Universal In Vitro Antifungal Resistance of Genetic Clades of the Fusarium solani Species Complex

Abstract

Eleven antifungal drugs were tested against representative isolates of the four phylogenetic clades of the Fusarium solani species complex obtained in a multilocus sequence analysis. They all showed very poor activity, with no differences among the clades. Amphotericin B was the most active drug.

Fusarium solani is a fungus that is widely distributed in nature and is able to produce many plant diseases with important economic impacts and, also, severe, usually fatal, human infections (7, 9, 15, 23). These infections can be characterized by their resistance to practically all available antifungal drugs (3, 21, 24). Primary therapy with voriconazole (VRC) or a lipid formulation of amphotericin B (AMB) is currently recommended (22). Although the response of Fusarium spp. to AMB is poor in general, it depends on the species involved, with F. solani being the most resistant species, at least in vitro (24). Recently, Zhang et al. (29) used a multilocus sequence analysis approach to demonstrate that under the generic name F. solani, at least 45 phylogenetically distinct species exist, most of which have not been described formally. It is unknown if antifungal susceptibility varies among these phylogenetic species. If such differences exist, knowing them could be useful for guiding clinical treatments. Due to the difficulties of testing representative strains of all the phylogenetic species, we tested isolates belonging to the four major clades, inferred from a combined phylogenetic analysis of fragments of three genes, i.e., the translation elongation factor 1α (EF-1α) and β-tubulin genes and the internal transcribed spacer (ITS) of the nuclear rRNA gene, against traditional and new antifungal drugs.

Fifty isolates from clinical or environmental sources, morphologically identified as F. solani (6), were included in the study (Table 1). Isolates were stored by lyophilization and submerged in slant cultures in mineral oil at room temperature. The procedures for DNA extraction, amplification, and sequencing of the different regions analyzed were described by Gilgado et al. (8). The annealing temperature was 55°C, and the primers used were EF-1H and EF-2T (16) for EF-1α, TUB-F (5) and T22 (17) for β-tubulin, and ITS5 and ITS4 (28) for the ITS. The phylogenetic analysis was performed using PAUP*, version 4.0b10 (27). Maximum parsimony trees were obtained after 100 heuristic searches with random sequence addition and tree bisection-reconnection branch-swapping algorithms, collapsing zero-length branches, and saving of all minimal-length trees (MulTrees). The results of the partition homogeneity test showed that the three locus sequence data sets were congruent (P = 0.2) and could therefore be combined. Sequences of the three genes were analyzed phylogenetically as separate (data not shown) and combined data sets.

TABLE 1.

