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Article

Assessing Differences between Clinical Isolates of Aspergillus fumigatus from Cases of Proven Invasive Aspergillosis and Colonizing Isolates with Respect to Phenotype (Virulence in Tenebrio molitor Larvae) and Genotype

by
Sam El-Kamand
1,
Martina Steiner
1,
Carl Ramirez
1,
Catriona Halliday
2,
Sharon C.-A. Chen
2,3,
Alexie Papanicolaou
4,* and
Charles Oliver Morton
1,*
1
Western Sydney University, School of Science, Campbelltown Campus, Campbelltown, NSW 2560, Australia
2
Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, New South Wales Health Pathology, Westmead Hospital, Westmead, NSW 2145, Australia
3
Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney, Sydney, NSW 2145, Australia
4
Hawkesbury Institute for the Environment, Western Sydney University, Hawkesbury Campus, NSW 2753, Australia
*
Authors to whom correspondence should be addressed.
Pathogens 2022, 11(4), 428; https://doi.org/10.3390/pathogens11040428
Submission received: 3 March 2022 / Revised: 26 March 2022 / Accepted: 29 March 2022 / Published: 31 March 2022
(This article belongs to the Special Issue Aspergillosis)
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Abstract

:
The fungus Aspergillus fumigatus, the cause of invasive aspergillosis (IA), is a serious risk to transplant patients and those with respiratory diseases. Host immune suppression is considered the most important factor for the development of IA. Less is known about the importance of fungal virulence in the development of IA including the significance of variation between isolates. In this study, isolates of A. fumigatus from cases diagnosed as having proven IA or colonisation (no evidence of IA) were compared in assays to measure isolate virulence. These assays included the measurement of radial growth and protease production on agar, sensitivity to UV light and oxidative stressors, and virulence in Tenebrio molitor (mealworm) larvae. These assays did not reveal obvious differences in virulence between the two groups of isolates; this provided the impetus to conduct genomic analysis. Whole genome sequencing and analysis did not allow grouping into coloniser or IA isolates. However, focused analysis of single nucleotide polymorphisms revealed variation in three putative genes: AFUA_5G09420 (ccg-8), AFUA_4G00330, and AFUA_4G00350. These are known to be responsive to azole exposure, and ccg-8 deletion leads to azole hypersensitivity in other fungi. A. fumigatus virulence is challenging, but the findings of this study indicate that further research into the response to oxidative stress and azole exposure are required to understand the development of IA.

1. Introduction

The fungus Aspergillus fumigatus is a globally distributed decomposer of organic matter in the environment. It produces vast numbers of conidia, which are easily distributed by wind currents; these can be inhaled by birds and mammals, leading to the development of several disease states [1]. In humans, the most severe disease state is invasive aspergillosis (IA), which usually affects immunocompromised individuals, particularly those that are neutropenic [2,3]. Not all at-risk patients develop IA, and the severity of host-damage can vary; the disease occurs in only 7% of acute myeloid leukemia patients [4]. The contribution of variation in fungal virulence to the development of IA is poorly understood. Variation in virulence is apparent in isolates of A. fumigatus from lower respiratory samples of individuals who have the risk factors for IA but show no evidence of IA; these isolates are referred to as colonisers [3,5,6,7]. To date, studies have had difficulty identifying the key differences between colonising and IA isolates using in vitro tests and model hosts [5].
Intraspecies variation in A. fumigatus isolates has been described in traits that may be important for pathogenesis [8]. These include the growth rate [8,9], pigmentation [10], resistance to oxidative stress [11], and azole antifungals [12]. Despite these studies, inferring the clinical significance of intraspecific variation in A. fumigatus remains challenging. Another approach is the use of invertebrate models to study fungal infections. These models include nematodes (Caenorhabditis elegans), fruit flies (Drosophila melanogaster), and the larvae of moths (Galleria mellonella) and beetles (Tenebrio molitor) [13,14]. An advantage of G. mellonella and T. molitor larvae compared to the other invertebrate models is that they can be reared at 37 °C, the internal body temperature of humans [13]. An issue with G. mellonella is that it is not readily available in Australia as it is defined as a pest, making T. molitor an attractive alternative. The validity of these invertebrate models has been strengthened since the patterns of A. fumigatus virulence are consistent between invertebrate and vertebrate models [15,16].
Another approach to examine variation in fungal virulence is whole genome sequencing (WGS), which can be employed to identify intraspecies genetic variation in known virulence factors [17,18]. The high-resolution nature of WGS has revealed clinically relevant heterogeneity in the fungal pathogens Candida albicans [19] and Cryptococcus neoformans [20]. C. albicans showed major differentiating genetic variants located on genes associated with biofilm production [19], and WGS analysis of C. neoformans identified intra-specific heterogeneity in 40 genes putatively associated with pathogenesis [20].
Most studies into A. fumigatus intraspecific variation have focused on comparisons between environmental isolates and clinical isolates. In this study, we examined intraspecific variation in only clinical isolates of A. fumigatus. These came from cases defined as having no evidence of IA (coloniser isolates) and cases defined as having IA (IA isolates); cases were defined according to the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) criteria [21]. This approach was similar to a study performed using A. fumigatus and A. terreus in Drosophila melanogaster [5]. We adapted T. molitor larvae as a model system [14] to measure A. fumigatus virulence and used this model along with other assays (radial growth rate, resistance to oxidative stress, and infection of T. molitor larvae) to assess the virulence. We also used WGS to identify fungal characteristics linked to IA. This will provide a foundation for future studies into the factors that determine the outcome of the host–fungus interaction.

2. Results

2.1. Phenotypic Characterisation of Clinical A. fumigatus Isolates

Intraspecific variation in 15 clinical isolates of A. fumigatus was assayed through exposure to UV light, oxidative stress, radial growth rate, and protease production (Figure 1 and Figure 2). These data indicated that IA isolates were not clearly different from coloniser isolates in the virulence-associated traits tested in this study. The coloniser isolates showed a trend towards greater fitness with respect to radial growth, UV resistance, and resistance to oxidative stress.
The radial growth rate of clinical A. fumigatus isolates was evaluated on potato dextrose agar (PDA) at 37 °C (Figure 1a). Growth of IA isolates displayed a trend towards slower radial growth than coloniser isolates. On average, coloniser isolates grew 124.1 (± 4.4) µm/h faster than IA isolates. The trend was consistent, with four of the five slowest growers being IA isolates (Figure 1a). There was variation in protease production between isolates (p < 0.0001) on skim milk agar (SMA), but there was no significant difference based on isolate origin (colonisers 997 µm ± 629 µm; IA 534 µm ± 506 µm). The production of proteases showed greater variability between isolates than radial growth rate (Figure 1c).
Coloniser and IA isolates showed similar levels of viability following one minute of UV irradiation 1.6 W/m2 (Figure 2a). These data suggest that conidia from IA isolates do not differ significantly from coloniser isolates with respect to UV irradiation. The response of A. fumigatus isolates to three hours of exposure to 50 mM H2O2 or 50 mM menadione indicated an increased resistance to oxidative stress in coloniser isolates compared to IA isolates, particularly with respect to menadione treatment (Figure 2c,e). After exposure to menadione, coloniser isolates had an average survival of 71.5% (±2.1) compared to 50.4 (±1.9) for IA isolates, whereas exposure to H2O2 led to 19% (±7.3) survival compared to 2.4% (±0.8%) for IA isolates. The data presented in Figure 1 and Figure 2 do not suggest that there are sufficient differences between colonizer and IA isolates to explain why the IA isolates were associated with cases of IA. Therefore, we also conducted virulence and genomic analyses.

2.2. Modelling A. fumigatus Virulence in T. molitor Larvae

2.2.1. Model Validation

The injection of mealworms with PBS-tween had an impact on the survival of mealworms that was similar for all injection sites (p = 0.96; Figure 3a). However, it was observed that injecting mealworms at the base of the fifth sternite led to the lowest frequency of hemolymph leakage, which was associated with better outcomes. The fifth sternite was chosen as the injection site for all future experiments.
Kaplan–Meier survival curves of mealworms inoculated with A. fumigatus (Af01) showed a dose-dependent response (Figure 3b). All inoculum sizes tested (5 × 101, 5 × 102, 5 × 103, 5 × 104, 5 × 105, and 5 × 106 conidia per larva) significantly decreased the survival rate relative to controls. Inoculation with 5 × 104 conidia consistently yielded mortality rates above 50%, which is required for the calculation of the median survival time, without killing at a rate so high that isolates of higher virulence would be difficult to resolve. All further experiments used an inoculum of 5 × 104 conidia per larva.

