Secondary metabolite profiles and antifungal drug susceptibility of Aspergillus fumigatus and closely related species, Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans

J Infect Chemother 21 (2015) 385e391

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Original article

Secondary metabolite profiles and antifungal drug susceptibility of Aspergillus fumigatus and closely related species, Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans Hiroyuki Tamiya a, b, c, *, Eri Ochiai a, 1, Kazuyo Kikuchi d, Maki Yahiro a, 1, Takahito Toyotome a, 1, Akira Watanabe a, c, e, 1, Takashi Yaguchi d, Katsuhiko Kamei a, c, e, 1 a

Division of Fungal Infection, Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan Division of Clinical Research, Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8677, Japan d Division of Bio-resources, Medical Mycology Research Center, Chiba University, Chiba, Japan e Division of Control and Treatment of Infectious Diseases, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba 260-8677, Japan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 August 2014 Received in revised form 5 January 2015 Accepted 13 January 2015 Available online 23 January 2015

The incidence of Aspergillus infection has been increasing in the past few years. Also, new Aspergillus fumigatus-related species, namely Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans, were shown to infect humans. These fungi exhibit marked morphological similarities to A. fumigatus, albeit with different clinical courses and antifungal drug susceptibilities. The present study used liquid chromatography/time-of-flight mass spectrometry to identify the secondary metabolites secreted as virulence factors by these Aspergillus species and compared their antifungal susceptibility. The metabolite profiles varied widely among A. fumigatus, A. lentulus, A. udagawae, and A. viridinutans, producing 27, 13, 8, and 11 substances, respectively. Among the mycotoxins, fumifungin, fumiquinazoline A/B and D, fumitremorgin B, gliotoxin, sphingofungins, pseurotins, and verruculogen were only found in A. fumigatus, whereas auranthine was only found in A. lentulus. The amount of gliotoxin, one of the most abundant mycotoxins in A. fumigatus, was negligible in these related species. In addition, they had decreased susceptibility to antifungal agents such as itraconazole and voriconazole, even though metabolites that were shared in the isolates showing higher minimum inhibitory concentrations than epidemiological cutoff values were not detected. These strikingly different secondary metabolite profiles may lead to the development of more discriminative identification protocols for such closely related Aspergillus species as well as improved treatment outcomes. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Keywords: Aspergillus fumigatus Aspergillus lentulus Aspergillus udagawae Aspergillus viridinutans Secondary metabolites Liquid chromatography/time-of-flight mass spectrometry

1. Introduction Aspergillus fumigatus is the most prevalent causal organism of invasive aspergillosis (IA) [1]. However, recently, closely related species in the section Fumigati, including Aspergillus lentulus, Neosartorya udagawae (anamorph, Aspergillus udagawae), and

* Corresponding author. Division of Fungal Infection, Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan. Tel.: þ81 043 222 7171; fax: þ81 043 226 2486. E-mail address: [email protected] (H. Tamiya). 1 Tel.: þ81 043 222 7171; fax: þ81 043 226 2486.

Aspergillus viridinutans, have been shown to cause aspergillosis [2e6]. These fungi are morphologically similar to A. fumigatus, which is problematic because their identification in clinical isolates largely depends on macroscopic and microscopic morphology [3,7]. The related species are clinically relevant because they have a different pathogenicity, such as a longer disease duration and progressive spread across anatomical planes in a contiguous manner for A. udagawae and A. viridinutans [5,6,8]. They also exhibit lower susceptibility to some antifungal agents compared with A. fumigatus. For example, Vinh et al. [8] examined the susceptibility of four A. udagawae isolates and found a decreased susceptibility to amphotericin B (AMPH-B), itraconazole (ITCZ), and

http://dx.doi.org/10.1016/j.jiac.2015.01.005 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

