Hidden allies: Decoding the core endohyphal bacteriome of Aspergillus fumigatus

Hidden allies: Decoding the core endohyphal bacteriome of Aspergillus fumigatus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hidden allies: Decoding the core endohyphal bacteriome of Aspergillus fumigatus Daryna Piontkivska, João M.P. Jorge, Dalila Mil-Homens, Tiago M. Martins, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4912975/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Bacterial-fungal interactions that influence the behavior of one or both organisms are common in nature. Well-studied systems include endosymbiotic relationships that range from transient (facultative) to long-term (obligate) associations. Diverse endohyphal bacteria associate with fungal hosts, emphasizing the need to better comprehend the fungal bacteriome. We evaluated the hypothesis that the human fungal pathogen Aspergillus fumigatus harbors an endohyphal community of bacteria that influence the host phenotype. We analyzed whether 39 A. fumigatus strains, mainly clinical isolates, show stable association with diverse endohyphal bacteria. All fungal strains analyzed were derived from single-conidium cultures that were subjected to antibiotic and heat treatments. Results The fungal bacteriome, inferred through analysis of bacterial diversity within the fungal strains (short- and long- read sequencing methods), revealed the presence of core endohyphal bacterial genera (≤ 19). Microscopic analysis further confirmed the presence of endobacteria within the hyphae of distinct fungal strains. The fungal strains exhibited high genetic diversity and phenotypic heterogeneity in terms of drug susceptibility and virulence (using Galleria mellonella as an infection model). No correlations were observed between genomic or functional traits and bacteriome diversity. However, the abundance of the Bryobacter genus exhibited a positive correlation with fungal virulence; and the presence of other bacteria genera was correlated with posaconazole susceptibility. Based on the genetic pool of the endobacteriome of A. fumigatus both the functional annotation of prokaryotic taxa and the metabolic space could be predicted showing functional roles in major categories, for example, nitrogen fixation and chemoheterotrophy. Conclusions These observations suggest intricated metabolic dependencies between fungal strains and their endohyphal bacteria partners. Our study emphasizes the existence of complex interactions between fungi and bacteria and the need to better understand the relationship between endohyphal bacteria and fungal virulence. Aspergillus fumigatus fungal virulence clinical isolates fungal bacteriome endohyphal bacteria endosymbionts Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Globally, invasive fungal infections are the cause of 1.5 million deaths each year —three times more than malaria and comparable to tuberculosis [ 1 , 2 ]. Recognizing the need for combating fungal infections, the World Health Organization (WHO) identified four fungal priority pathogens, namely Aspergillus fumigatus , Cryptococcus neoformans , Candida auris , and C. albicans [ 3 ]. Aspergillus fumigatus is widespread, in both its geographic and ecological distribution, and it is the primary causative agent of invasive pulmonary aspergillosis - a particularly dangerous condition for immunocompromised individuals [ 4 ]. Furthermore, the escalating prevalence of antifungal resistance and the growing diversity of fungal species that are overcoming host-specific barriers (increasing due to climate change) create major threats to human health [ 5 ]. This threat is further raised by the finding that environmental pollutants also increase the production of virulent A. fumigatus airborne spores [ 6 ]. The combination of all of these factors increases the complexity of treating fungal infections and means that we urgently need reinforced vigilance and innovative strategies for clinical management [ 7 ]. Significant progress has been made in recent years towards understanding the genetic, molecular, and ecological factors that determine the pathogenic potential of A. fumigatus [ 8 , 9 ]. Studies have revealed that the virulence of this species is intricately modulated by a variety of factors, including variations in secondary metabolite production [ 10 ], cell wall composition [ 11 ], and expression levels of virulence-associated genes [ 9 ]. Moreover, genetic polymorphisms in key regulatory genes and signaling pathways have been implicated in shaping the virulence profiles of different A. fumigatus strains [ 9 , 10 ], stressing the complex genetic architecture that underlies pathogenicity. Unlike phytopathogenic fungi, for which specific virulence factors often dictate pathogenicity [ 12 ], the genetic and ecological distinctions that separate pathogenic A. fumigatus strains from their non-pathogenic counterparts remain unknown [ 13 ]. Studies have demonstrated that genetically identical conidia ( i.e. asexual spores) exhibit substantial phenotypic diversity – this has been reported for multiple, different fungal species, including A. fumigatus [ 14 ]. The observed phenotypic plasticity raises questions about the environmental cues that play a crucial role in shaping the pathogenic potential of A. fumigatus - adding yet another layer of complexity to its virulence. Numerous, disparate studies have shown an increasingly intricate interplay between fungi and their bacterial counterparts. While traditionally viewed as independent axenic entities, it has been shown that fungi and bacteria coexist within the same microbial communities [ 15 , 16 ], and that bacteria can also inhabit the internal spaces of fungal hyphae [ 17 ]. These endohyphal bacteria can modulate various aspects of fungal biology, including growth, metabolism, and virulence [ 17 ], through direct physical interactions or through the secretion of bioactive molecules. Furthermore, it was suggested that endohyphal bacterial associates in fungi appear to be the rule rather than the exception, as demonstrated by the examination of 700 phylogenetically diverse fungal isolates, including more than 50 environmental Aspergillus strains [ 15 ]. However, to date, our understanding of the functional complexity of endohyphal bacteria in clinically relevant fungi remains limited. We addressed the hypothesis that A. fumigatus strains harbor endobacterial partners that can influence their hosts’ phenotypes. To validate this hypothesis, we evaluated a set of Aspergillus spp. clinical isolates (n = 40), mostly A. fumigatus (n = 37) and one environmental (soil) A. fumigatus isolate, to systematically analyze the presence of endobacteria. Bacteriome profiling was undertaken using short- and long-read sequencing approaches. All A. fumigatus strains were phenotypically characterized in terms of their drug susceptibility and virulence profiles, and a subset was visualized microscopically. Statistical analyses were used to explore potential correlations between specific endohyphal bacterial partners and the key phenotypic traits of the fungal host. Finally, bioinformatics methods were used to predict the functional niche space within the genetic pool of the endobacteria community. The structure of the core bacteriome, and its implications for the fungal host, are comprehensively discussed. Material and Methods Study design. In this study we tested the hypothesis that the bacterial partners of A. fumigatus strains contribute to their phenotypic variability. Fungal isolates were recovered from patients at the Hospital Santa Maria (HSM) in Lisbon; one isolate, originating from soil, was also included in the study [6]. The taxonomy of each strain was verified through Internal transcribed spacer (ITS) sequencing [6]. Initially, bacteriomes were profiled in three randomly selected strains using a nested PCR 16S touchdown strategy with sequencing of V4 region [18]. In all bacteriome profiling analyses, the Amplicon sequence variants (ASVs), computed using the DADA2 pipeline, were identified using the IDTaxa algorithm ( detailed below ). Each fungal strain was then subjected to both antibiotic and thermal treatment (60 ° C, 1h), followed by the isolation of a microcolony derived from a single conidium in solid medium to bypass any contribution from spores’ heterogeneity. Each individual conidium culture was then propagated whenever fresh spores were required. The strains were genotyped and phenotypically analyzed (antifungal susceptibility and virulence). The endobacteriome of each single conidium strain was systematically analyzed using V3-V4 amplification (n=38) and full length 16S MinIon sequencing (n= 9), using multiple quality controls. In the last subset of fungal strains, the relative proportion of 18S:16S was also quantified in DNA extracted from fungal samples. Bioinformatics analyses always considered the relative abundances of each ASV at distinct taxon levels up to genus. Complementary whole genome sequencing of fungal DNA was also performed (n=2). Attempts to isolate bacteria in aerobic standard conditions were carried out as well. Finally, the bacterial partners inside the hyphae of 20-h old mycelia were visualized through fluorescent microscopy and transmission electron microscopy. Culture media. Aspergillus Minimal Medium (AMM) containing glucose (10 g·L -1 ), thiamine (0,01 g·L -1 ), 5% (v/v) nitrate salts solution [NaNO 3 (120 g·L-1), KCL (10.4 g·L -1 ), MgSO 4 ·7H 2 O (10.4 g·L -1 ) and KH 2 PO 4 (30.4 g·L -1 )], 0.1% (v/v) trace element solution [ZnSO4·7H 2 O (22.0 g·L -1 ), H 3 BO 4 (11.0 g·L -1 ), MnCl 2 ·4H 2 O (5.0 g·L -1 ), FeSO 4 ·7H 2 O (5.0 g·L -1 ), CoCl 2 ·6H 2 O (1.7 g·L -1 ), CuSO4·5H 2 O (1.6 g·L -1 ), Na 2 MoO 4 ·2H 2 O (1.5 g·L -1 ) and Na 4 EDTA (50 g·L -1 )], the final pH was adjusted to 6.5 with NaOH. For solid media 1.5% (w/v) of agar was added. Clinical strain collection Aspergillus strains were isolated by the Laboratory of Microbiology of Hospital Santa Maria (HSM), Lisbon, Portugal, between August and October of 2018 (Table 1, Additional file 2: Dataset 1). The fungal isolates were obtained from sputum specimens or from bronchoalveolar lavage. Samples were inoculated on to Sabouraud Dextrose Agar (SDA) plates containing chloramphenicol and gentamicin (SGA, Biomérieux) and incubated at 35-37 °C for 7 to 10 days. Fungal isolates were collected during routine sampling of patients, and were provided anonymized, without any information that could be used to identify the patient. All fungal isolates were identified phenotypically, at the species level, based on macroscopic colony morphology and micromorphological characteristics. Additionally, fungal isolates were subjected to molecular identification by amplification and sequencing of ribosomal internal transcribed spacers (ITS) as previously reported [6]. For each fungal strain, conidia stocks were prepared and stored at -80 °C until required. In some assays, A. fumigatus reference laboratory strain Af293, purchased from the Fungal Genetics Stock Center (Kansas City, MO, United States), and a soil isolate AEM006 [6], referred as Af_SI.00, were used. Aspergillus single-conidium cultures To minimize inter-spore variability and eliminate transient bacteria, a single-spore culture technique was applied [19]. Additionally, spores underwent a high-temperature treatment. In brief, spore suspensions in 0.85% NaCl solution (100 conidia·mL -1 ) were incubated at 60 °C for 1h to kill most bacteria associated with the spores’ surface. Two bacterial controls were used: Escherichia coli and Hydrobacter perzbergensis . After the heat-treatment, no bacterial colonies were visible after 24h at 37 °C on Luria-Bertani (LB) agar and Reasoner's 2A(R2A) agar plates. Following heat-treatment, aliquots (100 µl) of the conidia were spread on solidified AMM supplemented with 100 mg·L -1 of ciprofloxacin (37 °C, 16-20h). One germinated spore was selected, carefully cut, and transferred to fresh medium (supplemented with 100 mg·L -1 of ciprofloxacin) and further incubated at 37 °C. Conidia were harvested after 5-7 days of growth using a saline solution (NaCl 8.5 g⋅L -1 ) containing 0.01 % Tween ® 20, washed two times with saline solution and collected after passing through three layers of miracloth. The resulting spore suspensions were conserved in saline solution containing 30% (v/v) glycerol at -80 °C until further use. Cultures conditions. Fungal biomass was obtained by growing ~10 8 spores·mL ‑1 in 4 mL of liquid AMM, supplemented with or without 100 mg·L ‑1 of ciprofloxacin, in 6-well plates for 48h. Each fungal strain was grown in a separate 6-well plate. Grown mycelia were removed from the medium surface, immediately frozen, and stored at -80 °C until DNA extraction. DNA extraction. The mycelium biomass was grinded in liquid nitrogen using mortar-pestle, followed by performing 1 cycle of heating (1 h at 90 °C) and sonication (each tube contained 1 g of glass beads per sample, with equal amounts of 0.5- and 0.1-mm beads) for 5 min, with the aid of an extraction buffer (50 mM NaH 2 PO4, 50 mM NaCl, 500 mM Tris-HCl, 5% SDS, pH 8; 1 mL per culture). Afterwards, the sample was mixed with an equal volume 25:24:1 (v) mixture of phenol, chloroform and isoamyl alcohol (solution A), shaken (2 min) and centrifuged (5 min, 2,400 g ) to recover the aqueous phase which was re-extracted with an equal volume 24:1 (v) mixture of chloroform and isoamyl alcohol (solution B) and recovered as described before. To the recovered aqueous phase, 20 µL of RNAse (10 mg·ml -1 ) were added and incubated at 37 ºC for 30 min. The mixture was re-extracted again with solution B as described before. To the recovered aqueous phase, 1/3 volume of 6M NaCl and 1/10 volume of 10% of cetyltrimethylammonium bromide (CTAB) in 0.7M NaCl were added, and the mixture was incubated for 30 min at 65 °C. After cooling to room temperature, an equal volume of solution B was added, shaken, and centrifuged (20 min, 1,400 g ) to recover the supernatant. Finally, DNA was precipitated in 2/3 volume of isopropanol and 1/10 volume of sodium acetate solution (3M) overnight at 4 °C. The precipitated DNA was recovered by centrifugation (20 min, 6,800 g ). The DNA pellet was washed with 200 µL of ethanol (75%), recovered by centrifugation as before, air dried and then eluted in 50-200 µL of TE buffer (Qiagen, Germany) and finally stored at -20 °C. When needed, DNA concentration was measured using a Nanodrop OneC (ThermoFisher, USA). Amplification of the V4 or V3-V4 regions of 16S rRNA gene. Bacteriome profiling relied on amplicon sequencing of the V4 and/or V3-V4 regions of the 16S rRNA gene using DNA extracted from 2-day old mycelium. Nested PCR was used to enhance the bacterial signal: the PCR product of the first amplification with universal primer set fD1/rP2 [20] was used as the template for a second amplification with the nested primer set 515F/806R [18] to amplify the V4 region or 341F/785R [21] to amplify the V3-V4 region of the 16S rRNA gene. The PCR mixture for the remaining amplifications contained 2.5 μL of 10x DreamTaq buffer (includes 20 mM MgCl 2 ), 0.5 μL of 50 mM MgCl2, 0.75 μL of 10 mM dNTPs, 1.5 μL of 0.15 mM BSA, 0.75 μL of DreamTaq DNA Polymerase (5 U·μL − 1 , Thermo Scientific™), 1 μL of 5 μM forward primer, 1 μL of 5 μM reverse primer and 50-400 ng of DNA template, adjusted to a final volume of 25 μL with ultrapure water. PCR amplifications were performed using a T100™ Thermal Cycler (Bio-Rad). The amplification using the 16S rRNA gene universal primers fD1 5’‑AGAGTTTGATCCTGGCTCAG-3’; rP2 5’‑ACGGCTACCTTGTTACGACTT-3’, was performed under the following conditions: 5 min at 96 °C, 10 cycles of 30s at 94 °C, 45s annealing at 62-52 °C (decrements of 1 °C per cycle) and 90s at 72 °C, followed by 20 cycles of 30s at 94 °C, 45 s annealing at 52 °C and 90s at 72 °C, with a final extension step at 72 °C for 5 min. For the primers 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′) the conditions used were: 5 min at 96 °C, 10 cycles of 30s at 94 °C, 45s annealing at 60-50 °C (decrements of 1 °C per cycle) and 90s at 72 °C, followed by 20 cycles of 30s at 94 °C, 45s annealing at 50 °C and 90s at 72 °C, with a final extension step at 72 °C for 5 min. The PCR obtained products were analyzed by agarose-gel electrophoresis. The “no-template” controls (amplification and extraction) were always handled simultaneously, and in a manner consistent with, the DNA samples derived from mycelia. Additionally, all work was conducted according to strict sterile technique ( e.g ., clean workspaces, barrier tips, pre- and post-PCR pipettes) to limit potential cross-contamination. Before further analyses, all PCR products were purified using GeneClean® Turbo Kit (MP Biomedicals™). All samples (region V3 or V3V4) were sequenced on Illumina MiSeq (2 × 300 bp paired-end reads) using the Illumina 16S Metagenomic Sequencing service of STAB Vida Lda., Portugal. Analysis of the V4 or V3-V4 regions of 16S rRNA gene amplicons. Amplicon reads were processed using the divisive amplicon denoising algorithm DADA2 [22] to infer ASVs present in each sample. Default settings were used for filtering and trimming. Built-in training models were utilized to learn error rates for the amplicon dataset. Identical sequencing reads were combined through DADA2’s dereplication functionality, and the DADA2 sequence–variant inference algorithm was applied to each dataset. Subsequently, paired-end reads were merged. Taxonomy assignment was performed using the DECIPHER package with the IDTAXA algorithm [23] that employs principles from machine learning to reduce over classification errors. The closest bacterial hit from the SILVA SSU database r138 [24] was considered, and sequences identified as nonbacterial were discarded. All bacterial genera represented by ASVs identified in DNA amplification or extraction controls were excluded from further analysis. Full-length 16S rRNA gene sequencing on the MinION™ platform. For the library construction 100-200 ng of genomic DNA was used and processed with the 16S barcoding kit (SQK-RAB204, ONT, Oxford, United