Effect of bacterial symbiosis on filamentous fungal adaption to environmental stressors

Effect of bacterial symbiosis on filamentous fungal adaption to environmental stressors | 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 Article Effect of bacterial symbiosis on filamentous fungal adaption to environmental stressors Lauben Kumureeba, Benson Musinguzi, Kennedy Kassaza, Anthony Kasajja, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6506101/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 Fungal-bacterial co-existence significantly influences the pathogenicity of challenging microbial infections. Traditional diagnostic methods typically focus on identifying a single pathogen, often overlooking fungi, particularly in resource-limited settings. This study investigates the interactions between fungi and their bacterial endosymbionts in infection contexts. A total of 152 fungal isolates (103 clinical and 49 environmental) were screened for bacterial endosymbionts via targeted Polymerase chain reaction (PCR) for the 16S rDNA gene. Only 8 (5.3%) of the isolates were found to possess bacterial symbionts, with 5 (3.3%) being clinical and 3 (2.0%) environmental. The study further examined how these fungi responded to environmental stressors such as elevated carbon dioxide, heat, pH, and antimicrobial exposure. Fungi containing the 16S gene demonstrated enhanced growth and better adaptation to these stressors compared to strains lacking bacterial symbionts or those treated with ciprofloxacin. The findings highlight the complex dynamics of fungal-bacterial symbiosis and suggest potential avenues for improving clinical management of fungal-bacterial co-infections, emphasizing the need for further exploration of these interactions in infection niches. Biological sciences/Microbiology Health sciences/Medical research Health sciences/Pathogenesis Fungal environmental bacterial symbiosis filamentous stressors BACKGROUND Historically, fungi have been known for their inconspicuous nature and cryptic life style in soils and or dead matter. For years we have known them as ecological engines through which their ability to decompose organic matter and; perform nutrients’ exchange and recycling has proved pivotal [ 1 ], [ 2 ]. However, over the past 30 years we have witnessed fungi transition into key aetiological agents and; responsible for some of the most difficult to treat infections. From this stand point, fungi are now known to affect about 1.2 billion and kill between 1.5-2.0 million people annually worldwide. Yet, despite their increased clinical importance, fungal infections remain among the most neglected diseases worldwide [ 3 ]. One key question that mycologists are dealing with of late is how exactly fungi transition from a saprophytic life style to pathogens. In an attempt to answer this question, several theories have been suggested including but not limited to their ability to produce adhesive compounds, toxins, pigments and enzymes [ 4 ]. In as much these indeed contribute to fungal pathogenicity, there is one subtle component that is often ignored and; that’s the ability of fungi to co-exist and interact with other organisms in the environment as well as in the infection niche. We now know that it’s not uncommon for fungi to coexist and interact with other organisms such as bacteria in the wide environments [ 5 ]. Through this it’s been established that indeed fungi can establish rather addictive and mutualistic relationships such as the one that is exhibited by the bacterial symbiosis among fungi [ 6 ]. The few bacterial symbionts associated with fungi have so far revealed subtle influences on fungal phenotypes such as virulence and metabolic adaptions [ 7 ], [ 8 ]. What we forget is that during a poly microbial co-existence, there is often a dominating and suppressed participants. Hitherto, complexity of fungal-bacterial interactions is not fully explored and the influences of the participating organisms tend to get masked during clinical manifestations. However, we should draw from what has been established in plant pathology. Where the bacteria such as Burkholderia rhizoxinica and Burkholderia endofungorum have been implicated in the blight disease of rice seedlings producing contamination and toxicity respectively [ 9 ], [ 10 ], [ 11 ]. Indeed the fungus Rhizopus microsporus formally believed as the aetiological agent for rice seedling blight disease, harbors the endosymbiont B. rhizoxinica that secrets a highly potent anti-beta tubulin toxin Rhizoxin [ 12 ], [ 13 ]. This is an antimitotic toxin that causes decay of plant tissue hence rice disease [ 14 ]. On the other hand drawing from this, Itabangi et al, also describes for the first time that a bacterial endosymbiont modulates fungal virulence, metabolism and antifungal response during infection by the Rhizopus microsporus in zebrafish, galleria, mice and amoebae models of infection [ 15 ]. Whether these observations can be a baseline for understanding fungal pathogenicity remains to be seen, but rather instigates a series of questions that need to be answered now. For instance, how do bacterial endosymbionts modulate fungal virulence. Do all filamentous fungi harbor endo-hyphal bacteria or do all fungal opportunists require a bacterial endo-hyphal bacterium to evade immune system and if so, can a combination therapy of antibiotics and antifungals improve patients’ prognosis. With this background, we have expanded our investigations to encompass routinely isolated clinical and environmental filamentous fungi. Specifically, at Mbarara Regional Referral Hospital (MRRH), routine isolation of fungal opportunists for instance Aspergillus species, dermatophytes, and Dematiaceous fungi is not uncommon in our clinical setting [ 16 ], [ 17 ]. Here, we aimed at screening for bacterial endosymbiosis among the clinically and environmentally isolated filamentous fungi. We also explored how bacterial symbiosis impacts fungal phenotypes including thermotolerance, growth, and response to antifungals. This way, we revealed that there is significant cross talk between co-existing fungi and bacterial endosymbionts that significantly influence metabolic and genomic evolution. MATERIALS AND METHODS Study design and samples This was a laboratory based, cross-sectional study that targeted clinically and environmentally isolated filamentous fungi from Mbarara Regional Referral Hospital (MRRH). A total of 152 isolates were recruited in that study. These clinical isolates had various sample origins including Ear, nose and throat (ENT), ophthalmology, dermatology, gynecology, surgical and internal medicine samples. The environmental samples were collected through culture plate exposure in the environment including, external open air, laboratory confinement and surgical ward confinement and around homestead confinement environments such as the refrigerators and food storage facilities. Sample processing Routine cultivation of fungal isolates was performed on Sabouraud Dextrose Agar (SDA) (Formedium) at 28 o C for 7 days as described by Nevalainen et al [ 18 ]. The isolates were then sub cultured on Potato Dextrose Agar (PDA) (Formedium) at 28 o C for 7 days as described by Nevalainen et al [ 18 ] to encourage fruiting for later identification. The isolates were identified microscopically through Lacto phenol Cotton Blue Staining (LPCB) (Sigma Aldrich). Fungal fermentation for detection of the 16S rDNA gene For detection of bacterial endosymbiont, fungi were aseptically fermented in 250 mL flasks containing VK media containing 1% corn starch (Sigma Aldrich), 0.5% glycerol (sigma Aldrich), 1% gluten meal (sigma Aldrich), 1% dried yeast (sigma Aldrich) and 1% corn steep liquor (sigma Aldrich), 1% CaCO 3 (sigma Aldrich) and pH adjusted to 6.5, incubated at 30 o c with shaking at 80 rpm using an incubated shaker for 7 days as described by [ 19 ], [ 20 ]. Fungal stocks were stored at − 80 o c in 5% (w/v) trehalose (Thermofisher) and 10% (v/v) glycerol (Thermofisher) as described by Rohad et al [ 21 ]. Generation of bacteria free fungal strains (Bacterial knockout fungal or cured fungal isolates) For the generation of bacteria-free fungal strains, the fungi were cultivated and maintained on PDA with 60 µg/mL of ciprofloxacin for up to 3 months. Absence of the bacteria was confirmed through polymerase chain reaction (PCR) amplification of the 1.5 kb band for bacterial 16S rDNA as previously described by [ 20 ]. Screening for bacterial symbiosis using target PCR PCR screening for the presence or absence of the endosymbionts was performed using universal primers ( 5′-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3′/5′-CCCGGGATCCAAGCTTACGGCTACCTTGTTACGACTT-3′) as applied by[ 22 ], [ 23 ] ; which amplify a 1.5 kb 16S rDNA product. Deoxyribonucleic acid (DNA) was obtained from fungal spores in a screw cup with beads and homogenized using a bead beater at a speed of 6500xg for 1 min. DNA was then extracted with the DNeasy Powerlyzer Microbial kit (Qiagen) following the manufacturer’s instructions. PCR was then performed using 2X Hot Start Taq PCR master mix (New England Biolabs Inc., USA) and reaction mixes prepared as follows; for each 25 µL PCR reaction consisted of 12.5 µL of 2X Hot Start Taq master mix, 1.0 µL of 10 µM forward and reverse primers each, 3 µL of template DNA, and 7.5 µL of RNase-free water in the final reaction mix. PCR cycling consisted of the following conditions; 96 o C for 5 min, followed by 35 cycles of 96 o C for 1min, 52 o C for 1min, 72 o C for 2min and a final extension at 72 o C for 5 min. PCR Amplicons were electrophoresed at 120V and 80mA for 1 hr using 0.8% agarose gel prepared in 1X Tris-Borate EDTA buffer (TBE) (Formedium), 5µL Safe View Classic™ DNA stain (cat # G108)/100mL of TBE (sigma Aldrich), 6X loading dye (Thermo Scientific #R0611) and run with a 1kb ladder (NEB-Biolabs). Bands were visualized using the Dark Reader Trans-illuminator (Clare Chemical Research, USA) to confirm the amplicon size. The Actin housekeeping gene (0.6kb) was used, a control also used by [ 20 ], [ 22 ]. The 16S rDNA gene isolated from Escherichia coli was used as a positive control while PCR water was used as a negative control. The gel was run on amplicons from at least 3 technical repeats. Effect of bacterial symbiosis on fungal phenotypes Fungi with (wild type) and without (knockout type) the 16S rDNA bacteria gene were evaluated for growth, adaptation to carbon dioxide (CO 2 ), response to antifungal drugs, pH and heat stresses. In total 3 fungal strains were tested; namely wild type (with the 16S rDNA gene), naïve type (negative for the 16S rDNA gene) and the knockout type (negative for the 16S rDNA following bacterial knockout). Accordingly, from freshly grown fungal plates, circular disc incisions of 8mm were made using sterile pipette tips and transferred on to new PDA plates with the incised disc upside down as described by [ 18 ]. These inoculums were made in 3 repeats. For effects on growth ; the plates were incubated at 30 o C for 7 days as described by Ali et al [ 24 ]. Growth of the fungi were monitored and recorded through serial measurements of the fungal colony diameter, with growth increment determined by subtracting the final diameter from the diameter to the disc incision. For thermoterelence (heat stress) ; inoculated plates were respectively incubated at 37 o C, 40 o C and 45 o C for 7 days as described by [ 25 ]; and growth rate monitored and recorded as described above. For response to (CO 2 ) stress; inoculated plates were incubated in both low (atmospheric) and high (5% CO 2 ) concentrations for 7 days as described by [ 26 ], [ 27 ]; and growth rate monitored and recorded as described earlier. For response to pH stress ; accordingly, fungi were inoculated on culture media adjusted to acidic (pH 3, 5 and 6) using 0.1M Hydrochloric (HCL) acid (Sigma Aldrich), neutral (pH7) and alkaline (pH 8) using 1 M sodium hydroxide (NaOH) (sigma Aldrich); and incubated for 7days as described by [ 25 ]. For response to antifungals; we used fluconazole (Pfizer Inc., New York, NY) at the strength of 200 times the normal, keeping in mind that filamentous fungi are intrinsically resistant to fluconazole. Accordingly, the spore inoculum size was adjusted to 5X10 6 conidia /mL as recommend by European committee for Antimicrobial Susceptibility Testing (EUCAST). The antifungal was thus in microtitre plates in a two-fold serial dilutions at 200 times the strength using Roswell Park Memorial Institute broth medium double strength with 2% glucose (RPMI 2% G) (sigma Aldrich) buffered with Morpholinepropanesulfunic acid (MOPs) (Thermofisher) as recommended by EUCAST (Arendrup et al., 2015). The titre plates were incubated at 37 o C and read macroscopically with the aid of a magnifying lens at 48 hours and 120 hours for fusarium, penicillium and Trichophyton species (spp) respectively. Data analysis Here we obtained mostly quantitative data which involved monitoring growth through serial measurement of growth diameters. These data were then cleaned using Excel spread sheet and analyzed using EPI-info software version 7.2 RESULTS Bacterial symbiosis among clinical and environmental filamentous fungal isolates We screened 152 isolates for bacterial symbiosis using universal primers that amplify a 1.5 kb bacterial 16S rDNA gene; out of these 67.8% (103/152) were of clinical while 42.2% (49/152) were of environmental origin. Bacterial symbiosis was established in 5.3% (8/152) of the isolates whilst clinical isolates contributed 3.3% (5/152), environmental isolates contributed 2.0% (3/152) as shown in Table 1 . The bacteria positive isolates included; Fusarium spp (n = 4/8), Penicillium spp (n = 2/8) and Trichophyton mentagrophytes (n = 2/8); with Bacterial symbiosis more prevalent among Fusarium spp. Table 1 Bacterial symbiosis among clinical and environmental filamentous fungal isolates Fungal isolate Category Number of isolates n (%) Bacterial symbiosis n (%) Clinical isolates 103(67.8) 5(3.3) Environmental isolates 49(42.2) 3(2.0) Total isolates 152 8(5.3) Effects of bacterial symbiosis on fungal phenotypes Here we explored how bacterial symbiosis among the different filamentous fungi does impact their phenotypes including growth, response to temperature, Carbon dioxide (CO 2 ) tension, pH and antifungal drugs. In our experiments, we challenge all the three strain types; Naïve type (negative for the 16S rDNA gene), wild type (positive for 16 S rDNA gene) and the knocked-out type or (16sS rDNA) cured strain under the different above-mentioned conditions. The naïve strains are those fungi that have naturally not associated with bacterial endosymbionts and thus negative for the 16S rDNA, the wild type strains are those that are associated with the bacterial endosymbiont and thus have tested positive for the 16S rDNA while the knocked-out type are the fungal strains that have test positive for bacterial endosymbiont but the bacteria has been knocked out or cured through treatment with ciprofloxacin. Thus, we trucked how the different strain types of fungi behave when exposed to the different above-mentioned conditions. Accordingly, a total of 24 fungal isolates were studied as shown in Table 2 below. Table 2 Showing fungal isolates studied in different environmental conditions Fungal species Naïve types Wild types Knocked out types Penicillium spp (n = 6) 2 2 2 Trychophyton mentagrophytes (n = 6) 2 2 2 Fusarium spp (n = 12) 4 4 4 Total isolates (n = 24) 8 8 8 Effects of bacterial symbiosis on fungal growth All types of microbial growth are impacted by environmental conditions. One of the most critical factors for microbial growth is availability of nutrients and an energy source. Like other walks of life fungi also need vitamins, carbohydrates, fats, proteins, essential metals, humidity and aerobic conditions among other requirements. Here we tracked the ability of the various fungal strains’ growth on Potato dextrose agar (PDA) medium. PDA is made of potato infusion and dextrose (also known as glucose) as a carbohydrate source that supports the luxuriant growth of fungi and bacteria and is observed to encourage mold sporulation and pigment production in certain filamentous fungi. Accordingly; following inoculation and incubation at 30 o C for 7 days. We noted an increased mean growth colony diameter (48.8 mm) exhibited by wild type fungi when compared with the naïve strains (34.3 mm) and the cured strains (36.6 mm). With this you could say that the mean fungal growth changes attributed to bacteria symbiosis was 12.2 mm; when the mean colony growth diameters of the cured strains are subtracted from the wildtype positive strains as demonstrated in Table 3 . Table 3 Effects of bacterial symbiosis on fungal growth Fugal species Bacterial symbiotic status Fungal growth changes attributed to bacterial symbiosis (mm) Naïve types mean colony diameters (mm) Wild types mean colony diameters (mm) Knocked out types mean colony diameters (mm) Penicillium spp (n = 6) 22.9 40.6 25.2 15.4 Trichophyton spp (n = 6) 28.9 34.5 31.2 3.3 Fusarium spp (n = 12) 51 71.2 53.4 17.8 Mean 34.3 48.8 36.6 12.2 Effects of bacterial symbiosis on fungal response to carbon dioxide (CO 2 ) tension The carbon cycle is one pivotal natural process of reusing carbon atoms, which travel from the atmosphere into different organisms on earth and then back into the atmosphere over and over again. However, different organisms will tolerate particular concentrations of CO 2 . This is akin to fungi’s ability to adopt to environmental CO 2 of say 5%, which is similar to the concentration of CO 2 found in the human host. Simply, for the fungi to be able to infect man they have to overcome the elevated redox potential which is contributed by oxygen and CO 2 levels (Sherrington et al., 2018a). Here, we tested against atmospheric and 5% of CO 2; the different fungal strains demonstrated different growth abilities. With naïve and cured strains showing similar but slower growth rates as compared to the wild type fungi. Under atmospheric CO 2 , the mean colony growth diameters for the naïve and cured strains were 33.1 mm and 35.2 mm respectively while that of the wild type positive strain was 47.1 mm. Under the 5% CO 2 , the naïve and cured strains demonstrated growth diameters of 15.7 mm and 16.1 mm respectively whilst the wild type demonstrated 23.3 mm, with the growth attributable to the endosymbiont being 11.9 mm and 7.2 mm under atmospheric and 5% conditions respectively as shown in Table 4 . Table 4 Effect of bacterial symbiosis on fungal response to carbon dioxide (CO 2 ) tension Fungal species CO 2 concentration Bacterial symbiotic status Fungal growth changes attributed to bacterial symbiosis (mm) Naïve types mean colony diameters (mm ) Wild types mean colony diameters (mm) Knocked out types mean colony diameters (mm ) Penicillium spp (n = 6) Atmospheric 20 38.5 23 15.5 5% 12.2 25.2 13.2 12 Trichophyton mentagrophytes (n = 6) Atmospheric 29.1 34.5 29.1 5.4 5% 20.7 22.7 20.1 2 .6 Fusarium spp (n = 12) Atmospheric 50.3 68.5 53.6 14.9 5% 14.3 22 15 7.0 Mean Atmospheric 33.1 47.1 35.2 11.9 5% 15.7 23.3 16.1 7.2 Effect of bacterial symbiosis on fungal response to temperature (thermotolerance) Thermotolerance is the ability of an organism to survive high temperatures. An organism's natural tolerance of heat is there basal thermotolerance (Boulanger et al., 2023, Larkindale et al., 2005, Sarkar et al., 2021). Meanwhile, acquired thermotolerance is defined as an enhanced level of thermotolerance after exposure to a heat stress. Multiple factors contribute to thermotolerance including signaling molecules like abscisic acid, salicylic acid, and pathways like the ethylene signaling pathway and; heat response pathway (Larkindale et al., 2005, Sarkar et al., 2021). Indeed, for filamentous fungi to succeed at infecting the human host must overcome the 37 o C temperature which is much higher than their basal temperature of 25 o C. Here, we exposed the three fungal strain types to 3 different temperatures of 37 o C (body temperature), 40 o C and 45 o C. as shown in Table 5 , we noted that both naïve and cured fungal strains struggled to grow as the temperature increased from 37 o C to 45 o C with no growth registered under 45 o C for all the strain types. In general growth attributed to fungal symbiosis was 11.3 mm and 12.3 mm at 37 o C and 40 o C respectively. Table 5 Effect of bacteria symbiosis on fungal response to heat stress Fungal species Temperature Bacterial symbiosis status Fungal growth changes attributed to bacterial symbiosis (mm) Naïve types mean colony diameters (mm) Wild types mean colony diameters (mm) Knocked out types mean colony diameters (mm) Penicillium spp (n = 6) 37 o C 15 32.5 16 16.5 40 o C 0 18.5 0 18.5 45 o C 0 0 0 0 Trychophyton mentagrophytes (n = 6) 37 o C 22 29.7 22.2 7.5 40 o C 17 24.4 16 8.4 45 o C 0 0 0 0 Fusarium spp (n = 12) 37 o C 17.2 27.8 18 9.8 40 o C 8.5 18.7 8.7 10 45 o C 0 0 0 0 Mean 37 o C 18 30 18.7 11.3 40 o C 8.5 20.5 8.2 12.3 45 o C 0 0 0 0 Effect of bacterial symbiosis on fungal response to pH stress Fungi and bacteria have a wide range of acid/alkaline needs for growth, and are often negatively correlated between pH 4.5 and 8.3 but generally range from pH 3.0 to more than pH 8.0, with an ideal pH of approximately pH 5.0, assuming that all nutritional requirements are met (Elzwai et al., 2018). In general, filamentous fungi such as Penicillium spp and Fusarium spp species appear more apt toward low pH, while bacteria are naturally neutrophiles growing best at pH 7.0 with a few acidophiles growing optimally at a pH near 3.0 whilst alkaliphiles grow optimally between a pH of 8 and 10.5. In this study we exposed the different fungal strains in question to pH 3.0, 5.0, 6.0, 7.0 and 8.0. We noted that all the strains registered their optimal growth at pH 6.0 and thereafter growth begins to drop as the pH turns alkaline. However, in all the strains, the wildtype positive for the endosymbiotic gene showed more adaptability for growth when compared with naïve and cured strains as demonstrated in Table 6 . Indeed, the growth rate attributable to bacterial symbiosis was as listed below; 9.8 mm at pH 3.0, 9.8 mm at pH 5.0, 11.7 mm at pH 6.0, 10.6 mm at pH 7.0 and 8.3 mm at pH 8.0 mm in a similar manner as noted above. Table 6 Effect of bacterial symbiosis on fungal response to pH stress Fungal species pH Bacterial symbiosis status Fungal growth changes attributed to bacterial symbiosis (mm) Naïve types mean colony diameters (mm) Wild types mean colony diameters (mm) Knocked out types mean colony diameters (mm) Penicillium spp (n = 6) 3.0 12.7 21.2 14.5 6.7 5.0 18.5 35 20.7 14.3 6.0 21.2 39.7 23.1 16.6 7.0 17.5 30.7 20.6 10.1 8.0 10.5 26.7 15.5 11.2 Trychophyton mentagrophytes (n = 6) 3.0 24 28 24 4 5.0 27.5 31 29 2 6.0 31.4 34.5 31 3.5 7.0 22 24.0 23 1.0 8.0 20.5 22.0 20 2.0 Fusarium spp (n = 12) 3.0 34.8 54.6 36 18.6 5.0 50.2 65.3 52.4 12.9 6.0 53.7 71 56.0 15 7.0 42.7 61.1 43.3 17.8 8.0 40.4 52 40.3 11.7 Mean 3.0 23.8 34.6 24.8 9.8 5.0 32.1 43.8 34 9.8 6.0 35.4 48.4 36.7 11.7 7.0 27.4 39.6 29 10.6 8.0 23.8 33.6 25.3 8.3 Effects of bacterial symbiosis on fungal response to antifungal drugs Filamentous fungi are intrinsically resistant to many of the available antifungal drugs including fluconazole. Here, we employed fluconazole at a strength of 200 times the normal concentrations. Though intrinsically resistant to fluconazole we noted inhibitory activity exhibited by majorly naïve and cured strains when compared with the endosymbiotic positive bacteria. When exposed to fluconazole concentrations of 2, 4, 8, 16, 32, 64, 128 and 256 mg/L, we noted both naïve and cured strains of Penicillium spp and Trichophyton mentagrophytes struggled to grow more than Fusarium spp when co-incubated with the antifungal when compared to the endosymbiotic bacteria positive fungi. Indeed, the mean growth rate attributable to bacterial symbiosis was 106.7 mm as shown in Table 7 . Table 7 Effect of bacterial symbiosis on fungal response to antifungal drugs Fungal species Fungal isolate bacteria status MIC changes (mg/L) attributed to bacterial symbiosis Naïve types mean MIC value (mg/L) Wild types mean MIC value (mg/L) Knocked out types mean MIC value (mg/L) Penicillium spp (n = 6) 16 128 32 96 Trychophyton mentagrophytes (n = 6) 32 128 32 96 Fusarium spp (n = 12) 64 256 128 128 Mean 37.3 170.7 64 106.7 Generally, all test isolates had reduced susceptibility to fluconazole (MIC ≥ 16 mg/L). Nonetheless, endo-bacteria positive fungi showed more resistance (mean MIC of 170.7 mg/L) than cured and naïve fungi (mean MIC of 64 and 37.3 mg/L) respectively. DISCUSSION The compartmentalization that exists between mycologists and bacteriologists has left us prone to infections by co-operating microbial partners from both domains. This is because such practice has over time encouraged the study of fungi and bacteria to be in axenic settings [ 28 ]. Depending on which of the compartment we come from, we tend to be biased and hence overlook the fact that fungi and bacteria co-exist and interact in many different environments and; on various physical, biochemical and phylogenetic levels. If we are to draw from existing literature in this area, it’s now evident enough that fungi and bacteria may form physically and metabolically charged