In vitro and in vivo interactions of minocycline and azoles, including itraconazole, voriconazole, and posaconazole, against filamentous pathogenic fungi were investigated. A total of 56 clinical isolates were studied in vitro via broth microdilution checkerboard technique, including 20 strains of Aspergillus fumigatus, 7 strains of Aspergillus flavus, 16 strains of Exophiala dermatitidis, 10 strains of Fusarium solani, and 3 strain s of Fusarium oxysporum.
KEYWORDS: minocycline, tetracycline, Aspergillus, Exophiala, Fusarium, synergy, fungi, antifungal, resistance, azole
ABSTRACT
In vitro and in vivo interactions of minocycline and azoles, including itraconazole, voriconazole, and posaconazole, against filamentous pathogenic fungi were investigated. A total of 56 clinical isolates were studied in vitro via broth microdilution checkerboard technique, including 20 strains of Aspergillus fumigatus, 7 strains of Aspergillus flavus, 16 strains of Exophiala dermatitidis, 10 strains of Fusarium solani, and 3 strain s of Fusarium oxysporum. The results revealed that minocycline did not exhibit any significant antifungal activity against any of the tested strains. However, favorable synergy of minocycline with itraconazole, voriconazole, or posaconazole was observed against 34 (61%), 28 (50%), and 38 (68%) isolates, respectively, including azole-resistant A. fumigatus and Fusarium spp. with inherently high MICs of azoles. Synergistic combinations resulted in 4-fold to 16-fold reduction of effective MICs of minocycline and azoles. No antagonism was observed. In vivo effects of minocycline-azole combinations were evaluated by survival assay in a Galleria mellonella model infected with E. dermatitidis strain BMU00034; F. solani strain FS9; and A. fumigatus strains AF293, AFR1, and AFR2. Minocycline acted synergistically with azoles and significantly increased larvae survival in all isolates (P < 0.001), including azole-resistant A. fumigatus and azole-inactive Fusarium spp. In conclusion, the results suggested that minocycline combined with azoles may help to enhance the antifungal susceptibilities of azoles against pathogenic fungi and had the potential to overcome azole resistance issues.
INTRODUCTION
Opportunistic fungal infection, especially invasive infection, has emerged as a growing threat for human health in recent decades, posing new challenges to health care professionals. Among filamentous fungal infections, invasive aspergillosis is the most common invasive fungal infection associated with relatively high mortality (1, 2). Fusarium spp., well-known plant pathogens and food contaminants, have also appeared as one of the most important groups of medically significant fungi. Fusariosis is, after aspergillosis, the second most common mold infection in humans, among which the Fusarium solani species complex and Fusarium oxysporum species complex are responsible for approximately 60% and 20% of the cases, respectively (3). Meanwhile, Exophiala dermatitidis, the leading cause of severe neurotropic phaeohyphomycosis and a common cause of chromoblastomycosis, is also being increasingly recognized and reported (4–7).
Prompt antifungal treatment is crucial to prevent life-threatening fungal disease. However, the antifungal armamentarium has long been insufficient. Azoles are the first-choice antifungal agent for management. However, research has indicated that the prevalence of azole-resistant Aspergillus fumigatus isolates has been constantly increasing in recent years, threatening the effectiveness of current antifungals (8). It is notable that azole-resistant strains harboring the association of a tandem repeat sequence and punctual mutation of the Cyp51A gene (TR34/L98H and TR46/Y121F/T289A) have become widely disseminated among both clinical and environmental settings across the world within a short time period (8). Additionally, Aspergillus species other than Aspergillus fumigatus that have less susceptibility to available antifungal agents constitute a significant proportion of invasive aspergillosis (9). As for infection with Exophiala spp., the success rate of treatment was only 40% to 70%, although in vitro susceptibility tests showed favorable antifungal activities of most available antifungal agents (10–12). In contrast, available antifungal drugs have shown poor in vitro activity against Fusarium spp. (3). Fusariosis is mostly refractory to treatment, with a high mortality rate for systemic disseminations (3). Given the paucity of newly developed antifungals, the investigation of a novel combination regimen appears to be a worthy endeavor.
Minocycline (MIN) is an expanded-spectrum, semisynthetic tetracycline antibiotic that also exerts a variety of nonantimicrobial properties, such as anti-inflammatory, antiapoptosis, immune modulatory, neuroprotective, and antiviral activity (13, 14). In addition, previous studies have demonstrated synergism between fluconazole and MIN against Candida albicans (15). Hence, it is reasonable to suspect that MIN might exert some antifungal effect and serve as an additional agent, which could potentially enhance the efficacy of common antifungal agents against filamentous fungi. In the present study, the in vitro and in vivo effects of MIN alone and combined with itraconazole (ITC), voriconazole (VRC), or posaconazole (POS) against pathogenic filamentous fungi were investigated.
RESULTS
In vitro interactions between MIN and azoles against Aspergillus spp.
The MICs of MIN alone against all strains were >64 μg/ml. As shown in Table 1, the MIC ranges of azoles alone against A. fumigatus, except for azole-resistant strains, were 1 to 2 μg/ml for ITC and 0.5 to 1 μg/ml for VRC and POS. The MIC ranges of all tested azoles against Aspergillus flavus were 1 to 2 μg/ml. The MICs of azoles were >16 μg/ml for ITC and 4 μg/ml for VRC and POS against AFR1(TR34/L98H); and >16 μg/ml for VRC and 4 μg/ml for ITC and POS against AFR2(TR46/Y121F/T 289A).
