10.6092/issn.2531-7342/12759
Aspergillosis is a fungal disease caused by different Aspergillus species and have high morbidity/mortality rates. Aspergillus fumigatus is the main aetiological agent related to most of Aspergillus infections. The use of triazoles is recommended as first line of treatment, however, the reports of azole-resistant A. fumigatus due to mutations in CYP51 have increased around the world. The goals were to determine the antifungal susceptibility patterns for triazoles in thirty A. fumigatus isolates from clinical and environmental samples and to detect mutation M220 in the resistant ones. Determination of minimum inhibitory concentrations of every isolate was performed by using the Etest commercial method for itraconazole, voriconazole and posaconazole. For the resistant isolates, a PCR was performed for amplifying a 173 bp fragment of cyp51A and the resulting amplicons were sequenced. Nine out of 30 isolates were resistant to itraconazole and none were resistant to voriconazole or posaconazole. The alignment of sequences with a CYP51 A. fumigatus wild type strain did not show any modifications at codon 220. Other molecular mechanisms of resistance are probably related to the resistant phenotypes isolated.
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Screening for M220 mutation in azole-resistant <em>Aspergillus fumigatus</em> isolates from clinical and environmental specimens in Cuba
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Verweij PE, Lestrade PPA, Melchers WJG, Meis JF (2016) Azole resistance surveillance in Aspergillus fumigatus: beneficial or biased? Journal of Antimicrobial Chemotherapy 71(8):2079-2082. https://doi.org/10.1093/jac/dkw259
Screening for M220 mutation in azole-resistant <em>Aspergillus fumigatus</em> isolates from clinical and environmental specimens in Cuba
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 50 (2021): 44-51
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Short note

                 Screening for M220 mutation in azole-resistant
                 Aspergillus fumigatus isolates from clinical and
                       environmental specimens in Cuba

                   Javier L. San Juan-Galán*, Carlos M. Fernández-Andreu,
                   Gerardo F. Martínez-Machín, Mayda R. Perurena-Lancha,
                     María T. Illnait-Zaragozí, Rosario E. Velar-Martínez
Department of Bacteriology-Mycology, Institute of Tropical Medicine “Pedro Kourí”, autopista “Novia del Mediodía”,
km 61/2, La Lisa, La Habana

* Corresponding author e-mail: jlsanjuan91@gmail.com


Article info
Received 22/4/2021; accepted 2/6/2021
https://doi.org/10.6092/issn.2531-7342/12759

Abstract
Aspergillosis is a fungal disease caused by different Aspergillus species and have high morbidity/mortality rates.
Aspergillus fumigatus is the main aetiological agent related to most of Aspergillus infections. The use of triazoles is
recommended as first line of treatment, however, the reports of azole-resistant A. fumigatus due to mutations in CYP51
have increased around the world. The goals were to determine the antifungal susceptibility patterns for triazoles in
thirty A. fumigatus isolates from clinical and environmental samples and to detect mutation M220 in the resistant ones.
Determination of minimum inhibitory concentrations of every isolate was performed by using the Etest commercial
method for itraconazole, voriconazole and posaconazole. For the resistant isolates, a PCR was performed for amplifying
a 173 bp fragment of cyp51A and the resulting amplicons were sequenced. Nine out of 30 isolates were resistant to
itraconazole and none were resistant to voriconazole or posaconazole. The alignment of sequences with a CYP51 A.
fumigatus wild type strain did not show any modifications at codon 220. Other molecular mechanisms of resistance are
probably related to the resistant phenotypes isolated.

Keywords
Triazoles, Etest, drug resistance, CYP51A, sequencing


Introduction
According to the data provided by the Leading International Fungal Education (LIFE), aspergillosis
has a significant impact on human health, with a global figure of 200,000 patients at risk of developing
an invasive form of aspergillosis and three million people worldwide to be affected by chronic
pulmonary aspergillosis (Brown et al., 2012; Anonymous, 2017). Despite there are a few Aspergillus
species that have been reported as cause of mycosis, A. fumigatus Fresen. is still the most relevant
species, related to 90% of all aspergillosis cases (Bonifaz, 2015). The management of this disease
comprehend the use of echinocandins, polyenes and triazoles within the therapy (Jenks et al., 2018).
This last group of antifungals is defined as the first line of treatment, however, in the last decades the
reports of A. fumigatus resistant isolates have increased and the resistance issue has evolved from
itraconazole resistance to panfungal resistance in just a few years (Sweileh et al., 2017). Several
researchers state that triazoles resistance in Aspergillus is not only related to intrinsic characteristics

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2021 The Authors. This work is licensed under CC BY 4.0
San Juan-Galán et al.                                           Italian Journal of Mycology 50 (2021): 44-51

