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.

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.

Survival of Salmonella Enteritidis on four types of stainless steel surface under a dry condition and recovery by swabbing.

The survival and recovery of Salmonella Enteritidis inoculated on stainless steel surfaces with different metal contents and surface finishes were examined. Two S. Enteritidis strains possessing different levels of biofilm productivity were inoculated with tryptone soya broth (TSB) and egg yolk emulsion (EY) on the surface of stainless steel squares (1 cm × 1 cm) and stored at 22℃ under a dry condition. After storage, cells were recovered from the stainless steel surfaces by swabbing with a cotton swab. The numbers of cells recovered by swabbing and the cells remaining on the stainless steel squares were counted. The survival ratio of the strain possessing high biofilm productivity was greater than that of the strain possessing low biofilm productivity. The survival ratio of S. Enteritidis suspended in TSB was often higher than that in EY. There were no significant differences in the survival and recovery ratios of S. Enteritidis based on stainless steel composition or surface finish. From all except one sample, more than 98% of viable cells of S. Enteritidis were recovered by swabbing with a cotton swab.

Microbial contamination associated with consumption and the growth in plastic bottled beverage.

Plastic bottles enable the storage of unfinished beverages, and most of microbial contamination has occurred in the unfinished beverage that was left. Therefore, we investigated microorganisms in various beverages contaminated by pouring and drinking directly by mouth from the bottle, and analyzed the growth of microorganisms in the beverages at room temperature. In the pouring test, microbial growth was detected in 60 of 320 samples, and 13 bacterial strains, 49 mold strains, and 8 yeast strains were isolated. Molds including Cladosporium spp., Tramets spp., Bjerkandera spp., and Penicillium spp. accounted for the majority of isolated microorganisms. In the drinking test, microbial growth was detected in 181 of 352 samples, and 225 bacterial strains, 27 mold strains and 77 yeast strains were isolated. Bacteria including Streptococcus spp. such as S. salivarius and Staphylococcus spp. such as S. aureus accounted for the majority of isolated microorganisms. Enterotoxin-producing S. aureus and Bacillus cereus were also isolated. The pH of the beverage influenced the growth of bacteria. The Brix values of the beverage did not correlate with the growth of microorganisms. These results revealed that various microorganisms including foodborne pathogens were able to grow in numerous types of beverages and that the storage of unfinished beverage in inappropriate condition, such as the storage at room temperature led microorganism to grow easily in beverage. Therefore, it is necessary to consume beverages as soon as possible after opening the bottle.

Decontamination of Vibrio parahaemolyticus in fish by washing with hygienic seawater and impacts of the high level contamination in the gills and viscera.

The effect of washing in Vibrio parahaemolyticus contaminated and hygienic seawater on fish, and the frequency and level of natural V. parahaemolyticus contamination in fish were investigated. In the first experiment, live horse mackerel was experimentally kept in seawater artificially contaminated with V. parahaemolyticus. After washing in contaminated and hygienic seawater, the contamination in fish was quantitatively analyzed. Washing fish in the seawater contaminated with V. parahaemolyticus increases the contamination level on the surface and in the gills of the fish. Washing in hygienic seawater was effective in reducing the contamination in fish and cutting board surfaces, but not in the gills or viscera. In the second experiment, natural V. parahaemolyticus contamination in various fish caught by us was analyzed. V. parahaemolyticus was detected in 6 of 28 gill samples and 10 of 28 viscera samples of naturally contaminated fish. The means of V. parahaemolyticus level on gills were 3.3 and 3.9 log cfu/g, and those in viscera were 2.6 and 4.4 log cfu/g by culture method and a real-time PCR assay, respectively. These results indicate that the gills and viscera are able to spread the pathogens to fish meat as well as fish surface contamination by washing in the contaminated seawater. Washing with hygienic seawater and control of contamination from gills and viscera are critically important to prevent V. parahaemolyticus infections.

Characteristics of a sharp decrease in Vibrio parahaemolyticus infections and seafood contamination in Japan.