microbial consortia that harbor properties different from those of their single components [ 29 ]. With this in mind, it’s with great hope that if we are to examine the effects of fungal-bacterial co-existence and subsequent interactive modalities, our approaches to microbial infection biology in medicine could be handled a little different and; so that it perhaps leads to better management results. Other disciplines including agriculture, dentistry, food technology and biotechnology have picked this up with a strong hold and their research effects seems to be progressing somewhat different [ 28 ], [ 30 ] when compared to medicine which still lags behind in that regard. Central to fungal-bacterial co-existence is the cross talk that takes place between the two partnering organisms. Indeed, several communication channels have been suggested including but not limited to trophic interactions, and interactions via antibiosis, via protein secretions and gene transfer, cooperative metabolism, via chemotaxis and cellular contacts, via modulation of the physiochemical environments and; via signal-based interactions [ 28 ]. However, through these communication channels, special fungal-bacterial relationships are also formed which have included but not limited to mutualism, commensalism, antagonistic, saprophytism and symbiotic relations. Through such establishments fungal-bacterial co-existence through various interactions may influence several phenotypic expressions of either partners. For instance, fungal bacterial interactions impact microbial physiology, pathogenicity, microbial growth and development, microbial survival, dispersal and colonization; microbial heritable changes and; microbial complexity in life cycles [ 28 ]. Indeed, here we explore one of such establishments in particular the bacterial symbiosis among filamentous fungi. The symbiotic bacteria associated with pathogenic fungi seem valuable for microbial resources and worthy of an in-depth investigation. It’s important that we analyze the community structure, succession of the symbiont through generations of fungal reproduction and various medical importance in pathogenic fungi. This can assist in isolation of symbiont strains that positively influence the harboring fungi and have an essential relationship with the fungal reproduction cycles as well as an impact on infection traits. In this regard, we assessed both clinical and environmental filamentous fungal isolates for symbiotic bacteria and the effect of these symbionts on fungal growth, response to temperature, carbon dioxide tension, antifungal and pH stresses. Through this evaluation we established a 5.3% prevalence of bacterial symbiosis among152 studied fungal subjects with only about 3 fungal species (i.e. Fusarium spp (n = 4), Penicillium spp (n = 2) and Trichophyton mentagrophytes (n = 2) known to be involved with the bacterial symbiosis Table 1 . When compared with other studies, our prevalence of bacterial symbiosis is on the lower end. For instance, Lastovetsky et al and Okrasińska et al established incidences of 90% and 20 percent respectively [ 14 ], [ 31 ]. However, this difference could originate from the experimental approach since both studies employed Burkholderia -specific primers that amplify a portion of the 23S rRNA gene and 16S rRNA gene [ 14 ]. Additionally, this being the first of such a study in our location, there is very little to compare with. But these results can be explained by environmental events suggesting that perhaps our weather and environmental conditions may not favor a lot of bacterial symbiosis among fungi but this remains to be determined later. Through this study, we have generally demonstrated that fungal strains with the bacterial endosymbiont possess enhanced growth rate, exhibits better survival when exposed to the various stress stimuli including temperature, carbon dioxide tension, pH, and antifungal drugs when compared with the endosymbiont free strains Tables 3 – 7 . Accordingly; the endosymbiotic bacteria contributed radial growth of 12 mm more than that of fungal species without the endosymbiont Table 3 . These results are similar with what Uehling et al determined when the fungus Mortierella elongata’s radial growth increased from 3.00 ± 0.03 mm/day without the endosymbiont to 4.38 ± 0.74 mm/day when it had bacterial endosymbiont Burkholderia strain BT03. The same fungus increased from 6.41 ± 1.27 mm/day without the endo-bacteria to 7.29 ± 0.39 mm/day with the endo-bacteria when grown on Malt extract agar (MEA) [ 32 ]. In another study by Vannini et al demonstrates that an endobacterium Candidatus G. gigasporarum enhances fungal growth [ 33 ]. Finally, Shaffer et al also demonstrates that bacterial endosymbiont Chitinophaga spp. (strain PS-EHB01) significantly increased growth of the fungus Fusarium spp compared to the endosymbiont free strain. With this in play, research labor to explain how this could be happening. For instance, it’s been suggested that bacterial endosymbionts are known to influence growth through modulation of the main fungal metabolic pathways in the mitochondria such as the adenosine triphosphate (ATP) synthesis, respiration and reactive oxygen species detoxification and thus increasing ASP production and respiration [ 12 ], [ 33 ], [ 34 ]. While others have suggested that certain endo bacteria may encourage production of certain compounds that may act as growth factors for the hosting fungus and or may detoxifying harmful waste products such as reactive oxygen species that may hinder growth [ 35 ]. It’s a little early to tell what could be happening in samples until further investigations are conducted. However, this is not the case for all fungi that harbor the endo bacteria as determined by Shaffer et al and Hoffman et al. Both of these studies gave contrasting findings, for instance Shaffer et al showed that growth by the fungus Pestalotiopsis spp (Strain 9143) was inhibited by the presence of endo-bacteria[ 6 ] whilst Hoffman et al found no difference in growth rates by both endosymbiont positive and negative fungal isolates [ 36 ]. There is no clear explanation for these events until more explorations are conducted. When tested against the CO 2, tension we established those fungi generally struggled to grow in an environment with high oxygen tension. However, the fungus positive for the endosymbiont generally survived better in high CO 2 concentrations. Accordingly; the mean radial growth attributed to bacterial symbiont was 11.9 mm at atmospheric CO 2 and 7.2 mm 5% CO 2 Table 4 . Though fungi generally struggle to grow under high CO 2 concentrations, their ability to grow here at almost similar rate with those under ideal conditions shows that the endosymbiont could be playing a key role in enabling the fungus survive better. But we know that Fungi can sense and adopt to elevated levels of CO 2 within the host environment and this is often attributable to the activity of fungal soluble adenylyl cyclase and carbonic anhydrase enzymes available in fungal cells [ 27 ]. These enzymes catalyze the bidirectional conversion of CO 2 and water into bicarbonate (HCO 3 ) and protons (H+)[ 37 ] thus creating a CO 2 free environment. However, it’s not clear how bacterial symbiotic fungi survive better in increased carbon dioxide that endosymbiotic free fungi. This remains to be explored further. Fungal response to the temperature stimuli is often constitutive just as it is with other microbes. In this regard filamentous fungi grow at an optimum temperature range of 25-30 o C [ 38 ]; and often begin to struggle when the temperature goes beyond 35 o C. This means that a human host temperature of 37 o C is one of the host defenses that filamentous fungi must overcome to manifest an infection. In this study we exposed the study subjects to the body temperature of 37 o C and a much higher temperature of 40 o C and 45 o C Table 5 . We demonstrated that though filamentous fungi struggled to grow under these temperature sets, the fungi positive for the endo bacterial gene grew with comfort under 37 o C and 40 o C when compared with the endobacteria naïve versions of the fungi registering mean radial growth rate attributed to endobacteria to be 11.3 mm at 37 o C, 11.2 mm at 40 o C and 0.00 mm 45 o C Table 5 . Note that we registered no growth under 45 o C for either version of the fungus. However, there is no data to support our claim here but Frey-Klett et al in his review put out a hypothesis that bacterial symbiosis could facilitate fungal thermotolerance [ 28 ]. There is limited data to explain how the endobacteria facilitate protection of the host fungus against heat stimuli. However, Corbin et al using some of his observations in entomological and plant studies suggested that endo-bacteria have the capacity to release metabolites which increase the expression stress resistant genes preparing the host against most of the stress stimuli [ 39 ]. Following these findings, we recommend that more investigations are done to exhaustively understand how endo bacteria benefit fungal hosts when it comes to thermotolerance. This study further examined fungal’s response towards pH stress. While fungi grow better at acidic pH range of 3–6 and; at alkaline pH or between 8 and 10.5. Our findings determined the optimum growth pH of 6 similar to a study by [ 24 ]. Generally, all fungal versions started slowly at low pH of 3 peaking at pH 6 and thereafter slowing again at pH7 and pH8 Table 6 . However, in all scenarios, endosymbiont free fungi showed low radial growth when compared with endosymbiont charged fungi. In the endosymbiont positive bacteria mean radial growth was more by 9.8 mm at pH3, 9.8 mm at pH5, 11.7 mm at pH 6, 10.6 mm at pH 7 and 8.83 mm at pH 8 all these increments were attributable to bacterial endosymbiont Table 6 . Still there is not much data to support our claim here but a few research including Yang et al and Brown et al have fronted an idea that filamentous fungi have the ability to modulate and create environmental pH that favors bacterial growth and as the bacteria thrives, it provides requirements that are essential for fungal growth and survival [ 40 ], [ 41 ], [ 42 ]. However, it’s still early to explain what is happening here and remains to be harnessed bit future studies. Finally, we evaluated these fungal versions against antifungal therapy. Though, its common knowledge that filamentous fungi are intrinsically resistant to fluconazole [ 43 ], [ 44 ], we defied this fact and used fluconazole anyway but at a concentration of 2, 4, 8, 16, 32, 64, 128 and 256 mg/L to offer some guidance. Although filamentous fungi exhibit less activity against fluconazole, we registered minimal activity which can be used as a basis for future evaluation. Interestingly, to our surprise we demonstrate a similar trend seen by others. For instance, Lupini et al showed that bacterial endosymbionts can increase fungal resistance towards heavy metals known to have antimicrobial activity [ 45 ]; Shao et al showed that a fungus Spiromastix spp. (strain SCSIO F190) while in a symbiotic relationship with endobacterium Alcaligenes feacalis resists most antifungal drugs including nystatin and the vice versa is true when the endo bacteria is knocked out [ 46 ]. Finally, Itabangi et al, demonstrated that endosymbiont free Rhizopus microsporus fungi are more sensitive to Amphotericin B than the endosymbiont charged Rhizopus spp [ 15 ]. Though not clear how the fungus utilizes the endosymbiont to resist against antimicrobials, Vannini et al and Shao et al tempt to give an insight. They suggest that there could be a vertical gene transfer between the fungus and the endosymbiont but this in addition to the fact that certain endobacterium such as the Candidatus Glomeribacter gigasporarum can elicit mechanisms to detoxify free radicles and also modulate fungal protein expression through influencing DNA replication and transcription. This leads to an accumulation of proteins that are involved in fungal cell wall rigidity hence fungal protection [ 33 ], [ 46 ]. This could serve as a basis to support our claim here but still leaves more questions than answers, which can only be got through future explorations. Conclusions Bacterial symbiosis among pathogenic and environmental fungi is a long-standing concept that we are only just discovering now. Its impact on clinical outcomes is not yet harnessed to full potential and yet could its exploration may improve patient prognosis. Here we have attempted to highlight the importance of bacterial symbiosis in fungal survival against some of the human host defense line in temperature, pH, antimicrobials and against CO 2 