LWT - Food Science and Technology 165 (2022) 113681 Contents lists available at ScienceDirect LWT journal homepage: www.elsevier.com/locate/lwt Repeated sub-inhibitory doses of cassia essential oil do not increase the tolerance pattern in Listeria monocytogenes cells M. Bermúdez-Capdevila, B.R.H. Cervantes-Huamán, J.J. Rodríguez-Jerez *, C. Ripolles-Avila Area of Human Nutrition and Food Science, Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Antimicrobial resistance in Listeria monocytogenes biofilms is considered a risk. When using disinfectants, the Listeria monocytogenes minimum inhibitory concentration (MIC) must be contemplated to avoid resistance. The objective of the present Biofilms study was to determine the MIC of four essential oils in four L. monocytogenes strains. Once the MICs were ob­ Resistance tained, the effect of subinhibitory doses of the most effective oil was determined. Strains were subsequently Essential oils Food safety subjected to increasing doses of cassia and the MICs were determined again to evaluate differences. The effect of subinhibitory doses for biofilm formation was evaluated using quantitative and observational methodologies. Last, it was studied whether the strains were more sensitive to antibiotics after being in contact with the oil. After continuous exposure to 1/2 MIC, a decrease in the initial MIC (P = 0.013) was observed, specifically for strains belonging to serotype 1/2a (P = 0.041). In contrast, the formation of biofilms did not show differences between the control and exposed groups (P > 0.05). The qualitative study showed that there were no differences in the structure of the biofilms before and after contact with cassia, except for the CECT 935 strain, indicating a strain- dependent trend. Moreover, species-dependent differences were observed in the conformation of the extracel­ lular matrix. 1. Introduction products be withdrawn from the market but biofilms damage the heat transfer processes and increase the corrosion of surfaces (Winkelstroter, Cross contamination has been identified as one of the main vehicles 2015). Furthermore, the settlement and persistence of pathogens in the of contamination of food by Listeria monocytogenes due to its ability to food industry is closely related to their response to factors such as hu­ form biofilms (Mazaheri et al., 2021), which are structured communities midity, temperature, pH, and the type of adhesion surface, among others of microbial cells adhered to a surface and embedded in a matrix of (Abdallah et al., 2014; Bridier et al., 2015; Simões et al., 2010; Win­ extracellular polymeric substances (EPS), characterized by presenting kelstroter, 2015). an altered phenotype and gene expression (Colagiorgi et al., 2017). The cells that make up a biofilm are more resistant to antimicrobial L. monocytogenes can form biofilms on the surface of different materials compounds than cells in a planktonic state (González-Rivas et al., 2018). used in the food industry, representing a serious concern in terms of food This fact is directly related to the architecture and physiology in the safety because of their possible relation with consequent sources of biofilm environment, which confers greater cell survival due to reduced contamination (Colagiorgi et al., 2017; González-Rivas et al., 2018). diffusion of compounds, anaerobic growth, physiological changes due to The presence of biofilms on industrial surfaces is an important factor reduced growth rates, or production of enzymes that degrade antimi­ in the persistence of pathogens in food processing environments. In crobial substances. (González-Rivas et al., 2018; Simões et al., 2010; addition, biofilms have a significant impact on public health since they Winkelstroter, 2015). Due to the greater cellular resistance in these can reach food as a result of cross contamination (Mazaheri et al., 2021). biofilms, their complete elimination is more complex. The strategies to This contamination can occur in any phase of food processing through combat biofilms are basically divided into two aspects: the inhibition of handlers, equipment, or contact surfaces (Abdallah et al., 2014; Win­ their formation and the application of a treatment to eliminate them kelstroter, 2015). In addition to the impact at the safety level, biofilms once they have formed. In this context, repeated efforts have been made increase the economic losses of these industries because not only can the to prevent and control their formation, with elimination strategies * Corresponding author. E-mail address: josejuan.rodriguez@uab.cat (J.J. Rodríguez-Jerez). https://doi.org/10.1016/j.lwt.2022.113681 Received 5 April 2022; Accepted 18 June 2022 Available online 22 June 2022 0023-6438/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). M. Bermúdez-Capdevila et al. LWT 