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voriconazole (VRCZ). Alhambra et al. [9] reported that A. lentulus also tends to be resistant to the same drugs. However, available data are limited and are inconsistent among studies. Therefore, characterization of these pathogens is important for better management of aspergillosis. Currently, approximately 400 fungal mycotoxins have been identified [10], and most studies focused on food intoxication and its prevention [11]. Mycotoxin studies addressing human infection are essential because some of them were identified as virulence factors. For instance, gliotoxin, an epipolythiodioxopiperazine toxin, has been implicated in A. fumigatus-mediated infection [12], particularly under non-neutropenic conditions [13,14]. A. fumigatus is the most common species causing IA, and over 90% strains isolated from cases of IA in tertiary care cancer centers were gliotoxin producers [15]. In contrast, less frequent pathogenic Aspergillus species such as Aspergillus terreus, Aspergillus flavus, and Aspergillus niger also produce gliotoxin, but at a much lower level than A. fumigatus [15]. Such differences in mycotoxin production may be a key factor in understanding the diversity of pathogenicity among these fungi. Whereas mycotoxin production by A. fumigatus sensu stricto is well documented, only a few reports have described the mycotoxins secreted by A. fumigatus-related species [16e18], and the information available is insufficient to support a relationship between their toxin production and pathogenicity. The present study compared A. fumigatus and A. fumigatusrelated species (A. lentulus, A. udagawae, and A. viridinutans) in terms of drug susceptibility. Also, the bioactive secondary metabolites reported during patient screening were identified by liquid chromatography time-of-flight mass spectrometry (LC/TOF-MS), and gliotoxin content was measured by liquid chromatography tandem mass spectrometry (LC/MS/MS), which is a promising candidate of virulence factor of the fungi. 2. Materials and methods 2.1. Fungal strains, growth conditions and media A total of 92 Aspergillus isolates were used in this study (Table 1): 71 isolates (66 clinical, 5 environmental) of A. fumigatus sensu stricto (hereafter described as “A. fumigatus”) and 21 of A. fumigatus-related species (hereafter “related species”), including 8 A. lentulus isolates (2 clinical, 5 environmental, 1 unknown), 9 A. udagawae isolates (5 clinical, 3 environmental, 1 unknown), and 4 A. viridinutans isolates (clinical). All isolates were stored and maintained at the Medical Mycology Research Center (Chiba University), where they were characterized and morphologically identified. Gene analyses, including those of b-tubulin and calmodulin, were performed when the isolate did not grow at 48  C [19]. Each isolate was cultured on potato dextrose agar (Difco Laboratories, Detroit, MI, USA) slants (7e10 days, 25  C) for the secondary metabolite extraction study and for 5 days or until good sporulation was obtained at 35  C for the drug susceptibility study. The conidia were harvested by gentle agitation after adding sterile distilled water containing 0.05% Tween 20 for secondary metabolite analysis. The resulting suspension was filtered through four layers of gauze and 3G3 glass filter (Asahi Glass Corporation, Ltd, Japan) to remove hyphal contaminants and washed two times in the same solutions. 2.2. Chloroform extraction protocol for culture filtrates Secondary metabolite production depends primarily on the culture conditions. Therefore, all isolates were cultured in RPMI 1640 medium containing L-glutamine and NaHCO3 (Sigma-Aldrich Japan) under oxygen-rich conditions to mimic the in vivo

