Microbial succession during button mushroom (Agaricus bisporus) production evaluated via high-throughput sequencing.

Button mushrooms (Agaricus bisporus), are one of the most widely consumed mushrooms in the world. However, changes within its microbial community as it relates to the use of different raw materials and cultivation methods, as well as potential points of microbial contamination throughout the production process have not been investigated extensively. In the present study, button mushroom cultivation was investigated in each of the four stages (raw materials, composting (phase I, Ⅱ, and Ⅲ), casing, and harvesting), and samples (n = 186) from mushrooms and their related environments were collected from four distinct mushroom-growing farms (A-D) in Korea. Shifts within the bacterial consortium during mushroom production were characterized with 16 S rRNA amplicon sequencing. The succession of bacterial communities on each farm was dependent on the raw material incorporated, aeration, and the farm environment. The dominant phyla of the compost stack at the four farms were Pseudomonadota (56.7%) in farm A, Pseudomonadota (43.3%) in farm B, Bacteroidota (46.0%) in farm C, and Bacillota (62.8%) in farm D. During the Phase Ⅰ, highly heat-resistant microbes, such as those from the phylum Deinococcota (0.6-65.5%) and the families Bacillaceae (1.7-36.3%), Thermaceae (0.1-65.5%), and Limnochordaceae (0.3-30.5%) greatly proliferated. The microbial diversity within compost samples exhibited a marked decline as a result of the proliferation of thermophilic bacteria. In the spawning step, there were considerable increases in Xanthomonadaceae in the pasteurized composts of farms C and D - both of which employed an aeration system. In the harvesting phase, beta diversity correlated strongly between the casing soil layer and pre-harvest mushrooms, as well as between gloves and packaged mushrooms. The results suggest that gloves may be a major source of cross-contamination for packaged mushrooms, highlighting the need for enhanced hygienic practices during the harvesting phase to ensure product safety. These findings contribute to the current understanding of the influence of environmental and adjacent microbiomes on mushroom products to benefit the mushroom industry and relevant stakeholders by ensuring quality production.

Efficacy of combined caprylic acid and thymol treatments for inactivation of Listeria monocytogenes on enoki mushrooms in household and food-service establishments.

Raw enoki mushroom is a high-risk vector for listeriosis, which led to foodborne outbreaks resulting in four deaths in the United States in 2020. This study aimed to investigate the washing method for the inactivation of L. monocytogenes in enoki mushrooms for household and food service establishments. Five methods of washing fresh agricultural products without using disinfectants were selected: (1) rinsing under running water (2 L/min, 10 min), (2-3) dipping in water (200 ml/20 g) at 22 or 40 °C for 10 min, and using (4) 10% NaCl or (5) 5% vinegar at 22 °C for 10 min. The antibacterial efficacy of each washing method along with the final rinse was tested with enoki mushrooms inoculated with a 3-strain cocktail of L. monocytogenes (ATCC 19111, 19115, 19117; ca. 6 log CFU/g). The 5% vinegar showed a significant difference in antibacterial effect compared to the other treatments except 10% NaCl (P < 0.05), with the maximum elimination of L. monocytogenes by 1.23 log CFU/g. Therefore, a disinfectant for enoki mushrooms that can complement the commonly used washing method was developed using antimicrobials (caprylic acid, CA: 0, 0.20, 0.40%; thymol, TM: 0, 0.075, 0.15%). By combined treatment of 0.40% CA and 0.15% TM at 22 °C for 10 min, L. monocytogenes was completely inactivated (>5.55 log reduction CFU/g) and did not recover after enrichment, although individual treatments of antimicrobials showed low bactericidal effects of <1.50 log reduction CFU/g. The bacterial membrane disintegration induced by the disinfectant was analyzed through flow cytometry. Additionally, the sensory scores (odor and appearance) and color parameters (L*, a*, and b*) of enoki mushrooms treated with the disinfectant were not significantly different from those of enoki mushrooms washed with water (P > 0.05). Our findings suggest a washing disinfectant consisting of low concentrations of CA and TM with synergistic antibacterial effects without quality deterioration that can ensure the safe consumption of raw enoki mushrooms in homes and food service establishments.

