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Biomedical subjects

T Miwatani

Publications and source records attributed to T Miwatani.

At least 145 records · Page 8Linked to original sources
PubMed1983

Effects of lincomycin and tetracycline on production and properties of enterotoxins of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli grown in the presence of lincomycin and tetracycline produced an increased amount of heat-labile enterotoxin (LT). These antibiotics increased the production of not only extracellular LT but also intracellular LT. On the other hand, lincomycin did not stimulate the production of heat-stable enterotoxin by enterotoxigenic E. coli. The extracellular LTs produced in the presence of lincomycin and tetracycline were purified and analyzed by sodium dodecyl sulfate-polyacrylamide gel disc electrophoresis. Results showed that the A subunits of the purified LTs were not nicked, unlike that of extracellular LT produced in the absence of the antibiotics.

Anti-Bacterial Agents↗
PubMed1983

Detection by a staphylococcal coagglutination test of heat-labile enterotoxin-producing enterotoxigenic Escherichia coli.

For detection of heat-labile enterotoxin-producing enterotoxigenic Escherichia coli, the staphylococcal coagglutination test reported by Brill et al. (J. Clin. Microbiol. 9:49-55, 1979) was modified to give better results. Staphylococcal cells were sensitized with anti-heat-labile enterotoxin antiserum and suspended in phosphate-buffered saline containing 0.5% bovine serum albumin, 0.05% Tween 80, 0.01% gelatin, and 0.02% NaN3. The test strain was cultured in 0.25 ml of Biken broth no. 2 in a test tube (12 by 100 mm) stood at an inclination of about 10 degrees to the horizontal. After incubation for 5 h at 37 degrees C, the cells were collected by centrifugation at 2,500 rpm for 15 min and suspended in 50 microliters of polymyxin B solution (20,000 IU/ml). The suspension was then incubated for 1 h at 37 degrees C and centrifuged at 2,500 rpm for 15 min, and 10 microliters of the supernatant was used for the test on a slide. The results of the modified test correlated completely with those obtained by the Biken test. The modifications of the staphylococcal coagglutination test described here allow for detection of heat-labile enterotoxin-producing enterotoxigenic E. coli within 6 to 7 h after inoculation of a test strain.

Agglutination Tests↗
PubMed1983

[The antibacterial activity of new cephem antibiotics against clinical isolates. A comparison of the antibacterial activity of cefotaxime with 6 other antibiotics].

During the period from May through July 1981, a comparative study was carried out on the antibacterial activities of cefotaxime (CTX) and ceftizoxime (CZX), cefoperazone (CPZ), latamoxef (LMOX), cefotiam (CTM), cefmetazole (CMZ) and cefazolin (CEZ). CTX and these other cephem antibiotics were tested against fresh clinical isolates which had been obtained from clinical materials by the laboratories of 14 participating medical institutions. 1. The clinical isolates were obtained from various clinical materials in the following decreasing order: urine, sputum and pus/discharge; 85.7% of the isolates came from these materials. 2. Concerning the sources of each species of clinical isolates, it was found that P. aeruginosa was isolated from the greatest number -9- of different clinical materials. This was followed by E. coli and E. cloacae, each isolated from 8 different clinical materials, and C. freundii and E. aerogenes, each found in 7 different clinical materials. 3. In relation to S. pyogenes, S. agalactiae and S. pneumoniae, CTX showed the best antibacterial activity; the second most potent antibiotic was CZX. CMZ and LMOX were found to show relatively high MIC values for those species. Against S. aureus, CEZ showed the best antibacterial activity, but 3 resistant strains had MICs of greater than 100 micrograms/ml. 4. With regard to Gram-negative bacteria, CTX and CZX showed the best antibacterial activities for all of the species, except for P. aeruginosa. These were followed, in order, by LMOX and CPZ. Compared with these 4 antibiotics, CTM, CMZ and CEZ were found to have inferior antibacterial activities against these bacteria. In relation to P. aeruginosa, the peak of the MIC distribution for CPZ was 6.25 micrograms/ml, and this was the best antibacterial activity detected with the various antibiotics tested. This was followed by CTX (25 micrograms/ml) LMOX (25 micrograms/ml) and CZX (50 micrograms/ml). CTM had an MIC of 100 micrograms/ml for 1 strain, and MICs of greater than 100 micrograms/ml for all of the other strains of P. aeruginosa, indicating them to be resistant to this antibiotic. All of the strains were resistant to CMZ and CEZ, showing MICs of greater than 100 micrograms/ml. 5. For each of the tested antibiotics, no correlation was found between the MIC and the serogroup for either P. aeruginosa or S. marcescens.

