Uniform Suspension of Heat-Killed Staphylococcus aureus for a Positive Control Used in the Monocyte-Activation Test.

Pyrogens, classified as bacterial endotoxins and non-endotoxin pyrogens (NEPs), induce fever or shock when released into the bloodstream or spinal fluid. Recently, a monocyte-activation test (MAT) involving human cell culture has been developed to detect pyrogens in injectable products. To evaluate the sensitivity of MAT, a reference standard endotoxin was used as a positive control; however, the reactivity differed between the endotoxins and NEPs, necessitating positive controls for NEPs. This study aimed to explore a preparation method for heat-killed Staphylococcus aureus (HKSA) as a positive control for NEPs in MAT. Because S. aureus forms grape-like clusters, nine types of glass filters with pore sizes of 0.5-2.7 µm were evaluated to obtain a uniform bacterial suspension. The suspension was then heat-treated to kill the bacteria, resulting in HKSA samples. Serial dilutions of HKSA were tested by MAT using peripheral blood mononuclear cells. The interleukin-6 concentrations in the culture supernatant were measured by enzyme-linked immuno-sorbent assay to assess pyrogenic activities of HKSA. The pore sizes of the glass filters affected the uniformity of HKSA, and GF/C filter was selected for HKSA preparation. Repeated filtration improved uniformity, and a uniform suspension of HKSA was obtained through double filtration using a GF/C filter. Despite the decrease in HKSA activity as filtration frequency increased, the detection limit remained consistently unchanged. This suggests that repeated filtration can adjust the activity of HKSA to a baseline level and that a uniform suspension of HKSA exhibiting low variation is suitable as a positive control in MAT.

[Detection of Salmonella and Enumeration of Hygienic Indicator Bacteria in Dried Wood Ear Mushrooms].

An outbreak of Salmonella Stanley in the United States associated with dried wood ear mushrooms imported from China prompted us to conduct serotyping of Salmonella isolated from dried wood ear mushrooms in voluntary testing, and quantitative test for Salmonella along with enumeration of hygienic indicator bacteria in positive samples in order to evaluate the risk of Salmonella outbreak from dried wood ear mushrooms. The major serovars of Salmonella isolates obtained from 20 samples were as follows: O3,10 group-London (n=3) and Weltevreden (n=5) etc, totaling 9 strains; O4 serogroup-Saintpaul (n=2), Stanley (n=1), Typhimurium (including monophasic variant; n=3), totaling 6 strains. O7 serogroup (Potsdam) and O8 serogroup (Newport) were one strain each. Qualitative and quantitative tests for Salmonella were conducted on 10 samples with remaining amounts. As a result, one sample was 220 MPN/g, six samples were<0.6 MPN/g, and three samples were negative for Salmonella per 25 g. The mean aerobic bacterial counts and coliforms in these samples were 7.8 and 6.1 log10 CFU/g, respectively. Furthermore, qualitative test for Salmonella and enumeration of hygienic indicator bacteria were conducted on dried wood ear mushroom products (33 domestic and 30 imported products) retailed in Japan. No samples showed positive for Salmonella per 25 g, and the mean aerobic bacterial counts and coliforms were approximately 2 log10 CFU/g lower than those in the 10 samples where Salmonella was isolated during voluntary testing. While no Salmonella was detected in domestically retailed wood ear mushrooms products, the serovars associated with foodborne diseases were isolated from voluntary testing samples. It indicates that potential for consumption of Salmonella contaminated wood ear mushrooms, which is at risk of causing food poisoning.

Atypical diarrhoeagenic Escherichia coli in milk related to a large foodborne outbreak.