2.2.2. Quantification of A. fumigatus in Infected T. molitor Larvae

Methods for monitoring fungal infection in the T. molitor larvae were evaluated (Figure 3c). Fungal quantification was attempted using both viable plate counts and quantitative PCR (qPCR). Viable plate counts (Figure 3d) showed the presence of greater amounts of the fungus, 1.3 × 104 (±5.4 × 103) CFU/larva earlier but lower CFU counts, 1.2 × 102 (±3 × 101) CFU/larva later in the experiment, suggesting clearance of the fungus despite increasing the larval mortality. qPCR analysis (Figure 2e) indicated that the fungal burden increased over time from 3.1 × 101 (±2 × 101) genomes/larva earlier to 4 × 103 (±6 × 102) genomes per larva later in the experiment, which matched the expectations of the experiment created by the mortality data (Figure 3c). The difference may be attributable to A. fumigatus being a filamentous fungus, leading to unreliable recovery of CFU from larvae after fungal germination and maturation into hyphae. Monitoring of infection may be resolved by microscopic analysis of the larvae, but that methodology is challenging and has not been optimized for this study.

2.2.3. Quantifying Virulence of Clinical A. fumigatus Isolates in T. molitor

The isolate origin had no significant effect on the survival of infected T. molitor larvae (Figure 4). There was a trend for shorter mean survival times in larvae inoculated with coloniser isolates, but this was not significant. The average survival rate was 2.52 (±0.25) days with coloniser isolates and 2.97 (±0.4) days with IA isolates (Figure 3b). In this study, the shorter survival time represents greater virulence. The ability of fungi to cause death in mealworms or other invertebrates such as Galleria mellonella is indicative of their virulence potential in mammalian models. From these data, there is not a clear distinction in virulence between the two groups of isolates. Inclusion of patient characteristics such as antifungal treatment would be required to fully resolve the differences between coloniser and IA isolates.

2.3. Genomic Analysis of Clinical A. fumigatus Isolates

2.3.1. Identifying Single Nucleotide Variants (SNV)

Following the assembly of the ten sequenced genomes (Table S1), the genomes were analysed to identify SNVs unique to IA isolates. Across the six coloniser and four IA isolates, a total of 981,551 SNV sites were identified in the core-genome of A. fumigatus when SNVs were called against the AF293 reference genome. Filtering for bi-allelic sites in unmasked regions where the locally collinear block (LCB) length is over 200 bp resulted in a final callset of 95,999 sites. SNPeff annotation revealed a total of 19,589 putative non-synonymous mutations.
Synonymous variants were filtered for those exclusively found in one group (coloniser or IA isolates). There were five variants present in all members of one group, which were absent in all members of the other (Table 1). These variants were all previously reported in FungiDB (release 48 beta) with an allele frequency greater than 40%, suggesting these variants are real.
The SNVs found in all IA isolates but absent in coloniser isolates were localized to three genes: AFUA_5G09420, AFUA_4G00330, and AFUA_4G00350 (Table 2). AFUA_5G09420 is annotated in FunCat as Clock controlled protein (ccg-8) and classified by InterProScan as Transcription factor opi1, a ccg-8 homolog in yeast (Table 2). No signal peptide or transmembrane domains were predicted by SignalP5 or TMHMM. AFUA_4G00350 is not annotated in FunCat but contains a metallopeptidase domain, and protein BLAST reveals similarity to fungal sequences annotated as archaemetzincin-2. Similarly, AFUA_4G00330 is not annotated in FunCat but does have several transmembrane domains.

2.3.2. Detecting Presence/Absence of Oxidative Stress Response Genes

Scanning the genome assemblies of 10 clinical A. fumigatus isolates for the presence or absence of nine genes involved in the melanin biosynthetic process (GO:0042438) revealed no inter-isolate variation. Presence/absence analysis of 135 oxidative stress response genes (GO:0006979) showed some variation across clinical isolates. Each isolate tested had 129–131 oxidative stress response genes present. Neither variation in the total number of genes present (Table 3) nor the occurrence patterns of individual genes (Table S2) were consistent with oxidative stress resistance.

3. Discussion

Many phenotypic properties of A. fumigatus with theoretical links to virulence show intra-specific heterogeneity [5,17,25,26,27]; however, the clinical relevance of this variation remains unclear. In this study, we tested the hypothesis that comparison of phenotypic traits related to virulence would elucidate differences between isolates that cause IA and those that just colonise patients. Fifteen clinical A. fumigatus were characterised with respect to the growth rate, protease production, resistance to oxidative stress, and virulence in T. molitor larvae. We then compared the genomes of isolates from patients that were colonised to isolates from patients that had proven IA.
The growth rate of A. fumigatus isolates is theorized to impact virulence as greater quantities of fungal biomass are more difficult for the immune system to clear [8,9]. In A. fumigatus deletion mutants, decreased virulence in murine models is often accompanied by a decreased growth rate [28,29,30]. Positive correlations between the growth rate and virulence in murine models have also been observed in populations of wild-type isolates [9,26,31]. These findings span growth rates calculated using solid and liquid cultures, and both minimal and nutrient rich media. The growth rate does not always positively correlate with virulence, as has been demonstrated by studies in both insect and murine models [32]. As in previous studies [8], the isolates examined here showed significant variability in the growth rate, as assayed on PDA (Figure 1a,b). There was no clear difference between coloniser and IA isolates, which was also observed for the growth and creation of zones of clearing on SM agar (Figure 1c,d).
Resistance of A. fumigatus conidia to solar UV radiation and UV-induced reactive oxygen species (ROS) is important for the survival of airborne conidia. However, there was very limited intraspecific variation in UV resistance observed in this study (Figure 2a,b). The limited significant variation in UV resistance is in coloniser isolates and therefore would not be considered essential for the development of IA [8,11]. Conidia do contain molecules associated with fungal pathogenesis such as melanin, which may be important for the establishment of infection by A. fumigatus [33]. Conidial cell wall melanin has been implicated in pathogenesis by promoting pre-germination concealment of immunogenic PAMPs [34,35], evasion of internalization by phagocytes [36], and persistence within immune and alveolar epithelial cells [37,38]. Further studies that focus on the role of melanin in infection would be required to determine the role of melanin in the differential development of IA. Analysis of the response to oxidative stress revealed greater resistance in coloniser isolates than in IA isolates (Figure 2c–f). A link between resistance to oxidative stress and virulence has been demonstrated previously in A. fumigatus; deletion of catalases and superoxide dismutases has been associated with reduced or delayed virulence in murine models, and sensitization to killing by phagocytes [39]. It seems counter-intuitive for our isolates to demonstrate the inverse association. However, this is similar to the overall trend in our observations of growth rate (Figure 1) and virulence in T. molitor larvae (Figure 4).
Hosts susceptible to IA are almost always immunodeficient; however, their immunological profiles can vary wildly [40]. IA is both uncommon and often misdiagnosed, making it difficult to obtain a large set of clinical isolates standardised with respect to potentially noise-creating host factors such as the primary condition, therapeutic history, or geographical region. The use of animal models (mouse and invertebrate) allows the virulence of A. fumigatus isolates from different hosts to be compared in an experimental system where host factors are standardized. In this study, we validated and optimized T. molitor larvae (mealworms) as a model for invasive fungal infection. Unlike Drosophila or nematodes, mealworms can be reared at 37 °C. They can also be inoculated via injection, allowing for precise control over the infective load. Advantages over G. mellonella include low maintenance and widespread availability, mitigating the requirement of maintaining in-house colonies. These advantages of T. molitor have also been reviewed elsewhere [41]. Here, we have shown that the injection of mealworms with A. fumigatus isolates causes mortality in a dose-dependent manner (Figure 3b). Furthermore, the fungus is present in the mealworm throughout the course of experimentation (Figure 3d,e). The mean survival time for G. mellonella injected with 105 conidia of wild-type A. fumigatus was 2–3 days [16], which compares well with the data from this experiment (Figure 4). Although our inoculum was 104 conidia, differences could be expected due to the isolates used and the immunity systems of the respective hosts.
Several studies comparing clinical and environmental A. fumigatus isolates have been conducted. In immunosuppressed mice, environmental isolates were found to be less virulent than clinical isolates using mortality-based metrics [42]. A similar trend was observed in mixed infection murine models, where mice were co-infected with a clinical and corresponding environmental isolate and relative virulence inferred from the ratio of recovery after the mice shows signs of pulmonary distress [43]. Importantly, this trend in virulence is not mammal specific. Clinical isolates are also more virulent than environmental isolates in G. mellonella larvae [25]. Taken together, these studies suggest that virulence data produced in animal models are clinically relevant and that either (1) some environmental A. fumigatus isolates possess phenotypic profiles more conducive to causing infection than others, or that (2) within a human host, virulence-enhancing micro-evolution occurs.
In T. molitor larvae, IA isolates could not be distinguished from coloniser isolates (Figure 4a,b). This suggests that clinically important fungal properties selected for use in human hosts were not selected for our invertebrate model. One possibility is that virulence factors important in overcoming a clinical barrier to infection have fitness costs that become visible when the selective pressure is lifted due to differences between the clinical environment and the experimental system used to assess virulence. The ability of an isolate to survive prophylaxis or response therapy is clinically important; this was not modelled in T. molitor larvae. Significant fitness trade-offs associated with resistance to common antifungals, including those used in prophylactic and first-line treatment of IA, have been observed in Candida [44,45]. Notably, some of the common azole-resistance conferring mutations do not appear detrimental to A. fumigatus fitness in either immunosuppressed [46] or immunocompetent mice [47].
To guide future work, we explored the genomic variation in clinical isolates and shortlisted variants likely to be of interest (Table 1). Previously, broad-scale phylogenetic comparison and even geneset-restricted SNV-based analysis has failed to resolve different clinical forms of aspergillosis [45,46]. In this study, we identified five non-synonymous SNVs that were found in all IA isolates but not in coloniser isolates. The SNVs are all common in FungiDB [48], suggesting that they are legitimate genetic variants and not sequencing artifacts. Identical allele frequencies for four of these variants implies that they represent a haplotype common in clinical isolates. The shortlisted variants affected three genes: AFUA_5G09420, AFUA_4G00330, and AFUA_4G00350.
These were further investigated through a literature search to determine their functions (Table 2). AFUA_5G09420 is transcription factor CCG-8. Knockout studies in Neurospora crassa and Fusarium verticillioides showed that loss of CCG-8 hypersensitizes cells to azole antifungals, and this can be rescued by transformation with a CCG-8 containing vector [22]. AFUA_4G00350 and AFUA_4G00330 are less well characterized. AFUA_4G00350 is likely to be a metallopeptidase (a possible archaemetzincin-2 homolog), as classified by InterProScan. AFUA_4G00330 contains several transmembrane domains and likely encodes a membrane-bound protein (Table 2). There is some evidence that itraconazole treatment leads to increased expression of both AFUA_4G00330 and AFUA_4G00350 [23,24].
The potential relationship between AFUA_5G09420, AFUA_4G00330, and AFUA_4G00350 with responses to azole fungicides led us to test the isolates against a range of fungicides. This analysis indicated potential differences in response to some fungicides by IA isolates (Figure S1). This will require further investigation since further replication and testing will be required to confirm a role for differential drug responses in IA isolates. The MIC methodology will also require optimization as differences in drug sensitivity will be subtle since the tested isolates did not appear to have obvious azole resistance alleles. Strong drug resistance may not be required to enable the development of IA since consistent drug concentrations are not always observed between or within patients [49,50]. There will be patients or regions of the body with sub-optimal drug concentrations that will allow the growth of fungal variants with low/intermediate drug resistance.
In addition to SNV analysis, we examined the presence/absence of genes involved in oxidative stress response or the melanin biosynthetic process in the genomes of clinical A. fumigatus isolates. The patterns of variation observed were not consistent with patterns of oxidative stress resistance (Table 3; Table S2). Thus, it is unlikely that gene presence/absence drives oxidative stress resistance. Previous studies have also failed to resolve A. fumigatus isolates of differing clinical significance based on the presence/absence of virulence-associated genes (including those involved in oxidative stress response) [51]. There is evidence that Saccharomyces cerevisiae has distinct responses to oxidative stress induced by menadione and H2O2 [52], and this has been supported by transcriptional analysis of Aspergillus oryzae [53]. In the A. oryzae study, similar oxidative stressors led to different transcriptional responses in the important transcription factors yap1 and skn7, as well as catalases and superoxide dismutase. The A. fumigatus yap1 is important for the oxidative stress response by controlling the expression of downstream genes. Understanding differences in oxidative stress response between fungal isolates will require a functional genomics approach that incorporates genomic, proteomic, and transcriptomic data.
Testing coloniser and IA isolates of A. fumigatus with assays for virulence was inconclusive, but genome comparison indicated that understanding the development of IA in at-risk patients will require further study into complete or partial azole resistance.