Bacteria↗
PubMed1983

The use of the Biken test to detect enterotoxigenic "Escherichia coli" producing heat labile enterotoxin.

For identification of enterotoxigenic Escherichia coli producing heat labile enterotoxin (LT), various assay methods have been developed. However, many of these assay methods are unsuitable for routine clinical purposes, especially in developing countries, because they need special materials or techniques or both, such as a large number of animals, stocks of special tissue culture cells, and radioisotopes. Recently, we developed a simple and reproducible assay method, Biken test, which can be widely used in clinical laboratories in both developing and developed countries (15). This method is based on the principles of the Elek test and Ouchterlony double gel diffusion test. The Biken test was used in a field study in Bangladesh with good results (14). Moreover, the Diarrheal Diseases Control Programme, WHO, organized a multi-laboratory study to evaluate the accuracy and reproducibility of the Biken test, in which 5 investigators tested an identical set of 100 unknown strains of E. coli, using the Biken test and at least one other assay method, which includes CHO cell assay, Y1 adrenal cell assay, GM1 ganglioside ELISA, DNA probe test and passive immune hemolysis. The results reported by Sutton (International Symposium on Bacterial Diarrheal Diseases, Osaka, 1982) showed that the Biken test was a reliable assay method. To evaluate further the Biken test, we tested a total of 2,229 strains of E. coli isolated from diarrheal patients. The results showed 99.0% coincidence with those obtained by CHO cell assay. The use of anti-LT antiserum gave sharper and clearer results than cholera antitoxin. Samples for heat stable enterotoxin (ST) assay were obtained from the agar plates used for the Biken test and the ST activities of these samples were compared with those of samples obtained from standard liquid cultures. Results on samples of 2,229 strains from the Biken agar plates and from liquid cultures were almost identical.

Animals↗
PubMed1982

Amino-acid sequence of a heat-stable enterotoxin produced by human enterotoxigenic Escherichia coli.

A heat-stable enterotoxin produced by a human strain of enterotoxigenic Escherichia coli was extensively purified by reverse-phase high-performance liquid chromatography. The minimum effective dose of the purified toxin to cause fluid accumulation in suckling mice was 2.5 ng. The amino acid sequence of the purified toxin was determined by Edman degradation and a combination of fast atom bombardment mass spectrometry and carboxypeptidase digestion to be Asn-Ser-Ser-Asn-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr. This sequence was identical to that deduced from the nucleotide sequence encoding a human heat-stable enterotoxin, reported by Moseley et al., except for the C-terminal Tyr residue.

Amino Acid Sequence↗
PubMed1982

Purification of a lethal toxin produced by Shigella dysenteriae.

A lethal toxin was purified from the culture supernatant of Shigella dysenteriae 1. The purification procedure utilized ammonium sulfate fractionation, column chromatography on DEAE-cellulose, CM-cellulose, hydroxylapatite and gel filtration on Sephadex G-200. About a 4760-fold purification was achieved, with a yield of 2.7%. The purified lethal toxin appeared homogeneous on polyacrylamide gel disc electrophoresis. The LD50 of the purified lethal toxin in mice following i.p. injection was about 7.5 micrograms/kg. Injection i.p. of 2 LD50 of the purified lethal toxin into mice caused paralysis of the limbs followed by death after 2-4 days.

Animals↗
PubMed1982

Molecular heterogeneity of heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli.

