Novel hypothesis for infant methemoglobinemia: Survival and metabolism of nitrite-producers from vegetables under gastrointestinal stress and intestinal adhesion.

Infants have digestive environments that are more favorable for microbial proliferation and subsequent endogenous nitrite production than those of adults, but direct evidence of this has been lacking. In this study, we propose a novel epidemiology of infant methemoglobinemia by demonstrating the risk posed by nitrite-producers in the gastrointestinal tract. Nitrite-producers from vegetables (n = 323) were exposed to stress factors of the gastrointestinal environment (gastric pH, intestinal bile salts, anaerobic atmosphere) reflecting 4 different postnatal age periods (Neonate, ≤1 month; Infant A, 1-3 months; Infant B, 3-6 months; Infant C, 6-12 months). "High-risk" strains with a nitrate-to-nitrite conversion rate of ≥1.3 %, the minimum rate corresponding to nitrite overproduction, under the Neonate stress condition were analyzed for intestinal adhesion. Among all the phyla, Pseudomonadota achieved the highest survival (P < 0.05; survival rate of 51.3-71.8 %). Possible cross-protection against bile resistance due to acid shock was observed for all the phyla. All the high-risk strains exhibited moderate autoaggregation (14.0-36.4 %), whereas only a few exhibited satisfactory surface hydrophobicity (>40 %). The Pantoea agglomerans strain strongly adhered to Caco-2 cells (7.4 ± 1.1 %). This study showed the ability of the Pantoea, Enterobacter, and Klebsiella strains to survive under gastrointestinal stress for ≤12 months, to excessively produce nitrite under neonatal stress conditions, and to settle in the human intestine. To our knowledge, this is the first study to reveal the role of the natural flora of vegetables in the epidemiology of infant methemoglobinemia through a multilateral approach.

Prevalence and phylogenetic traits of nitrite-producing bacteria in raw ingredients and processed baby foods: Potential sources of foodborne infant methemoglobinemia.

Nitrite, which has been mainly regarded as a chemical hazard, can induce infant methemoglobinemia. As for nitrite as a product of microbial metabolism, the contribution of the oral or gut microbiome has mostly received attention, whereas the role of nitrite-producing bacteria (NPBs) in food has been less elucidated. In this study, mesophilic NPBs were isolated from food samples (n = 320) composed of raw ingredients for weaning foods (n = 160; beetroot, broccoli, carrot, lettuce, rice powder, spinach, sweet potato, and honey) and processed baby foods (n = 160; cereal snack, cheese, yogurt, powdered infant formula, sorghum syrup, vegetable fruit juice, and weaning food). The phylogenetic diversity of the NPB strains was analyzed via 16S rRNA sequencing. All 15 food items harbored NPBs, with a prevalence of 71.9 % and 34.4 % for the raw ingredients and processed foods, respectively. The NPBs isolated from the foods were identified as Actinomycetota (Actinomycetes), Bacteroidota (Flavobacteriia, Sphingobacteriia), Bacillota (Bacilli), or Pseudomonadota (Alpha-, Beta-, and Gammaproteobacteria). Among the raw and processed foods, beetroot (85.0 %) and powdered infant formula (70.0 %) showed had the highest NPB prevalence (P > 0.05). Bacillota predominated in both types of food. The contamination source of Pseudomonadota, which was another major phylum present in the raw ingredients, was presumed to be the soil and endophytes in the seeds, whereas that of Bacillota was the manufacturing equipment used with the raw ingredients. Common species for probiotics, such as Lacticaseibacillus, Leuconostoc, Enterococcus, and Bacillus, were isolated and identified as NPBs. To our knowledge, this is the first study to reveal the taxonomical diversity and omnipresence of NPBs in food for babies. The results of this study highlight the importance of food-mediated microbiological risks of infant methemoglobinemia which are yet underrecognized.

One Health approach for prioritization of potential foodborne pathogens: Risk-ranking, Delphi survey, and criteria evaluation pre- and post-COVID-19 pandemic.

