Germlings of Rhizopus arrhizus, rather than sporangiospores, lead to rhino-orbito-cerebral mucormycosis acquired intranasally in a murine model of uncontrolled diabetes

Germlings of Rhizopus arrhizus, rather than sporangiospores, lead to rhino-orbito-cerebral mucormycosis acquired intranasally in a murine model of uncontrolled diabetes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Germlings of Rhizopus arrhizus, rather than sporangiospores, lead to rhino-orbito-cerebral mucormycosis acquired intranasally in a murine model of uncontrolled diabetes Rachna Singh, Jasdeep Kaur, Pavneet Kaur, Anayata Sharma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6817419/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 Mucormycosis is a rapidly fatal, angioinvasive fungal infection. The disease presents as distinct clinical entities, with each being linked to specific risk factors and routes of transmission. Rhino-orbito-cerebral (ROC) manifestation remains the predominant form of mucormycosis in the developing world, especially in India amongst patients with uncontrolled diabetes. Limited literature is available on experimental animal models to study mucormycosis, and a murine model of ROC mucormycosis that recapitulates the risk factor (diabetes) and mode of acquisition (intranasal) of this disease is largely undocumented. In this study, we demonstrate that sporangiospores of Rhizopus arrhizus (1x10 6 cfu) failed to establish infection in diabetic mice when administered intranasally although intracerebral and intrasinus (ethmoid) inoculations were successful. The diabetic mice instilled intranasally with R. arrhizus spores (1x10 6 , and even 1x10 7 cfu) showed 100% survival, monitored upto 30 days post-inoculation. Their internal organs exhibited normal gross morphologies; fungal microscopy and culture were negative. In contrast, intranasal administration of 1x10 6 germlings of R. arrhizus to diabetic mice led to successful development of the infection, with signs typical of ROC mucormycosis. 50% of the exposed animals became morbid within 48 h and died by 4–6 days of exposure. The fungus was recovered both in microscopy (KOH mounts and histopathology) and culture. These findings reveal that R. arrhizus germlings, rather than sporangiospores, are mainly responsible for the natural acquisition of ROC mucormycosis intranasally by a diabetic host. Further, the work establishes a clinically-relevant murine model of ROC mucormycosis that can be utilized for future studies. diabetes intranasal mice mucormycosis rhino-orbito-cerebral Rhizopus arrhizus Figures Figure 1 Figure 2 Figure 3 Introduction Mucorales such as Rhizopus , Mucor , Rhizomucor , Lichtheimia and Apophysomyces are opportunistic fungi that cause rapidly progressing, angioinvasive infections associated with high morbidity and mortality. The disease is often community-acquired, and transmitted by inhalation, traumatic implantation or occasionally by ingestion of these saprophytic molds [ 1 , 2 ]. Mucormycosis is classified into rhino-orbito-cerebral (ROC), pulmonary, cutaneous, gastrointestinal and disseminated types. Each of these forms is linked to specific predisposing conditions, with epidemiological differences existing between the developed and developing world in terms of prevalence, common risk factors and clinical manifestations [ 1 , 2 ]. While pulmonary mucormycosis associated with neutropenia or hematological malignancies is the most frequent clinical type in the West, ROC infection linked to uncontrolled diabetes with or without ketoacidosis remains the principal manifestation in India [ 1 , 2 ]. The emergence of mucormycosis has been reported across the globe, and a particularly remarkable surge has been seen in developing countries. The situation was exacerbated by COVID-19, with COVID-19 associated mucormycosis being declared an epidemic in India [ 1 , 3 ]. Notably, ROC mucormycosis remained the predominant clinical form during COVID-19, primarily in patients with diabetes and COVID-19-related or corticosteroid-associated glycemic imbalance [ 1 , 3 ]. Limited diagnostic and therapeutic interventions are available against this deadly fungal infection, and the fatality remains high, ranging from 50% to nearly 100%, especially in cases with disseminated infection, or pulmonary and cerebral involvement [ 1 – 5 ]. Despite such a significant impact, the literature on animal models to study the pathogenesis of mucormycosis and/or evaluate newer therapies is relatively scarce. Majority of the available models have focused on pulmonary, disseminated and/or cutaneous mucormycosis, developed in immunosuppressed, deferoxamine-treated or diabetic mice [ 6 – 16 ]. Few studies reported the establishment of primary cerebral infections in experimental animals through intracranial or intraethmoid inoculation of fungal spores or hematogenous dissemination [ 8 , 17 , 18 ]. Intriguingly, murine models of ROC mucormycosis that mimic the risk factor (diabetes) and transmission route (intranasal) observed in humans are largely undocumented. Considering the high incidence of ROC mucormycosis, the devastating consequences of this aggressive infection and the lack of appropriate in vivo models, the present work aimed to develop a murine model of ROC mucormycosis that recapitulates the risk factor, mode of acquisition and clinical manifestation of this disease. Materials and Methods Fungal strain and inoculum preparation Rhizopus arrhizus NCCPF 710004 was used as the test strain in this study. The strain was procured from the National Culture Collection of Pathogenic Fungi (NCCPF), Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India during our previous studies [ 19 , 20 ]. It was preserved in 15% glycerol (v/v) at -70°C. The sporangiospores were harvested from R. arrhizus cultures according to the method of Singh et al. (2011) [ 21 ]. Briefly, R. arrhizus was grown on Sabouraud dextrose agar at 37°C for 4–5 days. The plates were flooded with 10 ml normal saline (0.85% Sodium Chloride) and sporangiospores were harvested by repeated washing of the mycelia with the added saline. The resulting suspensions were centrifuged at 8000 rpm for 10 min; the pellets were washed twice with normal saline and finally suspended in 1 ml normal saline. The harvested spore suspensions were confirmed to be completely free from hyphal fragments by microscopy, and the spores were counted using a Neubauer hemocytometer. Their viability and CFU counts were verified by culturing on Sabouraud dextrose agar and Dichloran rose Bengal chloramphenicol agar containing 5 µg benomyl/ml (DRBC agar) [ 22 , 23 ], respectively at 37°C. Germlings were obtained by incubating 1x10 6 sporangiospores/ml in Sabouraud dextrose broth at 37°C for 5 h, followed by resuspension in normal saline to obtain the desired counts [ 12 , 19 ]. The median length of these germlings was 54 µm, as measured microscopically. Murine model of Type 1 Diabetes The work was performed on female LACA mice (age 6–8 weeks, weight 18–20 g) procured from the Central Animal House, Panjab University, Chandigarh, India. The mice were maintained in standard laboratory environment, housed in groups of 3–4 in pre-sterilized cages, and given irradiated feed and sterile water containing ciprofloxacin (50 µg/ml) ad libitum [ 6 ]. Type 1 diabetes was induced by treatment with streptozotocin [ 24 ]. The food was withdrawn from animal cages 4 h prior to streptozotocin injection. A dose of 210 mg streptozotocin/kg prepared freshly in ice-cold 50 mM citrate buffer (pH 4.5) was administered intraperitoneally per mouse. Control mice were injected citrate buffer as placebo (vehicle). To prevent lethal hypoglycemia, sucrose water (10%) was given orally after streptozotocin treatment, and then replaced with normal water [ 24 ]. The animals were observed for signs of diabetes including