Hospital Environment as a Source of Azole-Resistant Aspergillus fumigatus Strains with TR34/L98H and G448S Cyp51A Mutations

Azole-resistant Aspergillus fumigatus is an emerging worldwide problem with increasing reports of therapy failure cases produced by resistant isolates. A case of azole-resistant A. fumigatus hospital colonization in a patient is reported here. Investigations of the hospital environment led to the recovery of A. fumigatus strains harboring the TR34/L98H and the G448S Cyp51A azole resistance mechanisms. Isolate genotyping showed that one strain from the environment was isogenic with the patient strains. These are the first environmental A. fumigatus azole resistant strains collected in a hospital in Spain; it supports the idea of the hospital environment as a source of dissemination and colonization/infection by azole resistant A. fumigatus in patients. The isolation of an azole-resistant strain from an azole-naïve patient is an interesting finding, suggesting that an effective analysis of clinical and environmental sources must be done to detect azole resistance in A. fumigatus. The emergence and spread of these resistance mechanisms in A. fumigatus is of major concern because it confers high resistance to voriconazole and is associated with treatment failure in patients with invasive aspergillosis.

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Multi-resistance to non-azole fungicides in Aspergillus fumigatus TR 34 /L98H azole resistant isolates.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00642-21 ◽

2021 ◽

Author(s):

I Gonzalez-Jimenez ◽

R Garcia-Rubio ◽

S Monzon ◽

J Lucio ◽

I Cuesta ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Plant Pathogens ◽

Crop Protection ◽

Resistance Mechanisms ◽

Azole Resistance ◽

Sequencing Analysis ◽

Human Fungal Pathogen ◽

Evolution Pattern ◽

Environmental Setting ◽

Resistant Strains

Drug resistance is a worldwide problem affecting all pathogens. The human fungal pathogen Aspergillus fumigatus coexists in the environment with other fungi targeted by crop protection compounds being unintentionally exposed to the selective pressure of multiple antifungal classes leading to the selection of resistant strains. A. fumigatus azole resistant isolates are emerging in both the clinical and environmental setting. Since their approval, azole drugs have dominated the clinical treatment for aspergillosis infections, and the agriculture fungicide market. However, other antifungal classes are used for crop protection including benzimidazoles (MBC), strobilurins (QoIs) and succinate dehydrogenase inhibitors (SDHIs). Mutations responsible for resistance to these fungicides have been widely researched in plant pathogens, but it has not been explored in A. fumigatus . In this work, the genetic basis underlying resistance to MBCs, QoIs and SDHIs were studied in azole susceptible and resistant A. fumigatus strains. E198A/Q and F200Y mutations in the β-tubulin conferred resistance to MBCs, G143A and F129L substitutions in the Cytochrome b to QoIs and H270R/Y mutations in SdhB to SDHIs. Characterization of the susceptibility to azoles showed a correlation between strains resistant to these fungicides and the ones with TR-based azole resistance mechanisms. Whole genome sequencing analysis showed a genetic relationship among fungicide multi resistant strains, which grouped together into subclusters that only included strains carrying the TR-based azole resistance mechanisms, indicating a common ancestor/evolution pattern and confirming the environmental origin of this type of azole resistant A. fumigatus .

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Epidemiology and Molecular Characterizations of Azole Resistance in Clinical and Environmental Aspergillus fumigatus Isolates from China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01005-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5878-5884 ◽

Cited By ~ 32

Author(s):

Yong Chen ◽

Zhongyi Lu ◽

Jingjun Zhao ◽

Ziying Zou ◽

Yanwen Gong ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Antifungal Susceptibility ◽