Isolates included in this study

Isolatea	Source	GenBank accession no.
		EF-1α	β-Tubulin	ITS
FMR 4389	Human scrotum skin, United States	AM397191	AM419414	AM412634
FMR 4391	Human blood, United States	AM397184	AM419413	AM412635
FMR 7140	Aquarium sand, Spain	AM397227	AM419415	AM412636
FMR 7141	Aquarium sand, Spain	AM397228	AM419416	AM412637
FMR 7238	Human ulcer, Spain	AM397225	AM419417	AM412638
FMR 7240	Human skin lesion, Spain	AM397210	AM419418	AM412639
FMR 7242	Human skin lesion, Spain	AM397212	AM419419	AM412640
FMR 7985	Human keratitis, Brazil	AM397218	AM419406	AM412641
FMR 7988	Human keratitis, Brazil	AM397202	AM419420	AM412642
FMR 7989	Human keratitis, Brazil	AM397219	AM419421	AM412643
FMR 7991	Human keratitis, Brazil	AM397220	AM419412	AM412624
FMR 7992	Human keratitis, Brazil	AM397186	AM419422	AM412625
FMR 7993	Human keratitis, Brazil	AM397198	AM419423	AM412626
FMR 7994	Human keratitis, Brazil	AM397194	AM419424	AM412627
FMR 7995	Human keratitis, Brazil	AM397206	AM419425	AM412628
FMR 7996	Human keratitis, Brazil	AM397199	AM419376	AM412629
FMR 7997	Human keratitis, Brazil	AM397209	AM419377	AM412630
FMR 7998	Human keratitis, Brazil	AM397204	AM419378	AM412631
FMR 8000	Human keratitis, Brazil	AM397207	AM419379	AM412632
FMR 8013	Human keratitis, Brazil	AM397189	AM419380	AM412633
FMR 8014	Human keratitis, Brazil	AM397205	AM419381	AM412604
FMR 8016	Human keratitis, Brazil	AM397226	AM419382	AM412605
FMR 8017	Human keratitis, Brazil	AM397208	AM419383	AM412606
FMR 8019	Human keratitis, Brazil	AM397197	AM419384	AM412607
FMR 8021	Human keratitis, Brazil	AM397195	AM419385	AM412608
FMR 8023	Human keratitis, Brazil	AM397201	AM419386	AM412609
FMR 8024	Human keratitis, Brazil	AM397223	AM419409	AM412610
FMR 8027	Human keratitis, Brazil	AM397203	AM419387	AM412611
FMR 8028	Human keratitis, Brazil	AM397196	AM419388	AM412612
FMR 8030	Human keratitis, Brazil	AM397229	AM419389	AM412613
FMR 8031	Human keratitis, Brazil	AM397213	AM419407	AM412614
FMR 8032	Human keratitis, Brazil	AM397222	AM419410	AM412615
FMR 8036	Human keratitis, Brazil	AM397192	AM419390	AM412616
FMR 8037	Human keratitis, Brazil	AM397188	AM419391	AM412617
FMR 8038	Human keratitis, Brazil	AM397230	AM419392	AM412618
FMR 8039	Human keratitis, Brazil	AM397231	AM419393	AM412619
FMR 8040	Human keratitis, Brazil	AM397193	AM419394	AM412620
FMR 8207	Nematode, Spain	AM397190	AM419395	AM412621
FMR 8263	Nematode, Spain	AM397187	AM419396	AM412622
FMR 8281	Nematode, Spain	AM397215	AM419397	AM412623
FMR 8340	Human fungemia, Qatar	AM397232	AM419398	AM412594
FMR 8482	Human clinical source, Qatar	AM397224	AM419411	AM412595
FMR 8483	Human clinical source, Qatar	AM397221	AM419408	AM412596
FMR 8484	Nematode, Spain	AM397200	AM419399	AM412597
FMR 8631	Human clinical source, Spain	AM397185	AM419400	AM412598
FMR 8633	Human nail, Venezuela	AM397214	AM419401	AM412599
FMR 8634	Human diabetic foot sore, Spain	AM397233	AM419402	AM412600
FMR 8666	Nematode, Spain	AM397211	AM419403	AM412601
FMR 8673	Nematode, Spain	AM397216	AM419404	AM412602
FMR 8688	Nematode, Spain	AM397217	AM419405	AM412603

^aFMR, Facultat de Medicina i Ciències de la Salut, Reus, Spain.

We evaluated the in vitro activities of 11 antifungal drugs against 27 representative strains (22 clinical and 5 environmental) randomly chosen from the main clades obtained in the phylogenetic analysis. The isolates were grown on potato dextrose agar plates and incubated at 25°C for 7 days. We used a microdilution reference method (14), with some modification. The inocula were adjusted to a final concentration of 4 × 10³ to 5 × 10⁴ conidia/ml with a hemocytometer and verified by quantitative colony counts on potato dextrose agar plates. Paecilomyces variotii ATCC 36257 was included in each batch of tests as a quality control strain. The antifungal agents tested were AMB, albaconazole, VRC, itraconazole, ravuconazole, terbinafine, ketoconazole (KTC), posaconazole (PSC), micafungin (MFG), fluconazole (FLC), and flucytosine (5-FC). MFG, FLC, and 5-FC were diluted in sterile distilled water, and the rest were diluted in dimethyl sulfoxide. Final drug concentrations ranged from 64 to 0.12 μg/ml for FLC and 5-FC, from 128 to 0.25 μg/ml for MFG, and from 16 to 0.03 μg/ml for the rest. The MIC end point for the triazoles and AMB was defined as the lowest concentration that produced complete inhibition of growth, and that for FLC, KTC, 5-FC, and MFG was defined as the lowest concentration that produced a 50% inhibition of growth.