A foodborne outbreak related to milk cartons served in school lunches occurred in June 2021, which involved more than 1,800 cases from 25 schools. The major symptoms were abdominal pain, diarrhoea, vomiting, and fever. Although major foodborne toxins and pathogens were not detected, a specific Escherichia coli strain, serotype OUT (OgGp9):H18, was predominantly isolated from milk samples related to the outbreak and most patients tested. The strains from milk and patient stool samples were identified as the same clone by core genome multilocus sequence typing and single-nucleotide polymorphism analysis. The strain was detected in milk samples served for two days related to the foodborne outbreak at a rate of 69.6% and levels of less than ten most probable number/100 mL but not on days unrelated to the outbreak. The acid tolerance of the strain for survival in the stomach was similar to that of enterohaemorrhagic E. coli O157:H7, and the same inserts in the chu gene cluster in the acid fitness island were genetically revealed. The pathogenicity of the strain was not clear; however, it was indicated that the causative pathogen was atypical diarrhoeagenic E. coli OUT (OgGp9):H18.

[Suitability of Culture Broth and Conditions for Escherichia coli Growth and Gas Production as a Test for Food Additives in EC Broth].

The growth and gas production test for Escherichia coli in the microbiological examination of food additives is stipulated in the ninth edition of Japan's Specifications and Standards for Food Additives (JSFA) and described as a part of the "Confirmation Test for Escherichia coli" in "Microbial Limit Tests" in the same manuscript. The growth and gas production test for E. coli indicated that the positive or negative of "gas production and/or turbidity" in EC broth should be confirmed after incubating at 45.5±0.2℃ for 24±2 h. If both gas production and turbidity are negative, the culture is additionally incubated up to 48±2 h to determine E. coli contamination. The internationally referenced Bacteriological Analytical Manual of the U.S. FDA had revised the incubation temperature in tests for coliforms and E. coli from 45.5±0.2℃ to 44.5±0.2℃ in 2017. Therefore, we conducted research in anticipation of this temperature change being reflected in the microbiological examination of the JSFA. We used seven EC broth products and six food additives across eight products that are available in Japan in order to compare the growth and gas production at temperatures of 45.5±0.2℃ and 44.5±0.2℃ of E. coli NBRC 3972, which is designated as the test strain in JSFA. Both with/without food additives, the number of EC broth products in which medium turbidity and gas production by the strain were positive in three out of three tubes at all test times was greater at 44.5±0.2℃ than at 45.5±0.2℃. These results suggest that the growth and gas production test for E. coli could be more appropriately conducted by incubation at 44.5±0.2℃ in the "Confirmation Test for Escherichia coli" for E. coli in the JSFA in comparison to 45.5±0.2℃. Furthermore, there were differences in the growth and gas production of E. coli NBRC 3972 depending on the EC broth product used. Therefore, the importance of "Media growth promotion test" and "Method suitability test" in the ninth edition of the JSFA should be emphasized.

Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry Analysis for the Direct Detection of SARS-CoV-2 in Nasopharyngeal Swabs.

The most common diagnostic method used for coronavirus disease-2019 (COVID-19) is real-time reverse transcription polymerase chain reaction (PCR). However, it requires complex and labor-intensive procedures and involves excessive positive results derived from viral debris. We developed a method for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs, which uses matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF MS) to identify specific peptides from the SARS-CoV-2 nucleocapsid phosphoprotein (NP). SARS-CoV-2 viral particles were separated from biological molecules in nasopharyngeal swabs by an ultrafiltration cartridge. Further purification was performed by an anion exchange resin, and purified NP was digested into peptides using trypsin. The peptides from SARS-CoV-2 that were inoculated into nasopharyngeal swabs were detected by MALDI-ToF MS, and the limit of detection was 106.7 viral copies. This value equates to 107.9 viral copies per swab and is approximately equivalent to the viral load of contagious patients. Seven NP-derived peptides were selected as the target molecules for the detection of SARS-CoV-2 in clinical specimens. The method detected between two and seven NP-derived peptides in 19 nasopharyngeal swab specimens from contagious COVID-19 patients. These peptides were not detected in four specimens in which SARS-CoV-2 RNA was not detected by PCR. Mutated NP-derived peptides were found in some specimens, and their patterns of amino acid replacement were estimated by accurate mass. Our results provide evidence that the developed MALDI-ToF MS-based method in a combination of straightforward purification steps and a rapid detection step directly detect SARS-CoV-2-specific peptides in nasopharyngeal swabs and can be a reliable high-throughput diagnostic method for COVID-19.