Public Health Problem ◽

Wide Distribution ◽

Resistance Mechanisms ◽

Azole Resistance ◽

Common Mutation ◽

Content Type ◽

Environmental Resistance ◽

Resistant Strains

ABSTRACTAzole resistance inAspergillus fumigatushas emerged as a worldwide public health problem. We sought here to demonstrate the occurrence and characteristics of azole resistance inA. fumigatusfrom different parts of China. A total of 317 clinical and 144 environmentalA. fumigatusisolates from 12 provinces were collected and subjected to screening for azole resistance. Antifungal susceptibility,cyp51Agene sequencing, and genotyping were carried out for all suspected azole-resistant isolates and a subset of azole-susceptible isolates. As a result, 8 (2.5%) clinical and 2 (1.4%) environmentalA. fumigatusisolates were identified as azole resistant. Five azole-resistant strains exhibit the TR34/L98H mutation, whereas four carry the TR34/L98H/S297T/F495I mutation in thecyp51Agene. Genetic typing and phylogenetic analysis showed that there was a worldwide clonal expansion of the TR34/L98H isolates, while the TR34/L98H/S297T/F495I isolates from China harbored a distinct genetic background with resistant isolates from other countries. High polymorphisms existed in thecyp51Agene that produced amino acid changes among azole-susceptibleA. fumigatusisolates, with N248K being the most common mutation. These data suggest that the wide distribution of azole-resistantA. fumigatusmight be attributed to the environmental resistance mechanisms in China.

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Are Point Mutations in HMG-CoA Reductases (Hmg1 and Hmg2) a Step towards Azole Resistance in Aspergillus fumigatus?

Molecules ◽

10.3390/molecules26195975 ◽

2021 ◽

Vol 26 (19) ◽

pp. 5975

Author(s):

Irene Gonzalez-Jimenez ◽

Jose Lucio ◽

Alejandra Roldan ◽

Laura Alcazar-Fuoli ◽

Emilia Mellado

Keyword(s):

Aspergillus Fumigatus ◽

Structural Changes ◽

Survival Rates ◽

Point Mutations ◽

Resistance Mechanisms ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Recent Emergence ◽

Resistant Strains

Invasive aspergillosis, mainly caused by Aspergillus fumigatus, can lead to severe clinical outcomes in immunocompromised individuals. Antifungal treatment, based on the use of azoles, is crucial to increase survival rates. However, the recent emergence of azole-resistant A. fumigatus isolates is affecting the efficacy of the clinical therapy and lowering the success rate of azole strategies against aspergillosis. Azole resistance mechanisms described to date are mainly associated with mutations in the azole target gene cyp51A that entail structural changes in Cyp51A or overexpression of the gene. However, strains lacking cyp51A modifications but resistant to clinical azoles have recently been detected. Some genes have been proposed as new players in azole resistance. In this study, the gene hmg1, recently related to azole resistance, and its paralogue hmg2 were studied in a collection of fifteen azole-resistant strains without cyp51A modifications. Both genes encode HMG-CoA reductases and are involved in the ergosterol biosynthesis. Several mutations located in the sterol sensing domain (SSD) of Hmg1 (D242Y, G307D/S, P309L, K319Q, Y368H, F390L and I412T) and Hmg2 (I235S, V303A, I312S, I360F and V397C) were detected. The role of these mutations in conferring azole resistance is discussed in this work.

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Azole resistance among clinical isolates of Aspergillus fumigatus in Lima-Peru

Medical Mycology ◽

10.1093/mmy/myz032 ◽

2019 ◽

Vol 58 (1) ◽

pp. 54-60 ◽

Cited By ~ 1

Author(s):

Beatriz Bustamante ◽

Luis Ricardo Illescas ◽

Andrés Posadas ◽

Pablo E Campos

Keyword(s):

Aspergillus Fumigatus ◽

Latin American ◽

Pcr Amplification ◽

Sensu Stricto ◽

Clinical Isolates ◽

Azole Resistance ◽

Molecular Testing ◽

Naive Patient ◽

In Vitro Susceptibility

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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Differences in Interactions between Azole Drugs Related to Modifications in the 14-α Sterol Demethylase Gene (cyp51A) of Aspergillus fumigatus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.5.2119-2121.2005 ◽

2005 ◽

Vol 49 (5) ◽

pp. 2119-2121 ◽

Cited By ~ 23

Author(s):

G. Garcia-Effron ◽

E. Mellado ◽

A. Gomez-Lopez ◽

L. Alcazar-Fuoli ◽

M. Cuenca-Estrella ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Gene Knockout ◽

Resistance Mechanisms ◽

Knockout Strain ◽

Resistant Strains ◽

Gene Knockouts

ABSTRACT The combined activity of different azole drugs was investigated. Thirty-one Aspergillus fumigatus strains were tested, including two cyp51A − and one cyp51B − gene-knockout strain and azole-susceptible and -resistant strains with different resistance mechanisms. The combination of itraconazole and voriconazole was synergistic for all strains except for those with gene knockouts.

