so yeasts must be taken into that this resident yeast could be a good option to be used as a potential consideration when considering the residential microbiota and the biocontrol species in food industries in conjunction with other inhibitory ecological relation they can establish with certain pathogens. species of the pathogen. The search for a complementary and competi­ tive microbiota for the replacement of pathogens could be an alternative 4. Conclusions strategy for the microbial control of industrial surfaces in the future (Hascoët et al., 2019; Ripolles-Avila et al., 2019c). However, to this end Different ecological relationships were established between the two there is a need to study yeasts in greater depth, and specifically how they pathogens and the dominant microbiota evaluated, demonstrating no interact with other microbial species, since these microorganisms are the influence on their growth or an inhibition of it depending on the most abundant at the level of isolates and so it is essential to know their microorganism. P. fluorescens and P. fragi did not enhance or inhibit the interactions. growth of L. monocytogenes or S. enterica. However, both pathogens Regarding S. Typhimurium abundance, at the beginning of the in­ remained in the mixed biofilm, protected by the two species. B. safensis, cubation period (i.e. at 72 h) there was a higher percentage of B. megaterium, and C. zeylanoides inhibit the growth of L. monocytogenes C. zeylanoides, although the difference became much more evident at the to different degrees when forming dual-species biofilms, achieving a incubation period of 72 h + 24 h, when the population count of final reduction in the number of adhered cells. However, in the case of C. zeylanoides reached more than 90%, while S. Typhimurium abun­ S. enterica, B. safensis was the only microorganism that exerted this out- dance fell below 10% (Fig. 4-B). As the incubation period evolved, the competing effect. The results have a potential technological use in the opposite effect occurred, with the population of S. Typhimurium future to control foodborne pathogens and reduce the use of detergents beginning to increase, while that of C. zeylanoides decreased to the point and disinfectants. However, more studies must be carried out to where, in the last period (i.e. 72 h + 72 h), the S. Typhimurium popu­ corroborate the viability of this approach in terms of application, lation percentage reached that obtained at 72 h. This reduction in determine possible microorganisms with the same or greater inhibitory C. zeylanoides could be explained by the production by S. Typhimurium effect, and specify the best combination of microorganisms to control of determinate substances such as B complex vitamins (Fang et al., certain pathogens considered high risk. 2017). In this regard, it has been indicated that certain B-complex vi­ tamins such as Vitamin B2 (i.e., riboflavin), B3 (i.e. pantothenic acid), Funding sources and B9 (i.e. folic acid) have a partial antifungal effect against Candida albicans (Meir and Osherov, 2018). This study was supported by Research Project grants RTI2018- Moreover, the results in terms of cell counts are presented in Table 3. 098267-R-C32 from the Ministerio de Ciencia, Innovación y As can be observed, the number of S. Typhimurium cells when con­ Universidades. forming dual-species biofilms with C. zeylanoides in the first incubation period (i.e. 72 h) was significantly lower (P < 0.05) than that obtained in Declaration of competing interest the monospecies biofilm, with a difference in the population counts of approximately 0.60 log CFU/cm2. As the incubation periods elapsed, S. The authors declare that they have no known competing financial Typhimurium counts in the dual-species biofilms resembled those ob­ interests or personal relationships that could have appeared to influence tained in the monospecies biofilms, without presenting significant the work reported in this paper. 7 C. Ripolles-Avila et al. Food Microbiology 105 (2022) 104026 Acknowledgements and synergy and modification of matrix components. Front. Microbiol. 9, 1–15. https://doi.org/10.3389/fmicb.2018.01960. Haddad, S., Elliot, M., Savard, T., Deschênes, L., Smith, T., Ells, T., 2021. Variations in The authors would like to thank Dolors Busquets profusely for her biofilms harbouring Listeria monocytogenes in dual and triplex cultures with technical assistance. The authors alone are responsible for the content Pseudomonas fluorescens and Lactobacillus plantarum produced under a model system and writing of the paper. The authors acknowledge Ms. Sarah Davies for of simulated meat processing conditions, and their resistance to benzalkonium chloride. Food Control 123, 107720. https://doi.org/10.1016/j. the English grammar review. foodcont.2020.107720. Hascoët, A.-S., Ripolles-Avila, C., Guerrero-Navarro, A.E., Rodríguez-Jerez, J.J., 2019. 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