Kingdom), according to the manufacturer’s instruction with a few modifications. No-template controls (amplification and extraction) were always handled simultaneously and in a manner consistent with the fungal samples. The obtained bar-coded library was sequenced in a FLO-MIN106 flow cell using the MinION Mk1C device (ONT). MinKNOW version 21.05.25 (ONT) and Guppy 5.0.16 were used for data acquisition. A total of 2.79 M reads were generated. Analysis of full-length 16S rRNA gene. From the amplicon reads obtained, only those that contained both forward and reverse primers were used for further processing (in total 22,666 reads). To reduce redundancy and to increase the efficiency of downstream analyses, reads were clustered into OTUs based on phylogeny-derived distances, using the tip_glom function in the phyloseq R package [25] (tree-height threshold for similarity: h = 0.2). The OTUs were taxonomically identified against the SILVA SSU database r138 [24] using the blastn tool, the closest bacterial hit was considered, and sequences identified as nonbacterial were discarded. All bacterial OTUs identified in DNA amplification or extraction controls were excluded from further analysis (Additional file 1: Fig. S1). Microsatellite genotyping. Microsatellite genotyping was used to determine the genetic distances between the Aspergillus fumigatus clinical isolates. Genotyping was performed by CD Genomics (NY, USA) with a panel of nine short tandem repeats (TRs) as previously described [26]. In brief, three separate multiplex PCRs amplifications were performed to obtain three dinucleotide, three trinucleotide and three tetranucleotide loci fragments. Each PCR mixture contained 1x reaction buffer, 0.3 mM of corresponding amplification primers, 0.2 mM deoxynucleotide triphosphates, 0.5 U of Hot Start Taq DNA Polymerase and 10 ng of genomic DNA. Amplification was performed using the following thermal protocol: 5 min of initial denaturation at 95 °C, followed by 35 cycles of 30s of denaturation at 95 °C, 30s of annealing at 60 °C and 30s of extension at 72 °C, with final extension for 30 min at 60 °C. The fragments obtained were denatured at 95 °C for 3 min in a mixture containing 1.0 μl of PCR product, molecular weight internal standard and 0.05 % formamide. The fragment analysis was performed on the Applied Biosystems 3730xl DNA Analyzer, and the sizes determined using GeneMapper Software 5. In vivo infection with Galleria mellonella. Infection studies were performed as previously described using G. mellonella [6]. Briefly, G. mellonella larvae were reared in darkness at 25°C, from egg to last instar larvae, on a natural diet of beeswax and pollen grains. For the experiments, final instar larvae weighing 200 ± 25 mg were selected (n=30 per condition). Aliquots of the stock spore suspensions (derived from single-conidium cultures, see above) were used to adjust the inoculum to a concentration of 10 7 spores per larva for each tested condition. A microinjection system was used to control the volume of a microsyringe and inject 5 μl of spore suspension into each larva via the hindmost left proleg, previously sanitized with 70% (v/v) ethanol. Following injection, larvae were placed in Petri dishes and stored in the dark at 37 °C. Control larvae were injected with saline solution (pH 7.4). For each condition (n=30) larval survival was followed for 96 h. Caterpillars were considered dead when they displayed no movement in response to touch. Minimal Inhibitory Concentrations (MIC) of antifungals. The minimal inhibitory concentrations (MIC) were determined using a microbroth dilution format according to the EUCAST reference method [27]. Tests were performed using RPMI-1640 medium (R6504, with L-glutamine and without sodium bicarbonate) supplemented with glucose to a final concentration of 2% and 0.165 mol·L -1 of 3-(N-morpholino) propanesulfonic acid (MOPS) with pH adjusted to 7.0. The concentrations of antifungal drugs tested ranged from 16 to 0.03 mg·L -1 for amphotericin B and posaconazole, and from 8 to 0.016 mg·L -1 for voriconazole. Spore suspensions were prepared to a final working concentration of 10 6 conidia·mL -1 . Growth and negative controls were included in all tests. The microplates were incubated at 37 °C for 48h. The lowest concentration that exhibited no growth under microscopic observation was considered to be the MIC. Microscopy. Fluorescence microscopy . Hydrogel media discs were prepared for use in fluorescence microscopy. Discs were prepared using AMM followed by the addition of Phytagel to a final concentration of 4% w/v. The hydrogel slide preparation was adapted from methods described previously [28, 29]. Briefly, the media was autoclaved and kept in a water bath at 90 ºC. Afterwards the media was cast between two standard microscope slides (VWR, previously washed with ethanol and UV sterilized) and set to solidify under sterile conditions in a biosafety cabinet. The slides were then carefully separated using a sterile razor blade. Using a sterile 1 mL pipette tip, circular discs (~7 mm diameter) were stamped out. These discs were transferred to a 6-well plate, inoculated with 1 µL of a suspension of ~10 9 spores·mL -1 , then incubated at 37 °C. To avoid drying of the discs during the incubation period, UV sterilized paper towels saturated with sterile water were folded and placed in the space between the wells. After 20 h of incubation, the discs were fixed with 4% paraformaldehyde in PBS and incubated overnight at 4 °C. The fixed discs were washed three times with 1x PBS and stored in a solution of PBS with 0.1 U/µL Superase RNAse inhibitor with 10 mM Ribonucleoside Vanadyl Complex (RVC) at 4 °C until needed. Before use, the discs were washed three times with 1x PBS, dehydrated with ethanol-PBS solutions with 50 %, 75 %, 100 %, 75 %, 50 % ethanol, rinsed with PBS for 3 min at room temperature, and after removal of PBS were left to air dry for 10 min. For staining, each sample was first covered with 2 µM syto9 (Invitrogen, 5 min, room temperature, dark) and after washing with PBS, samples were covered with 20 µM calcofluor-white (Sigma, 3 min, room temperature, dark), washed again with PBS, then transferred to a microscope slide with a cover slip on the top of the disc. Some samples were stained with 5 µg/mL Hoechst 33342 (Thermo Fischer Scientific, dark, 5 min) before the calcofluor-white staining. Mycelia and bacteria were visualized using a Zeiss LSM 880 microscope equipped with a Fast Airyscan. ImageJ was used for image analysis. Transmission electron microscopy (TEM). Mycelia (SDA medium, 36 h, 37 ºC) were fixed with a solution of 2% (v/v) formaldehyde (Science Services), 2.5% glutaraldehyde (v/v) (Science Services) in 0.1 M cacodylate buffer (30 min, room temperature), then washed three times with 0.1 M cacodylate buffer. Fungal samples were sandwiched between 0.2 µm flat aluminum disks and frozen in a high-pressure freezer (Wohlwend Compact 2) using as filler 0.5% (w/v) low-melting point agarose (OmniPur) in 0.1 M cacodylate buffer at 37 ºC. These samples were placed in the freeze substitution device (Leica AFS2) at -90 ºC in a solution with 2% (w/v) osmium tetroxide (Science Services), 0.1% (w/v) uranyl acetate (Analar) with 1% distilled H 2 O in acetone. After 60 h, the samples were slowly warmed at a rate of 2 ºC/h: first to -60 ºC, kept at this temperature for 10 h, then to -30ºC and kept at this temperature for 10 h. Subsequently, using the maximum warming rate, samples were brought to 0 ºC and dehydrated with acetone (3 times, 10 min each). Dehydrated samples were infiltrated with increasing amounts of Embed-812 Epoxy Resin (Science Services) (5% 4 h, 10% overnight, 25% 4 h, 50% overnight, 75% 4 h, 100% overnight) and polymerized in fresh resin at 60 ºC for 48 h. Ultrathin sections (70 nm) were cut using an ultramicrotome (Leica UC7), picked-up in 1% formvar (Agar Scientific) in chloroform coated slot grids, and stained at room temperature with 1% (w/v) uranyl acetate and Reynolds lead citrate, for 5 min each. The grids were observed using a TEM (FEI Tecnai G2 Spirit BioTWIN with an Olympus-SIS Veleta CCD Camera) at 120 kV. Bioinformatics analysis. Phylogenetic distance was determined using the general time reversible model with the rate variation among sites described by a gamma distribution and the proportion of invariable sites (GTR + G + I model). The minimum spanning network, to determine genetic diversity between strains, was determined using Bruvo’s distance, and the discriminant analysis of principal components (DAPC) was performed using the R package poppr [30]. Data obtained in this study was compared with a subset of isolates from the databank for clinical and environmental A. fumigatus strains, which is based at the Canisius Wilhelmina Hospital in Nijmegen, Netherlands, and available at the afumID website [31]. Whole-genome sequencing . The quality and quantity of DNA were accessed by electrophoresis and using the Qubit dsDNA BR kit, following AMpureXP cleaning. For 150 bp pair-end DNA sequencing, libraries were generated using the Kapa HyperPrep kit (Roche) according to the manufacturer’s instructions and integrity accessed using a TapeStation 4200. Samples were indexed and sequenced on the lllumina Novaseq platform (≥30 million reads per sample). Quality control of reads was carried out using FastQC (v0.12.1) and trimmomatic (v0.39) [32]. The surviving reads (>99%) from each library were aligned to a library of three A. fumigatus reference genomes (for strains Af293, A1163 and CEA10, respectively ASM265v1, ASM15014v1 and CP097563 to CP097570) using segemehl (v0.3.4) [33]. Metagenomic analysis of the remaining reads (≤2%) was conducted using Kraken (v2.1.3) with PlusPF Refseq indexes as of June 5, 2023 [34] and Recentrifuge (v1.12.1) [35]. Reads were also aligned to specific bacterial genomes as described above, and genome coverage determined using Samtools (v1.17) [36]. Microbiome function prediction. FAPROTAX and PICRUSt2 were used to predict the ecologically relevant functions of the microbiomes from the fungal isolates [37, 38]. Both analyses employed default parameters using relative abundance ASVs from the bacterial V3-V4 hypervariable region of 16S rRNA sequences. For simplicity in the presentation of the PICRUSt2 prediction, MetaCyc pathways were categorized into class pathways. Results and Discussion Our main working hypothesis is that endohyphal bacteria contribute to the phenotypic heterogeneity observed in A. fumigatus . To address this hypothesis, Aspergillus clinical isolates were primarily isolated from patients at HSM in Lisbon, Portugal and were taxonomically classified as A. fumigatus (n=37), A. terreus , A. niger , and A. flavus (n=1, each) (Table 1). Additional strains used in some assay were an A. fumigatus soil isolate (Af_SI.00) [6] and the reference strain Af293. Setting-up a framework to analyze endohyphal bacterial partners of A. fumigatus clinical isolates The presence of endohyphal bacterial associates was initially tested in three randomly selected A. fumigatus strains to address two outstanding questions: i) if antibiotic treatment eliminates ephemeral bacterial associates; and ii) if cultures generated from a single conidium display a lower diversity of bacterial associates than those derived from multiple conidia. Bacterial profiling was systematically performed by amplicon sequencing of the hypervariable region V4 of 16S rRNA gene. The ASVs obtained were taxonomically identified through the IDTaxa algorithm [23] using the SILVA 16S database as a training set [24]. Due to the low taxonomic resolution of the short-read sequences analyzed here, the bacterial ASVs identified are displayed at class or family level. In order to evaluate the impact of antibiotic selection, the composition of the bacterial community in mycelia grown in media supplemented with high-dose ciprofloxacin (a broad-spectrum antibiotic) was compared to that from mycelia grown in media without antibiotic. A total of 233 unique ASVs were identified across the 6 samples examined (Additional file 1: Fig. S2A). Among these, 30 ASVs were present in the mycelia that were not subjected to the antibiotic selection, indicating that there are bacterial partners that could be eliminated from mycelia by treatment with antibiotic. This result suggests these bacteria are likely not stable endohyphal partners of A. fumigatus . One hundred and fifty-six ASVs were only detected after antibiotic treatment, suggesting that the antibiotic selection induced a very noticeable shift in the composition of the bacterial community, allowing detection of less abundant bacteria. Interestingly, 47 ASVs were found at relative high abundances, in mycelia cultivated in media both without and with antibiotic selection (Additional file 1: Fig. S2A). This observation implies a strong association between these ASVs and mycelia, suggesting the existence of protection mechanisms within hyphae. A phylogenetic analysis focusing on the top 100 most abundant ASVs was undertaken (representing ~98% and ~92% of the total relative abundance in the absence and presence of antibiotic, respectively) to better understand their distribution across different fungal strains and conditions. The results highlighted that there were major alterations in the composition of the bacterial community post-antibiotic selection, specifically a heightened community diversity with a clear dominance of ASVs belonging to the Bacteroidia, Alphaproteobacteria, and Polyangia classes (Fig. 1A). For a given fungal strain, no significant correlation was detected between the untreated control and the sample subjected to antibiotic selection. In contrast, a strong positive Pearson’s correlation ( p ≤0.0001) was identified among the three different fungal strains under the same conditions. These results suggest that the bacteriomes of the three fungal strains are similar under pre- or post-antibiotic treatment, but not across conditions (Additional file 1: Fig S2B). The complexity of the mycelial bacteriome led to the decision to isolate single-conidium cultures – with the aim of eliminating any contribution from conidia heterogeneity to the overall bacterial diversity. Additionally, as some bacteria outside of conidia may survive the antibiotic treatment, conidia were also subjected to heat stress (60°C, 1 h) in addition to the antibiotic pressure. The conditions were optimized using two axenic bacteria cultures ( E. coli and H. perzbergensis ). In contrast to some of the conidia, none of the bacteria could survive the heat treatment. We generated single-conidium cultures (n=3 per isolate) from three fungal isolates and analyzed the associated bacteriomes. The results showed similar bacteriomes across the single-conidium colonies from the same fungal strain and for the three different fungal strains tested (Fig. 1B). The resulting heatmap highlights the similar bacterial composition and the distribution of relative abundance across the sample set at the class level (Fig. 1B). Further examination, through phylogenetic analysis, focusing on the top 100 most abundant bacterial ASVs (representing ~95% of the total abundance), showed consistent results: all single-conidium colony cultures show a similar bacteriome, regardless of the parental fungal strain (Additional file 1: Fig. S3). The same inference can be derived from beta diversity analyses based on both weighted and unweighted Unifrac distances - also showing the similarity of the bacteriomes analyzed (Fig. 1C). Collectively, the results showed remarkable similarity for the hyphal bacteriomes across three distinct fungal strains, inoculated from conidia subjected to heat treatment in an antibiotic selection medium. All subsequent experiments used a randomly selected single-conidium colony per Aspergillus spp. strain. Determining the range of genotypic diversity and phenotypic heterogeneity of the A. fumigatus clinical isolates Most of the A. fumigatus clinical isolates (n=37 in total) originated from patients with cystic fibrosis (n=29, 78%). Overall, the bulk of the samples were isolated from sputum; the exceptions were from ear secretions (n=2), lung biopsies (n=2), and bronchoalveolar lavage (n=2). In some cases, two isolates were sourced from the same patient (Table 1, Fig. 2). We have used microsatellite genotyping to achieve a higher discriminatory power within A. fumigatus [26]. First, we tested if the geographic origin of the A. fumigatus strains from HSM in Lisbon impact on their genetic diversity. Therefore, our genotyping results (Additional file 3: Dataset 2) were compared with those of clinical isolates found in other countries [31]. The minimum spanning network displaying the relatedness between the isolates showed that the 37 clinical strains exhibit considerable genetic variability (Additional file 1: Fig. S4). This result suggests that genotypic diversity within isolates of A. fumigatus surpasses geographic boundaries, consistent with previous studies [31]. Distinct lineages within the A. fumigatus strains were identified through hierarchical cluster analysis using the Gower dissimilarity index (Fig. 2). The strains Af293 (lab strain) and Af_SI.00 (isolated from soil) were included for comparison purposes. Clustering with a maximum dissimilarity threshold of 10% revealed the presence of 27 clusters, highlighting the strains genotypic diversity. Four pairs of strains were found to be genotypically identical (Fig. 2) and just one of those pairs was isolated from the same patient (Af_CI.19 and Af_CI.44). We then analyzed the phenotypic heterogeneity of A. fumigatus strains, assessed in terms of their drug-resistance and virulence profiles (n=39). Minimal inhibitory concentrations (MICs) analyzed by the EUCAST method [27] ranged from 16 to 0.03 mg·L -1 for amphotericin B and voriconazole, and from 8 to 0.016 mg·L -1 for posaconazole (Additional file 1: Table S1). Overall, the results show significant heterogeneity in the drug-susceptibility profiles, with no apparent clustering to their lineage as depicted in Fig. 2. In vivo infection capacity of