Kazunori Tomono

Relationship between antimycobacterial activities of rifampicin, rifabutin and KRM-1648 and rpoB mutations of Mycobacterium tuberculosis.

We compared the in-vitro antimycobacterial activities of rifabutin and KRM-1648, two rifamycin derivatives, with that of rifampicin against 163 strains of Mycobacterium tuberculosis. We also evaluated the correlation between the level of resistance to rifampicin, rifabutin and KRM-1648 and genetic alterations in the rpoB gene. All 82 strains susceptible to rifampicin or resistant to rifampicin with MICs < or = 16 mg/L were susceptible to rifabutin and KRM-1648 with MICs < or = 1 mg/L. Seventy-six of 81 strains resistant to rifampicin with MICs > or = 32 mg/L were resistant to both rifabutin and KRM-1648, but with lower MICs than those of rifampicin. KRM-1648 showed more potent antimycobacterial activity than rifabutin against organisms with low MICs (< or = 1 mg/L), while rifabutin was more active than KRM-1648 against organisms with high MICs (> or = 2 mg/L). A total of 96 genetic alterations around the 69 bp core region of the rpoB gene were detected in 92 strains. Alterations at codons 515, 521 and 533 in the rpoB gene did not influence the susceptibility to rifampicin, rifabutin and KRM-1648. Point mutations at codons 516 and 529, deletion at codon 518 and insertion at codon 514 influenced the susceptibility to rifampicin but not that to rifabutin or KRM-1648. With the exception of one strain, all alterations at codon 513 and 531 correlated with resistance to the three test drugs. The resistant phenotype of strains with an alteration at codon 526 depended on the type of amino acid substitution. Our results suggest that analysis of genetic alterations in the rpoB gene might be useful not only for predicting rifampicin susceptibility, but also for deciding when to use rifabutin for treating tuberculosis. Further studies may be required to determine the usefulness of KRM-1648.

Azithromycin Inhibits MUC5AC Production Induced by the Pseudomonas aeruginosa Autoinducer N-(3-Oxododecanoyl) Homoserine Lactone in NCI-H292 Cells

ABSTRACT The features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus. Pseudomonas aeruginosa is one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease. The quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL)] not only regulates bacterial virulence but also is associated with the immune response. In this study, we investigated whether 3O-C12-HSL could stimulate the production of a major mucin core protein, MUC5AC. The effect of a macrolide on MUC5AC production was also studied. 3O-C12-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners. A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C12-HSL. 3O-C12-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-κB phosphorylation in cells, and this induction was suppressed by azithromycin. 3O-C12-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059. Our findings suggest that the P. aeruginosa autoinducer 3O-C12-HSL contributes to excessive mucin production in chronic bacterial infection. Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction.

Immunokinetics in severe pneumonia due to influenza virus and bacteria coinfection in mice

Coinfections of bacteria and influenza are a major cause of excessive mortality during influenza epidemics. However, the mechanism of the synergy between influenza virus and bacteria are poorly understood. In this study, mice were inoculated with influenza virus, followed 2 days later by inoculation with Streptococcus pneumoniae. The kinetics of viral titres, bacterial numbers and the immune response (cytokine and chemokine production) were also analysed. Short-term survival correlated with pathological changes in the lungs of infected mice. Influenza virus or S. pneumoniae infection alone induced moderate pneumonia; however, severe bronchopneumonia with massive haemorrhage in coinfected mice, which caused death of these mice ∼2 days after inoculation with S. pneumoniae, was noted. Intrapulmonary levels of inflammatory cytokines/chemokines, type‐1 T‐helper cell cytokines and Toll-like receptors, and the related mitogen-activated protein kinase signalling molecules (phosphorylated extracellular signal-regulated kinase ‐1 and ‐2, p38 and c‐Jun N‐terminal kinase), were increased in coinfected mice. These results suggest that immune mediators, including cytokines and chemokines, through Toll-like receptors/mitogen-activated protein kinase pathways, play important roles in the pathology of coinfection caused by influenza virus and Streptococcus pneumoniae.

Molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli

To clarify the molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli, the murA, glpT, uhpT, uhpA, ptsI and cyaA genes were sequenced from six fosfomycin-resistant isolates. Two strains were found to harbour a mutation in the murA gene that leads to an amino acid substitution (Asp369Asn or Leu370Ile) in the target protein. The remaining four strains carried specific mutations in the glpT gene; one strain possessed a mutation and the other three strains possessed truncated versions of the GlpT transporter owing either to the presence of insertion sequences or a deletion in the coding region of the gene. Two of the strains with truncated GlpT had also lost the entire uhpT gene, which encodes another fosfomycin transporter. Uptake of specific substrates for the transporters was either totally blocked or reduced in strains possessing truncated forms of GlpT or those lacking the uhpT gene. Escherichia coli strains expressing an amino-acid-substituted MurA were at least eight-fold more resistant to fosfomycin than the strain overproducing wild-type MurA. In conclusion, novel amino acid substitutions in MurA or the loss of function of transporters were identified as mechanisms of fosfomycin resistance in clinical isolates of E. coli.