Vibrio parahaemolyticus has been one of the most important foodborne pathogens in Japan since the 1960s, and a large epidemic was caused by the pandemic serotype O3:K6 from 1997 to 2001. V. parahaemolyticus infections, however, have sharply declined since that time. Data on serotypes isolated from 977 outbreaks were collected and analysed. Total and pathogenic, thermostable direct hemolysin (TDH) gene-positive V. parahaemolyticus were qualitatively and quantitatively detected in 842 seafood samples from wholesale markets in 2007-2009. Strains isolated from patients and seafood were analysed by serotyping, tdh-PCR, group-specific PCR for pandemic strains, and pulsed-field gel electrophoresis (PFGE). The sharp decrease in the infections from 1999 onwards was noted not only for O3:K6 infections but also for other serotypes. The change in the seafood contamination situation from 2001 to 2007-2009 was characterised by a decrease to three-fourths in the frequency of tdh-positive samples, although that decrease was small compared to the 18-fold decrease in the cases of V. parahaemolyticus outbreaks. PFGE detected the pandemic O3:K6 serotype in the same profile in seafood and patients from 1998 to the present. Because of no large decrease in seafood contamination by V. parahaemolyticus from the production to distribution stages and the presence of pandemic O3:K6 serotype in seafood to the present, it was suggested that the change of seafood contamination was unrelated to the sharp decrease in V. parahaemolyticus infections. V. parahaemolyticus infections might be prevented at the stages after the distribution stage.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Vibrio parahaemolyticus.

Loop-mediated isothermal amplification (LAMP) assays targeting the rpoD and toxR genes were developed to detect Vibrio parahaemolyticus. All 78 tested V. parahaemolyticus strains yielded positive results within 40 min, while negative results were obtained for 69 strains of other organisms even at 60 min. For V. parahaemolyticus ATCC 17802 in pure culture, the detection limits of LAMP assays targeting rpoD and toxR were 3.7 and 450 CFU per test, respectively. Due to the higher sensitivity of rpoD-LAMP, it was further evaluated for the ability to detect V. parahaemolyticus in seafood samples. V. parahaemolyticus populations spiked in short-necked clams were enumerated by the most-probable-number (MPN) method combined with the rpoD-LAMP assay and the MPN method with a culture method using agar medium. The MPN-rpoD-LAMP method had better sensitivity and was more rapid than the conventional method. These results indicate that the MPN-LAMP assay targeting the rpoD gene is a specific, sensitive, and rapid method to enumerate V. parahaemolyticus organisms.

Quantitative analysis of mycoflora on commercial domestic fruits in Japan.

A comprehensive and quantitative analysis of the mycoflora on the surface of commercial fruit was performed. Nine kinds of fruits grown in Japan were tested. Overall fungal counts on the fruits ranged from 3.1 to 6.5 log CFU/g. The mean percentages of the total yeast counts were higher than those of molds in samples of apples, Japanese pears, and strawberries, ranging from 58.5 to 67.0%, and were lower than those of molds in samples of the other six fruits, ranging from 9.8 to 48.3%. Cladosporium was the most frequent fungus and was found in samples of all nine types of fruits, followed by Penicillium found in eight types of fruits. The fungi with the highest total counts in samples of the various fruits were Acremonium in cantaloupe melons (47.6% of the total fungal count), Aspergillus in grapes (32.2%), Aureobasidium in apples (21.3%), blueberries (63.6%), and peaches (33.6%), Cladosporium in strawberries (38.4%), Cryptococcus in Japanese pears (37.6%), Penicillium in mandarins (22.3%), and Sporobolomyces in lemons (26.9%). These results demonstrated that the mycoflora on the surfaces of these fruits mainly consists of common pre- and postharvest inhabitants of the plants or in the environment; fungi that produce mycotoxins or cause market diseases were not prominent in the mycoflora of healthy fruits. These findings suggest fruits should be handled carefully with consideration given to fungal contaminants, including nonpathogenic fungi, to control the quality of fruits and processed fruit products.

Enumeration of fungi in fruits by the most probable number method.

