Yasuo Mizuguchi

Comparison of the nucleotide sequences of the genes for the thermostable direct hemolysin and the thermolabile hemolysin from Vibrio parahaemolyticus

The nucleotide sequences of genes encoding the thermostable direct (TSD) hemolysin and the thermolabile (TL) hemolysin of Vibrio parahaemolyticus were determined. From the nucleotide sequence of the TSD hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 189 amino acids and 165 amino acids, and that the molecular weights were 21.1 kDa or 18.5 kDa, respectively. Our data regarding TSD hemolysin were in complete agreement with previously published data. From the nucleotide sequence of the TL hemolysin gene, it was revealed that the preprotein and the mature protein consisted of 418 amino acids and 398 amino acids, and that the molecular weights were 47.5 kDa and 45.3 kDa, respectively. The GC content of the TSD hemolysin gene was 35.6%, while that of the TL hemolysin gene was 47.6% which is almost the same as that of V. parahaemolyticus genome. Maxicell analysis revealed that the molecular weights of the proteins encoded by the TSD hemolysin gene were 22.0 and 19.5 kDa, and that of the protein encoded by the TL hemolysin gene was 45.5 kDa, and that the promoters of these two hemolysin genes of V. parahaemolyticus were functional in Escherichia coli.

Mechanism of Antibiotic Resistance in Mycobacterium intracellulare

The mechanism of resistance of Mycobacterium intracellulare strain 103 and other clinical isolates to a variety of drugs including aminoglycoside and peptide antibiotics was investigated. Enzymatic inactivation of aminoglycoside and peptide antibiotics could not be demonstrated. Ribosomes of the strain were found to be sensitive to the antibiotics. The levels of resistance of strain 103 and other clinical isolates decreased dramatically when the culture medium was changed from Dubos agar to Tween 80‐containing agar. These results suggest that a permeability barrier is the reason for naturally occurring resistance in M. intracellulare.

Pathogenesis of Vibrio parahaemolyticus: Intraperitoneal and Orogastric Challenge Experiments in Mice

It has been noted that V. parahaemolyticus isolated from patients with food poisoning are almost always thermostable direct hemolysin (TDH)‐positive, whereas, isolates from foods or environmental sources are usually TDH‐negative. The virulence of V. parahaemolyticus in mice was examined by using intraperitoneal and orogastric challenge models, where the strains used were those isolated from patients and foods with food poisoning and included TDH‐positive and ‐negative. The LD50 in mice was estimated to be approximately 107 in the former and 108 in the latter challenge model. In the mice killed by challenge, either intraperitoneal or orogastric, the following pathological changes were almost always observed: swelling, redness and fluid accumulation in the small intestine, particularly the upper part of it. Histologically, congestion, edema, and vacuolation were observed in mucosal and submucosal tissues; furthermore, ulceration at tips of some villi was noticed. The lethality and pathological finding in mice due to V. parahaemolyticus were demonstrated similarly in both challenge models and irrespective of the TDH phenotype of the strain used. It will be suggested that the findings in challenged mice may be. attributed to enteropathogenic factor(s) other than TDH of V. par ahaemolyticus.

Altered Ribosomes in Antibiotic-Resistant Mutants of Mycobacterium smegmatis

Two alleles for viomycin-capreomycin resistance (vic) in Mycobacterium smegmatis affect ribosome structures. One (vicA) affects a component of 50S subunits and the other (vicB) affects a component of 30S subunits. The locus for neomycin-kanamycin resistance (nek), which is linked to vicA and vicB, affects a component of 30S subunits. Although the erythromycin resistance locus (ery) is linked to vic and nek, no ribosomal alterations could be detected. Mutations at the streptomycin locus (str) not linked to vic and nek caused alterations of 30S subunits.

Inhibitory Effect of Capsular Antigen of Vibrio vulnificus on Bactericidal Activity of Human Serum

Opaque (Op) and translucent (Tr) colonial variants were isolated from Vibrio vulnificus strains. Op‐type variants were more resistant than the isogenic Tr‐type variants, but the survival rate of the Op‐type variants varied with the strains. Antisera were prepared by immunizing rabbit with whole cells of Op and Tr variants of some strains, in which the difference of the sensitivity between Op and Tr cells was remarkable. Then agglutination tests with their living and heat‐killed cells were carried out. The results suggested the presence of capsular antigen in Op cells and its absence in Tr cells, with the exception of the existence of a slight amount of capsular material in Tr variants of strain L‐180. The thin capsular layer of Tr cells of strain L‐180 was also demonstrated electron microscopically, but the layer was thinner than that of the isogenic Op cells. Results of determination of sugar content in the extracted capsular fraction also showed that Op to Tr transformation was due to loss of capsular antigen of the cells. These results confirmed the morphological studies previously reported which suggested the prevention of host defense system by the capsular material of the vibrio.