165 (2022) 113681 becoming particularly prominent over time. This is why in the food in­ Table 1 dustry and after cleaning a wide variety of chemical disinfectants are Essential oils used and their corresponding active compounds (Bagheri et al., used, such as acid compounds, biocides based on aldehydes, caustic 2020). products such as chlorine, hydrogen peroxide, iodine, isothiazolinones, ESSENTIAL SCIENTIFIC NAME ACTIVE COMPOUND ozone, peracetic acid, phenolics, biguanides, halogen surfactants, and OIL quaternary ammonium compounds. However, factors such as interfer­ CASSIA Cinnamomum cassia Trans-cinnamaldehyde, cinnamyl acetate ence from inorganic substances, water hardness, chemical inhibitors, Blume concentration and contact time, among others, generally affect the CLOVE Eugenia spp. Eugenol, eugnyl acetate, caryophyllene OREGANO Thymus capitatus Carvacrol, ƿ-cimeno effectiveness of disinfectants and may make them insufficiently effective CINNAMON Cinnamomum Eugenol, cinnamaldehyde, cinnamyl to completely eliminate biofilms (Simões et al., 2010; Winkelstroter, zeylanicum acetate, β-phelandrene 2015). Likewise, the persistence of L. monocytogenes is related to the resis­ tance of strains to disinfectants, with studies showing that adaptation to 2.2. Bacterial strains disinfectants can occur when microorganisms are exposed to sublethal or sub-inhibitory concentrations (Carpentier & Cerf, 2011; In this study, four strains of Listeria monocytogenes of different origins González-Rivas et al., 2018). Therefore, it is important to consider the were used, two of them from the Spanish Collection of Type Cultures Minimum Inhibitory Concentration (MIC) in the use of disinfectants for (CECT, University of Valencia, Valencia, Spain) and belonging to sero­ the elimination of biofilms because their improper use can induce type 4b: CECT 935 and CECT 5672; and the other two, considered wild, resistance. In recent times it has also been suggested that there is a direct were isolated from surfaces of Iberian pig processing plants, with sero­ relationship between the phenomena of tolerance and adaptation of type 1/2a: S2-bac and EDG-e (Ripolles-Avila, Cervantes-Huaman, biocides and resistance to antibiotics because the mechanisms that favor Hascoët, Yuste, & Rodríguez-Jerez, 2019). The strains started in a the two phenomena are similar. In this regard, it has been shown that lyophilized state, so were rehydrated in Soy Tryptone Broth (TSB; Oxoid, exposure to sublethal concentrations of antimicrobials facilitates the Madrid, Spain) by incubation at 30 ◦ C for 40–48 h. After this initial selection of bacterial strains resistant to antibiotics, mainly through recovery period, the strains were streaked onto plates with Tryptone Soy mechanisms of cross-resistance or co-resistance (Capita & Agar (TSA; Oxoid, Madrid, Spain) and incubated at 37 ◦ C for 16–24 h. Alonso-Calleja, 2013; Molina-González et al., 2014). Resistance to an­ Colonies were isolated from the culture and re-seeded on TSA plates, and tibiotics thus becomes a complex problem that transcends international again incubated at 37 ◦ C for 16–24 h. Last, the re-seeded TSA plates were borders, particularly if the global rates of their constant use continue to stored as stock cultures in refrigeration (4 ◦ C) for a maximum period of increase. By 2050, antibiotic resistance is forecasted to cause around 10 one month. million deaths worldwide annually, costing the global economy 100 billion dollars (McCullough et al., 2016). Therefore, effective actions are 2.3. Determination of minimum inhibitory concentration (MIC) before urgently required by all sectors, including governments, researchers, exposure doctors, different types of industries and the public, to minimize anti­ biotic resistance by reducing the use of antibiotics or compounds that Bacterial inoculum was obtained from stock cultures kept in refrig­ favor the emergence of resistance (McCullough et al., 2016). In this eration. For each strain, a colony was recovered from the seeded TSA context, essential oils are gaining interest in the food industry for their plates and inoculated into a TSB tube, which was incubated at 37 ◦ C for antimicrobial properties (Dumas et al., 2021). Their use against different 24 h. After the incubation time, the inoculum was prepared at a con­ pathogenic microorganisms is therefore being investigated (Mutlu-Ingok centration of 105 CFU/mL in TSB. et al., 2020; Tariq et al., 2019). These substances’ ability to induce The MIC of the four essential oils was determined starting from a resistance must also be evaluated in the context of the strategy against concentration prepared in a previous screening carried out by the same pathogens such as L. monocytogenes. research group (data not shown). For this, 96-well microtiter plates This explains why