With the primers used, we were able to amplify and sequence 654, 461, and 573 bp of the EF-1α, β-tubulin, and ITS gene sequences, respectively. Parsimony analysis of the combined data set (1,688 bp) yielded three trees of 176 steps in length. Four main clades (I, II, III, and IV) with high bootstrap support were obtained, resulting in a total of 28 different haplotypes and numerous putative cryptic species (Fig. 1). We found no relationship between the biogeographical origins of the isolates and the molecular groups. In order to compare the topology of our trees with those obtained by Zhang et al. (29), we performed a new phylogenetic analysis based on ITS and EF-1α loci (data not shown). We included our isolates and several representative sequences, retrieved from GenBank, from Zhang et al.'s main groups. In general, our clades did not coincide with those of Zhang et al., which confirms the high genetic variability of this complex. However, several of our strains nested in their groups 3 and 4.

FIG. 1.

One of the three most parsimonious trees obtained from heuristic searches, based on a combined data set. Bootstrap support values above 70% are indicated at the nodes. CI, consistency index; RI, retention index; HI, homoplasy index.

Results of the in vitro susceptibility tests are shown in Table 2. In general, all drugs showed high MICs, with no remarkable differences among the clades. AMB was the most active drug, although in no case was the MIC lower than 1 μg/ml, followed by VRC. The latter drug showed the most variable results; sometimes the differences among MICs against different isolates in the same clade were >8-fold.

TABLE 2.

Distribution of 27 tested isolates from the four phylogenetic clades of F. solani, according to antifungal susceptibility

Antifungala	Cladeb	No. of isolates with MIC (μg/ml)
		1	2	4	8	16	32	64	128	256
AMB	I	1	8	2
	II		4		1
	III	1	4	2
	IV		1	3
VRC	I			3	4	1	2
	II			1	4	1
	III			1	1	4	1
	IV				3	1
ABC	I to IV						27
ITC	I to IV						27
KTC	I to IV						27
PSC	I to IV						27
RVC	I to IV						27
TBF	I to IV						27
FLC	I to IV								27
5-FC	I to IV								27
MFG	I to IV						1	3	10	13

^aAMB, amphotericin B; VRC, voriconazole; ABC, albaconazole; ITC, itraconazole; KTC, ketoconazole; PSC, posaconazole; RVC, ravuconazole; TBF, terbinafine; FLC, fluconazole; 5-FC, flucytosine; MFG, micafungin.

^bSee Fig. 1.

The poor in vitro activities of different antifungal drugs against Fusarium have been reported by other authors many times, with F. solani being more resistant than the other species of the genus (12, 19, 24, 26). In this study, AMB was revealed as the most active drug, but only one isolate from clade I and another from clade III showed a MIC of 1 μg/ml, with all others showing higher values. Although this drug is recommended for the treatment of fusariosis, it has poor clinical success (7, 11, 15). In cases involving neutropenic patients receiving corticosteroids, the survival rate is practically nil, despite aggressive treatment (15). The poor efficacy of AMB was also demonstrated in animal studies (10). VRC, the other recommended drug, is the only agent indicated for treating refractory fusariosis (7). However, in our study, the VRC MICs were always higher than 2 μg/ml, which agrees with the results of other studies (12, 26). This drug was effective in a few clinical cases of fusariosis (2, 4), although in none of them was the species involved referred to as F. solani. This is probably linked to the fact that in animal studies F. solani was clearly more virulent and more difficult to treat than F. oxysporum and F. verticillioides, the two other common species of the genus (13, 18). In a recent clinical trial, 45.5% (5/11) of patients responded satisfactorily to VRC (20), although unfortunately the isolates were not identified to the species level. Arikan et al. (1) demonstrated that the combination of caspofungin plus AMB was synergistic against 100% of strains of F. oxysporum but against only 25% of F. solani strains. In the case of PSC, we also obtained high MICs, again agreeing with other studies (19, 26). However, in a recent retrospective analysis, PSC as an aggressive treatment for invasive fusariosis gave a 48% successful outcome (25), but the species involved were not listed. In conclusion, the F. solani species complex constitutes a group of genetically diverse fungi with poor in vitro and in vivo responses to different antifungal drugs.