environment. Secondary metabolites extraction was performed as described previously [20] with slight modification. The mycotoxins produced during mycelial growth were collected from 7.5  106 conidia added to 150 mL of RPMI 1640 medium in a 500-mL flask and incubated at 37  C in 5% CO2 by shaking at 140 rotations per min (rpm) for 24 h. Of the related species, 3 isolates (A. lentulus IFM 47457, A. udagawae IFM 58400, and A. viridinutans IFM 59503) were incubated for 24 h, 72 h, and 120 h in RPMI 1640 medium to determine whether longer cultivation time facilitate metabolite production or not since the related species grow more slowly than A. fumigatus. The suspension was filtered through a 0.22-mm filter (Millipore, Bedford, MA, USA). Then, each culture filtrate was mixed with chloroform (2:1 vol/vol) and shaken vigorously for 30 s. After continuous mixing for 20 min, the aqueous fraction was collected and added to chloroform (2:1 vol/vol). After repeating this extraction step twice, the chloroform fraction was collected and evaporated to dryness at 40  C. 2.3. Methanol extraction protocol for conidia The conidial suspension obtained from the filtration step of the chloroform extraction was centrifuged (2500 rpm, 15 min), and the supernatant was filtered through an HPF Millex 0.45-mm filter (Millipore, Bedford, MA, USA). Then, 3 mL of 80% methanol was added. After vortexing, the solution was incubated (25  C, 24 h) and centrifuged (2500 rpm, 15 min). The methanol fraction was collected and evaporated to dryness at 40  C. 2.4. Analytical conditions of liquid chromatography/time-of-flight/ mass spectrometry The secondary metabolites were isolated and identified by LC/ TOF-MS. Although more than 200 bioactive secondary metabolites of Aspergillus spp. have been reported, we targeted the toxic metabolites reported previously by patient screening [16]. Sample analysis was conducted by liquid chromatography on an Agilent 1200 system (Agilent Technologies, Japan). Chromatographic separation was conducted at 40  C on an Agilent Zorbax Extend C18 column (2.1 mm  100 mm, 1.8-mm inner diameter size). The mobile phase included 0.1% HCOOH and 10 mM HCOONH4 in acetonitrile. The sample volume was 5 mL, and the flow rate was 0.2 mL/ min. A linear gradient was initiated with 10% acetonitrile and increased to 82% over 40 min. The TOF-MS analysis was conducted with an Agilent 6230 TOF/ LC/MS system in the positive electrospray ionization (ESI) mode, and the mass-to-charge ratio was 100e1000. The ESI conditions were as follows: drying gas flow rate, 10 L/min; drying gas temperature, 350  C; nebulizer gas pressure, 345 kPa; fragmentor voltage, 120 V. 2.5. Analytical conditions of liquid chromatography/tandem mass spectrometry Gliotoxin production was analyzed in 21 isolates of A. fumigatus, 8 of A. lentulus, and 8 of A. udagawae using LC/MS/MS. The liquid chromatography step was conducted as described above for LC/ TOF-MS, except that the linear gradient was 10%e80% acetonitrile over 40 min. The LC/MS analysis used an Agilent 6460 triple quadrupole LC/MS system in the Agilent jet stream mode. The ESI conditions were as follows: drying gas flow rate, 10 L/min; drying gas temperature, 325  C; nebulizer gas pressure, 345 kPa; and fragmentor voltage, 120 V. Gliotoxin production was quantified by measuring areas, followed by extrapolation from a calibration curve constructed using standard solutions of gliotoxin (Sigma-Aldrich, Japan).

H. Tamiya et al. / J Infect Chemother 21 (2015) 385e391 Table 1 Isolates of fungal strain used in this study (continues). Scientific name

Isolate no.

Source (underlying condition in human case if available)

A. fumigatus

IFM 41362

Blood (systemic aspergillosis of neonatal infant), Japan Lung (IPA, AML), Japan Lung (PA, pulmonary tuberculosis), Japan Pleural effusion (human), Japan Lung (PA), Japan Unknown (PA), Japan Bronchial brushing (pulmonary tuberculosis), Japan Pleural effusion (gastric cancer, lung abscess), Japan Sputum (IA, AML), Japan Sputum (human), Japan Unknown (lung empyema), Japan Bulla fluid (infectious bulla), Japan Sputum (ABPA), Japan Bronchial alveolar lavage fluid (PA, ABPA), Japan Bronchial alveolar lavage fluid (spontaneous pneumothorax), Japan Sputum (CNPA suspected, pneumoconiosis), Japan Bronchial alveolar lavage fluid (ABPA), Japan Lung (PA), Japan Sputum (CPA suspected, squamous cell carcinoma of lung, COPD), Japan Pulmonary bulla fluid (CPA suspected, pulmonary tuberculosis), Japan Chest wall/pleura (empyema), Japan Bronchial alveolar lavage fluid/pus (bronchogenic cyst), Japan Intratracheal tube (extremely low birth weight infant), Japan Sputum (ABPA suspected), Japan Sputum/pus (aspergillus empyema, chronic granulomatous disease), Japan Sputum (IPA suspected, interstitial pneumonia), Japan Bronchial aspiration (pneumonia, diabetes mellitus, hyperosmolar nonketotic coma), Japan Sputum/bronchial lavage fluid (human), Japan Unknown (human), Japan Bronchial aspiration (ABPA suspected), Japan Sputum from bronchoscopy (human), Japan Sputum (human), Japan Sputum (bronchial asthma, bronchiectasis, diabetes mellitus), Japan Sputum (human), Japan Bulla fluid (CPA suspected, nontuberculous mycobacterial infection), Japan Unknown (systemic aspergillosis), Japan Lung (lung abscess), Japan Unknown (human) Unknown, Japan Unknown, Japan Unknown, Japan Unknown (human), Japan

