Pyrosequencing analysis of the microbiota of kusaya gravy obtained from Izu Islands.

Kusaya is a salted, dried fish product traditionally produced on the Izu Islands in Japan. Fish are added to kusaya gravy repeatedly and intermittently, and used over several hundred years, which makes unique microbiota and unique flavors. In this study, we performed a metagenomic analysis to compare the composition of the microbiota of kusaya gravy between different islands. Twenty samples obtained from a total of 13 manufacturers on three islands (Hachijojima, Niijima, and Oshima Islands) were analyzed. The statistical analysis revealed that the microbiota in kusaya gravy maintain a stable composition regardless of the production steps, and that the microbiota are characteristic to the particular islands. The bacterial taxa common to all of the samples were not necessarily the dominant ones. On the other hand, the genera Halanaerobium and Tissierella were found to be characteristic to the microbiota of one or two islands. Because these genera are known to be present in the natural environment, it is likely that the bacterial strains peculiar to an island had colonized kusaya gravy for many years. The results of this study revealed an influence of geographical conditions on the microbiota in fermented food.

Risk of Listeria monocytogenes contamination of raw ready-to-eat seafood products available at retail outlets in Japan.

Examination of Listeria monocytogenes prevalence among ready-to-eat foods in Japan revealed frequent (5.7 to 12.1%) contamination of minced tuna and fish roe products, and the isolates had the same virulence levels as clinical isolates in terms of invasion efficiency and infectivity in cell cultures and a murine infection model, respectively. Premature stop codons in inlA were infrequent (1 out of 39 isolates). Cell numbers of L. monocytogenes in minced tuna and salmon roe increased rapidly under inappropriate storage temperatures (from a most probable number [MPN] of 10(0) to 10(1)/g to an MPN of 10(3) to 10(4)/g over the course of 2 days at 10 degrees C). Thus, regulatory guidelines are needed for acceptable levels of L. monocytogenes in these foods.

Development of a predictive program for Vibrio parahaemolyticus growth under various environmental conditions.

In this study, we developed a predictive program for Vibrio parahaemolyticus growth under various environmental conditions. Raw growth data was obtained with a V. parahaemolyticus O3:K6 strain cultured at a variety of broth temperatures, pH, and salt concentrations. Data were analyzed with our logistic model and the parameter values of the model were analyzed with polynomial equations. A prediction program consisting of the growth model and the polynomial equations was then developed. After the range of the growth environments was modified, the program successfully predicted the growth for all environments tested. The program could be a useful tool to ensure the bacteriological safety of seafood.

Real-time PCR and enrichment culture for sensitive detection and enumeration of Escherichia coli.

Rapid enumeration of Escherichia coli strains by quantitative real-time PCR targeting the uidA gene was developed and confirmed for minced beef, tuna and raw oyster. Higher sensitivity (1 CFU/g of E. coli in all three food samples) was obtained by incubating for 7 h in TSB. Colony-directed E. coli specific TaqMan PCR assay could effectively distinguish colonies grown on various selective media within 1.5-h. Inspection of E. coli in food testing laboratories is important, and our rapid E. coli detection strategy will contribute to quality control in food industries.

Difference of genotypic and phenotypic characteristics and pathogenicity potential of Photobacterium damselae subsp. damselae between clinical and environmental isolates from Japan.

Photobacterium damselae subsp. damselae has been known as an opportunistic pathogen in fish and mammals. Human infectious cases are often very serious and occasionally fatal. We previously reportedtwo fatal cases caused by this subspecies where the patients developed multiple organ failure within 20-36 h after the onset of initial symptoms. Despite its ability to cause serious infections in humans, this subspecies has not been well studied because human infectious cases caused by this subspecies are very rare. However, this subspecies has been reported to be present in a wide range with high incidence rate in aquatic environments. Thus, we investigated the genotypic and phenotypic differences between clinical and environmental strains of Photobacterium damselae subsp. damselae. Using molecular typing methods, such as ribotyping, AFLP (Amplified Fragment Length Polymorphism), and PFGE (Pulsed-Field Gel Electrophoresis) and sequencing analysis, we determined that thetwo clinical strains were genetically similar yet distinguishable from environmental strains, but not significantly so. On the other hand, phenotypic differences were clear; moreover, mouse assay and hemolytic assay indicated strong pathogenicity of only clinical isolates. Based on these data, we concluded that there are differences in pathogenicity potential among isolates of this subspecies, and some environmental isolates have the potential to become highly pathogenic.

Use of single-strand conformation polymorphism of amplified 16S rDNA for grouping of bacteria isolated from foods.