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of strains but also to the deep effect of selective pressure caused by demethylase inhibitors usually
used in agriculture and gardens to protect crops from plagues (Fisher et al., 2018).
   The pharmacological target of triazoles is the 14-α-sterol demethylase, which is involved in
ergosterol biosynthesis pathway and this enzyme belongs to the p450 cytochrome superfamily,
expressed by the cyp51 gene (Prasad et al., 2016). Aminoacidic substitution on the backbone of this
protein or modifications in the promotor region of the cyp51A allele can develop triazoles resistance in
A. fumigatus. Changes at codon 220 are usually related to a diminished susceptibility to posaconazole
and voriconazole and also drive to itraconazole resistance in the species (Rivero-Menendez et al.,
2016). Although this polymorphism is frequently identified in A. fumigatus isolated from patients
under azole therapy some researchers has described it at environmental screenings (Sanglard, 2016).
The impact of aspergillosis in Cuba remains unknown due to the lack of epidemiological data
and diagnostics methods. Few investigations suggest that the real burden of aspergillosis might
be underestimated in the country for which there should be a surveillance program for detection
of resistant A. fumigatus isolates (Beltrán et al., 2019). Although previous researches reported the
presence of itraconazole resistant phenotypes distributed in the country, there is no evidence of which
molecular mechanisms are involved (San Juan-Galán et al., 2017a). For that matter, the main goals of
this work were to determine the susceptibility patterns of A. fumigatus isolates against itraconazole,
voriconazole and posaconazole and to identify the M220 mutation in the resistant isolates.

Materials and methods
Isolates
Thirty A. fumigatus isolates preserved in the Fungal Pathogens Culture Collection (Institute of Tropical
Medicine “Pedro Kourí”, Cuba) were evaluated within the study (11 from clinical respiratory samples
and 19 from environmental screenings). All isolates were cultured in malt yeast extract agar and
Czapek yeast extract agar and incubated at 25 ºC and 50 ºC for 7 days to confirm correct identification
(Samson et al., 2007, 2014). Macro/micro-morphological features were checked as part of the identity
confirmation. A. fumigatus wild strain ATCC® MYA-3627 was used as reference strain for every test
and was processed using the same methodology as the isolates.

Susceptibility testing
For the determination of itraconazole, voriconazole and posaconazole minimum inhibitory
concentration (MIC), Etest (Biomeriéux, France) was performed. Conidial suspensions were
adjusted to approximately 106 CFU mL-1 using a Neubauer chamber. For the test, each suspension
was inoculated with a swab onto the surface of RPMI-1640 agar supplemented with MOPS
(morpholinepropanesulfonic acid) and 2% glucose (Sigma-Aldrich, Germany). Plates were incubated
at 35 ºC for 48 hours (BioMérieux).
    The MIC was defined as the lowest concentration of antifungal drug for which the tangent in the
elliptical zone of growth inhibition cuts the Etest strip perpendicularly. To establish susceptibility
patterns (wild-type and resistant-type) for itraconazole, voriconazole and posaconazole, the
epidemiological cut-off values used were 1.0 µg mL-1, 1.0 µg mL-1 and 0.5 µg mL-1 respectively.

DNA extraction
Every triazole-resistant isolate was cultured in Sabouraud-dextrose broth with agitation at 37 ºC for
5 days. Subsequently each culture was filtrated and 200 mg (wet weight) of mycelium was taken for
DNA extraction. Mycelium was washed two times with EDTA (0.5 M), shaked with 20-30 crystal
beads (0.30 mm) for 10 minutes and then incubated at 37 ºC for two and a half hours in 400 µL of

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spheroplasts buffer (Sorbitol, 1.0 M; EDTA, 0.1 M; Lyticase, 1.5 mg mL-1; ditiothreitol, 0.05 M).
After this pre-treatment QIAGEN UCP Pathogen MiniKit (QIAGEN Group, Germany) was used for
DNA extraction.

