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.


Introduction
Infections caused by the saprotrophic mold Aspergillus fumigatus and other Aspergillus spp. are due to the inhalation of conidia, which are present in the environment [1]. Daily, up to 200 conidia per person are inhaled, causing a wide spectrum of clinical affectations depending on the immunological status of the host [2]. In healthy immunocompetent individuals, the immune system is able to clean conidia from the lungs;however, immunosuppressed patients have a high predisposition to develop clinical manifestations associated with a worst outcome [1].
Clinical manifestations caused by A. fumigatus are encompassed under the name of aspergillosis, ranging from minor pathologies up to more severe forms, such as invasive pulmonary aspergillosis (IPA), with mortalityrates reaching 95% in immunocompromised hosts [3]. IPAand other forms of aspergillosis are currently being treated with a class of antifungal compounds named azoles [1,4]. Among azoles, the triazoles voriconazole, itraconazole, posaconazole, and isavuconazole are the most widely used drugs for both aspergillosis treatment andprophylaxis [5].
Lately, as is happening with a wide range of microorganisms worldwide, clinical isolates of A. fumigatus resistant to azoles are being acknowledged, and the prevalence is gradually increasing [6,7] involving a major concern due to its association with treatment failure in patients with IPA [8]. To date, A. fumigatus azole resistance is mostly associated with modifications of the azole target site, the enzyme Cyp51A encoded by the gene cyp51A, and its overexpression [9,10]. Triazole resistance can evolve during azole therapy in the clinical setting, but resistant isolates are also being detected in azole-naïve patients, suggesting an environmental origin of some resistance mechanisms [11].
So far, reports of modifications in cyp51A, or its expression, associated with azole resistance in A. fumigatus isolates can be distinguished in two categories. On the one hand, point mutations in the coding sequence of the gene involving amino acid changes in the protein (G54, P216, M220, G138, G448), and on the other hand tandem repeat insertions in the promoter region of the gene combined, or not, with point mutations in the coding sequence (TR34/L98H, TR53, TR34/R65K/L98H and TR46/Y121F/T289A) [10,12].Point mutations have usually been described in clinical isolates after long-term azole therapies while tandem repeat resistance mechanisms are more often isolated from environmental samples or azole-naïve patients [10].
Among all azole resistance mechanisms described to date, the most frequent is the TR34/L98H, associated with a multi-azole resistance phenotype to all clinical azoles. This mechanism was initially isolated in Europe, although it has spread worldwide since then [10,13,14]. Its isolation is normally linked to the use of azole fungicides to protect harvests by preventing crop damage [14] and, apart from the environment, strains harboring the TR34/L98H resistance mechanism have also been isolated from individuals, most of them azole-naïve patients that were originally infected by a strain carrying this mutation [13,15,16].
Here we report, for the first time, the isolation of environmental A. fumigatus azole resistantisolates in Spain. The azole resistance mechanismsof the isolates wereTR34/L98H and G448S inCyp51A. Moreover, the concomitant isolation of A. fumigatus azole resistant isogenic strains from a hospitalized patient and thehospital environment make the study more interesting.Whether the patient had a hospitalstrain acquisition or was the source of hospital contamination is discussed.

Aspergillus fumigatus Strains
Inthis study, a total offifteen A. fumigatus strains were analyzed, ten clinical and five environmental isolates.Strainsidentification was confirmed by amplification and sequencing of the ITS1-5.8S-ITS2 rDNA regions and a portion of β-tubulin gene [35].

Case Report and Environmental Search
In January 2019, a patient was admitted to the hospital with dyspnea, cough, and bronchial secretions. The patient had a background of hypertension, pneumoconiosis, and COPD. After ten days in the hospital, A. fumigatus was isolated in a sputum (15 January 2019) and no other pathogens were found in the sample. The patient had no obvious clinical signs of invasive aspergillosis, and this isolation was considered a colonization following the revised EORTC/MSG criteria [36]. Several colonies were analyzed (1003, 1003E, 1003E.2, 1004, 1004E, 1004E.2, 1005.1, 1005.2, 1005.3, and 1005.4). The calcofluor stain and lateral flow test were positive alerting the presence of Aspergillus species, and a quantitative real timePCR confirmed the identification of A. fumigatus. Two indoor environmental searches (23 January, 2019 and 5 February, 2019) of the patient hospital room and bathroom yielded A. fumigatus. On the first air sampling study 3 CFU/m 3 fungal isolates were obtained and 4 CFU/m 3 on the second. Five isolates in total were analyzed (TP1, TP2, TP3, TP4, and TP5). Volumetric air samples were obtained using a volumetric sampler (Merck Air Sampler MAS100) as previously described [37].
The full coding sequence of cyp51A including its promoter was amplified and sequenced. To exclude the possibility that any change identified in the sequences was due to PCR-induced errors, each isolate was independently analyzed twice. PCR reaction mixtures contained 0.5 µM of each primer, 0.2 µMofdeoxynucleoside triphosphate (Roche, Madrid, Spain), 5µLof PCR 10x buffer, 2 mMofMgCl 2 , DMSO 5.2%, 2.5 U of Taq DNA polymerase (Applied Biosystems, Foster City, CA, USA), and 100-200 ng of DNA in a final volume of 50µL. A DNA 1-kb molecular ladder (Promega, Madrid, Spain) was used for all electrophoresis analyses. Samples were amplified in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The parameters used were 1 cycle of 5 min at 94 • C and then 35 cycles of 30 s at 94 • C, 45 s at 56 • C for cyp51A promoter and 58 • C for cyp51A gene, and 2 min at 72 • C, followed by a 1 final cycle of 5 min at 72 • C. The amplified products were purified using IllustraExoProStar 1-step (GE Healthcare Life Science, Buckinghamshire, UK) and both strands were sequenced with the Big-Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) following manufacturer's instructions. All gene sequences were edited and assembled using Lasergene software package (DNAStar Inc., Madison, WI, USA). Primers used to amplify and sequence cyp51A and its promoter have been previously described [39].