tension in vitro. Whether, this can be extrapolated to explain clinical events remains to be explored. But in general, we conclude that bacterial symbionts are essential for fungal survival and where possible treatment approaches need to be revised to encompass poly microbial aetiology now that we have noted that they are more prevalent than before. Declarations Ethical approval This study has been approved by the Uganda National Council of Science and Technology (HS12976ES) and Mbarara University Research Ethics Committee (MUREC 08/10-20) and was conducted according to Uganda National legislation. Declaration of interests Authors declared no competing interests Funding : This study was funded through the Capacity building Research, Massachusetts general Hospital, USA (First Mile) award and; government of Uganda through the Busitema University Research and Innovation Fund (BURIF) Grant No.4/DGSRI/2022). Herbert Itabangi is also a fellow of the European Developing Countries Clinical Trials Partnership (EDCTP) in collaboration with the European Union (grant TMA 2019CDF-2789); and also provided additional support through this grant. Author Contributions KL, LA and HI conceptualized the study, BM and AK designed experiments, performed the work and analysis; and wrote the primary manuscript; , KL, JM and KK contributed to the collection, analysis and interpretation of the data. LA and BM reviewed the later versions of the manuscript; and BA, LA and HI critically revised the manuscript. All authors approved this manuscript for publication. Data availability The datasets collected during the study are available from the corresponding author upon request. Consent for publication Not applicable Acknowledgements We are grateful to Dr Elizabeth Ballou for University of Exeter, UK for providing most of the reagents used in the study through support by a welcome Trust award in medical mycology and fungal immunology (097377). We recognize Mwesigye James and the entire microbiology department of `Mbarara University for the isolation of all the clinical samples used in the study. We are grateful for different departments from which samples have been drawn for the isolation of the fungi used here including ophthalmology, Gynecology, ENT and dermatology departments. Finally, we appreciate the PI of Mbarara Akavurugye Eye Study (MAES), from which some of the isolates were obtained as well. References Bonugli-Santos, R. C. et al. Marine-derived fungi: Diversity of enzymes and biotechnological applications, Front Microbiol , vol. 6, no. MAR, (2015). 10.3389/fmicb.2015.00269 Kohout, P. et al. Forest Microhabitat Affects Succession of Fungal Communities on Decomposing Fine Tree Roots. Front. Microbiol. 12 10.3389/fmicb.2021.541583 (Jan. 2021). Bongomin, F., Gago, S., Oladele, R. O. & Denning, D. W. Global and multi-national prevalence of fungal diseases—estimate precision. Dec. 01 2017 MDPI AG 10.3390/jof3040057 Afroz Toma, M. et al. Fungal Pigments: Carotenoids, Riboflavin, and Polyketides with Diverse Applications, Apr. 01, MDPI . 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Methods for isolation and cultivation of filamentous fungi. Methods Mol. Biol. 1096 , 3–16. 10.1007/978-1-62703-712-9_1 (2014). Scherlach, K., Partida-Martinez, L. P., Danse, H. M. & Hertweck, C. Antimitotic rhizoxin derivatives from a cultured bacterial endosymbiont of the rice pathogenic fungus Rhizopus microsporus, J Am Chem Soc , vol. 128, no. 35, pp. 11529–11536, Sep. (2006). 10.1021/ja062953o Itabangi, H. et al. Environmental interactions with amoebae as drivers of bacterial-fungal endosymbiosis and pathogenicity. Mar 21 10.1101/584607 (2019). Rohadi, H., Ilyas, M. & Ekowati, N. Preservation Technique of Filamentous Fungi Based on Inactive Metabolism at Indonesian Culture Collection (InaCC), in IOP Conference Series: Earth and Environmental Science , IOP Publishing Ltd, Nov. (2020). 10.1088/1755-1315/593/1/012001 Ibrahim, A. S. et al. Oct., Bacterial endosymbiosis is widely present among zygomycetes but does not contribute to the pathogenesis of mucormycosis, Journal of Infectious Diseases , vol. 198, no. 7, pp. 1083–1090, (2008). 10.1086/591461 Partida-Martinez, L. P. & Hertweck, C. Pathogenic fungus harbours endosymbiotic bacteria for toxin production, Nature , vol. 437, no. 7060, pp. 884–888, Oct. (2005). 10.1038/nature03997 Ali, S. R. M., Fradi, A. J. & Al-Aaraji, A. M. Effect of some physical factors on growth of five fungal species, [Online]. Available: www.euacademic.org. Myers, R. R. et al. rtfA controls development, secondary metabolism, and virulence in Aspergillus fumigatus, PLoS One , vol. 12, no. 4, Apr. (2017). 10.1371/journal.pone.0176702 Saha, U. S., Misra, R., Tiwari, D. & Prasad, K. N. A cost-effective anaerobic culture method & its comparison with a standard method, Indian Journal of Medical Research , vol. 144, no. OCTOBER, pp. 611–613, (2016). 10.4103/0971-5916.200881 Sherrington, S. L., Kumwenda, P., Kousser, C. & Hall, R. A. Host Sensing by Pathogenic Fungi, in Advances in Applied Microbiology, vol. 102, Academic Press Inc., 159–221. doi: 10.1016/bs.aambs.2017.10.004 . (2018). Frey-Klett, P. et al. Bacterial-Fungal Interactions: Hyphens between Agricultural, Clinical, Environmental, and Food Microbiologists, Microbiology and Molecular Biology Reviews , vol. 75, no. 4, pp. 583–609, Dec. (2011). 10.1128/mmbr.00020-11 Bhatia, S. K. et al. Biotechnological potential of microbial consortia and future perspectives, Nov. 17, Taylor and Francis Ltd . (2018). 10.1080/07388551.2018.1471445 Hennig, S. et al. New approaches in bioprocess-control: Consortium guidance by synthetic cell-cell communication based on fungal pheromones, Jun. 01, Wiley-VCH Verlag . (2018). 10.1002/elsc.201700181 Okrasi, A. et al. New Endohyphal Relationships between Mucoromycota and Burkholderiaceae Representatives, (2021). 10.1128/AEM Uehling, J. K. et al. Microfluidics and Metabolomics Reveal Symbiotic Bacterial–Fungal Interactions Between Mortierella elongata and Burkholderia Include Metabolite Exchange. Front. Microbiol. 10 10.3389/fmicb.2019.02163 (Oct. 2019). Vannini, C. et al. An interdomain network: The endobacterium of a mycorrhizal fungus promotes antioxidative responses in both fungal and plant hosts. New Phytol. 211 (1), 265–275. 10.1111/nph.13895 (Jul. 2016). Salvioli, A. et al. Symbiosis with an endobacterium increases the fitness of a mycorrhizal fungus, raising its bioenergetic potential. ISME J. 10 (1), 130–144. 10.1038/ismej.2015.91 (Jan. 2016). Shaffer, J. P., U’Ren, J. M., Gallery, R. E., Baltrus, D. A. & Arnold, A. E. An endohyphal bacterium (Chitinophaga, Bacteroidetes) alters carbon source use by Fusarium keratoplasticum (F. solani species complex, Nectriaceae), Front Microbiol , vol. 8, no. MAR, Mar. (2017). 10.3389/fmicb.2017.00350 Hoffman, M. T., Gunatilaka, M. K., Wijeratne, K., Gunatilaka, L. & Arnold, A. E. Endohyphal Bacterium Enhances Production of Indole-3-Acetic Acid by a Foliar Fungal Endophyte, PLoS One , vol. 8, no. 9, Sep. (2013). 10.1371/journal.pone.0073132 Occhipinti, R. & Boron, W. F. Role of carbonic anhydrases and inhibitors in acid–base physiology: Insights from mathematical modeling. Int. J. Mol. Sci. 20 (15). 10.3390/ijms20153841 (Aug. 2019). Pietikäinen, J., Pettersson, M. & Bååth, E. Comparison of temperature effects on soil respiration and bacterial and fungal growth rates. FEMS Microbiol. Ecol. 52 (1), 49–58. 10.1016/j.femsec.2004.10.002 (Mar. 2005). Corbin, C., Heyworth, E. R., Ferrari, J. & Hurst, G. D. D. Heritable symbionts in a world of varying temperature. Jan 01 2017 Nat. Publishing Group. 10.1038/hdy.2016.71 Yang, P. & van Elsas, J. D. R. Oliveira da Rocha Calixto, and Migration of Paraburkholderia terrae BS001 Along Old Fungal Hyphae in Soil at Various pH Levels, Microb Ecol , vol. 76, no. 2, pp. 443–452, Aug. (2018). 10.1007/s00248-017-1137-1 Brown, H. E. et al. Aug., Identifying a novel connection between the fungal plasma membrane and pH-sensing, Mol Microbiol , vol. 109, no. 4, pp. 474–493, (2018). 10.1111/mmi.13998 Carrasco, J. & Preston, G. M. Growing edible mushrooms: a conversation between bacteria and fungi. Mar. 01 2020 Blackwell Publishing Ltd. 10.1111/1462-2920.14765 Peng, Y., Zhang, Q., Xu, C. & Shi, W. MALDITOF MS for the rapid identification and drug susceptibility testing of filamentous fungi, Exp Ther Med , Oct. (2019). 10.3892/etm.2019.8118 Halliday, C. L. et al. Antifungal susceptibilities of non-Aspergillus filamentous fungi causing invasive infection in Australia: support for current antifungal guideline recommendations. Int. J. Antimicrob. Agents . 48 (4), 453–458. 10.1016/j.ijantimicag.2016.07.005 (Oct. 2016). Lupini, S., Peña-Bahamonde, J., Bonito, G. & Rodrigues, D. F. Effect of Endosymbiotic Bacteria on Fungal Resistance Toward Heavy Metals. Front. Microbiol. 13 10.3389/fmicb.2022.822541 (Mar. 2022). Shao, M. et al. Upregulation of a marine fungal biosynthetic gene cluster by an endobacterial symbiont. Commun. Biol. 3 (1). 10.1038/s42003-020-01239-y (Dec. 2020). Additional Declarations No competing interests reported. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6506101","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":453871952,"identity":"055c0fe4-aa05-4d79-aae2-e6633427b0d0","order_by":0,"name":"Lauben Kumureeba","email":"","orcid":"","institution":"Mbarara University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lauben","middleName":"","lastName":"Kumureeba","suffix":""},{"id":453871953,"identity":"448d7229-f5a8-41b8-9c19-c6fa37af2155","order_by":1,"name":"Benson Musinguzi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACAwTJ2PgASPKQpKXZgAQtEMAmQZTDzBm40yR/FNyx23D+cFvVzbY7Mgzshx8w8/zCrcWygXebNI/Bs+QNBw623c5te8bDwJNmwMzbh8dhB4BaGAwOJxscbARpOQz0Sw4DM28Pfi2SP0BaDjO2FYO18L8hrEWCx+CwncExxjZmsBYJoC08P/D4pZl3szVQS4LkGcZm6Zxzh3nYJJ4ZHJzbgFuLOXvvxps//hy25zt//OHnnLLD9vz8yQ8fvPmDWwsDM4RKhBvLBsQHGNvwaIECezQ+PltGwSgYBaNgpAEAmsxOh0L5mrEAAAAASUVORK5CYII=","orcid":"","institution":"Muni University","correspondingAuthor":true,"prefix":"","firstName":"Benson","middleName":"","lastName":"Musinguzi","suffix":""},{"id":453871954,"identity":"a79df31a-b502-447d-b762-9d31cfe767c9","order_by":2,"name":"Kennedy Kassaza","email":"","orcid":"","institution":"Mbarara University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Kennedy","middleName":"","lastName":"Kassaza","suffix":""},{"id":453871955,"identity":"44995e42-a320-42a7-95ff-a9eb896416a7","order_by":3,"name":"Anthony Kasajja","email":"","orcid":"","institution":"Busitema University","correspondingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"","lastName":"Kasajja","suffix":""},{"id":453871956,"identity":"9384c861-f7f5-46c6-84eb-43cbca35dc10","order_by":4,"name":"Beatrice Achan","email":"","orcid":"","institution":"Makerere University","correspondingAuthor":false,"prefix":"","firstName":"Beatrice","middleName":"","lastName":"Achan","suffix":""},{"id":453871957,"identity":"b3c7150a-ab25-4134-87bc-4c5178ab9743","order_by":5,"name":"Lucas Ampaire","email":"","orcid":"","institution":"Mbarara University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Lucas","middleName":"","lastName":"Ampaire","suffix":""},{"id":453871958,"identity":"5cb1ea04-4a05-46e4-8019-cea278d9c599","order_by":6,"name":"Herbert Itabangi","email":"","orcid":"","institution":"Mbarara University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Herbert","middleName":"","lastName":"Itabangi","suffix":""}],"badges":[],"createdAt":"2025-04-22 16:23:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6506101/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6506101/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92396724,"identity":"4b32d730-a60b-400c-83fb-2d864e3c89c6","added_by":"auto","created_at":"2025-09-29 09:32:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1679241,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6506101/v1/a4021487-d465-4c48-bff3-63ed0994746c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of bacterial symbiosis on filamentous fungal adaption to environmental stressors","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eHistorically, fungi have been known for their inconspicuous nature and cryptic life style in soils and or dead matter. For years we have known them as ecological engines through which their ability to decompose organic matter and; perform nutrients\u0026rsquo; exchange and recycling has proved pivotal [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, over the past 30 years we have witnessed fungi transition into key aetiological agents and; responsible for some of the most difficult to treat infections. From this stand point, fungi are now known to affect about 1.2\u0026nbsp;billion