A total of 191 soil samples from Hangzhou, China, were submitted to detect non-wild-type (non-WT) Aspergillus fumigatus and its associated mechanisms. There were 2 (4.7%), 13 (12.4%), and 31 (23.1%) isolates identified as non-WT in 2014, 2016, and 2018, respectively. The resistant mutations of TR34/L98H, TR46/Y121F/T289A, and TR34/L98H/S297T/F495I were found in 3, 5, and 5 non-WT isolates. The G448S mutation, previously only found in clinical settings, was detected in A. fumigatus from soil samples.
KEYWORDS: Aspergillus fumigatus, environmental resistance, microsatellite typing, G448S
ABSTRACT
A total of 191 soil samples from Hangzhou, China, were submitted to detect non-wild-type (non-WT) Aspergillus fumigatus and its associated mechanisms. There were 2 (4.7%), 13 (12.4%), and 31 (23.1%) isolates identified as non-WT in 2014, 2016, and 2018, respectively. The resistant mutations of TR34/L98H, TR46/Y121F/T289A, and TR34/L98H/S297T/F495I were found in 3, 5, and 5 non-WT isolates. The G448S mutation, previously only found in clinical settings, was detected in A. fumigatus from soil samples.
INTRODUCTION
Aspergillus fumigatus, a globally distributed opportunistic pathogen, is the main cause of invasive aspergillosis (IA), which causes 40% to 90% mortality in immunocompromised patients (1). Triazole drugs (itraconazole, voriconazole, posaconazole, and isavuconazole) are the only oral drugs approved for clinical treatment and prevention of IA. Unfortunately, azole-resistant A. fumigatus (ARAF) has been increasingly found in patients who had received long-term antifungal treatment, azole-naive patients, and the environment (2–7). The emergence of ARAF is the primary cause of the increased numbers of treatment failures.
The mechanism of azole resistance is primarily characterized by alterations in cyp51A, the gene coding for sterol 14α-demethylase, which plays an important role in sterol synthesis. G54, G138, G434, M220, H147, Y121, G448, and P216 are common hot spot mutations that have been detected in ARAF from the patients under long-term antifungal treatment (8, 9). TR34/L98H and TR46/Y121F/T289A are the two most frequently identified resistance mechanisms found in environmental A. fumigatus strains and azole-naive patients and may result from the agricultural use of azole fungicides (9–11). Upregulation in intracellular sterol 14α-demethylase concentration because of overexpression of cyp51A is involved in resistance to triazole drugs. In addition, a decrease in intracellular drug concentration because of the overexpression of efflux pump genes can also result in resistance (9).
Immunocompromised patients may be infected by inhalation of airborne ARAF spores. Therefore, understanding the developmental processes and evolution of ARAF in the environment is significant for the treatment of patients with aspergillosis. Two resistant A. fumigatus soil isolates harboring mutations of TR34/L98H/S297T/F495I were identified in Beijing and Fuzhou, respectively (12). Ren et al. (6) reported the emergence of ARAF with the mutations TR46/Y121F/T289A and TR34/L98H/S297T/F495I in agricultural fields. We report here a 5-year survey concerning resistance in environmental A. fumigatus from Hangzhou in China.
A total of 191 soil samples were collected at a depth of 0 to 5 cm from the garden belt of eight hospitals during the 2014 to 2018 period from Hangzhou, Zhejiang, China (data not shown). A. fumigatus isolates were identified according to their microscopic and macroscopic morphology, by their capability to grow at 48°C, and by the internal transcribed spacer ribosomal DNA and β-tubulin gene (6, 13). Susceptibility testing of A. fumigatus against triazole medicines (voriconazole [VRC], itraconazole [ITZ], and posaconazole [POC]) was conducted according to the CLSI M38-A2 method (14). Isolates with a MIC of >1 mg/liter for ITZ and VRC and MIC of >0.25 mg/liter for POC were regarded as non-wild-type (non-WT) (15). To explore the underlying mutations of isolated non-WT A. fumigatus, the cyp51A and promoter regions were amplified and sequenced according to the method proposed by Ren et al. (6). For the non-WT strain without mutation, the mRNA expression levels of cyp51 (cyp51A and cyp51B) and two transporters (AfuMDR3 and AfuMDR4) were assessed according to the methods described by Cao et al. (16). The results were analyzed according to the 2−△△CT method (17). Additionally, genotyping of non-WT A. fumigatus isolates was realized by microsatellite typing using a panel of nine short tandem repeats (STR) as described previously (18). To determine the genotypic relationship between environmental non-WT A. fumigatus and the clinical isolates, a total of 29 non-WT A. fumigatus isolates were used. These data originated from China (environmental, n = 2; clinical, n = 15) (12, 19–21), Netherlands (environmental, n = 1; clinical, n = 6) (19, 22), Colombia (environmental, n = 1) (10), India (environmental, n = 3) (19), and Tanzania (environmental, n = 1) (19).
