Tateo Fujii

Cloning and Sequencing of the Histidine Decarboxylase Genes of Gram-Negative, Histamine-Producing Bacteria and Their Application in Detection and Identification of These Organisms in Fish

ABSTRACT The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.

Histamine formation by Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce

We examined histamine formation in cultures of Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce. T. muriaticus formed histamine in low acidity (pH 5.8), O2 limiting conditions with optimal NaCl and glucose concentrations of 5-7% (w/v) and above 1%, respectively. Histamine formation could not be prevented even at 20% (w/v) NaCl, indicating that NaCl could not prevent histamine formation by this bacterium. A conspicuous amount of histamine accumulated only during the late stationary phase regardless of the growth conditions. Studies of cell suspension experiments confirmed the results obtained from cultured cells.

Lysine decarboxylase of Vibrio parahaemolyticus: kinetics of transcription and role in acid resistance

Aim:u2002 The aim of this study was to investigate the detailed mechanisms of acid resistance in Vibrio parahaemolyticus.

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.

Purification and properties of a histidine decarboxylase from Tetragenococcus muriaticus, a halophilic lactic acid bacterium

Aims: A histidine decarboxylase from Tetragenococcus muriaticus, a halophilic histamine‐producing bacterium isolated from Japanese fermented squid liver sauce, was purified to homogeneity, for the first time.

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.

Photobacterium histaminum Okuzumi et al. 1994 is a later subjective synonym of Photobacterium damselae subsp. damselae (Love et al. 1981) Smith et al. 1991.

The type strain of Photobacterium histaminum, JCM 8968T (= ATCC 51805T), and that of Photobacterium damselae subsp. damselae, ATCC 33539T, exhibit 100% identity in their 16S rRNA sequence, more than 80% DNA-DNA homology and only one phenotypic difference. Also, like P. histaminum, P. damselae subsp. damselae was shown to excrete a large amount of histamine when cells were grown on medium containing excessive histidine under acidic conditions. Therefore, the name P. histaminum should be considered to be a later subjective synonym of P. damselae subsp. damselae.

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 Neis 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.

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.

Induction of the histidine decarboxylase genes of Photobacterium damselae subsp. damselae (formally P. histaminum) at low pH.

Aims:u2002 To elucidate the detailed mechanism of histamine production by Photobacterium damselae subsp. damselae.