Modeling the effect of pH, water activity, and ethanol concentration on biofilm formation of Staphylococcus aureus.

In this work, the effect of environmental factors on Staphylococcus aureus (ATCC 13150) biofilm formation in tryptic soy broth was investigated under different ranges of pH (3.0-9.5), ethanol concentration (EtOH 0.0-20.0%), and aw (NaCl, 0.866-0.992). Biofilm formation was quantified using the crystal violet staining method and optical density (OD: 590 nm) measurements. Biofilm formation was significantly stronger at pH and aw close to S. aureus optimal growth conditions, while it was high at EtOH around 2.5-3.5%. Data sets from the difference between the OD measurements of the test and control (ΔOD) were fitted to the cardinal parameter model (CPM) and cardinal parameter model with inflection (CPMI) to describe the effect of the environmental factors. The models showed good quality of fit for the experimental data in terms of calculated RMSE, with the latter ranging from 0.276 to 0.455. CPM gave a good quality of fit compared to CPMI for the environmental factors tested. Optimal pH was close to neutral (6.76-6.81) and biofilm formation was possible till pH = 3.81-3.78 for CPM and CPMI, respectively. Optimum EtOH and aw conditions for biofilm formation were in the range of 1.99-2.75 and 0.98-0.97, respectively. Predicted OD values observed using strain 13150 were very closely correlated to the OD values predicted with strain 12600 with R2 of 0.978, 0.991, and 0.947 for pH, EtOH, and aw, respectively. The cultivable bacterial cells within the biofilm were enumerated using standard plate counting and a linear model was applied to correlate the attached biofilm cells to ΔOD of biofilm formation. It was found that the biofilm formation correlated with S. aureus population growth. At 2.5-3.5% of EtOH the maximum population density was lower than that observed at 0.0% of EtOH. As 2.5-3.5% of EtOH initiated a stronger biofilm formation, biofilm formation seems to be induced by ethanol stress. The development of cardinal parameter models to describe the effect environmental factors of importance to biofilm formation, offers a promising predictive microbiology approach to decrypting the S. aureus population growth and survival ability on food processing surfaces.

Slightly acidic electrolyzed water combined with chemical and physical treatments to decontaminate bacteria on fresh fruits.

Effect of sequential combination of slightly acidic electrolyzed water (SAEW) with chemical and physical treatments on bacterial decontamination on fruits was investigated in this study. Effect of treatments on microbial and sensory quality was also analyzed after subsequent storage at 4 °C and room temperature (RT, 23 ± 0.15 °C). Whole apple and tomato fruits were inoculated with cocktail strains of Escherichia coli O157:H7 and Listeria monocytogenes. Uninoculated and inoculated fruits were washed first with distilled water (DW), calcium oxide (CaO), fumaric acid (FA), and SAEW at RT for 3 min. Combinations were performed by adding treatment one at a time to SAEW as following FA + SAEW, CaO + FA + SAEW, and CaO + FA + SAEW + ultrasonication (US) or microbubbles (MB). All the sanitizer treatments resulted in significant (p < 0.05) bacterial reduction compared to DW used as control. Increasing the treatments in combination from FA + SAEW to CaO + FA + SAEW + US resulted in an increased bacterial decontamination. The cavitation induced by ultrasonication in FA + SAEW solution resulted in a higher additive effect in decontamination of Escherichia coli O157:H7 and Listeria monocytogenes compare to the agitation generated by microbubble generator in FA + SAEW solution. CaO + FA + SAEW and CaO + FA + SAEW + US were effective in improving the microbial safety and quality of apple fruits. However, additional treatment of US impacted on the quality of tomato fruits during storage at RT. Therefore, a combination of SAEW with sanitizers (CaO and FA) and mechanical force (Ultrasonication) has the potential to be used in postharvest sanitation processing in the fresh fruit industry.

Impact of gamma radiation, potassium sorbate and low temperature on shrimp (Penaeus monodon) preservation.

The objective this study was to assess the effect of gamma radiation and 2% potassium sorbate with low temperature (4 °C) for shrimp (Penaeus monodon) preservation. Fresh samples were prepared for treatment with gamma radiation at 1.0 and 1.5 kGy doses and potassium sorbate (2%) soaked for 30 s (PS 30 s) and 60 s (PS 60 s). Organoleptic score (OS), biochemical composition and microbiological analysis were performed to evaluate the shelf-life extension and quality changes during the storage periods. Data showed that combination treatment of gamma irradiation at 1.5 kGy with low temperature was the most effective in extending shelf-life of shrimp. The isolated bacteria associated with shrimp samples were identified through PCR technique. Antibiotic sensitivity test was examined using ten commonly used antibiotics against these pathogenic isolates. Gentamicin and Imipenem showed up to 50% resistance on Gram-positive (Bacillus cereus and Staphylococcus aureus). This study indicates that gamma irradiation treatment with low temperature was most effective way for shelf-life extension of shrimp which might reduce the wastage of this important nutritional source.