IFM 41944 IFM 41945 IFM IFM IFM IFM

45915 46074 46075 46866

IFM 46894 IFM IFM IFM IFM IFM IFM

47439 47447 47448 47449 47450 47878

IFM 48051 IFM 49435 IFM 49824 IFM 49895 IFM 49896 IFM 50230 IFM 50886 IFM 50913 IFM 50916 IFM 50996 IFM 50999 IFM 51357 IFM 51505

IFM 51745 IFM 51746 IFM 51748 IFM 51941 IFM 51942 IFM 51977

IFM 51978 IFM 52108

IFM 53355 IFM 53543 IFM 53842 IFM 53869 IFM 53870 IFM 53872 IFM 54304 (KCH 2003012812) IFM 54364 IFM 54729 IFM 54771 IFM 54808

Sputum (renal transplantation, nocardiosis), Japan Unknown (IPA), Japan Lung (microscopic polyangiitis), Japan Lung (human), Japan

387

Table 1 (continued ) Scientific name

Isolate no.

Source (underlying condition in human case if available)

IFM 55369

Lung (PA, bronchial asthma, allergic rhinitis), Japan Blood, sputum (autoimmune hepatitis), Japan Unknown (CNPA), Japan Soil, China Soil, China Soil, China Soil, China Soil, China Lung (PA or CNPA, organizing pneumonia, bronchial asthma), Japan Unknown (pleuritis), Japan Sputum (IPA, rheumatoid arthritis, interstitial pneumonia), Japan Sputum (CPA, rheumatoid arthritis, interstitial pneumonia), Japan Sputum (CPA, rheumatoid arthritis, interstitial pneumonia), Japan Sputum (CNPA, rheumatoid arthritis, interstitial pneumonia), Japan Sputum (IPA, autoimmune hepatitis), Japan Sputum (IPA, lung cancer, old myocardial infarction, congestive heart failure), Japan Sputum (IPA, systemic sclerosis, fulminant hepatitis), Japan Sputum (CPA, bronchiectasis), Japan Sputum (CPA, bronchial asthma, pneumonia), Japan Sputum (CPA, pulmonary tuberculosis), Japan Sputum (CPA, interstitial pneumonia), Japan Sputum (IPA, liver transplantation), Japan Sputum (IPA, interstitial pneumonia), Japan Sputum (CPA, lung cancer), Japan Lung (PA), Japan Bronchial lavage fluid (human), Japan Soil, Venezuela Unknown, Japan Bronchial lavage fluid (human), USA Unknown (environment) Unknown (environment) Unknown (environment) Unknown (environment) Ocular bulb (ocular aspergillosis), Japan Bronchial lavage fluid (human), Japan Unknown (human), Japan Unknown (human), Japan Unknown (human), Japan

IFM 55548 IFM IFM IFM IFM IFM IFM IFM

58029 58236 58237 58238 58239 58240 58401

IFM 58816 IFM 58921 IFM 59056 IFM 59057 IFM 59073 IFM 59349 IFM 59354

IFM 59355 IFM 59358 IFM 59359 IFM 59361 IFM 59363 IFM 59364 IFM 59366

A. lentulus

A. udagawae

A. viridinutans

IFM 59369 IFM 59777 FH 5 IFM 41090 IFM 47063 IFM 47457 IFM 59077 IFM 59078 IFM 59079 IFM 59080 IFM 5058 IFM 51744 IFM 53867 IFM 53868 IFM 54302 (KCH 20020507107) IFM 58400 CBM FD 0143 CBM FA 0702 CBM FA 0703 IFM 54303 IFM 55266 IFM 59502 IFM 59503

Unknown, Japan Food, Japan Soil, Brazil Soil, Brazil Unknown (human), Japan Lung (human), Japan Unknown (human), Japan Unknown (human), Japan

IPA invasive pulmonary aspergillosis, AML acute myelogenous leukemia, PA pulmonary aspergilloma, IA invasive aspergillosis, ABPA allergic bronchopulmonary aspergillosis, COPD chronic obstructive pulmonary disease, CNPA chronic necrotizing pulmonary aspergillosis, CPA chronic pulmonary aspergillosis.

