Susceptibility Profiles of Amphotericin B and Posaconazole against Clinically Relevant Mucorales Species under Hypoxic Conditions

Abstract

The effect of hypoxic conditions on the in vitro efficacy of amphotericin B and posaconazole against Mucorales was evaluated by defining MICs with Etest and broth microdilution and identifying minimal fungicidal concentrations (MFCs). With Etest, oxygen-dependent changes were detected, while the MIC and the MFC determined with broth microdilution remained unaltered with reduced oxygen levels. The observed differences depended on the method used.

TEXT

The incidence of mucormycosis has increased within the last few decades in patients at risk and is associated with high mortality rates (1–3). Treatment for mucormycosis is complicated because Mucorales infection is resistant to most antifungal drugs (4) and clinical breakpoints are not available yet. Treatment with lipid formulations of amphotericin B in combination with surgery, if possible, is the first choice, but adjunctive therapeutic options, such as hyperbaric oxygen therapy, have also been explored (5–7). At the sites of infection, microenvironmental factors influence fungal growth and most likely also affect the efficacy of antifungal drugs (8). Hypoxia occurs during pulmonary fungal infections in vivo (9), and tissue oxygen concentrations can decrease to ≤1% (10, 11).

Simulating the host environment in in vitro testing will contribute to a better understanding of how conditions, such as hypoxia, might influence the susceptibility of Mucorales to antifungal drugs. Previous studies provided superior endpoints for caspofungin, micafungin (12), and anidulafungin (13) against Aspergillus species under hypoxic conditions. Additionally, amphotericin B MICs were reduced under hypoxic conditions, whereas no changes in itraconazole or micafungin MICs were detected (12). This study is the first to focus on hypoxia-driven changes in the susceptibility patterns of Mucorales. Therefore, we compared the in vitro activities of amphotericin B (Bristol-Myers Squibb, Austria) and posaconazole (Schering-Plough, Kenilworth, NJ) in normoxic and hypoxic conditions for the presence of clinically relevant Mucorales by standard methods.

All clinical isolates tested (n = 56) were identified by internal transcribed spacer sequencing, according to the methods of White et al. (14). The strain set comprised Lichtheimia corymbifera (n = 20), Lichtheimia ramosa (n = 6), Rhizomucor pusillus (n = 8), Rhizopus arrhizus (n = 7), Rhizopus microsporus (n = 12), and Mucor circinelloides (n = 3). The hypoxic conditions were set to 1% O₂, 5% CO₂, and 94% N₂ and were controlled throughout the duration of the experiments (Biospherix C-Chamber & Pro-Ox, Pro-CO₂ controller USA). All the experiments were done in parallel under normoxic conditions, which were considered general atmospheric levels (∼21% O₂). MICs were determined at 24 h by the agar diffusion method (Etest; bioMérieux, France) and broth microdilution according to EUCAST guidelines (15). For a better comparison, Etest MICs were raised to the next corresponding EUCAST concentration. These methods were chosen to verify the different impacts of hypoxic conditions on surface (exposure to actual 1% oxygen) or liquid cultures, where oxygen concentrations might vary also in the normoxic cultures. The MFCs were determined as described by Warn et al. (12); the MFC is defined as the lowest drug concentration resulting in a 99.9% killing rate. To check for normal distribution of the MICs, the D'Agostino-Pearson omnibus normality test was performed. The Kruskal-Wallis test was applied, as the data were not normally distributed. P values of ≤0.05 were regarded as statistically significant.

With Etest, the influence of hypoxic conditions on the susceptibility profile occurred in a species- and drug-dependent manner (Table 1). For Lichtheimia spp., differences due to hypoxic conditions were detected, with a statistically significant shift toward lower amphotericin B MICs. The same effect was observed for M. circinelloides and R. microsporus isolates. Rhizomucor pusillus and R. arrhizus isolates exhibited no significant changes in antifungal susceptibility to amphotericin B. A statistical comparison of the MIC distributions of posaconazole resulted in no significant differences for any Mucorales isolates.

TABLE 1.

In vitro susceptibility of amphotericin B and posaconazole against Mucorales species determined by Etest under normoxic and hypoxic growth conditions

Species (no. of isolates)	Antifungal agenta	MIC data (μg/ml) under:
		Normoxic conditions			Hypoxic conditions
		Range	MIC50	MIC90	Range	MIC50	MIC90
L. corymbifera (20)	AMB	0.03–2	0.25	1	0.03–0.25	0.125	0.125
	POS	0.125–32	0.25	8	0.125–32	0.25	16
L. ramosa (6)	AMB	0.25–2	0.25	2	0.06–0.125	0.06	0.125
	POS	0.125–1	0.25	1	0.06–0.5	0.125	0.5
R. pusillus (8)	AMB	0.03–0.25	0.125	0.25	0.03–0.25	0.125	0.25
	POS	0.25–0.5	0.5	0.5	0.125–1	1	1
R. microsporus (12)	AMB	0.25–32	2	32	0.125–32	0.5	16
	POS	0.5–32	2	32	0.5–32	4	32
R. arrhizus (7)	AMB	0.25–16	0.5	16	0.03–4	0.5	4
	POS	0.25–2	1	2	0.25–1	0.5	1
M. circinelloides (3)	AMB	0.03–0.06	0.03	0.06	0.03–0.06	0.03	0.06
	POS	32–32	32	32	32–32	32	32

^aAMB, amphotericin B; POS, posaconazole.