Amplification and sequencying of cyp51A
The PCR protocol followed for cyp51A amplification was the one described by Spiess et
al. (2012) with modifications. Briefly, for a total volume reaction of 50 µL there was added
5 µL of target DNA (~100 ng A. fumigatus DNA). Final concentration of 1X for TaqPol Buffer
with 1.5 mM MgCl2, 0.2 mM of dNTPs, 2.5 U of HotStar Taq Polymerase and 0.2 µM of
forward CypA-M220-fw (5’-GCCAGGAAGTTCGTTCCAA-3’) and reverse CypA-M220-rv
(5’-CTGATTGATGATGTCAACGTA-3’) primers. PCR was performed in a thermocycler (BIOER
Technology, China) using the following program conditions: denaturation for 2 minutes at 94 ºC;
followed by 40 cycles of denaturation at 94 ºC for 45 seconds, annealing at 52 ºC for 1 minute and
extension at 72 ºC for 1 minute; at the end, a final extension step at 72 ºC for 10 minutes. Amplicons
were checked in a 2% ethidium bromide agarose gel.
   For sequencying, PCR products were purified using QIAquick PCR Purification kit (QIAGEN
Group, Germany). Sanger sequencing was performed with CEQ DTCS QuickStart commercial kit
(Beckman Coulter, USA) for a final volume reaction of 20 µL. Amplification was performed in a
thermocycler (BIOER Technology, China) with the following protocol: 35 cycles of denaturation at
94 ºC for 20 seconds, annealing at 52 ºC for 20 seconds and extension at 72 ºC for 4 minutes. The
sequencer used was GenomeLab GeXP (Beckman Coulter, USA).

Sequence analysis
Sequences were edited using MEGA X ver. 10.0.5. For alignment of sequences and detection of
M220, a BLASTx ver. 2.9.0+ was performed. The reference sequence used for comparison was
Af293, an A. fumigatus wild-type 14-α-sterol demethylase (Accession Number: XP752137) available
in the GenBank database (http://www.ncbi.nlm.nih.gov/).

Statistical analysis
Antifungal susceptibility data was processed for descriptive analysis using GraphPad Prism software
ver. 6.01 (La Jolla, CA, United States). Also, a Kruskal-Wallis test was performed for determine
variations between the medians. Statistical significance was set at P < 0.05.

Results
From the A. fumigatus isolates evaluated, nine out of 30 were resistant to itraconazole and none were
resistant to voriconazole or posaconazole. Statistics from the descriptive analyses are presented in
Table 1. MIC values for each of the isolates in evaluation against all three azoles tested are presented
in Figure 1. Results from the Kruskal-Wallis showed a P value < 0.0001, confirming the differences
between the medians of the MIC values for the three tested triazoles. All PCR products from the
A. fumigatus itraconazole-resistant isolates were consistent with the cyp51A region selected for the
study.
   The BLASTx analysis performed did not show any evidence of modifications of methionine at
codon 220 neither at any other codon contained in the same 173 bp region.




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      Table 1 - Descriptive statistics of MICs data for A. fumigatus isolates evaluated against itraconazole,
      voriconazole and posaconazole.
        Isolation source                                          MIC50              MIC90          Geometric
                              Antifungal         Range           (µg/mL)            (µg/mL)           mean
         (No. of strains)
           Total (30)            ITZ            0.38–2.0           0.63                1.5             0.70
                                 VCZ           0.064–0.38          0.13               0.25             0.15
                                 PCZ           0.016–0.19          0.094              0.13             0.083


          Clinical (11)          ITZ            0.38–2.0            1.0               1.90             0.91
                                 VCZ           0.094–0.25          0.19               0.25             0.16
                                 PCZ           0.047–0.19          0.094              0.19             0.095


      Environmental (19)         ITZ            0.38–1.5           0.50                1.5             0.60
                                 VCZ           0.064–0.38          0.13               0.25             0.14
                                 PCZ           0.016–0.13          0.094              0.13             0.076




   Fig 1 - MIC values distribution for itraconazole (ITZ), voriconazole (VCZ) and posaconazole (PCZ) in Aspergillus
   fumigatus isolates. Epidemiological cut-off values used were 1.0 µg mL-1, 1.0 µg mL-1 and 0.5 µg mL-1 for
   itraconazole, voriconazole and posaconazole respectively. Isolates AF7 to AF17 are from clinical samples and the
   rest of isolates are from environmental screenings.




