Comparison of bio ﬁ lm formation between non-pathogenic Listeria strains under different stress conditions

Micro-organisms can attach to food surfaces and develop biofilms which present a concern in food and envi-ronmentalsafety.Themaingoalofthecurrentstudywastoinvestigatethebiofilmformationofsixnon-pathogenic Listeria strains under different stress conditions using a microplate assay. The effect of the weak bio ﬁ lm-forming non-pathogenic Listeria strains on the bio ﬁ lm formation of a strong bio ﬁ lm-forming pathogenic Listeria strain ( Listeria monocytogenes #8) was also examined. Listeria innocua CCM4030, Listeria innocua 2885 and Listeria seeligeri/welshimeri 292 showed the same patterns of bio ﬁ lm formation with increasing NaCl concentrations from 0.05 to 15%, butall theother strainsshowed acontinuously decreasing trend of OD 595 inthe same conditions. This study showed that in the case of non-pathogenic Listeria strains, higher concentrations of NaCl do not present a stress condition that enhances bio ﬁ lm formation. Decrease in pH inhibited bio ﬁ lm formation for all the non-pathogenic Listeria strains. The weak bio ﬁ lm forming non-pathogenic Listeria strains ( Listeria innocua 2885 and Listeriainnocua CCM4030)overgrewthestrongbio ﬁ lm-forming Listeria strain( Listeriamonocytogenes #8)during bio


INTRODUCTION
Food-borne diseases present a major problem throughout the world causing thousands of deaths each year from the consumption of food and water contaminated with toxins and pathogens. Contamination of food can occur at any point in the food chain, from the raw material to the consumers' table. Pathogens can enter and contaminate the food system through humans, animals, air, water, soil, and contaminated equipment. The increased level of knowledge on how, where, and when the contamination of food happens and how preventive measures should be applied to present a necessity for the food safety of the products we consume in our everyday life (Zaukuu et al., 2016).
Listeria species are widespread in the environment including in soil, raw foods, stream water, silage, sewage, plants, and animals. However, Listeria monocytogenes is a ubiquitous pathogen that can cause infections in humans, thus representing a major concern for public health and economic aspect. Listeria species commonly colonize the food processing environment and ready-to-eat products. Ready-to-eat foods (RTE) are products consumed without any heattreatment and are usually associated with listeriosis outbreaks (Mart ın et al., 2014;R€ uckerl et al., 2014). Novel nonthermal decontamination technologies, like high hydrostatic pressure treatment, have been recognized to have a positive effect on Listeria monocytogenes inactivation and preserve the quality attributes of RTE foods (Bajovic et al., 2012;Dalmadi et al., 2007).
However, elimination of this bacterium from ready-to-eat foods and food processing equipment is difficult. The main reason is because of the ability of this bacterium to form biofilms that protect it from stresses in food processing environments. The difficulty of biofilm removal is also due to the increased resistance against disinfectants caused by factors such as the age of biofilm and different stress responses (Di Ciccio et al., 2012;Van Houdt and Michiels, 2010). Environmental factors, including temperature, sugar, salt, pH, and nutrients, have been demonstrated to have impacts on the adhesion and biofilm formation of Listeria species in food (Renner and Weibel, 2011). To understand the susceptibility of Listeria species biofilms to different stress conditions, six biofilm-forming non-pathogenic Listeria strains were analyzed. The effect of the weak biofilm forming non-pathogenic Listeria strains on the biofilm formation of the strong biofilm-forming Listeria monocytogenes #8 was also examined. There are knowledge-gaps in literature for this particular topic, therefore the importance of this study lies in the possibility to develop new methods to control biofilm formation in the food industry and increase the knowledge on the role of the harmless bacteria in the food safety.

Culture preparation
A total of 7 previously identified Listeria strains were isolated from various sources and included in this study (Table 1). All the isolates were obtained in a previous study at the Department of Microbiology and Biotechnology, Faculty of Food Science, Szent Istv an University, Budapest, Hungary. The different bacteria strains were stored at À80 8C in a mixture of tryptic soy broth (TSB) and glycerol. They were later recovered on Brain Heart Infusion (BHI) Agar, cultivated at 37 8C for 24-48 h and then streaked onto Trypto-Casein Soy Agar (TSA) before cultivating again at 37 8C for 24 h.

