Architectural Features and Resistance to Food-Grade Disinfectants in Listeria monocytogenes-Pseudomonas spp. Dual-Species Biofilms

fmicb-13-917964 June 2, 2022 Time: 18:24 # 1 ORIGINAL RESEARCH published: 09 June 2022 doi: 10.3389/fmicb.2022.917964 Edited by: Avelino Alvarez-Ordóñez, Universidad de León, Spain Reviewed by: Efstathios D. Giaouris, University of the Aegean, Greece Tae Jin Cho, Korea University, South Korea *Correspondence: Pedro Rodríguez-López [email protected] Specialty section: This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology Received: 11 April 2022 Accepted: 04 May 2022 Published: 09 June 2022 Citation: Rodríguez-López P, Rodríguez-Herrera JJ and López Cabo M (2022) Architectural Features and Resistance to Food-Grade Disinfectants in Listeria monocytogenes-Pseudomonas spp. Dual-Species Biofilms. Front. Microbiol. 13:917964. doi: 10.3389/fmicb.2022.917964 Architectural Features and Resistance to Food-Grade Disinfectants in Listeria monocytogenes-Pseudomonas spp. Dual-Species Biofilms Pedro Rodríguez-López*, Juan José Rodríguez-Herrera and Marta López Cabo Laboratory of Microbiology and Technology of Marine Products (MICROTEC), Instituto de Investigacións Mariñas (IIM-CSIC), Vigo, Spain Listeria monocytogenes is considered a foodborne pathogen of serious concern capable of forming multispecies biofilms with other bacterial species, such as Pseudomonas spp., adhered onto stainless steel (SS) surfaces. In an attempt to link the biofilms’ morphology and resistance to biocides, dual-species biofilms of L. monocytogenes, in co-culture with either Pseudomonas aeruginosa,Pseudomonas fluorescens, or Pseudomonas putida, were assayed to ascertain their morphological characteristics and resistance toward benzalkonium chloride (BAC) and neutral electrolyzed water (NEW). Epifluorescence microscopy analysis revealed that each dual-species biofilm was distributed differently over the SS surface and that these differences were attributable to the presence of Pseudomonas spp. Confocal laser scanning microscopy (CLSM) assays demonstrated that despite these differences in distribution, all biofilms had similar maximum thicknesses. Along with this, colocalization analyses showed a strong trend of L. monocytogenes to share location within the biofilm with all Pseudomonas assayed whilst the latter distributed throughout the surface independently of the presence of L. monocytogenes, a fact that was especially evident in those biofilms in which cell clusters were present. Finally, a modified Gompertz equation was used to fit biofilms’ BAC and NEW dose-response data. Outcomes demonstrated that L. monocytogenes was less susceptible to BAC when co-cultured with P. aeruginosa or P. fluorescens, whereas susceptibility to NEW was reduced in all three dual-species biofilms, which can be attributable to both the mechanism of action of the biocide and the architectural features of each biofilm. Therefore, the results herein provided can be used to optimize already existing and develop novel target-specific sanitation treatments based on the mechanism of action of the biocide and the biofilms’ species composition and structure. Keywords: antimicrobial resistance, benzalkonium chloride, biofilm, CLSM, disinfectants, electrolyzed water, Listeria monocytogenes,Pseudomonas spp. Frontiers in Microbiology | www.frontiersin.org 1June 2022 | Volume 13 | Article 917964 fmicb-13-917964 June 2, 2022 Time: 18:24 # 2 Rodríguez-López et al. Listeria monocytogenes Binary Biofilms’ Resistance INTRODUCTION Listeria monocytogenes is currently considered an issue of concern for public health (Quereda et al., 2021). This Grampositive bacterial pathogen is the causative agent of human listeriosis, an uncommon disease causing high morbidity and mortality with symptoms that can vary from mild gastroenteritis and fever, to septicemia (Vázquez-Boland et al., 2001a,b). According to the latest One Health Report of the European Food Safety Authority (EFSA), the notification rate of listeriosis in the European Union was 0.42 per 100,000 population, representing a decrease of 7.1% in comparison with previous data (EFSA and CDC, 2021). Despite its low incidence, the mortality was 14.16% among confirmed cases and 20.48% among those patients that required hospitalization, especially among the so-called YOPIs (young, old, pregnant, and immunocompromised) group (EFSA and CDC, 2021). The common route for the transmission of the pathogen into the population is via foodstuffs, especially meat/meatbased (Fabrizio and Cutter, 2005;Kramarenko et al., 2013) and fish/fish-based ready-to-eat (RTE) products (Ghorban Shiroodi et al., 2016;EFSA, 2018), and contamination occurs mainly at a processing level (EFSA and CDC, 2021). Environmentally, this pathogen can persist and survive by adhering to