Phenotypic plasticity and the evolution of azole resistance in Aspergillus fumigatus; an expression profile of clinical isolates upon exposure to itraconazole

Abstract Azole resistance among Aspergillus fumigatus isolates, which is mainly related to mutations in the cyp51A gene, is a concern because it is rising, worldwide disseminated, and associated with treatment failure and death. Data on azole resistance of aspergillus from Latin American countries is very scarce and do not exist for Peru. Two hundred and seven Aspergillus clinical isolates collected prospectively underwent mycology and molecular testing for specie identification, and 143 isolates were confirmed as A. fumigatus sensu stricto (AFSS). All AFSS were tested for in vitro azole susceptibility, and resistant isolates underwent PCR amplification and sequencing of the whole cyp51A gene and its promoter. The in vitro susceptibility showed a minimal inhibitory concentration (MIC) range, MIC50 and MIC90 of 0.125 to >16, 0.25, and 0.5 μg/ml for itraconazole; 0.25 to 2, 0.5, and 0.5 μg/ml for voriconazole; and 0.003 to 1, 0.06, and 0.125 μg/ml for posaconazole. Three isolates (2%) showed resistance to itraconazole and exhibited different mutations of the cyp51A gene. One isolate harbored the mutation M220K, while a second one exhibited the G54 mutation plus a modification in the cyp51A gene promoter. The third isolate, from an azole naive patient, presented an integration of a 34-bp tandem repeat (TR34) in the promoter region of the gene and a substitution of leucine 98 by histidine (L98H). The three source patients had a diagnosis or suspicion of chronic pulmonary aspergillosis.

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Multiplecyp51A-Based Mechanisms Identified in Azole-Resistant Isolates of Aspergillus fumigatus from China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00003-15 ◽

2015 ◽

Vol 59 (7) ◽

pp. 4321-4325 ◽

Cited By ~ 30

Author(s):

Musang Liu ◽

Rong Zeng ◽

Lili Zhang ◽

Dongmei Li ◽

Guixia Lv ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Clinical Isolates ◽

Resistant Isolate ◽

Azole Resistance ◽

Content Type ◽

Resistant Strains

ABSTRACTSeventy-twoA. fumigatusclinical isolates from China were investigated for azole resistance based on mutations ofcyp51A. We identified four azole-resistant strains, among which we found three strains highly resistant to itraconazole, two of which exhibit the TR34/L98H/S297T/F495I mutation, while one carries only the TR34/L98H mutation. To our knowledge, the latter has not been found previously in China. The fourth multiazole-resistant isolate (with only moderate itraconazole resistance) carries a new G432A mutation.

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Genome-Wide Expression Profile Analysis Reveals Coordinately Regulated Genes Associated with Stepwise Acquisition of Azole Resistance in Candida albicans Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.4.1220-1227.2003 ◽

2003 ◽

Vol 47 (4) ◽

pp. 1220-1227 ◽

Cited By ~ 112

Author(s):

P. David Rogers ◽

Katherine S. Barker

Keyword(s):

Candida Albicans ◽

Expression Profile ◽

Profile Analysis ◽

Clinical Isolates ◽

Azole Resistance ◽

Down Regulation ◽

Human Fungal Pathogen ◽

Expression Profile Analysis ◽

Genome Wide ◽

First Time

ABSTRACT Candida albicans is an opportunistic human fungal pathogen and a causative agent of oropharyngeal candidiasis (OPC), the most frequent opportunistic infection among patients with AIDS. Fluconazole and other azole antifungal agents have proven effective in the management of OPC; however, with increased use of these agents treatment failures have occurred. Such failures have been associated with the emergence of azole-resistant strains of C. albicans. In the present study we examined changes in the genome-wide gene expression profile of a series of C. albicans clinical isolates representing the stepwise acquisition of azole resistance. In addition to genes previously associated with azole resistance, we identified many genes whose differential expression was for the first time associated with this phenotype. Furthermore, the expression of these genes was correlated with that of the known resistance genes CDR1, CDR2, and CaMDR1. Genes coordinately regulated with the up-regulation of CDR1 and CDR2 included the up-regulation of GPX1 and RTA3 and the down-regulation of EBP1. Genes coordinately regulated with the up-regulation of CaMDR1 included the up-regulation of IFD1, IFD4, IFD5, IFD7, GRP2, DPP1, CRD2, and INO1 and the down-regulation of FET34, OPI3, and IPF1222. Several of these appeared to be coordinately regulated with both the CDR genes and CaMDR1. Many of these genes are involved in the oxidative stress response, suggesting that reduced susceptibility to oxidative damage may contribute to azole resistance. Further evaluation of the role these genes and their respective gene products play in azole antifungal resistance is warranted.