the need to develop solutions and tools for the (Sudelab SL, Barcelona, Spain) were used, into which 20 μL of the control of biofilms, such as new disinfectants that do not induce the required dilution of essential oil were first inoculated in 3% Tween 80 generation of resistance to the biocide itself or cross-resistance to anti­ (Sigma-Aldrich, Madrid, Spain) together with 180 μL of the bacterial biotics, is becoming more urgent. To this end, the objectives of the inoculum (105 CFU/mL). At the same time, negative and positive con­ present study were: (1) To determine the MIC of four essential oils trols were included for each test performed. Once the wells were inoc­ (cassia, clove, oregano and cinnamon) in four different strains of ulated, in the case of cassia, the plates were incubated at 37 ◦ C for 48 h. L. monocytogenes (CECT 935, CECT 5672, S2-bac, EDG-e); (2) To For the essential oils clove, oregano, and cinnamon, and due to the determine the effect of exposure to sub-inhibitory doses on the MIC of impossibility of reading the wells due to the opacity derived from the the most effective oil, cassia oil, subjecting the 4 mentioned strains to color of the oil itself, an incubation was carried out at 37 ◦ C for 24 h, increasing doses of the essential oil; (3) To evaluate the effect of the followed by a dilution with 180 μL of TSB in new plates. These new application of these subinhibitory doses on the formation of biofilms of plates were incubated at 37 ◦ C for 24 h. After the incubation time, the L. monocytogenes; (4) To determine the effect of increasing subinhibitory microtiter plates were read and the concentration in the wells from concentrations applied on the pattern of susceptibility to various which there was no longer any growth determined as the MIC. antibiotics. 2.4. Exposure to increasing sub-inhibitory concentrations of cassia 2. Material and methods essential oil 2.1. Essential oils Once the MIC of the four essential oils was set, the most effective one was chosen (i.e. the one that presented a lower MIC for the four strains in A total of four essential oils were used: cassia, clove, oregano, and common), this being cassia. It was subsequently decided to use a MIC/2 cinnamon (Merck, Darmstadt, Germany) (Table 1). All the oils, except of this oil as a subinhibitory base dose (i.e. 206 ppm; see section 3.1.) to the oregano, were kept at room temperature in a cool, dry place pro­ adapt the L. monocytogenes strains to increasing concentrations of the oil tected from sunlight. The oregano oil was preserved at refrigeration (Molina-González et al., 2014). For this, 20 μL of the MIC/2 dilution of temperature (4 ◦ C) as per the product’s technical data sheet. the essential oil with 3% Tween 80 and 180 μL of the previously pre­ pared bacterial inoculum at 105 CFU/mL were inoculated into 96-well 2 M. Bermúdez-Capdevila et al. LWT 165 (2022) 113681 microtiter plates. Positive and negative controls were included in each From here on, serial dilutions were made in diluent (0.1% tryptone, experimental test. After inoculation, the plates were incubated at 37 ◦ C 0.85% sodium chloride) and were quantified by transferring 1 mL to the for 48 h. Once the incubation time had elapsed, 100 μL from these plates TEMPO vials, which contained the lyophilized nutrient medium previ­ were transferred to new microtiter plates into which 80 μL of TSB were ously rehydrated with 3 mL of sterile distilled water. The vials were also added as a nutrient to promote bacterial growth, as well as 20 μL of vortexed for homogenization before their contents were transferred to a the MIC/2 concentration of the cassia essential oil. The plates were then card using the TEMPO filler station. The barcodes on each card were incubated again at 37 ◦ C for 48 h, and this last step was repeated on new recorded and encoded using the TEMPO prep station. Last, the cards plates, making a total of two transfers at increasing doses of essential oil. were incubated at 30 ◦ C for 48 h and the reading was carried out using Last, 100 μL from random wells were transferred to 9.9 mL TSB tubes the TEMPO reader station. The necessary mathematical adjustments and incubated at 37 ◦ C for 24 h. These tubes with the exposed strains were made to express the results in log (CFU/cm2). were the ones that were subsequently used as starting inoculum in the evaluations determined in section 2.5. and 2.6. 