abiotic surfaces, such as stainless steel (SS), associated with other bacterial species forming multispecies biofilms (Elias and Banin, 2012;Willems et al., 2016;Møretrø and Langsrud, 2017). These are highly organized sessile structures of bacteria embedded in a selfproduced polymeric extracellular matrix, which confers bacteria therein a higher resistance to environmental aggression such as desiccation, radiation, and antimicrobials, compared with its planktonic (i.e., free) counterparts (Li et al., 2021). Among L. monocytogenes-accompanying species in biofilms, several authors have studied dual-species biofilms in co-culture with Pseudomonas spp. in terms of association capacity and resistance to biocides. Early studies in the field demonstrated that in L. monocytogenes Scott A–Pseudomonas sp. biofilms, the latter was able to predominate the structure, but that both were able to endure peracids, and the efficacy of such chemicals is negatively influenced by the presence of organic matter (Fatemi and Frank, 1999). Similarly, Saá Ibusquiza et al. (2012a) demonstrated that in 96-h-old biofilms grown on SS of L. monocytogenes CECT 4032 and CECT 5873 in co-culture with Pseudomonas putida, the resistance toward benzalkonium chloride (BAC) significantly increased for both species. Contrarily, Giaouris et al. (2013) reported that in L. monocytogenes–P. putida mixedspecies biofilms, only the latter presented an increased resistance to continuous exposure to BAC, even though no apparent adaptation was present in either of the strains. Abbreviations: AVC, adhered viable cells; AvT, average thickness; BAC, benzalkonium chloride; B52, Pseudomonas fluorescens B52; CECT110, Pseudomonas aeruginosa CECT 110; CECT324, Pseudomonas putida CECT 324; L34, Listeria monocytogenes L34; MxT, maximum thickness; M1, Manders’ co-occurrence coefficient 1; M2, Manders’ co-occurrence coefficient 2; NEW, neutral electrolyzed water; PCC, Pearson’s correlation coefficient; Ra, roughness coefficient; ROI, region of interest; SS, stainless steel; TAC, total available chlorine. More recently reported data demonstrate that L. monocytogenes ATCC 19114 and ATCC 19115 are able to associate and grow at 10◦C in polystyrene microplates with Pseudomonas aeruginosa and that this association allows both L. monocytogenes species to endure and survive against higher concentrations of ciprofloxacin and sodium hypochlorite (Yamakawa et al., 2018). In a similar way, Haddad et al. (2021) determined that the minimum biofilm eradication concentration (MBEC) toward BAC for L. monocytogenes increased from 6.2 ±1.4 to 58.3 ±7.5 µg ml−1when cocultured with Pseudomonas fluorescens, but that this MBEC did not significantly vary in co-culture with Lactobacillus plantarum, indicating that the first accompanying species provides L. monocytogenes some sort of protective effect that was not present with the latter. Considering the complexity of biofilms’ structures, microscopy approaches have been used to study their architectural features. For example, epifluorescence microscopy coped with image analysis has been used for numerical spatio– temporal characterization of monospecies L. monocytogenes biofilms grown on SS (Mosquera-Fernández et al., 2014; Rodríguez-López et al., 2015). Other authors have used this approach to evaluate the cleaning and disinfection potential of pronase–BAC combined strategies against L. monocytogenes–Escherichia coli dual-species biofilms (Rodríguez-López et al., 2017b) and the development of tolerances to those treatments after continuous sublethal exposures (Rodríguez-López and Cabo, 2017). In addition to the epifluorescence techniques, for indepth biofilm studies requiring tridimensional characterization, confocal laser scanning microscopy (CLSM) approaches are preferred (Relucenti et al., 2021). As an example, Bridier et al. (2010) elegantly described the biofilm architecture of different strains of E. coli,Enterococcus faecalis,L. monocytogenes, P. aeruginosa,Staphylococcus aureus, and Salmonella enterica. In a similar way, Mosquera-Fernández et al. (2016) described the structural biofilm dynamics of three different L. monocytogenes strains analyzing the resulting CLSM micrographs with three different software tools. In addition, CLSM has been used to determine the biovolume of singleand dual-species biofilms of different food-borne pathogens, as well as their three-dimensional distribution with some species, such as L. monocytogenes, with a clear preference toward the bottom layers (Puga et al., 2014, 2018). The latter has been recently hypothesized to be one of the potential factors that could explain the higher resistance to biocides in L. monocytogenescarrying biofilms. Despite the abovementioned, the relationship between the structure of L. monocytogenes mixed biofilms and the