{"paper_id":"021c39e2-218a-4bd4-998a-c3a99724a05b","body_text":"Identification of Microbial Community in Otomycosis by mNGS: Potential implication for treatment of this disorder with terbinafine | 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 Identification of Microbial Community in Otomycosis by mNGS: Potential implication for treatment of this disorder with terbinafine Shuai Xu, Xin Zhang, Qianqian Yang, Jianfeng Li, Zhaoyan Yu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2749783/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Sep, 2023 Read the published version in Mycopathologia → Version 1 posted 5 You are reading this latest preprint version Abstract The present study was designed to identify the microbial community as well as to analyze its diversity bymeans of metagenomic Next Generation Sequencing(mNGS)in 17 patients with otomycosis treated with terbinafinein the Department of Otolaryngology of Shandong Provincial Hospital from June 2021 to June 2022, so as to evaluate the relationship between microbial community and terbinafine resistance. Those 17 patients were divided into two groups, i.e., Terbinafine Effective Group (TEG, n = 14 cases) and Terbinafine Resistance Group(TRG, n = 3 cases) according to the therapy effect, whose microbial community of secretion of external auditory canal (EAC)was identified using mNGS. We found that the sequence of bacteria was significantly more than that of fungi and, whereas, the difference between the two groups of bacteria was not significant.There were significant differences in fungal community between the two groups. Aspergillus was the main pathogenic fungus of TEG patients while Malassezia was a dominant fungus in TRG patients. In conclusion, the results from this work indicate that Aspergillus terreusis the main pathogenic fungus in this cohortof otomycosis patients and MNGS sequencing can offer comprehensive information about the microbial community of otomycosis. The fungus community dominated by Malassezia is more likely to be resistant to terbinafine, which provides certain guidance for clinical treatment of otomycosis with terbinafine. Metagenomic Next Generation Sequencing Otomycosis Microbial community Terbinafine resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction In spite of the earlier emergence of fungal pathogens,for more than a century, it has been documented that the most common infections are caused by bacteria, viruses, and other parasites 1 . However, theprevalence of serious diseases resulted from fungi has increased inrecent decades due to the increasing number of immunocompromised individuals 2 . Meanwhile, in severely immunocompromised patients, some fungus can disseminateinto the bloodstream and colonize internal organs, resulting inlife-threatening systemic infections 3 , 4 . Otomycosis is a worldwide disease, in which appropriately 15–20% of external ear infections arecaused by mycelial fungi and yeasts 5 .Otomycosisis an infection that involves the EAC squamous epithelium, characterized by pruritus, erythema, scaling,otalgia,aural fullness and hypoacusis 5 , 6 . The most frequently reportedpathogens are Aspergillus and Candida species 6 – 8 . Fungal communities in otomycosis may vary in different areas. According to the latest literature,A. niger, A. terreus,A. tubingensisand A. awamori were the most frequent species in Aspergillus,while, C. albicans was by far the most common yeast in Candida 9 – 13 . In clinical practice, early identification of fungal pathogens is critical for thediagnosis and treatment of otomycosis. The traditional methods such as culture-based methods can only identify the main pathogens in most cases 14 ,whereas, molecular microbial tools, like next-generation sequencing (NGS), have the advantage that they are culture-independent and more sensitive. NGS uses anuntargeted sequencing approach, which can identify and quantify bacteria and fungi presentin a sample, including previously unknown microbes. Using untargeted sequencing method, NGS can not only identify and quantify common bacteria and fungi, but identify previously unknown microorganisms as well. As a kind of NGS, mNGS(also termed high-throughput sequencing technology) can sequence the sample microbial genes, which can realize the sequencing of all microbial genomes, assemble and obtain the microbial genome information, and carry out the annotation and difference comparison of microbial potential functions. Predisposing factors include residing in tropical and humid climates, repeated swimming, the insertion of foreign bodies, use of hearing aids, the presence of cerumen, lack of hygiene, the use of long-term antibiotic or steroid therapy, repeated cleaning of the EAC with swabs, genetic factors, seborrheic dermatitis, diabetes, and immune defects, all of which benefit the germination of the spores and conidia of the prevalent fungi 15 – 17 . Treatment mostly requires the use of topical antifungals such as clotrimazole,terbinafine,ketoconazole, econazole, ciclopiroxolamine, nystatin,tolnaftate, bifonazole, and miconazole for at least three or four weeks 7 , 18 , 19 . The fungal