2.6.3. Qualitative assessment of the biofilms structure and matrix Biofilm formation was also qualitatively evaluated to observe two 2.5. Biofilm formation different parameters, the first of which was the structure. For this pur­ pose, the discs were washed in duplicate with 3 mL of sterile distilled To carry out the tests on the biofilm formation ability, the strains water to remove any cells that had not adhered to the surface and which, were used once they had been subjected to the increasing concentrations therefore, were not part of the biofilm generated. The discs were then of cassia essential oil. In this case, 100 μL were removed from different placed on slides and 5 μL of the vital Live/Dead BacLight stain (Molec­ wells at random where there was growth after adaptation to the essential ular Probes, Oregon, USA) added before covering with coverslips. Due to oil and transferred to TSB tubes, which were incubated at 37 ◦ C for the sensitivity of the reagent, the procedure was carried out keeping the 16–24 h. After this time, serial dilutions were made in TSYEBgluc1%+ discs protected from sunlight. They were then incubated at room tem­ NaCl2% (TSB supplemented with 0.3% yeast extract (BD, Madrid, Spain), perature for 15 min (Ripolles-Avila et al., 2018). 1% glucose (BioLife, Barcelona, Spain) and 2% chloride sodium (Pan­ Second, the matrix produced was evaluated. For this, the FilmTracer reac, Castellar del Vallès, Spain)), an enriched culture medium specific SYPRO Ruby Biofilm Matrix Stain (Thermo Fisher Scientific, Barcelona, for the growth of L. monocytogenes (Pan et al., 2010; Ripolles-Avila, Spain) was used. Like the procedure for the previous evaluation, the 2018), until an inoculum concentration of 106 CFU/mL was obtained. discs were washed with 3 mL of sterile distilled water in duplicate and These inoculums were used for the formation of biofilms. For the for­ then placed on slides. They were stained with 200 μL of the stain and mation of biofilms from the controls (i.e. strains not exposed to the incubated for 30 min at room temperature, protected from sunlight. compound), the same procedure was followed with the only difference After this time, the reagent was removed and a coverslip placed on them. being the inoculation of between 3 and 5 colonies of each of the stock This type of staining stains biofilm matrices, marking most protein cultures of L. monocytogenes in tubes of TSYEBgluc1%+NaCl2% classes including glycoproteins, phosphoproteins, lipoproteins, calcium- Afterwards, the biofilms were formed following the model estab­ binding proteins, and fibrillar proteins (Ripolles-Avila et al., 2018). lished by Ripolles-Avila et al. (2018). For this, AISI 316 grade 2B The stained surfaces were evaluated by DEM, using an Olympus stainless steel surfaces 2 cm in diameter and 1 mm thick were used, prior BX51/BX52 direct epifluorescence microscope (Olympus, Tokyo, Japan) to which they were cleaned, disinfected and sterilized in accordance equipped with a 100 W mercury lamp (USH-103OL, Olympus) and a with the European standard UNE-EN 13697:2015, related to non-porous double-pass filter (U- M51004 F/R–V2, Olympus), and attached to a materials (AENOR, 2015). To form the biofilms, 30 μL of the suspension digital camera (DP73, Olympus). The discs were observed at 20X to described above were inoculated into the center of the stainless-steel examine the structure of the biofilm formed, to be able to evaluate the discs, resulting in a surface concentration of approximately 4 log vital status of the bacterial cells and the production of matrix. During the (CFU/cm2). Subsequently, the discs were placed in sterile Petri plates observational study, three photographs were taken at random of each and these, in turn, were placed in a humid chamber with saturated disk from three different fields to later be able to carry out a qualitative relative humidity for incubation at 30 ◦ C for 72 h to improve the growth evaluation in comparison with the controls of the strains that had not of adhered cells and promote biofilm formation. been exposed. 2.6. Evaluation of the cassia oil subinhibitory concentrations effect 2.6.4. Antibiotic susceptibility To determine the susceptibility to antibiotics of the strains after 2.6.1. Determination of MICs after adaptation having been in contact with cassia, antibiograms were performed using Once the experimental part described in section 2.4. was completed, 15 different antibiotic discs (Oxoid, Madrid, Spain) (Table 2) on the inoculum made from the strains exposed to the increasing doses of Mueller-Hinton agar (Oxoid, Madrid, Spain) using the disk diffusion cassia oil was used to again determine the MIC of the compound, and method described by Clinical and Laboratory Standards Institute (CLSI). thus be able to evaluate whether this subjection of the strains at Based on a bibliographic review, the 15 antibiotics of greatest interest to increasing sub-inhibitory concentrations of the oil had induced changes the study were selected (Baquero et al., 2020; Chow et al., 2021; Noll in the level of resistance/susceptibility. For this, the same procedure et al., 2018; Olaimat et al., 2018; Park et al., 2021; Wai et al., 2015). established in section 2.3. was