2022 Volume 28 Issue 6 Pages 521-527
This study illustrates the effectiveness of slightly acidic hypochlorous water (SAHW) in comparison with sodium hypochlorite (NaOCl) in reducing biomass and viable cells in biofilms established by the dual species, Listeria monocytogenes and Escherichia coli, on a microtiter plate and stainless-steel coupon. The SAHW and NaOCl treatments exhibited significant efficacy against biofilms (p < 0.05) on both surfaces. Additionally, compared with NaOCl treatment, SAHW treatment significantly reduced biofilm formation (p < 0.05). With its high antibiofilm activity, SAHW not only reduced the biomass of biofilms, but also significantly decreased viable biofilm cells to 5 log CFU/mL or ≤1 log CFU/cm2 on microtiter plates and stainless-steel surfaces, respectively. These results indicate that SAHW is a potential candidate for disinfectants against biofilms on various food contact surfaces.
Biofilms consist of adherent bacterial cells surrounded by an extracellular matrix on a surface and can be found in the natural, industrial, and hospital sectors (Hall-Stoodley et al., 2004). Biofilms are resistant to antibacterial agents, and certain physical treatments are also associated with infectious diseases (O'Toole et al., 2000). Additionally, foodborne pathogens, including Listeria monocytogenes, Escherichia coli, and Salmonella enterica, may grow predominantly as biofilms rather than in planktonic mode (Lindsay and Von Holy, 2006). Hence, biofilm formation is considered a critical hazard for food safety and hygiene because the survival of biofilm cells over long periods may lead to cross-contamination inside the food processing environment (Galié et al., 2018). Biofilm formation enhances the capacity of foodborne pathogens to withstand extreme conditions that are frequently encountered during food processing or disinfection (Berlanga and Guerrero, 2016). In the fish industry, biofilm formation may form a persistent source of product contamination, leading to hygiene problems and economic losses because of food spoilage and food poisoning (Mizan et al., 2015; Pandey et al., 2014; Vestby et al., 2009). Moreover, most biofilms in nature are formed by mixed species that represent the actual life processes of microbes (Rao et al., 2020). Mixed-species biofilms are stable and exhibit increased resistance to environmental conditions (Bridier et al., 2011). Consequently, the control of mixed-species biofilms has received considerable attention in recent years.
Listeria monocytogenes is ubiquitous in nature and attaches to a wide range of surfaces used in food processing (Da Silva and De Martinis, 2013). An important source of L. monocytogenes contamination in food is thought to be contaminated processing utensils (Rodríguez-López et al., 2018). Escherichia coli is a standard bacterium found in the intestines of healthy humans and animals. Moreover, E. coli is one of the most frequently isolated bacteria in the fish processing industry (Costa, 2013). Listeria monocytogenes and E. coli normally adhere to surfaces as part of complex multispecies biofilms (Rodríguez-López et al., 2015). These associations may increase resistance to chemical disinfectants in the food industry (Holah et al., 2002). Sodium hypochlorite (NaOCl) is one of the most commonly used disinfectants because of its broad-spectrum antimicrobial activity (Poggio et al., 2010). Nevertheless, NaOCl has negative effects on human health and aquatic environments (Slaughter et al., 2019). In this respect, as another potential antimicrobial agent, slightly acidic hypochlorous water (SAHW) shows a strong bactericidal effect on foodborne pathogens and their biofilms by disrupting the permeability of the cell membrane (Wang et al., 2018). Slightly acidic hypochlorous water is a chlorine-based solution at a near-neutral pH of 5–6 that contains a high concentration of hypochloric acid (HOCl), which is the most effective disinfectant of the chlorine family in dilute solutions (Okanda et al., 2019). It has been widely used to disinfect microorganisms in the food industry because of its strong disinfection ability, quick on-site production, low cost, and minimal health risks (Hussain et al., 2019). However, few studies have focused on the effects of this disinfectant on the removal of mixed-species biofilms. In the present study, the effectiveness of SAHW against L. monocytogenes-E. coli dual-species biofilm developed on a microtiter plate and stainless steel was studied.