resistance toward sanitizers still remains controversial. In this line, several authors have considered the biofilm complexity and its antimicrobial resistance as two related phenomena (Pan et al., 2006;Saá Ibusquiza et al., 2012a). On the contrary, Kocot et al. (2021) demonstrated that in L. monocytogenes–E. coli dualspecies biofilms cultured in a batch (i.e., polystyrene plates) or fed-batch (i.e. CDC biofilm reactor) system, the resistance toward BAC was not determined by the structural characteristics of the Frontiers in Microbiology | www.frontiersin.org 2June 2022 | Volume 13 | Article 917964 fmicb-13-917964 June 2, 2022 Time: 18:24 # 3 Rodríguez-López et al. Listeria monocytogenes Binary Biofilms’ Resistance biofilm itself but by the operational culture conditions in which the samples were grown. Therefore, the generation of such knowledge would contribute to (i) giving valuable information to unravel the mechanisms of action of disinfectants on biological systems involving Grampositive – Gram-negative mixed-species populations and the factors that influence the bactericidal potential of a given treatment and (ii) providing empirical data that could be used to optimize the amount of biocides deployed in a given sanitation treatment. The main aim of the present work was to attempt to ascertain the link between the architectural features and the resistance toward two food-grade biocides in L. monocytogenes– Pseudomonas spp. dual-species biofilms grown on SS. Firstly, the cross-influence of L. monocytogenes and Pseudomonas spp. in mixed communities in terms of adhesion and final biofilm morphology was assessed via classical agar plating and epifluorescence microscopy, respectively. Next, a numerical characterization of three different L. monocytogenesPseudomonas spp. biofilms was carried out using CLSM coupled with image analysis. Finally, the susceptibility of the biofilms toward BAC and neutral electrolyzed water (NEW) was assessed by modeling the obtained dose-response experimental data. MATERIALS AND METHODS Bacterial Strains All strains used in this study are listed in Table 1. The strains of L. monocytogenes used in this study were isolated from three different origins: A1, E1, and G1, which were from food industryrelated surfaces; L1, L7, and L34, which were isolated from fish products; and X1, X7, and X10, which were isolated from patients with human listeriosis. Regarding Pseudomonas spp., three different species were used as representatives of the same origins as for L. monocytogenes. Therefore, P. putida CECT 324, P. fluorescens B52, and P. aeruginosa CECT 110 were chosen as representatives for environmental (Wu et al., 2011;Meireles et al., 2013), food (Allison et al., 1998;Kumar et al., 2019), and clinical (Driscoll et al., 2007;Thi et al., 2020) sources, respectively. Bacterial stock cultures were kept at −80◦C in sterile brainheart infusion broth (BHI; Biolife, Milan, Italy) containing 50% (v v−1) sterile glycerol. Similarly, working cultures were maintained at −20◦C in sterile TSB (Cultimed, Barcelona, Spain) containing 50% (v v−1) sterile glycerol. Inocula Standardization Before the cultivation of the biofilms, reactivation cultures were prepared by transferring 100 µl of each of the working cultures to a tube containing 5 ml of sterile TSB, incubated overnight at 37◦C for L. monocytogenes and P. aeruginosa and 30◦C for P. fluorescens and P. putida, and subcultured twice for the proper revivification of the cells. Preinocula were prepared by adjusting the Abs700 to 0.100 ±0.001 in sterile phosphate-buffered saline (PBS) using a 3000 series scanning spectrophotometer (Cecil instruments, Cambridge, United Kingdom). This absorbance corresponds to a cellular density of approximately 108CFU ml−1according to previous calibrations. Biofilms Cultivation on Stainless Steel Both L. monocytogenes monospecies and L. monocytogenesPseudomonas spp. dual-species biofilms were cultivated on 10 mm ×10 mm ×1 mm AISI 316 SS coupons (Comevisa, Vigo, Spain) following a protocol described by Rodríguez-López et al. (2017a), with minor modifications. Preparation of the SS surfaces included washing with industrial soap to remove grease residues (Sutter Wash, Sutter Ibérica S.A., Madrid, Spain), thorough washing with tap water, final rinse with deionized water, and sterilization at 120◦C for 20 min. Next, coupons were individually placed in a 24-well polystyrene flat-bottomed plate (Falcon, Corning, NY, United States). In all cases, inocula were prepared by diluting the preinocula 1:10,000 in sterile TSB to obtain a final density of approximately 104CFU ml−1. For monospecies biofilms, 1 ml of the final suspension was used to inoculate each well containing a SS coupon. In the case of dual species biofilms, the corresponding inocula were 1:1 (v v−1) mixed, and 