2.6. Drug susceptibility testing The broth microdilution technique was performed according to the CLSI M38-A2 guidelines [21] with a slight modification, in which 0.05% Tween 80 was used in place of 0.05% Tween 20 in

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some isolates. Microtiter plates (Dry Plate; Eiken Chemicals, Tokyo, Japan) were used according to the manufacturer's instruction, with slight modification [19]. The concentration of conidial suspension was adjusted to 1  104 conidia/mL in RPMI 1640 with a hemocytometer. The inoculum suspension (0.1 mL) was added to each microtiter well, and the inoculated microdilution trays were incubated at 35  C. Visual examination of growth inhibition using a reading mirror was performed at 48 h for azoles and AMPH-B. For micafungin, the minimal effective concentration (MEC) was determined at 24 h or on the first day of culture confluence in the control well. The MIC ranges were within the limits recommended by recommended by the CLSI M38-A2 guidelines with the use of reference strain A. flavus ATCC 204304 (data not shown). 2.7. Statistical analysis All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, California, USA). KruskaleWallis tests were used for multiple group comparisons of median gliotoxin concentrations. A p-value of <0.05 was considered statistically significant. 3. Results 3.1. Species-specific secondary metabolite profiles The secondary metabolites were identified by LC/TOF-MS screening (Online resource Table S1). Table 2 shows that 27 secondary metabolites were identified from A. fumigatus, while only 13, 8, and 11 were identified from A. lentulus, A. udagawae, and A. viridinutans, respectively. In the related species, conidia contained a wider variety of secondary metabolites compared with culture filtrates. Fumigaclavine C, methyl-sulochrin, pyripyropene A and E, and trypacidin were commonly detected in the all four species. In contrast, 19 toxins were exclusively detected in A. fumigatus: 9 in

conidial extract (fumiquinazoline A/B and D, gliotoxin, fumifungin, fumitremorgin B, pseurotin B, sphingofungin C and D, and verruculogen) and 15 in culture filtrate (cyclopiazonic acid, fumagillin, fumigaclavine B, fumiquinazoline A/B and D, fumitremorgin A and C, gliotoxin, helvolic acid, neosartorin, methyl-sulochrin, pseurotin A/D and B, trypacidin, and verruculogen). Only A. lentulus produced toxins that were not found in A. fumigatus or the other related species: auranthine in conidia and culture filtrate. Eight metabolites (fumifungin, fumitremorgin A and B, pseurotin A/D and B, sphingofungin C and D, and verruculogen) were exclusively detected in clinical isolates (n ¼ 74) compared with environmental isolates (n ¼ 13) (Online resource Table S2). Metabolite profile did not differ substantially when cultivated for a longer period: trypacidin at 72 h in A. lentulus culture filtrate was the only metabolite newly detected after 24 h (Online resource Table S3). 3.2. Gliotoxin content in A. fumigatus-related species In culture filtrate, the A. fumigatus isolates produced significantly higher amounts of gliotoxin (median, 23.48 mg/mL) compared with A. lentulus and A. udagawae (both, 0.01 mg/mL) isolates (A. fumigatus vs. A. lentulus, p < 0.001; A. fumigatus vs. A. udagawae, p < 0.01). On the other hand, conidial extract contained very small amounts of gliotoxin in all three species (median: A. fumigatus, 0.01 mg/mL; A. lentulus, 0.01 mg/mL; A. udagawae, 0.001 mg/mL). 3.3. Drug susceptibility testing of A. fumigatus-related species The MIC50 and MIC90 of antifungal drugs were determined for A. fumigatus and all three related species (Table 3) on the basis of MIC distributions. Two A. fumigatus isolates were excluded because of insufficient growth on microtiter plates (Online resource Table S4). Amphotericin B was effective against all fungi (MIC90: 1e2 mg/mL). In contrast, there was considerable interspecies variability in

Table 2 Numbers (%) of isolates producing secondary metabolites. Metabolite

Auranthine Cyclopiazonic acid Fumagillin Fumifungin Fumigaclavine A Fumigaclavine B Fumigaclavine C Fumiquinazoline A/B Fumiquinazoline D Fumiquinazoline F/G Fumitremorgin A Fumitremorgin B Fumitremorgin C Gliotoxin Helvolic acid Methyl-sulochrin Neosartorin Pseurotin A or D Pseurotin B Pyripyropene A Pyripyropene E Pyripyropene O Pyripyropene S Sphingofungin C Sphingofungin D Trypacidin Verruculogen e: Not detectable.