Bacterial strains Listeria monocytogenes and E. coli isolated from two Vietnamese Pangasius fish-processing plants were used in this study (Phan et al., 2022). Each strain was stored in 30% glycerol at -80 °C, and working cultures were maintained on Tryptic soy agar (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) slants at 4 °C for no longer than 1 month. To prepare dual-species biofilms, equal volumes of L. monocytogenes and E. coli suspensions were mixed.
Biofilm formation on a microtiter plate and stainless-steel coupons Biofilm assays were performed on 96-well plates (Sanplatec Co., Ltd., Osaka, Japan) based on the methods previously described by Miyamoto et al. (2011) with some modifications. Stainless steel (SS) coupons (30 × 30 × 1 mm, type 304) were washed with soap (Mioraito, Kyoeisha Chemical Co., Ltd. Osaka, Japan), rinsed with tap water, and autoclaved at 121 °C for 20 min before use. Briefly, L. monocytogenes and E. coli strains were cultured overnight with shaking at 130 rpm in brain heart infusion broth (Oxoid Ltd., Hampshire, UK) at 30 °C and Luria broth (Becton, Dickinson, and Company, Sparks, MD, USA) at 37 °C. The culture was then diluted to 107–108 CFU/mL. The suspensions were added to 96-well plates and SS coupons individually placed in a sterilized Petri plate at 200 µL/well or SS coupon and grown at 30 °C for 3 d without agitation.
Effect of SAHW or NaOCl on the developed biofilm on the microtiter plate and SS coupons The efficacy of SAHW and NaOCl in removing viable cells in the biofilm and the biofilm mass of the dual-species biofilm formed by L. monocytogenes and E. coli was investigated. Slightly acidic hypochlorous water containing 40 mg/L available chlorine, pH 5.5, was kindly provided by Morinaga Milk Industry Co., Ltd. (Tokyo, Japan), and 100 and 200 mg/L NaOCl solutions were prepared by diluting a 10% NaOCl solution (Sigma-Aldrich, Tokyo, Japan) with sterilized water. Following biofilm formation, supernatants were removed, and the wells or coupons were washed three times with sterile water, mixed with 250 µL SAWH or NaOCl, and kept at 25 °C for 10 min. For the control, the same volume of phosphate-buffered saline (PBS, 1.47 mM KH2PO4, 8.10 mM Na2HPO4, 2.68 mM KCl, 137 mM NaCl, pH 7.4) was used instead of SAHW or NaOCl. After treatment, the supernatants were carefully removed from the wells, and the wells were gently rinsed twice with PBS. Biofilm cells were recovered in PBS by scraping the surface of the well with a pipette tip and rinsing with PBS and swabbing with sterile cotton swabs soaked in PBS for the microtiter plate and SS coupon, respectively. The recovered cell suspensions were serially diluted with PBS, and viable counts were determined by plating on CHROMagar Listeria (CHROMagar, Pais, France) and Pearlcore Desoxycholate Agar (Eiken Chemical, Tochigi, Japan) for the enumeration of L. monocytogenes and E. coli, respectively. The results are expressed as log CFU/mL or log CFU/cm2 for the microtiter plate and SS coupon, respectively. Furthermore, the biofilm mass was determined by crystal violet staining, as described previously (Miyamoto et al. 2011). The biofilm formation was obtained after 3 days, and then, the well and SS coupons were washed three times with PBS, dried at 25 °C, and stained with 1% crystal violet for 30 min. After removing unbound dye by washing with PBS twice, the crystal violet was dissolved in 99% ethanol at 25 °C for 15 min. The absorbance at 595 nm (A595) was measured using a Microplate Reader Infinite F50/Robotic (Tecan Trading AG, Switzerland).
For visualization under a microscope, the microtiter plate was stained with a LIVE/DEAD bacterial viability kit (Molecular Probes Inc., Eugene, Oregon, USA) to distinguish between cells with intact cell membranes (green fluorescence) and damaged cell membranes (red fluorescence). The staining solution was prepared by mixing 1.5 µL of Syto 9 and 1.5 µL of propidium iodide solutions in 1 mL of UltraPure Distilled water DNAse, RNAse-Free (Life Technologies, Eugene, USA). In each well of the microtiter plate, 250 µL of the staining solution was added and incubated for 15 min in the dark. The wells were then washed thrice with sterile water, air-dried, and visualized under a fluorescence microscope (BX53; Olympus Co., Tokyo, Japan).