polydipsia, urination frequency and hypo-activity, with their weight and body temperature being monitored daily. The urinary levels of glucose (glycosuria) and ketone bodies (acetoacetic acid; ketonuria) were measured regularly using keto-Diastix strips (Siemens Healthcare Pvt. Ltd., Gujarat, India). The urinary glucose level of more than 1000 mg/dl was considered as the standard indicator for diabetes [ 17 ]. Ketoacidosis was interpreted as trace, small, moderate, and large at urinary ketone levels of 5, 15, 40 and greater than 80 mg/dl respectively as per manufacturer’s instructions. Establishment of ROC mucormycosis For establishing the infection, spores (or germlings where specified) were introduced into the diabetic mice by intranasal instillation, intracerebral injection or intrasinus (ethmoid) inoculation after anesthetizing the animals intraperitoneally with 100 mg ketamine and 10 mg xylazine/kg) as per standard protocol [ 25 ]. For intranasal instillation, a volume of 5 µl containing 1x10 6 spores (or germlings) was slowly released per nostril by placing the mice in supine position, so as to deposit the inoculum in the nasal cavity. For intracerebral inoculation, 1x10 6 spores in 20 µl normal saline were injected by slight piercing of the cranium using disposable hypodermic syringe and 26G × 1/2″ needles [ 26 ]. Intrasinus inoculation was performed by introducing 1x10 6 spores into the ethmoid sinus using single lumen neonatal catheters (polyethylene; outer diameter 0.61 mm; internal diameter, 0.28 mm; length, 3 mm) [ 27 ]. Control groups comprising of non-diabetic mice inoculated with the fungus; un-inoculated, diabetic mice administered equivalent volumes of normal saline; and un-inoculated, non-diabetic mice administered normal saline were run in parallel. All experiments were conducted in groups of six mice each. The experiments involving intranasal instillation with spores or germlings were performed three times to confirm the observations. Ante-mortem and post-mortem examination Infected mice were inspected daily for body weight, temperature and behaviour, along with the disease signs. Urinary glucose levels were regularly monitored to confirm that the mice retained the diabetic state during the entire course of the experiment. Upon death, necropsies were promptly conducted within 1 h. The animals were subjected to initial examination for external signs of disease or injury, followed by removal of fur from abdominal and cranial regions. The skin was disinfected, and the internal organs (brain, lungs and kidneys) were removed aseptically, visually examined for gross morphology, and processed for microscopy [20% (w/v) aqueous KOH mount] and culturing on Sabouraud dextrose agar and DRBC agar at 37°C. For viable counting, a portion of the tissue samples was mildly homogenized, followed by ten-fold serial dilutions and spread plating. As the recovery of mucoralean fungi from tissue samples is arduous and may yield false negative results [ 5 ], the fungal hyphal load in the homogenized supernatants was also quantified (where specified) using hemocytometer. Additionally, sliced fragments of the tissue (10 to 16 in no.) were directly cultured in one set of the experiments, and the number of tissue fragments positive for fungal culture was enumerated [ 5 , 28 ]. The identification of the fungal isolate was based on morphological characteristics [ 5 ]. For histopathological examination, a section was fixed in 10% (v/v) buffered formalin and observed by hematoxylin-eosin staining. The challenged survivors were monitored for 30 days and then sacrificed by cervical dislocation and processed. Results Induction of Type 1 Diabetes With a single dose of 210 mg streptozotocin/kg, Type 1 diabetes was established in 85% of the mice within a week of administration. The urinary levels of glucose ranged from 1000–2000 mg/dl or more (median, 2000 mg/dl or more), and those of acetoacetic acid ranged from of 5–15 mg/dl (median, 5 mg/dl). Lethargy, weight loss (≈ 20%), polydipsia and increased urination frequency were observed in diabetic mice. Murine model of ROC mucormycosis The diabetic mice exposed to R. arrhizus spores by intracerebral inoculation began displaying signs of illness within 24 h of exposure, including reduced response to stimuli, decreased food and water intake, kyphosis (hunched posture) and hemiplegia. The infection was lethal in 100% of the exposed animals within 2 days of inoculation (Fig. 1 ). The fungus was recovered both by microscopy as well as by culturing and dilution plating of the homogenised tissue, with the viable counts being greater in the brain than in the kidneys and lungs (Fig. 1 ; brain, 5.23 ± 0.16; lungs, 3.53 ± 1.18; and kidneys, 2.63 ± 0.46 log 10 cfu/g tissue). Diabetic mice infected through the intrasinus route showed a similar but slower progression of the disease; the mice became morbid within 3–4 days and 72% died in 6–12 days (Fig. 1 ). In contrast, none of the diabetic mice instilled intranasally with R. arrhizus spores displayed any changes in behaviour, body temperature, or signs of the disease, even up to 30 days after inoculation (Fig. 1 ). Necropsies of these animals revealed normal gross morphologies of the internal organs. Fungal microscopy and culture were negative. The infection could not be established intranasally even by increasing the spore inoculum to 1x10 7 (survival, 100%). Instillation of 1x10 6 germlings in mice through the nasal route led to successful development of the infection, with signs typical of ROC mucormycosis (Figs. 1 and 2 ). The infected mice displayed lethargy, ruffled fur, reduced food and water intake, weight loss (41% and 23% versus the uninfected healthy controls and uninfected diabetic controls respectively), ptosis and impaired eye movements, conjunctival suffusion, purulent eye discharge, a hunched back, along with distressed behaviour, hypothermia and non-responsiveness to stimuli (Fig. 2 a). 50% of the exposed animals became morbid within 48 h and died by 4–6 days of exposure (Fig. 1 ). Upon autopsy, hemorrhage was observed in the brain and lungs, with the lobes shrunken in size and friable (Fig. 2 b). Necrosis was noted in the kidneys (Fig. 2 b). The fungus was recovered in both microscopy and culture. KOH mounts revealed the presence of broad aseptate hyphae in the tissue samples, indicative of mucoralean fungi (Fig. 2 c). Although the homogenised tissue samples did not yield any positive cultures upon dilution plating, direct inoculation of the brain tissue fragments on media plates revealed the fungal presence (Fig. 2 d and e), with the lactophenol cotton blue mounts of the cultured fungus clearly demonstrating the distinctive morphological features of R. arrhizus (Fig. 2 f). The fungal loads were found to be higher in the brain compared to the lungs and kidneys, based on the number of fungal-positive tissue fragments (brain, 88%; lungs, 19% and kidneys, 16%) as well as hemocytometric evaluation (Fig. 2 g). Histopathological examination of the cerebral cortex, cerebellum and olfactory bulb demonstrated the presence of mucoralean hyphae (Fig. 3 ), with a loss of architectural details, dilation and congestion of the blood vessels, infiltration of mononuclear cells, and necrosis. Degenerative changes in the molecular layer and necrosis of the Purkinje layer were observed. Furthermore, challenging the diabetic mice with an inoculum of 1x10 5 and 1x10 7 germlings intranasally resulted in 33.33% and 50% mortality respectively, indicating the lethal dose (LD) 50 to be 1x10 6 R. arrhizus germlings. No morbidity or mortality was seen in the inoculated non-diabetic mice and the control mice groups given sham inoculation. Discussion Mucormycosis is recognized as a highly devastating, aggressive fungal infection across the globe, and particularly in developing nations including