Strains Genotyping
All of the strains included in this study were genotyped following the previously described typing method TRESPERG [40]. Four markers were used: (i) Afu2g05150 encoding an MP-2 antigenic galactomannan protein (MP2); (ii) Afu6g14090 encoding a hypothetical protein with a CFEM domain (CFEM); (iii) Afu3g08990 encoding a cell surface protein A (CSP) and (iv) Afu1g07140 (ERG), which encodes a putative C-24(28) sterol reductase. The combination of the genotypes obtained with each marker has a discriminatory value (D) of 0.9972 using the Simpson index.

Clinical Antifungal Drugs Susceptibility Testing
Antifungal susceptibility testing (AFST) was performed following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution reference method 9.3.1 [41]. Antifungals used were amphotericin B (Sigma-Aldrich Química, Madrid, Spain) and the azoles itraconazole (Janssen Pharmaceutica, Madrid, Spain), voriconazole (Pfizer SA, Madrid, Spain), posaconazole (Schering-Plough Research Institute, Kenilworth, NJ, USA) and isavuconazole (BasileaPharmaceutica, Basel, Switzerland (tested from January 2017)). The final concentrations tested ranged from 0.03 to 16 mg/L for amphotericin B and 0.015 to 8 mg/L for the four azoles. A. flavus ATCC 204304 and A. fumigatus ATCC 204305 were used as quality control strains in all tests performed. Minimal inhibitory concentrations (MICs) were visually read after 24 and 48 h of incubation at 37 • C in a humid atmosphere. MICs were performed at least twice for each isolate. Clinical breakpoints for interpreting AFST results established by EUCAST [42] were used for classifying the A. fumigatus strains as susceptible or resistant.

Amplification and Sequence Analysis of cyp51A
Amplification and sequencing of cyp51A including its promoter revealed two azole resistance mechanisms present in most (14/15) of the A. fumigatus strainsincluded in this study ( Table 1). The first one consistingof a 34-bp tandem repeat insertion in the promoter region of cyp51A together with a L98H substitution in the coding sequence of the gene (TR34/L98H) that was present in all clinical samples and one environmental strain (TP3). The second one was a G448S substitution in cyp51A, which was harbored by three environmental samples (TP1, TP2, and TP4). Strain TP5 had no cyp51A promoter or the coding sequencemodifications (Table 1).

Antifungal Susceptibility Testing
AFST to clinical azoles showed azole resistant MIC values in all strains tested except for the TP5 environmental strain which showed an azole-susceptible profile with ranges between 0.06 and 1 mg/L for all azoles tested. MIC values for amphotericin B were considered susceptible for all fifteen isolates. Two different azole resistance profiles were identified among all azole resistant isolates. All clinical strains and the TP3 environmental isolate had the same susceptibility profile consisting of>8 mg/L to itraconazole, 4 mg/L to voriconazole, 0.5 mg/L to posaconazole and 8 mg/L to isavuconazole. The other three environmental isolates (TP1, TP2, and TP4) had a MIC profile of 1 mg/L to itraconazole, 8 mg/mL to voriconazole, 0.25-0.5 mg/L to posaconazole and 4 mg/L to isavuconazole (Table 1).