The grouping method for isolated strains from foods using single-strand conformation polymorphism (SSCP) after PCR amplification of a portion of 16S rDNA was developed. This method was able to group the strains from various food samples based on 16S rDNA sequence. As 97.8% of the isolated strains from various foods were grouped correctly, use of the PCR-SSCP method enables the prompt and labor-saving analysis of microbial population of food-derived bacterial strains. Advantages in speed and accuracy of bacterial population identification by the PCR-SSCP method have practical application for food suppliers and testing laboratories.

Development of a multilocus variable-number of tandem repeat typing method for Listeria monocytogenes serotype 4b strains.

Listeria monocytogenes serotype 4b strains have been identified as the causative agent in many human listeriosis epidemics as well as in a considerable number of sporadic cases. Due to the genetic homogeneity of serotype 4b isolates, development of rapid subtyping methods with high discriminatory power for serotype 4b isolates is required to allow for improved outbreak detection and source tracking. In this study, multilocus variable-number tandem repeat analysis (MLVA) was developed and used to characterize 60 serotype 4b isolates from various sources. All isolates were also characterized by automated EcoRI ribotyping, single enzyme pulsed-field gel electrophoresis (PFGE) with ApaI, and a multilocus sequence typing (MLST) scheme targeting six virulence and virulence-associated genes. Discriminatory power of MLVA (as determined by Simpson Index of Discrimination) was higher than the discriminatory power of any of the other three methods. MLVA markers targeted were found to be stable and did not change when three isolates were passaged daily for 70 days. Cluster analyses of MLVA, PFGE and MLST consistently grouped the same isolates into three major clusters, each of which includes one of the three major L. monocytogenes epidemic clones (i.e., ECI, ECIa and ECII). We conclude that the MLVA method described here (i) provides for more discriminatory subtyping of L. monocytogenes serotype 4b strains than the other three methods, (ii) identifies three major groups within the serotype 4b, which are consistent with the groups identified by other subtyping methods, and (iii) is easy to interpret. Use of MLVA may thus be recommended for subtyping of serotype 4b isolates, including as a secondary more discriminatory subtyping method that could be used after initial isolate characterization by PFGE or ribotyping.

Growth and toxin production of proteolytic Clostridium botulinum in aseptically steamed rice products at pH 4.6 to 6.8, packed under modified atmosphere, using a deoxidant pack.

Demand for aseptically steamed rice products has been increasing rapidly in Japan over the past 10 years. In our previous study, we showed that proteolytic Clostridium botulinum produce toxins in steamed rice products packaged under a modified atmosphere of < or =0.3% oxygen. In the present study, we examined the effect of pH to control botulism risk in steamed rice products packaged under modified atmosphere (5% CO2 and 95% N2 as the balance) with the inclusion of a deoxidant pack to produce an oxygen concentration of < or =0.3%. A mixture of 10 strains of proteolytic C. botulinum (5 type A strains and 5 type B strains) was inoculated into steamed rice products at pH values between 4.6 and 6.8 prior to packaging. All samples were stored at 30 degrees C for 24 weeks. Samples at higher pH showed earlier starts of neurotoxin production. Neurotoxin was detected after 2 weeks of incubation in samples at pH 5.4 or above, whereas it took 4 weeks for the toxin to be detected in samples at pH 5.2 to 5.3 and 12 weeks in samples at pH 5.0 to 5.1. In samples at pH 4.9 or below, no toxin was detected during the experimental period. Apparent sample spoilage did not occur before C. botulinum produced neurotoxin in most of the samples. Based on these results, we conclude that aseptically steamed rice products must be packaged at pH 4.9 or below under modified atmosphere containing < or =0.3% oxygen, with the inclusion of a deoxidant pack.

Multiple-locus variable-number of tandem-repeats analysis distinguishes Vibrio parahaemolyticus pandemic O3:K6 strains.

A specific serotype of Vibrio parahaemolyticus, O3:K6, has recently been linked to epidemics of gastroenteritis in Southeast Asia, Japan, and North America. These pandemic O3:K6 strains appear to have recently spread across continents from a single origin to reach global coverage, based on profiling of strains by several molecular typing methods. In this study, variable-number tandem repeats (VNTR)-based fingerprinting was applied to clinical and environmental V. parahaemolyticus O3:K6 strains in an attempt to develop a molecular method with increased sensitivity for discriminating strains; the relative discriminatory powers were compared with ribotyping and pulsed-field gel electrophoresis (PFGE). All clinical strains tested were independent human isolates obtained from different outbreaks or from sporadic cases in Tokyo during the period from 1996 to 2003. Multiple-locus VNTR analysis (MLVA) was shown to have high resolution and reproducibility for typing of V. parahaemolyticus clones. MLVA analysis of 28 pandemic V. parahaemolyticus O3:K6 strains isolated from human cases produced 28 distinct VNTR patterns. The VNTR loci displayed between 2 and 15 alleles at each of eight loci with Nei's diversity index ranging from 0.35 and 0.91. These data demonstrated that MLVA is useful for individual strain typing of new O3:K6 strains, which appear to be closely related by other molecular methods.