A. fumigatus (n ¼ 71)

A. lentulus (n ¼ 8)

A. udagawae (n ¼ 9)

A. viridinutans (n ¼ 4)

Conidia

Culture filtrate

Conidia

Culture filtrate

Conidia

Culture filtrate

Conidia

Culture filtrate

e e 62 (87) 2 (3) 70 (99) 42 (59) 69 (97) 61 (86) 68 (96) 66 (93) 14 (20) 10 (14) 65 (92) 2 (3) 59 (83) 57 (80) e e 4 (6) 70 (99) 39 (55) 51 (72) 67 (94) 2 (3) 9 (13) 36 (51) 1 (1)

e 1 (1) 55 (77) e 41 (58) 13 (18) 59 (83) 65 (92) 52 (73) 69 (97) 15 (21) e 60 (85) 61 (86) 21 (30) 24 (34) 1 (1) 37 (52) 3 (4) 58 (82) 27 (38) 38 (54) 56 (79) e e 22 (31) 16 (22)

7 (88) 6 (75) e e 4 (50) 2 (25) 3 (38) e e 4 (50) e e e e e 6 (75) 7 (88) e e 8 (100) 8 (100) 8 (100) 8 (100) e e 2 (25) e

6 (75) e e e 1 (13) e 2 (25) e e 6 (75) e e e e e e e e e e 3 (38) 1 (13) 3 (38) e e e e

e e e e 2 (22) e 3 (33) e e 1 (11) e e e e 7 (78) 4 (44) e e e 5 (55) 1 (11) e e e e 1 (11) e

e e e e e e e e e e e e e e e e e e e e e e e e e e e

e e 2 (50) e e e 1 (25) e e e 2 (50) e 2 (50) e 1 (25) 1 (25) e e e 2 (50) 2 (50) 2 (50) 2 (50) e e 1 (25) e

e e e e e e e e e e e e e e e e e e e 1 e e e e e e e

H. Tamiya et al. / J Infect Chemother 21 (2015) 385e391 Table 3 MIC50 and MIC90 (mg/ml) of Aspergillus fumigatus and the related species. Species (n)

A. A. A. A.

AMPH-B

a

fumigatus (69) lentulus (8) udagawae (9) viridinutans (4)

ITCZ

VRCZ

MIC50

MIC90

MIC50

MIC90

MIC50

MIC90

1 1 1 0.5

2 2 1 1

0.5 0.5 0.5 2

0.5 2 8 8

0.25 4 8 4

1 8 8 8

AMPH-B amphotericin B, ITCZ itraconazole, VRCZ voriconazole. a Two A. fumigatus isolates were excluded from this testing due to insufficient growth on microtiter plate.

susceptibility to azole antifungals. The three related species presented higher MICs to ITCZ, and VRCZ (MIC90: 2e16 mg/mL vs. 0.5e2.0 mg/mL). In all isolates, the MECs for micafungin were 0.015 mg/mL. Environmental isolates showed higher MICs to ITCZ (MIC90: 8 mg/mL vs. 1 mg/mL) and VRCZ (MIC90: 8 mg/mL vs. 2 mg/mL) than clinical isolates (Online resource Table S5). The susceptibility differences between A. fumigatus and the related species were even more evident when the epidemiological cut-off values (ECVs) for AMPH-B (2e4 mg/mL), ITCZ (1 mg/mL), and VRCZ (1 mg/mL) [22,23] were used as breakpoints. All A. fumigatus isolates were susceptible to all of these drugs. In contrast, the azoles generated MICs > ECVs for many related species isolates, which would be considered resistant: ITCZ, 2 (22%) A. udagawae isolates and all A. viridinutans isolates and VRCZ, 6 (66%) A. udagawae isolates, all A. lentulus isolates, and all A. viridinutans isolates. With regard to AMPH-B, all isolates of the four species would be considered susceptible. Interestingly, there was no secondary metabolite in common among all the resistant isolates of the related species. 4. Discussion The present study demonstrates that A. fumigatus and each related species possess different secondary metabolite profiles and antifungal susceptibility. A. fumigatus produces a wider variety of secondary metabolites compared with the related species. Furthermore, gliotoxin was abundant in the culture filtrate of most A. fumigatus isolates, whereas very low levels were detected in both culture filtrate and conidial extract of the related species. Most A. fumigatus isolates abundantly produced gliotoxin in culture filtrate, while a very few amount was detected both in conidial extract and culture filtrate of the related species. It is generally accepted that Aspergillus spp. secrete virulence factors that allow them to infect humans and that these factors are responsible for the variability in pathogenicity between Aspergillus spp. and other fungi [18,24]. Previous studies showed that Aspergillus-related species possessed features distinct from those of A. fumigatus in terms of drug susceptibility and disease course, although the mechanism remains largely elusive [5,6,8,9,17,25e27]. The differences in pathogenicity are likely to be caused, in part, by the different combinations of virulence factors among species. Mycotoxins are among these virulence factors, and many studies have shown that they contribute to the pathogenesis of aspergillosis [13,28e34]. However, analyses of the secondary metabolites secreted by the related species are limited [18,24]. Therefore, we conducted a detailed profile analysis of the secondary metabolites of A. fumigatus and the new closely related species responsible for misidentifications in clinical isolates, namely A. lentulus, A. udagawae, and A. viridinutans. The chemical composition of culture filtrate is expected to be more closely related to the invasion process compared with conidia during hyphae elongation into host tissues. Of the four species