Frequent foodborne illnesses with unknown causative agents highlight the need to explore zoonotic potential foodborne pathogens (PFPs). An effective PFP prioritization tool is indispensable, especially after experiencing the recent pandemic caused by zoonotic SARS-CoV-2. Risk information on pathogens (excluding 30 known foodborne pathogens) provided by governmental and international organizations was reviewed to generate a list of PFPs. Risk-ranking of PFPs was conducted based on a literature review of food poisoning or detection cases, and the ranks were determined with a decision tree. PFPs were prioritized by infectious disease (ID), veterinary medicine (VET), and food safety (FS) experts through a pre- and postpandemic Delphi survey, and key criteria in their decisions were illuminated. Among 339 PFPs, 32 rank-1 PFPs were involved in the foodborne outbreak(s). Discrepancies in opinions on prioritization between experts in different fields deepened after the pandemic. Only VET and FS experts valued the plausibility of foodborne transmission in evaluating bacteria and viruses, and a significant correlation between their selection of PFPs was found (p < .05). The impact of the pandemic induced all fields to focus more on human transmission and severity/fatality in prioritizing viruses, and only FS experts emphasized the plausibility of foodborne transmission after the pandemic. In contrast to prioritizing bacteria or viruses, ID and VET experts are unusually focused on foodborne transmission when prioritizing parasites. Criteria of consensus deduced by interdisciplinary experts with different interests and the criteria directly related to foodborne transmission should be acknowledged for adequate PFP prioritization.

Cultivation-free sample preparation and DNA purification for direct real-time qPCR of intracellular or spore-like Coxiella burnetii in beef, goat, and lamb meat.

Coxiella burnetii is a zoonotic pathogen that has been associated with foodborne outbreaks in products with ruminant origins. However, a method to detect C. burnetii in meat has been merely studied, and commercial kits cannot efficiently fulfill this purpose. In this study, an in-house preparation method for direct real-time qPCR of C. burnetii in beef, goat, and lamb meat was designed. In the sample preparation step (step 1), trypsin digestion and cell disruption techniques were introduced to target C. burnetii in an obligate intracellular or spore-like form. Afterward, 16 DNA purification protocols involving the following steps (steps 2-3) were assessed: the precipitation of meat proteins (step 2; using 2.5, 5.0 M NaCl or 1:1, 2:1 ethanol as the precipitant) and binding of DNA to silicon dioxide particles with chaotropic salts (step 3; using 2.5, 5.0 M NaCl or 2.5, 5.0 M guanidine thiocyanate as the salt). The protocols with superior performance in high-spiked loins (estimated 4-5 log cells/g) were verified in low-spiked (1-2 log cells/g) or Bacillus thuringiensis spore-inoculated (1-2 log CFU/g) loins, ribs, and hind legs. During the protein precipitation, 5.0 M NaCl induced significantly lower protein level as demonstrated by A280, when compared to 2.5 M NaCl or ethanol (P < 0.05). For the DNA binding step, Ct values were lowered in high-spiked goat or lamb loins (3.5-6.0▾; P < 0.05) when the concentration of NaCl was doubled or guanidine thiocyanate was introduced instead of NaCl as a chaotropic salt. Based on these results, two protocols using 5.0 M NaCl as the protein precipitant and 5.0 M NaCl (N2 + N2) or guanidine thiocyanate (N2 + G2) as the chaotropic salt were selected, which demonstrated successful detection in low-spiked (Ct values of N2 + N2, 32.9-35.6; N2 + G2, 32.3-36.4) or spore-inoculated meat (N2 + N2, 30.9-37.5; N2 + G2, 29.7-32.7). Verification in low-spiked meat showed that meat type/part significantly impacted the Ct values of N2 + G2 but not those of N2 + N2. To our knowledge, this is the first study that developed a highly accessible method for detecting C. burnetii in meat which could reveal the possibility of meat-borne Q fever in humans.

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.

Characterization of ready-to-eat fish surface as a potential source of contamination of Vibrio parahaemolyticus biofilms.