each A. fumigatus strains was assessed using G. mellonella as the infection model (n=38, plus the Af293 strain). This model is widely recognized for evaluating the virulence of microbial pathogens [39], particularly fungal pathogens [40], and demonstrates a reliable correlation with murine models [41]. Following a 96-hour post-infection period, the fungal strains exhibited varying degrees of infection capacity. According to the survival probability range, the infection index was classified as low (>75%); medium-low (75%-50%); medium-high (50%-25%); and high (<25%) (Fig. 2; Additional file 1: Fig S5). The virulence results did not exhibit any clustering with the isolates’ lineages. Collectively these results show that the 37 A. fumigatus clinical strains characterized in this study, sampled within close geographic proximity, exhibit substantial genotypic diversity and phenotypic heterogeneity in terms of their virulence and drug-resistance profiles, consistent with previous reports [42]. Neither antifungal susceptibility nor genotyping lineage correlated with virulence potential. Determining the core bacteriome of A. fumigatus clinical isolates To identify the A. fumigatus core bacteriome, all 37 clinical strains and the Af_SI.00 strain were profiled through sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. Prior to this analysis, we verified in a sub-set of strains (n=9) that the relative proportions of 18S:16S quantified via RT- q PCR were consistent in the DNA extracts of both spores and 48h-old mycelia (Additional file 1: Table S2). Bacterial DNA is consistently present in all DNA samples derived from either fungal source (except for AF_CI.08 that presents a slow growth rate), ranging from 0.35% to 1.6% of bacterial DNA in the total DNA. All analyzed A. fumigatus strains (n=38) exhibited a consistent bacteriome profile at the genus level (Fig. 3A). This result suggests stability in the bacterial community across all tested fungal strains. Such stability expands to the bacteriome profile of the other clinical Aspergillus spp. strains, namely A. terreus, A. niger, and A. flavus (n=1, each) (Additional file 1: Fig. S6). This observation supports the notion that the bacterial communities associated with distinct aspergilla originate from shared ecological niches and/or functional roles – this is deserving of a more focused analysis in the future. Evaluation of prevalence at the genus level highlighted the presence of 19 core bacteria genera, of which 11 were present in all A. fumigatus strains (Fig. 3B). Considering the relative abundance, Gemmata spp. was the most prevalent genus (5%), followed by the Burkholderia-Caballeronia-Paraburkholderia (BCP) group, Bradyrhizobium, Puia, Rastonia and Edaphobacter spp. (1%, each), Acidobacter and Sediminibacterium (0.5%, each), and the Methylobacterium-Methylorubrum (MM) group , Afipia , and Nevskia (<0.1%) (Fig. 3B). Most of the core bacterial genera match multiple ASVs identifications, likely due to the presence of closely related variants within each genus. Phylogenetic analysis of the core bacteriome tree suggests that these ASVs are closely related and likely represent the same bacterial species (Additional file 1: Fig S7). Importantly, bacteria detected in all quality controls (amplification and extraction negative controls) were systematically excluded from all bacteriome profiling analyses (Additional file 1: Fig. S1). To further test the presence of a core bacteriome, a subset of A. fumigatus strains (n=9, Additional file 1: Table S2) were profiled using long-read sequencing of 16S rRNA amplicons on the Oxford Nanopore MinION platform. The utilization of long-read sequencing techniques offers a deeper insight into microbial communities [43], and may potentially validate the identification performed using the shorter-amplified sequences. This approach yielded a total of 49 bacterial genera. However, the majority of these genera are associated with a few strains at remarkably low abundance (Fig. 4A). The potential roles of rare bacteria in the fungal bacteriome certainly warrant further investigation. In total, 11 core bacterial genera were identified, of which 7 were found consistently across all 9 isolates, including Rastonia , BCP group, MM group, Sphingomonas, Xylophilus, Bradyrhizobium, and Mucilaginibcater (Fig. 4B). The two core bacteriomes identified have 8 bacterial genera in common (Fig. 4C). This result highlights the stability of certain bacterial genera across different sequencing methods. Bacteria genera consistently identified within the core bacteriome may play key roles in the structure and function of this microbial community. Metabolic dependencies, promoting group survival in nutritionally challenging conditions is a well-established principle within metacommunities. Such dependencies are thought to play a significant role in species co-occurrence and are indicative of the presence of regular cooperative groups within microbial community architectures [44]. This concept also underlies the formation and structure of holobiont systems. Intercellular bacterial partners of eukaryotic hosts, including endosymbionts, can provide primary metabolic pathways (for example, photosynthesis) or expand the repertoire of secondary metabolism, while also influencing the host’s fitness, growth, development, behavior, and other functions [45, 46]. In this respect, most of the core endohyphal bacteria genera identified herein can be found in the soil habitat and demonstrate viability in acidic and low nutrient (oligotrophic) environments. Some are rare bacterial genera with only a few species known ( e.g. Gemmata , Puia , Acidibacter and Xylophilus ) [47-50], while others possess diverse genomic features. Many are known to form symbiotic relationships with eukaryotes and their chemoheterotrophy, suggesting the potential for biological N fixation, for example Ralstonia [51, 52], Bryobacter [53], Bradyrhizobium [54, 55], the BCP group [56] and Sphingomonas [57]. Based on the genetic pool of the endohyphal microbiota of A. fumigatus (inferred from the V3-V4 amplicons, Fig. 3), both the functional annotation of prokaryotic taxa (FAPROTAX) [37] and the metabolic space could be predicted (PICRUSt2 v2.1.4) [38, 58], as applied in other related studies [59]. The results show potential functional roles, mostly in categories such as chemoheterotrophy and nitrogen fixation, followed by human- pathogens/associated and animal parasites or symbionts (Fig. 3C), as well as Amino acid metabolism, Lipid metabolism, and Cofactor, Carrier and Vitamin biosynthesis (Additional file 1: Fig. S8). As performed in other studies [60-62], we subjected two A. fumigatus strains (Af_CI.002 and Af_CI.12) to whole-genome sequencing (WGS). The acquired data revealed a very low abundance of sequences not matching A. fumigatus (0.36-0.47 %) with the majority unclassified. This result is consistent with that inferred through the relative abundance of 18S/16S amplified from fungal DNA samples (Additional file 1: Table S2). It is however relevant that a predominant bacteria genus could be identified: Bradyrhizobium (Additional file 1: Fig S9), with several sequences aligning, at distinct regions, with a reference genome of B. guangzhouense (CCBAU 51670, acc. no. CP030053). This bacterium genus was consistently identified within the core bacteriome using other sequencing methods (Figs. 3 and 4), hence further validating its presence within the mycelia. Disclosing the full genome of predominant endohyphal bacteria would allow a better understanding of the core endobacteriome. However, due to the low abundance of bacteria DNA, such future studies will require multiple rounds of optimization to identify cultivation conditions and DNA processing methods that result in higher yields of endobacterial DNA. To further explore the fungal bacterial association, endohyphal bacteria were visualized under the microscope. Aspergillus fumigatus 20h-old hyphae (n=6, within those used for the long-read sequencing analyses) were stained with styo9, a dye commonly used to label nucleic acids and especially effective in staining endobacteria within fungi [63, 64]. The endobacteria were clearly observed within the hyphae of all analyzed fungal strains (Fig. 5A). None of these endohyphal bacteria could be cultured aerobically using standard growth protocols. Fungal nuclei, stained with Hoechst (blue), have a distinctive morphology and larger size than the syto9 stained endobacteria (green) (Fig. 5B). The direct visualization of endohyphal bacteria further validates the bacteriome profiling results. To further verify the presence of endobacteria, transmission electron microscopy of one strain of A. fumigatus was done, as applied before by others [65]. The mycelium from solid culture was frozen at high pressure and cryosubstituted in osmium tetroxide and uranyl acetate. Examination of the samples confirmed the presence of endohyphal bacteria within the cytosol of intact cells of A. fumigatus (Fig. 5C). Further assays are needed to identify conditions that allow them to grow outside of the fungal host. The long-read amplicons could match the identification of bacteria up to the species level, yet most identified species matched those of uncultivable bacteria (data not shown). Identifying endohyphal bacteria that potential impact the virulence and drug-resistance profiles of A. fumigatus clinical isolates To test the hypothesis that the endohyphal bacteriome contributes phenotypic heterogeneity of A. fumigatus virulence (i.e. in vivo infection capacity) and drug susceptibility, hierarchical clustering analyses were first applied using Bray-Curtis distances. Based on the data, the fungal bacteriome (genus level) did not correlate with either virulence or drug susceptibility (Fig. 6A, Additional file 1: Fig. S10A). This finding is consistent with the observation that all A. fumigatus strains (n=38) exhibited a clear core bacteriome regardless of their distinct phenotypic characteristics. The same conclusion – bacterial diversity did not correlate with the fungal phenotype – could be inferred from testing only the bacteria genera that contribute to variations in the bacteriome, i.e. only the rare endohyphal bacteria without the core bacteria (Additional file 1: Fig. S10B). Finally, we tested if the abundance of specific core endohyphal bacteria shows correlation with the analyzed fungal phenotypic traits using Spearman’s correlation analyses (Fig. 6B, Additional file 4: Dataset 3). The results of the correlation coefficients (r s ) suggest that endohyphal bacterial diversity did not correlate with susceptibility to either amphotericin B or voriconazole, with the exception of a negative correlation of MM abundance with voriconazole-susceptibility. However, posaconazole susceptibility showed positive correlations with abundance of Bryobacter (r s = 0.34) , Hydrobacter (r s = 0.34), Nevskia (r s = 0.39), and Brevundimonas (r s = 0.33), and negative correlations with the abundance of Singulisphaera (r s = -0.41), Aquabacterium (r s = ‑0.38), and Staphylococcus . This result suggests that specific bacterial genera may indeed influence the susceptibility of A. fumigatus to posaconazole treatment. Such protective mechanisms may be related to the bacterial chemoheterotrophy and ability to degrade the antifungal drug. Only Bryobacter exhibited a positive correlation (r s =0.45) with fungal virulence, possibly the genus abundance directly correlates with the in vivo infection capacity of the host. On the contrary, the abundance of either Stenotrophomonas (r s = -0.58) , Hydrobacter (r s = -0.43), Sphingobacterium (r s = ‑0.48), or Brevundimonas (r s = -0.33 ) genera displayed a negative correlation with fungal virulence. Overall, the correlational analyses highlighted a possible relationship between the abundance of a specific endohyphal bacteria genus within the core bacteriome of A. fumigatus and its virulence and drug susceptibility. Focused in-depth analyses are needed to better understand how core endohyphal bacteria impact the phenotypic traits of the fungal host, especially those that are clinically relevant. Conclusions We formulated a hypothesis that A. fumigatus strains harbor diverse core endohyphal bacteria that may contribute to its phenotypic heterogeneity. Amplicon sequencing of the 16S rRNA gene revealed a dynamic bacterial landscape, where antibiotic selection induced significant shifts in the composition of the community, with certain bacterial taxa persisting (Fig. 1 ). This initial result indicates that some bacteria taxa may exhibit more robust associations with their fungal hosts. Sequencing analyses, spanning both short- and long- read platforms, of single conidium-derived colonies of A. fumigatus strains (n = 38), which are genotypically diverse (Fig. 2 ), highlighted a core bacteriome (Figs. 3 and 4 ). This result makes it clear that conidia heterogeneity did not contribute to the bacteriome diversity observed. The A. fumigatus strains (n = 38) displayed high phenotypic heterogeneity in their antifungal susceptibility and virulence profiles, without clear correlation with their genotypic diversity (Fig. 2 ). This finding underscore, as is often reported, the multifaceted nature of A. fumigatus strains [ 14 ]. Microscopy analysis visually confirmed the presence of endobacteria - located within hyphae (Fig. 5 ). The role of the core bacteriome in expanding the nutrient assimilation capacity of A. fumigatus , as well as the ecological niches that it can occupy, remains hypothetical but is supported by the functional annotation of chemoheterotrophy and nitrogen fixation, among others (Fig. 3 C, Additional file 1: Fig. S8). The remarkable diversity of the fungal bacteriome at taxonomic, functional, and lifestyle levels has been reported in other studies [ 15 ]. Our data further challenges the paradigm of axenic fungi or limited association of fungi with only one [ 17 ] or two [ 57 ] endohyphal bacteria. Several reasons support the presence of a core bacteriome. Firstly, the genetic relationship of the fungal strains analyzed was similar to those from distant geographic locations (Additional file 1: Fig. S4 ) and was independent of proximity. Secondly, transient bacterial associates were efficiently removed through antibiotic pressure and heat-shock (Fig. 1 ). Thirdly, single-conidium cultures were consistently used in all assays to avoid heterogeneity within conidia populations (Fig. 2 ). The phylogenies were constructed from sequences obtained using culture-independent methods, ensuring an unbiased representation of endohyphal bacterial diversity. Rigorous bioinformatic methods were employed, and potential sequencing artefacts from cross-contaminants were systematically removed [ 66 ], in agreement with the highest standards for analyzing complex metagenomes [ 67 ]. Finally, the combined use of short- and long- read platforms is recognized as the optimal strategy for generating robust datasets [ 68 ]. Symbiosis drives the acquisition of adaptive traits, ecological range expansion and biodiversity. The transition to obligate interspecific mutualism marks a major evolutionary step [ 69 ], seen in fungal hosts unable to replicate without their symbionts [ 17 ]. While the roles of endohyphal bacteria in A. fumigatus are not fully determined, data suggest a close relationship between the fungi and their core bacterial associates. The presence of a core bacteriome across various aspergilla strains (n = 40) (Figs. 3 and 4 ; Additional file 1: Fig. S6) implies vertically transmitted endosymbionts. Gram-negative bacteria dominate this core bacteriome (Figs. 3 and 4 ) - a group crucial in shaping life on Earth and contributing to essential endosymbionts [ 70 ]. Fungi from all major phyla can harbor bacterial endosymbionts, primarily Gram-negative, with obligate associations in early diverging fungi such as Mucoromycota and facultative associations in more derived lineages [ 46 , 71 ]. Examples of symbiosis include intracellular bacterial symbionts in insects providing essential nutrients [ 72 ], and the Rhizopus microsporus and Burkholderia sp. partnership, where bacteria produce rhizoxin to aid fungal pathogenicity [ 73 ]. The identified core endohyphal bacteria likely constitute endosymbiotic partners of A. fumigatus . Although mutual dependence is not conclusively established, the inability to cure the host and culture the endohyphal bacteria, along with the observed metabolic enrichment (Fig. 3 C, Additional file 1: Fig. S8), supports this possibility. The prevalence of gram-negative endohyphal bacteria in the core bacteriome of A. fumigatus (Figs. 3 and 4 ) may be due to their ability to utilize the thick-walled structures of conidia as a temporary, protective habitat, similar to gram-negative bacterial residents in chlamydospores [ 74 ]. These bacteria have a thinner peptidoglycan layer between two membranes offering a superior functional capacity compared to that of gram-positive bacteria [ 75 ]. However, the stable bacterial diversity within mycelia, as well as the inability to remove or easily culture them outside of the host, suggests that these gram-negative bacteria are true endosymbionts. The fungal diversification timeline dates back to the Jurassic period, with aspergilli established in the Cretaceous period [ 76 ]. It is hypothesized that aspergilli acquired endohyphal bacteria around this time, occurring in parallel to the recruitment of fungi by plants. Fungal mutualisms with angiosperms allowed saprophytic fungi to diversify, providing water and mineral nutrients to plants through mycorrhizal root systems [ 54 , 77 ]. This relationship facilitated the expansion of the ectomycorrhizal fungal genera during the Cretaceous period and thus promoted angiosperm diversification. Although a conserved genetic toolkit for symbiotic nitrogen fixation exists in plants [ 78 ], no similar system has been identified in fungi, even in well-studied obligate endosymbiotic systems in the Mucoromycota phylum [ 79 ]. Our study provides important insights into the complex interplay between A. fumigatus and its associated endohyphal bacteria, establishing the existence of a potentially clinically relevant core bacteriome. The finding that Brydobacter bacteria may increase fungal virulence (Fig. 6 B) warrants further investigation. Several core endohyphal bacteria, such as Caulobacter sp. and Ralstonia sp ., have been linked to human diseases, including hospital-acquired meningitis [ 80 , 81 ]. This raises the important question of whether these partnerships enhance the infection capacity of A. fumigatus ; a species on the WHO’s list of fungal priority pathogens [ 3 ]. Our results challenge us to shift from a host-centric vision of fungal-bacterial partnerships to a bacteria-centric vision: focusing on the roles played by endohyphal bacteria and their interactions and adaptations. Understanding the molecular mechanisms governing the establishment and maintenance of these associations will also require a detailed analysis of host genetic processes, transmission mechanisms and population control. Declarations The samples (fungal isolates) were received anonymized, raising no ethical considerations to the ethics committee of ITQB NOVA. It is impossible to trace back the patients identities and clinical history. Ethics approval and consent to participate Not applicable. Note that all samples – fungal isolates - were provided anonymized. Consent for publication Not applicable Availability of data and material Additional File 1: Supplementary Information (MS Word), containing more detailed tables and figures that support the main figures panels at the main text. Additional File 2: Dataset1, Amplicon sequencing data of ITS regions (xls format) of the fungal strains. Additional File 3: Dataset 2, Micro-satellite genotyping data (xls format) of the fungal strains. Additional File 4: Dataset 3, Spearman's Correlation coefficients. The sequencing data has been deposited in the Sequence Read Archive (NCBI) with the accession code PRJNA1135973. Competing interests Not applicable Funding We acknowledge funding from Fundação para a Ciência e a Tecnologia (FCT) through the project “FATE” (PTDC/CTA-AMB/6587/2020), MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). This work was partially supported by PPBI - Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE - "Orçamento de Estado" and by european funds from FEDER - "Fundo Europeu de Desenvolvimento Regional". DP is grateful to FCT funding for the PhD scholarship PD/BD/138913/2018. T.M. and D.M. are grateful for the working contract financed by national funds under norma transitória D.L. n.