1 ml of the resulting mixture was used to inoculate the SS coupons. Next, plates containing coupons were placed at 25◦C in static conditions for 2 h to allow initial adhesion and then in constant shaking at 100 rpm until needed. Previous to any analysis performed, samples (SS coupons) were aseptically collected and immersed in 1 ml sterile PBS for 10 s to remove loosely attached cells. Epifluorescence Microscopy Assays Influence of Pseudomonas fluorescens B52 in Dual-Species Biofilms For this part of the study, all strains of L. monocytogenes (Table 1) were co-cultured with B52 strain and collected after 48 h, as described above. Then, the coupons were stained with FilmTracerTM LIVE/DEADR Biofilm Viability Kit (Life Technologies, Eugene, OR, United States) following manufacturer instructions to observe the final biofilm morphology and the distribution of live (green-emitting) cells and damaged/dead (red-emitting) cells. Biofilms were visualized under a Leica DM6000 epifluorescence microscope (Leica, Wetzlar, Germany) using a 40×dry objective and 10×ocular lenses. Representative images of each sample were acquired with a Leica DFC365 FX camera and the Metamorph MMAF software (Molecular Devices, Sunnyvale, CA, United States). Morphology of Listeria monocytogenes L34 Biofilms in Co-culture With Pseudomonas spp. In this second part of the microscopy assays, the variability of the resulting morphology of 48 h L34-Pseudomonas spp. dualspecies biofilms were assessed. Therefore, three different dualspecies biofilms, i.e., L34-CECT110, L34-CECT324, and L34B52, were cultured and collected as described above. Samples were then stained by means of ViaGramTM Red+ Bacterial Gram Stain and Viability Kit (Life Technologies) following Frontiers in Microbiology | www.frontiersin.org 3June 2022 | Volume 13 | Article 917964 fmicb-13-917964 June 2, 2022 Time: 18:24 # 4 Rodríguez-López et al. Listeria monocytogenes Binary Biofilms’ Resistance TABLE 1 | List of bacterial strains and codes used in this study. Bacterial species Strain code Representative source References Listeria monocytogenes A1 Environmental Rodríguez-López et al., 2015 E1 Environmental Rodríguez-López et al., 2015 G1 Environmental Leite et al., 2006 L1 Food Rodríguez-López et al., 2019a L7 Food Rodríguez-López et al., 2019a L34 Food Rodríguez-López et al., 2019a X1 Clinical Rodríguez-López et al., 2019a X7 Clinical Rodríguez-López et al., 2019a X10 Clinical Rodríguez-López et al., 2019a Pseudomonas fluorescens B52 Food Allison et al., 1998 Pseudomonas putida CECT 324 Environmental Reference strain Pseudomonas aeruginosa CECT 110 Clinical Reference strain the manufacturer’s instructions. This stain allows in a mixed population the distinction of Gram-positive cells (red-emitting) with a counterstain with DAPI. Finally, samples were visualized under the epifluorescence microscope using a 63×water immersion objective, and the images were acquired as described in the previous section. Confocal Laser Scanning Microscopy Assay To better determine the features of L34-Pseudomonas spp. biofilms, a CLSM approach was followed. For this, the same dual-species biofilms assayed in section “Morphology of Listeria monocytogenes L34 Biofilms in Co-culture With Pseudomonas spp.” were cultured and stained as described above. The biofilms’ CLSM images stacks were acquired in the Centre of Biological Engineering of the Universidade do Minho (Braga, Portugal) using the FluoroView application Software package (Olympus) and a FluoView FV1000 microscope (Olympus) equipped with a 60×oil immersion objective. The resulting stacks were analyzed using COMSTAT 2.1 (Heydorn et al., 2000;Vorregaard, 2008) to obtain biomass (BM), maximum thickness (MxT), average thickness (AvT), and roughness coefficient (Ra), and with Confocal Uniovi ImageJ v1.51 software1using the JaCoP plugin to obtain the Pearson’s correlation index (PCC) and Manders’ co-occurrence coefficients, M1and M2, to obtain information regarding the distribution of both bacterial species within the biofilm (Aaron et al., 2018). Effect of Disinfectants on L34-Pseudomonas spp. Biofilms The effects of two food-grade disinfectants, i.e. BAC (Guinama, Alboraya, Spain) and in-house made NEW, were tested on 48 h L34, L34 – CECT 110, L34 – CECT 324, and L34 – B52 samples. In all cases, quantification of the remaining adhered viable cells (AVC) after the antimicrobial treatments was assessed by recovering them with two sterile cotton swabs pre-moistened in buffered peptone water (BPW; Cultimed, Barcelona, Spain). 