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studied, 13 toxins were detected exclusively in culture filtrate of A. fumigatus, but did not in that of the others (Table 2). These mycotoxins are known to carry various biological activities [14,29,35e47] that may potentially work to the advantage of A. fumigatus by facilitating colonization, invasion, or sequestration from the host immune effector cells during tissue infection. In contrast, the present study shows that the related species are unable to produce significant amounts of gliotoxin, although Sugui et al. also detected gliotoxin production in A. udagawae using high performance liquid chromatography, as well as an amplicon with a sequence matching the gliP gene encoding the peptide synthase that catalyzes the first step in gliotoxin biosynthesis [17]. Therefore, it is of particular interest to determine how these A. fumigatus-related species cope with the host's defense systems without expressing so many toxins secreted by A. fumigatus. Further studies are required to elucidate the mechanism of their virulence. Larsen TO et al. [18] reported that A. fumigatus and A. lentulus do not share metabolites, except for pyripyropenes accumulating in conidia when they are cultured in the following agar media for a week at 25  C: Czapek yeast autolyzate agar, yeast extract sucrose agar, or yeast extract agar. However, the present study demonstrates that A. fumigatus and A. lentulus share pyripyropenes as well as fumifungin, methyl-sulochrin, trypacidin (conidia), fumigaclavines, and fumiquinazoline F/G (conidia and culture filtrate). Such discrepancy of metabolite profile may be explained by different culture conditions. In culture filtrate, A. lentulus produced auranthine, fumigaclavine A and C, fumiquinazoline F/G, and pyripyropene E, O, and S. A recent study showed that pyripyropene A, B, and D have anti-angiogenic and anti-proliferative effects on human umbilical vein endothelial cells [48]. The biological activities of pyripyropene E, O, and S have not been identified. However, if they have similar functions, they can affect pathogenicity by helping A. lentulus escape from the host immune cells. It remains unknown whether the other metabolites detected in A. lentulus culture filtrate are involved in the infection process. With regard to the other two A. fumigatus-related species, the production of secondary metablites was extremely low: only one secondary metabolite (pyripyropene A) was identified in culture filtrate of A. viridinutans, while none in that of A. udagawae. The most reasonable explanation for the small number of mycotoxins detected in these fungi is that they actually do not produce these mycotoxins, although they could be producing other metabolites not included in our screening panel. Drug susceptibility testing revealed that all three A. fumigatusrelated species have higher MICs to azoles compared with A. fumigatus, which is in agreement with previous reports [5,6,8,9,26]. In fact, when the ECVs of the azoles [22,23] are used as breakpoints, 22% A. udagawae isolates and all A. viridinutans isolates are considered resistant to ITCZ, whereas 66% of A. udagawae isolates, all A. lentulus isolates, and all A. viridinutans isolates are considered resistant to VRCZ. Therefore, the ECVs reveal that only a percentage of the A. fumigatus-related isolates are susceptible to azoles. In contrast, all A. fumigatus isolates were susceptible to azoles and AMPH-B. Approximaltely 4e5% of “A. fumigatus” isolated from patients have later turned out to be the related species [7]. These facts emphasize the importance of precise species identification and drug susceptibility testing when empirical therapy is ineffective against aspergillosis. We did not detect secondary metabolites shared by the isolates with MICs higher than ECVs. These data suggest that other factors may affect antifungal drug susceptibility, such as the expression of efflux pumps (in azoles) and the content or biosynthesis of cell surface ergosterol (in polyenes) [49,50]. Environmental isolates showed higher MICs to azoles than clinical isolates. These differences are partly explained by the dominance of

