Tadayuki Okitsu

Extended serotyping scheme forVibrio cholerae

Fifty-seven new O serogroups have been added to the existing serotyping scheme ofVibrio cholerae to extend the scheme from O84 to O140. Prominent new additions were serogroups O139 and O140. The reference strain of O139 was isolated from a patient from an epidemic of cholera-like diarrhea in Madras, Southern India. Serogroup O140 was assigned to a group ofV. cholerae strains which were tentatively named as the “Hakata” serogroup and which possessed the C (Inaba) factor but not the B (Ogawa) nor the A (major specific antigen of O1 serogroup ofV. cholerae). As all antisera against reference strains ofV. cholerae contained some amount of antibody to the rough (R) antigen, all diagnostic O antisera must be absorbed with the reference rough strain, CA385.

Evaluation of DNA Fingerprinting by PFGE as an Epidemiologic Tool for Salmonella Infections

To evaluate DNA fingerprinting as an epidemiologic tool, pulsed‐field gel electrophoresis (PFGE) was performed on isolates of Salmonella, including S. typhimurium, S. thompson, and S. enteritidis. Chromosomal DNA was digested with the restriction endonucleases Bln I and Xba I. The patterns of S. thompson and S. typhimurium isolates from various sources were different from one another. There was no correlation between the phage type and the digestion pattern of S. enteritidis isolates. Some strains belonging to one phage type were distinguished by their PFGE pattern in this study. These results suggest that the Bln I and Xba I digestion patterns of chromosomal DNA are useful for epidemiological analysis of an outbreak of Salmonella infection or food poisoning.

Two strains of Vibrio cholerae non-O1 possessing somatic (O) antigen factors in common with V. cholerae serogroup O139 synonym "Bengal"

Two strains (O22 reference strain, 169–68, and strain 490–93 isolated from a patient with diarrhea in Thailand) ofVibrio cholerae non-O1 possessing somatic (O) antigen factors in common withV. cholerae O139 synonym “Bengal” are described. The O antigens of these two strains were closely related to that ofV. cholerae O139 in an a,b-a,c type of relationship, but were not completely identical with serogroup O139. Therefore, both these strains are not classified into the O139 serogroup ofV. cholerae, because they have their own major antigens. As the strain 490–93 could not be placed into any of the 154 established O serogroups ofV. cholerae, this strain was assigned to a new serogroup, O155. For practical use, the diagnostic antiserum prepared against the O139 reference strain (MO45, ATCC 51394) ofV. cholerae must be absorbed with reference strains 169–68 and 490–93 representing serogroups O22 and O155 ofV. cholerae to remove cross-reacting agglutinins of the O22 and O155 strains, respectively.

Levels of thermostable direct hemolysin produced by Vibrio parahaemolyticus O3:K6 and other serovars grown anaerobically with the presence of a bile acid.

Twenty-three V. parahaemolyticus strains, including 12 pandemic O3:K6 strains, were examined for their growth and production of thermostable direct hemolysin (TDH) under an anaerobic culture condition with or without presence of a bile acid, taurocholic acid (TCA). Both bacterial growth and TDH production were markedly enhanced by TCA for a majority of the strains, but the scale of the TDH production was disproportionately greater than that of the corresponding growth for 14 strains. Such enhancement was, however, not specific to the pandemic strains.

Identification of Shiga Toxin-Producing Escherichia coli Possessing Insertionally Inactivated Shiga Toxin Gene

We have investigated the Shiga toxin genes of Shiga toxin‐producing Escherichia coli (STEC) strains, using polymerase chain reaction (PCR) amplifying the full lengths of these genes. As a result, we found the Shiga toxin 2 gene which was insertionally inactivated by an insertion sequence (IS). This IS element was identical to IS1203v which has been also found in inactivated Shiga toxin 2 genes, and was inserted at the same site as in the previous paper. On the other hand, both Shiga toxin 2 genes were different (98.3% identity). These suggested that IS1203v independently inserted into each Shiga toxin 2 genes, and STEC strains possessing the insertionally inactivated Shiga toxin genes are most likely to have a wide distribution. Amplification of the full length of the Shiga toxin gene is one of the effective methods to detect the gene no matter where the IS element is included, i.e., the insertion can be reflected in the size of amplicon.