1https://www10.uniovi.es/confocaluniovi/index.htm Swabs were placed in a tube containing 2 ml of sterile BPW and vigorously vortexed for 1 min to detach cells from the swab. The resulting suspension was serially diluted in sterile BPW and plated on tryptic soy agar (TSA; Cultimed, Barcelona, Spain). Results were expressed as the reduction in log CFU cm−2 compared to negative controls. Benzalkonium Chloride Benzalkonium chloride solutions were prepared in sterile deionized water at concentrations 50, 100, 150, and 200 µg ml−1 and kept at 4◦C for no longer than 14 days. Therefore, 1 ml of each solution was applied at room temperature on 48-h samples (n= 3) for a contact time of 10 min. For negative controls, 1 ml of sterile deionized water was used instead. After BAC treatment, samples were neutralized by immersing them for 30 s in 1 ml of LPT neutralizing broth (composition per liter: 10 ml of a 34 g L−1 KH2PO4buffer (pH 7.2); soybean lecithin: 3 g; Tween 80: 30 ml; Na2S2O3: 5 g; L-histidine: 1 g) and transferred to a fresh well. AVC quantification was performed as described above. Neutral Electrolyzed Water Neutral electrolyzed water was generated at room temperature using an Envirolyte EL-400 Unit model R-40 (Envirolyte Industries International Ltd., Estonia) according to the manufacturer’s instructions. Briefly, saturated NaCl solution and tap water were simultaneously pumped into the apparatus at an intensity of 20–25 A. Therefore, NEW was produced by appropriately mixing the anolyte solution [pH 2.0–3.0, oxidation–reduction potential (ORP) ≈1,200 mV] with the catholyte solution (pH 11.0–12.0, ORP ≈ −900 mV) after electrolysis. ORP and pH of the resulting solutions were determined using a portable pH and REDOX 26 multimeter (Crison Instruments S.A., Barcelona, Spain). Total available chlorine (TAC) was determined by iodometric titration (APHA., 2012). The resulting NEW had the following properties: TAC = 880 µg ml−1, pH 6.3, ORP = 940 mV. Next, TAC was adjusted with sterile deionized water to obtain working solutions of 100, 200, 400, 600, and 750 µg ml−1and kept protected from light at 4◦C for a maximum of 14 days. Frontiers in Microbiology | www.frontiersin.org 4June 2022 | Volume 13 | Article 917964 fmicb-13-917964 June 2, 2022 Time: 18:24 # 5 Rodríguez-López et al. Listeria monocytogenes Binary Biofilms’ Resistance For dose-response assays, 1 ml of each solution was applied at room temperature to 48-h samples (n= 3) for a contact time of 10 min, followed by neutralization and AVC quantification as described above. As in BAC assay for negative controls, 1 ml of sterile deionized water was used. Data Fitting and Determination of Lethal Dose 50 Lethal dose 50 (LD50) can be defined as the dose of an antimicrobial required to achieve a killing of half of the initial bacterial population and was used as a parameter to determine the effect of disinfectants on dual-species biofilms. To assess this, fitting of the experimental values into the dose-response model was performed utilizing a modified Gompertz equation proposed by Murado et al. (2002) using the least-squares method (quasiNewton) of the SOLVER function of Microsoft Excel 2016, as follows: R=Ke−e(b−cD)−e−eb(1) where R, biofilm reduction expressed in of log CFU cm−2; D, dose of disinfectant used; K, maximum logarithmic reduction (asymptote); b, parameter to be determined empirically (dimensionless); and c, specific inhibition coefficient (dimensions: inverse of the dose). Since Equation 1 modifies the resulting dose-response parameters by subtracting the intercept of the original equation, outcomes were further adjusted to obtain the new Kvalue (K0), as follows: K0=lim D→∞ R=K1−e−eb(2) Finally, the LD50 value was calculated using Equation 3: LD50 =1 cb−ln ln 2 1+e−cb (3) Statistical Analysis Two different tests were performed to assess significance among the obtained results at a confidence level of 95% (α= 0.05) or greater. Specifically, to statistically compare the number of AVC of L34 in monoculture or co-culture with Pseudomonas spp., a paired two-tailed Student’s t-test was performed using Microsoft Excel 2016 statistical analysis tool. Following this, to assess the significance of the CLSM parameters obtained, a one-way ANOVA with Bonferroni post hoc test was carried out using OriginPro 2021 v9.8.0.2000 (OriginLab corporation, Northampton, MA, United States). Finally, in LD50 assays, the correlation coefficient (r) was obtained to determine the discrepancy between the logarithmic reductions’ experimental values and those expected with respect to the model. RESULTS AND DISCUSSION Pseudomonas fluorescens B52 Influences the Final Biofilm Architecture Formed With Listeria monocytogenes Strains From Different Origin De visu analysis of the epifluorescence microscopy images of monospecies L. monocytogenes 48 h biofilms grown on AISI 316 SS revealed a variety of growth on the coupon (Figure 1). Of note, all the L. monocytogenes strains used in the present study were chosen since a previous work demonstrated no statistically significant differences in the level of