pathogens of otomycosis have been reported by researchers from most countries. But as yet, there have been only few studies on the co-existence and interaction of bacteria and fungi in otomycosis. With the widespread application of antifungal drugs to treat otomycosis, more and more studies of antifungal drug resistance have been reported. Antifungal resistance is emerging as a public health challenge that needs to be addressed concurrently with antimicrobial and antiviral resistance.Currently, people in the United States is heavily infected with a super fungus called Candida auralis, which has resistance to multiple antifungal drugs and causes about half of infected people to die within three months. In this study, 17 cases of otomycosis treated with terbinafine in Jinan, China, from June 2021 to June 2022 were analyzed retrospectively, and their microbial composition and possible interaction were studied by mNGS, with emphasis given on comparing the therapeutic effect of terbinafine, the relationship between different microbial communities and drug resistance to terbinafine. 2. Materials And Methods 2.1 Sample Collection The samples were collected from Shandong Provincial Hospital and were all the EAC irrigations that had been obtained in the past. Among them, 14 patients recovered with terbinafine treatment after 1 month, and 3 patients were ineffective after 1 month. 2.2 Sampling Method 17 patients wereall unilateral. The affected ear was washed with sterile physiological saline, and the washing solution was stored in a sterile sampling tube, and stored at − 80 ℃ for DNA detection. 2.3 Sample Inclusion and Exclusion Criteria 2.3.1 Inclusion Criteria: The age of patients was between 18 and 60 years old, all of whom were the first time to get sick. The patients hadat least one of the symptoms such as pruritus, tinnitus, otalgia, otorrhea,aural fullness and hypoacusis.Punctate, villous, or lumpy plaque could be seen in EAC under the otoscope(Fig. 1 A- 1 C). All fungal smears were positive, and fungal hyphae orsproes could be seen(Fig. 1 D- 1 F). 2.3.2 Exclusion Criteria The exclusion criteria were as follows: patients who had applied antibacterial drugs locally and systemically within 1 month; patients with serious systemic diseases and immune deficiency; a diagnosis different tootomycosis; and pregnant and lactating women. 2.4 DNA Extraction and Sequencing Analysis 2.4.1 DNA Extraction We used the QIAamp DNA Microbiome Kit(50) of Kaijie Company to extract DNA from each sample in strict accordance with the product operating instructions. The DNA degradation degree and potential pollution were monitored on the 1% agarose gel, and the purity and integrity of DNA were analyzed. The DNA concentration was accurately quantified with Qubit 4.0 fluorometer. 2.4.2 Construction of standard sequencing library The DNA samples were randomly broken into fragments with a length of about 350 bp using a Covaris ultrasonic crusher, and the library was constructed through terminal repair, A-tailed addition, splicing, purification, and PCR amplification. After the construction of the library, we used a Qubit 4.0 fluorometer for preliminary quantification, and diluted the library to 2 µ g/ µ l. Subsequently, the length of inserted fragments in the library was detected using an Aglent 2100 biological analyzer. 2.4.3 High-throughput Sequencing According to the standard scheme, the Illumina platform (PE150 sequencing method) was used for standardized sequencing to obtain the required DNA sequence. 2.5 Data processing We used FastQC (version v0.11.9) to perform base quality statistics on the original sequencing data, and used R statistical software to visualize the results.Trimmatic(Version 0.39)was used to cut the joint sequence, double-ended low-quality sequence, and double-ended pairing sequence greater than 36 bp was reserved. BMTagger (version 3.102) was used to remove the host genome sequence. 2.6 Beta Diversity Analysis Non-metric Multi-dimensional Scaling (NMDS) based on Bray-Curtis distancewas used to compare the composition of microbial community among different samples, and the difference of microbial community structure between different samples was evaluated by the distance between points. 3. Results 3.1 Raw Data and Quality Control The bacterial and fungal DNA in the EAC secretion of patients with otomycosis was sequenced with high flux, and the sequences obtained from each sample ranged from 29815652 to 87812501. After quality control, the sequence number of fungi varied from 11144 to 1746156, whilethe number of bacteria varied from 418491 to 8968300. The sequence number of bacteria were significantly higher than that of fungi. 