Taxonomic and Functional Differences in Cervical Microbiome Associated with Cervical Cancer Development.

The cervical microbiome is associated with cervical cancer risk, but how microbial diversity and functional profiles change in cervical cancer remains unclear. Herein, we investigated microbial-compositional and functional differences between a control group and a high-grade cervical intraepithelial neoplasia and cervical cancer (CIN2/3-CC) group. After retrospective collection of 92 cervical swab samples, we carried out 16S rRNA amplicon sequencing on 50 and 42 samples from the control and CIN2/3-CC groups, respectively. The EzBioCloud pipeline was applied to identify the genomic features associated with the groups using 16S rRNA data. A linear discriminant analysis effect size (LEfSe) was performed to assess the enrichment in the assigned taxonomic and functional profiles. We found a lower richness in the control group relative to the CIN2/3-CC group; however, the ß-diversity tended to be similar between the groups. The LEfSe analysis showed that a phylum Sacchaaribacteria_TM7, 11 genera, and 21 species were more abundant in the CIN2/3-CC group and that one uncharacterized Gardnerella species was more abundant only in the control group. Further characterization of the functional pathways using EzBioCloud showed that the 4 KEGG orthologs (Phosphotransferase system [PTS] sucrose-specific IIA, IIB, IIC components and PTS cellubiose-specific IIC component) were involved in the KEGG pathway of starch and sucrose metabolism. The two pathways of folate biosynthesis and oxidative phosphorylation were more abundant in the CIN2/3-CC group. Further confirmation of these results in larger samples can help to elucidate the potential association between the cervical microbiome and cervical cancer.

Disinfection Efficacy of Slightly Acidic Electrolyzed Water Combined with Chemical Treatments on Fresh Fruits at the Industrial Scale.

The objective of this study was to investigate the efficacy of slightly acidic electrolyzed water (SAEW) combined with fumaric acid (FA) and calcium oxide (CaO) treatment on the microbial disinfection of fresh fruits including apple, mandarin, and tomato at the industrial scale. The combined treatments can significantly (p < 0.05) reduce the population of natural microbiota from the fruit surfaces and the treated samples showed good sensory qualities during refrigeration storage. In addition, decontamination of inoculated foodborne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes) was carried out in the laboratory, and the combined treatments resulted in a reduction ranging from 2.85 to 5.35 log CFU/fruit, CaO followed by SAEW+FA treatment that resulted in significantly higher reduction than for SAEW+FA treatment. The technology developed by this study has been used in a fresh fruit industry and has greatly improved the quality of the products. These findings suggest that the synergistic properties of the combination of SAEW, FA, and CaO could be used in the fresh fruit industry as an effective sanitizer.

Development of antimicrobial edible coating based on modified chitosan for the improvement of strawberries shelf life.

Edible antimicrobial coating produced from chitosan (CS) and its derivative was applied to improve the shelf life of fresh strawberries at 10 °C. Fruits treated with coating solution was stored at 10 °C and evaluated for weight loss, visual decay and microbiological analysis. Results indicated that the percentage weight loss and the decay were significantly (p < 0.05) lower for chitosan-monomethyl fumaric acid (CS-MFA) than that of CS and control samples. The total aerobic count for CS-MFA was 3.32 log CFU/fruit and was considerably lowered (p < 0.05) than CS (3.83 log CFU/fruit) and control (5.31 log CFU/fruit) at the end of storage. Fruit coated with CS-MFA showed significantly lowered (p < 0.05) count of yeast and molds when compared with CS. In conclusion, the antimicrobial edible coating based on modified CS improved microbiological characteristics and increased the shelf life from 4 (control) to 8 days (coated fruits).

Sanitization Efficacy of Slightly Acidic Electrolyzed Water against pure cultures of Escherichia coli, Salmonella enterica, Typhimurium, Staphylococcus aureus and Bacillus cereus spores, in Comparison with Different Water Hardness.