adhesion in terms of CFU cm−2(Rodríguez-López et al., 2019a). Consequently, differences in the biofilm morphology, such as occupied area, would be attributed to factors other than the amount of viable-and-cultivable cells in the different biofilms. In environmental strains (i.e., A1, E1, and G1), cells in the biofilm were distributed on the coupon forming a dense network predominated by live (green-emitting) cells according to the LIVE/DEADR staining, even though some red-emitting cells were present in the samples, implying that even though at early maturation stages, a certain number of cells can appear either damaged or dead (Figure 1). This form of sessile growth forming a sort of honeycomb-like structure has been previously described as the predominant way in which L. monocytogenes adheres to surfaces (Pilchová et al., 2014). In a similar way, the biofilms formed by strains isolated from RTE fish products (i.e., L1, L8, and L34), were mainly characterized by a morphology in which the cells also formed a honeycomb-like structure but where the voids were visibly larger compared to the environmental strains (Figure 1). These voids were surrounded by dense groups of cells where live and damaged/dead cells were intermingled with a clear predominance of live cells in L8 and L34 strains, but not in L1 in which red-emitting cells were mostly present. Taking a closer look at the images of the biofilms formed by L8 and L34 strains, it can be observed that, even though similar, there is a trend in L34 to form clusters in which an accumulation of red-emitting cells is present (Figure 1). This observation is concomitant with the findings obtained in a study performed by Guilbaud et al. (2015) characterizing the biofilm morphological diversity of 96 L. monocytogenes strains. The authors demonstrated that not only L. monocytogenes mostly grows forming honeycomb-like shapes but clusters formed by a mixture of dead cells and matrix components are also common. Lastly, biofilms formed by strains isolated from patients with human listeriosis (i.e., X1, X8, and X10) were characterized by groups of undamaged cells and, to a lesser extent, damaged/dead cells that were distributed throughout the surface that were not fully interconnected between them forming quasi-honeycomb structures. This fact was especially remarkable in X1 strain, where cell clustering was almost absent (Figure 1). This way of growth in which L. monocytogenes appears as stochastically sparse cells on a SS surface has been previously observed in other Frontiers in Microbiology | www.frontiersin.org 5June 2022 | Volume 13 | Article 917964 fmicb-13-917964 June 2, 2022 Time: 18:24 # 6 Rodríguez-López et al. Listeria monocytogenes Binary Biofilms’ Resistance FIGURE 1 | Influence of the accompanying strain. Representative 40×-field fluorescence micrographs after LIVE/DEAD staining of different L. monocytogenes 48-h-old biofilm cultured either in monospecies or dual-species with P fluorescens B52 on AISI 316 SS coupons. Scale bar, 50 µm. strains, such as L. monocytogenes CECT 4032 and CECT 5873 (Mosquera-Fernández et al., 2014). The abovementioned structures were completely altered when all L. monocytogenes strains were co-cultured with Pseudomonas fluorescens B52 as displayed in Figure 1. The incorporation of the second strain deeply changed the resulting morphology of the biofilm turning it into a structure with an evident presence of big cell clusters surrounded by a network of mixed greenand red-emitting cells (Figure 1). These results are in line with those obtained previously with L. monocytogenes– P. fluorescens dual-species biofilms (Puga et al., 2014, 2016; Rodríguez-López et al., 2017a). It is well documented that P. fluorescens biofilms are characterized by the formation of microcolonies (Dynes et al., 2009;Wang et al., 2018). Thus, based on these results, it seems logical to think that P. fluorescens dominates the final structure in the dual-species biofilms, and L. monocytogenes would appear as a “passive” component of it. Of note, in mixed biofilms, there were a number of clusters present in the structure in which the local accumulation of damaged/dead cells was evident (Figure 1), being in line with previous observations (Rodríguez-López et al., 2017b). In this regard, Bayles (2007) described the role of dead/aged cells in biofilms, not only by acting as an anchoring strategy but also enhancing the stabilization of the final structure. Additionally, Jakubovics et al. (2013) demonstrated that dead and lysed cells can provide the biofilms with structural components, such as extracellular DNA (eDNA). This plays an essential role in L. monocytogenes biofilm formation as previously demonstrated (Harmsen et al., 2010a), pointing out that dead cells are not only a product of biofilm maturation/aging