Hisaaki Sato

Role of Pili in the Pathogenesis of Pseudomonas aeruginosa Burn Infection

The present study using three isogenic mutants (F+P−, F−P+, F−P−) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD50) value for burned mice inoculated with non‐piliated (P−) mutant was at least ten times higher than those inoculated with piliated (P+) bacteria. Meanwhile the LD50 value for burned mice inoculated with non‐flagellated (F−) mutant was at least 105 times higher than those inoculated with flagellated (F+) bacteria. At 24 hr after inoculation, the bacterial counts in burned skin of mice inoculated with P+ bacteria were ten times higher than those inoculated with P− bacteria; and at 48 hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24 hr after inoculation, P+ bacteria were isolated from blood, liver (F+P+), lung (F+P+), and kidney, while P− bacteria were not present in these tissues. And at 48 hr after inoculation, P+ bacteria were isolated from all tissues, while P− bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili‐mediated enhancement of virulence of P. aeruginosa was attributed to pili‐mediated enhanced colonization of the organisms at the burned skin surfaces.

Isolation of exfoliative toxin from Staphylococcus intermedius and its local toxicity in dogs

A rounding effect was demonstrated in cultured cells inoculated with the culture filtrates (CFs) of 60 strains of Staphylococcus intermedius derived from dogs affected with pyoderma. Exfoliative toxin (ET)-like toxin (ETLT) was isolated from the CF of S. intermedius strain D-52, which exhibited strong rounding activity and then was purified by gel filtration on a Sephadex G-75 column, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ETLT caused exfoliation in 1-day-old chickens, suckling Syrian hamsters, and dogs, but not in suckling mice. The ETLT was serologically different from exfoliative toxin A (ETA), exfoliative toxin B (ETB), exfoliative toxin C (ETC), S. hyicus exfoliative toxin A (SHETA), and SHETB, as shown by Western blot analysis. The molecular weight of the ETLT was estimated at 30 kDa by SDS-PAGE. In the present study, we propose the ETLT was a novel type of ET, S. intermedius exfoliative toxin (SIET).

Correlation between occurrence of exudative epidermitis and exfoliative toxin-producing ability of Staphylococcus hyicus

Staphylococcus hyicus was isolated from healthy pigs and pigs affected with exudative epidermitis (EE). Thirty seven strains (P-7 to P-43) were isolated from pigs affected with EE on 8 farms while 131 strains were isolated from healthy pigs bred on 2 farms in Japan. Isolation rate for pigs affected with EE was 100% while that for healthy pigs was 35.4%. The biochemical and cultural characteristics of the isolates from healthy and diseased pigs were identical except for the Voges-Proskauer reaction. The culture supernatant of many isolates caused skin exfoliation in 1-day-old chickens. Therefore, many isolates were considered to produce S. hyicus exfoliative toxin (shET). The rate of shET production by the isolates from piglets affected with EE was 87.5%, while that of the isolates from healthy pigs was 76.1%. shETs were divided in two serotypes by immunodiffusion. Piglets experimentally infected with shET-producing and nonproducing strains were observed. Local skin erythema at the inoculation site was observed with nonproducing strains and disappeared within 48 h, while the skin erythema at the sites inoculated with shET-producing strains did not disappear until 7 days after inoculation. Typical clinical signs, such as exfoliation, exudation and crusting were observed only in the piglets inoculated with shET-producing strains.

Isolation of exfoliative toxin from Staphylococcus hyicus subsp. hyicus and its exfoliative activity in the piglet

Exfoliative toxin was isolated from the sterile cell-free filtrate of 24 h culture of Staphylococcus hyicus subsp. hyicus strain P-1. The partial purification of exfoliative toxin produced by S. hyicus (shET) was performed by precipitation with 50-80% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column and column chromatography on DEAE-cellulose. Partially purified shET (pp-shET) caused exfoliation in piglets at 8 to 12 h after intradermal or subcutaneous injection. However, heat-treated pp-shET did not cause exfoliation in piglets for up to 24 h after injection. On histopathological examination of the skin at 12 h after injection of pp-shET, an intraepidermal cleavage plane was shown between the stratum corneum and stratum granulosum and at the stratum granulosum.

Chromosomal and Extrachromosomal Synthesis of Exfoliative Toxin from Staphylococcus hyicus

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by Staphylococcus hyicus (SHET) under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxins are distinct from each other is given. The molecular weights of SHET from plasmidless strain P-1 (SHETA) and from plasmid-carrying strains P-10 and P-23 (SHETB) were almost equal. Both of the serotypes of SHET exhibited exfoliation in 1-day-old chickens. The plasmid-cured (P(-)) substrains (P-23C1 and P-23C2) of S. hyicus P-23 did not cause exfoliation in 1-day-old chickens, whereas P(-) substrains (P-10C1 and P-10C2) of strain P-10 caused exfoliation, but they decreased their exfoliative activity. These findings suggest that SHETB was synthesized along with SHETA by strain P-10, whereas the P-23 strain synthesized SHETB alone. The plasmid-carrying strain (P-23) as well as the plasmidless strain (P-1) exhibited the typical clinical signs of exudative epidermitis in pigs. However, plasmid-cured (P(-)) substrains of P-23 (P23C1 and P23C2) did not exhibit the typical clinical signs of exudative epidermitis. These findings suggest that SHETA is synthesized under chromosomal control and SHETB is synthesized under plasmid control and that SHET-producing strains can be divided into three groups: SHETA-producing strains, SHETB-producing strains, and strains producing both toxins.