3.2 Community Composition of Bacteria and Fungi At the phylum level, the difference of bacteria between the two groups was not obvious. Three categories of microorganisms, namely Firmicutes, Actinobacteria and Proteobacteria occupied an absolute dominant position, and their proportions in different samples ranged from 89.9–99.8% (Fig. 2 A). At the phylum level, the fungal community structure of TEG and TRG was significantly different.Ascomycota was the main fungi in TEG, accounting for more than 95% in 8 samples, while Basidiomycota was dominant in TRG, accounting for more than 79% in 3 samples(Fig. 2 B). At the genus level of bacteria, Staphylococcus and Bacillus_ A were the most common bacteria in the two groups (Fig. 3 A). For fungi, Aspergillus of Ascomycetes was the main fungus in TEG, of which 7 cases accounted for more than 90%, while the fungi in TFG was mainly Malassezia, of which 2 samples accounted for more than 65% and 1 case accounted even for 97% (Fig. 3 B). At the bacterial species level, Staphylococcus aureus of Staphylococcus was dominant, while Staphylococcus epidermidis, Staphylococcus capitis and Bacillus_Abombysepticusin Bacillus_ A were also common bacteria (Fig. 4 A). For fungi, the most common fungus in TEG was Aspergillus terreus (Fig. 4 B), while the most common fungus in TRG was Malasseziarestricta, of which one case accounted for more than 90%. At the same time, in all samples, Staphylococcus aureus and Aspergillus terreus accounted for the largest proportion of bacteria and fungi respectively (Fig. 5 A and 5 B). 3.3 Analysis of Microbial Community Difference Beta diversity analysis was performed in order to show the difference between the two groups.Non-Metric Multi-Dimensional Scaling(NMDS) based on species level showed that the bacteria in two groups were not completely separated, and there was no significant difference between samples(Fig. 6 A), which indicated that there was no obvious difference in bacterial community structure inotomycosis, and bacteria were not the main factor causing fungal drug resistance. For fungi, there were significant differences between the two groups. The sample points of TEG and TRG were gathered respectively and separated significantly, indicating that there were significant differences in the fungal community structure between the two groups (Fig. 6 B). Samples of TRG were resistant to terbinafine because of their different fungal community structures. 4. Discussion Early identification of pathogens is essential for the diagnosis and treatment of otomycosis. In general, detection of pathogens of otomycosis mainly depends on traditional culture-based methods and modern sequencing technology. However,culture-negative strains or resident funguses could be hardly detected by regular culture conditions 14 . Some scholars had proved that thepositive culture rate of the agents causing fungal infections was 50%, especially when Malassezia species or Mucorales were highly suspected 20 . It had been reported thateight causative fungal genera were identified by ITS sequencing while five fungal genera were identified by culture in fungal keratitis. Moreover, Gu et al. identified the fungal community of otomycosis patients in Nanjing, Jiangsu Province, China, through ITS, and successfully detected some uncommon funguses such as Sagenomella and Cladosporium 21 . However, the limitation of ITS is that it is not suitable for the marking of species within the genus due to the small differences in the interval because of the evolutionary order and variation of fungi. MNGS is highly sensitive and informative, which possesses the ability to detect mixed infections of bacteria, fungi and virus. Therefore, it has the advantage of congenital thickening for diseases caused by multiple pathogens.In this work, fungi and bacterial communities in otomycosis were identified simultaneously by mNGS. Most researchers from different countries indicatedthat A. nigerserved as the most commonotomycosis agent 22 – 25 . However, through mNGS, we found that the most common fungus in otomycosis was Aspergillus terreus, which was consistent with the study of Zhang et al. 11 in Hangzhou, China. This indicates that the fungal community of otomycosis may vary in different region. In western China, Aspergillus tubingensiswas the most common fungi, followed by Aspergillus fumigatus and Aspergillus terreus 26 .Studies in Iran had found that Aspergillus flavus was the most common species of otomycosis, followed by Aspergillus tubingensis and Aspergillus niger 8 , while Aspergillus awamori was considered to be the most common fungi causing otomycosis in studies in southern Hungary 27 . In India, Aspergillus niger and Aspergillus fumigatus were dominant in otomycosis 17 .Candida albicans had also been reported as a common pathogen of otomycosis, which mainly occurred in patients with abnormal immune function 28 . To date, however, the bacterial community in otomycosis has been studied sparingly. In the present study, we found through mNGS that the most common bacteria in otomycosis patients was Staphylococcus aureus.At the same time, staphylococcus aureus is the main pathogen of bacterial external otitis, which can proliferate and infect the human body, leading to inflammation when human immunity is reduced and the environment of the ear canal changes 29 . Currently, the treatment of otomycosis currently relies mainly on topical use of antifungal drugs. Terbinafine is more effective against partial Aspergillus species in vitro than itraconazole or amphotericin B 30 . Meanwhile, terbinafine is considered