From 2014 to 2018, a total of 282 A. fumigatus isolates were recovered from 191 environment samples collected from gardens around eight hospitals in Hangzhou, China (Table 1 and data not shown). The prevalence rates of A. fumigatus were 45.8%, 64.7%, and 62.5% in environmental samples collected in 2014, 2016, and 2018, respectively. This was consistent with previous reports showing a range of 35 to 77.8% (6, 23, 24). Out of the obtained 282 isolates, 46 strains were determined as non-WT A. fumigatus (Table 2). The isolate rates of non-WT A. fumigatus were 4.7%, 12.4%, and 23.1% in 2014, 2016, and 2018, respectively. These results show that the proportion of non-WT A. fumigatus in soils around hospitals in Hangzhou seemly increased during the 5 years.
TABLE 1.
The frequency of isolation of total and non-wild-type A. fumigatus during 2014 to 2018a
| PS | TS | SCDAF | Rate of SCDAF/TS (%) | NIAF | NINAF | Rate of NINAF/NIAF (%) |
|---|---|---|---|---|---|---|
| Jun–Sep 2014 | 59 | 27 | 45.8 | 43 | 2 | 4.7 |
| Mar–May 2016 | 68 | 44 | 64.7 | 105 | 13 | 12.4 |
| Mar–May 2018 | 64 | 40 | 62.5 | 134 | 31 | 23.1 |
PS, period of sampling; TS, total samples; SCDAF, samples containing detectable A. fumigatus; NIAF, numbers of isolated A. fumigatus; NINAF, numbers of isolated non-wild-type A. fumigatus.
TABLE 2.
The MICs and mutations of non-wild-type A. fumigatus isolates from Chinaa
| Strain no. | Date of sample collection (day/mo/yr) | Location of sampling sites | MIC (mg/liter) |
cyp51A mutations | ||
|---|---|---|---|---|---|---|
| VRC | ITZ | POC | ||||
| H-13 | 18/06/2014 | ZPPH | 1 | 16 | 0.5 | TR34/L98H/S297T/F495I |
| H-20 | 12/07/2014 | HCH | 1 | 8 | 0.125 | TR34/L98H/S297T/F495I |
| H-58 | 02/03/2016 | ZPPH | 16 | 4 | 1 | TR46/Y121F/T289A |
| H-90 | 02/03/2016 | ZPPH | 2 | 0.5 | 0.125 | None |
| H-102 | 02/03/2016 | ZPPH | 2 | 0.5 | 0.25 | None |
| H-197 | 02/03/2016 | ZPPH | 2 | 0.125 | 0.0625 | None |
| H-85 | 02/03/2016 | AHFPH | 8 | 4 | 0.5 | G448S |
| H-96 | 02/03/2016 | AHFPH | 4 | 0.5 | 0.125 | G448S |
| H-98 | 02/03/2016 | AHFPH | 8 | 2 | 1 | G448S |
| H-103 | 02/03/2016 | AHFPH | 8 | 2 | 0.5 | G448S |
| H-107 | 02/03/2016 | AHFPH | 8 | 2 | 0.5 | G448S |
| H-111 | 02/03/2016 | AHFPH | 8 | 2 | 0.5 | G448S |
| H-114 | 27/03/2016 | ZPTCM | 8 | 2 | 0.5 | G448S |
| H-120 | 27/03/2016 | ZPTCM | 8 | 1 | 0.5 | G448S |
| H-126 | 27/03/2016 | ZPTCM | 8 | 2 | 0.5 | G448S |
| H-149 | 04/03/2018 | ZPPH | 2 | 0.5 | 0.0625 | None |
| H-152 | 04/03/2018 | ZPPH | 2 | 0.5 | 0.0625 | None |
| H-161 | 04/03/2018 | ZPPH | 2 | 0.5 | 0.0625 | None |
| H-190 | 04/03/2018 | ZPPH | >16 | 16 | 0.25 | TR46/Y121F/T289A |
| H-202 | 04/03/2018 | ZPPH | 8 | >16 | 1 | TR34/L98H |
| H-247 | 04/03/2018 | ZPPH | 8 | 4 | 0.125 | F46Y/G89G/M172V/N248T/D255E/L358L/E427K/C454C |
| H-234 | 26/03/2018 | HCH | >16 | 1 | 1 | TR46/Y121F/T289A |
| H-237 | 26/03/2018 | HCH | 4 | 0.25 | 0.0625 | None |
| H-264 | 26/03/2018 | HCH | 4 | >16 | 1 | TR34/L98H/S297T/F495I |
| H-176 | 04/03/2018 | TFHZP | 2 | 0.25 | 0.0625 | None |
| H-184 | 04/03/2018 | TFHZP | 2 | 0.25 | 0.0625 | None |
| H-186 | 04/03/2018 | TFHZP | 2 | 0.125 | 0.0625 | None |
| H-191 | 04/03/2018 | TFHZP | 2 | 0.25 | 0.0625 | None |
| H-197 | 04/03/2018 | TFHZP | 2 | 0.125 | 0.0625 | None |