The Development and Evaluation of a Selective Enrichment for the Detection of Escherichia albertii in Food.

Escherichia albertii is an emerging pathogen causing foodborne infections with diarrhea, abdominal pain, and fever. E. albertii has been isolated from various food sources, such as chicken and pork. Although many foodborne outbreaks of E. albertii have been reported, the causative food has not been identified. It is necessary to develop effective detection methods for E. albertii. Because enrichment procedure as the first step of food test is important for growing pathogens, this study aimed to develop a novel effective enrichment for E. albertii detection in food. In this study, we investigated the optimal concentration and combination of cefixime and tellurite for supplementing modified EC broth (mEC) to effectively isolate E. albertii from chicken meat. The results showed that mEC supplemented with 50 µg/L cefixime and 2.5 mg/L tellurite (CT-mEC) inhibited the growth of competitive bacteria in chicken meat but not that of E. albertii. Therefore, it was indicated that CT-mEC had strong potential to selectively grow E. albertii. In an E. albertii foodborne outbreak, CT-mEC was evaluated. E. albertii was successfully isolated from a food sample, a kind of salad, by enrichment with CT-mEC but not buffered peptone water and mEC. In this study, CT-mEC as a selective enrichment broth has been developed to detect E. albertii in chicken meat. It was demonstrated that the selective enrichment broth was effective for the efficient detection of E. albertii in food.

Development of a Novel Real-Time Polymerase Chain Reaction Assay to Detect Escherichia albertii in Chicken Meat.

Escherichia albertii is an emerging enteropathogen. Several foodborne outbreaks of E. albertii have been reported in Japan; however, foods associated with most outbreaks remain unidentified. Therefore, polymerase chain reaction (PCR) assays detecting E. albertii specifically and sensitively are required. Primers and probe for real-time PCR assays targeting E. albertii-specific gene (EA-rtPCR) was designed. With 74 strains, including 43 E. albertii strains and several of its close relatives, EA-rtPCR specifically amplified E. albertii; therefore, the sensitivity of EA-rtPCR was then evaluated. The detection limits were 2.8 and 2.0-3.2 log colony-forming unit (CFU)/mL for E. albertii culture and enriched chicken culture inoculated with the pathogen, respectively. In addition, E. albertii was detected from 25 g of chicken meat inoculated with 0.1 log CFU of the pathogen by EA-rtPCR. The detection of E. albertii from chicken meat by EA-rtPCR was also evaluated by comparing with the nested-PCR assay, and 28 retail chicken meat and 193 dissected body parts from 21 chicken carcass were tested. One and three chicken meat were positive in the nested-PCR assay and EA-rtPCR, respectively. Fourteen carcasses had at least one body part that was positive for EA-rtPCR, and 36 and 48 samples were positive for the nested-PCR assay and EA-rtPCR, respectively. A total of 37 strains of E. albertii were isolated from seven PCR-positive samples obtained from six chicken carcass. All E. albertii isolates harbored eae gene, and were classified as E. albertii O-genotype (EAOg)3 or EAOg4 by EAO-genotyping. The EA-rtPCR developed in this study has potential to improve E. albertii detection in food and advance research on E. albertii infection.

[Literature Review on the Type of Fish and Histamine-producing Bacteria Associated with Histamine Poisonings in Japan].