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First Clinical Isolation of an Azole-Resistant Aspergillus fumigatus Isolate Harboring a TR46 Y121F T289A Mutation in South America

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00872-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 8

Author(s):

Guillermina Isla ◽

Florencia Leonardelli ◽

Iris N. Tiraboschi ◽

Nicolás Refojo ◽

Alejandra Hevia ◽

...

Keyword(s):

United States ◽

South America ◽

Aspergillus Fumigatus ◽

Resistance Mechanism ◽

Resistance Mechanisms ◽

Azole Resistance ◽

Content Type

ABSTRACT One of the most recently described Aspergillus fumigatus CYP51A-mediated azole resistance mechanisms is TR46 Y121F T289A. Clinical A. fumigatus strains harboring these substitutions have been reported worldwide, with the exception of South America. We describe the first clinical A. fumigatus strain with this resistance mechanism isolated from an Argentinian patient. The strain was isolated in 2009 (1 year after the first-described mutant in United States), demonstrating that these alleles were scattered worldwide earlier than previously thought.

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Emerging aspergillosis by azole-resistant Aspergillus fumigatus at an intensive care unit in the Netherlands, 2010 to 2013

Eurosurveillance ◽

10.2807/1560-7917.es.2016.21.30.30300 ◽

2016 ◽

Vol 21 (30) ◽

Cited By ~ 30

Author(s):

Judith van Paassen ◽

Anne Russcher ◽

Astrid WM in 't Veld - van Wingerden ◽

Paul E Verweij ◽

Eduard J Kuijper

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

The Netherlands ◽

Aspergillus Fumigatus ◽

Resistance Mechanisms ◽

Antifungal Treatment ◽

University Hospital ◽

Resistant Isolate ◽

Azole Resistance ◽

Icu Patients

The prevalence of invasive aspergillosis (IA) at the intensive care unit (ICU) is unknown and difficult to assess since IA also develops in patients lacking specific host factors. In the Netherlands, increasing azole-resistance in Aspergillus fumigatus complicates treatment of patients with IA. The aim of this study was to determine the prevalence of IA by azole-resistant A. fumigatus at the ICU among patients receiving antifungal treatment and to follow their clinical outcome and prognosis. A retrospective cohort study was conducted in a university hospital ICU from January 2010 to December 2013. From all patients who received antifungal treatment for suspected IA, relevant clinical and microbiological data were collected using a standardised questionnaire. Of 9,121 admitted ICU-patients, 136 had received antifungal treatment for suspected IA, of which 38 had a positive A. fumigatus culture. Ten of the 38 patients harboured at least one azole-resistant isolate. Resistance mechanisms consisted of alterations in the cyp51A gene, more specific TR34/L98H and TR46/T289A/Y121F. Microsatellite typing did not show clonal relatedness, though isolates from two patients were genetically related. The overall 90-day mortality of patients with IA by azole-resistant A. fumigatus and patients with suspicion of IA by azole-susceptible isolates in the ICU was 100% (10/10) vs 82% (23/28) respectively. We conclude that the changing pattern of IA in ICU patients requires appropriate criteria for recognition, diagnosis and rapid resistance tests. The increase in azole resistance rates also challenges a reconsideration of empirical antifungal therapy.

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Genotypic Evolution of Azole Resistance Mechanisms in Sequential Candida albicans Isolates

Eukaryotic Cell ◽

10.1128/ec.00151-07 ◽

2007 ◽

Vol 6 (10) ◽

pp. 1889-1904 ◽

Cited By ~ 207

Author(s):

Alix Coste ◽

Anna Selmecki ◽

Anja Forche ◽

Dorothée Diogo ◽

Marie-Elisabeth Bougnoux ◽

...