| H-206 | 04/03/2018 | TFHZP | 2 | 0.125 | 0.0625 | None |
| H-208 | 04/03/2018 | TFHZP | 4 | 0.5 | 0.0625 | G170G |
| H-248 | 04/03/2018 | AHFPH | 2 | 0.5 | 0.0625 | None |
| H-252 | 04/03/2018 | AHFPH | 2 | 0.25 | 0.0625 | None |
| H-256 | 04/03/2018 | AHFPH | 2 | 0.5 | 0.25 | None |
| H-258 | 04/03/2018 | AHFPH | 4 | 0.25 | 0.125 | None |
| H-218 | 04/03/2018 | AHFPH | 2 | 0.5 | 0.0625 | None |
| H-242 | 26/03/2018 | ZPTCM | >16 | 0.5 | 0.5 | TR46/Y121F/T289A |
| H-244 | 26/03/2018 | ZPTCM | >16 | 16 | 0.25 | TR46/Y121F/T289A |
| H-260 | 26/03/2018 | ZPTCM | 2 | 0.25 | 0.0625 | None |
| H-261 | 26/03/2018 | ZPTCM | 2 | 0.125 | 0.0625 | None |
| H-266 | 26/03/2018 | ZPTCM | 2 | 0.125 | 0.0625 | None |
| H-259 | 26/03/2018 | HTH | 4 | >16 | 0.5 | TR34/L98H |
| H-263 | 26/03/2018 | HTH | 4 | >16 | 1 | TR34/L98H |
| H-272 | 01/05/2018 | THZP | 2 | >16 | 1 | TR34/L98H/S297T/F495I |
| H-277 | 01/05/2018 | THZP | 2 | 0.5 | 0.0625 | None |
| H-280 | 01/05/2018 | THZP | 2 | >16 | 1 | TR34/L98H/S297T/F495I |
ZPPH, Zhejiang Provincial People’s Hospital; HCH, Hangzhou Children’s Hospital; AHFPH, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine; ZPTCM, Zhejiang Provincial Hospital of TCM; HTH, Hangzhou Third Hospital; THZP, Tongde Hospital of Zhejiang Province; TFHZP, The First Hospital of Zhejiang Province.
Alterations in the cyp51A gene are usually the main causes of triazole resistance in A. fumigatus (9). There were six different mutations (Table 2 and data not shown) found in the isolated strains of non-WT A. fumigatus. The multiazole resistance mutations of TR34/L98H and TR46/Y121F/T289A were detected in 3 and 5 isolates, respectively. TR34/L98H and TR46/Y121F/T289A were first reported in Dutch patients and have been frequently found in azole-naive patients, clinics, and environmental samples worldwide (25). There is increasing evidence linking the occurrence and spread of these two resistance mechanisms to the use of triazole fungicides in agriculture (6, 26, 27). TR34/L98H/S297T/F495I, a widely reported mutation in China (12, 28), was found in 5 strains. The point mutations G448S and G170G and the mutations of F46Y/G89G/M172V/N248T/D255E/L358L/E427K/C454C were observed in 9, 1, and 1 strains, respectively.
In non-WT strains without mutations in cyp51A, the resistance may result from the decrease in intracellular toxin accumulation or an increase of target enzyme content. The apparent upregulation of AfuMDR3 and AfuMDR4 genes in the non-mutant, non-WT strains induced by ITZ was reported by Nascimento et al. (29). Recently, Cui et al. (30) reported the overexpression of AtrF, AfuMDR1, cyp51A, and cyp51B genes in non-WT strains HI-30 and HI-36 treated with tebuconazole. The expression of cyp51A, cyp51B, AfuMDR3, and AfuMDR4 in strains H-237 and H-258 was assessed by quantitative reverse transcription PCR, and the results are listed in Table 3. The expression of cyp51A and AfuMDR4 genes in H-237 were 4.12- and 3.12-fold greater, respectively, than those in the sensitive strains. Compared to the controls, 5.55- and 2.51- fold expression of cyp51A and AfuMDR3, respectively, was detected in strain H-258. These results imply that the overexpression of cyp51A and efflux pumps are responsible for the resistance in H-237 and H-258 to triazole drugs.