India [ 1 , 2 ]. The disease manifests as a range of clinical types, with ROC, pulmonary and cutaneous forms being common depending upon the risk factors, transmission routes and geographical distribution [ 1 , 2 ]. Uncontrolled diabetes mellitus remains the most frequent underlying disease associated with mucormycosis in many countries, especially India, wherein it leads to a disproportionately high number of cases with ROC mucormycosis [ 1 , 2 ]. The disease presents substantial diagnostic and therapeutic challenges, resulting in elevated mortality rates [ 5 ]. The morbidity associated with this infection is also significant, with survivors experiencing dilapidating outcomes, such as loss of vision and disfigurement in cases with ROC mucormycosis [ 1 ]. In spite of such a profound impact, there is a notable paucity of literature on animal models to study the pathogenesis of ROC mucormycosis or assess alternate treatment strategies. Majority of the available studies describe primary cerebral infections in experimental animals by administration of fungal spores through intracranial or intraethmoid route or hematogenous dissemination [ 8 , 17 , 18 ]. This, however, does not truly represent the natural course of infection transmission. In the present study, we therefore attempted to establish a murine model of ROC mucormycosis that mimics the risk factor (diabetes) and transmission route (intranasal) observed in humans. R. arrhizus was selected as the representative mucoralean species, as it is the predominant causative agent of mucormycosis in majority of the geographical locations worldwide [ 1 , 2 ]. Female LACA mice were made diabetic by intraperitoneal administration of a single high dose of streptozotocin as per standard protocol, and then challenged with R. arrhizus sporangiospores at a spore count of 1x10 6 by intranasal instillation. Experiments involving intracerebral and intrasinus (ethmoid) inoculations using the same spore inoculum dose were also performed for comparison. The diabetic mice that were subjected to intracerebral and intraethmoid inoculation with R. arrhizus spores became morbid within 24 h and 3–4 days of exposure, respectively. Whilst the mice inoculated intracerebrally had a mortality rate of 100% within 2 days of exposure, those inoculated via the intraethmoid route exhibited a mortality rate of 72% within 6–12 days. This relatively slower onset of the disease during intrasinus vs. intracerebral inoculation correlates with their anatomically location, and the observed mortality is consistent with the findings documented in the existing literature [ 8 , 17 , 18 ]. However, none of the diabetic mice instilled intranasally with R. arrhizus spores displayed signs of the disease, even up to 30 days after inoculation. Their internal organs exhibited normal gross morphologies, with fungal microscopy and culture being negative. The infection could not be established intranasally even by increasing the spore load to 1x10 7 (survival, 100%), although such high inocula are virtually non-existent in the environment. The observed results are intriguing, as intranasal acquisition is the primary route of transmission of ROC infection, and mice are known to be susceptible to both natural and experimental mucormycosis [ 8 ]. Considering that R. arrhizus hyphal forms show greater resilience to innate immunity and increased virulence in Galleria mellonella models compared with the spores [ 12 , 29 ], intranasal administration of 1x10 6 germlings in diabetic mice was then tested. It resulted in successful development of the infection, with signs typical of ROC mucormycosis. 50% of the exposed mice became morbid within 48 h and died by 4–6 days of inoculation. The recovery of R. arrhizus both in microscopy (KOH mounts and histopathology) and culture from the tissue samples corroborated these findings. Although the intranasal administration is likely to have deposited the fungus in both the nasal cavity and the lungs, the substantially higher fungal burdens in the brain compared to the lungs, along with the distinct ROC-specific signs noted in the infected mice suggest that the infection was primarily cerebral. Furthermore, pulmonary mucormycosis is often associated with risk factors such as neutropenia or malignancies, and development of primary pulmonary mucormycosis in murine models of uncontrolled diabetes has been reported to require more invasive procedures such as intratracheal administration in some studies [ 6 , 30 ]. The spread of infection to the other organs, such as the kidneys, is indicative of hematogenous dissemination. Although only brain, lung and kidney tissues were processed, the gross morphologies of the other organs were also indicative of the diseased state; for instance, the intestines were edematous and discolored. R. arrhizus inoculum of 1x10 6 germlings was also noted to be the LD 50 for establishment of murine ROC mucormycosis through intranasal instillation. According to the published literature, the LD 50 of R. arrhizus ranges from 10 3 to 10 6 spores depending upon the reported routes of inoculation and the clinical manifestations. [ 7 , 8 , 12 ], Taken together, the clinical signs, rapid onset and evolution of the disease, along with the presence of lesions and fungal load indicated that cerebral infection was the primary cause of death in the infected, diabetic mice. The findings further demonstrated that germlings of R. arrhizus lead to ROC mucormycosis in diabetic mice through intranasal acquisition. This also correlates with the fact that the germlings preferentially express the spore-coat protein CotH3, which serves as an invasin and facilitates the binding to the mucoralean receptor (glucose-regulated-protein 78) over-expressed on the nasal epithelium in diabetic patients [ 31 , 32 ]. Hyphal forms of R. arrhizus and Lichtheimia corymbifera have also been recently demonstrated to contribute to combat-relevant wound mucormycosis [ 33 ]. In contrast to the present study, Reinhardt et al. reported the development of cerebral infection in rabbits by intranasal instillation of R. arrhizus spores 3–4 days prior to induction of diabetes using alloxan [ 30 ]. This may, however, not accurately represent the disease cycle, as the infection is acquired in the backdrop of pre-existing diabetes. Furthermore, the inoculated sporangiospores are likely to have germinated in the animal host during that time period. A study by Bauer et al. published in the year 1955 demonstrated experimental cerebral mucormycosis acquired intranasally via spores in alloxan-treated diabetic rabbits [ 34 ]. Although this could reflect host-specific susceptibility, the possibility of germlings or hyphal forms in the spore inoculum used cannot be ruled out, as those were collected and separated from mycelial cultures using sterile gauze, whose mesh-size may not have fully filtered out the germlings and/or mycelial fragments. The failure of mucoralean spores to cause ROC mucormycosis in diabetic mice, as observed in the present study, likely reflects the reason why majority of the reported murine models involve alternate routes of inoculation or infection sites, even with diabetes as the underlying risk factor [ 6 , 8 , 12 – 15 , 17 , 18 , 35 ], despite a documented association of ROC with uncontrolled diabetes and intranasal acquisition [ 1 – 5 ]. In conclusion, the results of this study demonstrate the establishment of ROC mucormycosis by intranasal route in a murine model of uncontrolled diabetes. Germlings, but not sporangiospores, were found to be implicated in the successful establishment of infection. Further, the work reports a clinically-relevant murine model of mucormycosis that can be utilized in future studies for studying the pathogenesis and evaluating newer treatment strategies against this deadly fungal infection. Declarations