Hideaki Ohno

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.

Acute infection with influenza virus enhances susceptibility to fatal pneumonia following Streptococcus pneumoniae infection in mice with chronic pulmonary colonization with Pseudomonas aeruginosa

We established a mouse model in which fatal pneumonia was induced by pneumococcal superinfection following influenza virus infection in chronic Pseudomonas aeruginosa infected mice. In this mouse model, influenza virus infection caused a significant increase in inflammatory cells, cytokines and severe tissue damage in the lungs of these P. aeruginosa infected mice, before pneumococcal infection. Intrapulmonary virus titres were significantly increased in mice with chronic P. aeruginosa infection, compared with control mice. Neutrophil function analysis showed significant reduction of myeloperoxidase (MPO) activity and lysozyme secretion by influenza virus infection in these mice. Our results suggest that influenza virus infection may play an important role in inducing pneumococcal pneumonia in chronic P. aeruginosa infected mice. Our results suggested that our mouse model is useful for investigating the pathogenesis of influenza virus infection in patients with chronic lung infection.

Intrapulmonary concentrations of inflammatory cytokines in a mouse model of chronic respiratory infection caused by Pseudomonas aeruginosa

We investigated the role of inflammatory cytokines in a mouse model of chronic Pseudomonas aeruginosa infection mimicking diffuse panbronchiolitis (DPB), and determined the effects of clarithromycin therapy on the production of these cytokines. The concentrations of IL‐1β, IL‐2, IL‐4, IL‐5, interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) were measured serially in the lungs of mice with experimentally induced chronic respiratory P. aeruginosa infection until 60u2003days after inoculation. The concentrations of these cytokines during the course of the disease were significantly higher than baseline (before inoculation, Pu2003<u20030·01 for all cytokines). Clarithromycin significantly inhibited the production of IL‐1β and TNF‐α in the lung (Pu2003<u20030·01). The same treatment also reduced the levels of other cytokines, albeit insignificantly. Treatment with anti‐TNF‐α antibody significantly reduced the number of pulmonary lymphocytes and concentration of IL‐1β in the lung (Pu2003<u20030·01), but did not change the number of viable bacteria. Our findings resemble those detected in bronchoalveolar lavage fluid of patients with DPB and indicate that inflammatory cytokines play an important role in chronic P. aeruginosa lung infection. Our results also show that macrolides modulated the production of these cytokines, ultimately reducing lymphocyte accumulation in the lung. Our data suggest that anti‐TNF‐α antibody might be a useful new strategy for the treatment of chronic respiratory P. aeruginosa infection.

Combination therapy with fluconazole and flucytosine for pulmonary cryptococcosis.

We investigated, in vitro, the combined effects of fluconazole (FLCZ) and flucytosine (5-FC) against different strains of Cryptococcus neoformans, and retrospectively analyzed the clinical efficacy of combination therapy of FLCZ and 5-FC in patients with pulmonary cryptococcosis. The minimum inhibitory concentrations (MICs) of antifungal agents and drug interaction were determined by the broth microdilution method and checkerboard titration. FLCZ and 5-FC showed synergistic activity against 8 (32%) of 25 strains of C. neoformans. The clinical efficacy of the 2 drugs when combined together was good in 9 (90%) patients and fair in 1 (10%) patient with pulmonary cryptococcosis. Renal dysfunction occurred in 1 patient. Our results suggest that a combination therapy using FLCZ and 5-FC is clinically useful in patients with pulmonary cryptococcosis who otherwise show a limited response to monotherapy.

Quorum Sensing System Lactones Do Not Increase Invasiveness of a MexAB-OprM Efflux Mutant but Do Play a Partial Role in Pseudomonas aeruginosa Invasion

We studied the quorum sensing (QS) system and the related homoserine lactones (HSLs) observing Pseudomonas aeruginosa invasion using the epithelial cell monolayer penetration assay model. Compared to the PAO1 wild‐type, the QS mutants, ΔlasI and ΔrhlI, were compromised in their capacity to invade. The decreased invasiveness of ΔrhlI was restored by adding 100 μM exogenous C4‐HSL. However, the decreased invasiveness of an efflux mutant, AmexAB‐oprM, was not restored in the presence of exogenous HSLs. The QS system partially plays a role in P. aeruginosa invasion; however, C4‐HSL and 3‐O‐C12‐HSL are not the essential determinants for invasiveness for P. aeruginosa.

Community acquired pneumonia (CAP) caused by Cryptococcus neoformans in a healthy individual.

A 41-y-old male had been diagnosed as having community acquired pneumonia (CAP) with consolidations in the chest radiograph, fever and cough. Since clarithromycin and ß-lactam agents were not effective, bronchoscopic examination was performed. Indian ink staining of bronchial wash smears revealed yeast-like cells with a thick capsule, and Cryptococcus neoformans was isolated several d later. Serum glucuronoxylomannan antigen was≧×1024. The patient was treated with itraconazole for 16 weeks.

Combined Use of Clarithromycin and Antimycobacterial Agents in Mycobacterium avium Complex Chronic Pulmonary Infection

Clarithromycin, a new macrolide antibacterial agent, is effective against disseminatedMycobacterium avium complex (MAC) infection in AIDS patients. In this study, we evaluated the therapeutic effect of clarithromycin, used in combination with other antimycobacterial drugs, against chronic pulmonary MAC infections in non-AIDS patients. Patients were divided into two groups based on the antimycobacterial drugs used for treatment. Patients of group A (n=5) were recent cases diagnosed to have atypical mycobacteriosis. They were treated on diagnosis, with rifampicin (450 mg q.d.), isoniazid (400 mg q.d.), and clarithromycin (200 mg b.i.d.). Patients of group B (n=23) were treated by adding clarithromycin (200 mg b.i.d.) to an existing therapeutic regimen consisting of several antimycobacterial agents. Clinical improvement was observed in seven (25%) patients, including two (40%) from group A and five (22%) from group B. Treatment was associated with a total mycobacterial eradication rate of 100% (5/5) in group A and 22% (5/23) in group B. Clarithromycin was more effective in patients receiving the drug as the first-line therapy than when used in later therapy. Clarithromycin had a lower efficacy rate in this study compared with the reported effect of clarithromycin in AIDS patients with in disseminated MAC infection. Our results of the first-line use of clarithromycin in combination with other mycobacterial agents for the treatment of chronic pulmonary MAC infections indicate that this agent has a limited but encouraging effect on atypical pulmonary mycobacteriosis.