TABLE 1.
MIC and FICI results with the combinations of MIN and azoles against Aspergillus spp.
| Strain | MICa (μg/ml) for: |
||||||
|---|---|---|---|---|---|---|---|
| Agent alone |
Combinationb |
||||||
| MIN | ITC | VRC | POS | MIN/ITC | MIN/VRC | MIN/POS | |
| A. fumigatus | |||||||
| AF293 | >64 | 1 | 0.5 | 1 | 16/0.25(S) | 16/0.125(S) | 8/0.125(S) |
| AF001 | >64 | 1 | 0.5 | 1 | 16/0.5(I) | 16/0.125(S) | 16/0.25(S) |
| AF002 | >64 | 1 | 0.5 | 1 | 16/0.5(I) | 16/0.5(I) | 8/0.125(S) |
| AF003 | >64 | 2 | 0.5 | 1 | 8/0.25(S) | 16/0.5(I) | 16/0.125(S) |
| AF004 | >64 | 1 | 1 | 1 | 8/0.5(I) | 16/0.125(S) | 8/0.25(S) |
| AF005 | >64 | 1 | 0.5 | 1 | 16/0.25(S) | 8/0.5(I) | 8/0.125(S) |
| AF006 | >64 | 2 | 0.5 | 1 | 16/0.25(S) | 8/0.5(I) | 16/0.5(I) |
| AF007 | >64 | 1 | 0.5 | 1 | 16/0.5(I) | 8/0.5(I) | 8/0.125(S) |
| AF008 | >64 | 1 | 0.5 | 0.5 | 16/0.5(I) | 16/0.125(S) | 16/0.5(I) |
| AF009 | >64 | 1 | 0.5 | 1 | 16/0.25(S) | 8/0.125(S) | 16/0.5(I) |
| AF010 | >64 | 2 | 0.5 | 1 | 16/0.25(S) | 16/0.25(I) | 16/0.125(S) |
| AF011 | >64 | 1 | 1 | 1 | 8/0.25(S) | 8/0.25(S) | 8/0.25(S) |
| AF012 | >64 | 1 | 0.5 | 1 | 8/0.5(I) | 16/0.125(S) | 8/0.125(S) |
| AF013 | >64 | 1 | 0.5 | 1 | 8/1(I) | 8/0.5(I) | 16/0.5(I) |
| AF014 | >64 | 1 | 0.5 | 1 | 8/0.5(I) | 16/0.5(I) | 16/0.25(S) |
| AF015 | >64 | 1 | 0.5 | 1 | 16/0.25(S) | 16/0.5(I) | 8/0.25(S) |
| AF016 | >64 | 1 | 1 | 1 | 16/0.25(S) | 8/0.25(S) | 8/0.5(I) |
| AF017 | >64 | 1 | 0.5 | 1 | 8/0.25(S) | 4/0.25(I) | 4/0.125(S) |
| AF018 | >64 | 2 | 0.5 | 2 | 16/0.25(S) | 4/0.125(S) | 8/0.25(S) |
| R1(TR34/L98H) | >64 | >16 | 4 | 4 | 8/4(S) | 16/2(I) | 8/1(S) |
| R2(TR46/Y121F/T289A) | >64 | 4 | >16 | 4 | 16/1(S) | 16/8(I) | 16/1(S) |
| A. flavus | |||||||
| AFLA-1 | >64 | 1 | 1 | 1 | 8/0.5(I) | 4/0.25(S) | 4/0.25(S) |
| AFLA-2 | >64 | 1 | 1 | 1 | 8/0.5(I) | 8/0.25(S) | 8/0.125(S) |
| AFLA-3 | >64 | 1 | 2 | 2 | 4/0.125(S) | 4/0.125(S) | 8/0.25(S) |
| AFLA-4 | >64 | 2 | 2 | 1 | 8/0.25(S) | 4/0.25(S) | 4/0.125(S) |
| AFLA-5 | >64 | 2 | 1 | 1 | 8/0.25(S) | 4/0.5(I) | 8/0.5(I) |
| AFLA-6 | >64 | 1 | 1 | 1 | 8/0.5(I) | 4/1(I) | 8/0.25(S) |
| AFLA-7 | >64 | 2 | 1 | 1 | 4/0.25(S) | 8/0.25(S) | 4/0.125(S) |
The MIC is the concentration achieving 100% growth inhibition.
FICI results are shown in parentheses. S, synergy (FICI of ≤0.5); I, no interaction (indifference) (0.5 < FICI ≤ 4).