Microtiter plate biofilm formation assay
Biofilm formation was evaluated based on the measurement of the optical density (OD) of biofilms which is in correlation with the biofilm mass developed in microtiter plate wells, after crystal violet staining (Bakke et al., 2001;Djordjevic et al., 2002). The main aim of this investigation was to test the capability of Listeria strains to form biofilms in different stress conditions using the microtiter plate assay (Mouwakeh et al., 2019). For this purpose, Minimal Media M9 was used to assess the biofilm formation of Listeria strains under different sodium chloride (NaCl) concentrations (0.05, 5, 10, and 15%) and pH values of 4, 5, and 6 respectively. M9 Minimal media contained water, inorganic salts, and glucose as the sole carbon source. Constant pH of 4, 5 and 6 were obtained by adding hydrochloric acid (HCl 37%, Sigma Aldrich, USA) or sodium hydroxide (NaOH, Sigma Aldrich, USA) to the M9 Minimal Media. The first step of the microtiter plate assay was the preparation of an overnight culture of Listeria strains on Muller-Hinton Agar media. The next step was the adjustment of the OD 600 of Listeria strains to 0.3 in tubes with Minimal Media M9 using a McFarland densitometer. After that, 200 mL of the adjusted strain in M9 was added to each well in the same row of the 96 wells microtiter plates.
Similarly, in case of mixed-biofilm experiment, 100 mL of the adjusted Listeria pathogenic strain and 100 mL of the adjusted Listeria non-pathogenic strain were added to the wells of the microtiter plates. They were then incubated at 37 8C for 72 h.
The Crystal Violet method was applied for the detection of the total biofilms formed (Mouwakeh et al., 2019). For this purpose, the supernatant of the 96-wells plate was firstly removed and then each well was washed three times with 200 mL of phosphate-buffered saline (PBS) before discarding.
The formed biomass was stained by adding the Crystal Violet 0.4% [v/v] to each well and kept at

Statistical analysis
The significances of the differences between the groups were verified by one-way ANOVA, and the mean comparison were performed by Tukey's test using SPSS-20 software (SPSS Inc., IBM Company, USA). The results were considered significant when P ≤ 0.05.