Ethical approval All experiments were performed by following the guidelines of the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), India after due approval of the Institutional Animal Ethics Committee (IAEC), Panjab University, Chandigarh. (PU/45/99/CPCSEA/IAEC/2022/671). Competing interests: The author(s) declare that there are no conflicts of interest to declare that are relevant to the content of this article. Funding The study was supported by research funding from the Indian Council of Medical Research (ICMR), Govt. of India under extramural research grant scheme (Drug development initiative) (No. 67/2/2020-DDI/BMS) granted to RS. Author Contribution The study was designed by R.S. and J.K. The acquisition, analysis, and interpretation of the data were conducted by R.S., J.K., P.K and A.S. R.S., P.K. and A.S. drafted the manuscript. All authors agreed to the publication of the final manuscript. Data availability The data generated and analyzed in the current study are available from the corresponding author on reasonable request. References Hoenigl M, Seidel D, Sprute R, Cunha C, Oliverio M, Goldman GH et al (2022) COVID-19-associated fungal infections. Nat Microbiol 7:1127–1140. http://doi.org/10.1038/s41564-022-01172-2 Chakrabarti A, Singh R (2014) Mucormycosis in India: unique features. 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J Fungi (Basel) 9:9100958. http://doi.org/10.3390/jof9100958 Reinhardt DJ, Kaplan W, Ajello L (1970) Experimental cerebral zygomycosis in alloxan-diabetic rabbits I. Relationship of temperature tolerance of selected zygomycetes to pathogenicity. Infect Immun 2:404–413. http://doi.org/10.1128/iai.2.4.404-413.1970 Gebremariam T, Liu M, Luo G, Bruno V, Phan QT, Waring AJ et al (2014) CotH3 mediates fungal invasion of host cells during mucormycosis. J Clin Invest 124:237–250. http://doi.org/10.1172/JCI71349 Szebenyi C, Gu Y, Gebremariam T, Kocsube S, Kiss-Vetrab S, Jager O et al (2023) ) cotH genes are necessary for normal spore formation and virulence in Mucor lusitanicus . mBio 14:e0338622. http://doi.org/10.1128/mbio.03386-22 Thanapaul RJR, Alamneh YA, Finnegan DK, Antonic V, Abu-Taleb R, Czintos C et al (2024) Development of a combat-relevant murine model of wound mucormycosis: A platform for the pre-clinical investigation of novel therapeutics for wound-invasive fungal diseases. J Fungi (Basel) 10:10050364. http://doi.org/10.3390/jof10050364 Bauer H, Flanagan JF, Sheldon WH (1955) Experimental cerebral mucormycosis in rabbits with alloxan diabetes. Yale J Biol Med 28:29–36 Ibrahim AS, Avanessian V, Spellberg B, Edwards JE Jr (2003) Liposomal amphotericin B, and not amphotericin B deoxycholate, improves survival of diabetic mice infected with Rhizopus oryzae . Antimicrob Agents Chemother 47:3343–3344. http://doi.org/10.1128/AAC.47.10.3343-3344.2003 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6817419","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":467899399,"identity":"57a862de-eb6a-46b1-a466-cf8a5bd02c2c","order_by":0,"name":"Rachna Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYHACNhjJxvABKnSAaC2MM0jSAmIw8xDjKvP2w88eF9Qw2PNJt197bLvjMAN/+wHGwwV4tMicSTM3nnGMIbFN5ky5ce6ZwwwSZxIYDs/Ao0WCIYdNmoeNIYFNIidNOrftMAPDDQaGw/hcKMH/BqjlH4M9WIslUIs8QS0SQFt42xgY2yTSj0kzArUYENbyzEyat08isQ2oV7L3TDqP4ZnEBgIOS34mzfPNxl5+RvoziZ87rOXkjh8+/JmI0JYAYh4DBsYGBqBiIEkkYH9AguJRMApGwSgYSQAAJ7FAuQr3LVsAAAAASUVORK5CYII=","orcid":"","institution":"Panjab University","correspondingAuthor":true,"prefix":"","firstName":"Rachna","middleName":"","lastName":"Singh","suffix":""},{"id":467899400,"identity":"189a7707-6838-475b-937e-649ea49560a2","order_by":1,"name":"Jasdeep Kaur","email":"","orcid":"","institution":"Panjab University","correspondingAuthor":false,"prefix":"","firstName":"Jasdeep","middleName":"","lastName":"Kaur","suffix":""},{"id":467899401,"identity":"4efedd76-3ef8-4500-a3a0-3b9f3ccf7bda","order_by":2,"name":"Pavneet Kaur","email":"","orcid":"","institution":"Panjab University","correspondingAuthor":false,"prefix":"","firstName":"Pavneet","middleName":"","lastName":"Kaur","suffix":""},{"id":467899402,"identity":"58fc4dff-4477-4176-9f3e-55597f326a20","order_by":3,"name":"Anayata Sharma","email":"","orcid":"","institution":"Panjab University","correspondingAuthor":false,"prefix":"","firstName":"Anayata","middleName":"","lastName":"Sharma","suffix":""}],"badges":[],"createdAt":"2025-06-04 07:23:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6817419/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6817419/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86670430,"identity":"e1835e57-5586-4fc1-9a6f-6f14647ec715","added_by":"auto","created_at":"2025-07-14 11:28:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSurvival of diabetic mice after intracerebral (I/C), intrasinus (ethmoid; I/E) and intranasal (I/N) challenge with 1x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6 \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCFUs of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Rhizopus arrhizus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esporangiospores or germlings\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eas specified\u003c/strong\u003e. Control groups comprising of non-diabetic mice inoculated with the fungus; un-inoculated, diabetic mice administered equivalent volumes of normal saline; and un-inoculated, non-diabetic mice administered normal saline were run in parallel and showed 100% survival. The mice were monitored up to 30 days post inoculation.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6817419/v1/1a2594f17dceb10405b561e4.png"},{"id":86670444,"identity":"c5b72eff-b50a-4b60-9b69-989be5608868","added_by":"auto","created_at":"2025-07-14 11:28:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46142632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopment of rhino-orbito-cerebral mucormycosis in a murine model of uncontrolled diabetes after intranasal challenge with 1x10\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6 \u003c/strong\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eRhizopus arrhizus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egermlings. a\u003c/strong\u003e, Representative images of infected mice demonstrating ruffled fur, and demonstrable eye infection during initial stages of the disease compared with the uninfected controls. \u003cstrong\u003eb\u003c/strong\u003e, Gross morphologies of brain, kidneys and lungs of the infected mice versus the uninfected controls as observed after necropsies. \u003cstrong\u003ec,\u003c/strong\u003e 20% (w/v) aqueous potassium hydroxide mount of the infected brain showing broad, ribbon shaped hyphae indicative of mucoralean fungi (Magnification, 400x; Scale bar 20 µm). \u003cstrong\u003ed and e,\u003c/strong\u003e \u003cem\u003eRhizopus arrhizus\u003c/em\u003e recovered from the brain fragments of the infected mice after culturing on Sabouraud dextrose agar (d) and dichloran rose Bengal chloramphenicol agar with 5 µg benomyl/ml (e) following direct inoculation of the tissue fragments on the nutrient media. The cultures were positive within 24 h of incubation at 37 °C, and sporulation was noted within 7 days. \u003cstrong\u003ef,\u003c/strong\u003e Lactophenol cotton blue mount of the fungal culture recovered on Sabouraud dextrose agar from brain fragments, demonstrating \u003cem\u003eRhizopus arrhizus \u003c/em\u003e(Magnification, 100x; Scale bar 100 µm)\u003cem\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eg,\u003c/strong\u003e Fungal hyphal load in the brain, lungs and kidneys of the infected mice. