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Over the past two decades, the incidence of infections due to Candida glabrata, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly. Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species. In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of C. glabrata were evaluated. Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide. Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles. Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed. This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar. This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy. Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency. Finally, this mutant exhibited cross-resistance to all the azoles tested. In conclusion, blockage of respiration in C. glabrata induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA. This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain.

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The nature of the multinucleated cells involved in the resorption processes occurring inside macroporous calcium-phosphate biomaterials grafted into rabbit bone was studied using light microscopy, histomorphometric analysis, enzymatic detection of tartrate-resistant acid phosphatase (TRAP) activity, scanning, and electron microscopy. Samples were taken at days 7, 14, and 21 after implantation. As early as day 7, osteogenesis and resorption were observed at the surface of the biomaterials, inside the macropores. Resorption of both newly formed bone and calcium-phosphate biomaterials was associated with two types of multinucleated cells. Giant multinucleated cells were found only at the surface of the biomaterials; they showed a large number of nuclei, were TRAP negative, developed no ruffled border, and contained numerous vacuoles with large accumulation of mineral crystals from the biomaterials. Osteoclasts exhibited TRAP positivity and well-defined ruffled border. They were observed at the surface of both newly formed bone and biomaterials, around the implant, and inside the macropores. In contract with the biomaterials, infoldings of their ruffled border were observed between the mineral crystals, deeply inside the microporosity. The microporosity of the biomaterials (i.e., the noncrystalline spaces inside the biomaterials) increased underneath this type of cell as compared with underneath giant cells or to the depth of the biomaterials. These observations demonstrate that macroporous calcium-phosphate biomaterials implanted in bone elicit osteogenesis and the recruitment of a double multinucleated cell population having resorbing activity: giant multinucleated cells that resorb biomaterials and osteoclasts that resorb newly formed bone and biomaterials.

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