In this study, enumeration methods for fungi in foods were evaluated using fruits that are often contaminated by fungi in the field and rot because of fungal contaminants. As the test methods, we used the standard most probable number (MPN) method with liquid medium in test tubes, which is traditionally used as the enumeration method for bacteria, and the plate-MPN method with agar plate media, in addition to the surface plating method as the traditional enumeration method for fungi. We tested 27 samples of 9 commercial domestic fruits using their surface skin. The results indicated that the standard MPN method showed slow recovery of fungi in test tubes and lower counts than the surface plating method and the plate-MPN method in almost all samples. The fungal count on the 4th d of incubation was approximately the same as on the 10th d by the surface plating method or the plate-MPN method, indicating no significant differences between the fungal counts by these 2 methods. This result indicated that the plate-MPN method had a number agreement with the traditional enumeration method. Moreover, the plate-MPN method has a little laborious without counting colonies, because fungal counts are estimated based on the number of plates with growing colonies. These advantages demonstrated that the plate-MPN method is a comparatively superior and rapid method for enumeration of fungi.

Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification.

Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

Detection of Verotoxigenic Escherichia coli O157 and O26 in food by plating methods and LAMP method: a collaborative study.

In order to establish a rapid and sensitive method for the detection of Verotoxigenic Escherichia coli O157 and O26, a collaborative study was conducted focusing on a comparison of the efficiency of loop-mediated amplification (LAMP) assay targeting the Verocytotoxin (also called Shiga toxin) gene, utilizing a direct plating method and a plating method with immunomagnetic separation (IMS-plating method) using various agar media. In combination with enrichment with the modified EC supplemented with novobiocin, E. coli O157 was detected in most samples of ground beef and alfalfa sprouts by LAMP assay, the direct plating method and the IMS-plating method. E. coli O26 was detected in approximately 100% of the food samples by LAMP assay. However, the IMS-plating and direct plating methods recovered 80 and 50% in ground beef samples, respectively. As a result, it was demonstrated the LAMP assay is superior to the IMS-plating method. Based on these results, it appears LAMP assay is effective as a screening assay to detect E. coli O157 and O26 from positive samples.

Efficiency of real-time polymerase chain reaction assay to detect Vibrio vulnificus in seawater.

The growth of Vibrio vulnificus in an enriched culture of seawater during the summer in Japan was monitored by a plating technique used as the culture method and a real-time polymerase chain reaction (PCR) assay as the molecular method. V. vulnificus was detected by the real-time PCR assay in the samples of August and September but not by the culture method. Vibrio parahaemolyticus, however, was detected among all of the samples with both the culture method and real-time PCR assay. In the analysis of the bacterial populations in enrichment culture, it was demonstrated that the growth of V. vulnificus on agar media was inhibited by the rapid growth of V. parahaemolyticus after 4h of incubation and the 100 times larger initial populations of bacteria other than V. vulnificus and V. parahaemolyticus. These findings demonstrate that V. vulnificus detection by culture methods is a failure, and molecular methods are effective and detect V. vulnificus accurately.

Development of a quantitative real-time PCR method to enumerate total bacterial counts in ready-to-eat fruits and vegetables.

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the beta subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.

Development of a quantitative real-time PCR method for estimation of the total number of Vibrio parahaemolyticus in contaminated shellfish and seawater.

A real-time PCR method targeting the toxR gene of Vibrio parahaemolyticus was developed to quantify the number of V. parahaemolyticus cells, including those of both the hemolysin-producing and nonproducing strains. The specificity of the primer and probe set was confirmed using 25 strains of V. parahaemolyticus and 30 strains of other microbial species. We determined the threshold cycle number using the real-time PCR and the number of V. parahaemolyticus cells by plate count using serially diluted pure culture and developed a standard curve for quantification. Standard curves for V. parahaemolyticus in seawater and seafood were established using artificially inoculated samples. The threshold cycle number and the number of V. parahaemolyticus cells were correlated with 10(1) to 10(7) CFU/ml in pure culture, seawater, and shellfish homogenate. The real-time PCR method developed in this study was compared with the most-probable-number method in seafood samples that were naturally contaminated. The differences in the number of V. parahaemolyticus cells as determined by the culture method and the PCR method were less than 10-fold.

Development of a quantitative real-time polymerase chain reaction targeted to the toxR for detection of Vibrio vulnificus.