Statistical analysis All experiments were performed in triplicate. Results are presented as mean values and standard deviations of the mean. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test using the Statistical Package for the Social Sciences software (SPSS Inc., Chicago, IL, USA). All tests were performed with a confidence level of 95%.
As shown in Fig. 1, the absorbance of biomass of the dual-species biofilm was 0.75 for the microtiter plate and 0.05 for the SS coupon. There was a significant difference (p < 0.05) in biomass reduction after treatment with SAHW and NaOCl. Furthermore, a significant decrease in biomass was observed after treatment with SAHW compared to treatment with either 100 or 200 mg/L NaOCl on both surfaces (p < 0.05).
Effects of SAHW (40 mg/L available chlorine), or NaOCl (100 and 200 mg/L available chlorine) on biofilm mass 3-days dual-species biofilm of L. monocytogenes-E. coli in the microplate plate and stainless-steel coupon. Different letters (a-c) and (d-f) indicate significant differences (p < 0.05) among treatments in microtiter plate and stainless-steel coupon, respectively. Error bars show the standard deviation of the mean (n = 3).
As shown in Fig. 2A, the plate count method showed similar viable counts of biofilm cells for L. monocytogenes and E. coli on the microtiter plate at 7.34 and 7.26 log CFU/mL, respectively. After treatment, SAHW showed a stronger bactericidal effect on biofilm cells than NaOCl at the tested concentrations (p < 0.05). In the presence of SAHW, viable counts of L. monocytogenes and E. coli decreased by 5.52 and 5.61 log CFU/mL, respectively. The log reduction was 3.01 and 3.04 log CFU/mL for L. monocytogenes and 4.42 and 4.12 log CFU/mL for E. coli after treatment with 100 and 200 mg/L of NaOCl, respectively. For the SS coupon, as shown in Fig. 2B, there was a significantly higher viable count for L. monocytogenes than for E. coli, at 4.5 and 2.1 log CFU/cm2, respectively (p < 0.05). After treatment, both 100 and 200 mg/L SAHW and NaOCl exhibited significant antibacterial effects (p < 0.05) compared with the control.
Effects of SAHW (40 mg/L available chlorine), or NaOCl (100 and 200 mg/L available chlorine) on viable counts of 3-days dual-species biofilm of L. monocytogenes (□) – E.coli (■) biofilm in the microplate plate (A), and stainless-steel coupon (B). Different uppercase letters (A-D) and (E-H) indicate significant differences (p < 0.05) among treatments for L. monocytogenes in the microtiter plate and stainless-steel coupon, respectively. Different lowercase letters (a-d) and (e-f) indicate significant differences (p < 0.05) among treatments for E. coli in the microtiter plate and stainless-steel coupon, respectively. Error bars show the standard deviation of the mean (n = 3).
The fluorescence microscopy image in Fig. 3A shows blight green-fluorescent cells of L. monocytogenes and E. coli all over the plate surface, suggesting viable biofilm cells. The number of green-fluorescent cells largely decreased after treatment with SAHW and NaOCl compared to the control treatment. The number of green-fluorescent cells after treatment with SAHW (Fig. 3B) was less than that after treatment with NaOCl (Figs. 3C and D). These results were consistent with those determined by biofilm mass and viable counts shown in Figures 1 and 2, respectively.
Fluorescence microscopy images of dual species biofilm by L.monocytogenes and E.coli after different treatments.
3A, control sample; 3B, treatment with 40 mg/L SAHW; 3C, treatment with 100 mg/L NaOCl; 3D, treatment with 200 mg/L NaOCl. Scale bars correspond to 50 µm. Arrows show living cells.