4. Materials and Methods

4.1. Isolates of A. fumigatus and Media

All strains of A. fumigatus were provided by the Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital (Table 4). Each case was classified according to the criteria of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) [21]. Fifteen isolates of A. fumigatus were studied; ten isolates (Af1–Af10) were colonisers, from patients with no evidence of IA [3,5,6,7], and five (Af11–Af15) were invasive isolates, from patients with proven IA [5,21]. Cultures were grown on PDA for three days at 37 °C and conidia suspensions were prepared from each isolate as previously described [54]. Conidial concentrations were determined using a Neubauer chamber and viability was determined using CFU counts of ten-fold conidial dilutions on PDA (Sigma-Aldrich, Castle Hill, Australia) plates that were incubated for 24 h at 37 °C [55]. New conidial suspensions were prepared for each replicate experiment and concentrations were tested before the experiments.

4.2. Phenotypic Variation Amongst Clinical A. fumigatus Isolates

4.2.1. Radial Growth Rate and Proteolysis on SMA

The radial growth and growth rate have been associated with fungal virulence in several studies, with faster growth being indicative of greater virulence [26,56,57]. For each A. fumigatus isolate, a PDA plate (90 mm diameter) was spot-inoculated with 104 conidia. Cultures were incubated at 37 °C for 3 days. The colony diameter was measured at regular intervals. The radial growth rate was calculated by plotting the colony diameter (mm) versus time (h) from five linearly distributed data points; the growth rate was the slope of this line. Three independent experiments were performed.
Proteolytic activity has been associated with infection and tissue invasion in pathogenic fungi [58]. A simple method to determine the proteolytic activity is to grow microbes on a medium such as SMA where the proteolysis of milk proteins creates a zone of clearing around the colony [59,60]. SMA was prepared by adding 250 mL UHT skim milk to 250 mL autoclaved, still molten, 4% agar (Sigma-Aldrich). The milk and agar were mixed and poured into Petri dishes. These were inoculated with 104 conidia to the centre of the Petri dish. Cultures were incubated at 37 °C for 4 days. The zone of inhibition was measured daily to determine the time where the greatest discrimination between isolates was observed; a wider zone of clearing indicates greater proteolytic activity. Three independent experiments were performed.

4.2.2. Conidial UV Resistance

Conidial melanin has an important role in protection against UV light and the initial interactions between host and A. fumigatus, the conidial melanin providing protection against the activity of host phagocytes [61,62]. In this study, we used the response to UV to determine whether there were isolates with defects in conidial melanin. For each isolate, approximately 200 conidia were plated onto malt extract agar (MEA) (Sigma-Aldrich). Five plates inoculated with the same isolate were placed at different positions within a TopSafe PC2 Biosafety (Bio-Air, Pero, Italy) cabinet and UV irradiated (1.6 W/m2) for 1 min. This was repeated for all 15 isolates. Following irradiation of each isolate, the biosafety cabinet was vented for 5 min to prevent reactive oxygen species (ROS) accumulation. Colony forming units (CFUs) on control plates were counted following incubation at 37 °C for 24 h. An additional incubation for 24 h at 25 °C preceded CFU counting of UV-irradiated plates. The percent survival for each isolate was calculated relative to a non-irradiated control and based on the average CFU counts across the five irradiated plates. The experiment was repeated four times, with the irradiated isolates being changed to achieve a uniform average UV-order position amongst all isolates.

4.2.3. Measurement of Response to Oxidative Stress

To measure the effects of acute exposure to oxidative stress, isolates of A. fumigatus were exposed to H2O2 and menadione (both from Sigma-Aldrich) for three hours [63,64]. H2O2 (30% solution) and menadione (10 mM stock in ethanol) were added to A. fumigatus spore suspensions from each isolate; 1 × 106 conidia/mL was incubated for 3 h at 37 °C with 0 mM, 50 mM H2O2 or 50 mM of menadione. Acute exposure required greater exposure doses than used in other studies. After incubation, the conidial viability was determined using dilution plate counts on PDA (Sigma-Aldrich); plates were incubated at 37 °C for 24 h and counted to determine CFU/mL. The percent survival was calculated relative to the 0 mM control.

4.3. Using T. molitor to Measure A. fumigatus Virulence

The use of T. molitor larvae was based on a study using these larvae to monitor the virulence of C. albicans and C. neoformans [14]. Mealworms were purchased from BioSupplies (Biosupplies, Yagoona, Australia) and checked for uniform size (100–150 mg) and colour (dark brown to black indicates ill health) before use. In all experiments, mortality was determined by response to physical stimulation. Where not explicitly specified, mealworms were incubated in Petri dishes (rearing density: 10 mealworms/58 cm2 Petri dish) with 3 mL rearing diet and a slice of frozen carrot for moisture (0.4 cm3; 500 mg; changed daily). Rearing diet comprised of wheat bran and LSA (linseeds, sunflower seeds, and almonds) in a 5:1 v/v ratio.