Inactivation kinetics of slightly acidic electrolyzed water combined with benzalkonium chloride and mild heat treatment on vegetative cells, spores, and biofilms of Bacillus cereus.

Bacillus cereus can exist as vegetative cells, spores, and biofilms in food-processing environment, posing a big challenge for the food industry. The objective of this study was to examine the inactivation efficacy of slightly acidic electrolyzed water (SAEW) in combination with benzalkonium chloride (BAC) and mild heat treatment (50 and 60 °C) on B. cereus strains (ATCC 10987 and ATCC 14579). The inactivation efficacy of SAEW was found to be largely dependent on available chlorine concentration (ACC) level and exposure time as well as B. cereus strains and growth conditions. SAEW with ACC of 40 ppm reduced ATCC 10987 and ATCC 14579 vegetative cells to the non-detection limit within 30 s. and 1 min, respectively. Combination treatment with SAEW+60 °C for 10 min resulted in reductions of ATCC 10987 spores, ATCC 14579 spores, and ATCC 10987 biofilms at 0.76 logCFU/ml, 0.59 logCFU/ml, and 1.28 logCFU/cm2, respectively. While, treatment with SAEW+BAC + 60 °C for 10 min resulted in reductions of ATCC 10987 spores, ATCC 14579 spores, and ATCC 10987 biofilms at 1.91 logCFU/ml, 1.98 logCFU/ml, and 2.62 logCFU/cm2, respectively. The inactivation kinetics under different ACC of SAEW and in combination with BAC and mild treatment were determined by Weibull model. The calculated adjusted correlation coefficients (R2adj) and root mean sum of squared error (RMSE) values for all curves were found to be ranges from 0.95-0.99 and 0.04-0.23, respectively, indicating that the Weibull model precisely predicted the inactivation kinetics of B. cereus during SAEW in combination with BAC and mild heat treatments. These results suggest that SAEW in combination with BAC and mild heat may be used as an effective cleaning strategy against B. cereus in the food contact surfaces.

Impact of manganese and heme on biofilm formation of Bacillus cereus food isolates.

The objective of this study was to determine the impact of manganese (Mn2+) and heme on the biofilm formation characteristics of six B. cereus food isolates and two reference strains (ATCC 10987 and ATCC 14579). The data obtained from the crystal violet assay revealed that addition of a combination of Mn2+ and heme to BHI growth medium induced B. cereus biofilm formation. However, the induction of biofilm formation was strictly strain-dependent. In all of the induced strains, the impact of Mn2+ was greater than that of heme. The impact of these two molecules on the phenotypic characteristics related to biofilm formation, such as cell density, sporulation and swarming ability, was determined in a selected food isolate (GIHE 72-5). Addition of Mn2+ and heme to BHI significantly (p < 0.05) increased the number of cells, which was correlated with the results of crystal violet assays as well as scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analyses. In addition, induced biofilms showed higher numbers of spores and greater resistance to benzalkonium chloride. The swarming ability of B. cereus planktonic cells was increased in the presence of Mn2+ and heme in BHI. The expression levels of a number of selected genes, which are involved in mobility and extracellular polymeric substances (EPS) formation in B. cereus, were positively correlated with biofilm formation in the presence of Mn2+ and heme in BHI. These results further confirming the role of these molecules in swarming mobility and making matrix components related to B. cereus biofilm formation. These data indicate that signaling molecules present in the food environment might substantially trigger B. cereus biofilm formation, which could pose a threat to the food industry.

Effect of Electrolyzed Water on the Disinfection of Bacillus cereus Biofilms: The Mechanism of Enhanced Resistance of Sessile Cells in the Biofilm Matrix.

