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Biomedical subjects

Jun Terajima

Publications and source records attributed to Jun Terajima.

At least 19 recordsLinked to original sources
PubMed2006

Spontaneous recombination between homologous prophage regions causes large-scale inversions within the Escherichia coli O157:H7 chromosome.

It is known that XbaI-digested chromosomal DNAs of strains of Escherichia coli O157:H7 exhibit a wide variety in pulsed-field gel electrophoresis (PFGE) fragment patterns, which is used for epidemiological surveillance of this important pathogen. The variety in the restriction enzyme-digestion patterns suggests a wide genomic diversity, however, only a few studies have been conducted to investigate involvement of large-scale chromosomal rearrangements in development of the diversity. In this study, through rounds of subculturing E. coli O157:H7 strain EDL933, naturally occurring genome variation in the isolated derivatives was investigated. By comparing the PFGE patterns among clonally related derivatives, we found five types of large-scale inversions taking place within the chromosome. The five inversions found were across the replication axis and ranged from 250-kb to 1.4-Mb long, and all the corresponding recombination sites were associated with prophages or phage-like regions. Four inversions out of the five were resulted from recombination between pairs of lambda-like prophages disturbing the symmetry of the origin and terminus of the replication axis. These observations indicate that those prophage regions represent some of the hot spots for intrachromosomal recombination within the E. coli O157:H7 chromosome, where recombination between the prophage regions results not only in the large chromosomal inversions but might also in generation of chimeric phages.

Cells, Cultured↗
PubMed2006

Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the Whole Genome PCR scanning.

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 'variably absent or present' genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains.

Combinatorial Chemistry Techniques↗
PubMed2006

Effectiveness of pulsed-field gel electrophoresis for the early detection of diffuse outbreaks due to Shiga toxin-producing Escherichia coli in Japan.

We applied pulsed-field gel electrophoresis (PFGE) to the investigation of diffuse outbreaks of illness due to Shiga toxin?producing Escherichia coli O157:H7 (STEC O157) in Japan and used these data to develop a database of STEC O157 PFGE patterns and associated clinical and microbiologic information to facilitate the recognition of geographic and temporal clusters of cases based on their PFGE profiles. This project has evolved into a subtyping network called PulseNet Japan that is cooperatively run by National Institute of Infectious Diseases (NIID) and the local Health Institutes and the Ministry of Health, Labor and Welfare. Although our domestic PFGE network that utilized locally developed PFGE protocols was effective in recognizing diffuse outbreaks of STEC O157 within Japan, we decided to adopt the standardized PFGE protocols from PulseNet USA and collaborate closely with the Centers for Disease Control and Prevention (CDC) in the United States to facilitate recognition of international clusters of STEC O157 and their investigations.

Centers for Disease Control and Prevention, U.S.↗
PubMed2006

Development of a real-time PCR assay for detection of gyrA mutations associated with reduced susceptibility to ciprofloxacin in Salmonella enterica serovar typhi and paratyphi A.

A real-time PCR assay with the cycling probe method was used to detect mutations at codons 83 and 87 in the DNA gyrase A subunit encoded by gyrA in Salmonella enterica serovar Typhi and Paratyphi A clinical isolates. The susceptibility estimated from the results of the gyrA mutation assay was consistent with that identified by the culture method using an E-test. This assay allows rapid screening of S. enterica serovar Typhi and Paratyphi A with reduced susceptibility to ciprofloxacin.

Anti-Bacterial Agents↗
PubMed2006

Characterization of lysine decarboxylase-negative strains of Salmonella enterica serovar Enteritidis disseminated in Japan.

Salmonella enterica serovar Enteritidis is one of the leading causes of food-borne diseases in Japan. Typically, Salmonella spp. test positive for lysine-decarboxylase. However, the number of isolates of serovar Enteritidis without lysine-decarboxylase activity increased in Japan in 2003. Among 109 strains from distinct outbreaks, 10 lacked lysine-decarboxylase activity. Nine of the ten lysine-decarboxylase-negative strains showed quite similar pulsed-field gel electrophoresis profiles. Their lysine-decarboxylase phenotype was recovered by introduction of the cadBA locus from an lysine-decarboxylase-positive strain. Although the cad loci of the lysine-decarboxylase-negative strains seemed to be intact without any insertion sequences, cadC, a positive regulator of cadBA, had a single-base deletion at the same position, the 973rd base (cytosine), in all the nine lysine-decarboxylase-negative strains, whereas the wild-type cadC gene has a 1542 bp coding region (514 amino acids). This deletion was expected to produce a truncated (338 amino acids) form of CadC due to a frameshift. Because CadC senses environmental cues such as external pH and lysine through its putative C-terminal periplasmic domain, it is likely that the truncated CadC is not sensitive enough to external signaling to activate the cadBA operon, resulting in loss of the lysine-decarboxylase activity. Our results suggest that dissemination of these genetically closely related strains of serovar Enteritidis accounts for the unusual increase in the isolation of lysine-decarboxylase-negative strains.