The Influence of water source on the production of slightly acidic electrolyzed water (SAEW) and its sanitization efficacy were investigated. Two different water sources (tap water (TW) and underground water (UGW)) were applied to produce slightly acidic electrolyzed water (SAEW) at same setting current, with similar electrolyte flow rate (EFR) and concentration. Properties of SAEW were evaluated based on pH, Available chlorine concentration (ACC) and oxidation-reduction potential (ORP). Methods for the optimization of SAEW production process was examined to obtain high ACC value by implanting different types of electrolytes. Effect of ACC and pH of SAEW were evaluated in vitro towards inactivate foodborne pathogens. The results indicated that TW with hardness of 29 ppm produced effectively SAEW than through UGW (12 ppm) using electrolytes. Likewise, low water hardness could be reinforced by combining HCL with a salt (NaCl or KCL). The optimized SAEW production system was determined at 4% HCl + 2.0 M KCL with EFR of 2 mL/min and 4% HCl + 3.0 M KCL with EFR of 2 mL/min resulting in higher ACC value of 56.5 and 65.5 ppm, respectively using TW. Pathogenic vegetative cells were completely inactivated within 1 min of treatment in SAEW with 20 ppm. Viability observations using Confocal and TEM Microscopy, Flow cytometry, and antimicrobial activity were carried out to confirm the sanitizing effect and cell membrane disruption. Based on the experimental results obtained, it provides a foundation for future advancement towards commercial application of SAEW in the food and agricultural industries.

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.

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.

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.

Human microbiome restoration and safety.

The human gut microbiome consists of many bacteria which are in symbiotic relationship with human beings. The gut microbial metabolism, as well as the microbial-host co-metabolism, has been found to greatly influence health and disease. Factors such as diet, antibiotic use and lifestyle have been associated with alterations in the gut microbial community and may result in several pathological conditions. For this reason, several strategies including fecal microbiota transplant and probiotic administration have been applied and proven to be feasible and effective in restoring the gut microbiota in humans. Yet, safety concerns such as potential health risks that may arise from such interventions and how these strategies are regulated need to be addressed. Also, it will be important to know if these microbiome restoration strategies can have a profound impact on health. This review provides an overview of our current knowledge of the microbiome restoration strategies and safety issues on how these strategies are regulated.

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.

Evaluation of nisin-loaded chitosan-monomethyl fumaric acid nanoparticles as a direct food additive.

Nisin-loaded chitosan-monomethyl fumaric acid (CM-N) nanoparticles were evaluated as a novel, direct food additive. Chitosan (CS) was modified with monomethyl fumaric acid (MFA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). CS-loaded nisin (CS-N) and CM-N nanoparticles were produced through ionic interactions between the positive amino group of CS and CS-MFA and negative tripolyphosphate ions. The resultant materials were characterized by TNBS assay, X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, and zeta potential analysis. CS-MFA was successfully synthesized with 8.38 ±â€¯0.02% substitution of the amino groups, as confirmed by TNBS assay. The percentage yield of CS-N and CM-N nanoparticles was 81.64 and 76.83% and nisin encapsulation efficiency was 71.48 ±â€¯0.48 and 60.32 ±â€¯0.63%, respectively. The average particle size of CS-N and CM-N nanoparticles was 134.3 and 207.9 nm, while the zeta potential of CS-N and CM-N nanoparticles was +39.4 mV and +31.5 mV, respectively. Upon antibacterial activity against foodborne pathogens, CM-N significantly reduced bacterial counts compared to the other tested samples in orange juice after 48 h of incubation. Based on the preliminarily results, CM-N nanoparticles have shown impressive properties and can be used in the food industry as carriers and direct antimicrobial agents.

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.

Assessment of Enterotoxin Production and Cross-Contamination of Staphylococcus aureus between Food Processing Materials and Ready-To-Eat Cooked Fish Paste.

This study evaluated Staphylococcus aureus growth and subsequent staphylococcal enterotoxin A production in tryptone soy broth and on ready-to-eat cooked fish paste at 12 to 37 °C, as well as cross-contamination between stainless steel, polyethylene, and latex glove at room temperature. A model was developed using Barany and Roberts's growth model, which satisfactorily described the suitable growth of S. aureus with R(2)-adj from 0.94 to 0.99. Except at 12 °C, S. aureus cells in TSB presented a lag time lower (14.64 to 1.65 h), grew faster (0.08 to 0.31 log CFU/h) and produced SEA at lower cell density levels (5.65 to 6.44 log CFU/mL) compare to those inoculated on cooked fish paste with data of 16.920 to 1.985 h, 0.02 to 0.23 log CFU/h, and 6.19 to 7.11 log CFU/g, respectively. Staphylococcal enterotoxin type A (SEA) visual immunoassay test showed that primary SEA detection varied considerably among different storage temperature degrees and media. For example, it occurred only during exponential phase at 30 and 37 °C in TSB, but in cooked fish paste it took place at late exponential phase of S. aureus growth at 20 and 25 °C. The SEA detection test was negative on presence of S. aureus on cooked fish paste stored at 12 and 15 °C, although cell density reached level of 6.12 log CFU/g at 15 °C. Cross-contamination expressed as transfer rate of S. aureus from polyethylene surface to cooked fish paste surface was slower than that observed with steel surface to cooked fish paste under same conditions. These results provide helpful information for controlling S. aureus growth, SEA production and cross-contamination during processing of cooked fish paste.

