Antifungal Activity of Colistin against Mucorales Species In Vitro and in a Murine Model of Rhizopus oryzae Pulmonary Infection

Abstract

In immunosuppressed hosts, mucormycosis is a life-threatening infection with few treatment options. We studied the activity of colistin (polymyxin E) against Mucorales species in vitro and in a murine model of pulmonary Rhizopus oryzae infection. Colistin exhibited fungicidal activity in vitro against Mucorales spores and mycelia. At the colistin MIC, initial R. oryzae hyphal damage was followed by rapid regrowth; however, regrowth was prevented by combining colistin with a subinhibitory concentration of amphotericin B. Using electron microscopy and FM4-64 staining, we demonstrated that colistin disrupts R. oryzae cytoplasmic and vacuolar membranes, resulting in the leakage of intracellular contents. The prophylactic intranasal treatment of immunosuppressed mice with colistimethate significantly reduced the mortality rate and pulmonary fungal burden resulting from inhalational challenge with R. oryzae spores, whereas intraperitoneal colistimethate treatment had no effect. We conclude that colistin has modest in vitro and in vivo fungicidal activity against Mucorales spp. Further studies are warranted to assess the use of this drug in the prevention and treatment of mucormycosis.During the past decade, pulmonary mucormycosis (PMM) has emerged as an important life-threatening opportunistic infection in severely immunocompromised patients, such as those with prolonged neutropenia and recipients of allogeneic hematopoietic stem cell transplantation (12, 21). In contrast to diabetic patients who develop rhinocerebral mucormycosis, surgical debridement is impractical in patients with PMM, and the correction of the underlying immunosuppression is challenging (12, 21); therefore, antifungal chemotherapy remains the chief therapeutic intervention for many patients with PMM. Unfortunately, Mucorales species are inherently resistant to most clinically available antifungal agents except amphotericin B, which is associated with renal and infusional toxicity, and posaconazole, which is available only as an oral formulation and has variable absorption from the gastrointestinal tract (12, 21). Hence, there is an urgent need for novel drugs with activity against Mucorales spp.Polymyxins are cyclic, positively charged peptide antibiotics produced by the bacterium Bacillus polymyxa (16). Polymyxins bind to and disrupt the anionic outer cell membrane of Gramnegative bacteria and have a neutralizing effect on bacterial lipopolysaccharides (16). Interestingly, polymyxin B also has been shown to disrupt the cytoplasmic and vacuolar membranes of the model yeast Saccharomyces cerevisiae (23), suggesting that its antimicrobial activity extends to eukaryotic organisms. We studied the activity of polymyxin E (colistin) against Mucorales spp. in vitro and in a murine model of PMM. MATERIALS AND METHODSIsolates. We performed susceptibility testing on 21 clinical Mucorales isolates recovered from patients at The University of Texas M. D. Anderson Cancer Center (Houston). Isolates were identified by standard morphological criteria, and the gen...

The Calcineurin Pathway Inhibitor Tacrolimus Enhances the In Vitro Activity of Azoles against Mucorales via Apoptosis

“…We measured intracellular ATP efflux from germlings exposed to PCZ (R. oryzae, 0.125 to 0.5 g/ml; C. bertholletiae, 1 to 4 g/ml; M. circinelloides, 1 to 4 g/ml) or ICZ (R. oryzae, 0.06 to 0.25 g/ml; C. bertholletiae, 0.25 to 1 g/ml; M. circinelloides, 1 to 4 g/ml) in combination with TCR (0.015 g/ml) at 37°C for 3 h as an indication of cell membrane damage and cytoplasmic and mitochondrial membrane leakage (21). R. oryzae, C. bertholletiae, and M. circinelloides germlings were separated from the medium by centrifugation and resuspended in 1-ml aliquots of RPMI 1640.…”

“…R. oryzae, C. bertholletiae, and M. circinelloides germlings treated with TCR along with PCZ (0.125 to 4 g/ml) or ICZ (0.06 to 4 g/ml) for 3 h were stained with bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC; Molecular Probes) as previously described (21). PCZ and ICZ concentrations were decided on the basis of their MIC values.…”

Iron starvation induces apoptosis in rhizopus oryzae in vitro

“…11,20 The cells were removed by centrifugation, and the supernatants were assayed for ATP using the CellTiter-Glo luminescent kit (Promega, Madison, WI). Data were collected with a microplate luminometer (Spectramax M5; Molecular Devices, Sunnyvale, CA).…”

Aspergillus fumigatus Hyphal Damage Caused by Noninvasive Radiofrequency Field-Induced Hyperthermia

“…Immediately after exposure to hyperthermia (WBHT or RFHT), hyphal damage was evaluated using an XTT assay (Sigma-Aldrich) as described previously (8). XTT-treated hyphae were incubated at 37°C for 2 h in the dark.…”