4.3.1. Optimisation and Validation of T. molitor Larvae as Models of Fungal Infection

The site of injection into T. molitor larvae was optimized to reduce mortality due to physical trauma. Mealworms were injected between sternites with a maximum 5 µL of liquid using a Hamilton syringe (701 N, 10 μL capacity) [14]. Groups of 20 mealworms were injected ventrally with 5 μL of PBST at the base of one of five sternites. Sternites 2–6 were tested (Figure 1a). Survival was checked daily over 7 days of incubation at 37 °C. Each treatment group included 20 mealworms. Based on the findings of this experiment, mealworms were injected at the base of sternite 5 in all virulence assays.
To identify the fungal load with the greatest potential for resolving inter-isolate variation, a dose–response experiment was run. Groups of 20 mealworms were inoculated with 0, 5, 5 × 101, 5 × 102, 5 × 103, 5 × 104, 5 × 105, or 5 × 106 conidia of isolate AF01. Mortality was monitored daily over 7 days of incubation at 37 °C. The experiment was performed twice.
Groups of 30 mealworms were inoculated with 5 µL PBST (vehicle control), or 5 × 104 spores from coloniser AF03 or invasive isolate AF11. Each day, 5 mealworms were sampled from each cohort for fungal load quantification.

4.3.2. Quantification of A. fumigatus Infection of T. molitor Larvae

Both infected and uninfected larvae were frozen and stored at −20 °C after virulence had been measured. Frozen larvae were decapitated and cut below the 8th sternite to provide a section of worm that would be halved (longitudinally). One half was used for viable plate counts and the other was used for qPCR.
For viable plate counts the larval section was homogenized in PBS (Sigma-Aldrich) with a disposable pestle in a 1.5 mL microcentrifuge tube. The contents were serially diluted and 100 µL was spread using an L-shaped spreader on Petri dishes containing potato dextrose agar containing chloramphenicol (Himedia, Mumbai, India). Plates were prepared in duplicate and incubated at 37 °C until colonies were visible, approximately 20 h.
For qPCR DNA was isolated from the other half of the larval section. DNA was isolated using the ISOLATE II Genomic DNA Kit (Bioline, Eveleigh, Australia) with the following modifications: larval sections were placed in a 1.5 mL microcentrifuge tube with lysis buffer (GL), this was homogenized with a micro pestle. Proteinas K was added and the homogenate incubated at 55 °C for 2 h. Glass beads (710–1180 µm and 425–600 µm) were added to 250 µL in a 2 mL screw cap tube. The homogenate was added to the 2 mL tube and the sample was disrupted in a Mini-Beadbeater (BioSpec Products, Bartlesville, USA) for two cycles of 1 min of beadbeating and 1 min on ice. The manufacturer’s instructions were then followed with an elution volume of 50 µL.
qPCR was performed on a 7500-FAST real-time PCR system (ThermoFisher Scientific, North Ryde, Australia) using primers targeting the ITS region of A. fumigatus as previously described [65]. The number of fungal genomes was determined compared to a standard curve of genomic DNA isolated from 105 conidia.

4.3.3. Measuring Inter-Isolate Variation in Virulence of A. fumigatus

The virulence of all 15 clinical A. fumigatus isolates was evaluated. For each isolate, 20 mealworms were inoculated with 5 × 104 conidia (in 5 µL PBST) at the base of sternite 5. Larvae were incubated for 7 days at 37 °C. Each experimental replicate included three control groups: (1) mealworms injected with sterile PBST, (2) mealworms pierced at sternite 5 but with no solution injected, and (3) mealworms chilled on ice but otherwise untreated.

4.4. Genomic Variation Amongst Clinical A. fumigatus Isolates

4.4.1. DNA Isolation

For each of 10 clinical isolates, malt extract broth (20 mL) was inoculated with 104 conidia and incubated for 4–5 days at 37 °C with shaking. Fungal biomass was isolated through vacuum filtration and stored at −20°C until use. Genomic DNA was extracted from biomass using the ISOLATE II Genomic DNA Kit (Bioline) with pre-lysis steps supplemented by mechanical disruption and RNA degradation. Lysis buffer (180 µL) and 100 mg of biomass was added to FastPrep Lysing Matrix G (MP Biomedicals, Seven Hills, Australia) before bead milling in a FastPrep-24 (MP Biomedicals) (max speed; 30 s). RNase A (1 µL of a 20 mg/mL) solution was then added, and samples incubated at 37 °C for 30 min. RNase was degraded by adding 25 µL of Proteinase K solution and incubating at 56 °C for 1 h. Secondary lysis steps and column clean-up were conducted as per the standard protocol.

4.4.2. Library Preparation and Sequencing

WGS library preparation (TruSeq DNA PCR-Free, Opentrons, New York, NY, USA) and sequencing (Illumina NovaSeq 6000) was conducted by the Ramaciotti Center for Genomics.

4.4.3. Genome Assembly

FastQC1 (v0.11.2) was used to evaluate the read quality. The leading 7 bp, final base, Illumina adapters, and low-quality leading and trailing bases (phred < 30) were removed using trimmomatic2 (v0.38, http://www.usadellab.org/cms/index.php?page=trimmomatic (accessed on 1 June 2020)). Reads were error-corrected with LIGHTER3 (v1.1.2) [66]. SPAdes4 (v3.13) [67], was used to produce de novo assemblies of the 10 A. fumigatus isolates. SPAdes contigs were scaffolded into chromosomal level assemblies with Ragout6 (v2.2) [68]. Ragout scaffolding was based on synteny with the public AF293 reference genome (GCF_000002655.1), the draft genome of A1163 (GCA_000150145), and the contig-level isolate assemblies of the clinical isolates examined in this study. SPAdes contigs unplaced by Ragout were run through a nucleotide BLASTN (v2.6.0) similarity search [69]. Hits were filtered and sorted by e-value and percent identity (E < 1 × 10−10, >90% identity). Unplaced contigs longer than 200 bp with BLASTN top hits against A. fumigatus were incorporated into the assembly. The completeness of assemblies was evaluated using BUSCO, and contiguity using the n50stats.pl script packaged with the Just Annotate My genome5 (JAMg) pipeline (Apr 2016). Contiguity stats were generated assuming a true genome size of 29.4 Mb.

4.4.4. Variant Analysis

parSNP10 (v1.2) [70], was used to align the assembled A. fumigatus genomes AF293 reference and call SNPs in genomic regions that align across all genomes and the reference. Harvest tools (v1.2) [70], was used to convert data to VCF format. Variants were filtered using VCFtools (v0.1.15) [71], to include only bi-allelic sites and thin mutations less than 10 bp away from the nearest SNP. SNPs were annotated based on the RefSeq annotation of the AF293 reference genome using SnpEff (v 4.3t) [72].

4.4.5. Investigating Biological Function of Mutated Genes

To investigate the biological function of selected genes, associated FunCat annotations were identified using FungiFun (v2.2) [73]. Protein sequences were run through a protein BLAST. InterProScan (v5) [74], was used to classify proteins into families and predict protein domains, including signal peptide sequences using SignalP (v5) [75] and transmembrane domains using mobius and TMHMM.

4.4.6. Detecting Presence/Absence of Oxidative Stress Response Genes

To investigate whether gene presence/absence explains observed differences in the oxidative stress resistance of clinical A. fumigatus isolates, each genome assembly was scanned for the presence of genes involved in either melanin biosynthesis (GO:0042438) or oxidative stress response (GO:0006979). The nucleotide sequences of each gene in the AF293 reference genome were extracted using the GO search functionality of EuPathDB (release 49 beta) [76]. Gene presence/absence in each assembly was evaluated using ABRicate (v1.0.1, https://github.com/tseemann/ABRicate, (accessed on 10 march 2020)) [77]. Genes were considered present if both coverage and percent identity was greater than 90%.

4.5. MIC Determinations

Minimum inhibitory concentrations (MICs) of several antifungal drugs were determined using Yeast Sensititre YO10 plates (ThermoFisher Scientific) as per the manufacturer’s instructions [78]. In brief, fungal conidia were added to the included broth as described in the manufacturer’s instructions to achieve a density of 1.5–8 × 103 cells/mL; 100 µL of broth containing conidia was added to each well of the YO10 Sensititre plate (ThermoFisher Scientific). After inoculation, the plates were incubated at 37 °C for 24–48 h. The YO10 Sensititre plate contains the following drugs (concentration range): Amphotericin B (0.12–8 µg/mL), Anidulafungin (0.015–8 µg/mL), Caspofungin (0.008–8 µg/mL), Fluconazole (0.12–256 µg/mL), 5-Flucytosine (0.06–64 µg/mL), Itraconazole (0.015–16 µg/mL), Micafungin (0.008–8 µg/mL), Posazonazole (0.008–8 µg/mL), and Voriconazole (0.008–8 µg/mL).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens11040428/s1, Figure S1. MIC values determined for each of the coloniser (10 isolates) and IA (5 isolates) A. fumigatus isolates used in this study. The figure shows the drugs where there were differences in mean MIC between coloniser and IA isolates based on the Sensititre YO10 results. The horizontal lines represent the mean value of the isolates in each group; one Sensitrite YO10 plate was performed for each isolate. Table S1. Assembly Statistics for 10 A. fumigatus isolates and the public AF293 reference genome (GCF_000002655.1; ASM265v1). Table S2 Presence/absence of oxidative stress response genes (GO:0006979) in clinical A. fumigatus isolates.