Effects of sterilization methods on the survival of pathogenic bacteria in potting soil stored at various temperatures.

Fresh food products can be contaminated with pathogenic bacteria in various agricultural environments. Potting soil is sterilized by heat sterilization and then reused. This study evaluated the effects of three sterilization methods (non-sterilized, pasteurized, and sterilized) on the survival of pathogenic bacteria in potting soil during storage for 60 days at 5, 15, 25, and 35 °C. The reduction in Escherichia coli O157:H7, Salmonella Typhimurium, and Staphylococcus aureus in potting soil was higher at higher temperatures (25 and 35 °C) than at lower temperatures (5 and 15 °C). The population of pathogenic bacteria in pasteurized and sterilized potting soil was reduced below the detectable levels within 30 days at 35 °C. In contrast, the population of Bacillus cereus did not change in potting soil during storage for 60 days at all temperatures. These results indicate that sterilization and storage temperature of potting soil are critical factors influencing the survival of pathogenic bacteria.

Bacterial microbiota profiling of oyster mushrooms (Pleurotus ostreatus) based on cultivation methods and distribution channels using high-throughput sequencing.

The annual consumption and production of oyster mushrooms (Pleurotus ostreatus) have continued to rise due to its nutritive and health-promoting benefits. Cultivated mushrooms are mostly grown in small to medium-scaled scale production plants that present hygienic challenges which could, in turn, increase associated foodborne pathogenic outbreaks. The present study aimed to investigate the shift in microbial ecologies of oyster mushrooms from pre-distribution (cultivation in bottles or on shelves) to post-distribution at supermarkets and open-air markets. Aerobic plate counts and coliforms were quantified using traditional microbiological techniques, and the microbiome associated with oyster mushrooms (n = 70) was analyzed using 16S rRNA amplicon sequencing for an enhanced level of bacterial microbiota profiling. Overall, coliforms recovered from pre-distribution bottle-cultivated mushrooms were 1.9 log CFU/g higher (p < 0.05) than that of shelf-cultivated mushrooms. The mean aerobic plate counts of oyster mushrooms distributed to open-air markets was 1.2 log CFU/g higher (p < 0.05) than packaged mushrooms from supermarkets while there were no significant differences in coliform counts. The pattern of bacterial composition differed by post-distribution channels, with oyster mushrooms collected from the open-air markets demonstrating the richest microbiome diversity. An increase in the relative abundance of Enterobacteriaceae (55-68 %) and Pseudomonadaceae (27-35 %) was observed in pre- and post-distribution mushrooms, respectively. However, no distinct bacterial microbiota differences were observed for the different cultivation methods or different geographical locations for each market type. The current findings add to our understanding of the effects of cultivation methods and commercial distribution channels regarding the microbiome of oyster mushrooms and may inform potential intervention strategies for future production and distribution processes. Furthermore, the tandem analyses of culture-dependent and culture-independent methods can provide more comprehensive information than that obtained when using each approach independently.

Development of Strategies to Minimize the Risk of Listeria monocytogenes Contamination in Radish, Oriental Melon, and Carrots.