When MIN was combined with ITC, VRC, or POS, synergistic activity was observed in 13 (65%), 10 (50%), and 15 (75%) strains of A. fumigatus isolates; and in 4 (57%), 5 (71%), and 6 (86%) strains of A. flavus isolates (Table 1 and 2). Although AFR1 and AFR2 were resistant to ITC and VRC, respectively, the MIN-ITC and MIN-POS combination also showed favorable synergy against both strains. The MICs of MIN and ITC in the synergistic MIN-ITC combination decreased to 8 to 16 μg/ml and 0.25 μg/ml against azole-sensitive A. fumigatus isolates and 4 to 8 μg/ml and 0.125 to 0.25 μg/ml against A. flavus isolates, respectively (Table 1). The MICs of ITC against AFR1 and AFR2 in synergistic MIN-ITC combination were 4 μg/ml and 1 μg/ml, respectively. In synergistic MIN-POS combination, the MIC ranges of MIN and POS decreased to 4 to 16 μg/ml and 0.125 to 1 μg/ml against A. fumigatus isolates and 4 to 8 μg/ml and 0.125 to 0.25 μg/ml against A. flavus isolates, respectively. When MIN was combined with VRC, the effective working ranges of MIN and VRC were 4 to 16 μg/ml and 0.125 to 0.5 μg/ml against A. fumigatus isolates and 4 to 8 μg/ml and 0.125 to 0.25 μg/ml against A. flavus isolates, respectively (Table 1). No antagonism was observed in any combination.
TABLE 2.
Summary of drug interactions for the combination of MIN and azoles
| Species (n) | No. (%) of isolates showing synergism by combination of: |
||
|---|---|---|---|
| Minocycline + ITC | Minocycline + VRC | Minocycline + POS | |
| Aspergillus spp. (27) | 17 (63) | 15 (56) | 21 (78) |
| A. fumigatus (20) | 13 (65) | 10 (50) | 15 (75) |
| A. flavus (7) | 4 (57) | 5 (71) | 6 (86) |
| E. dermatitidis (16) | 11 (69) | 8 (50) | 11 (69) |
| Fusarium spp. (13) | 6 (46) | 5 (38) | 6 (46) |
| F. solani (10) | 5 (50) | 4 (40) | 4 (40) |
| F. oxysporum (3) | 1 (33) | 1 (33) | 2 (67) |
| Total (56) | 34 (61) | 28 (50) | 38 (68) |
In vitro interactions between MIN and azoles against E. dermatitidis.
The individual MIC ranges of tested agents against E. dermatitidis were >64 μg/ml, 1 to 2 μg/ml, 0.5 to 2 μg/ml, and 0.5 to 1 μg/ml for MIN, ITC, VRC, and POS, respectively (Table 3). When MIN was combined with ITC, VRC, or POS, synergy was observed in 11 (69%), 8 (50%), and 11 (69%) strains of E. dermatitidis isolates, respectively (Table 2 and 3). The MICs of MIN and ITC in the synergistic MIN-ITC combination decreased to 4 to 16 μg/ml and 0.25 to 0.5 μg/ml, respectively (Table 3). When MIN was combined with POS, the effective MIC ranges of MIN and POS decreased to 4 to 16 μg/ml, and 0.125 to 0.25 μg/ml, respectively. When MIN was combined with VRC, the effective working ranges of MIN and VRC were 8 to 16 μg/ml and 0.125 to 0.5 μg/ml, respectively (Table 3). No antagonism was observed in any combination.
TABLE 3.
MIC and FICI results with the combinations of MIN and azoles against E. dermatitidis
| Strain | MICa (μg/ml) for: |
||||||
|---|---|---|---|---|---|---|---|
| Agent alone |
Combinationb |
||||||
| MIN | ITC | VRC | POS | MIN/ITC | MIN/VRC | MIN/POS | |
| BMU00028 | >64 | 1 | 2 | 1 | 4/1(I) | 8/0.5(S) | 4/0.25(S) |
| BMU00029 | >64 | 1 | 0.5 | 1 | 16/1(I) | 16/0.25(I) | 16/0.25(S) |
| BMU00030 | >64 | 2 | 0.5 | 1 | 16/0.5(S) | 8/0.25(I) | 8/0.5(I) |
| BMU00034 | >64 | 2 | 0.5 | 0.5 | 8/0.5(S) | 8/0.125(S) | 4/0.25(I) |
| BMU00035 | >64 | 1 | 1 | 1 | 16/0.5(I) | 16/0.5(I) | 8/0.25(S) |
| BMU00036 | >64 | 1 | 1 | 0.5 | 8/0.25(S) | 8/0.5(I) | 16/0.125(S) |
| BMU00037 | >64 | 2 | 0.5 | 1 | 16/0.5(S) | 16/0.25(I) | 8/1(I) |
| BMU00038 | >64 | 1 | 0.5 | 1 | 8/1(I) | 8/0.125(S) | 8/0.25(S) |
| BMU00039 | >64 | 1 | 0.5 | 1 | 4/0.25(S) | 16/0.125(S) | 8/0.25(S) |
| BMU00040 | >64 | 1 | 0.5 | 0.5 | 8/0.25(S) | 8/0.5(I) | 8/0.5(I) |
| 109140 | >64 | 2 | 0.5 | 0.5 | 8/0.5(S) | 8/0.125(S) | 4/0.25(I) |
| 109144 | >64 | 1 | 1 | 1 | 4/0.25(S) | 8/0.25(S) | 8/0.125(S) |
| 109145 | >64 | 1 | 0.5 | 1 | 4/0.25(S) | 4/0.125(S) | 8/0.25(S) |
| 109148 | >64 | 1 | 0.5 | 0.5 | 4/0.25(S) | 8/0.25(I) | 4/0.25(I) |
| 109149 | >64 | 1 | 0.5 | 1 | 8/0.5(I) | 8/0.25(I) | 8/0.25(S) |
| 109152 | >64 | 1 | 0.5 | 1 | 4/0.25(S) | 4/0.125(S) | 4/0.125(S) |
The MIC is the concentration achieving 100% growth inhibition.