Author Contributions

Conceptualization, S.E.-K., C.H., S.C.-A.C., A.P. and C.O.M.; methodology, S.E.-K., C.R., M.S., A.P. and C.O.M.; validation, S.E.-K., C.R. and M.S.; formal analysis, S.E.-K., C.R., M.S., C.O.M. and A.P.; resources, C.H., S.C.-A.C., C.O.M. and A.P.; data curation, S.E.-K. and A.P.; writing—original draft preparation, S.E.-K., M.S., C.O.M. and A.P.; writing—review and editing, S.E.-K., C.H., S.C.-A.C., A.P. and C.O.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The majority of the data are contained within the article or Supplementary Materials. The genome data may be available on request but will require permission from Westmead Hospital.

Acknowledgments

The authors wish to acknowledge funding from Western Sydney University that enabled Sam El-Kamand, Martina Steiner, and Carl Ramirez to undertake this research. The authors would also like to thank Karena Gilroy, Chun Ho, and Smitha Parameswaran for technical support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Segal, B.H. Aspergillosis. N. Engl. J. Med. 2009, 360, 1870–1884. [Google Scholar] [CrossRef] [PubMed]
  2. Dagenais, T.R.; Keller, N.P. Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis. Clin. Microbiol. Rev. 2009, 22, 447–465. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Kosmidis, C.; Denning, D.W. The clinical spectrum of pulmonary aspergillosis. Thorax 2015, 70, 270–277. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Zhao, S.; Gibbons, J.G. A population genomic characterization of copy number variation in the opportunistic fungal pathogen Aspergillus fumigatus. PLoS ONE 2018, 13, e0201611. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Ben-Ami, R.; Lamaris, G.A.; Lewis, R.E.; Kontoyiannis, D.P. Interstrain variability in the virulence of Aspergillus fumigatus and Aspergillus terreus in a Toll-deficient Drosophila fly model of invasive aspergillosis. Med. Mycol. 2010, 48, 310–317. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Barberan, J.; Candel, F.J.; Arribi, A. How should we approach Aspergillus in lung secretions of patients with COPD? Rev. Esp. Quimioter. 2016, 29, 175–182. [Google Scholar] [PubMed]
  7. Shahi, M.; Ayatollahi Mousavi, S.A.; Nabili, M.; Aliyali, M.; Khodavaisy, S.; Badali, H. Aspergillus colonization in patients with chronic obstructive pulmonary disease. Curr. Med. Mycol. 2015, 1, 45–51. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Rinyu, E.; Varga, J.; Ferenczy, L. Phenotypic and genotypic analysis of variability in Aspergillus fumigatus. J. Clin. Microbiol. 1995, 33, 2567–2575. [Google Scholar] [CrossRef] [Green Version]
  9. Paisley, D.; Robson, G.D.; Denning, D.W. Correlation between in vitro growth rate and in vivo virulence in Aspergillus fumigatus. Med. Mycol. 2005, 43, 397–401. [Google Scholar] [CrossRef] [Green Version]
  10. Fuller, K.K.; Cramer, R.A.; Zegans, M.E.; Dunlap, J.C.; Loros, J.J. Aspergillus fumigatus Photobiology Illuminates the Marked Heterogeneity between Isolates. mBio 2016, 7, e01517-e16. [Google Scholar] [CrossRef] [Green Version]
  11. Hagiwara, D.; Sakai, K.; Suzuki, S.; Umemura, M.; Nogawa, T.; Kato, N.; Osada, H.; Watanabe, A.; Kawamoto, S.; Gonoi, T.; et al. Temperature during conidiation affects stress tolerance, pigmentation, and trypacidin accumulation in the conidia of the airborne pathogen Aspergillus fumigatus. PLoS ONE 2017, 12, e0177050. [Google Scholar] [CrossRef] [PubMed]
  12. Goncalves, S.S.; Souza, A.C.R.; Chowdhary, A.; Meis, J.F.; Colombo, A.L. Epidemiology and molecular mechanisms of antifungal resistance in Candida and Aspergillus. Mycoses 2016, 59, 198–219. [Google Scholar] [CrossRef] [PubMed]
  13. Arvanitis, M.; Glavis-Bloom, J.; Mylonakis, E. Invertebrate models of fungal infection. Biochim. Biophys. Acta 2013, 1832, 1378–1383. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. De Souza, P.C.; Morey, A.T.; Castanheira, G.M.; Bocate, K.P.; Panagio, L.A.; Ito, F.A.; Furlaneto, M.C.; Yamada-Ogatta, S.F.; Costa, I.N.; Mora-Montes, H.M.; et al. Tenebrio molitor (Coleoptera: Tenebrionidae) as an alternative host to study fungal infections. J. Microbiol. Methods 2015, 118, 182–186. [Google Scholar] [CrossRef] [Green Version]
  15. Amich, J.; Schafferer, L.; Haas, H.; Krappmann, S. Regulation of sulphur assimilation is essential for virulence and affects iron homeostasis of the human-pathogenic mould Aspergillus fumigatus. PLoS Pathog. 2013, 9, e1003573. [Google Scholar] [CrossRef] [Green Version]
  16. Slater, J.L.; Gregson, L.; Denning, D.W.; Warn, P.A. Pathogenicity of Aspergillus fumigatus mutants assessed in Galleria mellonella matches that in mice. Med. Mycol. 2011, 49 (Suppl. 1), S107–S113. [Google Scholar] [CrossRef] [Green Version]
  17. Hagiwara, D.; Takahashi, H.; Watanabe, A.; Takahashi-Nakaguchi, A.; Kawamoto, S.; Kamei, K.; Gonoi, T. Whole-genome comparison of Aspergillus fumigatus strains serially isolated from patients with aspergillosis. J. Clin. Microbiol. 2014, 52, 4202–4209. [Google Scholar] [CrossRef] [Green Version]
  18. Takahashi-Nakaguchi, A.; Muraosa, Y.; Hagiwara, D.; Sakai, K.; Toyotome, T.; Watanabe, A.; Kawamoto, S.; Kamei, K.; Gonoi, T.; Takahashi, H. Genome sequence comparison of Aspergillus fumigatus strains isolated from patients with pulmonary aspergilloma and chronic necrotizing pulmonary aspergillosis. Med. Mycol. 2015, 53, 353–360. [Google Scholar] [CrossRef] [Green Version]
  19. Cavalieri, D.; Di Paola, M.; Rizzetto, L.; Tocci, N.; De Filippo, C.; Lionetti, P.; Ardizzoni, A.; Colombari, B.; Paulone, S.; Gut, I.G.; et al. Genomic and Phenotypic Variation in Morphogenetic Networks of Two Candida albicans Isolates Subtends Their Different Pathogenic Potential. Front. Immunol. 2017, 8, 1. [Google Scholar] [CrossRef] [Green Version]
  20. Gerstein, A.C.; Jackson, K.M.; McDonald, T.R.; Wang, Y.; Lueck, B.D.; Bohjanen, S.; Smith, K.D.; Akampurira, A.; Meya, D.B.; Xue, C.; et al. Identification of Pathogen Genomic Differences That Impact Human Immune Response and Disease during Cryptococcus neoformans Infection. mBio 2019, 10, e01440-e19. [Google Scholar] [CrossRef] [Green Version]
  21. Donnelly, J.P.; Chen, S.C.; Kauffman, C.A.; Steinbach, W.J.; Baddley, J.W.; Verweij, P.E.; Clancy, C.J.; Wingard, J.R.; Lockhart, S.R.; Groll, A.H.; et al. Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin. Infect. Dis. 2020, 71, 1367–1376. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Sun, X.; Wang, K.; Yu, X.; Liu, J.; Zhang, H.; Zhou, F.; Xie, B.; Li, S. Transcription factor CCG-8 as a new regulator in the adaptation to antifungal azole stress. Antimicrob. Agents Chemother. 2014, 58, 1434–1442. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Furukawa, T.; van Rhijn, N.; Fraczek, M.; Gsaller, F.; Davies, E.; Carr, P.; Gago, S.; Fortune-Grant, R.; Rahman, S.; Gilsenan, J.M.; et al. The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus. Nat. Commun. 2020, 11, 427. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Irmer, H.; Tarazona, S.; Sasse, C.; Olbermann, P.; Loeffler, J.; Krappmann, S.; Conesa, A.; Braus, G.H. RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior. BMC Genom. 2015, 16, 640. [Google Scholar] [CrossRef] [Green Version]
  25. Alshareef, F.; Robson, G.D. Genetic and virulence variation in an environmental population of the opportunistic pathogen Aspergillus fumigatus. Microbiology 2014, 160, 742–751. [Google Scholar] [CrossRef] [Green Version]
  26. Amarsaikhan, N.; O’Dea, E.M.; Tsoggerel, A.; Owegi, H.; Gillenwater, J.; Templeton, S.P. Isolate-dependent growth, virulence, and cell wall composition in the human pathogen Aspergillus fumigatus. PLoS ONE 2014, 9, e100430. [Google Scholar] [CrossRef] [Green Version]
