Teizo Tsukamoto

Klebsiella pneumoniae produces no histamine: Raoultella planticola and Raoultella ornithinolytica strains are histamine producers.

ABSTRACT Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.

Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury.

ABSTRACT Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.

Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin‐producing Escherichia coli (VTEC), a sensitive bead‐enzyme‐linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo‐ and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.

Detection and Genetical Characterization of Shiga Toxin-Producing Escherichia coli from Wild Deer

Shiga toxin (Stx)‐producing Escherichia coli (STEC) strains isolated from wild deer in Japan were examined. A total of 43 fecal samples were collected 4 times from 4 different sites around Obihiro City, Hokkaido, Japan, in June and July 1997. Seven STEC strains were isolated by PCR screening, all of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing only active Stx type 2 (Stx2). Moreover, they seemed to carry the hemolysin and eaeA genes of STEC O157:H7, and some isolates harbored large plasmids which were similar to the 90‐kilobase virulence plasmid of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, PCR‐based DNA fingerprinting data obtained by using random amplified polymorphic DNA (RAPD) and the stx2 gene sequences, all isolates were divergent from each other except for 3 isolates from the first and second samplings. A DNA sequence analysis of representative isolates revealed that deer originating STEC strains were closely related to each other, but not to the Stx2‐producing STEC strains isolated from a mass outbreak in Obihiro at the same time. A phylogenic analysis of the deduced Stx2 amino acid sequences demonstrated that three distinct clusters existed in the deer originating STEC strains and that the Stx of deer originating STEC was closely associated with that originating from humans, but not those of STEC originating from other animals. These results suggest that STEC contamination of deer carcasses should be considered as a potential source of human infection and adequate sanitary inspection of meat for human consumption is also essential for wild animals.

Distribution of Virulence Factors in Escherichia coli Isolated from Urine of Cystitis Patients

The distribution of 7 urovirulence factors, such as type 1 pilus (pil), pilus associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin I (afaI), hemolysin (hly), aerobactin (aer) and cytotoxic necrotizing factor 1 (cnf1) was examined by a DNA colony hybridization test among 194 Escherichia coli strains isolated from the urine of cystitis patients and in 80 strains isolated from the stool specimens of healthy adults. All virulence factors examined, except pil, were significantly more frequently detected among the cystitis isolates than among the fecal isolates. When individual virulence factors were analyzed against the others, an association was discernible which was not apparent when all 7 virulence factors were considered collectively. There was an apparent correlation between the genotypes and serotypes of the E. coli strains from the cystitis patients. From the data presented, it was proposed that genetic detection of virulence factors would be useful for rapid diagnosis of cystitis, especially in patients without severe pyuria or bacteriuria.

Evaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses.

A one‐step reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT‐LAMP assay and compared with the results of routine RT‐PCR. The results of the RT‐LAMP assay corresponded well to that of RT‐PCR. These findings suggest the practical application of the RT‐LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT‐LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT‐LAMP system is able to detect NVs in clinical specimens within a wide range. J. Med. Virol. 79:326–334, 2007.

Precise Characterization of Norovirus (Norwalk-Like Virus)-Specific Monoclonal Antibodies with Broad Reactivity

ABSTRACT We have been characterizing monoclonal antibodies against Norovirus (Norwalk-like virus). In the course of our study, two monoclonal antibodies generated against Norovirus genogroup II capsid protein were found to react not only to genogroup II but also to genogroup I recombinant capsid proteins. In addition, we showed that these two monoclonal antibodies reacted to a 40-amino-acid-fragment located close to the N-terminal region of genogroup II Norovirus. Similar reactivity was observed with the equivalent region of genogroup I Norovirus. In this study, we confirmed that the epitopes of the two monoclonal antibodies existed within an 11-amino-acid peptide. To obtain an idea of the reactive ranges of the two monoclonal antibodies toward different strains of Norovirus, their reactivities were investigated using 16 types of peptide constructed according to the data in GenBank and 8 recombinant capsid proteins (7 whole capsid proteins and 1 short [80-amino-acid] protein fragment). A characteristic broad reactivity of the two monoclonal antibodies is clearly shown by the results of this study. Thus, these monoclonal antibodies could be useful tools for detecting a broad range of Norovirus strains.

CYTOLETHAL DISTENDING TOXIN (CDT): GENETIC DIVERSITY, STRUCTURE AND ROLE IN DIARRHEAL DISEASE

In 1987, cytolethal distending toxin (CDT) was discovered by Johnson and Lior as a new type of protein toxin produced by certain strains of Escherichia coli, which is different from heat-labile enterotoxin (LT) produced by enterotoxigenic E. coli. Although LT causes only cell elongation, CDT causes cell elongation, cell distention, irreversible cell cycle arrest, and consequently, death of the cultured mammalian cells. Recently, CDT was recognized as a new family of bacterial toxin, as a genotoxin, produced by a diverse group of gram-negative bacteria, all of which are related to mucosal infection. Although tremendous efforts have been made to study the structure and mode of action of CDT, its role in bacterial pathogenesis still remains unclear. In this review, we focus mainly on CDT produced by enteric bacteria and describe the history of CDT, their gene and protein structure, structure-function relationship, and its mode of action particularly how CDT contributes to the gastrointestinal infections.

Association of the Urease Gene with Enterohemorrhagic Escherichia coli Strains Irrespective of Their Serogroups

ABSTRACT Among various diarrheagenic Escherichia coli strains from clinical sources, we found that the urease gene was specifically associated with enterohemorrhagic E. coli (EHEC) strains irrespective of their serogroups. The results suggest that the urease gene can be a useful genetic marker for the detection of EHEC strains and for the diagnosis of infections caused by EHEC strains in the clinical situation.

Development and evaluation of a rapid, simple, and sensitive immunochromatographic assay to detect thermostable direct hemolysin produced by Vibrio parahaemolyticus in enrichment cultures of stool specimens

ABSTRACT Thermostable direct hemolysin (TDH) is considered to be a major virulence factor in Vibrio parahaemolyticus, and most cases of V. parahaemolyticus diarrhea in humans are caused by tdh gene-positive strains. In the present study, we developed an immunochromatographic assay to detect TDH (TDH-ICA) and evaluated the utility of TDH-ICA for the diagnosis of V. parahaemolyticus diarrhea. TDH-ICA allowed the detection of 0.2 ng/ml of TDH within 10 min. Fecal homogenates were spiked with various numbers of tdh-positive V. parahaemolyticus organisms, and their enrichment cultures were tested with TDH-ICA. The results of detection of TDH in the enrichment cultures by TDH-ICA were in accord with the results of recovery of the spiked V. parahaemolyticus organisms from the enrichment cultures by plating onto thiosulfate-citrate-bile salts-sucrose agar. When enrichment cultures of 217 stool specimens from patients with diarrhea were tested with TDH-ICA, the TDH-ICA results showed 100% sensitivity and specificity compared to the results of isolation of V. parahaemolyticus from the stool specimens by a conventional bacterial culture test. Since TDH-ICA was able to detect TDH in a fecal enrichment culture within 10 min, TDH-ICA testing of a fecal enrichment culture could be completed rapidly and easily within approximately 16 h, including incubation time for the fecal enrichment culture. These results indicate that TDH-ICA is a rapid, simple, and sensitive TDH detection method and that TDH-ICA testing of a fecal enrichment culture is useful as an adjunct to facilitate the early diagnosis of V. parahaemolyticus diarrhea.