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6817419/v1/ec32ce2a483dad16dd27997a.png"},{"id":86670433,"identity":"f249fbb1-4944-41c8-8fbf-5618d99f4f92","added_by":"auto","created_at":"2025-07-14 11:28:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":15469074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a and b), \u003c/strong\u003ePhotomicrographs of hematoxylin-eosin stained section of the cerebellum after establishment of rhino-orbito-cerebral mucormycosis by intranasal challenge with 1x10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003eRhizopus\u003c/em\u003e \u003cem\u003earrhizus\u003c/em\u003e germlings in a murine model of uncontrolled diabetes. Magnification, 400x; Scale bar 20 µm.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6817419/v1/b884c379f1d00687a69c5182.png"},{"id":98436011,"identity":"102d5740-81dc-4cc3-aa4e-a6acf69e0eac","added_by":"auto","created_at":"2025-12-17 16:54:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":67505999,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6817419/v1/172e0c22-1b52-4aea-8299-401400c438ca.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Germlings of Rhizopus arrhizus, rather than sporangiospores, lead to rhino-orbito-cerebral mucormycosis acquired intranasally in a murine model of uncontrolled diabetes","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eMucorales\u003c/em\u003e such as \u003cem\u003eRhizopus\u003c/em\u003e, \u003cem\u003eMucor\u003c/em\u003e, \u003cem\u003eRhizomucor\u003c/em\u003e, \u003cem\u003eLichtheimia\u003c/em\u003e and \u003cem\u003eApophysomyces\u003c/em\u003e are opportunistic fungi that cause rapidly progressing, angioinvasive infections associated with high morbidity and mortality. The disease is often community-acquired, and transmitted by inhalation, traumatic implantation or occasionally by ingestion of these saprophytic molds [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Mucormycosis is classified into rhino-orbito-cerebral (ROC), pulmonary, cutaneous, gastrointestinal and disseminated types. Each of these forms is linked to specific predisposing conditions, with epidemiological differences existing between the developed and developing world in terms of prevalence, common risk factors and clinical manifestations [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While pulmonary mucormycosis associated with neutropenia or hematological malignancies is the most frequent clinical type in the West, ROC infection linked to uncontrolled diabetes with or without ketoacidosis remains the principal manifestation in India [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The emergence of mucormycosis has been reported across the globe, and a particularly remarkable surge has been seen in developing countries. The situation was exacerbated by COVID-19, with COVID-19 associated mucormycosis being declared an epidemic in India [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, ROC mucormycosis remained the predominant clinical form during COVID-19, primarily in patients with diabetes and COVID-19-related or corticosteroid-associated glycemic imbalance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLimited diagnostic and therapeutic interventions are available against this deadly fungal infection, and the fatality remains high, ranging from 50% to nearly 100%, especially in cases with disseminated infection, or pulmonary and cerebral involvement [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite such a significant impact, the literature on animal models to study the pathogenesis of mucormycosis and/or evaluate newer therapies is relatively scarce. Majority of the available models have focused on pulmonary, disseminated and/or cutaneous mucormycosis, developed in immunosuppressed, deferoxamine-treated or diabetic mice [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Few studies reported the establishment of primary cerebral infections in experimental animals through intracranial or intraethmoid inoculation of fungal spores or hematogenous dissemination [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Intriguingly, murine models of ROC mucormycosis that mimic the risk factor (diabetes) and transmission route (intranasal) observed in humans are largely undocumented. Considering the high incidence of ROC mucormycosis, the devastating consequences of this aggressive infection and the lack of appropriate \u003cem\u003ein vivo\u003c/em\u003e models, the present work aimed to develop a murine model of ROC mucormycosis that recapitulates the risk factor, mode of acquisition and clinical manifestation of this disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFungal strain and inoculum preparation\u003c/h2\u003e\u003cp\u003e\u003cem\u003eRhizopus arrhizus\u003c/em\u003e NCCPF 710004 was used as the test strain in this study. The strain was procured from the National Culture Collection of Pathogenic Fungi (NCCPF), Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India during our previous studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. It was preserved in 15% glycerol (v/v) at -70\u0026deg;C. The sporangiospores were harvested from \u003cem\u003eR. arrhizus\u003c/em\u003e cultures according to the method of Singh \u003cem\u003eet al.\u003c/em\u003e (2011) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Briefly, \u003cem\u003eR. arrhizus\u003c/em\u003e was grown on Sabouraud dextrose agar at 37\u0026deg;C for 4\u0026ndash;5 days. The plates were flooded with 10 ml normal saline (0.85% Sodium Chloride) and sporangiospores were harvested by repeated washing of the mycelia with the added saline. The resulting suspensions were centrifuged at 8000 rpm for 10 min; the pellets were washed twice with normal saline and finally suspended in 1 ml normal saline. The harvested spore suspensions were confirmed to be completely free from hyphal fragments by microscopy, and the spores were counted using a Neubauer hemocytometer. Their viability and CFU counts were verified by culturing on Sabouraud dextrose agar and Dichloran rose Bengal chloramphenicol agar containing 5 \u0026micro;g benomyl/ml (DRBC agar) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], respectively at 37\u0026deg;C. Germlings were obtained by incubating 1x10\u003csup\u003e6\u003c/sup\u003e sporangiospores/ml in Sabouraud dextrose broth at 37\u0026deg;C for 5 h, followed by resuspension in normal saline to obtain the desired counts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The median length of these germlings was 54 \u0026micro;m, as measured microscopically.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMurine model of Type 1 Diabetes\u003c/h3\u003e\n\u003cp\u003eThe work was performed on female LACA mice (age 6\u0026ndash;8 weeks, weight 18\u0026ndash;20 g) procured from the Central Animal House, Panjab University, Chandigarh, India. The mice were maintained in standard laboratory environment, housed in groups of 3\u0026ndash;4 in pre-sterilized cages, and given irradiated feed and sterile water containing ciprofloxacin (50 \u0026micro;g/ml) \u003cem\u003ead libitum\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Type 1 diabetes was induced by treatment with streptozotocin [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The food was withdrawn from animal cages 4 h prior to streptozotocin injection. A dose of 210 mg streptozotocin/kg prepared freshly in ice-cold 50 mM citrate buffer (pH 4.5) was administered intraperitoneally per mouse. Control mice were injected citrate buffer as placebo (vehicle). To prevent lethal hypoglycemia, sucrose water (10%) was given orally after streptozotocin treatment, and then replaced with normal water [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The animals were observed for signs of diabetes including polydipsia, urination frequency and hypo-activity, with their weight and body temperature being monitored daily. The urinary levels of glucose (glycosuria) and ketone bodies (acetoacetic acid; ketonuria) were measured regularly using keto-Diastix strips (Siemens Healthcare Pvt. Ltd., Gujarat, India). The