to be an effective drug in the treatment of otomycosis 31 . It has been reported that the cure rate of Terbinafine in the treatment of otomycosis reaches 100% 32 , while, in this study, 14 of the 17 patients treated with Terbinafine have been cured, with a cure rate of 82.4%, which might be related to the increasingly severe fungal resistance at present. It is worth noting that Aspergillus is the main cause of cured cases in our study, beingconsistent with the above report 32 . Some Malassezia strains have been reported to be resistant to terbinafine 33 . We found that the fungal community dominated by Malassezia has resistance to terbinafine, and the precise mechanism of resistance needs to be further studied. In summary, the present study identifies by means of mNGSthat the most common fungal species among patients with otomycosis in the city of Jinan, Shandong Province, of the People’s Republic of China is Aspergillus terreus, and the most common bacterial species is Staphylococcus aureus. Fungi dominated by Malassezia are more resistant to terbinafine than fungi dominated by Aspergillus. Findings from this work have certain guiding significance for the clinical treatment of otomycosis with terbinafine. The specific mechanism underpinning drug resistance needs to be further researched. Declarations Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 82071039) and the National Natural Science Foundation of China (No. 82101215 ）. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Shuai Xu, Xin Zhang, Jianfeng Li and Zhaoyan Yu. The first draft of the manuscript was written by Shuai Xu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethical Approval This research was approved by the Biomedical Research Ethics Committee of Shandong Provincial Hospital. References Gupta A K , Venkataraman M . Antifungal resistance in Superficial mycoses[J]. Journal of Dermatological Treatment, 2021:1-25. Hazarika D, Jahan N, Sharma A. 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In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine. The British journal of dermatology. 2000;142:758-65. Cite Share Download PDF Status: Published Journal Publication published 19 Sep, 2023 Read the published version in Mycopathologia → Version 1 posted Reviewers invited by journal 06 Apr, 2023 Reviewers agreed at journal 01 Apr, 2023 Editor invited by journal 29 Mar, 2023 Editor assigned by journal 29 Mar, 2023 First submitted to journal 28 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-2749783\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":188302229,\"identity\":\"39b421f3-2f22-4654-868c-27916660a1b8\",\"order_by\":0,\"name\":\"Shuai Xu\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIie3QMQuCUBDA8XOxRavxRNBPECiCi2RfJRFybWxUhHN0besLNEej8sAWodW2XJpd3bKx6ekW9H7z/d/jDkAQfpUUA8izJGm7aYnCUgcnJYARLZUx02Z+drX+ysy51hIg+MYq5iRW89rpas1s0gN67iF03IKXIKt0iZg0JJmFUAQXXmIeS9J6YhvSSkJlTAL3tEKVWEAojUysRg49laKQlGA4sjViFzOv7UdP3vqU3dq2O/gGNwHcfn3KG/9YcB8VBEH4e2+RU0IzBZF3YgAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0000-0001-5987-1714\",\"institution\":\"Shandong Provincial Hospital\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Shuai\",\"middleName\":\"\",\"lastName\":\"Xu\",\"suffix\":\"\"},{\"id\":188302230,\"identity\":\"4a983212-d851-4e15-ab79-b3d26e7077ab\",\"order_by\":1,\"name\":\"Xin Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shandong Provincial Hospital\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xin\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":188302231,\"identity\":\"b94542ef-ffde-4229-bacc-f5d90f1426d8\",\"order_by\":2,\"name\":\"Qianqian Yang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Soochow University Affiliated No 1 People's Hospital: First Affiliated Hospital of Soochow University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Qianqian\",\"middleName\":\"\",\"lastName\":\"Yang\",\"suffix\":\"\"},{\"id\":188302232,\"identity\":\"af4bef97-f8b8-4e42-b799-4c861e14aeb1\",\"order_by\":3,\"name\":\"Jianfeng Li\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shandong Provincial Hospital\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jianfeng\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"},{\"id\":188302233,\"identity\":\"52a52a98-01b0-4863-a102-321bbca2f438\",\"order_by\":4,\"name\":\"Zhaoyan Yu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shandong Provincial Hospital\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhaoyan\",\"middleName\":\"\",\"lastName\":\"Yu\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-03-29 04:45:39\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-2749783/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-2749783/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1007/s11046-023-00791-5\",\"type\":\"published\",\"date\":\"2023-09-19T15:01:37+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":35281874,\"identity\":\"f669a6e1-e75a-4a2b-8717-d4bf0bf5ffca\",\"added_by\":\"auto\",\"created_at\":\"2023-04-04 16:27:51\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":988932,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A-C) Punctate, villous, and