The heat-labile enterotoxins produced by human enterotoxigenic Escherichia coli (LTh) and porcine enterotoxigenic E. coli (LTp) were purified to homogeneity, and their molecular properties were compared with those of purified cholera enterotoxin (CT). On polyacrylamide gel disk electrophoresis without sodium dodecyl sulfate, LTh, LTp, and CT differed in mobility, suggesting differences in their ionic charges. The pI values of LTh, LTp, and CT were 7.50, 8.10, and 6.80, respectively. On sodium dodecyl sulfate-polyacrylamide gel slab electrophoresis, the B subunit and A1 and A2 fragments of LTh, LTp, and CT differed in mobility, suggesting that they differed in molecular size. Their molecular sizes seemed to decrease in the following order: B subunit, LTh greater than LTp congruent to CT; A1 fragment, LTp greater than LTh congruent to CT; A2 fragment, LTh congruent to CT greater than LTp. Amino acid compositions of LTh, LTp, and CT were also compared.

Amino Acids↗
PubMed1982

Further evaluation of the Biken test (modified Elek test) for detection of enterotoxigenic Escherichia coli producing heat-labile enterotoxin and application of the test to sampling of heat-stable enterotoxin.

A total of 2,229 strains of Escherichia coli isolated from subjects with traveller's diarrhea were tested for heat-labile enterotoxin production by the Biken test (Honda et al., J. Clin. Microbiol. 13:1--5, 1981). Results of the Biken test showed a 99.0% coincidence with those obtained by Chinese hamster ovary cell assay. The use of antiserum against E. coli heat-labile enterotoxin gave sharper and clearer results than did the use of antiserum against cholera enterotoxin. Samples for heat-stable enterotoxin assay were obtained from the agar plates used for the Biken test, and the heat-stable enterotoxin activities of these samples were compared with those of samples obtained from standard liquid cultures. Results for samples of 2,229 strains from the Biken agar plates and from liquid cultures were almost identical.

Bacteriological Techniques↗
PubMed1982

Immunological detection of the Kanagawa phenomenon of vibrio parahaemolyticus on modified selective media.

Selective media for Vibrio parahaemolyticus, BTB-Teepol agar and modified arabinose-ammonium sulfate-cholate agar, were modified for use in immunological detection of the thermostable direct hemolysin produced by this organism. The modified BTB-Teepol agar and modified arabinose-ammonium sulfate-cholate agar were both found to be useful for the modified Elek test and immunohalo test with antiserum (or immunoglobulin G) against the thermostable direct hemolysin. With these modified media it is possible to isolate V. parahaemolyticus and identify the Kanagawa phenomenon on a single plate and thus save time in obtaining results.

Culture Media↗
PubMed1981

Vascular permeability activity in Escherichia coli heat-stable enterotoxin.

Purified heat-stable enterotoxin (ST) prepared from supernatants of Escherichia coli strain 53402 A-1, isolated from a patient with diarrhea, caused an increase in the permeability of the small blood vessels of the skin of adult rabbits after intracutaneous injection. Increased permeability was manifested by localized accumulation of intravenously injected blue dye at the injection sites. Permeability factor (PF) activity reached a peak 1 h after injection, and recovery of normal permeability was nearly complete in 3 h. Residual PF activity had disappeared by 24 h. Dose-response curves demonstrated a straight-line relationship between the logarithm of the dose and mean blueing diameter over a range of 3 to 12 mm. The PF assay was less sensitive that the suckling mouse assay, and adult rabbits varied in their sensitivity to the PF effect. Five to 100 mouse units of ST were required to consistently evoke strong and unequivocal blue lesions of 7 mm or more in diameter. PF activity was reduced 50 to 70% by heating at 70 degrees C for 30 min, 60 to 80% by boiling for 30 min, and 95 to 98% by autoclaving for 15 min, but it was unaffected by treatment with cholera antitoxin. These findings are consistent with the notion that PF activity is a property of the ST molecule itself. This PF assay cannot be used for the detection of ST in crude culture supernatant fluid because of low sensitivity and the presence of nonspecific PF effects of culture media and other E. coli products.