Contamination by Listeria monocytogenes in packaged produce is a major concern. The purpose of this study was to find natural and affordable sanitizers to reduce L. monocytogenes contamination in agricultural products. Organic acids, ultraviolet-C (UV-C), and ethanol were analyzed either alone or in combination to assess their ability to reduce L. monocytogenes population in radish, oriental melon, and carrot samples. In radish samples, 3% malic acid combined with UV-C at a dosage of 144 mj/cm2 significantly reduced (>4 log CFU/g) the population of L. monocytogenes (1.44 ± 0.5) compared to the control sample (5.14 ± 0.09). In the case of the melon samples, exposure to UV-C at a dosage of 144 mj/cm2 combined with 3% lactic acid (2.73 ± 0.75) or 50% ethanol (2.30 ± 0.01) was effective against L. monocytogenes compared to the control sample (5.10 ± 0.19). In carrot samples, 3% lactic acid combined with 144 mj/cm2 dosage UV-C reduced L. monocytogenes population (4.48 ± 0.25) more than in the control sample (5.85 ± 0.08). These results reveal that sanitizers that are effective for one crop are less effective for another crop indicating that effective prevention methods should be customized for each crop to prevent pathogen cross contamination during postharvest washing.

Colonization of Listeria monocytogenes in potting soils as affected by bacterial community composition, storage temperature, and natural amendment.

This study aimed to characterize the bacterial community of commercial potting soils with or without Listeria monocytogenes inoculation at 5-35 °C using 16S metagenomic sequencing and evaluate the effect of natural amendments on the reduction L. monocytogenes in non-sterile potting soils. An increase in the expected operational taxonomic units of each sample with or without L. monocytogenes was proportional to the increasing storage temperatures after 5 days. Biodiversity was distinct among all potting soils for Shannon and inverse Simpson indices, with the highest diversity being observed in a soil sample stored at 35 °C for 5 days with L. monocytogenes. An increase in richness and diversity of soil bacterial community structure positively correlated with less survival of the invading L. monocytogenes. Particularly, garlic extract was demonstrated as a promising soil-amendment substrate, reducing L. monocytogenes by ≥ 4.50 log CFU/g in potting soils stored at 35 °C. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00925-9.

A colorimetric Loop-mediated isothermal amplification (LAMP) assay based on HRP-mimicking molecular beacon for the rapid detection of Vibrio parahaemolyticus.

In the world wide, food poisoning accidents related to Vibrio spp. are on the rise, even numbers of food poisoning by other foodborne pathogens are decreasing. Therefore, the requirement of the rapid, sensitive and convenient detection method for V. parahaemolyticus has been grown. The objective of this study is to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay using a molecular beacon (HRPzyme connected with complementary oligonucleotides at the 5' and 3' ends) for the rapid, sensitive, and convenient detection of V. parahaemolyticus. The colorimetric LAMP assay optimized at 58.8°C showed a detection limit of 1 × 100 CFU/mL and was confirmed to be specific to V. parahaemolyticus. The colorimetric LAMP assay can be finished within 1 h including DNA extraction step. The method was successfully applied to flatfish samples artificially inoculated with known amount of V. parahaemolyticus, and its cut-off value for the flatfish samples was 1 × 101 CFU/g. In addition, the colorimetric LAMP assay developed in the study was found to be able to correct false-positive results, which are known to be a disadvantage of conventional LAMP assays. Therefore, these results indicated that the colorimetric LAMP method is a useful tool for the rapid, sensitive and convenient detection of V. parahaemolyticus in fishes and can also be used as a point-of-care molecular diagnostic technique since it does not require any expensive equipment such as a thermocycler and detectors.

Population changes and growth modeling of Salmonella enterica during alfalfa seed germination and early sprout development.

This study examined the effects of alfalfa seed germination on growth of Salmonella enterica. We investigated the population changes of S. enterica during early sprout development. We found that the population density of S. enterica, which was inoculated on alfalfa seeds was increased during sprout development under all experimental temperatures, whereas a significant reduction was observed when S. enterica was inoculated on fully germinated sprouts. To establish a model for predicting S. enterica growth during alfalfa sprout development, the kinetic growth data under isothermal conditions were collected and evaluated based on Baranyi model as a primary model for growth data. To elucidate the influence of temperature on S. enterica growth rates, three secondary models were compared and found that the Arrhenius-type model was more suitable than others. We believe that our model can be utilized to predict S. enterica behavior in alfalfa sprout and to conduct microbial risk assessments.