The TaqMan assay, a quantitative real-time polymerase chain reaction (PCR), was developed to target the ToxR gene (toxR) of Vibrio vulnificus. The toxR of V. vulnificus was cloned and sequenced. Based on these results, we designed specific primers and a probe for use in the quantitative PCR assay. Twenty-nine strains of V. vulnificus that were obtained from various sources produced a single PCR product. The amount of final amplification product and threshold cycle number were the same among the strains. We used the method to detect V. vulnificus in seawater and oyster samples. We developed standard curves to quantitate V. vulnificus numbers using the PCR from seawater and oyster samples. The standard curves were not different from that of the pure culture of V. vulnificus. We found the assay was very sensitive detecting as few as 10 microbes per milliliter of seawater and oyster homogenate. Moreover, we evaluated the TaqMan assay to detect V. vulnificus in seawater samples. The numbers of V. vulnificus counted by the TaqMan assay were similar to those by a culture method in almost samples. The TaqMan assay was performed within 2 h compared to days using the culture method. The results indicate the TaqMan assay method used in this study was rapid, effective and quantitative for monitoring V. vulnificus contamination in seawater and seafoods such as oysters.

Comparative evaluation of the Desoxycholate Agar Nissui Food Stamp and swab methods for estimating coliform organisms in poultry processing plants after cleaning in Japan.

Bacterial control in poultry processing plants is very important, but the swab method for estimating bacterial contamination is somewhat troublesome in routine work. We compared the Desoxycholate Agar Nissui Food Stamp (DA-NFS) based on the agar contact method with the swab method to estimate coliform organisms from various equipments in four poultry processing plants after cleaning. Overall 104 surfaces for coliform organisms were evaluated. The results from 98 (94.2%) surfaces for coliform organisms were equivalent by the DA-NFS and swab methods and there were no significant differences between two methods (P > 0.05). The correlation coefficient between the DA-NFS and swab methods was 0.91. We conclude that the DA-NFS could be useful for routine coliform organisms examination in poultry processing plants after cleaning in Japan.

Ribotyping and a study of transmission of Staphylococcus aureus collected from food preparation facilities.

Food poisoning from Staphylococcus aureus is sometimes caused by improper handling of food items in food preparation facilities. Prevention of contamination by employees is particularly important in facilities where a significant amount of food preparation is performed by hand. Some experiments have been performed to describe bacterial cross-contamination in the food preparation process, but there have been few studies of cross-contamination in actual food preparation facilities. Aiming to shed light on the transmission of S. aureus in food preparation facilities, this study collected samples of 66 strains of this bacterium from the fingers of food preparation staff, foodstuffs, prepared foods, cooking utensils, and cooking equipment and typed them with the ribotyping method. S. aureus from the same ribogroup was detected on the hands of a study participant, a faucet, knife, frying pan, and a salad, indicating that bacteria found on the hands of the study participant was transmitted to cooking utensils and prepared foods. Transmission (from a faucet to a frying pan handle) of bacteria by another person, a third party, was also detected.

Growth kinetics of Vibrio parahaemolyticus O3:K6 under varying conditions of pH, NaCl concentration and temperature.

The growth responses of Vibrio parahamolyticus to pH, NaCl concentration and temperature changes were studied using serotype O3:K6 and other strains. Growth curves were obtained for 27 different sets of conditions, comprised of three levels of NaCl concentration, pH and temperature. The temperature, pH and NaCl concentrations most favorable for growth were in the order of 25 degrees C, 20 degrees C and 15 degrees C, pH 8, 7 and 5.8, and 1%, 3% and 7%, respectively. The bacteria grew most rapidly at 25 degrees C, at a pH of 7 or 8 in the presence of 1% or 3% NaCl, with the population (initial, ca. 2.5 log CFU/mL) reaching a level log 7 CFU/mL at 12 h. A growth predictive model using the Gompertz equation was generated from the experimental data for any combination of NaCl concentration, pH and temperature within the range used in this study.

A multi-prefectural outbreak of Shigella sonnei infections associated with eating oysters in Japan.

Among roughly one thousand incidents of shigellosis annually in Japan, approximately 70% of the cases are estimated to be associated with overseas travel. However, at the end of 2001, reports of domestically acquired Shigella sonnei infections suddenly increased. We report here the first multi-prefectural outbreak of Shigella sonnei infections linked to the consumption of imported oysters in Japan at the end of 2001. Isolates of S. sonnei from patients epidemiologically linked to eating contaminated oysters and from the imported oysters themselves showed an indistinguishable pulsed-field gel electrophoresis pattern and drug resistance pattern.

Prevalence of pandemic thermostable direct hemolysin-producing Vibrio parahaemolyticus O3:K6 in seafood and the coastal environment in Japan.

Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.