lumpy plaque can be seen in EAC under the otoscope. (D-F) Scattered fungal hyphae and spores can be seen under microscope\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2749783/v1/ddedfd1044e314214d2f0c25.png\"},{\"id\":35281875,\"identity\":\"cf97853e-0e8d-4ddf-9a17-743c6fbe7496\",\"added_by\":\"auto\",\"created_at\":\"2023-04-04 16:27:51\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":267398,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRelative abundance of bacterial(A) and fungal(B) communities at phylum level in EAC of patients in TEG and TRG\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2749783/v1/6cb596401afafc8ffdb6a0ad.png\"},{\"id\":35282425,\"identity\":\"1a70490d-8972-4d47-ae28-84d0183454df\",\"added_by\":\"auto\",\"created_at\":\"2023-04-04 16:35:51\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":416123,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRelative abundance of bacterial(A) and fungal(B) communities at genus level in EAC of patients in TEG and TRG\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2749783/v1/79528857e8fd8148507fb4a5.png\"},{\"id\":35281873,\"identity\":\"145cbe76-f1e8-4b39-a3ce-772f9041d050\",\"added_by\":\"auto\",\"created_at\":\"2023-04-04 16:27:51\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":518325,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRelative abundance of bacterial(A) and fungal(B) communities at species level in EAC of patients in TEG and TRG\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2749783/v1/23fdb3e5ceb2f60b8e00f0f9.png\"},{\"id\":35281878,\"identity\":\"2d891b27-eb5c-4548-84fa-76b6fce24b6f\",\"added_by\":\"auto\",\"created_at\":\"2023-04-04 16:27:51\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1271426,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHeatmap of relative abundance of bacterial(A) and fungal(B) communities at species level in all patients\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2749783/v1/e4c44df0ab330be59b05da73.png\"},{\"id\":35282426,\"identity\":\"d43b4504-df4d-46cf-8546-8901314b5b1e\",\"added_by\":\"auto\",\"created_at\":\"2023-04-04 16:35:51\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":222906,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNon-Metric Multi-Dimensional Scaling (NMDS)based on species level in two groups. Each point in the diagram represents a sample, and samples from the same group are represented by the same color.For grouped samples, ellipses will be used to display the distinguishing areas of the sample group, and the distance between points indicates the degree of difference.Generally, when the Stress is less than 0.2, it indicates that NMDS analysis has certain reliability\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2749783/v1/73e5cc3f8d4464bddc427bfe.png\"},{\"id\":43640672,\"identity\":\"6cc721bf-6013-4d46-a2ea-a3145e115792\",\"added_by\":\"auto\",\"created_at\":\"2023-09-25 15:08:25\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3514206,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-2749783/v1/a2614129-55fd-45f2-b867-138dacff64bc.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Identification of Microbial Community in Otomycosis by mNGS: Potential implication for treatment of this disorder with terbinafine\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eIn spite of the earlier emergence of fungal pathogens,for more than a century, it has been documented that the most common infections are caused by bacteria, viruses, and other parasites\\u003csup\\u003e\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u003c/sup\\u003e. However, theprevalence of serious diseases resulted from fungi has increased inrecent decades due to the increasing number of immunocompromised individuals\\u003csup\\u003e\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e\\u003c/sup\\u003e. Meanwhile, in severely immunocompromised patients, some fungus can disseminateinto the bloodstream and colonize internal organs, resulting inlife-threatening systemic infections\\u003csup\\u003e\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eOtomycosis is a worldwide disease, in which appropriately 15\\u0026ndash;20% of external ear infections arecaused by mycelial fungi and yeasts\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e.Otomycosisis an infection that involves the EAC squamous epithelium, characterized by pruritus, erythema, scaling,otalgia,aural fullness and hypoacusis\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u003c/sup\\u003e. The most frequently reportedpathogens are Aspergillus and Candida species\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR7\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eFungal communities in otomycosis may vary in different areas. According to the latest literature,A. niger, A. terreus,A. tubingensisand A. awamori were the most frequent species in Aspergillus,while, C. albicans was by far the most common yeast in Candida\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR10 CR11 CR12\\\" citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eIn clinical practice, early identification of