Animals↗
PubMed1981

Production of cholera-like enterotoxin by a Vibrio cholerae non-O1 strain isolated from the environment.

Vibrio cholerae non-O1 strain E8498, isolated in 1978 from fresh water in Louisiana, produced a vascular permeability factor when cultured in shallow resting cultures of Casamino Acids-yeast extract-glucose medium for 24 h at 30 degrees C. Undiluted resting culture filtrates contained heat-labile permeability factor activity which was only partially neutralized by cholera antitoxin and GM1 ganglioside. Supernatants concentrated with PM-10 membranes caused hemorrhage and necrosis in rabbits within 1 h after intracutaneous injection, whereas appropriate dilutions of both filtrates and concentrates demonstrated delayed permeability factor activity, without hemorrhage or necrosis, which was indistinguishable in appearance from that caused by purified cholera enterotoxin produced by V. cholerae O1 Inaba strain 569B. Crude E8498 filtrates contained the biological equivalent of about 5 ng/ml of purified enterotoxin. Permeability factor activity in the fraction obtained by 20 to 50% saturation of filtrate concentrate with ammonium sulfate could be completely neutralized by reference standard cholera antitoxin prepared against purified 569 B enterotoxin. Hemorrhagic activity was unaffected by cholera antitoxin. A 5,000-fold concentrate of the culture supernatant yielded a line of identity with purified cholera enterotoxin in an agar gel double-diffusion test against cholera antitoxin purified by affinity column chromatography with BrCN-activated Sepharose 4B-linked purified cholera enterotoxin as the adsorbent. These findings indicate that V. cholerae non-O1 E8498 produces a permeability factor which is immunologically and biologically indistinguishable from that produced by a strain of V. cholerae O1 classical biotype.

Antitoxins↗
PubMed1981

Isolation of special antibodies which react only with homologous enterotoxins from Vibrio cholerae and Enterotoxigenic Escherichia coli.

Cholera enterotoxin (CT) and heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli share a common antigenic determinant. In addition, CT and LT each have a unique antigenic determinant. Antisera were prepared by immunoaffinity chromatography against these unique antigenic determinants, that is, antiserum that reacted with CT but not with LT and antiserum that reacted with LT but not with CT. Antiserum against the common antigenic determinant to CT and LT was also prepared by immunoaffinity chromatography. The specificities of these antisera were demonstrated by Ouchterlony double-gel diffusion tests and by neutralization of the activities of CT and LT to cause morphological changes in Chinese hamster ovary cells.

Animals↗
PubMed1981

Immunological nonidentity of heat-labile enterotoxins from human and porcine enterotoxigenic Escherichia coli.

The Ouchterlony double-gel diffusion test demonstrated that heat-labile enterotoxin (LT) from human strains of enterotoxigenic Escherichia coli was antigenically related but not identical to that from porcine E. coli. All human strains examined produced immunologically identical LTs, and all porcine strains examined produced immunologically identical LTs. The Biken test (modified Elek test), developed for detection of E. coli LT (Honda et al., J. Clin. Microbiol. 13: 1-5, 1981), was useful for confirmation of these results.

Animals↗
PubMed1981

In vitro formation of hybrid toxins between subunits of Escherichia coli heat-labile enterotoxin and those of cholera enterotoxin.

Heat-labile enterotoxin (LT) was purified from cells of enterotoxigenic Escherichia coli isolated from a patient with traveller's diarrhea. Purified LT was separated into A and B subunits by treatment with 6 M urea solution in 0.1 M propionic acid (pH 4.0). Biologically active toxin was reconstituted from isolated A and B subunits of LT. Hybrid toxins with biological activity were obtained in vitro from the A subunit of cholera enterotoxin and B subunit of LT, and from the A subunit of LT and B subunit of cholera enterotoxin. The hybrid toxins show a similar toxicity to that of the parent toxins from which the A subunits were derived. The in vitro formations of the hybrid toxins were confirmed by polyacrylamide gel disk electrophoresis.

Animals↗