urinary glucose level of more than 1000 mg/dl was considered as the standard indicator for diabetes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Ketoacidosis was interpreted as trace, small, moderate, and large at urinary ketone levels of 5, 15, 40 and greater than 80 mg/dl respectively as per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eEstablishment of ROC mucormycosis\u003c/h3\u003e\n\u003cp\u003eFor establishing the infection, spores (or germlings where specified) were introduced into the diabetic mice by intranasal instillation, intracerebral injection or intrasinus (ethmoid) inoculation after anesthetizing the animals intraperitoneally with 100 mg ketamine and 10 mg xylazine/kg) as per standard protocol [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For intranasal instillation, a volume of 5 \u0026micro;l containing 1x10\u003csup\u003e6\u003c/sup\u003e spores (or germlings) was slowly released per nostril by placing the mice in supine position, so as to deposit the inoculum in the nasal cavity. For intracerebral inoculation, 1x10\u003csup\u003e6\u003c/sup\u003e spores in 20 \u0026micro;l normal saline were injected by slight piercing of the cranium using disposable hypodermic syringe and 26G \u0026times; 1/2\u0026Prime; needles [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Intrasinus inoculation was performed by introducing 1x10\u003csup\u003e6\u003c/sup\u003e spores into the ethmoid sinus using single lumen neonatal catheters (polyethylene; outer diameter 0.61 mm; internal diameter, 0.28 mm; length, 3 mm) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Control groups comprising of non-diabetic mice inoculated with the fungus; un-inoculated, diabetic mice administered equivalent volumes of normal saline; and un-inoculated, non-diabetic mice administered normal saline were run in parallel. All experiments were conducted in groups of six mice each. The experiments involving intranasal instillation with spores or germlings were performed three times to confirm the observations.\u003c/p\u003e\n\u003ch3\u003eAnte-mortem and post-mortem examination\u003c/h3\u003e\n\u003cp\u003eInfected mice were inspected daily for body weight, temperature and behaviour, along with the disease signs. Urinary glucose levels were regularly monitored to confirm that the mice retained the diabetic state during the entire course of the experiment. Upon death, necropsies were promptly conducted within 1 h. The animals were subjected to initial examination for external signs of disease or injury, followed by removal of fur from abdominal and cranial regions. The skin was disinfected, and the internal organs (brain, lungs and kidneys) were removed aseptically, visually examined for gross morphology, and processed for microscopy [20% (w/v) aqueous KOH mount] and culturing on Sabouraud dextrose agar and DRBC agar at 37\u0026deg;C. For viable counting, a portion of the tissue samples was mildly homogenized, followed by ten-fold serial dilutions and spread plating. As the recovery of mucoralean fungi from tissue samples is arduous and may yield false negative results [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], the fungal hyphal load in the homogenized supernatants was also quantified (where specified) using hemocytometer. Additionally, sliced fragments of the tissue (10 to 16 in no.) were directly cultured in one set of the experiments, and the number of tissue fragments positive for fungal culture was enumerated [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The identification of the fungal isolate was based on morphological characteristics [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For histopathological examination, a section was fixed in 10% (v/v) buffered formalin and observed by hematoxylin-eosin staining. The challenged survivors were monitored for 30 days and then sacrificed by cervical dislocation and processed.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eInduction of Type 1 Diabetes\u003c/h2\u003e\u003cp\u003eWith a single dose of 210 mg streptozotocin/kg, Type 1 diabetes was established in 85% of the mice within a week of administration. The urinary levels of glucose ranged from 1000\u0026ndash;2000 mg/dl or more (median, 2000 mg/dl or more), and those of acetoacetic acid ranged from of 5\u0026ndash;15 mg/dl (median, 5 mg/dl). Lethargy, weight loss (\u0026asymp;\u0026thinsp;20%), polydipsia and increased urination frequency were observed in diabetic mice.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMurine model of ROC mucormycosis\u003c/h3\u003e\n\u003cp\u003eThe diabetic mice exposed to \u003cem\u003eR. arrhizus\u003c/em\u003e spores by intracerebral inoculation began displaying signs of illness within 24 h of exposure, including reduced response to stimuli, decreased food and water intake, kyphosis (hunched posture) and hemiplegia. The infection was lethal in 100% of the exposed animals within 2 days of inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The fungus was recovered both by microscopy as well as by culturing and dilution plating of the homogenised tissue, with the viable counts being greater in the brain than in the kidneys and lungs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; brain, 5.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16; lungs, 3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18; and kidneys, 2.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 log\u003csub\u003e10\u003c/sub\u003e cfu/g tissue). Diabetic mice infected through the intrasinus route showed a similar but slower progression of the disease; the mice became morbid within 3\u0026ndash;4 days and 72% died in 6\u0026ndash;12 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, none of the diabetic mice instilled intranasally with \u003cem\u003eR. arrhizus\u003c/em\u003e spores displayed any changes in behaviour, body temperature, or signs of the disease, even up to 30 days after inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Necropsies of these animals revealed normal gross morphologies of the internal organs. Fungal microscopy and culture were negative. The infection could not be established intranasally even by increasing the spore inoculum to 1x10\u003csup\u003e7\u003c/sup\u003e (survival, 100%).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eInstillation of 1x10\u003csup\u003e6\u003c/sup\u003e germlings in mice through the nasal route led to successful development of the infection, with signs typical of ROC mucormycosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The infected mice displayed lethargy, ruffled fur, reduced food and water intake, weight loss (41% and 23% versus the uninfected healthy controls and uninfected diabetic controls respectively), ptosis and impaired eye movements, conjunctival suffusion, purulent eye discharge, a hunched back, along with distressed behaviour, hypothermia and non-responsiveness to stimuli (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). 