Ryusuke Mizukane

Antimicrobial activity of superoxidized water

We tested the antimicrobial activity of superoxidized water against methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa and Burkholderia cepacia. The number of bacteria was reduced below detection limit following incubation in superoxidized water for 10 s. The bactericidal activity of superoxidized water was similar to that of 80% ethanol, but superior to that of 0.1% chlorhexidine and 0.02% povidone iodine. We conclude that superoxidized water is a low cost but powerful disinfectant.

Comparative in vitro exoenzyme-suppressing activities of azithromycin and other macrolide antibiotics against Pseudomonas aeruginosa.

The inhibitory effects of azithromycin (AZM), a new 15-membered macrolide antibiotic, on the production of exotoxin A, total protease, elastase, and phospholipase C by Pseudomonas aeruginosa were determined, and the virulence-suppressing effects of AZM were compared with those of erythromycin (EM), roxithromycin (RXM), and rokitamycin (RKM). The effect of exposure of P. aeruginosa PA103 or B16 in cultures to sub-MICs of these macrolide antibiotics on the production of exoenzymes was determined. AZM suppressed the in vitro production of extracellular and intracellular exotoxin A by P. aeruginosa PA103 more than did EM, even at a concentration of only 2 micrograms/ml. At concentrations of between 4 and 32 micrograms/ml, AZM also inhibited total protease, elastase, and phospholipase C production by P. aeruginosa B16 more than did EM, RXM, and RKM. AZM was effective in suppressing exotoxin A and total protease production through 24 h of incubation in the presence of drug at sub-MICs, but it had no significant effect on either the growth of P. aeruginosa or its total protein production. Moreover, at a concentration of 4 micrograms/ml, AZM suppressed exoenzyme production by other strains of P. aeruginosa more than did EM. These findings indicate that AZM, EM, RXM, and RKM each has an inhibitory effect on exoenzyme production separate from the antimicrobial effect and that, of these macrolides, AZM has the strongest virulence-suppressing effect.

Potential effects of erythromycin on host defense systems and virulence of Pseudomonas aeruginosa.

We evaluated several potential effects of erythromycin (EM) on host defense systems and the virulence of Pseudomonas aeruginosa. Peritoneal macrophages obtained from mice given 250 mg of EM per kg of body weight for 7 days by the intraperitoneal, intravenous, subcutaneous, or oral route produced significantly greater amounts of thymocyte-activating factors. These data suggest that EM enhances the in vivo production of cytokines, such as interleukins 1 and 6. Treatment of P. aeruginosa D4 with subinhibitory concentrations of EM enhanced the association of bacteria with murine Kupffer cells in vitro and increased bacterial clearance from the blood in mice. EM suppressed the in vitro production of exotoxin A, total protease, elastase, and phospholipase C by P. aeruginosa D4; exotoxin A production by P. aeruginosa PA-103; and total protease production by P. aeruginosa B16 and PAO1 in a generally dose-dependent manner. These data demonstrate that EM produces various effects in addition to its direct antimicrobial activity, suggesting that it has potential as an immunomodulator or bacterial virulence-suppressing agent against P. aeruginosa and other infections.

Trends in antimicrobial resistance of Streptococcus pneumoniae in Japan.

A total of 184 isolates of Streptococcus pneumoniae were recovered from the sputa of patients over a 5-year period in the Nagasaki area and were examined. A total of 30 strains were resistant to penicillin (MIC, > or = 0.10 micrograms/ml), 13 of which belonged to serotype 19B. These strains showed decreased susceptibility to other antimicrobial agents. Vancomycin, cefpirome, and imipenem were the most active agents tested.

In vitro and in vivo activities of macrolides against Mycoplasma pneumoniae.

We investigated the in vitro and in vivo activities of macrolides against Mycoplasma pneumoniae. In vitro MICs of azithromycin, erythromycin, clarithromycin, and roxithromycin were determined. Azithromycin was the most potent antimicrobial agent tested in vitro. Its MIC for 90% of the strains was 0.00024 micrograms/ml. MICs for 90% of the strains of erythromycin, clarithromycin, and roxithromycin were 0.0156, 0.0078, and 0.03125 micrograms/ml, respectively. In vivo activities were assessed in a pulmonary infection model with Syrian golden hamsters. We evaluated the in vivo effects on reduction of viable M. pneumoniae cell counts and on reduction of microscopic and macroscopic histopathologies for azithromycin, erythromycin, and clarithromycin given at 10 mg/kg once daily for 1 and 3 days and given at 15 mg/kg twice daily for 2.5 and 5 days. Azithromycin was significantly more effective than erythromycin or clarithromycin in the same regimens. Especially at 10 mg/kg once daily for 1 day, only azithromycin was significantly effective in the reduction of viable M. pneumoniae cells and histopathologies. These results show that azithromycin is more efficacious than the other drugs tested against M. pneumoniae pneumonia in hamsters. These data suggest that clinical studies of macrolides in human patients are warranted. Images

In vitro and in vivo activities of sparfloxacin against Mycoplasma pneumoniae

The in vitro and in vivo activities of sparfloxacin against Mycoplasma pneumoniae were compared with those of erythromycin, levofloxacin, ofloxacin, and minocycline. The MICs of sparfloxacin, erythromycin, levofloxacin, ofloxacin, and minocycline for 90% of the 43 M. pneumoniae strains tested were 0.063, 0.016, 0.5, 1, and 0.5 microgram/ml, respectively. In the experimental pulmonary M. pneumoniae infection model in Syrian golden hamsters, sparfloxacin was as effective as erythromycin when orally administered at 15 mg/kg twice daily for 5 days and more effective than erythromycin when orally administered at 10 mg/kg once daily for 5 days. Sparfloxacin was more effective than levofloxacin and ofloxacin in both dosing regimens. The peak concentrations of sparfloxacin in hamster sera after administration of single oral doses of 15 mg/kg were almost the same as those in human sera after administration of single oral doses of 200 mg (the usual clinical dose), and the half-life of sparfloxacin in hamster serum was shorter than that in human serum after administration of a single oral dose of 200 mg. These results suggest that sparfloxacin may be clinically useful for the treatment of M. pneumoniae infections.

