Relationship between respiration deficiency and azole resistance in clinical Candida glabrata

Candida glabrata has become a leading cause of invasive infections around the world and is exhibiting growing resistance to azole antifungals. To study the mechanism of its azole resistance, we analyzed the efflux pumps and found well known increased efflux expression and low metabolic state in all azole-resistant strains. The latter finding led us to further investigate the relationship between respiration status and azole antifungal susceptibility in clinical C.?glabrata by growing them on glycerol-containing agar, measuring the cellular ATP, reactive oxygen species (ROS) levels, oxygen consumption and transmission electron microscopy. All azole-resistant isolates were respiratory-deficient, with reduced generation of ATP and ROS and decreased oxygen consumption; two isolates grew as small colonies and exhibited mitochondrial deficiency. Spot assays and agarose disc diffusion tests were performed to evaluate the effects of respiratory chain inhibitors, sodium azide and salicylhydroxamic acid, on antifungal susceptibility. The results of antifungal susceptibility showed that inhibition of alternative respiration with salicylhydroxamic acid enhanced azole susceptibility of C.?glabrata. In conclusion, clinical azole-resistant C.?glabrata isolates harbor respiratory deficiency exhibiting petite mutant or normal phenotype. The alternative respiratory pathway plays an important role in the decreased susceptibility to azole antifungals.