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the related species in each group (14.9% in clinical isolates vs. 61.5% in environmental isolates). In conclusion, our study demonstrates remarkable differences in secondary metabolite profiles and antifungal susceptibilities between A. fumigatus and the related species, which may to the development of more discriminative identification protocols for clinical isolates and improvements in the efficiency of treatment for aspergillosis. Conflict of interest None. Acknowledgments We would like to express our gratitude to Dr. Masahiko Takino from Agilent Technologies (Japan) for the measurement of secondary metabolites. This research was supported by a Health and Labour Sciences Research Grant, the Cooperative Research Grants of NEKKEN (2010), H25-shinko-ippan-006, and the Aspergillus Project of Chiba University. The fungal isolates used in this study were supplied by the Culture Collection of the Medical Mycology Research Center, which was partly supported by a National BioResource Project grant. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jiac.2015.01.005. References [1] Latge JP. Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 1999;12: 310e50. [2] Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA. Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot Cell 2005;4:625e32. [3] Balajee SA, Nickle D, Varga J, Marr KA. Molecular studies reveal frequent misidentification of Aspergillus fumigatus by morphotyping. Eukaryot Cell 2006;5:1705e12. [4] Samson RA, Hong S, Peterson SW, Frisvad JC, Varga J. Polyphasic taxonomy of Aspergillus section Fumigati and its teleomorph Neosartorya. Stud Mycol 2007;59:147e203. [5] Vinh DC, Shea YR, Jones PA, Freeman AF, Zelazny A, Holland SM. Chronic invasive aspergillosis caused by Aspergillus viridinutans. Emerg Infect Dis 2009;15:1292e4. [6] Coelho D, Silva S, Vale-Silva L, Gomes H, Pinto E, Sarmento A, et al. Aspergillus viridinutans: an agent of adult chronic invasive aspergillosis. Med Mycol 2011;49:755e9. [7] Hong SB, Kim DH, Park IC, Choi YJ, Shin HD, Samson R. Re-identification of Aspergillus fumigatus sensu lato based on a new concept of species delimitation. J Microbiol 2010;48:607e15. [8] Vinh DC, Shea YR, Sugui JA, Parrilla-Castellar ER, Freeman AF, Campbell JW, et al. Invasive aspergillosis due to Neosartorya udagawae. Clin Infect Dis 2009;49:102e11. [9] Alhambra A, Catalan M, Moragues MD, Brena S, Ponton J, Montejo JC, et al. Isolation of Aspergillus lentulus in Spain from a critically ill patient with chronic obstructive pulmonary disease. Rev Iberoam Micol 2008;25:246e9. [10] Etzel RA. Mycotoxins. JAMA 2002;287:425e7. [11] Kamei K, Watanabe A. Aspergillus mycotoxins and their effect on the host. Med Mycol 2005;43:S95e9. [12] Watanabe A, Kamei K, Sekine T, Higurashi H, Ochiai E, Hashimoto Y, et al. Cytotoxic substances from Aspergillus fumigatus in oxygenated or poorly oxygenated environment. Mycopathologia 2004;158:1e7. [13] Stanzani M, Orciuolo E, Lewis R, Kontoyiannis DP, Martins SL, St John LS, et al. Aspergillus fumigatus suppresses the human cellular immune response via gliotoxin-mediated apoptosis of monocytes. Blood 2005;105:2258e65. [14] Sugui JA, Pardo J, Chang YC, Zarember KA, Nardone G, Galvez EM, et al. Gliotoxin is a virulence factor of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone. Eukaryot Cell 2007;6:1562e9. [15] Lewis RE, Wiederhold NP, Lionakis MS, Prince RA, Kontoyiannis DP. Frequency and species distribution of gliotoxin-producing Aspergillus isolates recovered from patients at a tertiary-care cancer center. J Clin Microbiol 2005;43: 6120e2.

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