° 57/2016. Funding received by the iBB-Institute for Bioengineering and Biosciences from the Portuguese Science and Technology Foundation (FCT) (UID/BIO/04565/2020) and by Programa Operacional Regional de Lisboa 2020 (Project N. 007317) is acknowledged. The project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy (i4HB) is also acknowledged. Authors’ contributions CSP supervised the project and the interpretation of data and prepared the final version of the manuscript. All authors have made substantial contributions to the acquisition, analysis and interpretation of data and contributed to the drafting of the manuscript: DP (fungal experiments, manuscript draft), DP, TM (bioinformatics); JMPJ, PC (microscopy); DM-H (virulence assays); DC, JMC (fungal isolation), CSP (conceptualization); CSP, RSL (supervision); CSP, RSL, GHG (resources). All authors read and approved the final version of the manuscript. Acknowledgments We extend our gratitude to all members of the Silva Pereira lab for their valuable discussions, with special thanks to alumnus researcher Celso Martins for his assistance in the initial data analyses and critical reading of the manuscript. We would like to acknowledge Dmitry A. Semchonok (ITQB NOVA) for his significant contributions, particularly in setting up appropriate electron microscopy methods and providing constructive criticism of the manuscript. Additionally, we recognize A.L. Sousa from the Electron Microscopy Facility at the Instituto Gulbenkian de Ciência for their technical expertise, sample processing, and imaging. Our thanks also go to Antonis Rokas and Matthew Mead (Vanderbilt University, USA) for their initial help with WGS and scientific discussions, as well as to Dean Morales (Center for Integrated Nanotechnologies, Los Alamos National Laboratory, USA) for his support in establishing microscopy methods. 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Proceedings of the National Academy of Sciences 2015, 112 (33):10112-10119. Rafiqi AM, Polo PG, Milat NS, Durmuş ZÖ, Çolak-Al B, Alarcón ME, Çağıl FZ, Rajakumar A: Developmental integration of endosymbionts in insects . Frontiers in Ecology and Evolution 2022, 10 :846586. Pawlowska TE, Gaspar ML, Lastovetsky OA, Mondo SJ, Real-Ramirez I, Shakya E, Bonfante P: Biology of fungi and their bacterial endosymbionts . Annu Rev Phytopathol 2018, 56 (1):289-309. Dial DT, Weglarz KM, Aremu AO, Havill NP, Pearson TA, Burke GR, von Dohlen CD: Transitional genomes and nutritional role reversals identified for dual symbionts of adelgids (Aphidoidea: Adelgidae) . The ISME Journal 2022, 16 (3):642-654. Partida-Martinez LP, Hertweck C: Pathogenic fungus harbours endosymbiotic bacteria for toxin production . Nature 2005, 437 (7060):884-888. Venkatesh N, Greco C, Drott MT, Koss MJ, Ludwikoski I, Keller NM, Keller NP: Bacterial hitchhikers derive benefits from fungal housing . Curr Biol 2022, 32 (7):1523-1533. e1526. Lake JA: Evidence for an early prokaryotic endosymbiosis . Nature 2009, 460 (7258):967-971. Steenwyk JL, Shen X-X, Lind AL, Goldman GH, Rokas A: A robust phylogenomic time tree for biotechnologically and medically important fungi in the genera Aspergillus and Penicillium . MBio 2019, 10 (4):10.1128/mbio. 00925-00919. Benton MJ, Wilf P, Sauquet H: The Angiosperm Terrestrial Revolution and the origins of modern biodiversity . New Phytol 2022, 233 (5):2017-2035. Oldroyd GE: Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants . Nature Reviews Microbiology 2013, 11 (4):252-263. Bonfante P, Desirò A: Who lives in a fungus? The diversity, origins and functions of fungal endobacteria living in Mucoromycota . The ISME journal 2017, 11 (8):1727-1735. Manchon R, Zarrouk V, Leflon V, Iakovlev G, Bert F: First case of bloodstream infection due to Caulobacter spp. associated with a postoperative meningitis . IDCases 2023, 32 :e01761. Ryan MP, Adley CC: Ralstonia spp.: emerging global opportunistic pathogens . Eur J Clin Microbiol Infect Dis 2014, 33 :291-304. Table Table 1 is available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Additionalfile1SUBMIT.pdf Dataset1.xlsx Dataset2.xlsx Dataset3.xlsx Table1SUBMIT.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4912975","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":347566663,"identity":"6b21d456-7222-4690-8cf2-568e0a85c63c","order_by":0,"name":"Daryna Piontkivska","email":"","orcid":"","institution":"Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"Daryna","middleName":"","lastName":"Piontkivska","suffix":""},{"id":347566664,"identity":"26e5c149-b873-4e6e-8bb0-d61ca7b94892","order_by":1,"name":"João M.P. Jorge","email":"","orcid":"","institution":"Universidade Nova de Lisboa","correspondingAuthor":false,"prefix":"","firstName":"João","middleName":"M.P.","lastName":"Jorge","suffix":""},{"id":347566665,"identity":"b36a6253-3a88-4041-9b71-d61c694cf039","order_by":2,"name":"Dalila Mil-Homens","email":"","orcid":"","institution":"Instituto Superior Técnico","correspondingAuthor":false,"prefix":"","firstName":"Dalila","middleName":"","lastName":"Mil-Homens","suffix":""},{"id":347566666,"identity":"d9fc22ff-f6c0-4018-b47b-d889166acb03","order_by":3,"name":"Tiago M. 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(\u003cstrong\u003eA\u003c/strong\u003e) Maximum likelihood midpoint rooted tree of the 100 most abundant bacterial ASVs across the sample set. The phylogenetic tree was constructed using the general time reversible model with the rate variation among sites described by a gamma distribution and the proportion of invariable sites (GTR + G + I model). Background colors indicate bacterial ASVs assigned at class level. Tree constriction was based on the hypervariable V4 region of 16S rRNA gene sequences, applying 1000 bootstrap replications to estimate confidence. Bootstrap values are indicated above or below the branches. The scale bar indicates nucleotide substitutions per site. Heatmap shows the relative abundances of bacterial ASVs found in tested fungal clinical isolates without (no) and with (yes) antibiotic treatment. The color intensity shows the ASV percentage in each sample (note that in the color key the dark blue corresponds to 5%). (\u003cstrong\u003eB\u003c/strong\u003e) Heat-map diagram bacteriome composition at class level of the 3 single spore colonies (marked as C1, C2 or C3) of the three tested fungal clinical isolates. (\u003cstrong\u003eC\u003c/strong\u003e) PCoA plot of beta diversity of the single spore cultures based on weighted and unweighted Unifrac distances.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/b06cb2a597b47d9b901971c9.png"},{"id":64067379,"identity":"67cdad01-5419-4de1-bf2e-ac8640e63c9a","added_by":"auto","created_at":"2024-09-06 05:28:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrosatellite genotyping and phenotypic heterogeneity of 47 A. fumigatusstrains assessed in terms of their drug-resistance and virulence profiles\u003c/strong\u003e. Hierarchical cluster dendrogram of microsatellite genotypes of A. fumigatus isolates constructed based on the Gower dissimilarity index, with the laboratory model strain Af293 and the soil isolate (Af_SI.00) for comparison. Dots at the end of the dendrogram indicate isolates obtained from the same patient (color-coded accordingly). The source of isolation and whether it originated from a cystic fibrosis patient are indicated below the dendrogram (detailed in Table 1). Antifungal susceptibility profiles, assessed via the EUCAST method, and in vivo infection capacity using Galleria mellonella as the infection model (96h) are represented in the heatmap (Supplementary Table 1).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/b2db039a42e3f40d52a2ef7b.png"},{"id":64067381,"identity":"54733b1c-3b7f-4046-b8bf-f4865001ae62","added_by":"auto","created_at":"2024-09-06 05:28:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe core endohyphal bacteriome of the 48 A. fumigatus strains profiled through sequencing of the V3-V4 hypervariable region of the 16S rRNA gene, and the derived functional annotation of prokaryotic taxa\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Stacked bar chart showing the relative abundance ASVs from the bacterial V3-V4 hypervariable region of 16S rRNA sequences, taxonomically classified at genus level. Low abundance taxa were deleted from the visualization. The order of bacterial genus in the legend is according with its position in the chart. (\u003cstrong\u003eB\u003c/strong\u003e) Heat map of the core bacteria at family level across the sample set (n = 38), based upon 75% prevalence with at least 0.1% detection threshold. The y-axis represents the detection thresholds (indicated as relative abundance), color shading indicates the prevalence of each bacterial genus among samples for each abundance threshold. (\u003cstrong\u003eC\u003c/strong\u003e) The annotation of prokaryotic taxa (FAPROTAX) predicted from the genetic pool of the core endobacteria (75% prevalence with at least 0.1% detection threshold) using the relative abundance ASVs from the bacterial V3-V4 hypervariable region of 16S rRNA sequences, show potential functional roles, mostly in categories such as chemoheterotrophy and nitrogen fixation, followed by human- pathogens/associated and animal parasites or symbionts.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/3a1d0f69e2d0bf6c89c6f563.png"},{"id":64067389,"identity":"5cd72345-f1ae-49e2-aa79-237b43f01499","added_by":"auto","created_at":"2024-09-06 05:28:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":385021,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe core endohyphal bacteriome of 9 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains profiled through long-read sequencing of 16S rRNA amplicons.\u003c/strong\u003e (A) Maximum likelihood tree of V1-V9 16S rRNA gene bacterial ASVs identified at genus level, with the respective relative abundance (log\u003csub\u003e10\u003c/sub\u003e) in each \u003cem\u003eA. fumigatus\u003c/em\u003e isolate. The genus order in the legend is the same found in the tree, counterclockwise. (\u003cstrong\u003eB\u003c/strong\u003e) Heat map of the core bacteria at family level across the sample set (n = 9), based upon 75% prevalence with at least 0.1% detection threshold. The y-axis represents the detection thresholds (indicated as relative abundance), color shading indicates the prevalence of each bacterial genus among samples for each abundance threshold. (\u003cstrong\u003eC\u003c/strong\u003e) Ven diagram showing the number of ASVs at genus level found in common in both long length (V1-V9) and short length (V3-V4) analysis.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/abc01b75e7e1e03cad650ead.png"},{"id":64067390,"identity":"339e8396-abc6-4459-ac38-ba4ae3546ffa","added_by":"auto","created_at":"2024-09-06 05:28:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":390659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisualization of endohyphal bacteria in Aspergillus fumigatus strains by microscopy\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Fluorescent microscopy, where mycelia were stained with CFW (blue) and bacteria DNA with SYTO9 (green) (Scale bar = 10 µm). Representatives photographs were selected, from left to right: Af_SI.00; Af_CI.01; Af_CI.02; Af_CI.03; Af_CI.12 and Af_CI.18. (\u003cstrong\u003eB\u003c/strong\u003e) Fluorescent microscopy, where mycelia were stained with calcofluor-white (blue), bacterial DNA with syto9 (green) and fungal nuclei with Hoechst (blue) (Scale bar = 10 µm). A representative example is shown: Af_CI.12. The fungal nuclei (↓) enable a clear distinction between fungal and bacterial DNA. (\u003cstrong\u003eC\u003c/strong\u003e) Transmission electron micrograph of fungal mycelium of A. fumigatus strain Af_CI.06 containing endohyphal bacteria (B), nucleus (N) and mitochondria (M).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/f7f956ae8852a9d646e9373e.png"},{"id":64067387,"identity":"a8d0a318-8b70-428b-bffa-ed96e689b2d7","added_by":"auto","created_at":"2024-09-06 05:28:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":232477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentifying endohyphal bacteria potentially impacting the virulence and drug-resistance profiles of A. fumigatusstrains\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Hierarchical clustering heat map of fungal bacteriome using Bray-Curtis distance. Samples were clustered with maximum 0.15 dissimilarity. (\u003cstrong\u003eB\u003c/strong\u003e) Spearman’s correlation, asterisks indicate significant correlations *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Based on region V3V4 of the 16S rRNA gene.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/8d9bbadd85b4d69a1cbce56d.png"},{"id":64068399,"identity":"75e71e0e-df43-4524-898e-ad6e7ebfd65f","added_by":"auto","created_at":"2024-09-06 06:00:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4552671,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/bf679436-71ff-4567-8c4d-e7ba6775da90.pdf"},{"id":64067380,"identity":"3480f347-5166-4fa4-a0c7-803073db5893","added_by":"auto","created_at":"2024-09-06 05:28:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1890573,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1SUBMIT.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/03efc9bd5fdd2d21141e75fc.pdf"},{"id":64067521,"identity":"718185b2-4522-440c-ad70-cea7f5de9a3f","added_by":"auto","created_at":"2024-09-06 05:36:51","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10990,"visible":true,"origin":"","legend":"","description":"","filename":"Dataset1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/883d1accc6f71439630ed24f.xlsx"},{"id":64067520,"identity":"3f445926-4bb1-4967-b32f-b5cf2ead3dbc","added_by":"auto","created_at":"2024-09-06 05:36:50","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16314,"visible":true,"origin":"","legend":"","description":"","filename":"Dataset2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/10be56030f5d15183f94c651.xlsx"},{"id":64067383,"identity":"bf6139c8-deba-4579-b5de-c8cfb5db2afd","added_by":"auto","created_at":"2024-09-06 05:28:51","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11787,"visible":true,"origin":"","legend":"","description":"","filename":"Dataset3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/8c40ee29aba3fc80efd73db7.xlsx"},{"id":64067388,"identity":"577a89a9-faa0-40c6-b961-16b8d69ad305","added_by":"auto","created_at":"2024-09-06 05:28:52","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15754,"visible":true,"origin":"","legend":"","description":"","filename":"Table1SUBMIT.docx","url":"https://assets-eu.researchsquare.com/files/rs-4912975/v1/10c0e32a71b460f065f28877.