and kill between 1.5-2.0\u0026nbsp;million people annually worldwide. Yet, despite their increased clinical importance, fungal infections remain among the most neglected diseases worldwide [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. One key question that mycologists are dealing with of late is how exactly fungi transition from a saprophytic life style to pathogens. In an attempt to answer this question, several theories have been suggested including but not limited to their ability to produce adhesive compounds, toxins, pigments and enzymes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In as much these indeed contribute to fungal pathogenicity, there is one subtle component that is often ignored and; that\u0026rsquo;s the ability of fungi to co-exist and interact with other organisms in the environment as well as in the infection niche. We now know that it\u0026rsquo;s not uncommon for fungi to coexist and interact with other organisms such as bacteria in the wide environments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Through this it\u0026rsquo;s been established that indeed fungi can establish rather addictive and mutualistic relationships such as the one that is exhibited by the bacterial symbiosis among fungi [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The few bacterial symbionts associated with fungi have so far revealed subtle influences on fungal phenotypes such as virulence and metabolic adaptions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. What we forget is that during a poly microbial co-existence, there is often a dominating and suppressed participants. Hitherto, complexity of fungal-bacterial interactions is not fully explored and the influences of the participating organisms tend to get masked during clinical manifestations. However, we should draw from what has been established in plant pathology. Where the bacteria such as \u003cem\u003eBurkholderia rhizoxinica\u003c/em\u003e and \u003cem\u003eBurkholderia endofungorum\u003c/em\u003e have been implicated in the blight disease of rice seedlings producing contamination and toxicity respectively [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Indeed the fungus \u003cem\u003eRhizopus microsporus\u003c/em\u003e formally believed as the aetiological agent for rice seedling blight disease, harbors the endosymbiont \u003cem\u003eB. rhizoxinica\u003c/em\u003e that secrets a highly potent anti-beta tubulin toxin Rhizoxin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This is an antimitotic toxin that causes decay of plant tissue hence rice disease [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. On the other hand drawing from this, Itabangi et al, also describes for the first time that a bacterial endosymbiont modulates fungal virulence, metabolism and antifungal response during infection by the \u003cem\u003eRhizopus microsporus\u003c/em\u003e in zebrafish, galleria, mice and amoebae models of infection [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Whether these observations can be a baseline for understanding fungal pathogenicity remains to be seen, but rather instigates a series of questions that need to be answered now. For instance, how do bacterial endosymbionts modulate fungal virulence. Do all filamentous fungi harbor endo-hyphal bacteria or do all fungal opportunists require a bacterial endo-hyphal bacterium to evade immune system and if so, can a combination therapy of antibiotics and antifungals improve patients\u0026rsquo; prognosis. With this background, we have expanded our investigations to encompass routinely isolated clinical and environmental filamentous fungi. Specifically, at Mbarara Regional Referral Hospital (MRRH), routine isolation of fungal opportunists for instance \u003cem\u003eAspergillus\u003c/em\u003e species, dermatophytes, and \u003cem\u003eDematiaceous\u003c/em\u003e fungi is not uncommon in our clinical setting [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Here, we aimed at screening for bacterial endosymbiosis among the clinically and environmentally isolated filamentous fungi. We also explored how bacterial symbiosis impacts fungal phenotypes including thermotolerance, growth, and response to antifungals. This way, we revealed that there is significant cross talk between co-existing fungi and bacterial endosymbionts that significantly influence metabolic and genomic evolution.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and samples\u003c/h2\u003e \u003cp\u003eThis was a laboratory based, cross-sectional study that targeted clinically and environmentally isolated filamentous fungi from Mbarara Regional Referral Hospital (MRRH). A total of 152 isolates were recruited in that study. These clinical isolates had various sample origins including Ear, nose and throat (ENT), ophthalmology, dermatology, gynecology, surgical and internal medicine samples. The environmental samples were collected through culture plate exposure in the environment including, external open air, laboratory confinement and surgical ward confinement and around homestead confinement environments such as the refrigerators and food storage facilities.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample processing\u003c/h3\u003e\n\u003cp\u003eRoutine cultivation of fungal isolates was performed on Sabouraud Dextrose Agar (SDA) (Formedium) at 28\u003csup\u003eo\u003c/sup\u003eC for 7 days as described by Nevalainen et al [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The isolates were then sub cultured on Potato Dextrose Agar (PDA) (Formedium) at 28\u003csup\u003eo\u003c/sup\u003eC for 7 days as described by Nevalainen et al [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] to encourage fruiting for later identification. The isolates were identified microscopically through Lacto phenol Cotton Blue Staining (LPCB) (Sigma Aldrich).\u003c/p\u003e\n\u003ch3\u003eFungal fermentation for detection of the 16S rDNA gene\u003c/h3\u003e\n\u003cp\u003eFor detection of bacterial endosymbiont, fungi were aseptically fermented in 250 mL flasks containing VK media containing 1% corn starch (Sigma Aldrich), 0.5% glycerol (sigma Aldrich), 1% gluten meal (sigma Aldrich), 1% dried yeast (sigma Aldrich) and 1% corn steep liquor (sigma Aldrich), 1% CaCO\u003csub\u003e3\u003c/sub\u003e (sigma Aldrich) and pH adjusted to 6.5, incubated at 30\u003csup\u003eo\u003c/sup\u003ec with shaking at 80 rpm using an incubated shaker for 7 days as described by [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Fungal stocks were stored at \u0026minus;\u0026thinsp;80\u003csup\u003eo\u003c/sup\u003ec in 5% (w/v) trehalose (Thermofisher) and 10% (v/v) glycerol (Thermofisher) as described by Rohad et al [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eGeneration of bacteria free fungal strains (Bacterial knockout fungal or cured fungal isolates)\u003c/h3\u003e\n\u003cp\u003eFor the generation of bacteria-free fungal strains, the fungi were cultivated and maintained on PDA with 60 \u0026micro;g/mL of ciprofloxacin for up to 3 months. Absence of the bacteria was confirmed through polymerase chain reaction (PCR) amplification of the 1.5 kb band for bacterial 16S rDNA as previously described by [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eScreening for bacterial symbiosis using target PCR\u003c/h3\u003e\n\u003cp\u003ePCR screening for the presence or absence of the endosymbionts was performed using universal primers \u003cb\u003e(\u003c/b\u003e5\u0026prime;-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3\u0026prime;/5\u0026prime;-CCCGGGATCCAAGCTTACGGCTACCTTGTTACGACTT-3\u0026prime;) as applied by[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] ; which amplify a 1.5 kb 16S rDNA product. Deoxyribonucleic acid (DNA) was obtained from fungal spores in a screw cup with beads and homogenized using a bead beater at a speed of 6500xg for 1 min. DNA was then extracted with the DNeasy Powerlyzer Microbial kit (Qiagen) following the manufacturer\u0026rsquo;s instructions. PCR was then performed using 2X Hot Start Taq PCR master mix (New England Biolabs Inc., USA) and reaction mixes prepared as follows; for each 25 \u0026micro;L PCR reaction consisted of 12.5 \u0026micro;L of 2X Hot Start Taq master mix, 1.0 \u0026micro;L of 10 \u0026micro;M forward and reverse primers each, 3 \u0026micro;L of template DNA, and 7.5 \u0026micro;L of RNase-free water in the final reaction mix. PCR cycling consisted of the following conditions; 96\u003csup\u003eo\u003c/sup\u003eC for 5 min, followed by 35 cycles of 96\u003csup\u003eo\u003c/sup\u003eC for 1min, 52\u003csup\u003eo\u003c/sup\u003eC for 1min, 72\u003csup\u003eo\u003c/sup\u003eC for 2min and a final extension at 72\u003csup\u003eo\u003c/sup\u003eC for 5 min. PCR Amplicons were electrophoresed at 120V and 80mA for 1 hr using 0.8% agarose gel prepared in 1X Tris-Borate EDTA buffer (TBE) (Formedium), 5\u0026micro;L Safe View Classic\u0026trade; DNA stain (cat # G108)/100mL of TBE (sigma Aldrich), 6X loading dye (Thermo Scientific #R0611) and run with a 1kb ladder (NEB-Biolabs). Bands were visualized using the Dark Reader Trans-illuminator (Clare Chemical Research, USA) to confirm the amplicon size. The Actin housekeeping gene (0.6kb) was used, a control also used by [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The 16S rDNA gene isolated from \u003cem\u003eEscherichia coli\u003c/em\u003e was used as a positive control while PCR water was used as a negative control. The gel was run on amplicons from at least 3 technical repeats.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of bacterial symbiosis on fungal phenotypes\u003c/h2\u003e \u003cp\u003eFungi with (wild type) and without (knockout type) the 16S rDNA bacteria gene were evaluated for growth, adaptation to carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e), response to antifungal drugs, pH and heat stresses. In total 3 fungal strains were tested; namely wild type (with the 16S rDNA gene), na\u0026iuml;ve type (negative for the 16S rDNA gene) and the knockout type (negative for the 16S rDNA following bacterial knockout). Accordingly, from freshly grown fungal plates, circular disc incisions of 8mm were made using sterile pipette tips and transferred on to new PDA plates with the incised disc upside down as described by [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These inoculums were made in 3 repeats.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFor effects on growth\u003c/b\u003e; the plates were incubated at 30\u003csup\u003eo\u003c/sup\u003eC for 7 days as described by Ali et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Growth of the fungi were monitored and recorded through serial measurements of the fungal colony diameter, with growth increment determined by subtracting the final diameter from the diameter to the disc incision.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFor thermoterelence (heat stress)\u003c/b\u003e; inoculated plates were respectively incubated at 37\u003csup\u003eo\u003c/sup\u003eC, 40\u003csup\u003eo\u003c/sup\u003eC and 45\u003csup\u003eo\u003c/sup\u003eC for 7 days as described by [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]; and growth rate monitored and recorded as described above.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFor response to (CO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e) stress;\u003c/b\u003e inoculated plates were incubated in both low (atmospheric) and high (5% CO\u003csub\u003e2\u003c/sub\u003e) concentrations for 7 days as described by [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]; and growth rate monitored and recorded as described earlier.