Reduction of the amphotericin B MICs can be partly explained by impaired growth, as reduced growth ability under hypoxic conditions occurred in species with decreased MICs, especially in Lichtheimia spp. (25% to 40% growth reduction in colony diameters) and M. circinelloides isolates (35% to 40% growth reduction in colony diameters). This, in addition to the fact that ergosterol biosynthesis is oxygen dependent (16), leads to the hypothesis that Mucorales infections are confronted with two stressors—antifungal exposure and the maintenance of cell membrane components—which may further explain the shift to lower MICs under hypoxic conditions. In addition to forming pores in fungal membranes, amphotericin B was previously shown to induce reactive oxygen species in amphotericin B-susceptible species (17, 18). This effect on mitochondrial respiration might further contribute to the observed shift to lower MICs for amphotericin B under hypoxic conditions.

In the broth microdilution assays, the MICs of amphotericin B and posaconazole were not altered under hypoxic conditions for any of the Mucorales isolates tested (Table 2); consequently, no significant differences in the MIC distributions were detected. Similarly, no significant oxygen-dependent differences in the MFCs for either antifungal agent were observed (Table 2). Amphotericin B was shown to be fungicidal in both oxygen conditions, with a close correlation between the MICs and MFCs. The only exception was its testing against L. ramosa, for which no MFCs were determined, even though the MIC₅₀ was 0.25 μg/ml. Regarding posaconazole, the MFCs exceeded the highest concentration tested (16 μg/ml) except for L. corymbifera and R. pusillus, for which the median MFCs were 4 μg/ml and 8 μg/ml, respectively. Unlike with amphotericin B, no correlation between the MICs and MFCs were observed for posaconazole against any Mucorales isolates.

TABLE 2.

Antifungal activity of amphotericin B and posaconazole against Mucorales species, reported as MICs^a and MFCs under normoxic and hypoxic growth conditions^a

Species (no. of isolates)	Antifungal drugb	Activity (μg/ml) under normoxic conditions					Activity (μg/ml) under hypoxic conditions
		MIC range	MIC50	MIC90	MFC range	MFCc	MIC range	MIC50	MIC90	MFC range	MFC
L. corymbifera (20)	AMB	0.25–1	0.5	0.5	1–16	4	0.125–1	0.25	0.5	0.5–16	1
	POS	0.5–4	1	1	1–16	4	0.5–1	1	1	4–16	>16
L. ramosa (6)	AMB	0.25–0.5	0.25	0.5	4–16	>16	0.125–0.25	0.125	0.5	8–16	>16
	POS	1–2	1	2	16–16	>16	0.5–2	1	2	16–16	>16
R. pusillus (8)	AMB	0.25–1	0.25	0.5	0.25–16	0.5	0.125–0.25	0.25	0.25	0.25–16	0.25
	POS	1–16	1	16	1–16	8	0.5–16	1	16	1–16	>16
R. microsporus (12)	AMB	0.25–0.5	0.5	1	0.25–1	0.5	0.25–0.5	0.25	0.5	0.25–1	0.5
	POS	1–2	1	2	1–16	>16	1–2	1	2	16–16	>16
R. arrhizus (7)	AMB	0.25–1	0.5	1	0.5–1	0.5	0.25–0.5	0.25	1	0.5–1	0.5
	POS	1–1	1	1	1–16	>16	0.5–1	1	1	16–16	>16
M. circinelloidies (3)	AMB	0.125–0.5	0.25	0.5	0.5–1	1	0.125–0.25	0.25	0.25	0.5–1	1
	POS	2–2	2	2	16–16	>16	1–2	1	2	16–16	>16

^aMICs were determined according to the EUCAST method.

^bAMB, amphotericin B; POS, posaconazole.

^cMFC, median of the minimum fungicidal concentration.

Various in vitro factors, such as medium, inoculum, and temperature, affect MICs significantly (8, 19). Additionally, hypoxic conditions were shown to have a significant impact on the targets of antifungal drugs, such as ergosterol biosynthesis, and on β-glucan in Aspergillus fumigatus (16, 20), so one would expect severe hypoxia-driven effects on antifungal activity. Here, in the first study to investigate the effect of hypoxic conditions on clinically relevant Mucorales isolates, changes in the MICs were due in part to the growth ability of fungal species under hypoxic conditions and were further influenced by the method of choice for determining them.

The minor differences in the MICs or MFCs under hypoxic conditions closely correlate with our own data and already published data of Aspergillus spp. and Candida spp. (12, 13). Generally, available data on the MFCs of Mucorales species are limited; therefore, the data presented herein contribute to a better overview of the antifungal activities of amphotericin B and posaconazole. The major finding of this study was the discrepancy between the posaconazole MICs and MFCs found for all Mucorales species, resulting in the in vitro classification of posaconazole as a fungistatic compound against Mucorales species in vitro.

In conclusion, hypoxic conditions have only a marginal influence on the in vitro antifungal susceptibility pattern of Mucorales species. The minor differences observed were most pronounced with the Etest method, which is not the method of choice for MIC determinations in Mucorales species due to difficulties in MIC reading caused by the cottony overgrowth of these fungi.