TABLE 3.
The expression levels of cyp51 and efflux transporter genes of nonmutant strains (H-237 and H-258) and susceptible strain (HT)
| Strain | Relative mRNA gene expression levela |
|||
|---|---|---|---|---|
| cyp51A | cyp51B | AfuMDR3 | AfuMDR4 | |
| HT | 1.00 (0.73–1.19) | 1.00 (0.81–1.22) | 1.00 (0.68–1.22) | 1.00 (0.80–1.16) |
| H-237 | 4.12 (3.53–5.12) | 1.53 (1.27–1.70) | 0.81 (0.66–1.10) | 3.12 (2.45–3.32) |
| H-258 | 5.55 (4.81–6.61) | 1.10 (1.05–1.33) | 2.51 (2.07–3.12) | 0.63 (0.61–0.77) |
HT values are averages from 5 randomly selected wild-type A. fumigatus isolates.
STR genotyping has been widely used to study the genetic similarity of A. fumigatus strains due to its reproducibility and high resolution (12, 18). The genotyping data showed a large genotypic diversity in TR34/L98H (S297T/F495I), TR46/Y121F/T289A, and no-mutation genotypes, whereas most of the G448S mutations were closely clustered with similarity observed at 6 to 9 of the nine loci studied (Fig. 1). Analysis of the genetic similarity between the present environmental isolates and Chinese clinical strains (n = 14) with TR34/L98H (S297T/F495I) showed that the genotypes of all environmental TR34/L98H (S297T/F495I) isolates were distinct from those of the clinical TR34/L98H (S297T/F495I) strains. The genotypes of TR46/Y121F/T289A from environmental and clinical isolates showed two major clusters. The clade in the upper part of the phylogenetic tree mainly contained TR46/Y121F/T289A genotypes from China (environmental isolates, n = 5; clinical isolates, n = 1) and other Eurasian countries, the Netherlands (environmental isolates, n = 1), and Colombia (environmental isolates, n = 1). The clade in the lower part of the tree reveals a single microsatellite cluster and was mainly composed of TR46/Y121F/T289A genotypes from the Netherlands (clinical isolates, n = 6), Tanzania (environmental isolates, n = 1), and India (environmental isolates, n = 3). No genetic similarity was found in F46Y/G89G/M172V/N248T/D255E/L358L/E427K/C454C and G170G genotypes. Strikingly, non-WT strains containing the resistance-related mutation of G448S isolated from different hospitals in China had a nearly identical genotype. Although G448S has not been isolated from a clinic, its spread is likely to increase the azole resistance risks to immunocompromised patients. Recently, there is increasing evidence the concerning development of resistance in A. fumigatus through its sexual cycle (31–33). The sexual crossing experiments conducted by Camps et al. (32) demonstrated that TR34/L98H strains could outcross with azole-susceptible isolates of diverse genetic backgrounds. Zhang et al. (34) found that the new mutation of TR463/Y121F/T289A could arise via sexual crossing between isolates with TR46/Y121F/T289A. Sexual reproduction leads to new microsatellite genotypes. Considering the diverse STR types among non-WT isolates in this study, sexual reproduction may be involved in the development and spread of resistant A. fumigatus.
FIG 1.
Genotypic relationships of selected non-wild-type A. fumigatus isolates from environmental samples with non-wild-type strains from China (environmental resistant, n = 2; clinical resistant, n = 15), the Netherlands (environmental resistant, n = 1; clinical resistant, n = 6), Colombia (environmental resistant, n = 1), India (environmental resistant, n = 3), and Tanzania (environmental resistant, n = 1). E, environmental; C, clinical. The dendrogram is based on a categorical analysis of STR repeat numbers in combination with unweighted pair group method using average linkages clustering.
In conclusion, this study revealed an increasing incidence of non-WT A. fumigatus in Hangzhou over a 5-year survey. The G448S mutation, previously reported only in clinical settings, was recovered in environmental isolates. The high prevalence (>20%) of non-WT A. fumigatus in hospital environments suggests that susceptible patients will inhale spores, resulting in azole-resistant aspergillosis. These results demonstrate that regular resistance surveillance of A. fumigatus is necessary to obtain sufficient helpful data to avoid clinical failures.
Data availability.
cyp51A sequences were deposited in the NCBI database under GenBank accession numbers MT418853–MT418878.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (no. 21777141, 41977342, and 21477112).
We have no competing interests to declare.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
cyp51A sequences were deposited in the NCBI database under GenBank accession numbers MT418853–MT418878.