FICI results are shown in parentheses. S, synergy (FICI of ≤ 0.5); I, no interaction (indifference) (0.5 < FICI ≤ 4).
In vitro interactions between MIN and azoles against Fusarium spp.
The individual MIC ranges of tested agents against Fusarium spp. were 16 to 32 μg/ml, ≥32 μg/ml, 1 to 4 μg/ml, and 8 to 32 μg/ml for MIN, ITC, VRC, and POS, respectively (Table 4). When MIN was combined with ITC, VRC, or POS, synergistic activity was observed in 6 (46%), 5 (38%), and 6 (46%) strains of Fusarium spp., respectively (Table 2 and 4). The MICs of MIN and ITC in synergistic MIN-ITC combination decreased to 4 to 8 μg/ml and 8 to 16 μg/ml, respectively (Table 4). When MIN was combined with POS, the effective MIC ranges of MIN and POS decreased to 8 μg/ml and 2 to 8 μg/ml, respectively. When MIN was combined with VRC, the effective working ranges of MIN and VRC were 8 μg/ml and 0.5 to 1 μg/ml, respectively (Table 4). No antagonism was observed in any combination.
TABLE 4.
MIC and FICI results with the combinations of MIN and azoles against Fusarium spp.
| Strain | MICa (μg/ml) for: |
||||||
|---|---|---|---|---|---|---|---|
| Agent alone |
Combinationb |
||||||
| MIN | ITC | VRC | POS | MIN/ITC | MIN/VRC | MIN/POS | |
| F. solani | |||||||
| FS1 | 32 | 32 | 2 | 16 | 4/8(S) | 8/0.5(S) | 16/4(I) |
| FS2 | 32 | 32 | 1 | 8 | 16/16(I) | 16/1(I) | 16/4(I) |
| FS3 | 32 | >64 | 2 | 16 | 8/16(S) | 8/0.5(S) | 8/2(S) |
| FS4 | 16 | >64 | 2 | 16 | 8/16(I) | 8/0.5(I) | 8/4(I) |
| FS5 | 16 | 32 | 2 | 16 | 16/16(I) | 8/1(I) | 8/8(I) |
| FS6 | 32 | 32 | 1 | 32 | 8/8(S) | 16/1(I) | 8/8(S) |
| FS7 | 32 | >64 | 4 | 16 | 8/8(S) | 8/1(S) | 8/4(S) |
| FS8 | 16 | 32 | 2 | 8 | 16/16(I) | 8/0.5(I) | 8/4(I) |
| FS9 | 32 | 32 | 4 | 8 | 8/8(S) | 8/1(S) | 8/2(S) |
| FS10 | 16 | 32 | 2 | 16 | 8/8(I) | 16/1(I) | 8/4(I) |
| F. oxysporum | |||||||
| FO1 | 16 | 32 | 2 | 8 | 8/16(I) | 8/2(I) | 8/8(I) |
| FO2 | 32 | 32 | 2 | 16 | 8/8(S) | 16/1(I) | 8/4(S) |
| FO3 | 32 | 32 | 4 | 8 | 16/16(I) | 8/1(S) | 8/2(S) |
The MIC is the concentration achieving 100% growth inhibition.
FICI results are shown in parentheses. S, synergy (FICI of ≤0.5); I, no interaction (indifference) (0.5 < FICI ≤ 4).
In vivo effect of MIN alone and combined with azoles against A. fumigatus.
The survival of larvae infected with AF293 in groups treated with VRC, ITC, POS, MIN, MIN with VRC, MIN with ITC, and MIN with POS was 33.3%, 30%, 30%, 0%, 53.3%, 43.3%, and 58.3%, respectively. Treatments with azoles alone and combined with MIN all significantly (P < 0.001) prolonged the survival of larvae infected with AF293 (Fig. 1A). The combination of MIN with VRC or POS act synergistically against AF293 infection, compared with VRC and POS alone, respectively (P < 0.05). There is no significance difference between the ITC-alone group and the MIN-ITC group.
FIG 1.
Survival curve of G. mellonella infected with A. fumigatus. (A) AF293, (B) AFR1, (C) AFR2. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.
As for AFR1 infection, the survival in groups treated with VRC, ITC, POS, MIN, MIN with VRC, MIN with ITC, and MIN with POS was 20%, 5%, 10%, 0%, 47.5%, 15%, and 41.7%, respectively. ITC or MIN alone failed to improve the survival of larvae, compared with the conidia group. However, treatment with POS alone, VRC alone, and the combination with azoles and MIN all significantly (P < 0.001) prolonged the survival of larvae infected with AFR1 (Fig. 1B). The survival rates of the groups treated with MIN combined with POS or VRC were significantly higher than groups treated with POS and VRC alone, respectively (P < 0.001).