The worldwide consumption of ready-to-eat seafood products has steadily increased due to a range of health benefits. However, depending on the handling or cutting process of raw fish, ready-to-eat sashimi can be exposed to microbiological risks that can lead to foodborne infection by marine pathogens. Since surface characteristics are key factors for microbial adhesion and biofilm formation, the present study aims to determine the correlation between raw fish skin properties and Vibrio parahaemolyticus biofilm formation. We analyzed V. parahaemolyticus biofilms (ATCC 17802; initially inoculated ca. 2 or 4 log CFU/cm2) on fish skin (gizzard shad, pomfret, red snapper, and mackerel; fish species served as sashimi without peeling the skin) formed under simulated marine environments (incubating in artificial seawater with rocking motion at 30 °C, the maximum temperature of seasonal seawater) for 24 h. The characteristics of fish skin were determined using confocal laser scanning microscopy/scanning electron microscopy. V. parahaemolyticus showed higher biofilm counts on fish skins than on stainless steel, which was used as a control (P < 0.05). In particular, V. parahaemolyticus formed biofilms with significantly higher levels of bacterial populations on gizzard shad and pomfret (ca. 5 log CFU/cm2; P < 0.05), highlighting the relationship between the biofilm formation level and the characteristics of gizzard shad and pomfret skins. The surface roughness of fish skins, including the main roughness parameters (Ra, Rq, and Rz), influenced the attachment of V. parahaemolyticus (P < 0.05). Additionally, images of V. parahaemolyticus biofilms suggested that different topographical profiles of fish species (e.g., mucus, unique structural features, etc.) could cause V. parahaemolyticus to exhibit different biofilm phenotypes, such as sticking to or entangling on the fish skin surface. The major findings of this study provide various phenotypic adhesions of V. parahaemolyticus to fish skin in considerations of the potential hazard for the consumption of ready-to-eat sashimi served with its skin.

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.

Prevalence and phylogenetic analysis of human enteric emerging viruses in porcine stool samples in the Republic of Korea.

Emerging infectious diseases (EID) in humans and animals are proving to be a serious health concern. This study investigated the prevalence of emerging or re-emerging human enteric viruses in porcine stools and swabs. Eleven enteric EID viruses were selected as target viruses for the current study and ranked based on their impact on public health and food safety: enterovirus (EV), hepatitis E virus, norovirus GI and GII, sapovirus (SaV), adenovirus (AdV), astrovirus, rotavirus, hepatitis A virus, aichivirus, and bocavirus. Using real-time RT-PCR or real-time PCR, EID viruses were detected in 129 (86.0%) of 150 samples. The most prevalent virus was EV, which was detected in 68.0% of samples, followed by AdV with a detection rate of 38.0%. In following sequencing and phylogenetic analyses, 33.0% (58/176) of the detected viruses were associated with human enteric EID viruses, including AdV-41, coxsackievirus-A2, echovirus-24, and SaV. Our results show that porcine stools frequently contain human enteric viruses, and that few porcine enteric viruses are genetically related to human enteric viruses. These findings suggest that enteric re-emerging or EID viruses could be zoonoses, and that continuous monitoring and further studies are needed to ensure an integrated "One Health" approach that aims to balance and optimize the health of humans, animals, and ecosystems.

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.

Improvement of nested reverse transcription-polymerase chain reaction (RT-PCR) with high specificity and sensitivity detection of sapovirus in food matrix.

Sapovirus (SaV) is a causative agent of human gastroenteritis in both community outbreaks and sporadic cases worldwide. Shellfish accumulate a variety of pathogens during filter feeding. In particular, the contamination of shellfish by SaV has caused several outbreaks. As reported previously, nested RT-PCR (nRT-PCR) has been widely used in clinical samples, but has not proven suitable for food samples, such as oysters. This study aimed to identify a primer set for the detection of SaV with high specificity and sensitivity in food samples. To accomplish this, primers were improved in RNA-dependent RNA polymerase (RdRp) regions of SaV whole genome sequences. The sensitivity of the improved nRT-PCR was 100-1000 times higher than that of previous nRT-PCR and > 10 times higher than that of the previous real-time RT-PCR assay. Notably, cross-reaction with other viruses or food matrices was not observed by the specificity test. This study improved the reliable primer set to detect SaV in various food matrices with high sensitivity.