Efficacy of FK463, a (1,3)-β-d-Glucan Synthase Inhibitor, in Disseminated Azole-Resistant Candida albicans Infection in Mice

ABSTRACT The efficacy of FK463, a new (1,3)-β-d-glucan synthase inhibitor, against azole-resistant Candida albicans strains has been studied. The MIC of FK463 was lower than those of azoles and amphotericin B againstCDR1-expressing C26 and CaMDR-expressing C40 strains. All mice treated with FK463 (1 mg/kg) survived disseminated murine candidiasis. The fungal burden in the kidney after 6 days was markedly reduced after therapy with FK463 and amphotericin B sodium deoxycholate, and plasma (1,3)-β-d-glucan concentration was found to be lower in FK463-treated mice. In our study, FK463 was found to be a potent antifungal agent against disseminated infection with azole-resistant C. albicans.

Clinical Practice Guidelines for Nursing- and Healthcare-associated Pneumonia (NHCAP) [Complete translation]

Shigeru Kohno, Yoshifumi Imamura, Yuichiro Shindo, Masafumi Seki, Tadashi Ishida, Shinji Teramoto, Junichi Kadota, Kazunori Tomono, Akira Watanabe Unit of Molecular Microbiology and Immunology, Nagasaki University, Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, 852-8501 Japan Institute for Advanced Research, Nagoya University, and Department of Respiratory Medicine, Nagoya University, Graduate School of Medicine, Japan Division of Infection Control and Prevention, Osaka University Hospital, Japan Department of Respiratory Medicine, Kurashiki Central Hospital, Japan Hitachinaka Medical Education and Research Center, University of Tsukuba, Japan Department of Internal Medicine II, Oita University Faculty of Medicine, Japan Division of Anti-Infective Agents, Institute of Development, Aging and Cancer, Tohoku University, Japan

Azithromycin Exhibits Bactericidal Effects on Pseudomonas aeruginosa through Interaction with the Outer Membrane

ABSTRACT The aim of the present study was to elucidate the effect of the macrolide antibiotic azithromycin on Pseudomonas aeruginosa. We studied the susceptibility to azithromycin in P. aeruginosa PAO1 using a killing assay. PAO1 cells at the exponential growth phase were resistant to azithromycin. In contrast, PAO1 cells at the stationary growth phase were sensitive to azithromycin. The divalent cations Mg2+ and Ca2+ inhibited this activity, suggesting that the action of azithromycin is mediated by interaction with the outer membranes of the cells, since the divalent cations exist between adjacent lipopolysaccharides (LPSs) and stabilize the outer membrane. The divalent cation chelator EDTA behaved in a manner resembling that of azithromycin; EDTA killed more PAO1 in the stationary growth phase than in the exponential growth phase. A 1-N-phenylnaphthylamine assay showed that azithromycin interacted with the outer membrane of P. aeruginosa PAO1 and increased its permeability while Mg2+ and Ca2+ antagonized this action. Our results indicate that azithromycin directly interacts with the outer membrane of P. aeruginosa PAO1 by displacement of divalent cations from their binding sites on LPS. This action explains, at least in part, the effectiveness of sub-MICs of macrolide antibiotics in pseudomonal chronic airway infection.

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.

Efficacy of Linezolid against Methicillin-Resistant or Vancomycin-Insensitive Staphylococcus aureus in a Model of Hematogenous Pulmonary Infection

ABSTRACT We investigated the activities of linezolid, vancomycin, and teicoplanin in a murine model of hematogenous pulmonary infection with Staphylococcus aureus. Our results demonstrate that linezolid clearly reduced bacterial numbers in the methicillin-resistant S. aureus hematogenous infection model and significantly improved the survival rate of immunocompromised mice infected with vancomycin-insensitive S. aureus compared with vancomycin and teicoplanin. The pharmacokinetic profiles also reflected the effectiveness of linezolid.

Efficacy of erythromycin lactobionate for treating Pseudomonas aeruginosa bacteremia in mice.

We induced endogenous Pseudomonas aeruginosa bacteremia by administering cyclophosphamide and ampicillin to specific pathogen-free mice fed P. aeruginosa. Using this model, we evaluated the efficacy of erythromycin lactobionate (EML) in treating P. aeruginosa bacteremia. Treatment with EML at 50 and 100 mg/kg of body weight per day twice a day for 14 days significantly increased the survival rate. The most effective dose was 100 mg/kg/day, with a survival rate of 80% compared with a 20% survival rate in the control. However, the administration of EML at 500 mg/kg/day rather decreased the survival rate. In a model of intravenous infection, treatment with EML at 100 mg/kg/day twice a day for 7 days before the bacterial challenge also enhanced the survival rate. EML levels in serum, liver, and stool were apparently lower than the MIC (512 micrograms/ml). These observations suggest that EML is effective against P. aeruginosa bacteremia despite a lack of specific activity for this pathogen. Although the protective mechanism is still unclear, it is possible that a subinhibitory level of EML may affect the virulence of P. aeruginosa and enhance the host defense system.