An Elderly Patient with Hemophagocytic Syndrome due to Severe Mycoplasma Pneumonia with Marked Hypercytokinemia

We present an extremely rare case of hemophagocytic syndrome (HPS) induced by fulminant Mycoplasma pneumoniae(Mp) pneumonia in an elderly adult. Erythrocytopenia and thrombocytopenia were observed in a patient with acute respiratory failure, liver dysfunction and renal failure. Mp-associated HPS was diagnosed in this case by clinical and laboratory findings, including a bone marrow aspiration specimen and serum Mp antibody titer. High serum levels of soluble interleukin-2 receptor, interleukin-6, human interleukin-10 and macrophage-colony stimulating factor were observed. Hypercytokinemia is a useful marker of disease activity and prognosis. Combined treatment with methylprednisolone and erythromycin was successful and led to a favorable outcome. Physicians should be aware of HPS as a complication in Mp infection.

Cloning and expression of human defensin HNP-1 genomic DNA in Escherichia coli

We amplified a 181-bp DNA fragment encoding HNP-1 from genomic DNA of human polymorphonucleated neutrophils by PCR. Sequencing of this fragment revealed that it only contained the mature protein coding region of HNP-1 and no intron was found in the sequences. The PCR product of HNP-1 genomic DNA was then cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase (the GST-HNP-1 fusion protein). The GST-HNP-1 fusion protein was expressed as insoluble inclusion bodies, so they were collected and solubilized in 8 M urea. Thus, reasonably pure GST-HNP-1 fusion protein was obtained, with which rabbits were immunized to raise polyclonal antibody against HNP-1. Ouchterlony immunodiffusion and immunoblotting showed that the antiserum reacted with chemically synthesized HNP-1, -2 and human PMN extracts and did not cross react with human GST in these assays. We demonstrated that human defensin HNP-1, a small cationic protein, was expressed in E. coli as an insoluble GST fusion protein, and polyclonal antibody raised by immunizing rabbits with this fusion protein did not cross react with human GST and was specific to defensins.

In Vitro and In Vivo Activity of AM-1155 against Mycoplasma pneumoniae

Mycoplasma pneumoniae is a major cause of pneumonia, accounting for as many as 20% of the total number of cases of this disease. The recommended therapy is erythromycin, which is effective in reducing the duration of symptoms_ However, the increasing resistance of conventional bacteria to penicillin and cephems may lead to more frequent use ofthe new quinolones, and the resistance of M. pneumoniae to erythromycin has also been reported (George & Frank 1991). Recently, it has been reported that the new quinolone antibiotics are effective against Mycoplasma (Robert et al. 1967); thus, it will become increasingly important to know the comparative susceptibilities of M. pneumoniae to various quinolones_ This study examines the in vitro activity of AM-1155 (a new quinolone with a broad spectrum of antimicrobial activity, and synthesised by Kyorin Pharmaceutical Co. Ltd) compared with ofloxacin, ciprofloxacin, lomefloxacin, tosufloxacin, erythromycin and minocycline, against 30 M. pneumoniae strains (27 clinical isolates, and standard strains, FH, Mac and MI29). The MIC values of these drugs were determined by the microbroth dilution method. Table I. In vitro activity of AM-1155 against Mycoplasma pneumoniae

Comparison of Complement Fixation and Microimmunofluorescence Tests in Respiratory Infections Caused by Chlamydia and an Evaluation of the Serum Amyloid A Protein in Chlamydial Infections

We retrospectively analyzed serum samples for antibodies to chlamydia species using the micro-immunofluorescence (MIF) test from 72 patients previously tested by the clamydia complement fixation (CF) test, which is used to detect pulmonary psittacosis. Nineteen patients were positive for chlamydia with the CF test. Of these, 9 patients (47.4%) were also positive forC. psittaci infection, and 7 patients (36.8%) were positive forC. pneumoniae infection by the MIF test. Five (9.4%) and (11.3%) of the 53 CF-negative patients were positive for psittacosis andC. pneumoniae infection by the MIF test, respectively. Our results indicate that the serum of patients suspected of pulmonary psittacosis should be examined by the MIF test as well as CF in order to distinguish between infections caused byC. psittaci, C. pneumoniae andC. trachomatis, and to improve the sensitivity of serodiagnosis. Serum samples of all 7 patients positive by both CF and the MIF test forC. pneumoniae infection showed a diagnostic IgG antibody change toC. pneumoniae without IgM antibody changes, which suggested a primary infection. These results suggest that the diagnostic CF antibody response occasionally appears in reinfection, especially inC. pneumoniae infection. There was a positive correlation (r=0.858) between C-reactive protein and serum amyloid protein A, a sensitive acutephase serum reactant, over a wide range of concentrations in 19 patients with chlamydia respiratory infections.