Discussion
Azole-resistant A. fumigatus isolates from clinical and environmental origin are being acknowledged worldwide [10,14]. Resistant strainsharbordifferent resistance mechanisms that confer particular susceptibility profiles to antifungal drugs used in the clinical setting and also in the environment [16,43]. To date, the most prevalent azole resistance mechanism is the TR34/L98H combination in Cyp51A, the target for azole drugs, which has been mostly described from environmental isolates conferring multi-azole resistance [10]. In clinical samples the substitution G448S is frequently associated withvoriconazole and isavuconazoleresistance [19][20][21][22][23]34].
In this study, we isolated environmental azole-resistant samples of A. fumigatus for the first time in Spain. These strains were obtained from the environment of a hospital patient's room, identifying two different resistance mechanisms (TR34/L98H and G448S) with two different genotypes. Out of the five samples obtained from the patient's room four were azole-resistant and, out of these four, three harbored the resistance mechanism G448S and were isogenic.Theremaining strain had the resistance mechanism TR34/L98H and a different genotype. This confirms that two different azole-resistant A. fumigatus strains were isolated from the hospital room environment. The two azole resistance profiles found in this study are in agreement with studies previously performed for strains harboring the same Cyp51A resistance mechanisms [10,13,23].
Previous studies in Spain have analyzed A. fumigatus strains from clinical samples and, to date, only a few environmental samples have been studied [44,45]. Although no previous azole-resistant samples have been identified in the Spanish environment, environmental samples harboring the same TR34/L98H Cyp51A alteration have been previously collected in other geographic regions being the most common resistance mechanism found worldwide [15]. This resistance mechanism has been detected in many European countries (Germany, Denmark, France, The Netherlands, Italy, Ireland, UK, and Switzerland), Asia (China, India, Iran, Japan, North Korea, Thailand, and Taiwan), Africa (Tanzania), and America (Colombia, USA) [10].
The most remarkable result of this study is that all clinical strains obtained from the patient and the TP3 environmental sample obtained from the patient's bathroom were isogenic, had the same MIC profile and Cyp51A resistance mechanism (TR34/L98H). Thissuggests that the patient had a hospital environmental acquisition of the strain, given that the houses and hospital environment can be contaminated by A. fumigatus azoleresistant strains [46,47]. Alternatively, the spread from patient to environment isa possibilityand this theory has been recently proposed by other authors [48,49]. A study performed in 2019 [49] was able to recover A. fumigatus from cough aerosolsof colonized patients with cystic fibrosis isogenic to those A. fumigatus obtained from the sputum of the same patient, suggesting environmental contamination through aerosols. Moreover, the case of a hospital patient acting as a source of A. fumigatus contamination of a hospital room environment after being infected in the same hospital, but different room has been reported recently [48].Other case reports from the beginning of the 2000s have described situations in which patients diagnosed with IPAhad isogenic strains with those isolated from the ICUs where they were hospitalized [50,51]. These studies bring to light the possibility of conidia being released through aerosols produced by aspergillosis patients, contaminating the air and causing patient-to-patient infection. Whether the patient of this studybecame colonized at the hospital by an A. fumigatus multi-azole resistant strain present in the environment, or if the patient was the source of an environmental contamination needs to be further investigated in order to elucidate the relation between these isogenic isolates.
Environmental strains harboring the point mutation G448S were also isolated in this study. This substitution is one of the most reported Cyp51A azole resistance mechanisms in patients treated with voriconazole (VCZ) [21,22,34,[52][53][54].To date, the G448Smutation has been mainly reportedfrom the clinical setting all over the world including Europe, Japan, USA, and Australia [10,55].However, recent reports of environmental strains harboring this substitution are being acknowledged (Table 2), and a mutation believed to be unique to the clinical setting is now also observed in the environment in Spain and other geographical regions, suggesting a possible environmental origin which is starting to be proposed by some authors [56]. The selection of this G448S substitution in response to antifungal pressure has been reported in combination with a TR46/Y121F/M172I/T298A azole resistance mechanism [30,31] and also, the same substitution at the corresponding position has been detected in Cyp51As proteins from plant pathogen fungi that were exposed to azole drugs used in agriculture, conferring resistance to them [10]. All of these data suggest that the origin of the G448S mutation needs to be clarified since the clinical origin previously proposed is no longer so certain. In fact, the G448S mutations could have a dual selection, emerging under VCZ pressure in clinical settings or under triazole drugs used for crop protection.

Conclusions
In conclusion, our study demonstrates that azole resistant A. fumigatus are present in the hospital environment. Research on an A. fumigatus colonized patient room environment showed, for the first time in Spain, the isolation of A. fumigatus azole resistant strains, with two different genotypes and different resistance mechanisms (TR34/L98H and G448S). Strains bearing the azole resistance mechanism TR34/L98H, environmental or clinical, were isogenic. This interesting finding suggests that an effective analysis of environmental sources needs to be done in order to detect azole resistant A. fumigatus. Whether the patient had a hospital strain acquisition or was the source of hospital contamination is being investigated. If azole resistance spreads through cough aerosols from patient to patient, the spread from patient to environment is also a possibility. In addition, the isolation of