Fumaric Acid and Slightly Acidic Electrolyzed Water Inactivate Gram Positive and Gram Negative Foodborne Pathogens.

Sanitizing effectiveness of slightly acidic electrolyzed water (SAEW) and fumaric acid (FA) at different dipping temperatures (25-60 °C), times (1-5 min), and concentrations (5-30 ppm for SAEW and 0.125%-0.5% for FA) on pure cultures of two Gram positive pathogens Staphylococcus aureus (SA) and Listeria monocytogenes (LM) and two Gram negative pathogens Escherichia coli O157:H7 (EC) and Salmonella Typhimurium (ST) was evaluated. FA (0.25%) showed the strongest sanitizing effect, demonstrating complete inactivation of EC, ST, and LM, while SA was reduced by 3.95-5.76 log CFU/mL at 25-60 °C, respectively, after 1 min of treatment. For SAEW, the complete inactivation was obtained when available chlorine concentration was increased to 20 ppm at 40 °C for 3 and 5 min. Moreover, Gram positive pathogens have been shown to resist to all treatment trends more than Gram negative pathogens throughout this experiment. Regardless of the different dipping temperatures, concentrations, and times, FA treatment was more effective than treatment with SAEW for reduction of foodborne pathogens. This study demonstrated that application of FA in food systems may be useful as a method for inactivation of foodborne pathogens.

Modeling of Bacillus cereus growth in brown rice submitted to a combination of ultrasonication and slightly acidic electrolyzed water treatment.

The combined effects of ultrasonication and slight acidic electrolyzed water were investigated to improve the microbial safety of brown rice against Bacillus cereus infection and to evaluate the growth kinetics of these bacteria during storage of untreated and treated rice at various temperatures (5, 10, 15, 20, 25, 30, and 35°C). The results indicate that this combination treatment was bactericidal against B. cereus, resulting in an approximately 3.29-log reduction. Although B. cereus can be efficiently reduced by treatment, temperature abuse during storage can allow B. cereus to recover and grow. A primary growth model (Baranyi and Roberts equation) was fitted to the raw growth data from untreated (control) and treated samples to estimate growth rate, lag time, and maximum population density, with a low standard error of the residuals (≤0.140) and high adjusted coefficient of determination (>0.990). The growth curves obtained from the Baranyi and Roberts model indicated that B. cereus grew more slowly on treated brown rice than on untreated brown rice. Secondary models predicting the square root of the maximum growth rate and the natural logarithm of the lag time as a function of temperature were satisfactory (bias factor = 0.993 to 1.013; accuracy factor = 1.290 to 1.352; standard error of prediction = 18.828 to 36.615%). Inactivation results and the model developed and validated in this study provided reliable and valuable growth kinetics information for quantitative microbiological risk assessment studies of B. cereus on brown rice.

Bacteriological quality of vegetables from organic and conventional production in different areas of Korea.

Foods grown in organic production systems have been described as representing an increased risk to public health compared with foods from conventional production. Leafy vegetables (spinach, romaine lettuce, and green sesame leaves) grown in organic and conventional systems were collected from various areas in Korea and examined using standard culture methods to compare the microbiological quality of the produce grown in the two agricultural systems. The 354 samples of these leafy vegetables were analyzed for levels of indicator bacteria (aerobic bacteria, coliforms, and Escherichia coli) and the prevalence of the pathogens Staphylococcus aureus, E. coli O157:H7, Listeria monocytogenes, Bacillus cereus, and Salmonella. Aerobic bacteria and coliforms were detected in all vegetable types, but nonpathogenic E. coli was below the limit of detection in all samples. B. cereus was the most prevalent pathogen, found on 7 (11.1%) of the 63 organic spinach samples. The prevalence of S. aureus was highest in organic sesame leaves; it was found on 5 (8.0%) of the 63 samples. The prevalence of L. monocytogenes was highest on organic romaine lettuce and spinach; it was found in 4 (6.4%) of 63 samples of each type of vegetable. E. coli O157:H7 found on only 1 (1.58%) of 55 conventional spinach samples. These results suggest that farming type at most only slightly affects the hygienic quality of leafy vegetables, and no effect was found for sample collection area. Salmonella was not isolated from any of the conventional or organic leafy vegetables. These results do not support the hypothesis that organic produce poses a substantially greater risk of pathogen contamination than does conventional produce.