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Discussion
In the last decade the reports on azole-resistant A. fumigatus research have increased exponentially
while new molecular mechanisms of resistance to these antifungals have come into sight. This fact
has become a world-wide subject, however, there are few studies of antifungal susceptibility in
A. fumigatus in Latin America and lesser in the Caribbean region (Gonçalves, 2016). Cuba has a
broad fungi diversity and Aspergillus genus is vastly represented all across its capital city, Havana
(Almaguer et al., 2013). Previous studies performed by researchers from the Institute of Tropical
Medicine “Pedro Kourí” stated the presence of azole-resistant phenotypes in A. fumigatus and other
species isolated from environmental and clinical specimens (San Juan-Galán et al., 2017a, b).
   In the present study, the number of itraconazole-resistant A. fumigatus isolates (30%) was similar to
reports from other countries. A susceptibility study performed by Badiee et al. (2012) at the University
of Medical Sciences in Shiraz, Iran, showed MIC50 and MIC90 values for itraconazole of 0.75 and 1.00
µg mL-1, respectively during the evaluation of thirty A. fumigatus isolates. In Netherlands, a group of
researchers found a 37% (155/420) of azole resistant A. fumigatus isolates and a high prevalence of
mutations on cyp51 gene within a screening on clinical samples (Buil et al., 2018).
   In Cuba, the clinical triazole available for the treatment of aspergillosis is itraconazole (Morales
Ojeda, 2018). This might be related to the absence of resistant phenotypes to voriconazole or
posaconazole because of the lack of selective pressure. Also commercialization and development
of these latter drugs has been limited for longer than for itraconazole, under patent conditions and
quality controls required by international regulatory entities, thus restraining their employment in
therapy (Maertens, 2004). On the other hand, these triazoles have other alternative pharmacological
targets that reinforce their antifungal effect, as it is the case of voriconazole which inhibits different
enzymes from the ergosterol biosynthesis pathway besides the 14-α sterol demethylase (Chandrasekar
and Manavathu, 2009).
   The M220 mutation comprises several different aminoacidic changes and emerges mostly in A.
fumigatus species isolated from patients with chronic infections who are under long-terms azole
therapy. Furthermore, it is associated to itraconazole resistance and wide-ranging susceptibility to
voriconazole and posaconazole (Arikan-Akdagli et al., 2018). In this work, six out of nine resistant
isolates came from clinical samples, specifically from patients with pulmonary infections caused by
A. fumigatus, reason why it was decided to search for M220 instead of other mutations like TR34/
L98H with higher frequencies, especially in environmental isolates.
   Although there is no evidence of M220 in the present study, the isolation of itraconazole resistant
phenotypes implies the existence of molecular mechanisms linked to them. While cyp51 mutations
are the most common cause of reduced susceptibility against triazoles there are other causes with a
probable association to this issue such as the expression of MFS (Major Facilitators Superfamiliy)
and ABC (ATP-binding cassettes) transporters (Cannon et al., 2009). Both of them inhibit the effect
of antifungals by pumping the drug out from the cytoplasm, in the first case through an antiporter
ion H+ exchange and in the second case through an efflux pump ATP-dependent. Within the MFS,
Mfs56 have been described with an important role on triazole resistance and some authors suggests
that cluster 10 of antifungal/H+ antiporter transporters may contain other molecules with similar
functions (Costa et al., 2014). There is very few data on ABC transporters in A. fumigatus: cdr1B
gene expression has a proven connection to triazole resistance and ABC transporters like AtrF, AtrI
are suggested to be involved in antifungal resistance (Berger et al., 2017).
   There are only two conjectures about the causes for the detection of A. fumigatus resistant isolates:
i) different cyp51 mutations such as TR34/L98H and TR46/Y121F/T289A, which are related to


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antifungal overuse in the environment, are expressed in Cuban phenotypes; ii) resistant phenotypes
were naturally introduced in the country via airborne or by accidental importation through people,
animals, plants or traded goods.
   As a reference for the first outline, according to official documentation published by the Cuban
Health and Agriculture Ministries there are 11 different demethylation inhibitors approved and
distributed all across the country which also can lead to cross-resistance to clinical triazoles (Snelders
et al., 2012; Cuba, 2016). There is no certainty about which of these chemicals have been used in
the sampled areas or in what proportions they have been used, however this is a hypothesis that can
lead to a high selective pressure in the environment and, consequently, to emergence of resistant
phenotypes. Thus this approach should be explored.
   As for the second case, A. fumigatus is an anemophilous fungus that can be spread through large
distances, and spores can keep viability for long periods of time (Magyar et al., 2016). Cuba is an
archipelago located in the middle of the three regions that constitute the American Continent. For
species different from Aspergillus, such as Peronospora tabacina D.B. Adam, there are published
articles about its natural introduction in the western region of Cuba due to strong winds from
hurricanes in the South and Central American coasts (Lopetegui et al., 2006). Reports on antifungal
resistance linked to TR34/L98H in A. fumigatus isolates from United States and Colombia indicate
that resistant genotypes are circulating in the nearby area (Gonҫalves et al., 2016; Verweij et al.,
2016). We suggest that resistant phenotypes could have a natural and accidental influx favoured by
atmospheric phenomena like hurricanes, so often in the Mexican Gulf. Multi-locus sequence typing,
microsatellite analysis or other molecular tools for epidemiological studies are needed in order to
shed light on this statement.
   This work is the first step for future investigations on molecular epidemiology and resistance
distribution in Cuba. It also supports the inclusion of voriconazole and posaconazole to the national
treatment scheme for fungal infections. As a final conclusion, new mechanisms of resistance need to
be studied focusing on those related to environmental sources.

Acknowledgments
The authors thanks to Liset Pérez, MSc. and Yanet Pintos, MSc. for the laboratory assistance on the
sequencing procedures.

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