but also an Frontiers in Microbiology | www.frontiersin.org 6June 2022 | Volume 13 | Article 917964 fmicb-13-917964 June 2, 2022 Time: 18:24 # 7 Rodríguez-López et al. Listeria monocytogenes Binary Biofilms’ Resistance integral part of the normal structure in L. monocytogenes dualspecies communities. Influence of Pseudomonas spp. in the Final Morphology of Listeria monocytogenes L34 Dual-Species Biofilms: Adhesion and Two-Dimensional Analysis Firstly, the composition in terms of AVC of L34 biofilms alone and in combination with Pseudomonas spp. was compared (Figure 2). Despite being subtle, significant (p<0.05) amount of AVC in L34 was observed when co-cultured with CECT110 when compared with the monoculture. In the rest of the samples, the level of adhesion of L34 did not present significant differences (Figure 2). Epifluorescence images of the resulting biofilms were therefore analyzed to visualize the influence of a given Pseudomonas strain and compared it with the previous experiment (the reader is kindly referred to section “Pseudomonas fluorescens B52 Influences the Final Biofilm Architecture Formed With Listeria monocytogenes Strains From Different Origin” and Figure 1 for further details). As can be seen in Figure 3, the resulting morphologies of the dual-species biofilm were remarkably different depending on the accompanying Pseudomonas sp. Co-culture with B52 gave rise to a structure with big cellular aggregates surrounded by sparsely distributed cells attached to the surfaces. Of note, these clusters presented a high red signal, meaning a local accumulation of L34 mixed with the accompanying species. This resulting morphology not only corroborated the results previously obtained with LIVE/DEAD staining (Figure 1) but also is in line with those previously obtained by Puga et al. (2014), observing that in L. monocytogenes–P. fluorescens biofilms, there is a tendency of the first to locally accumulate forming microcolonies with a high cellular density, whereas the rest of the biofilm is mainly occupied by the latter with cells randomly distributed all over the surface. Similarly, a certain cellular clustering was present in L34CECT324 samples, yet with some appreciable differences, as portrayed in Figure 3. In this particular case, cellular clumping was also evident although each cluster occupied a smaller surface compared to those in L34–B52 biofilms (Figure 3). This manner of growth has been previously observed by Hassan et al. (2004) in L. monocytogenes–P. putida dual-species biofilms on SS surfaces, concluding that in the absence of the latter, no cellular aggregation was present over the surface. Similarly, Saá Ibusquiza et al. (2012a) also observed this clusterization in dualspecies biofilms of L. monocytogenes and P. putida grown on SS in comparison to polypropylene where, in the latter, much denser and compacted structures were formed. Finally, the biofilm formed by L34-CECT110 was visually the one presenting the biggest differences compared to the two previous ones. This was characterized by a monolayer of cells in which both species were uniformly intermingled all over the surface (Figure 3). It has been previously reported that P. aeruginosa biofilms’ growth is mainly characterized by the formation of a dense and thick monolayer (Harmsen et al., 2010b;Rasamiravaka et al., 2015). Therefore, the fact that this structure remained unaltered despite the presence of L34 strain also strengthens the hypothesis that, in binary biofilms, L. monocytogenes does not influence the resulting structure, but adapts to the adhesion pattern of the accompanying species, as previously reported (Rodríguez-López et al., 2017a). Following this first approach on the different structures of L. monocytogenes–Pseudomonas spp. biofilms, a CLSM analysis was performed to analyze their three-dimensional structures. Confocal Microscopy Analysis of Listeria monocytogenes L34 – Pseudomonas spp. Dual-Species Biofilms In this second part of the study, the three-dimensional structural features of the three different L. monocytogenes–Pseudomonas spp. dual-species biofilms were assessed by CLSM coupled with image analysis. As depicted in Figure 4, the CLSM zenital (xy plane) micrographs show how the different architectures of the biofilms obtained in the previous assay were repeated (see section “Influence of Pseudomonas spp. in the Final Morphology of Listeria monocytogenes L34 Dual-Species Biofilms: Adhesion and Two-Dimensional Analysis” and Figure 3), confirming once more the influence of Pseudomonas spp. in the final architecture of the biofilm itself and showing the abovementioned patterns of distribution. Additionally, orthogonal xz and yz planes clearly showed that L34, with respect to Pseudomonas spp., was located in the lower layers of the structure in all