Morphological changes induced by beta-lactam antibiotics in Mycobacterium avium-intracellulare complex.

In vitro activity of seven beta-lactam antibiotics against strains of Mycobacterium avium-intracellulare was evaluated by the agar dilution method. The activity was influenced by the presence or absence of Tween 80 in Dubos medium, and cephazolin and cefotaxime were effective against most strains in the presence of Tween 80. beta-Lactam antibiotics at low concentrations induced long filamentous cells with branching. In contrast to the filaments induced by ampicillin, in which septation was rarely observed, filaments induced by cephazolin had many septa, suggesting that the mechanisms of filament induction were different from the drugs used. At high concentrations, ampicillin and cephazolin induced osmotically sensitive cells with bulging at polar end of the cells. Analysis of penicillin binding proteins (PBPs) of the organism showed that there were at least nine PBPs with molecular weights between 32,000 and 94,000 in the cytoplasmic membrane. Ampicillin showed the highest affinity for PBPs 1a or 1b, or both, and also PBPs 3a or 3b, or both. In contrast, there was very little specificity of binding of cephazolin for any of the PBPs. Images

Antibiotic Susceptibility of Legionella pneumophila Philadelphia- 1 in Cultured Guinea-pig Peritoneal Macrophages

The effect of antimicrobial agents on the intracellular multiplication of Legionella pneumophila in cultured guinea-pig peritoneal macrophages was measured. Beta-lactam antibiotics at concentrations 5 to 400 times the MIC in vitro did not inhibit the intracellular growth of the organism. Gentamicin inhibited the growth considerably but failed to eliminate the organism from the phagocytic mixture. Chloramphenicol or tetracycline at 10 micrograms ml-1 (40 or 5 times the MIC in vitro respectively) did not eliminate the organism. At a higher concentration (30 micrograms ml-1), however, these drugs eliminated the bacterium from the mixture. Only erythromycin and rifampin were effective in killing the organism at very low concentration (1 microgram ml-1). Intracellular multiplication of L. pneumophila was observed clearly by light microscopy using Wright-Giemsa staining.

Isolation and characterization of a filamentous phage, Vf33, specific for Vibrio parahaemolyticus.

Phage Vf33, a filamentous phage about 1,400 nm long and 7 nm wide, specific for Vibrio parahaemolyticus, was isolated and characterized. The buoyant density of Vf33 in CsCl was 1.292 g/cm3. As with other filamentous phages, the lytic activity of Vf33 was resistant to heating below 80 C and to treatment with diethylether, acetone or methanol but sensitive to chloroform. The nucleic acid of this phage is single‐stranded circular DNA 8.4 kb in size. The viral genome was converted to a double‐stranded replicative form in the host cell. Among the strains tested, only V. parahaemolyticus strains possessing K38 antigen was sensitive to the phage.

Interaction between 30 S Ribosomal Components in a Viomycin Resistant Mutant of Mycobacterium smegmatis

A high level viomycin resistant mutant of Mycobacterium smegmatis ATCC 14468 (AC16) was analyzed genetically and biochemically in an attempt to understand the mechanisms of expression of high level viomycin resistance and co‐resistance to kanamycin and streptomycin. Genetic analysis has shown that at least three different genes (vicC, str, and kan) were involved in the phenotypic expression of drug resistance in AC16, and high level resistance to viomycin was due to interactions between the products of these genes.

Plasmid profiles of penicillinase-producing Neisseria gonorrhoeae isolated in Fukuoka, Japan

Shin-ichi YOISHIDA,*,1 Hiromi OHTA,1 Hatsumi TANIGUCHI,1 Midori OGAWA,1 Yasuo MIZUGUCHI,1 Yasumasa KUNIFUJI,2 Shiro CHIHARA,3 and Shinji URABE4 1 Department of Microbiology, School of Medicine, 2Division of Clinical Chemistry, and 3Division of Microbiology, School of Medical Technology, University of Occupational and Environmental Health, Japan, Kitakyushu, Fukuoka 807, and 4Urabe Hospital , Nishi-Nakasu, Fukuoka, Fukuoka 810