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Azole Resistance in Aspergillus fumigatus Clinical Isolates from an Italian Culture Collection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02234-15 ◽

2015 ◽

Vol 60 (1) ◽

pp. 682-685 ◽

Cited By ~ 20

Author(s):

Cristina Lazzarini ◽

Maria Carmela Esposto ◽

Anna Prigitano ◽

Massimo Cogliati ◽

Gabriella De Lorenzis ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Culture Collection ◽

Resistance Rate ◽

Clinical Isolates ◽

Resistant Isolate ◽

Azole Resistance ◽

Content Type ◽

Italian Culture

ABSTRACTThe aims of the study were to investigate the prevalence of azole resistance amongAspergillus fumigatusclinical isolates. A total of 533 clinical isolates that had been collected between 1995 and 2006, from 441 patients, were screened. No resistance was detected in isolates collected between 1995 and 1997. Starting in 1998, the resistance rate was 6.9%; a total of 24 patients (6.25%) harbored a resistant isolate. The TR34/L98H substitution was found in 21 of 30 tested isolates.

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Azole Resistance of Environmental and Clinical Aspergillus fumigatus Isolates from Switzerland

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02088-17 ◽

2018 ◽

Vol 62 (4) ◽

Cited By ~ 14

Author(s):

Arnaud Riat ◽

Jérôme Plojoux ◽

Katia Gindro ◽

Jacques Schrenzel ◽

Dominique Sanglard

Keyword(s):

Aspergillus Fumigatus ◽

Opportunistic Pathogen ◽

Clinical Isolates ◽

Resistant Isolate ◽

Azole Resistance ◽

Clinical Settings ◽

Content Type ◽

Azole Antifungals ◽

First Time

ABSTRACT Aspergillus fumigatus is a ubiquitous opportunistic pathogen. This fungus can acquire resistance to azole antifungals due to mutations in the azole target ( cyp51A ). Recently, cyp51A mutations typical for environmental azole resistance acquisition (for example, TR 34 /L98H) have been reported. These mutations can also be found in isolates recovered from patients. Environmental azole resistance acquisition has been reported on several continents. Here we describe, for the first time, the occurrence of azole-resistant A. fumigatus isolates of environmental origin in Switzerland with cyp51A mutations, and we show that these isolates can also be recovered from a few patients. While the TR 34 /L98H mutation was dominant, a single azole-resistant isolate exhibited a cyp51A mutation (G54R) that was reported only for clinical isolates. In conclusion, our study demonstrates that azole resistance with an environmental signature is present in environments and patients of Swiss origin and that mutations believed to be unique to clinical settings are now also observed in the environment.

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In VitroSusceptibility of Aspergillus fumigatus to Isavuconazole: Correlation with Itraconazole, Voriconazole, and Posaconazole

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01141-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5778-5780 ◽

Cited By ~ 50

Author(s):

Lea Gregson ◽

Joanne Goodwin ◽

Adam Johnson ◽

Laura McEntee ◽

Caroline B. Moore ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Amphotericin B ◽

Clinical Isolates ◽

Cross Resistance ◽

Azole Resistance ◽

First Line ◽

Content Type

ABSTRACTTriazoles are first-line agents for treating aspergillosis. The prevalence of azole resistance inAspergillus fumigatusis increasing, and cross-resistance is a growing concern. In this study, the susceptibilities of 40A. fumigatusclinical isolates were tested by using the CLSI method with amphotericin B, itraconazole, voriconazole, posaconazole, and the new triazole isavuconazole. Isavuconazole MICs were higher in strains with reduced susceptibilities to other triazoles, mirroring changes in voriconazole susceptibility. Isavuconazole MICs differed depending on the Cyp51A substitution.