Genotypes of Blastocystis sp. among elderly health checkup people in South Korea with a questionnaire on risk factors.

Blastocystis sp. is a common zoonotic intestinal parasite of humans and animals, and has been classified into at least 17 distinct subtypes. Despite its potential impact on public health, the prevalence and subtype distribution of Blastocystis sp. have seldom been the study subject in South Korea. To determine the prevalence and subtype distribution of Blastocystis sp. and to obtain information on risk factors, we performed a cross-sectional study targeting elderly health checkup people, who visited Seoul Western Branch of the Korea Association of Health Promotion (KAHP) in October 2019. Stool samples were collected from 293 participants consisting of 128 males and 165 females with a mean age of 64.7 years (from 50 to 88 years) with a questionnaire on potential risk factors. All the samples were tested by PCR targeting the SSU rRNA gene of Blastocystis sp., and nucleotide sequences of positive samples were used to identify the subtypes. The overall prevalence of Blastocystis sp. was 9.2% (27/293). Among the positive samples, subtype 3 was predominant (59%; 16/27), followed by subtype 1 (41%; 11/27). No other subtypes were detected. In the univariable analysis, the age, sex, presence of digestive symptoms, source of drinking water, and history of drug intake were not significantly associated with Blastocystis sp. infection. Two parameters, including the Enterococcus hirae bacterial infection and the frequency of intake of cooked or boiled vegetables less than twice a week, showed statistical significance. However, the multivariable regression analysis revealed that only the latter parameter was statistically significant. The results suggested that subtypes 3 and 1 are the 2 major genotypes of Blastocystis sp. among elderly people in South Korea, and low frequency of consuming cooked or boiled vegetables is a potential risk factor.

Pathogenicity and seasonal variation of Aeromonas hydrophila isolated from seafood and ready-to-eat sushi in South Korea.

Aeromonas hydrophila is an emerging foodborne pathogen capable of causing human gastroenteritis, and the main reservoir is the aquatic environment. In this study, the prevalence and virulence of A. hydrophila in seafoods and ready-to-eat (RTE) sushi distributed in various conditions (refrigerated, dried, or frozen) or seasons was investigated. Strains were isolated from seafood (refrigerated or frozen oysters, sashimi, and processed fish; n = 333) and RTE sushi (n = 88) samples collected in South Korea and then genetically analyzed for gastroenteritis-related virulence genes (aer, ast, and alt). Raw oysters showed the highest prevalence of A. hydrophila (57.1%; 47/91) among all seafoods. Among the sashimi samples, flatfish sashimi (54.8%; 34/62) and salmon sushi (51.4%; 18/35) were the most prevalent. A. hydrophila was not detected in the oysters or anchovies distributed as either frozen or dried products. Seasonal investigations of sashimi and sushi showed that the summer prevalence of A. hydrophila with putative virulence genes was significantly lower in sashimi but highest in sushi. These results indicated that sushi could have been contaminated from several sources during the manufacturing or distribution processes. Significant correlations among the prevalence of putative virulence genes were confirmed, although no combination of genes presented a Phi correlation coefficient above 0.5 (0.26-0.43). To our knowledge, this is the first study to investigate the prevalence of A. hydrophila in various types of retail seafoods and RTE sushi in the East Asia region and then relate the prevalence to the distribution conditions of the samples. This study provides background information on the level of potential risk posed by A. hydrophila in retail seafoods and RTE sushi.

Microbial ecology of alfalfa, radish, and rapeseed sprouts based on culture methods and 16S rRNA microbiome sequencing.