The survival of larvae infected with AFR2 in groups treated with VRC, ITC, POS, MIN, MIN with VRC, MIN with ITC, and MIN with POS was 5%, 16.7%, 23.3%, 0%, 18.3%, 38.3%, and 43.3%, respectively. Infections of AFR2 showed resistance to VRC or MIN treatment alone. However, treatment with ITC alone, POS alone, and the combination of MIN and azoles all significantly prolonged the survival of larvae infected with AFR2 (P < 0.01) (Fig. 1C). The survival rates of larvae in the combination groups were significantly higher than azoles or MIN treatment alone (P < 0.01).
In vivo effect of MIN alone and combined with azoles against E. dermatitidis.
The survival in groups treated with VRC, ITC, POS, MIN, MIN with VRC, MIN with ITC, and MIN with POS was 55%, 58.3%, 63.3%, 6.7%, 76. 7%, 68.3%, and 81.7%, respectively. Treatment with azoles alone or combined with MIN all significantly (P < 0.0001) prolonged the survival of larvae infected with E. dermatitidis (Fig. 2A). Treatment with MIN alone had no effect on E. dermatitidis infection. However, in groups that received MIN combined with VRC or POS, the survival of larvae was significantly (P < 0.05) prolonged compared with groups that received VRC or POS only, respectively (Fig. 2A).
FIG 2.
Survival curve of G. mellonella infected with E. dermatitidis (A) and F. solani (B). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.
In vivo effect of MIN alone and combined with azoles against F. solani.
The survival in groups treated with VRC, ITC, POS, MIN, MIN with VRC, MIN with ITC, and MIN with POS was 26.7%, 10%, 10%, 1.7%, 46.7%, 15%, and 21.7%, respectively. Treatment with azoles alone or combined with MIN all significantly (P < 0.0001) prolonged the survival of larvae infected with F. solani (Fig. 2B). Treatment with MIN alone showed no effect on F. solani infection. However, the combination of VRC or POS with MIN significantly (P < 0.05) prolonged the survival of larvae compared with groups that received VRC or POS only (Fig. 2B).
DISCUSSION
Tetracyclines are bacteriostatic antibiotics that are active against a wide spectrum of microorganisms. MIN has been demonstrated to be the most effective tetracycline derivative and has a better pharmacokinetic profile (13). It has been used in daily clinical practice for over 30 years and shows excellent tolerability in human beings even when used chronically. In the last decades, tetracyclines, especially MIN, have gained much interest due to their nonantibiotic effects (16).
The antifungal effects of tetracyclines alone or in association with antifungals against Candida spp. have been reported for decades. MIN has been demonstrated to exert antifungal effects against C. albicans (17). Tetracycline, doxycycline, and minocycline have been reported to act synergistically with fluconazole or amphotericin B (AMB) against Candida spp. (15, 18, 19). In addition, synergy of AMB-tetracycline and AMB-minocycline have also been observed in A. fumigatus and Cryptococcus neoformans (18, 20). However, few studies reported the effect of tetracyclines in combination with triazoles against filamentous fungi. In the present study, MIN was tested alone and as an adjunct to azoles against clinically important filamentous fungi Aspergillus spp., Fusarium spp., and E. dermatitidis.
A total of 56 isolates of pathogenic fungi were studied in vitro. Although MIN alone failed to show any antifungal activity in vitro, MIN acted synergistically with ITC, VRC, and POS against 34 (61%), 28 (50%), and 38 (68%) isolates tested, demonstrating promising synergistic effects with azoles against pathogenic fungi, including azole-resistant A. fumigatus and azole-inactive Fusarium spp. It was noteworthy that MIN resulted in the reversion of ITC and POS resistance of both AFR1 and AFR2. As shown in Table 1, we observed a 4-fold reduction of the MICs of ITC and POS in the combination with MIN against azole-resistant A. fumigatus. Similarly, an up to 16-fold reduction of the MICs of azoles against pathogenic fungi was observed in synergistic combinations. The effective MICs of MIN in synergistic combinations were mostly within the range of 4 to 8 μg/ml, which was within the range of the frequently achieved MIC of MIN therapeutic doses in humans. Unlike a previous study that reported a decrease in terbinafine susceptibility of C. albicans in the presence of tetracycline (18), we did not observe any antagonism between MIN and azoles.
Our in vitro data were confirmed in vivo as the antifungal therapy of azoles in combination with MIN showed significant (P < 0.01) improvement in larvae survival in all isolates. No significant increase in survival due to MIN application alone could be detected. All azoles applied alone significantly (P < 0.01) increased larvae survival in all isolates, except that AFR1 and AFR2 infections were resistant to ITC and VRC treatment, respectively. However, MIN combined with ITC or VRC had a significant positive effect on survival of AFR1- and AFR2-infected larvae (P < 0.01), respectively, demonstrating synergism between MIN and ITC or VRC against azole-resistant Aspergillus infection in vivo. In addition, the combination of MIN and VRC or POS significantly enhanced the effect of VRC or POS alone against larvae infection in all isolates (P < 0.05), respectively. Although not statistically significant, a higher survival of larvae treated with MIN and ITC than groups treated with ITC alone was observed in AF293-, BMU00034-, AFR1-, and FS9-infected groups. As for AFR2-infected larvae, application with MIN and ITC resulted in a significant increase in survival compared with ITC application alone (P < 0.05). In contrast to the in vitro susceptibility that showed relatively higher MICs of azoles against FS9, all azoles significantly increased the survival of larvae infected with FS9 (P < 0.01). However, the survival rates of larvae infected with FS9 were much lower than those infected with BMU00034 or AF293, in concordance with in vitro susceptibility results that azoles alone exhibited higher MICs against FS9 than BMU00034 or AF293.