This study examined the disinfection efficacy and mechanism of electrolyzed water (EW) on Bacillus cereus biofilms. B. cereus strains, ATCC 14579 and Korean Collection for Type Cultures (KCTC) 13153 biofilms, were formed on stainless steel (SS) and plastic slide (PS) coupons. Mature biofilms were treated with slightly acidic EW (SAEW), acidic EW (AEW), and basic EW (BEW). SAEW (available chlorine concentration, 25 ± 1.31 mg L-1; pH 5.71 ± 0.16; and oxidation reduction potential, 818 to 855 mV) reduced ATCC 14579 biofilms on plastic slides to below the detection limit within 30 s. However, biofilms on SS coupons showed a higher resistance to the SAEW treatment. When the disinfection activities of three types of EW on biofilms were compared, AEW showed a higher bactericidal activity, followed by SAEW and BEW. In contrast, BEW showed a significantly ( P < 0.05) higher biofilm dispersal activity than AEW and SAEW. SAEW disinfection of the B. cereus biofilms was due to the disruption of the B. cereus plasma membrane. The higher resistance of biofilms formed on the SS coupon might be due to the higher number of attached cells and extracellular polymeric substances formation that reacts with the active chlorine ions, such as hypochlorous acid and hypochlorite ion of SAEW, which decreased the disinfection efficacy of SAEW. This study showed that the EW treatment effectively disinfected B. cereus biofilms, providing insight into the potential use of EW in the food processing industry to control the biofilm formation of B. cereus.

Microbiological Quality and Safety of Fresh Fruits and Vegetables at Retail Levels in Korea.

The aim of this study was to evaluate the microbiological quality and safety of fresh produce at retail level in Korea in order to periodically update information and establish available risks associated with consumption of fresh fruits and vegetables. The samples from different markets located in 3 provinces of South Korea were collected. The protocol in the Korean Food Standards Codex was applied and generic Escherichia coli, coliforms, aerobic mesophilic bacteria (AMB), and yeast and mold (YM) in 360 packaged and unpackaged fresh fruits and vegetables were analyzed. Presence of pathogens was examined using real-time polymerase chain reaction (q-PCR) after enrichment of samples. For all, the microbial counts ranged from 1.7 to 10.6 log cfu/g for AMB, 2.2 to 7.9 log cfu/g for coliforms, and 5.5 to 7.9 log cfu/g for YM. Three lettuce samples were contaminated by E. coli with a bacterial load ranging from 2 to 4 log cfu/g. Salmonella spp. were not detected in any fresh produce. Listeria monocytogenes, E. coli O157:H7, and Staphylococcus aureus were found in 1 (0.6%), 3 (0.8%), and 5 (1.4%) fresh produce samples, respectively. Bacillus cereus (50.3%) and Clostridium perfringens (13.3%) had the highest prevalence. These results indicate the need for employing strict control measures and developing preventive strategies to improve the quality and safety of fresh produce in Korea.

Preservative effect of Chinese cabbage (Brassica rapa subsp. pekinensis) extract on their molecular docking, antioxidant and antimicrobial properties.

This study aimed at investigating the antimicrobial activity of different solvent extracts of Chinese cabbage Brassica rapa subsp. pekinensis (BRARP) and their antioxidant and cytotoxicity properties. Of the different solvents extracts, the chloroform extracts (CE) were significantly inhibited the bacterial pathogens at minimum inhibitory concentration (MIC) of 16.5 mg.mL-1. Biochemical analysis revealed that total phenol (62.6 ± 0.05 mg GAE.g-1) and flavonoids (27.6 ± 0.04 mg QE.g-1) were higher in the extracts of BRARP, which resulted in enhanced antioxidant activity in CE. A total of eight dominant compounds were detected in the potent antimicrobial extract from BRARP based on GC-MS analysis. The molecular interactions study revealed that, among the screened compounds the 1,2-benzenedicarboxylic acid and 2,3-dicyanopropionamide interacted with the active site of pathogenicity and survival related protein with lipopolysaccharide (LpxC) with higer binding energy. This work concluded that the 1, 2-Benzenedicarboxylic acid and 2, 3-Dicyanopropionamide from BRARP was reported to be good non-cytotoxic and antioxidant antimicrobials against bacterial pathogens.

Biofilm formation of Bacillus cereus under food-processing-related conditions.

This study aims to understand the biofilm formation abilities of eight Bacillus cereus strains under food-industry-related conditions. Biofilms were grown in microtiter plates in tryptic soy broth (TSB) or brain heart infusion (BHI) at 30 °C for 24 or 48 h and quantified via the crystal violet assay. A significantly larger of biofilm was formed in TSB than in BHI after 48 h. Selected strains were used to test biofilm formation under food-related conditions produced by different surfaces (e.g., stainless steel, plastic, or glass), temperatures (25 or 30 °C), carbon sources, (glucose or glycerol) and NaCl. Biofilm formation appeared to be affected by surface properties, temperature, and carbon sources. A larger biofilm was formed on stainless steel at 30 °C compared to plastic and glass surfaces at 25 and 30 °C. Moreover, addition of glucose in combination with NaCl in TSB produced significantly larger biofilm than glucose, glycerol and/or NaCl. These results indicate that food-industry-related conditions could promote B. cereus biofilm formation, which is relevant to food safety.