Biofilm formation is considered a serious concern in the food industry because of its relevance as a potential source of cross-contamination. Cells attached to various food-contact surfaces may have increased resistance to sanitizers. In the present study, two trends were observed: the dominant growth of L. monocytogenes in comparison to that of E. coli on the SS coupon and the growth of both species on the microtiter plates. The different results can be related to the interactions between the bacterial cells and an inorganic surface (hydrophobic or hydrophilic surfaces) that are different for adhesion (Sommer et al., 1999). The two surfaces possess different surface properties: the SS coupon is hydrophilic, electronegative, and has a high surface energy, whereas the microtiter plate is hydrophobic, electrostatic, and has a low surface energy (Pawar et al., 2005). Moreover, the production of extracellular matrix components by E. coli increases the hydrophobicity of the cell surface, resulting in the inhibition of the attachment of E. coli cells to the SS coupon (Ryu et al., 2004). The results of the present study are contrary to those of other studies wherein E. coli was reported to be dominant in dual-species biofilms with L. monocytogenes (Almeida et al., 2011; Rodríguez-López et al., 2017). However, owing to various mechanisms affecting attachment, including interactions between bacteria and strain-dependent characteristics, further studies are needed to define the growth status of both L. monocytogenes and E. coli during biofilm formation.
Despite its several side effects on human health and the environment, NaOCl is one of the most frequently used disinfectants in industrial settings because of its low cost and broad-spectrum antimicrobial activity (Lineback et al., 2018; Luddin and Ahmed, 2013). The results revealed that NaOCl at both tested concentrations (100 and 200 mg/L) significantly decreased the number of biofilm cells on both surface types. Park et al. (2012) reported that after 50 min exposure to aerosolized 100 mg/L NaOCl the population of biofilm cells of E. coli and L. monocytogenes on a polyvinyl chloride coupon exhibited 2–3 log reductions. In recent years, SAHW has attracted attention as an alternative disinfectant with a wide range of applications because it overcomes the drawbacks of NaOCl (Rahman et al., 2016). The bactericidal mechanism of SAHW is attributed to the disruption of cell wall permeability, leakage of intracellular components, and inhibition of enzymatic activities for synthesis, metabolism, and transportation (Rahman et al., 2016). Specifically, SAHW can damage cell wall integrity and disrupt the intracellular structure of S. aureus and E. coli (Ding et al., 2016; Liao et al., 2017). Consistent with the present results, previous findings have revealed a 3–6 log CFU reduction in biofilm cells by SAHW, indicating good antibacterial efficacy (Hussain et al., 2019; Wang et al., 2018; Zhang et al., 2021).
Moreover, with removal of more than 40% of biomass, this study confirmed that SAHW had a significant effect on the removal of biofilm mass. This finding was consistent with those of other reports that indicated that SAHW treatment greatly decreased the biomass by 40–85% at a range of food contact surfaces, suggesting it as a useful disinfectant in the food industry (Hao et al., 2022; Pianpian et al., 2022; Zhang et al., 2021). These results may be owing to chlorine disrupting the structure matrix of exopolysaccharides, causing splitting of the biofilm (Liu et al., 2016; Ronner and Wong, 1993; Toté et al., 2010).
In the present study, SAHW was significantly more effective than NaOCl (p < 0.05). Slightly acidic hypochlorous water contains more than 95% of HOCl, which is an uncharged molecule with a relatively low molecular weight. HOCl can penetrate cell walls more easily and rapidly than any other chlorine-based disinfectant by entering the cytoplasm through the cell membrane and oxidizing DNA and proteins, leading to cell death (Hakim et al., 2016). Furthermore, NaOCl contains approximately 95% of hypochlorite ion (OCl-), which is negatively charged and has a relatively poor bactericidal effect because the ion cannot penetrate the cell membrane and enter the cytoplasm (Fukuzaki, 2006). Compared with the planktonic cells, biofilms cells have been reported to be significantly resistant to either SAHW or NaOCl treatment (Hao et al., 2022; Pianpian et al., 2022; Rodríguez-Melcón et al., 2019). Therefore, efficient and sustainable cleaning and disinfection of biofilms is necessary during food processing.
In conclusion, the use of SAHW as a sanitizer could be effective in reducing bacterial contamination in addition to its safe handling and eco-friendliness, which would be an advantage for the food industry. The effects of SAHW should be investigated in combination with mild heat at different time intervals on mixed-species biofilms for application of the method to naturally exiting biofilms in the actual processing machinery and plants of the food industry.
Acknowledgements This work was supported by the Can Tho University Improvement Project VN14-P6 and supported by a Japanese ODA loan.
Conflict of interest There are no conflicts of interest to declare.