  27. Kowalski, C.H.; Beattie, S.R.; Fuller, K.K.; McGurk, E.A.; Tang, Y.W.; Hohl, T.M.; Obar, J.J.; Cramer, R.A., Jr. Heterogeneity among Isolates Reveals that Fitness in Low Oxygen Correlates with Aspergillus fumigatus Virulence. mBio 2016, 7, e01515–e01516. [Google Scholar] [CrossRef] [Green Version]
  28. Fortwendel, J.R.; Zhao, W.; Bhabhra, R.; Park, S.; Perlin, D.S.; Askew, D.S.; Rhodes, J.C. A fungus-specific ras homolog contributes to the hyphal growth and virulence of Aspergillus fumigatus. Eukaryot. Cell 2005, 4, 1982–1989. [Google Scholar] [CrossRef] [Green Version]
  29. Mellado, E.; Aufauvre-Brown, A.; Gow, N.A.; Holden, D.W. The Aspergillus fumigatus chsC and chsG genes encode class III chitin synthases with different functions. Mol. Microbiol. 1996, 20, 667–679. [Google Scholar] [CrossRef]
  30. Zhao, W.; Panepinto, J.C.; Fortwendel, J.R.; Fox, L.; Oliver, B.G.; Askew, D.S.; Rhodes, J.C. Deletion of the regulatory subunit of protein kinase A in Aspergillus fumigatus alters morphology, sensitivity to oxidative damage, and virulence. Infect. Immun. 2006, 74, 4865–4874. [Google Scholar] [CrossRef] [Green Version]
  31. Mavridou, E.; Meletiadis, J.; Jancura, P.; Abbas, S.; Arendrup, M.C.; Melchers, W.J.; Heskes, T.; Mouton, J.W.; Verweij, P.E. Composite survival index to compare virulence changes in azole-resistant Aspergillus fumigatus clinical isolates. PLoS ONE 2013, 8, e72280. [Google Scholar] [CrossRef] [PubMed]
  32. Li, H.; Barker, B.M.; Grahl, N.; Puttikamonkul, S.; Bell, J.D.; Craven, K.D.; Cramer, R.A., Jr. The small GTPase RacA mediates intracellular reactive oxygen species production, polarized growth, and virulence in the human fungal pathogen Aspergillus fumigatus. Eukaryot. Cell 2011, 10, 174–186. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Braga, G.U.; Rangel, D.E.; Fernandes, E.K.; Flint, S.D.; Roberts, D.W. Molecular and physiological effects of environmental UV radiation on fungal conidia. Curr. Genet. 2015, 61, 405–425. [Google Scholar] [CrossRef] [PubMed]
  34. Bayry, J.; Beaussart, A.; Dufrene, Y.F.; Sharma, M.; Bansal, K.; Kniemeyer, O.; Aimanianda, V.; Brakhage, A.A.; Kaveri, S.V.; Kwon-Chung, K.J.; et al. Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response. Infect. Immun. 2014, 82, 3141–3153. [Google Scholar] [CrossRef] [Green Version]
  35. Chai, L.Y.; Netea, M.G.; Sugui, J.; Vonk, A.G.; van de Sande, W.W.; Warris, A.; Kwon-Chung, K.J.; Kullberg, B.J. Aspergillus fumigatus conidial melanin modulates host cytokine response. Immunobiology 2010, 215, 915–920. [Google Scholar] [CrossRef] [Green Version]
  36. Mech, F.; Thywissen, A.; Guthke, R.; Brakhage, A.A.; Figge, M.T. Automated image analysis of the host-pathogen interaction between phagocytes and Aspergillus fumigatus. PLoS ONE 2011, 6, e19591. [Google Scholar] [CrossRef] [Green Version]
  37. Amin, S.; Thywissen, A.; Heinekamp, T.; Saluz, H.P.; Brakhage, A.A. Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells. Int. J. Med. Microbiol. 2014, 304, 626–636. [Google Scholar] [CrossRef]
  38. Thywissen, A.; Heinekamp, T.; Dahse, H.M.; Schmaler-Ripcke, J.; Nietzsche, S.; Zipfel, P.F.; Brakhage, A.A. Conidial Dihydroxynaphthalene Melanin of the Human Pathogenic Fungus Aspergillus fumigatus Interferes with the Host Endocytosis Pathway. Front. Microbiol. 2011, 2, 96. [Google Scholar] [CrossRef] [Green Version]
  39. Warris, A.; Ballou, E.R. Oxidative responses and fungal infection biology. Semin. Cell Dev. Biol. 2019, 89, 34–46. [Google Scholar] [CrossRef]
  40. Hohl, T.M. Immune responses to invasive aspergillosis: New understanding and therapeutic opportunities. Curr. Opin. Infect. Dis. 2017, 30, 364–371. [Google Scholar] [CrossRef] [Green Version]
  41. Canteri de Souza, P.; Custodio Caloni, C.; Wilson, D.; Sergio Almeida, R. An Invertebrate Host to Study Fungal Infections, Mycotoxins and Antifungal Drugs: Tenebrio molitor. J. Fungi 2018, 4, 125. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Mondon, P.; De Champs, C.; Donadille, A.; Ambroise-Thomas, P.; Grillot, R. Variation in virulence of Aspergillus fumigatus strains in a murine model of invasive pulmonary aspergillosis. J. Med. Microbiol. 1996, 45, 186–191. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Aufauvre-Brown, A.; Brown, J.S.; Holden, D.W. Comparison of virulence between clinical and environmental isolates of Aspergillus fumigatus. Eur. J. Clin. Microbiol. Infect. Dis. 1998, 17, 778–780. [Google Scholar] [CrossRef] [PubMed]
  44. Sasse, C.; Dunkel, N.; Schafer, T.; Schneider, S.; Dierolf, F.; Ohlsen, K.; Morschhauser, J. The stepwise acquisition of fluconazole resistance mutations causes a gradual loss of fitness in Candida albicans. Mol. Microbiol. 2012, 86, 539–556. [Google Scholar] [CrossRef]
  45. Vincent, B.M.; Lancaster, A.K.; Scherz-Shouval, R.; Whitesell, L.; Lindquist, S. Fitness trade-offs restrict the evolution of resistance to amphotericin B. PLoS Biol. 2013, 11, e1001692. [Google Scholar] [CrossRef]
  46. Valsecchi, I.; Mellado, E.; Beau, R.; Raj, S.; Latge, J.P. Fitness Studies of Azole-Resistant Strains of Aspergillus fumigatus. Antimicrob. Agents Chemother. 2015, 59, 7866–7869. [Google Scholar] [CrossRef] [Green Version]
  47. Lackner, M.; Rambach, G.; Jukic, E.; Sartori, B.; Fritz, J.; Seger, C.; Hagleitner, M.; Speth, C.; Lass-Florl, C. Azole-resistant and -susceptible Aspergillus fumigatus isolates show comparable fitness and azole treatment outcome in immunocompetent mice. Med. Mycol. 2018, 56, 703–710. [Google Scholar] [CrossRef]
  48. Basenko, E.Y.; Pulman, J.A.; Shanmugasundram, A.; Harb, O.S.; Crouch, K.; Starns, D.; Warrenfeltz, S.; Aurrecoechea, C.; Stoeckert, C.J., Jr.; Kissinger, J.C.; et al. FungiDB: An Integrated Bioinformatic Resource for Fungi and Oomycetes. J. Fungi 2018, 4, 39. [Google Scholar] [CrossRef] [Green Version]
  49. Vena, A.; Munoz, P.; Mateos, M.; Guinea, J.; Galar, A.; Pea, F.; Alvarez-Uria, A.; Escribano, P.; Bouza, E. Therapeutic Drug Monitoring of Antifungal Drugs: Another Tool to Improve Patient Outcome? Infect. Dis. Ther. 2020, 9, 137–149. [Google Scholar] [CrossRef] [Green Version]
  50. Sinnollareddy, M.G.; Roberts, J.A.; Lipman, J.; Akova, M.; Bassetti, M.; De Waele, J.J.; Kaukonen, K.M.; Koulenti, D.; Martin, C.; Montravers, P.; et al. Pharmacokinetic variability and exposures of fluconazole, anidulafungin, and caspofungin in intensive care unit patients: Data from multinational Defining Antibiotic Levels in Intensive care unit (DALI) patients Study. Crit. Care. 2015, 19, 33. [Google Scholar] [CrossRef] [Green Version]
  51. Puértolas-Balint, F.; Rossen, J.W.A.; Oliveira dos Santos, C.; Chlebowicz, M.M.A.; Raangs, E.C.; van Putten, M.L.; Sola-Campoy, P.J.; Han, L.; Schmidt, M.; García-Cobos, S. Revealing the Virulence Potential of Clinical and Environmental Aspergillus fumigatus Isolates Using Whole-Genome Sequencing. Front. Microbiol. 2019, 10, 1970. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Jamieson, D.J. Saccharomyces cerevisiae has distinct adaptive responses to both hydrogen peroxide and menadione. J. Bacteriol. 1992, 174, 6678–6681. [Google Scholar] [CrossRef] [Green Version]