carried out, with the difference that this Zones of inhibition were measured and rated as sensitive (S), interme­ time it was based on the inoculum of the exposed bacterial strains. diate susceptibility (I), and resistant (R) according to the CLSI (2018) and EUCAST (2021) guidelines. A total of 720 tests were carried out 2.6.2. Biofilms quantitative analysis considering all the combinations of strains and antibiotics, plus the The quantitative analysis was carried out using the TEMPO system. comparative controls. First, the discs where the biofilms were formed were washed in duplicate with 3 mL of sterile distilled water to discard the non-adhered cells. The 2.7. Statistical analysis discs were then placed in a sterile bottle with 3.5 g of sterile glass beads, 10 mL of neutralizer [0.1% tryptone (BD, Madrid, Spain), 0.85% sodium In this study, all trials were performed in duplicate in three separate chloride (Panreac, Castellar del Vallés, Spain), and 3% of Tween 80]. experiments (n = 6). Regarding the quantitative evaluation of the bio­ Once the samples were introduced into the flasks, they were vortexed for films, the bacterial counts obtained were converted into decimal loga­ 90 s at 40 Hz to detach the adhered cells from the surface and thus be rithmic values to coincide with the assumption of a normal distribution. able to quantify them (Ripolles-Avila, Cervantes-Huaman et al., 2019). The IBM SPSS Statistics version 28 program was used for the statistical 3 M. Bermúdez-Capdevila et al. LWT 165 (2022) 113681 Table 2 Likewise, Chahbi et al., (2020), Kim et al. (2021), and Mutlu-Ingok et al. Antibiotic disks used for susceptibility screening, with their respective concen­ (2020) point out that oils that contain cinnamaldehyde among their trations and pharmacological families (Obando Pacheco et al., 2020). composition, such as oregano in whose composition carvacrol stands ANTIBIOTICS CONCENTRATION FAMILY out, are more effective compared to other essential oils. However, in the (МG) present study, the results obtained for cinnamon oil showed a lower AMPICILLIN (AMP) 10 Beta-lactams antibacterial activity. In this case, it is important to highlight that the GENTAMICIN (CN) 10 Aminoglycosides composition of the oil is a fundamental factor that directly influences its TRIMETHOPRIM- 25 Sulfonamides inhibitory capacity (de Oliveira et al., 2012). Cinnamaldehyde is one of SULFAMETHOXAZOLE (SXT) the main components in the essential oils of cassia and cinnamon TETRACYCLINE (TE) 30 Tetracyclines CHLORAMPHENICOL (C) 30 Amphenicols (Bagheri et al., 2020). In this regard, cinnamon oil obtained a signifi­ CIPROFLOXACIN (CIP) 5 Quinolones cantly higher MIC value (P < 0.001) (i.e. 4562.5 ppm) than cassia oil (i.e. CEFOXITIN (FOX) 30 Cephalosporins 412.5 ppm), which could be related to a different concentration of its CEFOTAXIME (CTX) 30 Cephalosporins active components or a different steric conformation of the molecules OXACILLIN (OX) 1 Beta-lactams ERYTHROMYCIN (E) 15 Macrolides because cassia oil contains cinnamaldehyde in trans-form. Regarding VANCOMYCIN (VA) 30 Glycopeptides this last point, Hill et al. (2013) indicated that the steric conformation of RIFAMPIN (RD) 5 Rifamycins the molecules of a compound can influence the solubility of the oil, ENROFLOXACIN (ENR) 5 Quinolones which would improve the distribution of the compound in the solution CEFEPIME (FEP) 30 Cephalosporins obtained with Tween 80. In the study mentioned, MIC values around NITROFURANTOIN (F) 300 Nitrofurans 500 ppm were obtained for Listeria innocua, consistent with those ob­ tained in the present study for L. monocytogenes. In parallel, the main analysis of the data. The “One Way ANOVA” test was used for the active compound in cinnamon oil is usually eugenol (Bagheri et al., comparison of the mean of the four oils, with a posteriori contrast using 2020). This compound has been shown to have a lower MIC than the “Tukey” test to observe whether there were statistically significant carvacrol, the majority compound in oregano oil (Walsh et al., 2019). differences between the MIC concentrations obtained for each oil. In However, it was questioned whether the degree of sensitivity could addition, the data obtained from the study of exposure to increasing sub- vary between strains. In this case, for the cassia, clove and cinnamon inhibitory concentrations of essential oil and the quantitative analysis of oils, no significant differences were observed between the strains (P > biofilms was analyzed using a general univariate linear model with a 0.05). However, oregano oil did present statistically significant differ­ posteriori contrast using the "Tukey" test. In all cases, a significance level ences between the CECT 5672 strain and the CECT 935 (P = 0.044), S2- of P < 0.05 was set. bac (P = 0.007), and EDG-e (P = 