Azole resistance of and azole inactive against pathogenic fungi are associated with treatment failure and death, demonstrating a growing public health concern. In the present study, MIN was shown to exert synergistic effects with azoles against pathogenic fungi in vitro and in vivo and had the potential to reverse azole resistance. In addition, considering that MIN easily penetrates the blood-brain barrier and has been shown to accumulate both in the cerebrospinal fluid and central nervous system (21), it is promising to assume the possible combinational use of MIN with azole in the treatment of neurotrophic fungal infection.
A previous study attributed the mechanisms of MIN-fluconazole synergy against fluconazole-resistant C. albicans to the enhancement of fluconazole penetrating biofilm as well as interrupting the cellular calcium balance (15). The synergy of fluconazole and doxycycline against C. albicans was associated with iron chelation (22). In addition, tetracycline effects on C. albicans were demonstrated to be associated with direct inhibition of mitochondrial function (18). It is well known that the mechanism of action for tetracyclines is the inhibition of translation through binding to the bacterial 30S ribosomal unit. It is also known that tetracycline also affects protein synthesis in the mitochondria of eukaryotic cells, due to the fact that the mitochondrial ribosome of eukaryotic cells is related to bacterial ribosomes in structure and function (21). Therefore, we suspected that synergy between MIN and azoles against filamentous fungi might result from the interference of cellular electrolyte balance and a loss of mitochondrial function. However, further investigations are warranted to elucidate the underlying mechanism and to determine their possible reliable and safe application in clinical practice.
In conclusion, our results expanded the knowledge regarding synergy between MIN and azoles against filamentous fungi and suggested that MIN combined with azoles may help to enhance the antifungal susceptibilities of azoles against filamentous fungi and had the potential to overcome azole resistance issues. The underlying mechanism remains to be elucidated. Further studies are warranted to investigate these combination effects in more isolates and more species and to evaluate the potential for concomitant use of these agents in humans.
MATERIALS AND METHODS
Fungal strains.
A total of 20 strains of A. fumigatus, including two azole-resistant A. fumigatus strains harboring the association of a tandem repeat sequence and punctual mutation of the Cyp51A gene (TR34/L98H and TR46/Y121F/T289A), 7 strains of A. flavus, 16 strains of E. dermatitidis, 10 strains of F. solani, and 3 strains of F. oxysporum were studied. Candida parapsilosis (ATCC 22019) and A. flavus (ATCC 204304) were included to ensure quality control. All tested strains were clinical isolates and were identified by microscopic morphology and by molecular sequencing of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) (23). For identification of Aspergillus spp., an additional molecular sequence of β-tubulin and calmodulin was required (24, 25). For identification of Fusarium spp., an additional molecular sequence of translation elongation factor 1α (TEF-1α) was required (26).
Antifungals and chemical agents.
All tested agents, including MIN, ITC, VRC, and POS, were purchased in powder form from Selleck Chemicals, Houston, TX, and were diluted in dimethyl sulfoxide as stock solutions (3,200 μg/ml).
In vitro interactions of MIN and azoles against pathogenic fungi.
The in vitro interactions between MIN and azoles against pathogenic fungi were tested via the microdilution chequerboard technique, adapted from the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method M38-A2 (27). Conidia harvested from cultures grown for 7 days on Sabouraud dextrose agar (SDA) were suspended in sterile distilled water containing 0.03% Triton and diluted to a concentration of 1 × 106 to 5 × 106 spores/ml, which were than diluted 100 times in RPMI 1640 to achieve a 2-fold suspension more concentrated than the density needed or to approximately 1 × 104 to 5 × 104 spores/ml (27). Serial diluents of tested agents were prepared as outlined in the M38-A2 by dilution with RPMI 1640 (27). The working concentration ranges were 0.06 to 32 μg/ml for ITC, 0.125 to 64 μg/ml for VRC and POS, and 1 to 64 μg/ml for MIN. As described, 50 μl of MIN with serial dilutions was inoculated in horizontal direction and another 50 μl of azoles with serial dilutions was inoculated in a vertical direction on the 96-well plate, which contained 100-μl prepared inoculum suspensions. The interpretation of results was performed after incubation at 35°C for 48 h for Aspergillus spp. and Fusarium spp. and 72 h for Exophiala spp. The MICs were determined as the lowest concentration resulting in complete inhibition of growth (27). Drug combination interaction was classified on the basis of the fractional inhibitory concentration index (FICI). The FICI as calculated by the formula FICI = (Ac/Aa) + (Bc/Ba), where Ac and Bc are the MICs of antifungal drugs in combination and Aa and Ba are the MICs of antifungal drugs A and B alone (28). A FICI of ≤0.5 indicates synergy, a FICI of >0.5 to ≤4 indicates no interaction (indifference), and an FICI of >4 indicates antagonism (29). All tests were performed in triplicate.
In vivo effect of MIN alone and combined with azoles in Galleria mellonella.