Sprouts harbor high populations of bacteria and cause numerous foodborne disease outbreaks, yet little is known about their microbial composition. The present study aimed to define the microbiological ecology of sprouts using 16S rRNA microbiome sequencing and culture-dependent methods. Different types (radish, alfalfa, and rapeseed), brands (A, B, and C), and distribution routes (online and offline) of sprouts (n = 70) were considered for microbiome analysis, as well as quantitative (aerobic plate count and coliforms) and qualitative analyses (Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium). The aerobic plate count ranged from 7 to 8 CFU/g, and the coliforms ranged from 6 to 7 log CFU/g. Microbiome analysis revealed that Proteobacteria was the dominant phylum, accounting for 79.0% in alfalfa sprouts, 68.5% in rapeseed sprouts, and 61.9% in radish sprouts. Enterobacteriaceae was the dominant family in alfalfa sprouts (33.9%) and rapeseed sprouts (14.6%), while Moraxellaceae (11.9%) were prevalent on radish sprouts. The majority of the dominant genera were common in the environment, such as soil or water. Alfalfa sprouts yielded the lowest aerobic plate count but the highest relative abundance of Enterobacteriaceae compared to the other sprouts. These results could explain why alfalfa sprouts are a leading cause of sprout-related foodborne disease outbreaks. Alpha-diversity results (Chao1 and Shannon indices) suggested that species richness was greater on radish sprouts than the other sprout types. Beta-diversity results showed samples were clustered by types, indicating dissimilarity in microbial communities. However, the distribution route had a limited influence on microbial composition. The present study provides a comparative examination of the microbial profiles of sprouts. Microbiome analyses contribute to an in-depth understanding of the microbial ecology of sprouts, leading to potential control measures for ensuring food safety.

Contamination of Clostridium perfringens in soy sauce, and quantitative microbial risk assessment for C. perfringens through soy sauce consumption.

The objective of this study was to conduct QMRA (quantitative microbial risk assessment) of Clostridium perfringens through soy sauce consumption. Four hundred and ninety soy sauce samples from markets were analyzed to detect C. perfringens. Temperature and time were also measured during transportation and display of soy sauce. A primary model was developed by fitting the Weibull model to the C. perfringens cell counts in soy sauce at 7-35°C, and δ (the time needed to decrease 1 log CFU/ml) and ρ (curve shape) were calculated. The parameters were analyzed, using the Exponential model (secondary model) as a function of temperature. The consumption amount and percentage of soy sauce were surveyed, and a dose-response model was searched. Using all collected data, a simulation model was prepared in the @RISK program to estimate the probability of C. perfringens foodborne illness by soy sauce consumption. C. perfringens were negative in 490 samples. Thus, the initial contamination level was estimated to be -2.9 log CFU/ml. The developed predictive models showed that C. perfringens cell counts decreased during transportation and display. The average consumption amounts, and the percentage of soy sauce were 7.81 ml and 81.2%, respectively. The simulation showed that the probability of C. perfringens foodborne illness by consumption of soy sauce was 1.7 × 10-16 per person per day. Therefore, the risk of C. perfringens by consumption of soy sauce is low in Korea.

Toxic potential of Bacillus cereus isolated from fermented alcoholic beverages.

Pathogenicity of Bacillus cereus is associated with the production of various toxins. This study investigated the distribution of toxin genes encoding haemolysin BL (hblA, hblC, and hblD), nonhemolytic enterotoxin (nheA, nheB, and nheC), enterotoxin FM (entFM), cytotoxin K (cytK), and cereulide (ces) in 185 B. cereus strains isolated from draft beer, microbrewed beer, pasteurized beer, grape wine, other fruit wine, refined rice wine, traditional Korean pure liquor, and traditional Korean turbid rice wines. A total of 93.0% (172 isolates) of these isolates possessed at least one toxin gene. The nonhemolytic enterotoxin-encoding genes were highly prevalent in the isolates; the detection rate of enterotoxins was 91.4% for nheC, 81.6% for entFM, 62.7% for nheB, 57.3% for nheA, 53.0% for hblC, 48.6% for cytK, 36.8% for hblA, and 36.2% for hblD. Overall, 54.6% and 33.0% of strains carried the integrated Nhe-encoding gene cluster (nheA, nheB, nheC at the same time) and had the Hbl-encoding gene cluster, respectively. The cereulide synthetase gene was detected in only 2.2% of isolates. Toxin gene distribution patterns could be classified into 8 major profile clusters, and the most prevalent profile was the presence of enterotoxin genes only and no emetic toxin genes. Therefore, B. cereus in fermented alcoholic beverages was predominantly of the diarrhoeagenic type. Our results may provide important basic information when considering microbial standards and regulations for B. cereus in related products.