0.044) strains, showing a lower sus­ ceptibility of the CECT 5672 strain by requiring more concentration of 3. Results and discussion the compound to obtain an effect. This strain in particular is known as Scott A and tends to have greater resistance to different types of treat­ 3.1. MIC determination in 4 essential oils ments (Bucur et al., 2018). It has been recognized that the ability of L. monocytogenes to survive stress conditions may be strain-dependent, Table 3 shows the MIC values of each essential oil studied for the the variations being more related to the genetic lineage than to the different strains of L. monocytogenes. Cassia was the oil with the highest origin of the strain (Dumas et al., 2021). Kawacka et al. (2021) also show effectiveness as it obtained a lower MIC value (i.e. 412.5 ppm) and differences in MICs of distinct strains when in contact with essential oils, presented significant differences (P < 0.05) from the rest of the oils. In unlike the results obtained in the present study, apart from those for turn, it was shown that all the oils presented significant differences oregano oil. Last, in the study carried out by Dumas et al. (2021) and in between them (P < 0.05) in relation to their inhibitory capacity. These the present study, no significant differences were observed between results agree with those reported by Oussalah et al. (2007) and Mith L. monocytogenes strains when faced with the essential oil of cassia. As et al. (2014) in relation to the maximum effectiveness found for the there were no differences, the average MIC value between strains was essential oil cassia. Cinnamon-based oils, such as cassia and cinnamon used as a reference value for the continuation of the study. itself, have been shown to have a very effective antimicrobial spectrum against L. monocytogenes (Bagheri et al., 2020; Barbosa et al., 2021). Table 3 MIC values of the evaluated essential oils obtained for four strains of Listeria monocytogenes (n = 6) together with the corresponding standard error of the mean (SEM). Essential MIC (ppm) Average MIC oils (ppm) CECT CECT 935 S2-bac EDG-e Species level 5672 Cassia 400 ± 00 350 ± 50 450 ± 50 450 ± 412.5 ± Aa Aa Aa 50 Aa 22.66 A Clove 4000 ± 3000 ± 3000 ± 3500 ± 3375 ± 500 Ba 00 Ba 500 Ba 00 Ba 205.94 B Oregano 3250 ± 2500 ± 2000 ± 2500 ± 2562.5 ± 250Ca 00 Cb 00 Cb 00 Cb 175.19 C Cinnamon 5250 ± 4500 ± 4000 ± 4500 ± 4562.5 ± 250 Da 500 Da 00 Da 00 Da 199.05 D Fig. 1. MIC determination for L. monocyotgenes strains before (Control ) and a-b Values in a row that lack a common lowercase superscript differ significantly after (Exposed ) exposure to increasing concentrations of cassia. Each value (P < 0.05). corresponds to the mean of two replications carried out on three different days A− D Values in a column lacking a common uppercase superscript differ signifi­ (n = 6). The error bars represent the standard error of the mean. a-b indicate the cantly (P < 0.05). significant differences (P < 0.05) between the two groups. 4 M. Bermúdez-Capdevila et al. LWT 165 (2022) 113681 3.2. MIC comparison after exposure to subinhibitory concentrations of ensure optimal conditions for growth and to favor the formation of cassia biofilms of the pathogen (Kadam et al., 2013). Statistically, the counts obtained in the biofilms of the exposed group did not present significant Fig. 1 shows the MIC values obtained before and after exposure to differences from those of the control group (P = 0.222), and there were increasing concentrations of cassia oil, previously described as the most also no significant differences between strains (P = 0.240). The count of effective. Significant differences were observed at the species level be­ the biofilms obtained in this study agrees with the findings of other in­ tween the values obtained in the control and the values obtained after vestigations in which quantitative methods were used to evaluate the exposure (P = 0.013). More specifically, the strains that showed sig­ biofilm formation (Koo et al., 2014; Ripolles-Avila et al., 2018). nificant differences between the MIC required in the control and the MIC Since there were no significant differences between the biofilm manifested after adaptation were the S2-bac and EDG-e strains counts before and after exposure to increasing doses of cassia, but sig­ belonging to serotype 1/2a (P = 0.041), as opposed to the strains of nificant differences were obtained between the MICs of the control and serotype 4b (P = 0.605 and P = 0.195, respectively for CECT 5672 and species-exposed groups, this suggests that L. monocytogenes can form CECT 935). In this case, after adaptation the serotype 1/2a strains biofilms as a protection against the stress agent. Thus, although the showed a lower MIC than the serotype 4b strains. To reinforce this planktonic cells were