The efficacy of MIN alone and combined with azoles in G. mellonella infected with E. dermatitidis strain BMU00034; F. solani strain FS9; and A. fumigatus strains AF293, AFR1, and AFR2 were evaluated by survival assay as described previously (30), using sixth instar larvae (∼300 mg; Sichuan, China). Groups of 20 larvae were maintained in wood shavings in the dark at room temperature before use. Suspensions of E. dermatitidis, F. solani, and A. fumigatus that had been grown on SDA for 72 h at 37°C were harvested by gentle scraping of colony surfaces with sterile plastic loops, washed twice, and adjusted to 1 × 107 spores/ml for E. dermatitidis and F. solani and to 1 × 108 spores/ml for A. fumigatus in sterile saline. The following control groups were included: larvae injected with sterile saline, larvae injected with conidia suspension, and untouched larvae. A Hamilton syringe (25 gauge, 50 μl) was used to inoculate larvae with the conidia suspension and for introduction of treatments or control solutions into the larvae. To determine the in vivo effects of MIN alone and in combination with azoles against pathogenic fungi, a total of seven intervention therapy groups were included, namely, MIN treated group, ITC treated group, POS treated group, VRC treated group, MIN with ITC treated group, MIN with POS treated group, and MIN with VRC treated group. Larvae were infected with a conidia suspension (5 μl for E. dermatitidis and 10 μl for F. solani and A. fumigatus per larvae) and injected with tested agents (0.5 μg per agent) 2 h postinfection. The survival of the larvae was monitored every 24 h over 120 h after the infection. The experiment was repeated on 3 independent occasions. The G. mellonella survival curves were analyzed by the Kaplan-Meier method. Differences were considered significant at P values of <0.05.
ACKNOWLEDGMENTS
We thank Ruoyu Li and Wei Liu from Peking University First Hospital, Research Center for Medical Mycology, Peking University, Beijing; Qiangqiang Zhang from Fudan University Huashan Hospital, Shanghai, China, and G. Sybren de Hoog from CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, for kindly provided us with the isolates studied.
This work was supported by the Hubei Province Health and Family Planning Scientific Research Project (WJ2018H178) and the Natural Science Foundation of Hubei Province (2019CFB567).
The funders had no role in study design, data analysis, decision to publish, or preparation of the manuscript.
We declare no conflicts of interest.
REFERENCES
- 1.Kontoyiannis DP, Bodey GP. 2002. Invasive aspergillosis in 2002: an update. Eur J Clin Microbiol Infect Dis 21:161–172. doi: 10.1007/s10096-002-0699-z. [DOI] [PubMed] [Google Scholar]
- 2.Steinbach WJ, Marr KA, Anaissie EJ, Azie N, Quan SP, Meier-Kriesche HU, Apewokin S, Horn DL. 2012. Clinical epidemiology of 960 patients with invasive aspergillosis from the PATH Alliance registry. J Infect 65:453–464. doi: 10.1016/j.jinf.2012.08.003. [DOI] [PubMed] [Google Scholar]
- 3.Guarro J. 2013. Fusariosis, a complex infection caused by a high diversity of fungal species refractory to treatment. Eur J Clin Microbiol Infect Dis 32:1491–1500. doi: 10.1007/s10096-013-1924-7. [DOI] [PubMed] [Google Scholar]
- 4.Li DM, Li RY, de Hoog GS, Sudhadham M, Wang DL. 2011. Fatal Exophiala infections in China, with a report of seven cases. Mycoses 54:e136–e142. doi: 10.1111/j.1439-0507.2010.01859.x. [DOI] [PubMed] [Google Scholar]
- 5.Hagiya H, Maeda T, Kusakabe S, Kawasaki K, Hori Y, Kimura K, Ueda A, Yoshioka N, Sunada A, Nishi I, Morii E, Kanakura Y, Tomono K. 2019. A fatal case of Exophiala dermatitidis disseminated infection in an allogenic hematopoietic stem cell transplant recipient during micafungin therapy. J Infect Chemother 25:463–466. doi: 10.1016/j.jiac.2018.12.009. [DOI] [PubMed] [Google Scholar]
- 6.Sato E, Togawa A, Masaki M, Shirahashi A, Kumagawa M, Kawano Y, Ishikura H, Yamashiro Y, Takagi S, To H, Kobata K, Takeshita M, Kusaba K, Sueoka E, Tamura K, Takamatsu Y, Takata T. 2019. Community-acquired disseminated Exophiala dermatitidis mycosis with necrotizing fasciitis in chronic graft-versus-host disease. Intern Med 58:877–882. doi: 10.2169/internalmedicine.1706-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Klasinc R, Riesenhuber M, Bacher A, Willinger B. 2019. Invasive fungal infection caused by Exophiala dermatitidis in a patient after lung transplantation: case report and literature review. Mycopathologia 184:107–113. doi: 10.1007/s11046-018-0275-4. [DOI] [PubMed] [Google Scholar]
- 8.Hagiwara D, Watanabe A, Kamei K, Goldman GH. 2016. Epidemiological and genomic landscape of azole resistance mechanisms in Aspergillus fungi. Front Microbiol 7:1382. doi: 10.3389/fmicb.2016.01382. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Lass-Flörl C, Cuenca-Estrella M. 2017. Changes in the epidemiological landscape of invasive mould infections and disease. J Antimicrob Chemother 72:i5–i11. doi: 10.1093/jac/dkx028. [DOI] [PubMed] [Google Scholar]