Histamine poisoning has been reported worldwide. Improvements in refrigeration technology have led to a reduction in this food poisoning; however, it continues to occur. Misdiagnosis of fish allergies has compounded this problem and the number of patients subjected to histamine poisoning that are transported to the emergency ward because of anaphylactic shock-like symptoms should not be underestimated. We investigated incidents of histamine food poisoning in Japan from 1998 to 2020, and found that there were a mean 9.7 incidents/year and 195.3 cases/year. Facility-wise occurrence of the incidents per year was the highest in restaurants followed by lunch facilities, and these together accounted for approximately 70% of the incidents. Facility-wise total number of cases was the highest in lunch facilities followed by restaurants, and these together accounted for 80% of the cases. Fish associated with histamine poisoning were mainly tuna, marlin, and mackerel. Based on the current literature review, 23 genera of histamine-producing bacteria were isolated from fish purchased in Japan. The most frequently reported bacteria were Morganella morganii and Photobacterium damselae. Psychrophilic bacteria such as Morganella psychrotolerans and Photobacterium phosphoreum were also isolated. To prevent histamine poisoning, freezing or fast handling of fish and the products during processing and consuming is important because only refrigeration of fish is enough.

Amine skeleton-based c-di-GMP derivatives as biofilm formation inhibitors.

Bacteria can form a biofilm composed of diverse bacterial microorganism, which work as a barrier to protect from threats, such as antibiotics and host immunity system. The formation of biofilms significantly impairs the efficacy of antibiotics against pathogenic bacteria. It is also a serious problem to be solved that the emergence of multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus, MRSA) accelerated by the overuse of antibiotics. Therefore, the usage of biofilm inhibition agents has attracted immense interest as a novel strategy for treatment of diseases related to bacterial infection. From the difference of mode of action against bacterial cells, biofilm inhibition agents are expected to circumvent the emergence of multidrug-resistant bacteria. In this study, we have developed the derivatives of c-di-GMP, a kind of cyclic dinucleotide that is expected to have the effect of inhibiting bacterial biofilm formation. Some of the synthesized derivatives were found to inhibit biofilm formation of Gram-positive bacteria.

[Contamination of Pathogenic Escherichia coli in Retail Fresh Vegetables in the Mekong Delta, Vietnam].

From July 2017 to January 2019, total of 645 retail fresh vegetables collected from 19 retail shops and markets was investigated to know the contamination of enterohemorrhagic Escherichia coli (EHEC) and enterotoxigenic E. coli (ETEC). Of 645 samples, 2 samples (0.3%) were positive for pathogenic E. coli. Of 2 pathogenic E. coli positive samples, 1 was EHEC (stx2 positive) and the other was ETEC (sta positive). Two pathogenic E. coli strains were isolated from crisphead lettuce. EHEC strain was not serotyped by commercial antisera and ETEC was serotyped as O20. EHEC and ETEC strains showed multi-drug resistance against 4 and 7 antibiotics, respectively. These results indicate that retail fresh vegetables seem to be not an important source of human EHEC and ETEC infection in the Mekong Delta, Vietnam.

[Assessment of Modified Forms of Fumonisins in Corn-Based Products Retailed in Japan by an Alkaline Hydrolysis Method].

Fumonisins, which are secondary metabolites produced by some Fusarium species, are detected mainly in corn and corn-based products. Recently, the presence of modified forms of fumonisins in fumonisin-contaminated food products has been reported. In order to evaluate the health risk of modified forms of fumonisins to the Japanese population, we analyzed modified forms of fumonisins in corn-based products retailed in Japan. The modified and free forms of fumonisins in food samples were hydrolyzed by alkaline treatment. The resulting hydrolyzed fumonisins were quantified by LC-MS/MS, and total fumonisins (sum of modified and free forms) was calculated. A total of 166 samples of corn-based products were analyzed over two years. The relative ratios of mean total fumonisins to mean free fumonisins in the cornflakes, corn snacks, corn flour and powdered corn soup samples were 4.7, 2.8, 2.1 and 1.2, respectively. Total fumonisins in the residual solid of five cornflake and three corn snack samples obtained after extraction with methanol-water (3 : 1) were quantified. In the cornflakes and corn snacks samples, 56-72 and 83-98% of the modified forms of fumonisins were present in the residual solid, respectively. The average daily intake of fumonisins from cornflakes and corn snacks by the Japanese population was estimated at 1.1 to 3.9 ng/kg body weight/day when the results of free fumonisins were used for the estimate, but when the results of total fumonisins were used, average daily intake increased about three times and was estimated at 3.3 to 12.5 ng/kg body weigh/day. These results indicate that a risk assessment of fumonisins, including the modified forms of fumonisins, is necessary in order to evaluate the true risk of fumonisins to Japanese people.