Urea Hydrolysis and Suppressed Production of Thermostable Direct Hemolysin (TDH) by Vibrio parahaemolyticus Associated with Presence of TDH-Related Hemolysin Genes

Abstract. A total of 18 strains of V. parahaemolyticus isolated from patients of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were assayed for presence of the thermostable direct hemolysin (TDH) gene and the TDH-related hemolysin (TRH) genes (trh 1 and trh 2) with specific reference to their ability to hydrolyze urea and TDH production. A polymerase chain reaction assay revealed that all urea-hydrolyzing strains (9 strains) carried either trh 1 gene or trh 2 gene. The strains carrying the trh genes as well as the tdh gene produced TDH less by a factor of 4 to 16 than those carrying only the tdh gene, suggesting the expression of the tdh gene was suppressed by the presence of trh gene through a mechanism yet to be defined.

Spontaneous reactivation of Shiga toxins in Escherichia coli O157:H7 cells caused by transposon excision.

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes (stx2) of Escherichia coli O157:H7. Using PCR amplification, we detected the wild-type stx2 genes in colonies of E. coli O157:H7 which possessed stx2 genes inactivated by insertion of IS1203v. This suggests that IS1203v is excised from the inactivated stx2 genes in E. coli O157:H7. We isolated the cells possessing the wild-type stx2 genes, and confirmed Stx2 productivities by reversed passive latex agglutination. We also analyzed the frequency of the appearance of the Stx2-producing cells using a quantitative PCR method. As a result, the frequency was 3.00 x 10(-6) with culturing for 24 h at 37 degrees C, and this increased to 8.83 x 10(-5) when E. coli O157:H7 possessing the inactivated stx2 genes was transformed by an expression plasmid harboring the IS1203v transposase. These results showed that some Stx2-nonproducing E. coli O157:H7 strains could be spontaneously changed into Stx2-producing cells.

Host-dependent activation of IS1203v excision in shiga toxin-producing Escherichia coli

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.

Aeromonas hydrophila の主要O群株からの耐熱性リパーゼ遺伝子の検出

On the basis of DNA hybridization data and phenotypes, most pathogenic strains of Aeromonas hydrophila are grouped into hybridization group 1 (HG1). These pathogenic strains secret the theromostable lipase, and its gene (lipAH) has been cloned and sequenced. The present study was performed to identify the pathogenic strains of A. hydrophila by the polymerase chain reaction (PCR) technique to detect the lipAH. Synthetic oligonucleotide primers (lipAH-ns-1 and -na-1) were used in the PCR. The PCR identified 80% of lipAH-positive strains, consisting of seven of the 11 O11 strains (64%), 13 of the 15 O16 strains (87%) and 25 of the 30 O34 strains (83%) in clinical isolates of A. hydrophila used in this study.

Isolation of Aeromonas Species from Patients with Sporadic Diarrhea and Characterization of Aeromonas hydrophila Isolates

A total of 16 strains of Aeromonas species were isolated from feces of 348 patients with sporadic diarrhea in western Kanagawa, Japan from 1996 to 1998. Of the 16 isolates, 7 were Aeromonas hydrophila, 1 was A. sobria and 8 were A. caviae. The strains of A. hydrophila were examined for hemolytic activities, hemolysin gene types and O-serogroups. Although all 7 strains of A. hydrophila showed hemolytic activities on sheep blood agar, in the test for hemolytic activities in culture supernatant, only 1 of the these strains showed no hemolytic activity against sheep erythrocytes. From the results of PCR assay, the tested strains of A. hydrophila were grouped into 2 hemolysin gene types of [ahh1 + ahh3 + aerA] (n = 6) and [ahh1 + aerA] (n = 1) both of which are recognized to be enteropathogenic. Five of the 7 strains of A. hydrophila belonged to serogroup O11. These results suggest that 7 strains of A. hydrophila isolates are recognized to be enteropathogenic strains and serogroup O11 is the major O-serogroup of enteropathogenic A. hydrophila in humans.