Changes in Consumers' Food Purchase and Transport Behaviors over a Decade (2010 to 2019) Following Health and Convenience Food Trends.

Although consumers' food purchase/transport have been reported as causes of food safety risks, there is a lack of empirical data that are feasible to identify persistent and emerging risky behaviors of consumers. This longitudinal trend study consists of individual consumer surveys in 2010 (n = 609) and 2019 (n = 605) to analyze changes in risky behaviors linked to food purchase/transport over a decade. Overall, the results identified purchase/transport time and purchase order as the emerging and unchanged risk factors, respectively. Consumers' preferences into channels for purchase (large discount stores rather than small/traditional markets) and transport (using cars or delivery) implied the convenience as the noticeable trend. Whereas, unexpected increases in purchase/transport time highlighted the underestimated risks in long-term exposure of foods under inadequate temperature. Food should not be exposed to danger zones > 1-2 h, but consumers might be unaware of the risk especially for preferred channels (e.g., 77 and 36 min. are required for purchase and transport from large discount stores, respectively). In the case of unchanged risky behavior, more than half of consumers in both surveys did not follow proper purchasing orders. Our findings highlight the necessity for novel countermeasures and the improvement of current consumer guidelines against emerging and unchanged risky behaviors, respectively.

Author Correction: Risk factors influencing contamination of customized cosmetics made on-the-spot: Evidence from the national pilot project for public health.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Labchip-based diagnosis system for on-site application: Sensitive and easy-to-implement detection of single recoverable Cronobacter in infant formula without post-enrichment treatment.

Microfluidic labchips have achieved much advancement in the molecular diagnosis of foodborne pathogens. Whereas difficulties in the flow control during the transportation of liquid fluids can occur and should be overcome. Manipulations of reaction temperature and the complex procedures from sample pre-treatment to analysis in a single chip device are major obstacles for the on-site application. Thus, the efficient temperature control of samples without any flow of reaction fluids in microfluidic channels of plastic chip and the simplest protocol omitting post-enrichment processing steps may overcome these limitations represented by the stability and the complexity, respectively. This study aims to develop a novel type of labchip and thermocycler specialized for the gene amplification in microfluidic channels and to evaluate the detectability by sensing the minimum recoverable level of Cronobacter in powdered infant formula (PIF). We developed a thermocycling device accelerating reactions through dual heating-blocks optimized to control temperatures of samples in microfluidic-channels by direct contact with labchip sequentially and repetitively. The structural design of microfluidic channels was to eliminate interference factors associated with the optical detection of fluorescent signals (without distortion due to air bubbles in the reaction chamber). To improve the applicability, a portable device and simplified operation to allow direct loading of samples in the chip without post-enrichment procedures were also adopted. Detection performance was evaluated by a sensitivity/specificity tests using 50 isolates of Cronobacter. Cross-reactivity tests for non-Cronobacter organisms and gDNA [human, raw materials of PIF (cow, soybean)] showed that there was no interference-factor causing false-positive results. In terms of the applied research conducted by using PIF, the enrichment of samples without broth medium (distilled water) displayed outstanding performance and 12 h of incubation facilitated detecting target at concentration as low as 1 CFU/300 g PIF (as initial contamination level) without post-enrichment treatment. Validation of the operation conditions using 30 commercial PIF products was also consistent. The present study presents a novel approach of microfluidic technology with perspective to not only the performance and the practicability [easy-to-implement protocol, portable materials, cost-effectiveness (the use of a miniaturized plastic chip requires a minimum level of materials)] for on-site diagnosis.