[Discovery of Putative Anti-Kudoa septempunctata Chemical Agents by Comprehensive Assay].

We screened 360 chemicals and discovered that 71 chemicals had anti-Kudoa septempunctata effect. Especially 19 and seven of 71 chemicals were antibiotics and antibacterial agents/disinfectants, respectively. The other 45 chemicals were pesticides, natural toxins, industrial chemicals and medicines for non-infectious diseases. Nineteen antibiotics that possessed anti-Kudoa effect contained four tetracyclines, one steroid, two macrolides, one aminoglycoside, three ß-lactams, one quinolone, two rifamycines, one polyene, one novobiocine, one sulfonamide and two nitroimidazoles. To use these drugs for prevention of Kudoa infection, the further study is need for the determination of effective dose.

Host Range-Associated Clustering Based on Multilocus Variable-Number Tandem-Repeat Analysis, Phylotypes, and Virulence Genes of Atypical Enteropathogenic Escherichia coli Strains.

Atypical enteropathogenic Escherichia coli (aEPEC) strains (36 Japanese and 50 Bangladeshi) obtained from 649 poultry fecal samples were analyzed by molecular epidemiological methods. Clermont's phylogenetic typing showed that group A was more prevalent (58%, 50/86) than B1 (31%, 27/86). Intimin type ß1, which is prevalent among human diarrheal patients, was predominant in both phylogroups B1 (81%, 22/27) and A (70%, 35/50). However, about 95% of B1-ß1 strains belonged to virulence group I, and 77% of them were Japanese strains, while 17% (6/35) of A-ß1 strains did. Multilocus variable-number tandem-repeat analysis (MLVA) distributed the strains into 52 distinct profiles, with Simpson's index of diversity (D) at 73%. When the data were combined with those of 142 previous strains from different sources, the minimum spanning tree formed five zones for porcine strains, poultry strains (excluding B1-ß1), strains from healthy humans, bovine and human patient strains, and the B1-ß1 poultry strains. Antimicrobial resistance to nalidixic acid was most common (74%) among the isolates. Sixty-eight percent of them demonstrated resistance to ≥3 antimicrobial agents, and most of them (91%) were from Bangladesh. The strains were assigned into two groups by hierarchical clustering. Correlation matrix analysis revealed that the virulence genes were negatively associated with antimicrobial resistance. The present study suggested that poultry, particularly Japanese poultry, could be another reservoir of aEPEC (phylogroup B1, virulence group I, and intimin type ß1); however, poultry strains seem to be apart from patient strains that were closer to bovine strains. Bangladeshi aEPEC may be less virulent for humans but more resistant to antibiotics.IMPORTANCE Atypical enteropathogenic Escherichia coli (aEPEC) is a diarrheagenic type of E. coli, as it possesses the intimin gene (eae) for attachment and effacement on epithelium. Since aEPEC is ubiquitous even in developed countries, we previously used molecular epidemiological methods to discriminate aEPEC as a human pathogen. The present study assessed poultry as another source of human diarrheagenic aEPEC. Poultry could be the source of aEPEC (phylogroup B1, virulence group I, and intimin type ß1) found among patient strains in Japan. However, the minimum spanning tree (MST) suggested that the strains from Japanese poultry were far from Japanese patient strains compared with the distance between bovine and patient strains. Bangladeshi avian strains seemed to be less diarrheagenic but are hazardous as a source of drug resistance genes.

[DNA Extraction for Sensitive Detection of Shiga Toxin-Producing Escherichia coli in Food by Real-Time PCR Assays].