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFor response to pH stress\u003c/b\u003e; accordingly, fungi were inoculated on culture media adjusted to acidic (pH 3, 5 and 6) using 0.1M Hydrochloric (HCL) acid (Sigma Aldrich), neutral (pH7) and alkaline (pH 8) using 1 M sodium hydroxide (NaOH) (sigma Aldrich); and incubated for 7days as described by [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eFor response to antifungals;\u003c/b\u003e we used fluconazole (Pfizer Inc., New York, NY) at the strength of 200 times the normal, keeping in mind that filamentous fungi are intrinsically resistant to fluconazole. Accordingly, the spore inoculum size was adjusted to 5X10\u003csup\u003e6\u003c/sup\u003e conidia /mL as recommend by European committee for Antimicrobial Susceptibility Testing (EUCAST). The antifungal was thus in microtitre plates in a two-fold serial dilutions at 200 times the strength using Roswell Park Memorial Institute broth medium double strength with 2% glucose (RPMI 2% G) (sigma Aldrich) buffered with Morpholinepropanesulfunic acid (MOPs) (Thermofisher) as recommended by EUCAST (Arendrup et al., 2015). The titre plates were incubated at 37\u003csup\u003eo\u003c/sup\u003eC and read macroscopically with the aid of a magnifying lens at 48 hours and 120 hours for fusarium, penicillium and Trichophyton species (spp) respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eHere we obtained mostly quantitative data which involved monitoring growth through serial measurement of growth diameters. These data were then cleaned using Excel spread sheet and analyzed using EPI-info software version 7.2\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBacterial symbiosis among clinical and environmental filamentous fungal isolates\u003c/h2\u003e \u003cp\u003eWe screened 152 isolates for bacterial symbiosis using universal primers that amplify a 1.5 kb bacterial 16S rDNA gene; out of these 67.8% (103/152) were of clinical while 42.2% (49/152) were of environmental origin. Bacterial symbiosis was established in 5.3% (8/152) of the isolates whilst clinical isolates contributed 3.3% (5/152), environmental isolates contributed 2.0% (3/152) as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The bacteria positive isolates included; \u003cem\u003eFusarium\u003c/em\u003e spp (n\u0026thinsp;=\u0026thinsp;4/8), \u003cem\u003ePenicillium\u003c/em\u003e spp (n\u0026thinsp;=\u0026thinsp;2/8) and \u003cem\u003eTrichophyton mentagrophytes\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2/8); with Bacterial symbiosis more prevalent among \u003cem\u003eFusarium\u003c/em\u003e spp.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBacterial symbiosis among clinical and environmental filamentous fungal isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFungal isolate Category\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of isolates n (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacterial symbiosis\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical isolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e103(67.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5(3.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnvironmental isolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49(42.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3(2.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal isolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8(5.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffects of bacterial symbiosis on fungal phenotypes\u003c/h2\u003e \u003cp\u003eHere we explored how bacterial symbiosis among the different filamentous fungi does impact their phenotypes including growth, response to temperature, Carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) tension, pH and antifungal drugs. In our experiments, we challenge all the three strain types; Na\u0026iuml;ve type (negative for the 16S rDNA gene), wild type (positive for 16 S rDNA gene) and the knocked-out type or (16sS rDNA) cured strain under the different above-mentioned conditions. The na\u0026iuml;ve strains are those fungi that have naturally not associated with bacterial endosymbionts and thus negative for the 16S rDNA, the wild type strains are those that are associated with the bacterial endosymbiont and thus have tested positive for the 16S rDNA while the knocked-out type are the fungal strains that have test positive for bacterial endosymbiont but the bacteria has been knocked out or cured through treatment with ciprofloxacin. Thus, we trucked how the different strain types of fungi behave when exposed to the different above-mentioned conditions. Accordingly, a total of 24 fungal isolates were studied as shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e below.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eShowing fungal isolates studied in different environmental conditions\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFungal species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa\u0026iuml;ve types\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWild types\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKnocked out types\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePenicillium\u003c/em\u003e spp (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTrychophyton mentagrophytes\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusarium\u003c/em\u003e spp (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal isolates (n\u0026thinsp;=\u0026thinsp;24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of bacterial symbiosis on fungal growth\u003c/h2\u003e \u003cp\u003eAll types of microbial growth are impacted by environmental conditions. One of the most critical factors for microbial growth is availability of nutrients and an energy source. Like other walks of life fungi also need vitamins, carbohydrates, fats, proteins, essential metals, humidity and aerobic conditions among other requirements. Here we tracked the ability of the various fungal strains\u0026rsquo; growth on Potato dextrose agar (PDA) medium. PDA is made of potato infusion and dextrose (also known as glucose) as a carbohydrate source that supports the luxuriant growth of fungi and bacteria and is observed to encourage mold sporulation and pigment production in certain filamentous fungi. Accordingly; following inoculation and incubation at 30\u003csup\u003eo\u003c/sup\u003eC for 7 days. We noted an increased mean growth colony diameter (48.8 mm) exhibited by wild type fungi when compared with the na\u0026iuml;ve strains (34.3 mm) and the cured strains (36.6 mm). With this you could say that the mean fungal growth changes attributed to bacteria symbiosis was 12.2 mm; when the mean colony growth diameters of the cured strains are subtracted from the wildtype positive strains as demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of bacterial symbiosis on fungal growth\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFugal species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eBacterial symbiotic status\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFungal growth changes attributed to bacterial symbiosis (mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa\u0026iuml;ve types mean colony diameters (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWild types mean colony diameters (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKnocked out types mean colony diameters (mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePenicillium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTrichophyton\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;12)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e48.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffects of bacterial symbiosis on fungal response to carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) tension\u003c/h2\u003e \u003cp\u003eThe carbon cycle is one pivotal natural process of reusing carbon atoms, which travel from the atmosphere into different organisms on earth and then back into the atmosphere over and over again. However, different organisms will tolerate particular concentrations of CO\u003csub\u003e2\u003c/sub\u003e. This is akin to fungi\u0026rsquo;s ability to adopt to environmental CO\u003csub\u003e2\u003c/sub\u003e of say 5%, which is similar to the concentration of CO\u003csub\u003e2\u003c/sub\u003e found in the human host. Simply, for the fungi to be able to infect man they have to overcome the elevated redox potential which is contributed by oxygen and CO\u003csub\u003e2\u003c/sub\u003e levels (Sherrington et al., 2018a). Here, we tested against atmospheric and 5% of CO\u003csub\u003e2;\u003c/sub\u003e the different fungal strains demonstrated different growth abilities. With na\u0026iuml;ve and cured strains showing similar but slower growth rates as compared to the wild type fungi. Under atmospheric CO\u003csub\u003e2\u003c/sub\u003e, the mean colony growth diameters for the na\u0026iuml;ve and cured strains were 33.1 mm and 35.2 mm respectively while that of the wild type positive strain was 47.1 mm. Under the 5% CO\u003csub\u003e2\u003c/sub\u003e, the na\u0026iuml;ve and cured strains demonstrated growth diameters of 15.7 mm and 16.1 mm respectively whilst the wild type demonstrated 23.3 mm, with the growth attributable to the endosymbiont being 11.9 mm and 7.2 mm under atmospheric and 5% conditions respectively as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of bacterial symbiosis on fungal response to carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) tension\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFungal species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eBacterial symbiotic status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFungal growth changes attributed to bacterial symbiosis (mm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eNa\u0026iuml;ve types mean colony diameters (mm\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eWild types mean colony diameters (mm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eKnocked out types mean colony diameters (mm\u003c/b\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003ePenicillium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAtmospheric\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eTrichophyton mentagrophytes\u003c/b\u003e \u003cb\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAtmospheric\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 .6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;12)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAtmospheric\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAtmospheric\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of bacterial symbiosis on fungal response to temperature (thermotolerance)\u003c/h2\u003e \u003cp\u003eThermotolerance is the ability of an organism to survive high temperatures. An organism's natural tolerance of heat is there basal thermotolerance (Boulanger et al., 2023, Larkindale et al., 2005, Sarkar et al., 2021). Meanwhile, acquired thermotolerance is defined as an enhanced level of thermotolerance after exposure to a heat stress. Multiple factors contribute to thermotolerance including signaling molecules like abscisic acid, salicylic acid, and pathways like the ethylene signaling pathway and; heat response pathway (Larkindale et al., 2005, Sarkar et al., 2021). Indeed, for filamentous fungi to succeed at infecting the human host must overcome the 37\u003csup\u003eo\u003c/sup\u003eC temperature which is much higher than their basal temperature of 25 \u003csup\u003eo\u003c/sup\u003eC. Here, we exposed the three fungal strain types to 3 different temperatures of 37 \u003csup\u003eo\u003c/sup\u003eC (body temperature), 40 \u003csup\u003eo\u003c/sup\u003eC and 45 \u003csup\u003eo\u003c/sup\u003eC. as shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, we noted that both na\u0026iuml;ve and cured fungal strains struggled to grow as the temperature increased from 37 \u003csup\u003eo\u003c/sup\u003eC to 45 \u003csup\u003eo\u003c/sup\u003eC with no growth registered under 45 \u003csup\u003eo\u003c/sup\u003eC for all the strain types. In general growth attributed to fungal symbiosis was 11.3 mm and 12.3 mm at 37 \u003csup\u003eo\u003c/sup\u003eC and 40 \u003csup\u003eo\u003c/sup\u003eC respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eEffect of bacteria symbiosis on fungal response to heat stress\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFungal species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eBacterial symbiosis status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFungal growth changes attributed to bacterial symbiosis (mm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNa\u0026iuml;ve types mean colony diameters (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWild types mean colony diameters (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKnocked out types mean colony diameters (mm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003ePenicillium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eTrychophyton mentagrophytes\u003c/b\u003e \u003cb\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;12)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45\u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of bacterial symbiosis on fungal response to pH stress\u003c/h2\u003e \u003cp\u003eFungi and bacteria have a wide range of acid/alkaline needs for growth, and are often negatively correlated between pH 4.5 and 8.3 but generally range from pH 3.0 to more than pH 8.0, with an ideal pH of approximately pH 5.0, assuming that all nutritional requirements are met (Elzwai et al., 2018). In general, filamentous fungi such as \u003cem\u003ePenicillium\u003c/em\u003e spp and \u003cem\u003eFusarium\u003c/em\u003e spp species appear more apt toward low pH, while bacteria are naturally neutrophiles growing best at pH 7.0 with a few acidophiles growing optimally at a pH near 3.0 whilst alkaliphiles grow optimally between a pH of 8 and 10.5. In this study we exposed the different fungal strains in question to pH 3.0, 5.0, 6.0, 7.0 and 8.0. We noted that all the strains registered their optimal growth at pH 6.0 and thereafter growth begins to drop as the pH turns alkaline. However, in all the strains, the wildtype positive for the endosymbiotic gene showed more adaptability for growth when compared with na\u0026iuml;ve and cured strains as demonstrated in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Indeed, the growth rate attributable to bacterial symbiosis was as listed below; 9.8 mm at pH 3.0, 9.8 mm at pH 5.0, 11.7 mm at pH 6.0, 10.6 mm at pH 7.0 and 8.3 mm at pH 8.0 mm in a similar manner as noted above.