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hidden allies: Decoding the core endohyphal bacteriome of Aspergillus fumigatus","fulltext":[{"header":"Background","content":"\u003cp\u003eGlobally, invasive fungal infections are the cause of 1.5\u0026nbsp;million deaths each year \u0026mdash;three times more than malaria and comparable to tuberculosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Recognizing the need for combating fungal infections, the World Health Organization (WHO) identified four fungal priority pathogens, namely \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, \u003cem\u003eCryptococcus neoformans\u003c/em\u003e, \u003cem\u003eCandida auris\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eAspergillus fumigatus\u003c/em\u003e is widespread, in both its geographic and ecological distribution, and it is the primary causative agent of invasive pulmonary aspergillosis - a particularly dangerous condition for immunocompromised individuals [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, the escalating prevalence of antifungal resistance and the growing diversity of fungal species that are overcoming host-specific barriers (increasing due to climate change) create major threats to human health [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This threat is further raised by the finding that environmental pollutants also increase the production of virulent \u003cem\u003eA. fumigatus\u003c/em\u003e airborne spores [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The combination of all of these factors increases the complexity of treating fungal infections and means that we urgently need reinforced vigilance and innovative strategies for clinical management [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSignificant progress has been made in recent years towards understanding the genetic, molecular, and ecological factors that determine the pathogenic potential of \u003cem\u003eA. fumigatus\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Studies have revealed that the virulence of this species is intricately modulated by a variety of factors, including variations in secondary metabolite production [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], cell wall composition [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and expression levels of virulence-associated genes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, genetic polymorphisms in key regulatory genes and signaling pathways have been implicated in shaping the virulence profiles of different \u003cem\u003eA. fumigatus\u003c/em\u003e strains [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], stressing the complex genetic architecture that underlies pathogenicity. Unlike phytopathogenic fungi, for which specific virulence factors often dictate pathogenicity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the genetic and ecological distinctions that separate pathogenic \u003cem\u003eA. fumigatus\u003c/em\u003e strains from their non-pathogenic counterparts remain unknown [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Studies have demonstrated that genetically identical conidia (\u003cem\u003ei.e.\u003c/em\u003e asexual spores) exhibit substantial phenotypic diversity \u0026ndash; this has been reported for multiple, different fungal species, including \u003cem\u003eA. fumigatus\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The observed phenotypic plasticity raises questions about the environmental cues that play a crucial role in shaping the pathogenic potential of \u003cem\u003eA. fumigatus\u003c/em\u003e - adding yet another layer of complexity to its virulence.\u003c/p\u003e \u003cp\u003eNumerous, disparate studies have shown an increasingly intricate interplay between fungi and their bacterial counterparts. While traditionally viewed as independent axenic entities, it has been shown that fungi and bacteria coexist within the same microbial communities [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and that bacteria can also inhabit the internal spaces of fungal hyphae [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These endohyphal bacteria can modulate various aspects of fungal biology, including growth, metabolism, and virulence [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], through direct physical interactions or through the secretion of bioactive molecules. Furthermore, it was suggested that endohyphal bacterial associates in fungi appear to be the rule rather than the exception, as demonstrated by the examination of 700 phylogenetically diverse fungal isolates, including more than 50 environmental \u003cem\u003eAspergillus\u003c/em\u003e strains [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, to date, our understanding of the functional complexity of endohyphal bacteria in clinically relevant fungi remains limited.\u003c/p\u003e \u003cp\u003eWe addressed the hypothesis that \u003cem\u003eA. fumigatus\u003c/em\u003e strains harbor endobacterial partners that can influence their hosts\u0026rsquo; phenotypes. To validate this hypothesis, we evaluated a set of \u003cem\u003eAspergillus spp.\u003c/em\u003e clinical isolates (n\u0026thinsp;=\u0026thinsp;40), mostly \u003cem\u003eA. fumigatus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;37) and one environmental (soil) \u003cem\u003eA. fumigatus\u003c/em\u003e isolate, to systematically analyze the presence of endobacteria. Bacteriome profiling was undertaken using short- and long-read sequencing approaches. All \u003cem\u003eA. fumigatus\u003c/em\u003e strains were phenotypically characterized in terms of their drug susceptibility and virulence profiles, and a subset was visualized microscopically. Statistical analyses were used to explore potential correlations between specific endohyphal bacterial partners and the key phenotypic traits of the fungal host. Finally, bioinformatics methods were used to predict the functional niche space within the genetic pool of the endobacteria community. The structure of the core bacteriome, and its implications for the fungal host, are comprehensively discussed.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStudy design. \u003c/em\u003e\u003c/strong\u003eIn this study we tested the hypothesis that the bacterial partners of \u003cem\u003eA. fumigatus\u003c/em\u003e strains contribute to their phenotypic variability. Fungal isolates were recovered from patients at the Hospital Santa Maria (HSM) in Lisbon; one isolate, originating from soil, was also included in the study [6]. The taxonomy of each strain was verified through Internal transcribed spacer (ITS) sequencing [6]. Initially, bacteriomes were profiled in three randomly selected strains using a nested PCR 16S touchdown strategy with sequencing of V4 region [18]. In all bacteriome profiling analyses, the Amplicon sequence variants (ASVs), computed using the DADA2 pipeline, were identified using the IDTaxa algorithm (\u003cem\u003edetailed below\u003c/em\u003e). Each fungal strain was then subjected to both antibiotic and thermal treatment (60 \u003csup\u003e°\u003c/sup\u003eC, 1h), followed by the isolation of a microcolony derived from a single conidium in solid medium to bypass any contribution from spores’ heterogeneity. Each individual conidium culture was then propagated whenever fresh spores were required. The strains were genotyped and phenotypically analyzed (antifungal susceptibility and virulence). The endobacteriome of each single conidium strain was systematically analyzed using V3-V4 amplification (n=38) and full length 16S MinIon sequencing (n= 9), using multiple quality controls. In the last subset of fungal strains, the relative proportion of 18S:16S was also quantified in DNA extracted from fungal samples. Bioinformatics analyses always considered the relative abundances of each ASV at distinct taxon levels up to genus. Complementary whole genome sequencing of fungal DNA was also performed (n=2). Attempts to isolate bacteria in aerobic standard conditions were carried out as well. Finally, the bacterial partners inside the hyphae of 20-h old mycelia were visualized through fluorescent microscopy and transmission electron microscopy. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCulture media. \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eAspergillus\u003c/em\u003e Minimal Medium (AMM) containing glucose (10 g·L\u003csup\u003e-1\u003c/sup\u003e), thiamine (0,01 g·L\u003csup\u003e-1\u003c/sup\u003e), 5% (v/v) nitrate salts solution [NaNO\u003csub\u003e3\u003c/sub\u003e (120 g·L-1), KCL (10.4 g·L\u003csup\u003e-1\u003c/sup\u003e), MgSO\u003csub\u003e4\u003c/sub\u003e·7H\u003csub\u003e2\u003c/sub\u003eO (10.4 g·L\u003csup\u003e-1\u003c/sup\u003e) and KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (30.4 g·L\u003csup\u003e-1\u003c/sup\u003e)], 0.1% (v/v) trace element solution [ZnSO4·7H\u003csub\u003e2\u003c/sub\u003eO (22.0 g·L\u003csup\u003e-1\u003c/sup\u003e), H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e4\u003c/sub\u003e (11.0 g·L\u003csup\u003e-1\u003c/sup\u003e), MnCl\u003csub\u003e2\u003c/sub\u003e·4H\u003csub\u003e2\u003c/sub\u003eO (5.0 g·L\u003csup\u003e-1\u003c/sup\u003e), FeSO\u003csub\u003e4\u003c/sub\u003e·7H\u003csub\u003e2\u003c/sub\u003eO (5.0 g·L\u003csup\u003e-1\u003c/sup\u003e), CoCl\u003csub\u003e2\u003c/sub\u003e·6H\u003csub\u003e2\u003c/sub\u003eO (1.7 g·L\u003csup\u003e-1\u003c/sup\u003e), CuSO4·5H\u003csub\u003e2\u003c/sub\u003eO (1.6 g·L\u003csup\u003e-1\u003c/sup\u003e), Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e·2H\u003csub\u003e2\u003c/sub\u003eO (1.5 g·L\u003csup\u003e-1\u003c/sup\u003e) and Na\u003csub\u003e4\u003c/sub\u003eEDTA (50 g·L\u003csup\u003e-1\u003c/sup\u003e)], the final pH was adjusted to 6.5 with NaOH. For solid media 1.5% (w/v) of agar was added. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eClinical strain collection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e strains were isolated by the Laboratory of Microbiology of Hospital Santa Maria (HSM), Lisbon, Portugal, between August and October of 2018 (Table 1, Additional file 2: Dataset 1). The fungal isolates were obtained from sputum specimens or from bronchoalveolar lavage. Samples were inoculated on to Sabouraud Dextrose Agar (SDA) plates containing chloramphenicol and gentamicin (SGA, Biomérieux) and incubated at 35-37 °C for 7 to 10 days. Fungal isolates were collected during routine sampling of patients, and were provided anonymized, without any information that could be used to identify the patient. All fungal isolates were identified phenotypically, at the species level, based on macroscopic colony morphology and micromorphological characteristics. Additionally, fungal isolates were subjected to molecular identification by amplification and sequencing of ribosomal internal transcribed spacers (ITS) as previously reported [6]. For each fungal strain, conidia stocks were prepared and stored at -80 °C until required. In some assays, \u003cem\u003eA. fumigatus\u003c/em\u003e reference laboratory strain Af293, purchased from the Fungal Genetics Stock Center (Kansas City, MO, United States), and a soil isolate AEM006 [6], referred as Af_SI.00, were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAspergillus single-conidium cultures \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo minimize inter-spore variability and eliminate transient bacteria, a single-spore culture technique was applied [19]. Additionally, spores underwent a high-temperature treatment. In brief, spore suspensions in 0.85% NaCl solution (100 conidia·mL\u003csup\u003e-1\u003c/sup\u003e) were incubated at 60 °C for 1h to kill most bacteria associated with the spores’ surface. Two bacterial controls were used: \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eHydrobacter perzbergensis\u003c/em\u003e. After the heat-treatment, no bacterial colonies were visible after 24h at 37 °C on Luria-Bertani (LB) agar and Reasoner's 2A(R2A) agar plates. Following heat-treatment, aliquots (100 µl) of the conidia were spread on solidified AMM supplemented with 100 mg·L\u003csup\u003e-1\u003c/sup\u003e of ciprofloxacin (37 °C, 16-20h). One germinated spore was selected, carefully cut, and transferred to fresh medium (supplemented with 100 mg·L\u003csup\u003e-1\u003c/sup\u003e of ciprofloxacin) and further incubated at 37 °C. Conidia were harvested after 5-7 days of growth using a saline solution (NaCl 8.5 g⋅L\u003csup\u003e-1\u003c/sup\u003e) containing 0.01 % Tween\u003csup\u003e®\u003c/sup\u003e 20, washed two times with saline solution and collected after passing through three layers of miracloth. The resulting spore suspensions were conserved in saline solution containing 30% (v/v) glycerol at -80 °C until further use. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCultures conditions. \u003c/em\u003e\u003c/strong\u003eFungal biomass was obtained by growing ~10\u003csup\u003e8\u003c/sup\u003e spores·mL\u003csup\u003e‑1\u003c/sup\u003e in 4 mL of liquid AMM, supplemented with or without 100 mg·L\u003csup\u003e‑1\u003c/sup\u003e of ciprofloxacin, in 6-well plates for 48h. Each fungal strain was grown in a separate 6-well plate. Grown mycelia were removed from the medium surface, immediately frozen, and stored at -80 °C until DNA extraction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDNA extraction. \u003c/em\u003e\u003c/strong\u003eThe mycelium biomass was grinded in liquid nitrogen using mortar-pestle, followed by performing 1 cycle of heating (1 h at 90 °C) and sonication (each tube contained 1 g of glass beads per sample, with equal amounts of 0.5- and 0.1-mm beads) for 5 min, with the aid of an extraction buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO4, 50 mM NaCl, 500 mM Tris-HCl, 5% SDS, pH 8; 1 mL per culture). Afterwards, the sample was mixed with an equal volume 25:24:1 (v) mixture of phenol, chloroform and isoamyl alcohol (solution A), shaken (2 min) and centrifuged (5 min, 2,400\u003cem\u003eg\u003c/em\u003e) to recover the aqueous phase which was re-extracted with an equal volume 24:1 (v) mixture of chloroform and isoamyl alcohol (solution B) and recovered as described before. To the recovered aqueous phase, 20 µL of RNAse (10 mg·ml\u003csup\u003e-1\u003c/sup\u003e) were added and incubated at 37 ºC for 30 min. The mixture was re-extracted again with solution B as described before. To the recovered aqueous phase, 1/3 volume of 6M NaCl and 1/10 volume of 10% of cetyltrimethylammonium bromide (CTAB) in 0.7M NaCl were added, and the mixture was incubated for 30 min at 65 °C. After cooling to room temperature, an equal volume of solution B was added, shaken, and centrifuged (20 min, 1,400\u003cem\u003eg\u003c/em\u003e) to recover the supernatant. Finally, DNA was precipitated in 2/3 volume of isopropanol and 1/10 volume of sodium acetate solution (3M) overnight at 4 °C. The precipitated DNA was recovered by centrifugation (20 min, 6,800\u003cem\u003eg\u003c/em\u003e). The DNA pellet was washed with 200 µL of ethanol (75%), recovered by centrifugation as before, air dried and then eluted in 50-200 µL of TE buffer (Qiagen, Germany) and finally stored at -20 °C. When needed, DNA concentration was measured using a Nanodrop OneC (ThermoFisher, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAmplification of the V4 or V3-V4 regions of 16S rRNA gene. \u003c/em\u003e\u003c/strong\u003eBacteriome profiling relied on amplicon sequencing of the V4 and/or V3-V4 regions of the 16S rRNA gene using DNA extracted from 2-day old mycelium. Nested PCR was used to enhance the bacterial signal: the PCR product of the first amplification with universal primer set fD1/rP2 [20] was used as the template for a second amplification with the nested primer set 515F/806R [18] to amplify the V4 region or 341F/785R [21] to amplify the V3-V4 region of the 16S rRNA gene. The PCR mixture for the remaining amplifications contained 2.5 μL of 10x DreamTaq buffer (includes 20 mM MgCl\u003csub\u003e2\u003c/sub\u003e), 0.5 μL of 50 mM MgCl2, 0.75 μL of 10 mM dNTPs, 1.5 μL of 0.15 mM BSA, 0.75 μL of DreamTaq DNA Polymerase (5 U·μL\u003csup\u003e− 1\u003c/sup\u003e, Thermo Scientific™), 1 μL of 5 μM forward primer, 1 μL of 5 μM reverse primer and 50-400 ng of DNA template, adjusted to a final volume of 25 μL with ultrapure water. PCR amplifications were performed using a T100™ Thermal Cycler (Bio-Rad). The amplification using the 16S rRNA gene universal primers fD1 5’‑AGAGTTTGATCCTGGCTCAG-3’; rP2 5’‑ACGGCTACCTTGTTACGACTT-3’, was performed under the following conditions: 5 min at 96 °C, 10 cycles of 30s at 94 °C, 45s annealing at 62-52 °C (decrements of 1 °C per cycle) and 90s at 72 °C, followed by 20 cycles of 30s at 94 °C, 45 s annealing at 52 °C and 90s at 72 °C, with a final extension step at 72 °C for 5 min. For the primers 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′) the conditions used were: 5 min at 96 °C, 10 cycles of 30s at 94 °C, 45s annealing at 60-50 °C (decrements of 1 °C per cycle) and 90s at 72 °C, followed by 20 cycles of 30s at 94 °C, 45s annealing at 50 °C and 90s at 72 °C, with a final extension step at 72 °C for 5 min. The PCR obtained products were analyzed by agarose-gel electrophoresis. The “no-template” controls (amplification and extraction) were always handled simultaneously, and in a manner consistent with, the DNA samples derived from mycelia. Additionally, all work was conducted according to strict sterile technique (\u003cem\u003ee.g\u003c/em\u003e., clean workspaces, barrier tips, pre- and post-PCR pipettes) to limit potential cross-contamination. Before further analyses, all PCR products were purified using GeneClean® Turbo Kit (MP Biomedicals™). All samples (region V3 or V3V4) were sequenced on Illumina MiSeq (2 × 300 bp paired-end reads) using the Illumina 16S Metagenomic Sequencing service of STAB Vida Lda., Portugal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalysis of the V4 or V3-V4 regions of 16S rRNA gene amplicons. \u003c/em\u003e\u003c/strong\u003eAmplicon reads were processed using the divisive amplicon denoising algorithm DADA2 [22] to infer ASVs present in each sample. Default settings were used for filtering and trimming. Built-in training models were utilized to learn error rates for the amplicon dataset. Identical sequencing reads were combined through DADA2’s dereplication functionality, and the DADA2 sequence–variant inference algorithm was applied to each dataset. Subsequently, paired-end reads were merged. Taxonomy assignment was performed using the DECIPHER package with the IDTAXA algorithm [23] that employs principles from machine learning to reduce over classification errors. The closest bacterial hit from the SILVA SSU database r138 [24] was considered, and sequences identified as nonbacterial were discarded. All bacterial genera represented by ASVs identified in DNA amplification or extraction controls were excluded from further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFull-length 16S rRNA gene sequencing on the MinION™ platform. \u003c/em\u003e\u003c/strong\u003eFor the library construction 100-200 ng of genomic DNA was used and processed with the 16S barcoding kit (SQK-RAB204, ONT, Oxford, United Kingdom), according to the manufacturer’s instruction with a few modifications. No-template controls (amplification and extraction) were always handled simultaneously and in a manner consistent with the fungal samples. The obtained bar-coded library was sequenced in a FLO-MIN106 flow cell using the MinION Mk1C device (ONT). MinKNOW version 21.05.25 (ONT) and Guppy 5.0.16 were used for data acquisition. A total of 2.79 M reads were generated. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalysis of full-length 16S rRNA gene. \u003c/em\u003e\u003c/strong\u003eFrom the amplicon reads obtained, only those that contained both forward and reverse primers were used for further processing (in total 22,666 reads). To reduce redundancy and to increase the efficiency of downstream analyses, reads were clustered into OTUs based on phylogeny-derived distances, using the \u003cem\u003etip_glom\u003c/em\u003e function in the \u003cem\u003ephyloseq\u003c/em\u003e R package [25] (tree-height threshold for similarity: h = 0.2). The OTUs were taxonomically identified against the SILVA SSU database r138 [24] using the \u003cem\u003eblastn\u003c/em\u003e tool, the closest bacterial hit was considered, and sequences identified as nonbacterial were discarded. All bacterial OTUs identified in DNA amplification or extraction controls were excluded from further analysis (Additional file 1: Fig. S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMicrosatellite genotyping. \u003c/em\u003e\u003c/strong\u003eMicrosatellite genotyping was used to determine the genetic distances between the \u003cem\u003eAspergillus fumigatus\u003c/em\u003e clinical isolates. Genotyping was performed by CD Genomics (NY, USA) with a panel of nine short tandem repeats (TRs) as previously described [26]. In brief, three separate multiplex PCRs amplifications were performed to obtain three dinucleotide, three trinucleotide and three tetranucleotide loci fragments. Each PCR mixture contained 1x reaction buffer, 0.3 mM of corresponding amplification primers, 0.2 mM deoxynucleotide triphosphates, 0.5 U of Hot Start Taq DNA Polymerase and 10 ng of genomic DNA. Amplification was performed using the following thermal protocol: 5 min of initial denaturation at 95 °C, followed by 35 cycles of 30s of denaturation at 95 °C, 30s of annealing at 60 °C and 30s of extension at 72 °C, with final extension for 30 min at 60 °C. The fragments obtained were denatured at 95 °C for 3 min in a mixture containing 1.0 μl of PCR product, molecular weight internal standard and 0.05 % formamide. The fragment analysis was performed on the Applied Biosystems 3730xl DNA Analyzer, and the sizes determined using GeneMapper Software 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e infection with \u003cem\u003eGalleria mellonella. \u003c/em\u003e\u003c/strong\u003eInfection studies were performed as previously described using \u003cem\u003eG. mellonella \u003c/em\u003e[6]. Briefly, \u003cem\u003eG. mellonella\u003c/em\u003e larvae were reared in darkness at 25°C, from egg to last instar larvae, on a natural diet of beeswax and pollen grains. For the experiments, final instar larvae weighing 200 ± 25 mg were selected (n=30 per condition). Aliquots of the stock spore suspensions (derived from single-conidium cultures, see above) were used to adjust the inoculum to a concentration of 10\u003csup\u003e7\u003c/sup\u003e spores \u003cem\u003eper \u003c/em\u003elarva for each tested condition. A microinjection system was used to control the volume of a microsyringe and inject 5 μl of spore suspension into each larva via the hindmost left proleg, previously sanitized with 70% (v/v) ethanol. Following injection, larvae were placed in Petri dishes and stored in the dark at 37 °C. Control larvae were injected with saline solution (pH 7.4). For each condition (n=30) larval survival was followed for 96 h. Caterpillars were considered dead when they displayed no movement in response to touch.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMinimal Inhibitory Concentrations (MIC) of antifungals. \u003c/strong\u003eThe minimal inhibitory concentrations (MIC) were determined using a microbroth dilution format according to the EUCAST reference method [27]. Tests were performed using RPMI-1640 medium (R6504, with L-glutamine and without sodium bicarbonate) supplemented with glucose to a final concentration of 2% and 0.165 mol·L\u003csup\u003e-1\u003c/sup\u003e of 3-(N-morpholino) propanesulfonic acid (MOPS) with pH adjusted to 7.0. The concentrations of antifungal drugs tested ranged from 16 to 0.03 mg·L\u003csup\u003e-1\u003c/sup\u003e for amphotericin B and posaconazole, and from 8 to 0.016 mg·L\u003csup\u003e-1\u003c/sup\u003e for voriconazole. Spore suspensions were prepared to a final working concentration of 10\u003csup\u003e6\u003c/sup\u003e conidia·mL\u003csup\u003e-1\u003c/sup\u003e. Growth and negative controls were included in all tests. The microplates were incubated at 37 °C for 48h. The lowest concentration that exhibited no growth under microscopic observation was considered to be the MIC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy. \u003c/strong\u003e\u003cem\u003eFluorescence microscopy\u003c/em\u003e. Hydrogel media discs were prepared for use in fluorescence microscopy. Discs were prepared using AMM followed by the addition of Phytagel to a final concentration of 4% w/v. The hydrogel slide preparation was adapted from methods described previously [28, 29]. Briefly, the media was autoclaved and kept in a water bath at 90 ºC. Afterwards the media was cast between two standard microscope slides (VWR, previously washed with ethanol and UV sterilized) and set to solidify under sterile conditions in a biosafety cabinet. The slides were then carefully separated using a sterile razor blade. Using a sterile 1 mL pipette tip, circular discs (~7 mm diameter) were stamped out. These discs were transferred to a 6-well plate, inoculated with 1 µL of a suspension of ~10\u003csup\u003e9\u003c/sup\u003e spores·mL\u003csup\u003e-1\u003c/sup\u003e, then incubated at 37 °C. To avoid drying of the discs during the incubation period, UV sterilized paper towels saturated with sterile water were folded and placed in the space between the wells. After 20 h of incubation, the discs were fixed with 4% paraformaldehyde in PBS and incubated overnight at 4 °C. The fixed discs were washed three times with 1x PBS and stored in a solution of PBS with 0.1 U/µL Superase RNAse inhibitor with 10 mM Ribonucleoside Vanadyl Complex (RVC) at 4 °C until needed. Before use, the discs were washed three times with 1x PBS, dehydrated with ethanol-PBS solutions with 50 %, 75 %, 100 %, 75 %, 50 % ethanol, rinsed with PBS for 3 min at room temperature, and after removal of PBS were left to air dry for 10 min. For staining, each sample was first covered with 2 µM syto9 (Invitrogen, 5 min, room temperature, dark) and after washing with PBS, samples were covered with 20 µM calcofluor-white (Sigma, 3 min, room temperature, dark), washed again with PBS, then transferred to a microscope slide with a cover slip on the top of the disc. Some samples were stained with 5 µg/mL Hoechst 33342 (Thermo Fischer Scientific, dark, 5 min) before the calcofluor-white staining. Mycelia and bacteria were visualized using a Zeiss LSM 880 microscope equipped with a Fast Airyscan. ImageJ was used for image analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTransmission electron microscopy (TEM). \u003c/em\u003eMycelia (SDA medium, 36 h, 37 ºC) were fixed with a solution of 2% (v/v) formaldehyde (Science Services), 2.5% glutaraldehyde (v/v) (Science Services) in 0.1 M cacodylate buffer (30 min, room temperature), then washed three times with 0.1 M cacodylate buffer. Fungal samples were sandwiched between 0.2 µm flat aluminum disks and frozen in a high-pressure freezer (Wohlwend Compact 2) using as filler 0.5% (w/v) low-melting point agarose (OmniPur) in 0.1 M cacodylate buffer at 37 ºC. These samples were placed in the freeze substitution device (Leica AFS2) at -90 ºC in a solution with 2% (w/v) osmium tetroxide (Science Services), 0.1% (w/v) uranyl acetate (Analar) with 1% distilled H\u003csub\u003e2\u003c/sub\u003eO in acetone. After 60 h, the samples were slowly warmed at a rate of 2 ºC/h: first to -60 ºC, kept at this temperature for 10 h, then to -30ºC and kept at this temperature for 10 h. Subsequently, using the maximum warming rate, samples were brought to 0 ºC and dehydrated with acetone (3 times, 10 min each). Dehydrated samples were infiltrated with increasing amounts of Embed-812 Epoxy Resin (Science Services) (5% 4 h, 10% overnight, 25% 4 h, 50% overnight, 75% 4 h, 100% overnight) and polymerized in fresh resin at 60 ºC for 48 h. Ultrathin sections (70 nm) were cut using an ultramicrotome (Leica UC7), picked-up in 1% formvar (Agar Scientific) in chloroform coated slot grids, and stained at room temperature with 1% (w/v) uranyl acetate and Reynolds lead citrate, for 5 min each. The grids were observed using a TEM (FEI Tecnai G2 Spirit BioTWIN with an Olympus-SIS Veleta CCD Camera) at 120 kV. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatics analysis. \u003c/strong\u003ePhylogenetic distance was determined using the general time reversible model with the rate variation among sites described by a gamma distribution and the proportion of invariable sites (GTR + G + I model). The minimum spanning network, to determine genetic diversity between strains, was determined using Bruvo’s distance, and the discriminant analysis of principal components (DAPC) was performed using the R package \u003cem\u003epoppr \u003c/em\u003e[30]. Data obtained in this study was compared with a subset of isolates from the databank for clinical and environmental \u003cem\u003eA. fumigatus\u003c/em\u003e strains, which is based at the Canisius Wilhelmina Hospital in Nijmegen, Netherlands, and available at the afumID website [31].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWhole-genome sequencing\u003c/em\u003e. The quality and quantity of DNA were accessed by electrophoresis and using the Qubit dsDNA BR kit, following AMpureXP cleaning. For 150 bp pair-end DNA sequencing, libraries were generated using the Kapa HyperPrep kit (Roche) according to the manufacturer’s instructions and integrity accessed using a TapeStation 4200. Samples were indexed and sequenced on the lllumina Novaseq platform (≥30 million reads per sample). Quality control of reads was carried out using FastQC (v0.12.1) and trimmomatic (v0.39) [32]. The surviving reads (\u0026gt;99%) from each library were aligned to a library of three \u003cem\u003eA. fumigatus\u003c/em\u003e reference genomes (for strains Af293, A1163 and CEA10, respectively ASM265v1, ASM15014v1 and CP097563 to CP097570) using segemehl (v0.3.4) [33]. Metagenomic analysis of the remaining reads (≤2%) was conducted using Kraken (v2.1.3) with PlusPF Refseq indexes as of June 5, 2023 [34] and Recentrifuge (v1.12.1) [35]. Reads were also aligned to specific bacterial genomes as described above, and genome coverage determined using Samtools (v1.17) [36].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicrobiome function prediction. \u003c/em\u003eFAPROTAX and PICRUSt2 were used to predict the ecologically relevant functions of the microbiomes from the fungal isolates [37, 38]. Both analyses employed default parameters using relative abundance ASVs from the bacterial V3-V4 hypervariable region of 16S rRNA sequences. For simplicity in the presentation of the PICRUSt2 prediction, MetaCyc pathways were categorized into class pathways.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eOur main working hypothesis is that endohyphal bacteria contribute to the phenotypic heterogeneity observed in\u003cem\u003e A. fumigatus\u003c/em\u003e. To address this hypothesis, \u003cem\u003eAspergillus \u003c/em\u003eclinical isolates were primarily isolated from patients at HSM in Lisbon, Portugal and were taxonomically classified as \u003cem\u003eA. fumigatus\u003c/em\u003e (n=37), \u003cem\u003eA. terreus\u003c/em\u003e, \u003cem\u003eA. niger\u003c/em\u003e, and \u003cem\u003eA. flavus \u003c/em\u003e(n=1, each) (Table 1). Additional strains used in some assay were an \u003cem\u003eA. fumigatus\u003c/em\u003e soil isolate (Af_SI.00) [6] and the reference strain Af293. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSetting-up a framework to analyze endohyphal bacterial partners of A. fumigatus clinical isolates\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of endohyphal bacterial associates was initially tested in three randomly selected \u003cem\u003eA. fumigatus\u003c/em\u003e strains to address two outstanding questions: i) if antibiotic treatment eliminates ephemeral bacterial associates; and ii) if cultures generated from a single conidium display a lower diversity of bacterial associates than those derived from multiple conidia. Bacterial profiling was systematically performed by amplicon sequencing of the hypervariable region V4 of 16S rRNA gene. The ASVs obtained were taxonomically identified through the IDTaxa algorithm [23] using the SILVA 16S database as a training set [24]. Due to the low taxonomic resolution of the short-read sequences analyzed here, the bacterial ASVs identified are displayed at class or family level. \u003c/p\u003e\n\u003cp\u003eIn order to evaluate the impact of antibiotic selection, the composition of the bacterial community in mycelia grown in media supplemented with high-dose ciprofloxacin (a broad-spectrum antibiotic) was compared to that from mycelia grown in media without antibiotic. A total of 233 unique ASVs were identified across the 6 samples examined (Additional file 1: Fig. S2A). Among these, 30 ASVs were present in the mycelia that were not subjected to the antibiotic selection, indicating that there are bacterial partners that could be eliminated from mycelia by treatment with antibiotic. This result suggests these bacteria are likely not stable endohyphal partners of \u003cem\u003eA. fumigatus\u003c/em\u003e. One hundred and fifty-six ASVs were only detected after antibiotic treatment, suggesting that the antibiotic selection induced a very noticeable shift in the composition of the bacterial community, allowing detection of less abundant bacteria. Interestingly, 47 ASVs were found at relative high abundances, in mycelia cultivated in media both without and with antibiotic selection (Additional file 1: Fig. S2A). This observation implies a strong association between these ASVs and mycelia, suggesting the existence of protection mechanisms within hyphae. \u003c/p\u003e\n\u003cp\u003eA phylogenetic analysis focusing on the top 100 most abundant ASVs was undertaken (representing ~98% and ~92% of the total relative abundance in the absence and presence of antibiotic, respectively) to better understand their distribution across different fungal strains and conditions. The results highlighted that there were major alterations in the composition of the bacterial community post-antibiotic selection, specifically a heightened community diversity with a clear dominance of ASVs belonging to the Bacteroidia, Alphaproteobacteria, and Polyangia classes (Fig. 1A). For a given fungal strain, no significant correlation was detected between the untreated control and the sample subjected to antibiotic selection. In contrast, a strong positive Pearson’s correlation (\u003cem\u003ep\u003c/em\u003e≤0.0001) was identified among the three different fungal strains under the same conditions. These results suggest that the bacteriomes of the three fungal strains are similar under pre- or post-antibiotic treatment, but not across conditions (Additional file 1: Fig S2B). \u003c/p\u003e\n\u003cp\u003eThe complexity of the mycelial bacteriome led to the decision to isolate single-conidium cultures – with the aim of eliminating any contribution from conidia heterogeneity to the overall bacterial diversity. Additionally, as some bacteria outside of conidia may survive the antibiotic treatment, conidia were also subjected to heat stress (60°C, 1 h) in addition to the antibiotic pressure. The conditions were optimized using two axenic bacteria cultures (\u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eH. perzbergensis\u003c/em\u003e). In contrast to some of the conidia, none of the bacteria could survive the heat treatment. We generated single-conidium cultures (n=3 per isolate) from three fungal isolates and analyzed the associated bacteriomes. The results showed similar bacteriomes across the single-conidium colonies from the same fungal strain and for the three different fungal strains tested (Fig. 1B). The resulting heatmap highlights the similar bacterial composition and the distribution of relative abundance across the sample set at the class level (Fig. 1B). Further examination, through phylogenetic analysis, focusing on the top 100 most abundant bacterial ASVs (representing ~95% of the total abundance), showed consistent results: all single-conidium colony cultures show a similar bacteriome, regardless of the parental fungal strain (Additional file 1: Fig. S3). The same inference can be derived from beta diversity analyses based on both weighted and unweighted Unifrac distances - also showing the similarity of the bacteriomes analyzed (Fig. 1C). \u003c/p\u003e\n\u003cp\u003eCollectively, the results showed remarkable similarity for the hyphal bacteriomes across three distinct fungal strains, inoculated from conidia subjected to heat treatment in an antibiotic selection medium. All subsequent experiments used a randomly selected single-conidium colony per \u003cem\u003eAspergillus\u003c/em\u003e spp. strain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermining the range of genotypic diversity and phenotypic heterogeneity of the A. fumigatus clinical isolates\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMost of the \u003cem\u003eA. fumigatus\u003c/em\u003e clinical isolates (n=37 in total) originated from patients with cystic fibrosis (n=29, 78%). Overall, the bulk of the samples were isolated from sputum; the exceptions were from ear secretions (n=2), lung biopsies (n=2), and bronchoalveolar lavage (n=2). In some cases, two isolates were sourced from the same patient (Table 1, Fig. 2).