  53. Shao, H.; Tu, Y.; Wang, Y.; Jiang, C.; Ma, L.; Hu, Z.; Wang, J.; Zeng, B.; He, B. Oxidative Stress Response of Aspergillus oryzae Induced by Hydrogen Peroxide and Menadione Sodium Bisulfite. Microorganisms 2019, 7, 225. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Morton, C.O.; Varga, J.J.; Hornbach, A.; Mezger, M.; Sennefelder, H.; Kneitz, S.; Kurzai, O.; Krappmann, S.; Einsele, H.; Nierman, W.C.; et al. The temporal dynamics of differential gene expression in Aspergillus fumigatus interacting with human immature dendritic cells in vitro. PLoS ONE 2011, 6, e16016. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Morton, C.O.; Loeffler, J.; De Luca, A.; Frost, S.; Kenny, C.; Duval, S.; Romani, L.; Rogers, T.R. Dynamics of extracellular release of Aspergillus fumigatus DNA and galactomannan during growth in blood and serum. J. Med. Microbiol. 2010, 59, 408–413. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Schmalhorst, P.S.; Krappmann, S.; Vervecken, W.; Rohde, M.; Muller, M.; Braus, G.H.; Contreras, R.; Braun, A.; Bakker, H.; Routier, F.H. Contribution of galactofuranose to the virulence of the opportunistic pathogen Aspergillus fumigatus. Eukaryot. Cell 2008, 7, 1268–1277. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Rhodes, J.C. Aspergillus fumigatus: Growth and virulence. Med. Mycol. 2006, 44 (Suppl. 1), S77–S81. [Google Scholar] [CrossRef] [Green Version]
  58. Yike, I. Fungal proteases and their pathophysiological effects. Mycopathologia 2011, 171, 299–323. [Google Scholar] [CrossRef]
  59. Botelho, N.S.; de Paula, S.B.; Panagio, L.A.; Pinge-Filho, P.; Yamauchi, L.M.; Yamada-Ogatta, S.F. Candida species isolated from urban bats of Londrina-Parana, Brazil and their potential virulence. Zoonoses Public Health 2012, 59, 16–22. [Google Scholar] [CrossRef]
  60. Rajamani, S.; Hilda, A. Plate Assay to Screen Fungi for Proteolytic Activity. Curr. Sci. 1987, 56, 1179–1181. [Google Scholar]
  61. Ferling, I.; Dunn, J.D.; Ferling, A.; Soldati, T.; Hillmann, F. Conidial Melanin of the Human-Pathogenic Fungus Aspergillus fumigatus Disrupts Cell Autonomous Defenses in Amoebae. mBio 2020, 11, e00862-e20. [Google Scholar] [CrossRef] [PubMed]
  62. Heinekamp, T.; Thywissen, A.; Macheleidt, J.; Keller, S.; Valiante, V.; Brakhage, A.A. Aspergillus fumigatus melanins: Interference with the host endocytosis pathway and impact on virulence. Front. Microbiol. 2012, 3, 440. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Kurucz, V.; Kruger, T.; Antal, K.; Dietl, A.M.; Haas, H.; Pocsi, I.; Kniemeyer, O.; Emri, T. Additional oxidative stress reroutes the global response of Aspergillus fumigatus to iron depletion. BMC Genom. 2018, 19, 357. [Google Scholar] [CrossRef] [PubMed]
  64. Ma, Y.; Qiao, J.; Liu, W.; Wan, Z.; Wang, X.; Calderone, R.; Li, R. The sho1 sensor regulates growth, morphology, and oxidant adaptation in Aspergillus fumigatus but is not essential for development of invasive pulmonary aspergillosis. Infect. Immun. 2008, 76, 1695–1701. [Google Scholar] [CrossRef] [Green Version]
  65. Morton, C.O.; de Luca, A.; Romani, L.; Rogers, T.R. RT-qPCR detection of Aspergillus fumigatus RNA in vitro and in a murine model of invasive aspergillosis utilizing the PAXgene(R) and Tempus RNA stabilization systems. Med. Mycol. 2012, 50, 661–666. [Google Scholar] [CrossRef] [Green Version]
  66. Song, L.; Florea, L.; Langmead, B. Lighter: Fast and memory-efficient sequencing error correction without counting. Genome Biol. 2014, 15, 509. [Google Scholar] [CrossRef] [Green Version]
  67. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [Green Version]
  68. Kolmogorov, M.; Raney, B.; Paten, B.; Pham, S. Ragout-a reference-assisted assembly tool for bacterial genomes. Bioinformatics 2014, 30, i302–i309. [Google Scholar] [CrossRef]
  69. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
  70. Treangen, T.J.; Ondov, B.D.; Koren, S.; Phillippy, A.M. The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biol. 2014, 15, 524. [Google Scholar] [CrossRef] [Green Version]
  71. Danecek, P.; Auton, A.; Abecasis, G.; Albers, C.A.; Banks, E.; DePristo, M.A.; Handsaker, R.E.; Lunter, G.; Marth, G.T.; Sherry, S.T.; et al. The variant call format and VCFtools. Bioinformatics 2011, 27, 2156–2158. [Google Scholar] [CrossRef] [PubMed]
  72. Cingolani, P.; Platts, A.; Wang, L.L.; Coon, M.; Nguyen, T.; Wang, L.; Land, S.J.; Lu, X.; Ruden, D.M. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 2012, 6, 80–92. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Priebe, S.; Linde, J.; Albrecht, D.; Guthke, R.; Brakhage, A.A. FungiFun: A web-based application for functional categorization of fungal genes and proteins. Fungal Genet. Biol. 2011, 48, 353–358. [Google Scholar] [CrossRef] [PubMed]
  74. Jones, P.; Binns, D.; Chang, H.Y.; Fraser, M.; Li, W.; McAnulla, C.; McWilliam, H.; Maslen, J.; Mitchell, A.; Nuka, G.; et al. InterProScan 5: Genome-scale protein function classification. Bioinformatics 2014, 30, 1236–1240. [Google Scholar] [CrossRef] [Green Version]
  75. Almagro Armenteros, J.J.; Tsirigos, K.D.; Sonderby, C.K.; Petersen, T.N.; Winther, O.; Brunak, S.; von Heijne, G.; Nielsen, H. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 2019, 37, 420–423. [Google Scholar] [CrossRef]
  76. Aurrecoechea, C.; Barreto, A.; Basenko, E.Y.; Brestelli, J.; Brunk, B.P.; Cade, S.; Crouch, K.; Doherty, R.; Falke, D.; Fischer, S.; et al. EuPathDB: The eukaryotic pathogen genomics database resource. Nucleic. Acids. Res. 2017, 45, D581–D591. [Google Scholar] [CrossRef]
  77. Seemann, T.; Grüning, B. ABRicate. Available online: https://github.com/tseemann/abricate (accessed on 10 March 2020).
  78. El-Khoury, M.; Ligot, R.; Mahoney, S.; Stack, C.M.; Perrone, G.G.; Morton, C.O. The in vitro effects of interferon-gamma, alone or in combination with amphotericin B, tested against the pathogenic fungi Candida albicans and Aspergillus fumigatus. BMC Res. Notes 2017, 10, 364. [Google Scholar] [CrossRef] [Green Version]
Pathogens 11 00428 g001 550
Figure 1. Phenotypic comparison of clinical A. fumigatus isolates; isolates 1–10 originated from patients with no evidence of IA, and isolates 11–15 were isolated from patients diagnosed as having IA. (a) Radial growth rate of coloniser and IA isolates on PDA at 37 °C; (c) diameter of the zone of inhibition on SMA made by colonizer and IA isolates. Blue data points are for coloniser isolates and red data points are for IA isolates. Variation amongst isolates was examined using a Welch’s ANOVA with Dunnet’s T3 post hoc analysis. Results of all vs. all post hoc testing are shown in tile plots (b,d).
Figure 1. Phenotypic comparison of clinical A. fumigatus isolates; isolates 1–10 originated from patients with no evidence of IA, and isolates 11–15 were isolated from patients diagnosed as having IA. (a) Radial growth rate of coloniser and IA isolates on PDA at 37 °C; (c) diameter of the zone of inhibition on SMA made by colonizer and IA isolates. Blue data points are for coloniser isolates and red data points are for IA isolates. Variation amongst isolates was examined using a Welch’s ANOVA with Dunnet’s T3 post hoc analysis. Results of all vs. all post hoc testing are shown in tile plots (b,d).
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Pathogens 11 00428 g002 550
Figure 2. Phenotypic comparison of clinical A. fumigatus isolates, isolates 1–10 originated from patients with no evidence of IA and isolates 11–15 were isolated from patients diagnosed as having IA. (a) Survival of A. fumigatus conidia following 1 min of 1.6 W/m2 UV irradiation; (c) Response of A. fumigatus conidia to acute treatment (3 h exposure) with 50 mM menadione (e) Response of A. fumigatus conidia to acute treatment (3 h exposure) with 50 mM H2O2. Blue data points are for coloniser isolates and red data points are for IA isolates. Variation amongst isolates was examined using a Welch’s ANOVA with Dunnet’s T3 post hoc analysis. Results of all vs. all post hoc testing are shown in tile plots (b,d,f).