Development of multilocus single strand conformation polymorphism (MLSSCP) analysis of virulence genes of Listeria monocytogenes and comparison with existing DNA typing methods.

Development of rapid and simple typing methods is required for analyzing the distribution and contamination routes of food-borne pathogens. We established a simple typing method for Listeria monocytogenes using MLSSCP (Multilocus Single Strand Conformation Polymorphism) analysis. Four virulence genes, hlyA, iap, actA and inlB were amplified by PCR, digested with endonucleases and applied to gels for SSCP. As banding patterns have been shown to reflect even a single nucleotide difference, this method has a potential discriminatory power comparable to that of sequencing analysis. The 64 strains isolated from five meat processing plants were divided into 18 groups by this MLSSCP. Additionally, clustering obtained with this method showed strong correspondence with phylogenetic lineages I and II, and was achieved with much less expenditure in time and cost than is required for other methods, such as MLST. The validity of the MLSSCP lineage classification was confirmed by PFGE, AFLP and ribotyping results. This newly developed MLSSCP method is suitable when obtaining accurate results quickly and simply is crucial.

Nonsense-mutated inlA and prfA not widely distributed in Listeria monocytogenes isolates from ready-to-eat seafood products in Japan.

InlA is a surface protein participating in the entry of Listeria monocytogenes into mammalian non-phagocytic cells. PrfA is a positive regulatory factor that regulates the expression of a set of virulence genes. Recent studies revealed that some L. monocytogenes strains have a truncated form of these proteins because of nonsense mutations in their sequences, and these truncations contribute to the significant reduction in virulence of this pathogen. In this study, sequence analyses of inlA and prfA among L. monocytogenes isolated from ready-to-eat seafood revealed that only one out of 59 isolates had a nonsense-mutated inlA and all had non-mutated prfA. This indicated that these strains could be fully virulent based on the sizes of these proteins.

Evaluation of PCR-single-strand conformational polymorphism analysis for identification of gram-negative histamine-producing bacteria isolated from fish.

In this study, the previously established PCR-single-strand conformation polymorphism (SSCP) system for detecting and identifying gram-negative histamine-producing bacteria was evaluated. This system can detect and identify histamine-producing bacteria directly from seafood by the use of sequence polymorphisms of the histidine decarboxylase gene (hdc). First, we isolated 81 histamine-producing strains of bacteria from fish samples and analyzed the hdc gene by the PCR-SSCP system. The 22 newly obtained SSCP banding patterns were added to our database, and the utility of our modified database was tested in a second experiment consisting of 18 strains of histamine-producing bacteria isolated from 25 fish samples. Approximately 80% of the histamine-producing strains corresponded to those in the new database. Use of the database for PCR-SSCP analysis, including the band patterns newly added in this study, for the hdc gene makes it possible to more accurately identify histamine producers.

Quantitative duplex PCR of Clostridium botulinum types A and B neurotoxin genes.

A duplex quantitative polymerase chain reaction (PCR) assay for Clostridium botulinum types A and B was developed. The sensitivity and specificity of the assay were verified by using 6 strains of type A, 7 strains of type B, and 14 genera of 42 non-C. botulinum types A and B strains, including C. botulinum types C, D, E, F, and G. In pure culture, the detection limit was 10(2) CFU/ mL for type A and 10(3) CFU/mL for type B. In mushroom broth, increases in the amounts of C. botulinum types A and B could be monitored separately (the quantifiable range was 10(2) to 10(6) for type A and 10(2) to 10(7) for type B) from each sample that contained a large number of background bacteria, and toxin could be detected much earlier than with mouse assay. These results suggest that duplex quantitative PCR methods are useful to detect and quantify C. botulinum types A and/ or B toxin genes.

[Development of a predictive program for microbial growth under various temperature conditions].

A predictive program for microbial growth under various temperature conditions was developed with a mathematical model. The model was a new logistic model recently developed by us. The program predicts Escherichia coli growth in broth, Staphylococcus aureus growth and its enterotoxin production in milk, and Vibrio parahaemolyticus growth in broth at various temperature patterns. The program, which was built with Microsoft Excel (Visual Basic Application), is user-friendly; users can easily input the temperature history of a test food and obtain the prediction instantly on the computer screen. The predicted growth and toxin production can be important indices to determine whether a food is microbiologically safe or not. This program should be a useful tool to confirm the microbial safety of commercial foods.

