Tateo Fujii

Tetragenococcus muriaticus sp. nov., a New Moderately Halophilic Lactic Acid Bacterium Isolated from Fermented Squid Liver Sauce

A total of 11 strains of moderately halophilic histamine-producing bacteria isolated from fermented squid liver sauce were studied phenotypically, genotypically, and phylogenetically. These strains are considered members of the genus Tetragenococcus based on their physiological, morphological, and chemotaxonomic characteristics. A 16S rRNA gene sequence analysis showed that these strains clustered with, but were separate from, Tetragenococcus halophilus. The results of DNA-DNA hybridization experiments indicated that the new isolates represent a new Tetragenococcus species, for which we propose the name Tetragenococcus muriaticus; strain X-1 (= JCM 10006) is the type strain of this species.

Photobacterium histaminum sp. nov., a Histamine-Producing Marine Bacterium

A total of 23 strains of halophilic histamine-producing bacteria previously isolated from marine fish and seawater were studied phenotypically, genotypically, and phylogenetically. These organisms are facultatively anaerobic, gram-negative, short rods that are motile by means of one to three unsheathed polar flagella. They are mesophilic and nonluminescent, produce oxidase, utilize d-glucose but not d-mannitol as a carbon and energy source, and accumulate poly-β-hydroxybutyrate. The guanine-plus-cytosine contents of the DNAs are ca. 41 mol%. Genomic DNA hybridization experiments revealed that some of the isolates exhibited low levels of reassociation (less than 40%) with previously described Photobacterium species. 16S rRNA gene sequence information confirmed the phylogenetic position of one of the isolates, strain C-8T (T = type strain), as a member of the genus Photobacterium. Therefore, we concluded that these organisms are members of a new species of the genus Photobacterium and propose the name Photobacterium histaminum sp. nov. for them. The type strain is strain C-8 (= JCM 8968).

Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish.

ABSTRACT A rapid, quantitative PCR assay (TaqMan assay) which quantifiesClostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains ofC. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 102 to 108 CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.

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

ABSTRACT 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 100 to 101/g to an MPN of 103 to 104/g over the course of 2 days at 10°C). Thus, regulatory guidelines are needed for acceptable levels of L. monocytogenes in these foods.

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).

Differentiation of Tetragenococcus populations occurring in products and manufacturing processes of puffer fish ovaries fermented with rice-bran

Tetragenococcus strains isolated from the manufacturing process of Japanese puffer fish ovaries fermented with rice-bran were characterized and differentiated phenotypically and genotypically. A total of 413 Tetragenococcus isolates were evaluated. On the basis of five representative substrates, the isolates were grouped into seven groups. An RFLP (restriction fragment length polymorphisms) analysis of the 16S rRNA gene of representative strains of major groups revealed that they could be grouped into two groups: one was identified as the most prominent halophilic lactic acid coccus, Tetragenococcus halophilus, and the other as T. muriaticus, which has recently been added to the genus Tetragenococcus as a new species. Physiologically, the major differences between the two groups were found in the ability to grow in medium not supplemented with NaCl and the fermentation of L-arabinose, sucrose and D-mannitol, and several other carbohydrates.

Marinospirillum gen. nov., with descriptions of Marinospirillum megaterium sp. nov., isolated from kusaya gravy, and transfer of Oceanospirillum minutulum to Marinospirillum minutulum comb. nov.

Two strains of helical, halophilic, Gram-negative, heterotrophic bacteria were isolated from kusaya gravy which is a traditional Japanese fermented brine. These strains were motile by means of a single polar or bipolar tuft flagellum. They had a large cell size, were helical, formed coccoid bodies, were microaerophilic and had quinone type Q-8. The DNA G + C content of the strains was 44-45 mol %. A detailed investigation of the phenotypic, chemotaxonomic and phylogenetic characteristics of the strains revealed that they represent a new species of halophilic helical bacteria. The sequence of the 16S rRNA gene of strain H7T, designated the type strain of the new isolates, and all of the Oceanospirillum species except for Oceanospirillum linum were determined. Phylogenetic analysis indicated that these strains were closely related to Oceanospirillum minutulum, with enough distance to separate the O. minutulum/new isolate H7T cluster from Oceanospirillum sensu stricto on the genus level. It is proposed that a new genus, Marinospirillum, be created; this genus should include Marinospirillum minutulum ATCC 19193T (formerly Oceanospirillum minutulum) as the type species, as well as Marinospirillum megaterium JCM 10129T (= H7T).

Strictly anaerobic halophiles isolated from canned Swedish fermented herrings (Surstromming)

Strictly anaerobic halophiles were isolated from canned Swedish fermented herrings (Surströmming). All isolates were phenotypically uniform with some exceptions and were identified as the genus Haloanaerobium and assigned to either Haloanaerobium praevalens or Haloanaerobiuim alcaliphilum. A comparative analysis of 16S rDNA sequences revealed that the representative strain S-8 of the isolates was identical to that of Haloanaerobium praevalens DSM 2228T. Furthermore, this strain exhibited high levels (> 80%) of DNA-DNA homology with Haloanaerobium praevalens DSM 2228T. This is a novel report of halophilic anaerobes isolated from a food product. Such anaerobes may contribute to the intense flavor and the swollen can characteristics of Swedish fermented herring.

Viability and Histidine Decarboxylase Activity of Halophilic Histamine-Forming Bacteria During Frozen Storage

The specific activity of histidine-decarboxylase of halophilic histamine-forming bacteria, Photobacterium phosphoreum and Photobacterium histaminum sp. nov., remained 27~53% of the initial value after seven days storage at -20°C, while the viable cells decreased by more than 6 log cycles of the initial counts. This finding suggests the possibility that outbreaks of scombroid fish poisoning are caused by the ingestion of frozen-thawed fish and its products, even when the viable count of histamine-forming bacteria is low.

Haloanaerobium fermentans sp. nov., a strictly anaerobic, fermentative halophile isolated from fermented puffer fish ovaries.

A strain of strictly anaerobic and moderately halophilic bacteria isolated from salted puffer fish ovaries was studied phenotypically, genotypically and phylogenetically. On the basis of its physiological and morphological characteristics, the new isolate is considered to be a member of the genus Haloanaerobium. It is a motile, rod-shaped, non-spore-forming, gram-negative, obligate anaerobe that grows in the presence of 25% (w/v) NaCl. The optimum salt concentration for growth is 10% (w/v). It grows well at 15 and 45 degrees C, but not at 10 or 50 degrees C. The optimum temperature for growth is 35 degrees C. It grows at pH 6.0-9.0 and the optimum pH for growth is 7.5. It ferments N-acetylglucosamine, cellobiose, fructose, galactose, D-glucose, lactose, maltose, D-mannose, raffinose, D-ribose, sucrose and D-xylose. It ferments D-glucose with the production of hydrogen, carbon dioxide, ethanol and organic acids such as acetate, formate and lactate. 16S rRNA gene sequence information confirmed the phylogenetic position of the new isolate, strain R-9T, as a member of the genus Haloanaerobium. DNA-DNA hybridization data revealed that isolate R-9T exhibited low levels of reassociation (less than 30%) with previously described Haloanaerobium species. Based on these results, the new isolate appears to represent a new Haloanaerobium species, for which the name Haloanaerobium fermentans sp. nov. is proposed. The type strain is R-9T (= JCM 10494T).