Alkali-heat DNA extraction, a rapid and economical method, was evaluated for use in the detection of Shiga toxin-producing Escherichia coli in food using real-time PCR assays. Alkali-heat DNA extracts led to highly sensitive detection (102-104 CFU/mL) of stx and O-antigen genes in beef liver, ground beef, sliced pork, cheese, lettuce, radish sprouts, tomato, and spinach, equivalent to the sensitivity obtained using a commercial DNA extraction kit that utilizes proteinase K lysis, and silica membrane purification. Although there were differences in DNA concentration and purity between DNA extraction methods, the sensitivity of real-time PCR assays was similar. These results indicate that alkali-heat DNA extraction is a viable method when testing food products with real-time PCR assays for the presence of stx and O-antigen genes.

[Study of Tea and Coffee on Inactivation of Kudoa septempunctata].

The inhibition of Kudoa septempunctata by green tea extract, black tea extract, and coffee extract were studied. Incubation of about 104 Kudoa spores with green tea extract, black tea extract, or coffee extract at 25℃ for 4 hr reduced the survival ratio of Kudoa to 0%. While coffee extract and green tea extract contain approximately 2 and 1 mM of caffeine, respectively, the incubation of Kudoa spores with 2 and 1 mM of caffeine reduced its survival ratio to 68.2 and 93.3%, respectively. Although green tea extract and black tea extract contain over 1 mM of catechin, incubation with 0.01 mM of catechin was enough to reduce the survival ratio of Kudoa to 20%. These results suggested that green tea extract, black tea extract, and coffee extract have strong inhibitory effects on Kudoa and the effects of green tea extract and black tea extract are mainly manifested through catechin.

[Major Vehicles and O-Serogroups in Foodborne Enterotoxigenic Escherichia coli Outbreaks in Japan, and Effective Detection Methods of the Pathogen in Food Associated with An Outbreak].

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen in developing countries, and causes foodborne infections through contaminated vegetables and water. ETEC also caused some foodborne infections in developed countries, though the vehicles are often unclear. We analyzed ETEC foodborne outbreaks in Japan based on the National Food Poisoning Statistics. Vegetables and private well water accounted for 50% and 22.2% of vehicles, respectively. The main vehicles were similar to those in developing countries. Serogroups of ETEC were also analyzed, and O6, O25, O27, O148, O153, O159, and O169 were the seven major O-serogroups. We investigated suitable detection methods for the pathogen (O148) in food samples associated with an outbreak of ETEC in Japan in 2011. We show that ETEC O148 could be effectively detected in cut leeks by means of a two-step enrichment and real-time PCR assay targeting heat-stable enterotoxin gene. Our survey of the vehicles and the major O-serogroups of ETEC outbreaks in Japan indicates that ETEC survives in the environment in Japan.

Epidemiological evidence of lesser role of thermostable direct hemolysin (TDH)-related hemolysin (TRH) than TDH on Vibrio parahaemolyticus pathogenicity.

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

[Molecular Detection Methods for Vibrio parahaemolyticus in Seafood].

To detect Vibrio parahaemolyticus in seafood, we evaluated efficient combinations of molecular methods with DNA extraction methods using heat extraction and alkaline heat extraction, and PCR, real-time PCR and loop-mediated isothermal amplification (LAMP) assays were performed targeting V parahaemolyticus species-specific genes (tlh and rpoD) and pathogenic factors genes (tdh and trh). The species-specific genes were detected in all combinations of two strains (a tdh * trh1-positive strain and a trh2-positive strain), two kinds of shellfish (oyster and bloody clams) and molecular methods with tlh-real time PCR or rpoD-LAMP assays with DNA of alkaline heat extraction at 85-145cfu/test level. tdh was detected in both seafoods with real time PCR assay with DNA of heat extraction at 85cfu/test level, and detected with the LAMP and real time PCR assays with DNA of alkaline heat extraction at 85cfu/test level. Detection of both trh1 and trh2 with the PCR assay with DNA of alkaline heat extraction was comparatively high though trh2 was detected with the LAMP assay with DNA of alkaline heat extraction at 145cfu/test level. It, however, is necessary to investigate more sensitive trh-detection methods. In this study, the results indicated that tlh-real time PCR or rpoD-LAMP, tdh-real time PCR and tdh-LAMP assays with DNA of alkaline heat extraction are relatively-sensitive methods to detect V. parahaemolyticus in seafood.