RESULTS AND DISCUSSION
From literature, Listeria strains prefer different temperatures and media when forming biofilms (Pan et al., 2010). However, there is a deficit of studies in the literature showing the biofilm formation of non-pathogenic Listeria strains. According to our results, Listeria ivanovii 204 formed larger quantities of biofilms than the other non-pathogenic Listeria strains when incubated in M9 Minimal Media at 0.05% NaCl concentration (without adjusting NaCl concentration) at temperature 37 8C (Fig. 1). Nevertheless, it can be concluded that most of the studied non-pathogenic Listeria strains formed lower amounts of biofilms at the mentioned conditions compared to the pathogenic Listeria monocytogenes #8 (Fig. 3). From Fig. 1, the OD 595 of the Listeria ivanovii 204 incubated in M9 Minimal Media at 37 8C with increasing NaCl concentration from 0.05 to 10% dropped faster from 0.145 to 0.090 respectively, probably because the bacteria growth was inhibited. When NaCl concentration is adjusted to 15%, biofilm formation of the Listeria ivanovii 204 did not decrease any further. With the exception of Listeria seeligeri/welshimeri 292 (P ≤ 0.05), all the other strains in the study showed no significant differences of OD 595 values at 10 and 15% NaCl concentrations.
Similar patterns of biofilm formation (OD 595 ) were observed in Listeria innocua CCM4030, Listeria innocua 2885, and Listeria seeligeri/welshimeri 292 when grown on 0.05-15% NaCl Fig. 1. Biofilm formation (OD 595 ) comparison between the studied Listeria strains following 72 h incubation at 37 8C under different NaCl concentrations (0.05, 5, 10, and 15%) using M9 Minimal Media concentrations. A continuously decreasing trend of OD 595 was detected for all the other strains, indicating that more biofilm was formed at 0.05 and 5% than at 10 and 15% NaCl concentrations. From these results, it can be concluded that the higher concentrations of NaCl did not present stress conditions that enhanced biofilm formation from non-pathogenic Listeria strains. Similar results were obtained when biofilm formation of Listeria monocytogenes strains in different NaCl concentrations were studied (Pan et al., 2010;Xu et al., 2010).
From Fig. 2, all the Listeria strains incubated in M9 Minimal Media at 37 8C at different pH values (4, 5, and 6) showed a continuously decreasing trend of OD 595 , except Listeria ivanovii 204. Therefore, more biofilm amounts were detected at pH of 6 than at pH of 4 and 5, respectively, which proves the inhibition effect of acidic conditions for the biofilm formation of Listeria strains.
The lowest amounts of biofilms were observed at pH 5 compared to pH 6 in M9 Minimal Media for all the strains (P ≤ 0.05). At pH 4, Listeria ivanovii 204 and Listeria denitrificans 1157 were found to produce the most biofilm thus showing resistance to low pH. The most sensitive strain at pH 4 was Listeria welshimeri CCM3971 that showed weak biofilm formation. At pH 5 5 the lowest amounts of biofilm formed were observed from Listeria ivanovi 204 and Listeria innocua CCM4030, these were the weakest biofilms forming strains.
The biofilm formation (OD 595) from strong biofilm-forming strain Listeria monocytogenes #8 and its interaction with the weak biofilm forming non-pathogenic Listeria strains, were also studied. Significantly, (P ≤ 0.05) more biomass was produced by Listeria monocytogenes #8 (OD 595 5 0.251 ± 0.015) compared to Listeria innocua 2885 (OD 595 5 0.185 ± 0.02) and Listeria innocua CCM4030 strains (OD 595 5 0.182 ± 0.01). The OD 595 values of the biofilms formed when Listeria monocytogenes #8 was mixed with the non-pathogenic Listeria strains were significantly (P ≤ 0.05) less than that of the biofilms formed by Listeria monocytogenes #8 only (Fig. 3). Biofilm formation of the strong biofilm-forming Listeria monocytogenes #8 was overgrown when mixed with the weak biofilm-forming Listeria innocua 2885 strain among the conditions described above. The same results were also obtained when Listeria monocytogenes #8 was grown in presence of Listeria innocua CCM4030, thus reducing its biofilm formation. From Fig. 3, the OD 595 values for mixedbiofilm Listeria monocytogenes #8 with Listeria innocua 2885 strain were significantly (P ≤ 0.05) lower compared to mixed-biofilm Listeria monocytogenes #8 with Listeria innocua CCM4030 strain.
The same inhibition phenomenon was obtained from the studies of Noorwoord and Gilmour (2001) Zhao et al. (2006Zhao et al. ( , 2013 have shown promising results on the use of Lactoccocus lactis against Listeria monocytogenes biofilm in food facility-testing. Contrarily, other studies showed a strong effect that the microorganisms present in food facilities can play on the number of adhered cells of Listeria monocytogenes in the biofilm. This can correspond to either mixed biofilm enhancement or inhibition (Carpentier and Chassaing, 2004;Fox et al., 2014). Nevertheless, a recent study by Tan et al. (2019) proved that the composition and diversity of microbiota from food processing surfaces indicates the persistence of the Listeria monocytogenes. This reiterates the need for further investigation on microbiota characterization particularly, on the role of the harmless bacteria in the food safety.

CONCLUSIONS
After studying growth conditions of non-pathogenic Listeria strains, higher concentrations of sodium chloride did not present a stress condition that enhanced biofilm formation. Decrease in pH however, exhibited some inhibition effect for biofilm formation in all of the non-pathogenic Listeria strains. The results also showed that the biofilm formation of the strong biofilm-forming Listeria monocytogenes #8 was overgrown when mixed with the weak biofilm-forming Listeria strains. Biofilm inhibition was 17.5 and 39.1% for Listeria monocytogenes #8, when it was grown in the presence of Listeria innocua CCM4030 or Listeria innocua 2885 non-pathogenic strains. The future perspective of these findings is the use of potential non-pathogenic strains of Listeria as biocontrol in the food industry. Therefore, biofilm formation of the pathogenic Listeria could be inhibited by the application of non-pathogenic strains on the food processing facilities as a Fig. 3. Biofilm formation of Listeria monocytogenes #8 and its mixed biofilm formation with Listeria innocua 2885 and Listeria innocua CCM4030 following 72 h incubation at 378 C using M9 Minimal Media. Error bars represent the standard deviations of the means, from three individual measurements. Different letters above the bars indicate a significant difference (P ≤ 0.05) part of sanitation procedures. However, further investigation on the biofilm formation of Listeria strains and their interaction under different environmental conditions is still necessary. Their antagonistic effects could provide more information on conditions that may inhibit biofilm formation and could further be used to control the production of Listeria monocytogenes biofilms in the food industry.

ACKNOWLEDGMENT
The Project was supported by the European Union and co-financed by the European Social Fund (grant agreement no. EFOP-3.6.3-VEKOP-16-2017-00005).