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Genome-Wide Association for Itraconazole Sensitivity in Non-Resistant Clinical Isolates of Aspergillus fumigatus

10.1101/2020.08.31.275297 ◽

2020 ◽

Author(s):

Shu Zhao ◽

Wenbo Ge ◽

Akira Watanabe ◽

Jarrod R. Fortwendel ◽

John G. Gibbons

Keyword(s):

Aspergillus Fumigatus ◽

Complex Traits ◽

Genetic Basis ◽

Molecular Mechanisms ◽

Clinical Isolates ◽

Genome Wide Association ◽

Azole Resistance ◽

Genome Wide ◽

Population Structure Analysis ◽

Recent Emergence

AbstractAspergillus fumigatus is a potentially lethal opportunistic pathogen that infects over ∼200,000 people and causes ∼100,000 deaths per year globally. Treating A. fumigatus infections is particularly challenging because of the recent emergence of azole-resistance. The majority of studies focusing on the molecular mechanisms underlying azole resistance have examined azole-resistant isolates. However, isolates that are susceptible to azoles also display variation in their sensitivity, presenting a unique opportunity to identify genes contributing to azole sensitivity. Here, we used genome-wide association (GWA) analysis to identify loci involved in azole sensitivity by analyzing the association between 68,853 SNPs and itraconazole (ITCZ) minimum inhibitory concentration (MIC) in 76 clinical isolates of A. fumigatus from Japan. Population structure analysis suggests the presence of four distinct populations, with ITCZ MICs distributed relatively evenly across populations. We independently conducted GWA when treating ITCZ MIC as a quantitative trait and a binary trait and identified two SNPs with strong associations that were identified in both analyses. These SNPs fell within the coding regions of Afu2g02220 and Afu2g02140. We functionally validated Afu2g02220 by knocking it out using a CRISPR/Cas-9 approach, because orthologs of this gene are involved in sterol modification and ITCZ targets the ergosterol pathway. Knockout strains displayed no difference in growth compared to the parent strain in minimal media, yet a minor but consistent inhibition of growth in the presence of 0.15 ug/ml ITCZ. Our results suggest that GWA paired with efficient gene deletion is a powerful and unbiased strategy for identifying the genetic basis of complex traits in A. fumigatus.ImportanceAspergillus fumigatus is a pathogenic mold that can infect and kill individuals with compromised immune systems. The azole class of drugs provide antifungal activity against A. fumigatus infections and have become an essential treatment strategy. Unfortunately, A. fumigatus azole resistance has recently emerged and rapidly risen in frequency making treatment more challenging. Our understanding of the molecular basis of azole sensitivity has been shaped mainly through candidate gene studies. Unbiased approaches are necessary to understand the full repertoire of genes and genetic variants underlying azole resistance and sensitivity. Here, we provide the first application of genome-wide association analysis in A. fumigatus in the identification of a gene (Afu2g02220) that contributes to itraconazole susceptibility. Our approach, which combines association mapping and CRISPR/Cas-9 for functional validation of candidate genes, has broad application for investigating the genetic basis of complex traits in fungal systems.