- 10.Revankar SG, Sutton DA. 2010. Melanized fungi in human disease. Clin Microbiol Rev 23:884–928. doi: 10.1128/CMR.00019-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kondori N, Gilljam M, Lindblad A, Jonsson B, Moore ER, Wenneras C. 2011. High rate of Exophiala dermatitidis recovery in the airways of patients with cystic fibrosis is associated with pancreatic insufficiency. J Clin Microbiol 49:1004–1009. doi: 10.1128/JCM.01899-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Patel AK, Patel KK, Darji P, Singh R, Shivaprakash MR, Chakrabarti A. 2013. Exophiala dermatitidis endocarditis on native aortic valve in a postrenal transplant patient and review of literature on E. dermatitidis infections. Mycoses 56:365–372. doi: 10.1111/myc.12009. [DOI] [PubMed] [Google Scholar]
- 13.Garrido-Mesa N, Zarzuelo A, Gálvez J. 2013. Minocycline: far beyond an antibiotic. Br J Pharmacol 169:337–352. doi: 10.1111/bph.12139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Nagarakanti S, Bishburg E. 2016. Is minocycline an antiviral agent? A review of current literature. Basic Clin Pharmacol Toxicol 118:4–8. doi: 10.1111/bcpt.12444. [DOI] [PubMed] [Google Scholar]
- 15.Shi W, Chen Z, Chen X, Cao L, Liu P, Sun S. 2010. The combination of minocycline and fluconazole causes synergistic growth inhibition against Candida albicans: an in vitro interaction of antifungal and antibacterial agents. FEMS Yeast Res 10:885–893. doi: 10.1111/j.1567-1364.2010.00664.x. [DOI] [PubMed] [Google Scholar]
- 16.Garrido-Mesa N, Zarzuelo A, Gálvez J. 2013. What is behind the non-antibiotic properties of minocycline? Pharmacol Res 67:18–30. doi: 10.1016/j.phrs.2012.10.006. [DOI] [PubMed] [Google Scholar]
- 17.Waterworth PM. 1974. The effect of minocycline on Candida albicans. J Clin Pathol 27:269–272. doi: 10.1136/jcp.27.4.269. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Oliver BG, Silver PM, Marie C, Hoot SJ, Leyde SE, White TC. 2008. Tetracycline alters drug susceptibility in Candida albicans and other pathogenic fungi. Microbiology 154:960–970. doi: 10.1099/mic.0.2007/013805-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Gu W, Yu Q, Yu C, Sun S. 2018. In vivo activity of fluconazole/tetracycline combinations in Galleria mellonella with resistant Candida albicans infection. J Glob Antimicrob Resist 13:74–80. doi: 10.1016/j.jgar.2017.11.011. [DOI] [PubMed] [Google Scholar]
- 20.Hughes CE, Harris C, Peterson LR, Gerding DN. 1984. Enhancement of the in vitro activity of amphotericin B against Aspergillus spp. by tetracycline analogs. Antimicrob Agents Chemother 26:837–840. doi: 10.1128/AAC.26.6.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Chopra I, Roberts M. 2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 65:232–260. doi: 10.1128/MMBR.65.2.232-260.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Fiori A, Van Dijck P. 2012. Potent synergistic effect of doxycycline with fluconazole against Candida albicans is mediated by interference with iron homeostasis. Antimicrob Agents Chemother 56:3785–3796. doi: 10.1128/AAC.06017-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol 61:1323–1330. doi: 10.1128/AEM.61.4.1323-1330.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Hong SB, Go SJ, Shin HD, Frisvad JC, Samson RA. 2005. Polyphasic taxonomy of Aspergillus fumigatus and related species. Mycologia 97:1316–1329. doi: 10.3852/mycologia.97.6.1316. [DOI] [PubMed] [Google Scholar]
- 25.Samson RA, Varga J. 2009. What is a species in Aspergillus? Med Mycol 47:S13–S20. doi: 10.1080/13693780802354011. [DOI] [PubMed] [Google Scholar]
- 26.Geiser DM, del Mar Jiménez-Gasco M, Kang S, Makalowska I, Veeraraghavan N, Ward TJ, Zhang N, Kuldau GA, O'donnell K. 2004. FUSARIUM-ID v. 1.0: a DNA sequence database for identifying Fusarium. Eur J Plant Pathol 110:473–479. doi: 10.1023/B:EJPP.0000032386.75915.a0. [DOI] [Google Scholar]
- 27.Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard, 2nd ed CLSI document M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
- 28.Tobudic S, Kratzer C, Lassnigg A, Graninger W, Presterl E. 2010. In vitro activity of antifungal combinations against Candida albicans biofilms. J Antimicrob Chemother 65:271–274. doi: 10.1093/jac/dkp429. [DOI] [PubMed] [Google Scholar]
- 29.Odds FC. 2003. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 52:1. doi: 10.1093/jac/dkg301. [DOI] [PubMed] [Google Scholar]
- 30.Maurer E, Browne N, Surlis C, Jukic E, Moser P, Kavanagh K, Lass-Flörl C, Binder U. 2015. Galleria mellonella as a host model to study Aspergillus terreus virulence and amphotericin B resistance. Virulence 6:591–598. doi: 10.1080/21505594.2015.1045183. [DOI] [PMC free article] [PubMed] [Google Scholar]