[Growth inhibition of Vibrio parahaemolyticus in seafood by tabletop dry ice cooler].

Tabletop dry ice coolers (three types; dome model, cap model and tripod model), which are used in kitchens and hotel banquet halls to refrigerate fresh seafood, were investigated to determine whether growth of Vibrio parahaemolyticus was inhibited by their use. On TSA plates containing 1.8% NaCl and fresh seafood (fillets of squid, pink shrimp and yellowtail), V. parahaemolyticus (O3:K6, TDH+) inoculated at 4 to 5 log CFU/sample and left at ambient temperature (25 degrees C) grew by 1.0 to 2.8 orders in 4 hours. In contrast, with tabletop coolers no significant increase in viable count occurred in 3 to 4 hours, confirming that tabletop coolers inhibited the growth of V. parahaemolyticus. The temperature in each tabletop cooler was kept below 10 degrees C for 80 to 135 min, though the CO2 gas concentration in them remained high for only a short time (0 to 75 min). It was presumed that the refrigeration function mainly contributed to growth inhibition. Our results indicate that tabletop dry ice coolers are helpful for prevention of food-borne disease due to V. parahaemolyticus in food-service locations, such as kitchens and banquet halls.

Growth and toxin production by Clostridium botulinum in steamed rice aseptically packed under modified atmosphere.

Sales and consumption of ready-to-eat aseptic steamed rice products have increased manyfold in Japan over the past 10 years. To determine the safety of steamed rice (water content 60%, pH 6.5) aseptically packaged under modified atmosphere, challenge studies were performed using a mixture of Clostridium botulinum proteolytic strains (five strains of type A and five strains of type B). Atmospheric conditions of 0 and 15% oxygen (with 5% CO2 and 5% N2 as the balance) were used. No neurotoxins were detected, and organoleptically acceptable conditions persisted for 24 weeks at 15% oxygen conditions. However, botulinum neurotoxin was found in one of three samples at 12 weeks and in one of two samples at 24 weeks at 0% oxygen and 30 degrees C. When samples were inoculated with C. botulinum with amylase (0% oxygen), neurotoxin and sample spoilage was detected after only 1 week of storage. Challenge studies using proteolytic strains of C. botulinum mixed with Bacillus subtilis (amylase formers) also were performed with atmosphere conditions of oxygen at 0, 5, 10, and 15% (with 5% CO2 and 5% N2 as the balance). Under 10 and 15% oxygen conditions, neurotoxin was not detected after 1 week of storage, but sample spoilage was detected after the same period. Under 0% oxygen conditions, neurotoxin was detected at 1 week, but the sample remained organoleptically acceptable even after 2 weeks of storage. Both neurotoxin and sample spoilage were detected at 1 week of storage under 5% oxygen conditions. Based on these results, cocontamination of amylase-producing Bacillus with C. botulinum would increase the risk of foodborne botulism when aseptic rice samples are packed under low-oxygen conditions (<5%). Therefore, to ensure the safety of these products, packing under atmospheric containing more than 10% oxygen is recommended.

Incidence of Listeria monocytogenes in raw seafood products in Japanese retail stores.

The incidence of Listeria monocytogenes in raw fish, shellfish, and fish roe was investigated in seafood products collected from randomly selected retail stores in and around Tokyo, Japan. Of the 10 samples of 208 examined found positive for L. monocytogenes by mini-VIDAS LMO, seven were fish roe (cod, salmon) and three were minced tuna. Three serotypes (1/2a, 1/2b, 3b) were detected among the isolated strains; serotype 1/2a was predominant (8 of 10).

Direct detection and identification of lactic acid bacteria in a food processing plant and in meat products using denaturing gradient gel electrophoresis.

We established a novel system using denaturing gradient gel electrophoresis (DGGE) to quickly identify bacteria known to be responsible for spoilage in meat processing plants and meat products. We extracted bacterial DNA from swabbed samples at various locations in the plant and from meat products and performed PCR amplification, targeting 16S rDNA from the dominant organisms. The amplification products were subjected to DGGE, and the contaminating bacteria in the meat products and the plant were analyzed. This analysis indicated that lactic acid bacteria and spoilage-causing bacteria are widely distributed within the meat processing plant. We developed molecular size markers to identify the dominant organisms obtained from the plant and meat products. The establishment of the present method allows quick and simple identification of bacteria causing the possible deterioration of products and contamination and thus permits constant monitoring of any harmful bacteria within meat processing plants.