Amino Acid Sequence↗
PubMed2006

Assessment of genes associated with Streptococcus mutans biofilm morphology.

Streptococcus mutans, the major pathogen responsible for dental caries in humans, is a biofilm-forming bacterium. In the present study, 17 different pulsed-field gel electrophoresis patterns of genomic DNA were identified in S. mutans organisms isolated clinically from whole saliva. The S. mutans isolates showed different abilities to form biofilms on polystyrene surfaces in semidefined minimal medium cultures. Following cultivation in a flow cell system in tryptic soy broth with 0.25% sucrose and staining using a BacLight LIVE/DEAD system, two strains, designated FSC-3 and FSC-4, showed the greatest and least, respectively, levels of biofilm formation when examined with confocal laser scanning microscopy. Further, image analyses of spatial distribution and architecture were performed to quantify the merged green (live cells) and red (dead cells) light. The light intensity of the FSC-3 biofilm was greater than that of the FSC-4 biofilm in the bottom area but not in the top area. S. mutans whole-genome array results showed that approximately 3.8% of the genes were differentially expressed in the two strains, of which approximately 2.2%, including bacitracin transport ATP-binding protein gene glrA and a BLpL-like putative immunity protein gene, were activated in FSC-3. In addition, about 1.6% of the genes, including those associated with phosphotransferase system genes, were repressed. Analyses of the glrA-deficient strains and reverse transcription-PCR confirmed the role of the gene in biofilm formation. Differential assessment of biofilm-associated genes in clinical strains may provide useful information for understanding the morphological development of streptococcal biofilm, as well as for colonization of S. mutans.

Adult↗
PubMed2006

OspE2 of Shigella sonnei is required for the maintenance of cell architecture of bacterium-infected cells.

The OspE2 product of Shigella spp., the expression of which is regulated by the mxiE gene, is secreted through a type III secretion system into host cells. We investigated the function of OspE2 of Shigella sonnei by using cultured epithelial cells. Cells invaded by an ospE2 deletion mutant altered their morphology into the rounding shape, which was not due to cell death, whereas cells invaded by the wild-type strain kept their cell shape intact. The ospE2 mutation did not affect initial cell entry and multiplication in cells, but the mutant formed smaller-than-normal plaques on cell monolayers, indicating a deficiency in cell-to-cell spread by the bacteria. An mxiE deletion mutant also showed changes in cell morphology and deficiency in bacterial spread to adjacent cells. In cells invaded by the ospE2 mutant, disturbance of actin stress fibers was prominent at 3 h after invasion. Analysis of OspE2 localization indicated that the OspE2 protein accumulated on focal contact-like structures in the infected host cells. These results suggest that colocalization of the OspE2 protein in the focal contacts of infected cells may function to maintain an intact cell morphology. The morphological change induced by invasion of the ospE2 mutant may affect secondary bacterial transmission.

Amino Acid Sequence↗
PubMed2005

Antimicrobial susceptibility of Shigella sonnei isolates in Japan and molecular analysis of S. sonnei isolates with reduced susceptibility to fluoroquinolones.

We performed susceptibility testing with Shigella sonnei isolates from imported and domestic cases of infection in Japan during 2001 and 2002. Some S. sonnei isolates were resistant to nalidixic acid, tetracycline, and trimethoprim-sulfamethoxazole. Most of the nalidixic acid-resistant strains showed reduced susceptibility to fluoroquinolones but did not show fluoroquinolone resistance.

Drug Resistance, Bacterial↗
PubMed2005

Characterization of isolates of Salmonella enterica serovar typhimurium displaying high-level fluoroquinolone resistance in Japan.

Strains of the multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium isolated in Japan were examined for high-level fluoroquinolone resistance. Since the first isolation in 2000 (described in reference 13), we have identified 12 human and 5 nonhuman isolates with high-level fluoroquinolone-resistance (ciprofloxacin MIC of 24 microg/ml or more). Most of these isolates shared some features including definitive phage type (DT 12/193), resistance type (ACSSuTNCp; resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, nalidixic acid, and ciprofloxacin), and genotype on pulsed-field gel electrophoresis that were different from those of the MDR S. enterica Typhimurium DT 104. Mutations in quinolone resistance-determining regions of gyrA and parC were also conserved in almost all of the isolates despite the absence of any apparent epidemiological relationships among cases. This suggests that a specific clonal group of the serovar Typhimurium with high levels of fluoroquinolone resistance is disseminating among animals and humans in Japan.

