Eiko Yabuuchi

Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and Two Genospecies of the Genus Sphingomonas

Based on the partial nucleotide sequence analysis of 16S ribosomal ribonucleic acid (rRNA), presence of unique sphingoglycolipids in cellular lipid, and the major type of ubiquinone (Q10), we propose Sphingomonas gen. nov. with the type species Sphingomonas paucimobilis (Holmes et al, 1977) comb. nov. From the homology values of deoxyribonucleic acid‐deoxyribonucleic acid hybridization and the phenotypic characteristics, three new species, Sphingomonas parapaucimobilis, Sphingomonas yanoikuyae, Sphingomonas adhaesiva, and one new combination, Sphingomonas capsulata, are described. S. par apaucimobilis JCM 7510 (=GIFU 11387), S. yanoikuyae JCM 7371 (=GIFU 9882), and S. adhaesiva JCM 7370 (=GIFU 11458) are designated as the type strains of the three new species. Emended description of the type strain of S. capsulata is presented.

Cytopathogenic effect of Salmonella typhi GIFU 10007 on M cells of murine ileal Peyer's patches in ligated ileal loops: an ultrastructural study.

An electron microscopic study revealed that, within 30 min after inoculation into the ligated ileal loop of anesthetized mice. cells of Salmonella typhi GIFU 10007 adhered to the M cell surface of Peyers patch lymphoid follicle epithelium, and induced almost complete destruction of M cells. The M cell cytoplasms were pinched off and extruded from the epithelial lining into the luminal space together with the lymphoid cells primarily enfolded into the corresponding M cells. When two or more M cells were destroyed, a large defect in the epithelial lining was apparent, and a number of bacteria appeared near the basal lamina of the epithelial lining. These findings suggest, as far as anesthetized murine ileal loops and strain 10007 are concerned, that ileal M cells are the target cell at an early stage of S. typhi infection and the infection may further progress to deeper tissues and to the general circulation.

Small-Scale DNA Preparation for Rapid Genetic Identification of Campylobacter Species without Radioisotope

A simplified and rapid genetic identification method for Campylobacter species without radioisotope was established. Three different amounts of DNA (200, 50, and 12.5 ng) extracted from each type strain of Campylobacter species with standard Marmurs procedure were spotted on a nitrocellulose filter. DNA obtained from one ml bacterial suspension at a concentration of McFarland standard turbidity No. 1 of Campylobacter fetus, C. jejuni, C. coli, and C. pylori isolates were sufficiently labeled with photo‐biotin within 15 min and clearly hybridized with the type strain of the corresponding species within four to six hours. Hybridized spots were visualized with alkaline‐phosphatase‐conjugated streptavidin color‐detection method. The reaction was usually stopped within 30 min. Atypical clinical isolates such as a nitrate‐negative C. jejuni, two nalidixic acid‐resistant C. jejuni, and two strains of C. fetus able to grow at 42 C, which were tentatively identified as such, were definitely identified by the simplified DNA hybridization method presented here. This method will be applicable routinely for the definite identification of atypical strains of Campylobacter species and other gram‐negative bacteria difficult to identify biochemically.

Burkholderia uboniae Sp. Nov., l‐Arabinose‐Assimilating but Different from Burkholderia thailandensis and Burkholderia vietnamiensis

A polar multitrichous Gram‐negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA‐DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B. thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9%. The strain was clearly differentiated from the type strain of B. thailandensis by physiological, biochemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition. Based on the results of 16S rDNA sequence analysis, DNA‐DNA hybridization and phenotypic characterization, Burkholderia uboniae sp. nov. is herein proposed. The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand. Major respiratory quinone is ubiquinone‐8(Q8). G+C content of DNA is 69.71%.

Evaluation of the microplate hybridization method for rapid identification ofLegionella species

Rapid genetic identification of 23Legionella species was performed using microdilution plate hybridization. When an isolate appeared to be a strain of theLegionella species, its DNA was quickly extracted, labelled with photobiotin and distributed to microdilution wells where 23 different DNAs had previously been immobilized. After hybridization for 90 min at 45 °C, the microdilution well which emitted the maximum fluorescence was determined. The criterion for interpreting results of this genetic identification method is given.

Invasiveness of Salmonella typhi strains in HeLa S3 monolayer cells.

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 × 1.2 μm in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 × 3.0 μm and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)‐containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM‐containing medium by both light‐microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40‐fold within 24 hr after inoculation, whereas in the four other less or non‐infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.

An Ultrastructural Study of HeLa Cell Invasion with Salmonella typhi GIFU 10007

Scanning electron micrograph of HeLa S3 monolayered cells, inoculated with viable bacteria of a Salmonella typhi strain GIFU 10007, revealed that the extended microvilli tangled the bacteria within 10 min after inoculation. The micrographs of HeLa cells, at 1 hr after inoculation, indicate the following: shortening of bacterium‐attached microvilli, subsiding of tangled bacteria into microvilli bush, and then attachment of bacterial soma to cell surface making the cell membrane depressed. The transmission electron micrographs, at 1 hr after inoculation, demonstrated the findings of interaction between HeLa cell and S. typhi 10007, similar to those observed on scanning electron micrographs. Hair‐like fine structures from the soma of challenge organisms were also observed. They were in contact with HeLa cell microvilli and cell membrane. The bacteria were first partially and then totally surrounded by the HeLa cell plasma membrane. One, two, or several bacteria with intact outer membrane were enclosed in intracytoplasmic membrane‐bound vacuoles. Fragmented vacuolar membrane was still visible around the intracellularly accumulated bacteria at 24 hr after inoculation. The viable cells of S. typhi 10007 are regarded as internalizing into HeLa cells by a process of endocytosis and to multiply within the membrane‐bound vacuoles.

Effects of Biocidal Treatments to Inhibit the Growth of Legionellae and Other Microorganisms in Cooling Towers

The effects of biocidal treatments for cooling towers were examined through the use of chemicals and ultraviolet irradiation to inhibit the growth of legionellae and other microorganisms. In the water of cooling towers without continuous biocidal treatments, heterotrophic bacteria and bacterivorous protozoan first appeared, and then legionellae increased up to 104 CFU/100 ml. When a UV sterilizer was connected to the cooling tower, the legionellae count was 1/10 or 1/100 of that in the nontreated tower water. In the water of towers supplemented continuously with the biocidal chemicals, legionellae were not found during a 4‐month period. The biocidal treatments tested were proved to suppress the increase of legionellae in cooling‐tower water, and thus are useful in preventing the outbreak of legionellosis due to inhalation of contaminated aerosol from the cooling tower system.

Genotypic and Phenotypic Differentiation of Flavobacterium indologenes Yabuuchi et al 1983 from Flavobacterium gleum Holmes et al 1984

The type and eight strains of Flavobacterium indologenes were clearly differentiated from the type and two reference strains of Flavobacterium gleum by deoxyribonucleic acid‐deoxyribonucleic acid homology data and phenotypic characteristics. Phenotypic characteristics useful to differentiate the two species are presented.

Ecological Studies of Legionella Species

The occurrence and viable counts of Legionella pneumophila in acid‐treated water samples of 62 cooling towers on the main island of Japan were determined by inoculating them onto plates of Wadowsky‐Yee‐Okuda (WYO) agar medium. WYO plate cultures of 39 (63%) of the samples yielded L. pneumophila with viable counts ranging from 10 to 104 colony‐forming units per 100 ml.