\u003c/p\u003e\n\u003cp\u003eWe have used microsatellite genotyping to achieve a higher discriminatory power within \u003cem\u003eA. fumigatus\u003c/em\u003e [26]. First, we tested if the geographic origin of the \u003cem\u003eA. fumigatus\u003c/em\u003e strains from HSM in Lisbon impact on their genetic diversity. Therefore, our genotyping results (Additional file 3: Dataset 2) were compared with those of clinical isolates found in other countries [31]. The minimum spanning network displaying the relatedness between the isolates showed that the 37 clinical strains exhibit considerable genetic variability (Additional file 1: Fig. S4). This result suggests that genotypic diversity within isolates of \u003cem\u003eA. fumigatus\u003c/em\u003e surpasses geographic boundaries, consistent with previous studies [31]. Distinct lineages within the \u003cem\u003eA. fumigatus\u003c/em\u003e strains were identified through hierarchical cluster analysis using the Gower dissimilarity index (Fig. 2). The strains Af293 (lab strain) and Af_SI.00 (isolated from soil) were included for comparison purposes. Clustering with a maximum dissimilarity threshold of 10% revealed the presence of 27 clusters, highlighting the strains genotypic diversity. Four pairs of strains were found to be genotypically identical (Fig. 2) and just one of those pairs was isolated from the same patient (Af_CI.19 and Af_CI.44).\u003c/p\u003e\n\u003cp\u003eWe then analyzed the phenotypic heterogeneity of \u003cem\u003eA. fumigatus\u003c/em\u003e strains, assessed in terms of their drug-resistance and virulence profiles (n=39). Minimal inhibitory concentrations (MICs) analyzed by the EUCAST method [27] ranged from 16 to 0.03 mg·L\u003csup\u003e-1\u003c/sup\u003e for amphotericin B and voriconazole, and from 8 to 0.016 mg·L\u003csup\u003e-1\u003c/sup\u003e for posaconazole (Additional file 1: Table S1). Overall, the results show significant heterogeneity in the drug-susceptibility profiles, with no apparent clustering to their lineage as depicted in Fig. 2. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e infection capacity of each \u003cem\u003eA. fumigatus\u003c/em\u003e strains was assessed using \u003cem\u003eG. mellonella\u003c/em\u003e as the infection model (n=38, plus the Af293 strain). This model is widely recognized for evaluating the virulence of microbial pathogens [39], particularly fungal pathogens [40], and demonstrates a reliable correlation with murine models [41]. Following a 96-hour post-infection period, the fungal strains exhibited varying degrees of infection capacity. According to the survival probability range, the infection index was classified as low (\u0026gt;75%); medium-low (75%-50%); medium-high (50%-25%); and high (\u0026lt;25%) (Fig. 2; Additional file 1: Fig S5). The virulence results did not exhibit any clustering with the isolates’ lineages.\u003c/p\u003e\n\u003cp\u003eCollectively these results show that the 37 \u003cem\u003eA. fumigatus\u003c/em\u003e clinical strains characterized in this study, sampled within close geographic proximity, exhibit substantial genotypic diversity and phenotypic heterogeneity in terms of their virulence and drug-resistance profiles, consistent with previous reports [42]. Neither antifungal susceptibility nor genotyping lineage correlated with virulence potential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDetermining the core bacteriome of A. fumigatus clinical isolates \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the \u003cem\u003eA. fumigatus\u003c/em\u003e core bacteriome, all 37 clinical strains and the Af_SI.00 strain were profiled through sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. Prior to this analysis, we verified in a sub-set of strains (n=9) that the relative proportions of 18S:16S quantified via RT-\u003cem\u003eq\u003c/em\u003ePCR were consistent in the DNA extracts of both spores and 48h-old mycelia (Additional file 1: Table S2). Bacterial DNA is consistently present in all DNA samples derived from either fungal source (except for AF_CI.08 that presents a slow growth rate), ranging from 0.35% to 1.6% of bacterial DNA in the total DNA. \u003c/p\u003e\n\u003cp\u003eAll analyzed \u003cem\u003eA. fumigatus\u003c/em\u003e strains (n=38) exhibited a consistent bacteriome profile at the genus level (Fig. 3A). This result suggests stability in the bacterial community across all tested fungal strains. Such stability expands to the bacteriome profile of the other clinical \u003cem\u003eAspergillus spp.\u003c/em\u003e strains, namely \u003cem\u003eA. terreus, A. niger, and A. flavus\u003c/em\u003e (n=1, each) (Additional file 1: Fig. S6). This observation supports the notion that the bacterial communities associated with distinct aspergilla originate from shared ecological niches and/or functional roles – this is deserving of a more focused analysis in the future. \u003c/p\u003e\n\u003cp\u003eEvaluation of prevalence at the genus level highlighted the presence of 19 core bacteria genera, of which 11 were present in all \u003cem\u003eA. fumigatus\u003c/em\u003e strains (Fig. 3B). Considering the relative abundance, \u003cem\u003eGemmata spp.\u003c/em\u003e was the most prevalent genus (5%), followed by the \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e (BCP) group, \u003cem\u003eBradyrhizobium, Puia, Rastonia \u003c/em\u003eand\u003cem\u003e Edaphobacter spp.\u003c/em\u003e (1%, each), \u003cem\u003eAcidobacter \u003c/em\u003eand \u003cem\u003eSediminibacterium \u003c/em\u003e(0.5%, each), and the \u003cem\u003eMethylobacterium-Methylorubrum\u003c/em\u003e (MM) group\u003cem\u003e, Afipia\u003c/em\u003e, and \u003cem\u003eNevskia \u003c/em\u003e(\u0026lt;0.1%) (Fig. 3B). Most of the core bacterial genera match multiple ASVs identifications, likely due to the presence of closely related variants within each genus. Phylogenetic analysis of the core bacteriome tree suggests that these ASVs are closely related and likely represent the same bacterial species (Additional file 1: Fig S7). Importantly, bacteria detected in all quality controls (amplification and extraction negative controls) were systematically excluded from all bacteriome profiling analyses (Additional file 1: Fig. S1).\u003c/p\u003e\n\u003cp\u003eTo further test the presence of a core bacteriome, a subset of \u003cem\u003eA. fumigatus\u003c/em\u003e strains (n=9, Additional file 1: Table S2) were profiled using long-read sequencing of 16S rRNA amplicons on the Oxford Nanopore MinION platform. The utilization of long-read sequencing techniques offers a deeper insight into microbial communities [43], and may potentially validate the identification performed using the shorter-amplified sequences. This approach yielded a total of 49 bacterial genera. However, the majority of these genera are associated with a few strains at remarkably low abundance (Fig. 4A). The potential roles of rare bacteria in the fungal bacteriome certainly warrant further investigation. \u003c/p\u003e\n\u003cp\u003eIn total, 11 core bacterial genera were identified, of which 7 were found consistently across all 9 isolates, including \u003cem\u003eRastonia\u003c/em\u003e, BCP group, MM group, \u003cem\u003eSphingomonas, Xylophilus, Bradyrhizobium, \u003c/em\u003eand \u003cem\u003eMucilaginibcater\u003c/em\u003e (Fig. 4B). The two core bacteriomes identified have 8 bacterial genera in common (Fig. 4C). This result highlights the stability of certain bacterial genera across different sequencing methods. \u003c/p\u003e\n\u003cp\u003eBacteria genera consistently identified within the core bacteriome may play key roles in the structure and function of this microbial community. Metabolic dependencies, promoting group survival in nutritionally challenging conditions is a well-established principle within metacommunities. Such dependencies are thought to play a significant role in species co-occurrence and are indicative of the presence of regular cooperative groups within microbial community architectures [44]. This concept also underlies the formation and structure of holobiont systems. Intercellular bacterial partners of eukaryotic hosts, including endosymbionts, can provide primary metabolic pathways (for example, photosynthesis) or expand the repertoire of secondary metabolism, while also influencing the host’s fitness, growth, development, behavior, and other functions [45, 46]. In this respect, most of the core endohyphal bacteria genera identified herein can be found in the soil habitat and demonstrate viability in acidic and low nutrient (oligotrophic) environments. Some are rare bacterial genera with only a few species known (\u003cem\u003ee.g.\u003c/em\u003e \u003cem\u003eGemmata\u003c/em\u003e, \u003cem\u003ePuia\u003c/em\u003e, \u003cem\u003eAcidibacter\u003c/em\u003e and \u003cem\u003eXylophilus\u003c/em\u003e) [47-50], while others possess diverse genomic features. Many are known to form symbiotic relationships with eukaryotes and their chemoheterotrophy, suggesting the potential for biological N fixation, for example \u003cem\u003eRalstonia\u003c/em\u003e [51, 52], \u003cem\u003eBryobacter \u003c/em\u003e[53], \u003cem\u003eBradyrhizobium\u003c/em\u003e [54, 55], the BCP group [56] and \u003cem\u003eSphingomonas\u003c/em\u003e [57]. Based on the genetic pool of the endohyphal microbiota of \u003cem\u003eA. fumigatus\u003c/em\u003e (inferred from the V3-V4 amplicons, Fig. 3), both the functional annotation of prokaryotic taxa (FAPROTAX) [37] and the metabolic space could be predicted (PICRUSt2 v2.1.4) [38, 58], as applied in other related studies [59]. The results show potential functional roles, mostly in categories such as chemoheterotrophy and nitrogen fixation, followed by human- pathogens/associated and animal parasites or symbionts (Fig. 3C), as well as Amino acid metabolism, Lipid metabolism, and Cofactor, Carrier and Vitamin biosynthesis (Additional file 1: Fig. S8).\u003c/p\u003e\n\u003cp\u003eAs performed in other studies [60-62], we subjected two \u003cem\u003eA. fumigatus\u003c/em\u003e strains (Af_CI.002 and Af_CI.12) to whole-genome sequencing (WGS). The acquired data revealed a very low abundance of sequences not matching \u003cem\u003eA. fumigatus\u003c/em\u003e (0.36-0.47 %) with the majority unclassified. This result is consistent with that inferred through the relative abundance of 18S/16S amplified from fungal DNA samples (Additional file 1: Table S2). It is however relevant that a predominant bacteria genus could be identified: \u003cem\u003eBradyrhizobium\u003c/em\u003e (Additional file 1: Fig S9), with several sequences aligning, at distinct regions, with a reference genome of \u003cem\u003eB. guangzhouense \u003c/em\u003e(CCBAU 51670, acc. no. CP030053). This bacterium genus was consistently identified within the core bacteriome using other sequencing methods (Figs. 3 and 4), hence further validating its presence within the mycelia. Disclosing the full genome of predominant endohyphal bacteria would allow a better understanding of the core endobacteriome. However, due to the low abundance of bacteria DNA, such future studies will require multiple rounds of optimization to identify cultivation conditions and DNA processing methods that result in higher yields of endobacterial DNA. \u003c/p\u003e\n\u003cp\u003eTo further explore the fungal bacterial association, endohyphal bacteria were visualized under the microscope. \u003cem\u003eAspergillus fumigatus\u003c/em\u003e 20h-old hyphae (n=6, within those used for the long-read sequencing analyses) were stained with styo9, a dye commonly used to label nucleic acids and especially effective in staining endobacteria within fungi [63, 64]. The endobacteria were clearly observed within the hyphae of all analyzed fungal strains (Fig. 5A). None of these endohyphal bacteria could be cultured aerobically using standard growth protocols. Fungal nuclei, stained with Hoechst (blue), have a distinctive morphology and larger size than the syto9 stained endobacteria (green) (Fig. 5B). The direct visualization of endohyphal bacteria further validates the bacteriome profiling results. To further verify the presence of endobacteria, transmission electron microscopy of one strain of \u003cem\u003eA. fumigatus\u003c/em\u003e was done, as applied before by others [65]. The mycelium from solid culture was frozen at high pressure and cryosubstituted in osmium tetroxide and uranyl acetate. Examination of the samples confirmed the presence of endohyphal bacteria within the cytosol of intact cells of \u003cem\u003eA. fumigatus\u003c/em\u003e (Fig. 5C). Further assays are needed to identify conditions that allow them to grow outside of the fungal host. The long-read amplicons could match the identification of bacteria up to the species level, yet most identified species matched those of uncultivable bacteria (data not shown). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIdentifying endohyphal bacteria that potential impact the virulence and drug-resistance profiles of A. fumigatus clinical isolates\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the hypothesis that the endohyphal bacteriome contributes phenotypic heterogeneity of \u003cem\u003eA. fumigatus\u003c/em\u003e virulence (i.e. \u003cem\u003ein vivo\u003c/em\u003e infection capacity) and drug susceptibility, hierarchical clustering analyses were first applied using Bray-Curtis distances. Based on the data, the fungal bacteriome (genus level) did not correlate with either virulence or drug susceptibility (Fig. 6A, Additional file 1: Fig. S10A). This finding is consistent with the observation that all \u003cem\u003eA. fumigatus\u003c/em\u003e strains (n=38) exhibited a clear core bacteriome regardless of their distinct phenotypic characteristics. The same conclusion – bacterial diversity did not correlate with the fungal phenotype – could be inferred from testing only the bacteria genera that contribute to variations in the bacteriome, \u003cem\u003ei.e.\u003c/em\u003e only the rare endohyphal bacteria without the core bacteria (Additional file 1: Fig. S10B). Finally, we tested if the abundance of specific core endohyphal bacteria shows correlation with the analyzed fungal phenotypic traits using Spearman’s correlation analyses (Fig. 6B, Additional file 4: Dataset 3). The results of the correlation coefficients (r\u003csub\u003es\u003c/sub\u003e) suggest that endohyphal bacterial diversity did not correlate with susceptibility to either amphotericin B or voriconazole, with the exception of a negative correlation of MM abundance with voriconazole-susceptibility. However, posaconazole susceptibility showed positive correlations with abundance of \u003cem\u003eBryobacter \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= 0.34)\u003cem\u003e, Hydrobacter \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= 0.34),\u003cem\u003e Nevskia \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= 0.39), and \u003cem\u003eBrevundimonas \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= 0.33), and negative correlations with the abundance of \u003cem\u003eSingulisphaera \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= -0.41),\u003cem\u003e Aquabacterium \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= ‑0.38), and \u003cem\u003eStaphylococcus\u003c/em\u003e. This result suggests that specific bacterial genera may indeed influence the susceptibility of \u003cem\u003eA. fumigatus \u003c/em\u003eto posaconazole treatment. Such protective mechanisms may be related to the bacterial chemoheterotrophy and ability to degrade the antifungal drug. Only \u003cem\u003eBryobacter\u003c/em\u003e exhibited a positive correlation (r\u003csub\u003es\u003c/sub\u003e=0.45) with fungal virulence, possibly the genus abundance directly correlates with the \u003cem\u003ein vivo\u003c/em\u003e infection capacity of the host. On the contrary, the abundance of either \u003cem\u003eStenotrophomonas \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= -0.58)\u003cem\u003e, Hydrobacter \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= -0.43),\u003cem\u003e Sphingobacterium \u003c/em\u003e(r\u003csub\u003es \u003c/sub\u003e= ‑0.48), or \u003cem\u003eBrevundimonas\u003c/em\u003e (r\u003csub\u003es \u003c/sub\u003e= -0.33\u003cem\u003e)\u003c/em\u003e genera displayed a negative correlation with fungal virulence. Overall, the correlational analyses highlighted a possible relationship between the abundance of a specific endohyphal bacteria genus within the core bacteriome of \u003cem\u003eA. fumigatus\u003c/em\u003e and its virulence and drug susceptibility. Focused in-depth analyses are needed to better understand how core endohyphal bacteria impact the phenotypic traits of the fungal host, especially those that are clinically relevant.