Animals↗
PubMed2005

Evaluation of pulsed-field gel electrophoresis analysis performed at selected prefectural institutes of public health for use in PulseNet Japan.

In order to evaluate reliability of pulsed-field gel electrophoresis (PFGE) analysis performed at different prefectural public health institutes (PHIs) for use in the PulseNet Japan surveillance system to detect enterohemorrhagic Escherichia coli O157, we compared the results of PFGE-typing of 14 selected strains of O157 performed at 8 selected PHIs to evaluate the reliability of different experimental protocols used in these PHIs. PFGE was performed for 14 strains for which there were 14 PFGE types in 3 PHIs, and 13 PFGE types in 5 PHIs by using their own protocols and/or those of the National Institute of Infectious Diseases (NIID). PFGE fingerprints from 5 out of the 8 PHIs were successfully genotyped for all of the 14 strains. A PFGE fingerprint from one PHI was successfully genotyped when the NIID pulsing protocol was used, but was not genotyped when the PHI's own protocols were used. PFGE fingerprints from 2 PHIs failed to be genotyped for one each of the strains. The cause of this genotyping failure was considered to be inappropriate PFGE pulsing protocols or inadequate digestion of chromosomal DNA. These results suggest that PFGE protocols should be standardized for the establishment of PulseNet Japan.

Electrophoresis, Gel, Pulsed-Field↗
PubMed2004

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.

Animals↗
PubMed2004

PCR-based identification of pandemic group Vibrio parahaemolyticus with a novel group-specific primer pair.

A PCR-based assay to identify pandemic group Vibrio parahaemolyticus has been developed. The assay employs an oligonucleotide primer pair derived from the group-specific sequence of an arbitrarily primed-PCR fragment, which is located in the genome encoding a "hypothetical protein," approximately 80% homologous to the Mn2+ and Fe2+ transporter of the NRAMP family of V. vulnificus. The assay distinguished the pandemic group from other V. parahaemolyticus strains by yielding a 235-bp specific amplicon, and can be a useful diagnostic tool for identification of pandemic group strains.

Bacterial Proteins↗
PubMed2004

Development of a PCR-restriction fragment length polymorphism assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli.

Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T. Sato, T. Shimizu, M. Watarai, M. Kobayashi, S. Kano, T. Hamabata, Y. Takeda, and S. Yamasaki, Gene 309:35-48, 2003). Region V, which is ca. 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli (STEC) on the basis of the diversity of region V. When region V was amplified by long and accurate-PCR (LA-PCR) with five control E. coli strains carrying six different Stx phages such as E. coli strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained. Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains. When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained. Two STEC O157 strains, however, did not produce any bands. Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups. The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area. To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay. In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern. Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks.

Disease Outbreaks↗
PubMed2003

Effects of lysogeny of Shiga toxin 2-encoding bacteriophages on pulsed-field gel electrophoresis fragment pattern of Escherichia coli K-12.

Escherichia coli K-12 lysogens of three different Shiga toxin 2 (Stx2)-encoding bacteriophages were examined for variability in their pulsed-field gel electrophoresis (PFGE) fragment patterns. The PFGE fragment patterns could be classified into three types (i.e., PFGE types B, C, and D). For the PFGE type D, a 255-kbp fragment present in the original K-12 strain was apparently shifted by the size of Stx 2-encoding phage genomic DNA (ca. 65 kbp) to the position at 320 kbp. In contrast, the types B and C showed the above fragment shift plus further 6- and 10-fragment differences, respectively, from the original K-12 strain. The evidence suggests that even a single genetic event like lysogeny can cause marked genotypic modification of the host strain.

Bacteriophages↗
PubMed2003

IpaC of Shigella binds to the C-terminal domain of beta-catenin.

IpaC of Shigella is essential for initial bacterial entry into epithelial cells. We report here that IpaC interacts with beta-catenin and destabilizes the cadherin-mediated cell adhesion complex. Using a yeast two-hybrid system, we identified beta-catenin as a binding partner of IpaC within the host cell after cell entry, but not in the initial entry. Co-immunoprecipitation, confocal microscopy, and GST pull-down experiments confirmed the intracellular and cell-free interactions between these two proteins. The interaction sites were mapped to the ninth armadillo repeat of beta-catenin and to the C-terminus of IpaC. IpaC-associated beta-catenin was phosphorylated at tyrosine residues. This phosphorylation led to the destabilization of the functional cadherin-catenin complex, which could be a mechanism whereby the epithelial cell-cell tight adhesion is disrupted. These events may facilitate the further basolateral invasion of bacteria through the disrupted space and/or modulate the cell-to-cell spread of Shigella.

Antigens, Bacterial↗