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Genome-Wide Association for Itraconazole Sensitivity in Non-resistant Clinical Isolates of Aspergillus fumigatus

Frontiers in Fungal Biology ◽

10.3389/ffunb.2020.617338 ◽

2021 ◽

Vol 1 ◽

Author(s):

Shu Zhao ◽

Wenbo Ge ◽

Akira Watanabe ◽

Jarrod R. Fortwendel ◽

John G. Gibbons

Keyword(s):

Aspergillus Fumigatus ◽

Complex Traits ◽

Molecular Mechanisms ◽

Clinical Isolates ◽

Genome Wide Association ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Genome Wide ◽

Population Structure Analysis ◽

Recent Emergence

Aspergillus fumigatus is a potentially lethal opportunistic pathogen that infects over ~200,000 people and causes ~100,000 deaths per year globally. Treating A. fumigatus infections is particularly challenging because of the recent emergence of azole-resistance. The majority of studies focusing on the molecular mechanisms underlying azole resistance have examined azole-resistant isolates. However, isolates that are susceptible to azoles also display variation in their sensitivity, presenting a unique opportunity to identify genes contributing to azole sensitivity. Here, we used genome-wide association (GWA) analysis to identify loci involved in azole sensitivity by analyzing the association between 68,853 SNPs and itraconazole (ITCZ) minimum inhibitory concentration (MIC) in 76 clinical isolates of A. fumigatus from Japan. Population structure analysis suggests the presence of four distinct populations, with ITCZ MICs distributed relatively evenly across populations. We independently conducted GWA when treating ITCZ MIC as a quantitative trait and a binary trait, and identified two SNPs with strong associations in both analyses. These SNPs fell within the coding regions of Afu2g02220 and Afu2g02140. We functionally validated Afu2g02220 by knocking it out using a CRISPR/Cas9 approach, because orthologs of this gene are involved in sterol modification and ITCZ targets the ergosterol biosynthesis pathway. Knockout strains displayed no difference in growth compared to the parent strain in minimal media, yet a minor but consistent inhibition of growth in the presence of 0.15 μg/ml ITCZ. Our results suggest that GWA paired with efficient gene deletion is a powerful and unbiased strategy for identifying the genetic basis of complex traits in A. fumigatus.

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Aspergillus fumigatus Clinical Isolates Carrying CYP51A with TR34/L98H/S297T/F495I Substitutions Detected after Four-Year Retrospective Azole Resistance Screening in Brazil

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02059-19 ◽

2019 ◽

Vol 64 (3) ◽

Cited By ~ 1

Author(s):

Laís Pontes ◽

Caio Augusto Gualtieri Beraquet ◽

Teppei Arai ◽

Guilherme Leite Pigolli ◽

Luzia Lyra ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Tandem Repeat ◽

Antifungal Susceptibility ◽

Antifungal Resistance ◽

Clinical Isolates ◽

Azole Resistance ◽

Resistance Screening ◽

Environmental Isolates ◽

Content Type ◽

Susceptibility Tests

ABSTRACT Azole antifungal resistance in Aspergillus fumigatus is a worldwide concern. As in most public hospitals in Brazil, antifungal susceptibility tests are not routinely performed for filamentous fungi at our institution. A 4-year retrospective azole antifungal resistance screening revealed two azole-resistant A. fumigatus clinical isolates carrying the CYP51A TR34 (34-bp tandem repeat)/L98H (change of L to H at position 98)/S297T/F495I resistance mechanism mutations, obtained from two unrelated patients. Broth microdilution antifungal susceptibility testing showed high MICs for itraconazole, posaconazole, and miconazole. Short tandem repeat (STR) typing analysis presented high levels of similarity between these two isolates and clinical isolates with the same mutations reported from the Netherlands, Denmark, and China, as well as environmental isolates from Taiwan. Our findings might indicate that active searching for resistant A. fumigatus is necessary. They also represent a concern considering that our hospital provides tertiary care assistance to immunocompromised patients who may be exposed to resistant environmental isolates. We also serve patients who receive prophylactic antifungal therapy or treatment for invasive fungal infections for years. In these two situations, isolates resistant to the antifungal in use may be selected within the patients themselves. We do not know the potential of this azole-resistant A. fumigatus strain to spread throughout our country. In this scenario, the impact on the epidemiology and use of antifungal drugs will significantly alter patient care, as in other parts of the world. In summary, this finding is an important contribution to alert hospital laboratories conducting routine microbiological testing to perform azole resistance surveillance and antifungal susceptibility tests of A. fumigatus isolates causing infection or colonization in patients at high risk for systemic aspergillosis.

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