In vitro antifungal activity of aminosterols against moulds isolated from cystic fibrosis patients

Abstract

Sir, We have recently demonstrated that aminosterol derivatives (ASDs) possess an interesting in vitro antibacterial activity against multidrug-resistant bacteria recovered from cystic fibrosis (CF) patients. However, mould infections are also a serious threat for CF patients. Indeed, the fragile pulmonary environment of these patients facilitates the development of bacterial and fungal infections. Repeated bacterial infections, intensive antibiotic therapy and lung transplantation are considered as risk factors for airway infection and/or colonization by moulds in CF patients. Major problems are related to Aspergillus spp. and Scedosporium spp. that may cause chronic disorders such as asthma, bronchitis and aspergilloma. Amphotericin B, caspofungin and azoles are currently used in the management of fungal infections. However, in addition to acquired resistance resulting from antifungal treatment pressure as noted for Aspergillus spp., intrinsic resistance to available antifungals reported in some fungi such as Fusarium, Rhizopus, Rhizomucor and Scedosporium spp. represents a major issue, highlighting the need to develop new antifungal compounds. ASDs have been evaluated only against reference fungal strains. We evaluated herein for the first time the in vitro antifungal activity of squalamine and one of our compounds (ASD 1; the chemical structure is available in Alhanout et al.) possessing an in vitro antibacterial effect, against a panel of clinical mould isolates recovered from sputa of CF patients. All tested isolates were identified at the species level using partial internal transcribed spacer PCR amplification, and the sequencing procedure and sequences have been deposited in GenBank under accession numbers GU594733– GU594779. The in vitro antifungal activities of currently available systemic antifungal drugs were evaluated for comparison purposes. MICs of all tested compounds were determined by the reference broth dilution method as previously reported, and found to be reproducible and in agreement with previous reports for standard antifungal agents as noted using reference fungal strains including four yeasts and one mould (Aspergillus brasiliensis ATCC 16404) (Table 1). Squalamine and ASD 1 displayed MICs for all tested isolates ranging from 8 to 16 mg/L and from 2 to 4 mg/L, respectively. Since no internationally agreed antifungal resistance breakpoints are currently available, we adopted breakpoints proposed in a recent work suggesting MICs .2 mg/L for azoles to designate resistance in Aspergillus spp. For simplicity, we used this MIC to assign resistance to other antifungal drugs for all fungal isolates. In keeping with previous studies, Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus isolates were susceptible to all tested antifungals. Conversely, Aspergillus terreus isolates were resistant to amphotericin B as previously reported and, surprisingly, to itraconazole for our CF clinical isolates, which is unusual. As has been documented for other Aspergillus spp., the resistance of A. terreus to itraconazole probably may be acquired since this fungus chronically colonizes the airways of CF patients that are exposed to various antimicrobial treatments. According to previous reports, Aspergillus ustus was resistant to itraconazole, voriconazole and caspofungin. Resistance to caspofungin and voriconazole was noted for Emericella quadrilineata. Antifungal resistance in Penicillium and Cladosporium spp. was previously reported. Accordingly, two out of four Penicillium griseofulvum isolates were resistant to voriconazole and caspofungin while the other two isolates were resistant to all tested available antifungals. Moreover, Cladosporium spp. were resistant to all antifungals except amphotericin B, while Alternaria spp. were susceptible to all tested antifungals. In accordance with previous findings, resistance to all antifungals was found for all tested isolates of Fusarium proliferatum, Scedosporium prolificans, Pseudallescheria boydii, Scedosporium apiospermum, Rhizopus oryzae and Rhizomucor tauricus. Hence, airway colonization with these moulds is quite worrying, calling for an effective therapy to be available. Conversely, tested aminosterols and particularly ASD 1 demonstrated a homogeneous in vitro activity against all susceptible and resistant isolates. This finding suggests that novel mechanistic aspects, distinct from those of available drugs, underlie the antifungal activity of ASDs. While it is known that squalamine acts by disrupting bacterial membranes, the antifungal mechanism of action of ASDs remains unknown. Squalamine and ASD 1 previously demonstrated comparable activities against various bacterial isolates, whilst the present data interestingly show that ASD 1 was superior to squalamine, indicating a probable different effect due to a different chemical structure of the two tested compounds. While the safety of squalamine has been demonstrated in clinical studies, no in vitro or in vivo toxicity studies have been reported for synthesized aminosterols. Nevertheless, since ASDs were reported to act as membrane disruptors, such a wide antimicrobial spectrum may encompass a potential toxicity that should be further investigated. Overall, we have demonstrated herein that ASDs possess an interesting in vitro antifungal activity, advocating their development for local administration, for example as aerosols especially in the context of CF. Further studies are warranted in order to evaluate their in vivo antimicrobial activities in animal models.