cases. Nevertheless, subtle differences in the way of the red and blue signals that were distributed along the images on the z-axis could still be appreciated. That is, whereas in co-culture with CECT110 and CECT324, the red signal corresponding to L34 strain did not exactly coincide with the blue signal corresponding to Pseudomonas spp., and appeared to be randomly intermingled, and in L34-B52 biofilms the red signal was almost coincident with the blue signal and located below it. This phenomenon of cellular distribution, known as blanketing, initially described in P. aeruginosa–Agrobacterium tumefaciens dual-species biofilms (An et al., 2006), has been previously observed in a study conducted by Puga et al. (2014) with L. monocytogenes Scott A co-cultured with P. fluorescens ATCC 948. The authors demonstrated that, regardless of the temperature and the age of biofilms, L. monocytogenes appears located in the bottom layers of the biofilms. This way of growth is not only limited to Pseudomonas spp., but also in mixed culture with other Gram-negative species such as E. coli and/or Salmonella spp. (Almeida et al., 2010). Early studies dealing with L. monocytogenes mixed-species biofilms, attributed this fact to the already described slow growth of the pathogen with respect to other Gram-negative species, making Listeria become somehow covered or even masked by classical detection methods (such as selective agar plating) by the accompanying strain (Almeida et al., 2010;Langsrud et al., 2016). Nevertheless, a recent study demonstrated how planktonic cells of L. monocytogenes are able to invade and locate Frontiers in Microbiology | www.frontiersin.org 7June 2022 | Volume 13 | Article 917964 fmicb-13-917964 June 2, 2022 Time: 18:24 # 8 Rodríguez-López et al. Listeria monocytogenes Binary Biofilms’ Resistance FIGURE 2 | Plate count values of adhered viable cells (AVC) corresponding to 48 h control biofilms grown on AISI 316 SS coupons. Bars represent mean values (n= 15) expressed in CFU cm-2of L. monocytogenes L34 mono-species biofilms (void) and in dual-species biofilms with Pseudomonas spp. (gray). Error bars represent the samples’ standard error. Asterisk indicates statistical significance (two-tailed Student’s t-test, α= 0.05). FIGURE 3 | Representative epifluorescence 63×-field overlay images of the three dual-species biofilms assayed in this study, stained with ViaGramTM Red + Bacterial Gram Stain (ThermoFisher). Red signal: Listeria monocytogenes L34. Blue signal: Pseudomonas spp. Scale bar, 50 µm. themselves in the bottom layers of an pre-formed P. fluorescens biofilm, taking advantage of the voids between the cellular aggregates left by the latter, and that this colonization was more successful at low temperatures (Puga et al., 2018). In addition to this, Maggio et al. (2021) showed that in dual biofilm with P. fluorescens,L. monocytogenes is not only able to better Frontiers in Microbiology | www.frontiersin.org 8June 2022 | Volume 13 | Article 917964 fmicb-13-917964 June 2, 2022 Time: 18:24 # 9 Rodríguez-López et al. Listeria monocytogenes Binary Biofilms’ Resistance FIGURE 4 | Orthogonal projections of CLSM of the three dual-species biofilms assayed in this study. The images were obtained using a 60×oil immersion objective. Red signal: Listeria monocytogenes L34. Blue signal: Pseudomonas spp. Scale bar, 20 µm. adhere to but over-produce extracellular matrix components compared with monospecies biofilms. Taken together, these data suggest that L. monocytogenes distribution within multispecies communities is not a random phenomenon, but appears to be actively regulated, also taking advantage of its facultative anaerobe and psychrotrophic characteristics, allowing the pathogen to associate with a wide diversity of microorganisms in a very structured manner (Rodríguez-López et al., 2015, 2019b). Biofilms were further analyzed using COMSTAT 2.1. (Heydorn et al., 2000;Vorregaard, 2008) to get quantitative data on the mixed structures. The parameters obtained were BM (volume per area unit of field, representing an estimation of the amount of biofilm adhered to the coupon), AvT (mean of the heights in the biofilm), MxT (maximum value of thickness over the analyzed stacks), and Ra (variations of thickness throughout all the analyzed surface, giving information about the heterogeneity of the structure). These parameters have been described and used previously as good descriptors and can be easily interpreted in biological terms (Rodrigues and Elimelech, 2010). Biomass values exhibited by the three dual-species biofilms did not present significant differences among them (Table 2). This indicates that, regardless of the Pseudomonas spp., the amount of biofilm attached to the surface of the SS coupon is similar. 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