50% of the exposed animals became morbid within 48 h and died by 4\u0026ndash;6 days of exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUpon autopsy, hemorrhage was observed in the brain and lungs, with the lobes shrunken in size and friable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Necrosis was noted in the kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The fungus was recovered in both microscopy and culture. KOH mounts revealed the presence of broad aseptate hyphae in the tissue samples, indicative of mucoralean fungi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Although the homogenised tissue samples did not yield any positive cultures upon dilution plating, direct inoculation of the brain tissue fragments on media plates revealed the fungal presence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and e), with the lactophenol cotton blue mounts of the cultured fungus clearly demonstrating the distinctive morphological features of \u003cem\u003eR. arrhizus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The fungal loads were found to be higher in the brain compared to the lungs and kidneys, based on the number of fungal-positive tissue fragments (brain, 88%; lungs, 19% and kidneys, 16%) as well as hemocytometric evaluation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Histopathological examination of the cerebral cortex, cerebellum and olfactory bulb demonstrated the presence of mucoralean hyphae (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), with a loss of architectural details, dilation and congestion of the blood vessels, infiltration of mononuclear cells, and necrosis. Degenerative changes in the molecular layer and necrosis of the Purkinje layer were observed. Furthermore, challenging the diabetic mice with an inoculum of 1x10\u003csup\u003e5\u003c/sup\u003e and 1x10\u003csup\u003e7\u003c/sup\u003e germlings intranasally resulted in 33.33% and 50% mortality respectively, indicating the lethal dose (LD)\u003csub\u003e50\u003c/sub\u003e to be 1x10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003eR. arrhizus\u003c/em\u003e germlings. No morbidity or mortality was seen in the inoculated non-diabetic mice and the control mice groups given sham inoculation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMucormycosis is recognized as a highly devastating, aggressive fungal infection across the globe, and particularly in developing nations including India [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The disease manifests as a range of clinical types, with ROC, pulmonary and cutaneous forms being common depending upon the risk factors, transmission routes and geographical distribution [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Uncontrolled diabetes mellitus remains the most frequent underlying disease associated with mucormycosis in many countries, especially India, wherein it leads to a disproportionately high number of cases with ROC mucormycosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The disease presents substantial diagnostic and therapeutic challenges, resulting in elevated mortality rates [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The morbidity associated with this infection is also significant, with survivors experiencing dilapidating outcomes, such as loss of vision and disfigurement in cases with ROC mucormycosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In spite of such a profound impact, there is a notable paucity of literature on animal models to study the pathogenesis of ROC mucormycosis or assess alternate treatment strategies. Majority of the available studies describe primary cerebral infections in experimental animals by administration of fungal spores through intracranial or intraethmoid route or hematogenous dissemination [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This, however, does not truly represent the natural course of infection transmission. In the present study, we therefore attempted to establish a murine model of ROC mucormycosis that mimics the risk factor (diabetes) and transmission route (intranasal) observed in humans. \u003cem\u003eR. arrhizus\u003c/em\u003e was selected as the representative mucoralean species, as it is the predominant causative agent of mucormycosis in majority of the geographical locations worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFemale LACA mice were made diabetic by intraperitoneal administration of a single high dose of streptozotocin as per standard protocol, and then challenged with \u003cem\u003eR. arrhizus\u003c/em\u003e sporangiospores at a spore count of 1x10\u003csup\u003e6\u003c/sup\u003e by intranasal instillation. Experiments involving intracerebral and intrasinus (ethmoid) inoculations using the same spore inoculum dose were also performed for comparison. The diabetic mice that were subjected to intracerebral and intraethmoid inoculation with \u003cem\u003eR. arrhizus\u003c/em\u003e spores became morbid within 24 h and 3\u0026ndash;4 days of exposure, respectively. Whilst the mice inoculated intracerebrally had a mortality rate of 100% within 2 days of exposure, those inoculated via the intraethmoid route exhibited a mortality rate of 72% within 6\u0026ndash;12 days. This relatively slower onset of the disease during intrasinus vs. intracerebral inoculation correlates with their anatomically location, and the observed mortality is consistent with the findings documented in the existing literature [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, none of the diabetic mice instilled intranasally with \u003cem\u003eR. arrhizus\u003c/em\u003e spores displayed signs of the disease, even up to 30 days after inoculation. Their internal organs exhibited normal gross morphologies, with fungal microscopy and culture being negative. The infection could not be established intranasally even by increasing the spore load to 1x10\u003csup\u003e7\u003c/sup\u003e (survival, 100%), although such high inocula are virtually non-existent in the environment. The observed results are intriguing, as intranasal acquisition is the primary route of transmission of ROC infection, and mice are known to be susceptible to both natural and experimental mucormycosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConsidering that \u003cem\u003eR. arrhizus\u003c/em\u003e hyphal forms show greater resilience to innate immunity and increased virulence in \u003cem\u003eGalleria mellonella\u003c/em\u003e models compared with the spores [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], intranasal administration of 1x10\u003csup\u003e6\u003c/sup\u003e germlings in diabetic mice was then tested. It resulted in successful development of the infection, with signs typical of ROC mucormycosis. 50% of the exposed mice became morbid within 48 h and died by 4\u0026ndash;6 days of inoculation. The recovery of \u003cem\u003eR. arrhizus\u003c/em\u003e both in microscopy (KOH mounts and histopathology) and culture from the tissue samples corroborated these findings. Although the intranasal administration is likely to have deposited the fungus in both the nasal cavity and the lungs, the substantially higher fungal burdens in the brain compared to the lungs, along with the distinct ROC-specific signs noted in the infected mice suggest that the infection was primarily cerebral. Furthermore, pulmonary mucormycosis is often associated with risk factors such as neutropenia or malignancies, and development of primary pulmonary mucormycosis in murine models of uncontrolled diabetes has been reported to require more invasive procedures such as intratracheal administration in some studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The spread of infection to the other organs, such as the kidneys, is indicative of hematogenous dissemination. Although only brain, lung and kidney tissues were processed, the gross morphologies of the other organs were also indicative of the diseased state; for instance, the intestines were edematous and discolored. \u003cem\u003eR. arrhizus\u003c/em\u003e inoculum of 1x10\u003csup\u003e6\u003c/sup\u003e germlings was also noted to be the LD\u003csub\u003e50\u003c/sub\u003e for establishment of murine ROC mucormycosis through intranasal instillation. According to the published literature, the LD\u003csub\u003e50\u003c/sub\u003e of \u003cem\u003eR. arrhizus\u003c/em\u003e ranges from 10\u003csup\u003e3\u003c/sup\u003e to 10\u003csup\u003e6\u003c/sup\u003e spores depending upon the reported routes of inoculation and the clinical manifestations. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e],\u003c/p\u003e\u003cp\u003eTaken together, the clinical signs, rapid onset and evolution of the disease, along with the presence of lesions and fungal load indicated that cerebral infection was the primary cause of death in the infected, diabetic mice. The findings further demonstrated that germlings of \u003cem\u003eR. arrhizus\u003c/em\u003e lead to ROC mucormycosis in diabetic mice through intranasal acquisition. This also correlates with the fact that the germlings preferentially express the spore-coat protein CotH3, which serves as an invasin and facilitates the binding to the mucoralean receptor (glucose-regulated-protein 78) over-expressed on the nasal epithelium in diabetic patients [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Hyphal forms of \u003cem\u003eR. arrhizus\u003c/em\u003e and \u003cem\u003eLichtheimia corymbifera\u003c/em\u003e have also been recently demonstrated to contribute to combat-relevant wound mucormycosis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn contrast to the present study, Reinhardt \u003cem\u003eet al.\u003c/em\u003e reported the development of cerebral infection in rabbits by intranasal instillation of \u003cem\u003eR. arrhizus\u003c/em\u003e spores 3\u0026ndash;4 days prior to induction of diabetes using alloxan [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This may, however, not accurately represent the disease cycle, as the infection is acquired in the backdrop of pre-existing diabetes. Furthermore, the inoculated sporangiospores are likely to have germinated in the animal host during that time period. A study by Bauer \u003cem\u003eet al.\u003c/em\u003e published in the year 1955 demonstrated experimental cerebral mucormycosis acquired intranasally via spores in alloxan-treated diabetic rabbits [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although this could reflect host-specific susceptibility, the possibility of germlings or hyphal forms in the spore inoculum used cannot be ruled out, as those were collected and separated from mycelial cultures using sterile gauze, whose mesh-size may not have fully filtered out the germlings and/or mycelial fragments. The failure of mucoralean spores to cause ROC mucormycosis in diabetic mice, as observed in the present study, likely reflects the reason why majority of the reported murine models involve alternate routes of inoculation or infection sites, even with diabetes as the underlying risk factor [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], despite a documented association of ROC with uncontrolled diabetes and intranasal acquisition [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn conclusion, the results of this study demonstrate the establishment of ROC mucormycosis by intranasal route in a murine model of uncontrolled diabetes. Germlings, but not sporangiospores, were found to be implicated in the successful establishment of infection. Further, the work reports a clinically-relevant murine model of mucormycosis that can be utilized in future studies for studying the pathogenesis and evaluating newer treatment strategies against this deadly fungal infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical approval\u003c/h2\u003e\u003cp\u003e All experiments were performed by following the guidelines of the Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), India after due approval of the Institutional Animal Ethics Committee (IAEC), Panjab University, Chandigarh. (PU/45/99/CPCSEA/IAEC/2022/671).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003cp\u003eThe author(s) declare that there are no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe study was supported by research funding from the Indian Council of Medical Research (ICMR), Govt. of India under extramural research grant scheme (Drug development initiative) (No. 67/2/2020-DDI/BMS) granted to RS.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe study was designed by R.S. and J.K. The acquisition, analysis, and interpretation of the data were conducted by R.S., J.K., P.K and A.S. R.S., P.K. and A.S. drafted the manuscript. All authors agreed to the publication of the final manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data generated and analyzed in the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHoenigl M, Seidel D, Sprute R, Cunha C, Oliverio M, Goldman GH et al (2022) COVID-19-associated fungal infections. Nat Microbiol 7:1127\u0026ndash;1140. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1038/s41564-022-01172-2\u003c/span\u003e\u003cspan address=\"10.1038/s41564-022-01172-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChakrabarti A, Singh R (2014) Mucormycosis in India: unique features. 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Antimicrob Agents Chemother 47:3343\u0026ndash;3344. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1128/AAC.47.10.3343-3344.2003\u003c/span\u003e\u003cspan address=\"10.1128/AAC.47.10.3343-3344.2003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"diabetes, intranasal, mice, mucormycosis, rhino-orbito-cerebral, Rhizopus arrhizus","lastPublishedDoi":"10.21203/rs.3.rs-6817419/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6817419/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMucormycosis is a rapidly fatal, angioinvasive fungal infection. The disease presents as distinct clinical entities, with each being linked to specific risk factors and routes of transmission. Rhino-orbito-cerebral (ROC) manifestation remains the predominant form of mucormycosis in the developing world, especially in India amongst patients with uncontrolled diabetes. Limited literature is available on experimental animal models to study mucormycosis, and a murine model of ROC mucormycosis that recapitulates the risk factor (diabetes) and mode of acquisition (intranasal) of this disease is largely undocumented. In this study, we demonstrate that sporangiospores of \u003cem\u003eRhizopus arrhizus\u003c/em\u003e (1x10\u003csup\u003e6\u003c/sup\u003e cfu) failed to establish infection in diabetic mice when administered intranasally although intracerebral and intrasinus (ethmoid) inoculations were successful. The diabetic mice instilled intranasally with \u003cem\u003eR. arrhizus\u003c/em\u003e spores (1x10\u003csup\u003e6\u003c/sup\u003e, and even 1x10\u003csup\u003e7\u003c/sup\u003e cfu) showed 100% survival, monitored upto 30 days post-inoculation. Their internal organs exhibited normal gross morphologies; fungal microscopy and culture were negative. In contrast, intranasal administration of 1x10\u003csup\u003e6\u003c/sup\u003e germlings of \u003cem\u003eR. arrhizus\u003c/em\u003e to diabetic mice led to successful development of the infection, with signs typical of ROC mucormycosis. 50% of the exposed animals became morbid within 48 h and died by 4\u0026ndash;6 days of exposure. The fungus was recovered both in microscopy (KOH mounts and histopathology) and culture. These findings reveal that \u003cem\u003eR. arrhizus\u003c/em\u003e germlings, rather than sporangiospores, are mainly responsible for the natural acquisition of ROC mucormycosis intranasally by a diabetic host. Further, the work establishes a clinically-relevant murine model of ROC mucormycosis that can be utilized for future studies.\u003c/p\u003e","manuscriptTitle":"Germlings of Rhizopus arrhizus, rather than sporangiospores, lead to rhino-orbito-cerebral mucormycosis acquired intranasally in a murine model of uncontrolled diabetes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 11:28:12","doi":"10.21203/rs.3.rs-6817419/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":"b934365f-ae80-47fb-84ef-84a378775872","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T22:38:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 11:28:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6817419","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6817419","identity":"rs-6817419","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}