Development of a Screening Method and Device for the Detection of Escherichia coli from Agri-Food Production Environments and Fresh Produce.

This study was conducted to develop a screening method using Colilert-18 and a device for the detection of E. coli from agri-food production environments and fresh vegetables. The specificity and sensitivity of Colilert-18 by temperature (37°C and 44°C) were evaluated with 38 E. coli and 78 non-E. coli strains. The false-positive rate was 3.8% (3/78) and 0% (0/78) at 37°C and 44°C, respectively. The detection limit of E. coli at 37°C at <1.0 log CFU/250 ml was lower than that at 44°C. The efficiency of the developed device, which comprised an incubator equipped with a UV lamp to detect E. coli in the field, was evaluated by measuring the temperature and UV lamp brightness. The difference between the set temperature and actual temperature of the developed device was about 1.0°C. When applying the developed method and device to various samples, including utensils, gloves, irrigation water, seeds, and vegetables, there were no differences in detection rates of E. coli compared with the Korean Food Code method. For sanitary disposal of culture samples after experiments, the sterilization effect of sodium dichloroisocyanurate (NaDCC) tablets was assessed for use as a substitute for an autoclave. The addition of one tablet of NaDCC per 50 ml was sufficient to kill E. coli cultured in Colilert-18. These results show that the developed protocol and device can efficiently detect E. coli from agri-food production environments and vegetables.

Enrichment Broth for the Detection of Campylobacter jejuni and Campylobacter coli in Fresh Produce and Poultry.

Although campylobacteriosis caused by Campylobacter jejuni and Campylobacter coli has been increasingly reported worldwide owing to the consumption of contaminated poultry and fresh produce, the current detection protocols are not selective enough to inhibit unspecific microbes other than these pathogens. Five antibiotics were separately added to Bolton broth, and the survival rates of 18 Campylobacter spp. and 79 non-Campylobacter spp. were evaluated. The survival rate of the non-Campylobacter spp. was the lowest in Bolton broth with rifampin (6.3%), followed by cefsulodin (12.7%), novobiocin (16.5%), and potassium tellurite and sulfamethozaxole (both 17.7%). Also the most effective concentration of rifampin was found to be 12.5 mg/L, which markedly inhibited non-Campylobacter strains while not affecting the survival of Campylobacter strains. After the Campylobacter spp. were enriched in Bolton broth supplemented with 12.5 mg/L rifampin (R-Bolton broth), CampyFood Agar (CFA) was found to be better in selectively isolating the pathogens in the enrichment broth than the International Organization for Standardization method of using modified charcoal cefoperazone deoxycholate agar (mCCDA) for this step. When applied to natural food samples-here, romaine lettuce, pepper, cherry tomato, Korean leek, and chicken-the R-Bolton broth-CFA combination decreased the number of false-positive results by 50.0, 4.2, 20.8, 50.0, and 94.4%, respectively, compared with the International Organization for Standardization method (Bolton broth-mCCDA combination). These results demonstrate that the combination of R-Bolton broth and CFA is more efficient in detecting C. jejuni and C. coli in poultry and fresh produce and thus should replace the Bolton broth-mCCDA combination.

Genetic Diversity and Antibiotic Resistance Patterns of Staphylococcus Aureus Isolated from Leaf Vegetables in Korea.