Keyword(s):

Candida Albicans ◽

Resistance Genes ◽

Resistance Mechanisms ◽

High Expression ◽

Azole Resistance ◽

Polymorphism Analysis ◽

Comparative Genome Hybridization ◽

Single Nucleotide ◽

Copy Numbers ◽

Resistant Strains

ABSTRACT TAC1 (for transcriptional activator of CDR genes) is critical for the upregulation of the ABC transporters CDR1 and CDR2, which mediate azole resistance in Candida albicans. While a wild-type TAC1 allele drives high expression of CDR1/2 in response to inducers, we showed previously that TAC1 can be hyperactive by a gain-of-function (GOF) point mutation responsible for constitutive high expression of CDR1/2. High azole resistance levels are achieved when C. albicans carries hyperactive alleles only as a consequence of loss of heterozygosity (LOH) at the TAC1 locus on chromosome 5 (Chr 5), which is linked to the mating-type-like (MTL) locus. Both are located on the Chr 5 left arm along with ERG11 (target of azoles). In this work, five groups of related isolates containing azole-susceptible and -resistant strains were analyzed for the TAC1 and ERG11 alleles and for Chr 5 alterations. While recovered ERG11 alleles contained known mutations, 17 new TAC1 alleles were isolated, including 7 hyperactive alleles with five separate new GOF mutations. Single-nucleotide-polymorphism analysis of Chr 5 revealed that azole-resistant strains acquired TAC1 hyperactive alleles and, in most cases, ERG11 mutant alleles by LOH events not systematically including the MTL locus. TAC1 LOH resulted from mitotic recombination of the left arm of Chr 5, gene conversion within the TAC1 locus, or the loss and reduplication of the entire Chr 5. In one case, two independent TAC1 hyperactive alleles were acquired. Comparative genome hybridization and karyotype analysis revealed the presence of isochromosome 5L [i(5L)] in two azole-resistant strains. i(5L) leads to increased copy numbers of azole resistance genes present on the left arm of Chr 5, among them TAC1 and ERG11. Our work shows that azole resistance was due not only to the presence of specific mutations in azole resistance genes (at least ERG11 and TAC1) but also to their increase in copy number by LOH and to the addition of extra Chr 5 copies. With the combination of these different modifications, sophisticated genotypes were obtained. The development of azole resistance in C. albicans is therefore a powerful instrument for generating genetic diversity.

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The Strength of Synergistic Interaction between Posaconazole and Caspofungin Depends on the Underlying Azole Resistance Mechanism of Aspergillus fumigatus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04469-14 ◽

2015 ◽

Vol 59 (3) ◽

pp. 1738-1744 ◽

Cited By ~ 15

Author(s):

Eleftheria Mavridou ◽

Joseph Meletiadis ◽

Antony Rijs ◽

Johan W. Mouton ◽

Paul E. Verweij

Keyword(s):

Aspergillus Fumigatus ◽

Resistance Mechanism ◽

Fungal Growth ◽

Resistance Mechanisms ◽

Azole Resistance ◽

Wild Type ◽

Content Type ◽

Synergistic Interactions ◽

Drug Concentrations

ABSTRACTThe majority of azole resistance mechanisms inAspergillus fumigatuscorrespond to mutations in thecyp51Agene. As azoles are less effective against infections caused by multiply azole-resistantA. fumigatusisolates, new therapeutic options are warranted for treating these infections. We therefore investigated thein vitrocombination of posaconazole (POSA) and caspofungin (CAS) against 20 wild-type and resistantA. fumigatusisolates with 10 different resistance mechanisms. Fungal growth was assessed with the XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] method. Pharmacodynamic interactions were assessed with the fractional inhibitory concentration (FIC) index (FICi) on the basis of 10% (FICi-0), 25% (FICi-1), or 53 0% (FICi-2) growth, and FICs were correlated with POSA and CAS concentrations. Synergy and antagonism were concluded when the FICi values were statistically significantly (ttest,P< 0.05) lower than 1 and higher than 1.25, respectively. Significant synergy was found for all isolates with mean FICi-0 values ranging from 0.28 to 0.75 (median, 0.46). Stronger synergistic interactions were found with FICi-1 (median, 0.18; range, 0.07 to 0.47) and FICi-2 (0.31; 0.07 to 0.6). The FICi-2 values of isolates with tandem-repeat-containing mutations or codon M220 were lower than those seen with the other isolates (P< 0.01). FIC-2 values were inversely correlated with POSA MICs (rs= −0.52,P= 0.0006) and linearly with the ratio of drug concentrations in combination over the MIC of POSA (rs= 0.76,P< 0.0001) and CAS (rs= 0.52,P= 0.0004). The synergistic effect of the combination of POSA and CAS (POSA/CAS) againstA. fumigatusisolates depended on the underlying azole resistance mechanism. Moreover, the drug combination synergy was found to be increased against isolates with elevated POSA MICs compared to wild-type isolates.

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