damaged by the effect of the oil, the moment they argument, significant differences were observed between serotypes (P < came into contact with the study surface they began to structure 0.05). The results obtained differ from those reported for other active themselves as biofilm, forming microcolonies and producing extracel­ compounds such as eugenol and carvacrol, for which previous exposure lular substances that act as a protective polymeric matrix (Fuster-Valls of L. monocytogenes to sublethal doses conditioned an increase in its et al., 2008). resistance (Souza et al., 2015). Furthermore, it is known that there is variability regarding the Thus, a trend is observed where the difference in MIC between the ability to form biofilms between strains that belong to the same species two serotypes is evident. To this effect, after adaptation, serotype 1/2a (Borges et al., 2018; Ripolles-Avila, 2018), but in this study biofilm shows a proportionally greater decrease in MIC. In addition, a trend was resistance did not show a strain dependent trend, but rather a species also observed for the CECT 935 strain, which showed a lower MIC dependent one. Likewise, the results obtained do not allow us to compared to the other strains in both the control group and the exposed conclude whether there is a relationship between strain and biofilm group. According to Álvarez-Ordóñez et al. (2008), exposure to anti­ formation capacity. There were also no significant differences between microbials in sublethal concentrations can result in the development of the counts obtained by the serotype 1/2a strains and the serotype 4b greater tolerance to the same factors, an adaptation called homologous. strains (P = 0.381). Therefore, although a differentiation between the This tolerance was not demonstrated in the results of the present study, two serotypes has been previously observed, the same is not shown in but rather a sensitization of the population or a lack of influence. It could the formation of biofilms. However, several researchers have associated be that the decrease in MIC occurs for two reasons: (1) That the sensi­ serotypes with various attributes of L. monocytogenes, including the tized strains are inhibited and have difficulty growing at the same initial formation of biofilms (Orsi et al., 2011; Wang et al., 2017; Zoz et al., concentrations of MIC; (2) or that this second MIC has had a lethal effect 2017). However, the relationship between L. monocytogenes serotype on them and therefore they do not show growth. In the second case, MIC and biofilm formation remains unresolved (Kadam et al., 2013; Ripol­ would be considered what is known as Minimum Bactericidal Concen­ les-Avila et al., 2019) tration (MBC), which is defined as a concentration that eliminates 99.9% or more of the initial inoculums (Tariq et al., 2019). Nonetheless, further 3.4. Subinhibitory doses impact on the structure and matrix of biofilms studies are needed to expand the number of strains evaluated to be able produced by L. monocytogenes to confirm the trend described here, since in most experimental studies no observations have been made between strains of the same species. Fig. 3 shows the biofilms generated by the four L. monocytogenes strains under study before and after exposure to subinhibitory doses of 3.3. Effect of cassia subinhibitory doses on biofilm formation cassia essential oil. In both study groups (i.e. control and exposed), the shape of the cells in terms of the distribution of the colonies on the Biofilm counts of L. monocytogenes obtained from subjection to sub­ surface can be observed, in some cases establishing themselves in geo­ inhibitory doses of cassia can be observed in Fig. 2. The ability of the metric shapes, and in other cases being more dispersed. In this regard, it strains of L. monocytogenes to form biofilms on stainless steel was eval­ has been indicated that the formation of biofilms can be determined uated during a 72-h incubation period. This process was established to based on the organization of the cells that comprise it, through obser­ vation by the DEM method (Ripolles-Avila et al., 2018). One charac­ teristic that shows that a biofilm has reached its state of maturity is the presence of water channels (Abdallah et al., 2014). These formations promote the constant circulation of nutrients and the elimination of waste within the biofilm (Ripolles-Avila et al., 2018). Taking this into account, it can be confirmed that the CECT 5672, S2-bac, and EDG-e strains used in the study formed biofilms in a 72-h incubation period in the same way before and after being subjected to increasing doses of cassia oil, although with a different organization level. Following this line, the images obtained are similar to those presented in the studies by Marsh et al. (2003) and Ripolles-Avila