Staphylococcus aureus is an important foodborne pathogen on global basis. The current study investigated the genetic patterns in S. aureus isolates from leaf vegetables (n = 53). Additional isolates from livestock (n = 31) and humans (n = 27) were compared with the leaf vegetable isolates. Genes associated with toxins, antibiotic resistance, and pulsed-field gel electrophoresis (PFGE) patterns were analyzed. At least 1 enterotoxin-encoding gene (sea, seb, sec, sed, and see) was detected in 11 of 53 (20.75%) leaf vegetable isolates. When the agr (accessory gene regulator) grouping was analyzed, agr II was the major group, whereas agr IV was not present in leaf vegetable isolates. All S. aureus isolates from leaf vegetables were resistant to more than one of the antibiotics tested. Nineteen of 53 (35.85%) isolates from leaf vegetables exhibited multidrug-resistance, and 11 of these were MRSA (methicillin-resistant S. aureus). A dendrogram displaying the composite types of S. aureus isolates from 3 origins was generated based on the combination of the toxin genes, agr genes, antibiotic resistance, and PFGE patterns. The isolates could be clustered into 8 major composite types. The genetic patterns of S. aureus isolates from leaf vegetables and humans were similar, whereas those from livestock had unique patterns. This suggests some S. aureus isolates from leaf vegetables to be of human origin.

Gene-related strain variation of Staphylococcus aureus for homologous resistance response to acid stress.

This study investigated the effect of adaptation of Staphylococcus aureus strains to the acidic condition of tomato in response to environmental stresses, such as heat and acid. S. aureus ATCC 13565, ATCC 14458, ATCC 23235, ATCC 27664, and NCCP10826 habituated in tomato extract at 35°C for 24 h were inoculated in tryptic soy broth. The culture suspensions were then subjected to heat challenge or acid challenge at 60°C and pH 3.0, respectively, for 60 min. In addition, transcriptional analysis using quantitative real-time PCR was performed to evaluate the expression level of acid-shock genes, such as clpB, zwf, nuoF, and gnd, from five S. aureus strains after the acid habituation of strains in tomato at 35°C for 15 min and 60 min in comparison with that of the nonhabituated strains. In comparison with the nonhabituated strains, the five tomato-habituated S. aureus strains did not show cross protection to heat, but tomato-habituated S. aureus ATCC 23235 showed acid resistance. In quantitative real-time-PCR analysis, the relative expression levels of acid-shock genes (clpB, zwf, nuoF, and gnd) were increased the most in S. aureus ATCC 23235 after 60 min of tomato habituation, but there was little difference in the expression levels among the five S. aureus strains after 15 min of tomato habituation. These results indicate that the variation of acid resistance of S. aureus is related to the expression of acid-shock genes during acid habituation.

Comparison of sample preparation methods for the recovery of foodborne pathogens from fresh produce.

Sample preparation methods (pummeling, pulsifying, sonication, and shaking by hand) were compared for achieving maximum recovery of foodborne pathogens from iceberg lettuce, perilla leaves, cucumber, green pepper, and cherry tomato. Antimicrobial and dehydration effects also were examined to investigate causes of poor recovery of pathogens. Each produce type was inoculated with Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus at 6.0 log CFU/cm(2), and samples were prepared using the four methods. Bacterial populations recovered from the five types of produce were significantly different (P < 0.05) according to sample preparation methods and produce type. The bacterial populations recovered from pummeled and pulsified samples were higher (P < 0.05) than those recovered from sonicated and hand-shaken samples, except for cherry tomato. The number of bacteria recovered from produce was reduced (P < 0.05) from that of the inoculum by 0.16 to 2.69 log CFU/cm(2). Although extracts of iceberg lettuce, perilla leaves, cucumber, and green pepper had no antimicrobial activity, the populations of E. coli O157:H7, Salmonella Typhimurium, B. cereus, and L. monocytogenes in cherry tomato extract were slightly reduced after these treatments (P < 0.05). The pathogen populations on perilla leaves and cherry tomatoes decreased by >2 log CFU/cm(2) after exposure to 40% relative humidity for 1 h. No reduction was observed when the five pathogens were exposed to 90% relative humidity. These data suggest that pummeling and pulsifying are optimal sample preparation methods for detection of microorganisms. Acidic produce such as cherry tomato should be treated with a method that does not cause sample breakdown so that acid stress on the bacteria can be minimized. Dehydration stress also affects recovery of pathogens from produce.