Figure 2. Phenotypic comparison of clinical A. fumigatus isolates, isolates 1–10 originated from patients with no evidence of IA and isolates 11–15 were isolated from patients diagnosed as having IA. (a) Survival of A. fumigatus conidia following 1 min of 1.6 W/m2 UV irradiation; (c) Response of A. fumigatus conidia to acute treatment (3 h exposure) with 50 mM menadione (e) Response of A. fumigatus conidia to acute treatment (3 h exposure) with 50 mM H2O2. Blue data points are for coloniser isolates and red data points are for IA isolates. Variation amongst isolates was examined using a Welch’s ANOVA with Dunnet’s T3 post hoc analysis. Results of all vs. all post hoc testing are shown in tile plots (b,d,f).
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Pathogens 11 00428 g003 550
Figure 3. Validation of T. molitor larvae (mealworms) as a model of invasive fungal infections. (a) Survival of mealworms injected with sterile PBS-Tween (0.05% v/v) at the base of 5 different sternites; (b) dose-dependent survival of T. molitor larvae infected with A. fumigatus (Af01) conidia; (c) Kaplan–Meier survival plot of mealworms injected with 5 × 104 A. fumigatus spores from coloniser isolate Af03, IA isolate AF11, or PBST. There were five worms at each time point for only one replicate experiment to enable optimization of fungal quantification. (d) Corresponding fungal load per T. molitor larva, expressed as A. fumigatus CFU per larva or (e) A. fumigatus genomes per larva measured by qPCR. Data presented for CFU and qPCR are mean and standard error for five worms.
Figure 3. Validation of T. molitor larvae (mealworms) as a model of invasive fungal infections. (a) Survival of mealworms injected with sterile PBS-Tween (0.05% v/v) at the base of 5 different sternites; (b) dose-dependent survival of T. molitor larvae infected with A. fumigatus (Af01) conidia; (c) Kaplan–Meier survival plot of mealworms injected with 5 × 104 A. fumigatus spores from coloniser isolate Af03, IA isolate AF11, or PBST. There were five worms at each time point for only one replicate experiment to enable optimization of fungal quantification. (d) Corresponding fungal load per T. molitor larva, expressed as A. fumigatus CFU per larva or (e) A. fumigatus genomes per larva measured by qPCR. Data presented for CFU and qPCR are mean and standard error for five worms.
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Pathogens 11 00428 g004 550
Figure 4. Virulence of clinical A. fumigatus isolates in T. molitor larvae. (a) Kaplan–Meier curve of larval infection by A. fumigatus (infection by both coloniser and IA isolates). The chart is a representative replicate from three replicate experiments. Control represents larvae injected with 5 µL PBS-T; the p-value was determined by log-rank test run on coloniser and IA isolate data. (b) Median survival time of T. molitor larvae injected with 5 × 104 spores and incubated at 37 °C for 7 days. Blue data points are for coloniser isolates and red data points are for IA isolates. Data shown are mean and standard error from three replicate experiments, variation amongst isolates was examined using a Welch’s ANOVA with Dunnet’s T3 post hoc analysis (p = 0.6).
Figure 4. Virulence of clinical A. fumigatus isolates in T. molitor larvae. (a) Kaplan–Meier curve of larval infection by A. fumigatus (infection by both coloniser and IA isolates). The chart is a representative replicate from three replicate experiments. Control represents larvae injected with 5 µL PBS-T; the p-value was determined by log-rank test run on coloniser and IA isolate data. (b) Median survival time of T. molitor larvae injected with 5 × 104 spores and incubated at 37 °C for 7 days. Blue data points are for coloniser isolates and red data points are for IA isolates. Data shown are mean and standard error from three replicate experiments, variation amongst isolates was examined using a Welch’s ANOVA with Dunnet’s T3 post hoc analysis (p = 0.6).
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Table
Table 1. SNVs that occur in IA isolates but not in Coloniser.
Table 1. SNVs that occur in IA isolates but not in Coloniser.
ChromosomePosition 1Mutation 2GeneTranscriptClassAA ChangeAF 3
NC_007198.12422543T>CAFUA_5G09420rna-XM_748604.1missensep.Thr502Ala0.49
NC_007197.189009C>TAFUA_4G00330rna-XM_741330.1missensep.Gly11Glu0.41
NC_007197.195331G>AAFUA_4G00350rna-XM_741328.1missensep.His142Tyr0.41
NC_007197.195364C>GAFUA_4G00350rna-XM_741328.1missensep.Glu131Gln0.41
NC_007197.195399G>AAFUA_4G00350rna-XM_741328.1missensep.Ala119Val0.41
1 1-based; 2 AF293 allele > alternative allele; 3 frequency of alternate allele in FungiDB.
Table
Table 2. Investigating the function of genes potentially important for development of IA.
Table 2. Investigating the function of genes potentially important for development of IA.
PropertyAFUA_5G09420AFUA_4G00350AFUA_4G00330
FunCat ProteinClock controlled protein
(CCG-8)		NoneNone
FunCat Category Cell type differentiationNoneNone
InterProScan Protein familyTranscription factor OPI1Peptidase M54, archaemetzincin-2.
Metallopeptidase domain		None predicted.
LiteratureKnockouts in N. crassa and Fusarium verticillioides
hypersensitise to azoles [22] 		Increased expression following itraconazole treatment [23,24].Increased expression following itraconazole treatment [23,24].
Phobius/TMHMMNoneNone3 TMhelix, 4 Phobius transmembrane domains predicted
Table
Table 3. Presence/absence of genes involved in oxidative stress response (GO:0006979) and melanin biosynthesis (GO:0042438) in the genomes of 10 clinical A. fumigatus isolates.
Table 3. Presence/absence of genes involved in oxidative stress response (GO:0006979) and melanin biosynthesis (GO:0042438) in the genomes of 10 clinical A. fumigatus isolates.
Clinical OriginIsolateNumber of Genes Present in Assembly
Oxidative Stress ResponseMelanin Biosynthesis
ColoniserAf011309
Af021309
Af031309
Af041319
Af061319
Af101299
Proven IAAf111319
Af121309
Af131319
Af141309
Table
Table 4. Isolates of A. fumigatus used in the study sourced from Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital.
Table 4. Isolates of A. fumigatus used in the study sourced from Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital.
Isolate NamePatient ClassificationIsolate Origin
Af01Coloniser 1Sputum
Af02ColoniserSputum
Af03ColoniserBAL 3
Af04ColoniserBAL
Af05ColoniserSputum
Af06ColoniserTissue 4
Af07ColoniserTissue
Af08ColoniserSputum
Af09ColoniserSputum
Af10ColoniserSputum
Af11Proven IA 2Tissue
Af12Proven IABAL
Af13Proven IATissue
Af14Proven IABAL
Af15Proven IATissue
1 Coloniser cases had no evidence of IA [3,6,7]. 2 Patients classified according to the EORTC/MSG criteria [21]. 3 Bronchoalveolar lavage. 4 Cultured from a tissue biopsy.
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El-Kamand, S.; Steiner, M.; Ramirez, C.; Halliday, C.; Chen, S.C.-A.; Papanicolaou, A.; Morton, C.O. Assessing Differences between Clinical Isolates of Aspergillus fumigatus from Cases of Proven Invasive Aspergillosis and Colonizing Isolates with Respect to Phenotype (Virulence in Tenebrio molitor Larvae) and Genotype. Pathogens 2022, 11, 428. https://doi.org/10.3390/pathogens11040428

AMA Style

El-Kamand S, Steiner M, Ramirez C, Halliday C, Chen SC-A, Papanicolaou A, Morton CO. Assessing Differences between Clinical Isolates of Aspergillus fumigatus from Cases of Proven Invasive Aspergillosis and Colonizing Isolates with Respect to Phenotype (Virulence in Tenebrio molitor Larvae) and Genotype. Pathogens. 2022; 11(4):428. https://doi.org/10.3390/pathogens11040428

Chicago/Turabian Style

El-Kamand, Sam, Martina Steiner, Carl Ramirez, Catriona Halliday, Sharon C.-A. Chen, Alexie Papanicolaou, and Charles Oliver Morton. 2022. "Assessing Differences between Clinical Isolates of Aspergillus fumigatus from Cases of Proven Invasive Aspergillosis and Colonizing Isolates with Respect to Phenotype (Virulence in Tenebrio molitor Larvae) and Genotype" Pathogens 11, no. 4: 428. https://doi.org/10.3390/pathogens11040428

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