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of bacterial symbiosis on fungal response to pH stress\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFungal species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eBacterial symbiosis status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFungal growth changes attributed to bacterial symbiosis (mm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNa\u0026iuml;ve types mean colony diameters (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWild types mean colony diameters (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKnocked out types mean colony diameters (mm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003ePenicillium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eTrychophyton mentagrophytes\u003c/b\u003e \u003cb\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;12)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffects of bacterial symbiosis on fungal response to antifungal drugs\u003c/h2\u003e \u003cp\u003eFilamentous fungi are intrinsically resistant to many of the available antifungal drugs including fluconazole. Here, we employed fluconazole at a strength of 200 times the normal concentrations. Though intrinsically resistant to fluconazole we noted inhibitory activity exhibited by majorly na\u0026iuml;ve and cured strains when compared with the endosymbiotic positive bacteria. When exposed to fluconazole concentrations of 2, 4, 8, 16, 32, 64, 128 and 256 mg/L, we noted both na\u0026iuml;ve and cured strains of \u003cem\u003ePenicillium spp\u003c/em\u003e and \u003cem\u003eTrichophyton mentagrophytes\u003c/em\u003e struggled to grow more than \u003cem\u003eFusarium spp\u003c/em\u003e when co-incubated with the antifungal when compared to the endosymbiotic bacteria positive fungi. Indeed, the mean growth rate attributable to bacterial symbiosis was 106.7 mm as shown in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of bacterial symbiosis on fungal response to antifungal drugs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFungal species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eFungal isolate bacteria status\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMIC changes (mg/L) attributed to bacterial symbiosis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa\u0026iuml;ve types mean MIC value (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWild types mean MIC value (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKnocked out types mean MIC value (mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePenicillium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTrychophyton mentagrophytes\u003c/b\u003e \u003cb\u003e(n\u0026thinsp;=\u0026thinsp;6)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003espp (n\u0026thinsp;=\u0026thinsp;12)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e170.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e106.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGenerally, all test isolates had reduced susceptibility to fluconazole (MIC\u0026thinsp;\u0026ge;\u0026thinsp;16 mg/L). Nonetheless, endo-bacteria positive fungi showed more resistance (mean MIC of 170.7 mg/L) than cured and na\u0026iuml;ve fungi (mean MIC of 64 and 37.3 mg/L) respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe compartmentalization that exists between mycologists and bacteriologists has left us prone to infections by co-operating microbial partners from both domains. This is because such practice has over time encouraged the study of fungi and bacteria to be in axenic settings [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Depending on which of the compartment we come from, we tend to be biased and hence overlook the fact that fungi and bacteria co-exist and interact in many different environments and; on various physical, biochemical and phylogenetic levels. If we are to draw from existing literature in this area, it\u0026rsquo;s now evident enough that fungi and bacteria may form physically and metabolically charged microbial consortia that harbor properties different from those of their single components [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. With this in mind, it\u0026rsquo;s with great hope that if we are to examine the effects of fungal-bacterial co-existence and subsequent interactive modalities, our approaches to microbial infection biology in medicine could be handled a little different and; so that it perhaps leads to better management results. Other disciplines including agriculture, dentistry, food technology and biotechnology have picked this up with a strong hold and their research effects seems to be progressing somewhat different [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] when compared to medicine which still lags behind in that regard. Central to fungal-bacterial co-existence is the cross talk that takes place between the two partnering organisms. Indeed, several communication channels have been suggested including but not limited to trophic interactions, and interactions via antibiosis, via protein secretions and gene transfer, cooperative metabolism, via chemotaxis and cellular contacts, via modulation of the physiochemical environments and; via signal-based interactions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, through these communication channels, special fungal-bacterial relationships are also formed which have included but not limited to mutualism, commensalism, antagonistic, saprophytism and symbiotic relations. Through such establishments fungal-bacterial co-existence through various interactions may influence several phenotypic expressions of either partners. For instance, fungal bacterial interactions impact microbial physiology, pathogenicity, microbial growth and development, microbial survival, dispersal and colonization; microbial heritable changes and; microbial complexity in life cycles [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIndeed, here we explore one of such establishments in particular the bacterial symbiosis among filamentous fungi. The symbiotic bacteria associated with pathogenic fungi seem valuable for microbial resources and worthy of an in-depth investigation. It\u0026rsquo;s important that we analyze the community structure, succession of the symbiont through generations of fungal reproduction and various medical importance in pathogenic fungi. This can assist in isolation of symbiont strains that positively influence the harboring fungi and have an essential relationship with the fungal reproduction cycles as well as an impact on infection traits. In this regard, we assessed both clinical and environmental filamentous fungal isolates for symbiotic bacteria and the effect of these symbionts on fungal growth, response to temperature, carbon dioxide tension, antifungal and pH stresses. Through this evaluation we established a 5.3% prevalence of bacterial symbiosis among152 studied fungal subjects with only about 3 fungal species (i.e. \u003cem\u003eFusarium\u003c/em\u003e spp (n\u0026thinsp;=\u0026thinsp;4), \u003cem\u003ePenicillium\u003c/em\u003e spp (n\u0026thinsp;=\u0026thinsp;2) and \u003cem\u003eTrichophyton mentagrophytes\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) known to be involved with the bacterial symbiosis Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. When compared with other studies, our prevalence of bacterial symbiosis is on the lower end. For instance, Lastovetsky et al and Okrasińska et al established incidences of 90% and 20 percent respectively [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, this difference could originate from the experimental approach since both studies employed \u003cem\u003eBurkholderia\u003c/em\u003e-specific primers that amplify a portion of the 23S rRNA gene and 16S rRNA gene [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, this being the first of such a study in our location, there is very little to compare with. But these results can be explained by environmental events suggesting that perhaps our weather and environmental conditions may not favor a lot of bacterial symbiosis among fungi but this remains to be determined later.\u003c/p\u003e \u003cp\u003eThrough this study, we have generally demonstrated that fungal strains with the bacterial endosymbiont possess enhanced growth rate, exhibits better survival when exposed to the various stress stimuli including temperature, carbon dioxide tension, pH, and antifungal drugs when compared with the endosymbiont free strains Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAccordingly; the endosymbiotic bacteria contributed radial growth of 12 mm more than that of fungal species without the endosymbiont Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. These results are similar with what Uehling et al determined when the fungus \u003cem\u003eMortierella elongata\u0026rsquo;s\u003c/em\u003e radial growth increased from 3.00 \u0026plusmn; 0.03 mm/day without the endosymbiont to 4.38 \u0026plusmn; 0.74 mm/day when it had bacterial endosymbiont \u003cem\u003eBurkholderia\u003c/em\u003e strain BT03. The same fungus increased from 6.41 \u0026plusmn; 1.27 mm/day without the endo-bacteria to 7.29 \u0026plusmn; 0.39 mm/day with the endo-bacteria when grown on Malt extract agar (MEA) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In another study by Vannini et al demonstrates that an endobacterium Candidatus G. gigasporarum enhances fungal growth [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Finally, Shaffer et al also demonstrates that bacterial endosymbiont \u003cem\u003eChitinophaga\u003c/em\u003e spp. (strain PS-EHB01) significantly increased growth of the fungus \u003cem\u003eFusarium\u003c/em\u003e spp compared to the endosymbiont free strain. With this in play, research labor to explain how this could be happening. For instance, it\u0026rsquo;s been suggested that bacterial endosymbionts are known to influence growth through modulation of the main fungal metabolic pathways in the mitochondria such as the adenosine triphosphate (ATP) synthesis, respiration and reactive oxygen species detoxification and thus increasing ASP production and respiration [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. While others have suggested that certain endo bacteria may encourage production of certain compounds that may act as growth factors for the hosting fungus and or may detoxifying harmful waste products such as reactive oxygen species that may hinder growth [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It\u0026rsquo;s a little early to tell what could be happening in samples until further investigations are conducted. However, this is not the case for all fungi that harbor the endo bacteria as determined by Shaffer et al and Hoffman et al. Both of these studies gave contrasting findings, for instance Shaffer et al showed that growth by the fungus \u003cem\u003ePestalotiopsis\u003c/em\u003e spp (Strain 9143) was inhibited by the presence of endo-bacteria[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] whilst Hoffman et al found no difference in growth rates by both endosymbiont positive and negative fungal isolates [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. There is no clear explanation for these events until more explorations are conducted.