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe formulated a hypothesis that \u003cem\u003eA. fumigatus\u003c/em\u003e strains harbor diverse core endohyphal bacteria that may contribute to its phenotypic heterogeneity. Amplicon sequencing of the 16S rRNA gene revealed a dynamic bacterial landscape, where antibiotic selection induced significant shifts in the composition of the community, with certain bacterial taxa persisting (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This initial result indicates that some bacteria taxa may exhibit more robust associations with their fungal hosts. Sequencing analyses, spanning both short- and long- read platforms, of single conidium-derived colonies of \u003cem\u003eA. fumigatus\u003c/em\u003e strains (n\u0026thinsp;=\u0026thinsp;38), which are genotypically diverse (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e), highlighted a core bacteriome (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This result makes it clear that conidia heterogeneity did not contribute to the bacteriome diversity observed. The \u003cem\u003eA. fumigatus\u003c/em\u003e strains (n\u0026thinsp;=\u0026thinsp;38) displayed high phenotypic heterogeneity in their antifungal susceptibility and virulence profiles, without clear correlation with their genotypic diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This finding underscore, as is often reported, the multifaceted nature of \u003cem\u003eA. fumigatus\u003c/em\u003e strains [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Microscopy analysis visually confirmed the presence of endobacteria - located within hyphae (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The role of the core bacteriome in expanding the nutrient assimilation capacity of \u003cem\u003eA. fumigatus\u003c/em\u003e, as well as the ecological niches that it can occupy, remains hypothetical but is supported by the functional annotation of chemoheterotrophy and nitrogen fixation, among others (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Additional file 1: Fig. S8). The remarkable diversity of the fungal bacteriome at taxonomic, functional, and lifestyle levels has been reported in other studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Our data further challenges the paradigm of axenic fungi or limited association of fungi with only one [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] or two [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] endohyphal bacteria.\u003c/p\u003e \u003cp\u003eSeveral reasons support the presence of a core bacteriome. Firstly, the genetic relationship of the fungal strains analyzed was similar to those from distant geographic locations (Additional file 1: Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) and was independent of proximity. Secondly, transient bacterial associates were efficiently removed through antibiotic pressure and heat-shock (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thirdly, single-conidium cultures were consistently used in all assays to avoid heterogeneity within conidia populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The phylogenies were constructed from sequences obtained using culture-independent methods, ensuring an unbiased representation of endohyphal bacterial diversity. Rigorous bioinformatic methods were employed, and potential sequencing artefacts from cross-contaminants were systematically removed [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], in agreement with the highest standards for analyzing complex metagenomes [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Finally, the combined use of short- and long- read platforms is recognized as the optimal strategy for generating robust datasets [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSymbiosis drives the acquisition of adaptive traits, ecological range expansion and biodiversity. The transition to obligate interspecific mutualism marks a major evolutionary step [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], seen in fungal hosts unable to replicate without their symbionts [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. While the roles of endohyphal bacteria in \u003cem\u003eA. fumigatus\u003c/em\u003e are not fully determined, data suggest a close relationship between the fungi and their core bacterial associates. The presence of a core bacteriome across various aspergilla strains (n\u0026thinsp;=\u0026thinsp;40) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Additional file 1: Fig. S6) implies vertically transmitted endosymbionts. Gram-negative bacteria dominate this core bacteriome (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e) - a group crucial in shaping life on Earth and contributing to essential endosymbionts [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Fungi from all major phyla can harbor bacterial endosymbionts, primarily Gram-negative, with obligate associations in early diverging fungi such as Mucoromycota and facultative associations in more derived lineages [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Examples of symbiosis include intracellular bacterial symbionts in insects providing essential nutrients [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], and the \u003cem\u003eRhizopus microsporus\u003c/em\u003e and \u003cem\u003eBurkholderia sp.\u003c/em\u003e partnership, where bacteria produce rhizoxin to aid fungal pathogenicity [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The identified core endohyphal bacteria likely constitute endosymbiotic partners of \u003cem\u003eA. fumigatus\u003c/em\u003e. Although mutual dependence is not conclusively established, the inability to cure the host and culture the endohyphal bacteria, along with the observed metabolic enrichment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Additional file 1: Fig. S8), supports this possibility.\u003c/p\u003e \u003cp\u003eThe prevalence of gram-negative endohyphal bacteria in the core bacteriome of \u003cem\u003eA. fumigatus\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e) may be due to their ability to utilize the thick-walled structures of conidia as a temporary, protective habitat, similar to gram-negative bacterial residents in chlamydospores [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. These bacteria have a thinner peptidoglycan layer between two membranes offering a superior functional capacity compared to that of gram-positive bacteria [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. However, the stable bacterial diversity within mycelia, as well as the inability to remove or easily culture them outside of the host, suggests that these gram-negative bacteria are true endosymbionts. The fungal diversification timeline dates back to the Jurassic period, with aspergilli established in the Cretaceous period [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. It is hypothesized that aspergilli acquired endohyphal bacteria around this time, occurring in parallel to the recruitment of fungi by plants. Fungal mutualisms with angiosperms allowed saprophytic fungi to diversify, providing water and mineral nutrients to plants through mycorrhizal root systems [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. This relationship facilitated the expansion of the ectomycorrhizal fungal genera during the Cretaceous period and thus promoted angiosperm diversification. Although a conserved genetic toolkit for symbiotic nitrogen fixation exists in plants [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], no similar system has been identified in fungi, even in well-studied obligate endosymbiotic systems in the Mucoromycota phylum [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study provides important insights into the complex interplay between \u003cem\u003eA. fumigatus\u003c/em\u003e and its associated endohyphal bacteria, establishing the existence of a potentially clinically relevant core bacteriome. The finding that Brydobacter bacteria may increase fungal virulence (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) warrants further investigation. Several core endohyphal bacteria, such as \u003cem\u003eCaulobacter sp.\u003c/em\u003e and \u003cem\u003eRalstonia sp\u003c/em\u003e., have been linked to human diseases, including hospital-acquired meningitis [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. This raises the important question of whether these partnerships enhance the infection capacity of \u003cem\u003eA. fumigatus\u003c/em\u003e; a species on the WHO\u0026rsquo;s list of fungal priority pathogens [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Our results challenge us to shift from a host-centric vision of fungal-bacterial partnerships to a bacteria-centric vision: focusing on the roles played by endohyphal bacteria and their interactions and adaptations. Understanding the molecular mechanisms governing the establishment and maintenance of these associations will also require a detailed analysis of host genetic processes, transmission mechanisms and population control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe samples (fungal isolates) were received anonymized, raising no ethical considerations to the ethics committee of ITQB NOVA. It is impossible to trace back the patients identities and clinical history.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable. Note that all samples – fungal isolates - were provided anonymized. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional File 1: Supplementary Information (MS Word), containing more detailed tables and figures that support the main figures panels at the main text.\u003c/p\u003e\n\u003cp\u003eAdditional File 2: Dataset1, Amplicon sequencing data of ITS regions (xls format) of the fungal strains.\u003c/p\u003e\n\u003cp\u003eAdditional File 3: Dataset 2, Micro-satellite genotyping data (xls format) of the fungal strains.\u003c/p\u003e\n\u003cp\u003eAdditional File 4: Dataset 3, Spearman's Correlation coefficients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sequencing data has been deposited in the Sequence Read Archive (NCBI) with the accession code PRJNA1135973.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge funding from Fundação para a Ciência e a Tecnologia (FCT) through the project “FATE” (PTDC/CTA-AMB/6587/2020), MOSTMICRO-ITQB R\u0026amp;D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020). This work was partially supported by PPBI - Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE - \"Orçamento de Estado\" and by european funds from FEDER - \"Fundo Europeu de Desenvolvimento Regional\". DP is grateful to FCT funding for the PhD scholarship PD/BD/138913/2018. T.M. and D.M. are grateful for the working contract financed by national funds under norma transitória D.L. n.° 57/2016. Funding received by the iBB-Institute for Bioengineering and Biosciences from the Portuguese Science and Technology Foundation (FCT) (UID/BIO/04565/2020) and by Programa Operacional Regional de Lisboa 2020 (Project N. 007317) is acknowledged. The project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy (i4HB) is also acknowledged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCSP supervised the project and the interpretation of data and prepared the final version of the manuscript. All authors have made substantial contributions to the acquisition, analysis and interpretation of data and contributed to the drafting of the manuscript: DP (fungal experiments, manuscript draft), DP, TM (bioinformatics); JMPJ, PC (microscopy); DM-H (virulence assays); DC, JMC (fungal isolation), CSP (conceptualization); CSP, RSL (supervision); CSP, RSL, GHG (resources). All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to all members of the Silva Pereira lab for their valuable discussions, with special thanks to alumnus researcher Celso Martins for his assistance in the initial data analyses and critical reading of the manuscript. We would like to acknowledge Dmitry A. Semchonok (ITQB NOVA) for his significant contributions, particularly in setting up appropriate electron microscopy methods and providing constructive criticism of the manuscript. Additionally, we recognize A.L. Sousa from the Electron Microscopy Facility at the Instituto Gulbenkian de Ciência for their technical expertise, sample processing, and imaging. Our thanks also go to Antonis Rokas and Matthew Mead (Vanderbilt University, USA) for their initial help with WGS and scientific discussions, as well as to Dean Morales (Center for Integrated Nanotechnologies, Los Alamos National Laboratory, USA) for his support in establishing microscopy methods. Finally, we sincerely thank James Yates (ITQB NOVA) for his meticulous proofreading of this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFiracative C: \u003cstrong\u003eInvasive fungal disease in humans: are we aware of the real impact?\u003c/strong\u003e \u003cem\u003eMem Inst Oswaldo Cruz\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e115\u003c/strong\u003e:e200430.\u003c/li\u003e\n \u003cli\u003eBongomin F, Gago S, Oladele RO, Denning DW: \u003cstrong\u003eGlobal and multi-national prevalence of fungal diseases\u0026mdash;estimate precision\u003c/strong\u003e. \u003cem\u003eJournal of fungi\u0026nbsp;\u003c/em\u003e2017, \u003cstrong\u003e3\u003c/strong\u003e(4):57.\u003c/li\u003e\n \u003cli\u003eFisher MC, Denning DW: \u003cstrong\u003eThe WHO fungal priority pathogens list as a game-changer\u003c/strong\u003e. \u003cem\u003eNature Reviews Microbiology\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e21\u003c/strong\u003e(4):211-212.\u003c/li\u003e\n \u003cli\u003eLatg\u0026eacute; J-P, Chamilos G: \u003cstrong\u003eAspergillus fumigatus and Aspergillosis in 2019\u003c/strong\u003e. \u003cem\u003eClin Microbiol Rev\u0026nbsp;\u003c/em\u003e2019, \u003cstrong\u003e33\u003c/strong\u003e(1):10.1128/cmr. 00140-00118.\u003c/li\u003e\n \u003cli\u003eFisher MC, Gurr SJ, Cuomo CA, Blehert DS, Jin H, Stukenbrock EH, Stajich JE, Kahmann R, Boone C, Denning DW: \u003cstrong\u003eThreats posed by the fungal kingdom to humans, wildlife, and agriculture\u003c/strong\u003e. \u003cem\u003eMBio\u0026nbsp;\u003c/em\u003e2020, \u003cstrong\u003e11\u003c/strong\u003e(3):10.1128/mbio. 00449-00420.\u003c/li\u003e\n \u003cli\u003eMartins C, Piontkivska D, Mil-Homens D, Guedes P, Jorge JM, Brinco J, B\u0026aacute;rria C, Santos AC, Barras R, Arraiano C: \u003cstrong\u003eIncreased production of pathogenic, airborne fungal spores upon exposure of a soil mycobiota to chlorinated aromatic hydrocarbon pollutants\u003c/strong\u003e. \u003cem\u003eMicrobiology Spectrum\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e11\u003c/strong\u003e(4):e00667-00623.\u003c/li\u003e\n \u003cli\u003eEvans TJ, Lawal A, Kosmidis C, Denning DW: \u003cstrong\u003eChronic Pulmonary Aspergillosis: Clinical Presentation and Management\u003c/strong\u003e. 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The diversity, origins and functions of fungal endobacteria living in Mucoromycota\u003c/strong\u003e. \u003cem\u003eThe ISME journal\u0026nbsp;\u003c/em\u003e2017, \u003cstrong\u003e11\u003c/strong\u003e(8):1727-1735.\u003c/li\u003e\n \u003cli\u003eManchon R, Zarrouk V, Leflon V, Iakovlev G, Bert F: \u003cstrong\u003eFirst case of bloodstream infection due to Caulobacter spp. associated with a postoperative meningitis\u003c/strong\u003e. \u003cem\u003eIDCases\u0026nbsp;\u003c/em\u003e2023, \u003cstrong\u003e32\u003c/strong\u003e:e01761.\u003c/li\u003e\n \u003cli\u003eRyan MP, Adley CC: \u003cstrong\u003eRalstonia spp.: emerging global opportunistic pathogens\u003c/strong\u003e. \u003cem\u003eEur J Clin Microbiol Infect Dis\u0026nbsp;\u003c/em\u003e2014, \u003cstrong\u003e33\u003c/strong\u003e:291-304.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aspergillus fumigatus, fungal virulence, clinical isolates, fungal bacteriome, endohyphal bacteria, endosymbionts","lastPublishedDoi":"10.21203/rs.3.rs-4912975/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4912975/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial-fungal interactions that influence the behavior of one or both organisms are common in nature. Well-studied systems include endosymbiotic relationships that range from transient (facultative) to long-term (obligate) associations. Diverse endohyphal bacteria associate with fungal hosts, emphasizing the need to better comprehend the fungal bacteriome. We evaluated the hypothesis that the human fungal pathogen \u003cem\u003eAspergillus fumigatus\u003c/em\u003e harbors an endohyphal community of bacteria that influence the host phenotype. We analyzed whether 39 \u003cem\u003eA. fumigatus\u003c/em\u003e strains, mainly clinical isolates, show stable association with diverse endohyphal bacteria. All fungal strains analyzed were derived from single-conidium cultures that were subjected to antibiotic and heat treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fungal bacteriome, inferred through analysis of bacterial diversity within the fungal strains (short- and long- read sequencing methods), revealed the presence of core endohyphal bacterial genera (≤ 19). Microscopic analysis further confirmed the presence of endobacteria within the hyphae of distinct fungal strains. The fungal strains exhibited high genetic diversity and phenotypic heterogeneity in terms of drug susceptibility and virulence (using \u003cem\u003eGalleria mellonella\u003c/em\u003e as an infection model). No correlations were observed between genomic or functional traits and bacteriome diversity. However, the abundance of the \u003cem\u003eBryobacter\u003c/em\u003e genus exhibited a positive correlation with fungal virulence; and the presence of other bacteria genera was correlated with posaconazole susceptibility. Based on the genetic pool of the endobacteriome of \u003cem\u003eA. fumigatus\u003c/em\u003e both the functional annotation of prokaryotic taxa and the metabolic space could be predicted showing functional roles in major categories, for example, nitrogen fixation and chemoheterotrophy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese observations suggest intricated metabolic dependencies between fungal strains and their endohyphal bacteria partners. Our study emphasizes the existence of complex interactions between fungi and bacteria and the need to better understand the relationship between endohyphal bacteria and fungal virulence.\u003c/p\u003e","manuscriptTitle":"Hidden allies: Decoding the core endohyphal bacteriome of Aspergillus fumigatus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-06 05:28:45","doi":"10.21203/rs.3.rs-4912975/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2e5c54e7-dba2-4064-adac-015178023e5c","owner":[],"postedDate":"September 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-06T05:28:46+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-06 05:28:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4912975","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4912975","identity":"rs-4912975","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}