et al. (2018). In contrast, a disaggregated cell arrangement indicates that the biofilm has not formed (Chmielewski & Frank, 2003). Following these indications, in the images it can be seen that the control strain CECT 935 Fig. 2. Count of cells that make up the biofilms of L. monocytogenes strains (Fig. 3-B1) does not follow the same organization, but rather shows a before (Control ) and after (Exposed ) exposure to increasing subinhibitory greater dispersion. This behavior was also observed in the study by doses of cassia essential oil. Each value corresponds to the mean of two repli­ Marsh et al. (2003), at 72 h of incubation by a L. monocytogenes strain cations carried out on three different days (n = 6). The error bars represent the that also corresponded to serotype 4b. Thus, at the count level this strain standard error of the mean. a indicates the absence of significant differences (P does not present differences, but differences are observed at an obser­ < 0.05) between the two groups. vational structural level. In this aspect, the CECT 935 strain shows an 5 M. Bermúdez-Capdevila et al. LWT 165 (2022) 113681 Fig. 3. Images obtained by DEM of L. monocytogenes biofilms: (A) CECT 5672, (B) CECT 935, (C) S2-bac and (D) EDG-e, before (1) and after (2) exposure to subinhibitory does of cassia essential oil, stained with Live/Dead Baclight. 20× magnification. adaptation after being in contact with cassia oil. staining most of the protein classes (Ripolles-Avila et al., 2018). Biofilms, as mentioned above, are described as microbial aggrega­ Furthermore, this parameter also indicates the maturity of the biofilm tions that grow on surfaces and excrete different substances such as and contributes greatly to the final architecture of the community polysaccharides, proteins, and eDNA among others, all embedded in the (Branda et al., 2005). The results obtained show that after subjection to extracellular matrix (ECM) (Colagiorgi et al., 2017). Thus, the ECM increasing doses of cassia all the strains generated a greater production generated to form the biofilms was studied using DEM. Fig. 4 shows the of protein substances that gave the matrix consistency. Based on these difference in ECM production of the four study strains, before and after results, it can be deduced that a considerably greater amount of extra­ exposure to cassia oil subinhibitory doses (i.e. control and exposed, cellular protein is generated. On the other hand, the images obtained respectively). This qualitative study, which has been used in various from the control group agree and present similarities with those pre­ investigations (Dapa et al., 2013; Frank & Patel, 2007; Sanchez et al., sented in the study by Ripolles-Avila et al. (2018). 2013), was carried out to be able to observe the biofilm matrices by 6 M. Bermúdez-Capdevila et al. LWT 165 (2022) 113681 Fig. 4. Images obtained by DEM of L. monocytogenes biofilms: (A) CECT 5672, (B) CECT 935, (C) S2-bac, and (D) EDG-e, before (1) and after (2) exposure to subinhibitory doses of cassia essential oil, stained with FilmTracer SYPRO Ruby Biofilm Matrix Stain. 20× magnification. 3.5. Susceptibility profiles derived from continuous exposure to antibiotics As seen in Table 2, the FEP and CTX antibiotics belong to the ceph­ alosporin family, and two of their three induced changes in the sus­ Table 4 shows the susceptibility of the studied strains to 15 different ceptibility pattern are related to an increase in resistance. In contrast, antibiotics, before and after exposure to increasing doses of cassia oil. CIP and ENR, which belong to the quinolone family, induced an increase For most antibiotics, no changes were observed in the susceptibility/ in sensitivity. These differences in the susceptibility pattern can there­ resistance pattern after this exposure. To this effect, there were only five fore be explained by the different mechanism of action of the different changes: (1) from R to I for FEP in strain CECT 5672; (2) from R to S for families (Reygaert, 2018). On the one hand, cephalosporins are bacte­ CIP in strain S2-bac; (3) changes from I to R for CTX in the EDG-e strain; ricidal antibiotics that act by inhibiting the synthesis of the bacterial cell (4) from I to S for ENR; and (5) from S to I for FEP. Of these five changes, wall. They inhibit transpeptidation in the final stages of peptidoglycan three show an increase in sensitivity and, conversely, the other two show synthesis, an essential polymer for the bacterial wall (Georgopapadakou an increase in resistance to antibiotics. & Bertasso, 1993). 7 M. Bermúdez-Capdevila et al. LWT 165 (2022) 113681 Table 4 References Susceptibility of L. monocytogenes against 15 antibiotics, before (CNT) and after (EXP) contact with cassia essential oil. 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