Characteristics of bacterial and fungal growth in plastic bottled beverages under a consuming condition model.

Microbial contamination in unfinished beverages can occur when drinking directly from the bottle. Various microorganisms, including foodborne pathogens, are able to grow in these beverages at room temperature or in a refrigerator. In this study, we elucidated the characteristics of microorganism growth in bottled beverages under consuming condition models. Furthermore, we provide insight into the safety of partially consumed bottled beverages with respect to food hygiene. We inoculated microorganisms, including foodborne pathogens, into various plastic bottled beverages and analysed the dynamic growth of microorganisms as well as bacterial toxin production in the beverages. Eight bottled beverage types were tested in this study, namely green tea, apple juice drink, tomato juice, carbonated drink, sport drink, coffee with milk, isotonic water and mineral water, and in these beverages several microorganism types were used: nine bacteria including three toxin producers, three yeasts, and five moulds. Following inoculation, the bottles were incubated at 35°C for 48 h for bacteria, 25°C for 48 h for yeasts, and 25°C for 28 days for moulds. During the incubation period, the number of bacteria and yeasts and visible changes in mould-growth were determined over time. Our results indicated that combinations of the beverage types and microorganism species correlated with the degree of growth. Regarding factors that affect the growth and toxin-productivity of microorganisms in beverages, it is speculated that the pH, static/shaking culture, temperature, additives, or ingredients, such as carbon dioxide or organic matter (especially of plant origin), may be important for microorganism growth in beverages. Our results suggest that various types of unfinished beverages have microorganism growth and can include food borne pathogens and bacterial toxins. Therefore, our results indicate that in terms of food hygiene it is necessary to consume beverages immediately after opening the bottle.

Effective Decontamination Methods for Shiga Toxin-producing Escherichia coli on Beef Surfaces for Application in Beef Carcass Hygiene.

Effective methods for decontamination of Shiga toxin-producing Escherichia coli (STEC) on beef were evaluated by 48 mL spraying, 100 mL, and 500 mL flushing with ethanol, hydrogen peroxide, peracetic acid, acidified sodium chlorite, and sodium hypochlorite in this study. The flushing with 500 mL of 1,000 ppm peracetic acid was most effective, reducing pathogens by 2.8 log CFU/cm2, followed by 1,200 ppm acidified sodium chlorite. The spraying with 1,000 ppm peracetic acid reduced pathogens by 1.6 log CFU/cm2. The flushing with 500 mL of 200 and 500 ppm acidified sodium chlorite, and 50, 100, 200, and 500 ppm peracetic acid significantly reduced the STEC population compared with those treated with distilled water (p < 0.05), reducing pathogens by 2.1, 2.4, 1.6, 1.8, 2.1 and 2.4 log CFU/cm2, respectively. Additionally, the flushing with 500 mL of 200 and 500 ppm acidified sodium chlorite significantly changed the color of beef samples (p < 0.05), whereas 100-500 ppm peracetic acid did not significantly change the color (p > 0.05). The flushing with 500 mL of 200 and 500 ppm acidified sodium chlorite and 200 and 500 ppm peracetic acid significantly changed the odor of beef samples compared with those treated with distilled water (p < 0.05). There was no difference in the reduction of STEC population between peracetic acid treatment at 25 °C and 55 °C, with or without washing with sterilized distilled water after decontamination. Washing with distilled water after flushing with peracetic acid tended to reduce the odor of the samples. These results suggest that treatment with 100, 200, and 500 ppm peracetic acid, followed by washing with distilled water, might reduce the STEC population without retaining the odor of the sanitizer.