\u003c/p\u003e \u003cp\u003eWhen tested against the CO\u003csub\u003e2,\u003c/sub\u003e tension we established those fungi generally struggled to grow in an environment with high oxygen tension. However, the fungus positive for the endosymbiont generally survived better in high CO\u003csub\u003e2\u003c/sub\u003e concentrations. Accordingly; the mean radial growth attributed to bacterial symbiont was 11.9 mm at atmospheric CO\u003csub\u003e2\u003c/sub\u003e and 7.2 mm 5% CO\u003csub\u003e2\u003c/sub\u003e Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Though fungi generally struggle to grow under high CO\u003csub\u003e2\u003c/sub\u003e concentrations, their ability to grow here at almost similar rate with those under ideal conditions shows that the endosymbiont could be playing a key role in enabling the fungus survive better. But we know that Fungi can sense and adopt to elevated levels of CO\u003csub\u003e2\u003c/sub\u003e within the host environment and this is often attributable to the activity of fungal soluble adenylyl cyclase and carbonic anhydrase enzymes available in fungal cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These enzymes catalyze the bidirectional conversion of CO\u003csub\u003e2\u003c/sub\u003e and water into bicarbonate (HCO\u003csub\u003e3\u003c/sub\u003e) and protons (H+)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] thus creating a CO\u003csub\u003e2\u003c/sub\u003e free environment. However, it\u0026rsquo;s not clear how bacterial symbiotic fungi survive better in increased carbon dioxide that endosymbiotic free fungi. This remains to be explored further.\u003c/p\u003e \u003cp\u003eFungal response to the temperature stimuli is often constitutive just as it is with other microbes. In this regard filamentous fungi grow at an optimum temperature range of 25-30\u003csup\u003eo\u003c/sup\u003eC [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]; and often begin to struggle when the temperature goes beyond 35\u003csup\u003eo\u003c/sup\u003eC. This means that a human host temperature of 37\u003csup\u003eo\u003c/sup\u003eC is one of the host defenses that filamentous fungi must overcome to manifest an infection. In this study we exposed the study subjects to the body temperature of 37\u003csup\u003eo\u003c/sup\u003eC and a much higher temperature of 40\u003csup\u003eo\u003c/sup\u003eC and 45\u003csup\u003eo\u003c/sup\u003eC Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. We demonstrated that though filamentous fungi struggled to grow under these temperature sets, the fungi positive for the endo bacterial gene grew with comfort under 37\u003csup\u003eo\u003c/sup\u003eC and 40\u003csup\u003eo\u003c/sup\u003eC when compared with the endobacteria na\u0026iuml;ve versions of the fungi registering mean radial growth rate attributed to endobacteria to be 11.3 mm at 37\u003csup\u003eo\u003c/sup\u003eC, 11.2 mm at 40\u003csup\u003eo\u003c/sup\u003eC and 0.00 mm 45\u003csup\u003eo\u003c/sup\u003eC Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Note that we registered no growth under 45 \u003csup\u003eo\u003c/sup\u003eC for either version of the fungus. However, there is no data to support our claim here but Frey-Klett et al in his review put out a hypothesis that bacterial symbiosis could facilitate fungal thermotolerance [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. There is limited data to explain how the endobacteria facilitate protection of the host fungus against heat stimuli. However, Corbin et al using some of his observations in entomological and plant studies suggested that endo-bacteria have the capacity to release metabolites which increase the expression stress resistant genes preparing the host against most of the stress stimuli [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Following these findings, we recommend that more investigations are done to exhaustively understand how endo bacteria benefit fungal hosts when it comes to thermotolerance.\u003c/p\u003e \u003cp\u003eThis study further examined fungal\u0026rsquo;s response towards pH stress. While fungi grow better at acidic pH range of 3\u0026ndash;6 and; at alkaline pH or between 8 and 10.5. Our findings determined the optimum growth pH of 6 similar to a study by [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Generally, all fungal versions started slowly at low pH of 3 peaking at pH 6 and thereafter slowing again at pH7 and pH8 Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. However, in all scenarios, endosymbiont free fungi showed low radial growth when compared with endosymbiont charged fungi. In the endosymbiont positive bacteria mean radial growth was more by 9.8 mm at pH3, 9.8 mm at pH5, 11.7 mm at pH 6, 10.6 mm at pH 7 and 8.83 mm at pH 8 all these increments were attributable to bacterial endosymbiont Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Still there is not much data to support our claim here but a few research including Yang et al and Brown et al have fronted an idea that filamentous fungi have the ability to modulate and create environmental pH that favors bacterial growth and as the bacteria thrives, it provides requirements that are essential for fungal growth and survival [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, it\u0026rsquo;s still early to explain what is happening here and remains to be harnessed bit future studies.\u003c/p\u003e \u003cp\u003eFinally, we evaluated these fungal versions against antifungal therapy. Though, its common knowledge that filamentous fungi are intrinsically resistant to fluconazole [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], we defied this fact and used fluconazole anyway but at a concentration of 2, 4, 8, 16, 32, 64, 128 and 256 mg/L to offer some guidance. Although filamentous fungi exhibit less activity against fluconazole, we registered minimal activity which can be used as a basis for future evaluation. Interestingly, to our surprise we demonstrate a similar trend seen by others. For instance, Lupini et al showed that bacterial endosymbionts can increase fungal resistance towards heavy metals known to have antimicrobial activity [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]; Shao et al showed that a fungus \u003cem\u003eSpiromastix\u003c/em\u003e spp. (strain SCSIO F190) while in a symbiotic relationship with endobacterium \u003cem\u003eAlcaligenes feacalis\u003c/em\u003e resists most antifungal drugs including nystatin and the vice versa is true when the endo bacteria is knocked out [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Finally, Itabangi et al, demonstrated that endosymbiont free Rhizopus microsporus fungi are more sensitive to Amphotericin B than the endosymbiont charged Rhizopus spp [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Though not clear how the fungus utilizes the endosymbiont to resist against antimicrobials, Vannini et al and Shao et al tempt to give an insight. They suggest that there could be a vertical gene transfer between the fungus and the endosymbiont but this in addition to the fact that certain endobacterium such as the \u003cem\u003eCandidatus Glomeribacter gigasporarum\u003c/em\u003e can elicit mechanisms to detoxify free radicles and also modulate fungal protein expression through influencing DNA replication and transcription. This leads to an accumulation of proteins that are involved in fungal cell wall rigidity hence fungal protection [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This could serve as a basis to support our claim here but still leaves more questions than answers, which can only be got through future explorations.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBacterial symbiosis among pathogenic and environmental fungi is a long-standing concept that we are only just discovering now. Its impact on clinical outcomes is not yet harnessed to full potential and yet could its exploration may improve patient prognosis. Here we have attempted to highlight the importance of bacterial symbiosis in fungal survival against some of the human host defense line in temperature, pH, antimicrobials and against CO\u003csub\u003e2\u003c/sub\u003e tension in vitro. Whether, this can be extrapolated to explain clinical events remains to be explored. But in general, we conclude that bacterial symbionts are essential for fungal survival and where possible treatment approaches need to be revised to encompass poly microbial aetiology now that we have noted that they are more prevalent than before.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been approved by the Uganda National Council of Science and Technology (HS12976ES) and Mbarara University Research Ethics Committee (MUREC 08/10-20) and was conducted according to Uganda National legislation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declared no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eThis study was funded through the Capacity building Research,\u0026nbsp;Massachusetts general Hospital, USA (First Mile) award and; government of Uganda through the Busitema University Research and Innovation Fund (BURIF) Grant No.4/DGSRI/2022). Herbert Itabangi is also a fellow of the European Developing Countries Clinical Trials Partnership (EDCTP) in collaboration with the European Union (grant TMA 2019CDF-2789);\u0026nbsp;and also provided additional support through this grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKL, LA and HI conceptualized the study, BM and AK designed experiments, performed the work and analysis; and wrote the primary manuscript; , KL, JM and KK contributed to the collection,\u0026nbsp;analysis and interpretation of the data. LA and BM reviewed the later versions of the manuscript; and BA, LA and HI critically revised the manuscript. All authors approved this manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets collected during the study are available from the corresponding author upon request.\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\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Dr Elizabeth Ballou for University of Exeter, UK for providing most of the reagents used in the study through support by a welcome Trust award in medical mycology and fungal immunology (097377). We recognize Mwesigye James and the entire microbiology department of `Mbarara University for the isolation of all the clinical samples used in the study. 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Biol.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s42003-020-01239-y\u003c/span\u003e\u003cspan address=\"10.1038/s42003-020-01239-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Dec. 2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Fungal, environmental, bacterial, symbiosis, filamentous, stressors","lastPublishedDoi":"10.21203/rs.3.rs-6506101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6506101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFungal-bacterial co-existence significantly influences the pathogenicity of challenging microbial infections. Traditional diagnostic methods typically focus on identifying a single pathogen, often overlooking fungi, particularly in resource-limited settings. This study investigates the interactions between fungi and their bacterial endosymbionts in infection contexts. A total of 152 fungal isolates (103 clinical and 49 environmental) were screened for bacterial endosymbionts via targeted Polymerase chain reaction (PCR) for the 16S rDNA gene. Only 8 (5.3%) of the isolates were found to possess bacterial symbionts, with 5 (3.3%) being clinical and 3 (2.0%) environmental. The study further examined how these fungi responded to environmental stressors such as elevated carbon dioxide, heat, pH, and antimicrobial exposure. Fungi containing the 16S gene demonstrated enhanced growth and better adaptation to these stressors compared to strains lacking bacterial symbionts or those treated with ciprofloxacin. The findings highlight the complex dynamics of fungal-bacterial symbiosis and suggest potential avenues for improving clinical management of fungal-bacterial co-infections, emphasizing the need for further exploration of these interactions in infection niches.\u003c/p\u003e","manuscriptTitle":"Effect of bacterial symbiosis on filamentous fungal adaption to environmental stressors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 07:27:22","doi":"10.21203/rs.3.rs-6506101/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":"7aa09206-6c9b-42e5-8f32-6f0058042c74","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48266067,"name":"Biological sciences/Microbiology"},{"id":48266068,"name":"Health sciences/Medical research"},{"id":48266069,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-09-29T09:24:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-09 07:27:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6506101","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6506101","identity":"rs-6506101","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}