Antifungal susceptibility of Aspergillus sp., Scedosporium sp. and Exophiala sp. isolated from patients with cystic fibrosis.

BRITO-DEVOTO T; POLA S; RUBEGLIO E; FINQUELIEVICH J; GAMARRA, S.; GARCIA, G.; CUESTA ML

Buenos Aires

Congreso; XVIII Congreso Argentino de la Sociedad Argentina de Infectología/XVIII Congress of the international society of Infectious Diseases. 2018.; 2018

SADI

PurposeMolds are frequently recovered from respiratory samples of cystic fibrosis (CF) patients. During the last decade, antifungal resistance in non-Candida species is increasingly reported. The emergence of azole resistance in Aspergillus fumigatus is an increasing problem. Azole resistance in filamentous fungi is mostly mediated by point mutations in the cyp51A gene, which encodes lanosterol 14α-demethylase, the target of azole antifungals. The aim of this work is to overview the susceptibility profile from different fungal species isolated from respiratory secretions of Argentinean CF patients and to analyze the underlying molecular mechanisms of azole resistance in the isolated Aspergillus fumigatus strains.Methods and MaterialsAmphotericin B (AMB), itraconazole (ITC), posaconazole (PSC) and voriconazole (VCZ) minimum inhibitory concentrations (MICs) for 70 fungal strains (53 Aspergillus sp., 12 Scedosporium sp. and 5 Exophiala sp.) recovered from 32 CF patients were determined by using the CLSI M38-A2 broth dilution method. The mechanism of azole resistance in A. fumigatus, was investigated by PCR amplification and sequence analysis of the full Cyp51A coding sequences and its 5´UTR region.ResultsFor AMB, MICs were above the epidemiological cut-off values (ECV) in 26%, 50% and 60% of the Aspergillus sp., Scedosporium sp. and Exophiala sp., respectively. For VCZ, MICs were above the ECV in 10% and 42% of Aspergillus and Scedosporium species, respectively. For PSC, MICs were above the ECV in 8%, 17% and 20% of Aspergillus, Scedosporium and Exophiala species, respectively. For ITC, MICs were above the ECV in 8% and 83% of Aspergillus and Scedosporium species, respectively (Table 1). Those A. fumigatus strains with MICs values higher than their corresponding ECVs for azole drugs (Table 2) were checked for mutations in theCyp51A gene. Results showed that the three isolates with high azole MIC values harbor 34-bp tandem repeat of 34 (TR34) (Figure 1) in the promoter region, a L98H mutation at codon 98 (TR34/L98H) and a S297T amino acid substitution (Figure 2). Moreover, Isolate # 37 showed the described Cyp51Ap substitutions together with a R65K mutation. This last strain showed a MIC value for VCZ higher than ECV (Table 3). Isolates (n)n/drugFrequencies of MICs (µg/µl)A. flavus (7)784210,50,250,120,060,030,015AMB4002100000VCZ0001600000PSC0000220012ITC0000310003A. fumigatus (27)2784210,50,250,120,060,030,015AMB41161500000VCZ001214100000PSC0120633417ITC3002711742A. niger (5)584210,50,250,120,060,030,015AMB0102200000VCZ0000320000PSC0000310100ITC0021100100A. terreus (10)1084210,50,250,120,060,030,015AMB2112300000VCZ0033111000PSC0000022005ITC0000101025Exophiala sp. (5)584210,50,250,120,060,030,015AMB0211010000VCZ0012110000PSC0011102000ITC0001002110Scedosporium spp. (12)1284210,50,250,120,060,030,015AMB6211100000VCZ0233210000PSC2431010000ITC10000000001A. calidoustus (2)284210,50,250,120,060,030,015AMB0110000000VCZ2000000000PSC2000000000ITC2000000000A. lentulus (1)184210,50,250,120,060,030,015AMB1000000000VCZ1000000000PSC0001000000ITC1000000000A. parasiticus (1)184210,50,250,120,060,030,015AMB1000000000VCZ0000100000PSC0000010000ITC0000010000Table 2 ? Frequency of CIM distribution of isolates. Cells in red shading correspond to values above the ECV. Percentage of isolates above the ECVTotal = 70AMBVCZPSCITCA. fumigatus5133A. flavus4000A. niger1000A. terreus2300A. lentulus1111Scedosporium spp.65210Exophiala spp.3010Sub-Total2210714%32,8%14,9%10,4%20,9%% Aspergillus spp.26%10%8%8%% Scedosporium spp.50%42%17%83%% Exophiala spp.60%0%20%0%Table 1 ? Percentage of strains with MICs above ECV.Isolate #MICs (µg/µl)Genotype (Cyp51Ap)Cyp51Ap aa. Substitutions at residueAMBVCZPSCITCCYP51A PromotorR65KL98S29750.501.002.008.00TR34WTL98HS297T370.502.004.008.00TR34R65KL98HS297T600.501.002.008.00TR34WTL98HS297TTable 3 ? MICs values and mutation profiles for the three non-WT A. fumigatus. Red shaded MIC values are higher than the described ECVs. AMB: amphotericin B, VCZ: voriconazole, PSC: posaconazole, ITC: itraconazole. WT: wild type. The known TR34 associated with L98H mutation was found in all isolates, along with S297T. Cuiorusly, only isolate #37,that also harboured the R65K point mutation showed a MIC value for VCZ higher than it ECV.. Figure 1 ? Alignment of promoter region of the three non-WT A. fumigatus with reference sequence showing the presence of the TR34. Figure 2 ? Alignment of the translated coding sequence ofCyp51Aof the three non-WT A. fumigatus with reference sequence (AF338659.1). Point mutations can be observed in amino acid positions65, 98 and 297 with respect to reference sequence. ConclusionThis is the first susceptibility profile studies for antifungal drugs in molds from CF patients in Argentina. We found that some Aspergillus spp. isolates have a reduced susceptibility to ITC (8%), PSC (8%) and VCZ (10%). Also, we found that 26% of the strains showed high AMB MIC values. Similarly, Scedosporium spp. showed high MIC values for all tested drugs in 50%, 42%, 17%, 83% of the strains for AMB, VCZ, PSC and ITC, respectively. In contrast, azoles showed good in vitro activity against Exophiala spp.. All ITC/PSC-non-wild type A. fumigatus isolates showed one of the most frequently described resistance mechanism in A. fumigatus worldwide (TR34/L98H). Since theTR34/L98H mutant strains are reported to be of environmental origin and to be a consequence of the use of fungicides in agriculture, further studies are needed to confirm this environmental hypothesis in our country. The R65K amino acid substitution in Cyp51Ap role in azole resistance phenotype should be studied. Finally, azole-resistance is an emerging problem that should be continuously monitored in CF patients.References?Espinel-Ingroff, A., Cuenca-Estrella, M., Fothergill, A., Fuller, J., Ghannoum, M., Johnson, E., ? Turnidge, J. (2011). Wild-Type MIC Distributions and Epidemiological Cutoff Values for Amphotericin B and Aspergillus spp. for the CLSI Broth Microdilution Method (M38-A2 Document). Antimicrobial Agents and Chemotherapy, 55(11), 5150?5154. http://doi.org/10.1128/AAC.00686-11?Lackner, M., de Hoog, G. S., Verweij, P. E., Najafzadeh, M. J., Curfs-Breuker, I., Klaassen, C. H., & Meis, J. F. (2012). 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