Antimicrobial resistance of Staphylococcus pseudintermedius isolates from healthy dogs and dogs affected with pyoderma in Japan

Staphylococcus pseudintermedius strains were isolated from healthy dogs and dogs with pyoderma in 2000-2002 and 2009. All the isolates from dogs with pyoderma in 1999-2000 and from healthy dogs in 2000-2002 and 2009 were susceptible to cefalexin and/or other cephalosporins and oxacillin. However, 7.1-12.5 and 11.4% of S. pseudintermedius isolates from dogs with pyoderma in 2009 were resistant to cephalosporins and oxacillin, respectively. All S. pseudintermedius isolates from dogs with pyoderma in 1999-2000 and those from healthy dogs in 2000-2002 were susceptible to fluoroquinolones; however, 50% of the S. pseudintermedius strains isolated from dogs with pyoderma in 2009 and 30% of the S. pseudintermedius strains isolated from healthy dogs in 2009 were resistant to fluoroquinolones. Of the 21 oxacillin-resistant S. pseudintermedius (MRSP) isolates, 11 carried SCCmec type V and 10 carried hybrid SCCmec types II-III. Staphylococcus pseudintermedius strains that were resistant to only one of three fluoroquinolones had a mutation in the quinolone resistance determination region of grlA, whereas S. pseudintermedius strains that were resistant to two or more fluoroquinolones had mutations in the quinolone resistance determination regions of both grlA and gyrA.

Cloning of the gene coding for Staphylococcus hyicus exfoliative toxin B and its expression in Escherichia coli.

The Staphylococcus hyicus exfoliative toxin B (SHETB) gene was cloned into pUC118 and expressed in Escherichia coli. The nucleotide sequence of the SHETB gene consists of a coding region of 804 bp specifying a polypeptide of 268 amino acid residues, which included a putative 20-residue signal sequence.

Susceptibility of various animals and cultured cells to exfoliative toxin produced by Staphylococcus hyicus subsp. hyicus

In piglets inoculated with partially purified exfoliative toxin (pp-shET) produced by Staphylococcus hyicus subsp. hyicus, exfoliation was observed at 12 h after injection. Chickens inoculated with the same dose of pp-shET also showed exfoliation within 30 min of injection. However, exfoliation was not demonstrated in mouse, rat, guinea pig, hamster, dog or cat inoculated with pp-shET until 24 h after injection. In cultured cell lines, especially L-929 and Hep-2, the rounding effect occurred after incubation with pp-shET for 1 h. The rounding effect was also seen in five other cultured cells (NCTC 2544, HeLa/S3, HmLu-1, CHO and BHK-21) 6-24 h after exposure to pp-shET. These round cells survived for 72 h after inoculation and formed a monolayer 24 h after changeover to a toxin-free medium. The rounding effect was observed in cells after the formation of the monolayer, but not before. It was suggested that the rounding effect was not caused by the increase in cyclic AMP in cells inoculated with pp-shET but by the cleavage of intracellular contacts.

Cloning of the gene coding for Staphylococcus intermedius exfoliative toxin and its expression in Escherichia coli

An exfoliative toxin (SIET)-producing strain (D-52) of Staphylococcus intermedius derived from canine pyoderma did not possess large plasmids. Therefore, the gene coding for SIET was considered to be located on the chromosomal DNA. The SIET gene was cloned from the chromosomal DNA of S. intermedius and was expressed in Escherichia coli. The nucleotide sequence of the SIET gene consists of a coding region of 990 bp specifying a polypeptide of 330 amino acid residues, which included a putative 42-residue signal sequence.

Immunological characterization of protective antigens prepared by alkaline treatment of whole cells and from the culture filtrate of Erysipelothrix rhusiopathiae

Culture filtrate and alkaline-extracted antigens from whole cells of an attenuated strain of Erysipelothrix rhusiopathiae (strain Koganei: serovar 1a) were fractionated with ammonium sulfate; both induced protective immunity in mice. Sephadex G-200 gel filtration revealed three protein fractions in the alkaline-extracted antigen and four protein fractions in the culture filtrate antigen. A fraction in the alkaline extract (NaOH P-2) and in the culture filtrate (CF P-2) induced protection in mice against challenge with a different serovar strain (strain Agata: serovar 5). Anti-NaOH P-2 and anti-CF P-2 mouse sera were protective against different serovars. Glycoprotein fraction derived from CF P-2 antigen by affinity chromatography with Con A-Sepharose 4B did not show protective activity. Western blotting between the antisera (anti-NaOH P-2, Anti-CF P-2 and anti-Koganei strain) and the antigens (NaOH P-2, and sonicated antigens of Agata, Fujisawa and Koganei strains) showed strong recognition of the same bands at 62, 42 and 41 kDa.