fungal pathogens is critical for thediagnosis and treatment of otomycosis. The traditional methods such as culture-based methods can only identify the main pathogens in most cases\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e,whereas, molecular microbial tools, like next-generation sequencing (NGS), have the advantage that they are culture-independent and more sensitive. NGS uses anuntargeted sequencing approach, which can identify and quantify bacteria and fungi presentin a sample, including previously unknown microbes. Using untargeted sequencing method, NGS can not only identify and quantify common bacteria and fungi, but identify previously unknown microorganisms as well. As a kind of NGS, mNGS(also termed high-throughput sequencing technology) can sequence the sample microbial genes, which can realize the sequencing of all microbial genomes, assemble and obtain the microbial genome information, and carry out the annotation and difference comparison of microbial potential functions.\\u003c/p\\u003e \\u003cp\\u003ePredisposing factors include residing in tropical and humid climates, repeated swimming, the insertion of foreign bodies, use of hearing aids, the presence of cerumen, lack of hygiene, the use of long-term antibiotic or steroid therapy, repeated cleaning of the EAC with swabs, genetic factors, seborrheic dermatitis, diabetes, and immune defects, all of which benefit the germination of the spores and conidia of the prevalent fungi\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR16\\\" citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eTreatment mostly requires the use of topical antifungals such as clotrimazole,terbinafine,ketoconazole, econazole, ciclopiroxolamine, nystatin,tolnaftate, bifonazole, and miconazole for at least three or four weeks\\u003csup\\u003e\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe fungal pathogens of otomycosis have been reported by researchers from most countries. But as yet, there have been only few studies on the co-existence and interaction of bacteria and fungi in otomycosis. With the widespread application of antifungal drugs to treat otomycosis, more and more studies of antifungal drug resistance have been reported. Antifungal resistance is emerging as a public health challenge that needs to be addressed concurrently with antimicrobial and antiviral resistance.Currently, people in the United States is heavily infected with a super fungus called Candida auralis, which has resistance to multiple antifungal drugs and causes about half of infected people to die within three months. In this study, 17 cases of otomycosis treated with terbinafine in Jinan, China, from June 2021 to June 2022 were analyzed retrospectively, and their microbial composition and possible interaction were studied by mNGS, with emphasis given on comparing the therapeutic effect of terbinafine, the relationship between different microbial communities and drug resistance to terbinafine.\\u003c/p\\u003e\"},{\"header\":\"2. Materials And Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Sample Collection\\u003c/h2\\u003e \\u003cp\\u003eThe samples were collected from Shandong Provincial Hospital and were all the EAC irrigations that had been obtained in the past. Among them, 14 patients recovered with terbinafine treatment after 1 month, and 3 patients were ineffective after 1 month.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Sampling Method\\u003c/h2\\u003e \\u003cp\\u003e17 patients wereall unilateral. The affected ear was washed with sterile physiological saline, and the washing solution was stored in a sterile sampling tube, and stored at \\u0026minus;\\u0026thinsp;80 ℃ for DNA detection.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Sample Inclusion and Exclusion Criteria\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.1 Inclusion Criteria:\\u003c/h2\\u003e \\u003cp\\u003eThe age of patients was between 18 and 60 years old, all of whom were the first time to get sick. The patients hadat least one of the symptoms such as pruritus, tinnitus, otalgia, otorrhea,aural fullness and hypoacusis.Punctate, villous, or lumpy plaque could be seen in EAC under the otoscope(Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA-\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). All fungal smears were positive, and fungal hyphae orsproes could be seen(Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD-\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.2 Exclusion Criteria\\u003c/h2\\u003e \\u003cp\\u003eThe exclusion criteria were as follows: patients who had applied antibacterial drugs locally and systemically within 1 month; patients with serious systemic diseases and immune deficiency; a diagnosis different tootomycosis; and pregnant and lactating women.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 DNA Extraction and Sequencing Analysis\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.1 DNA Extraction\\u003c/h2\\u003e \\u003cp\\u003e We used the QIAamp DNA Microbiome Kit(50) of Kaijie Company to extract DNA from each sample in strict accordance with the product operating instructions. The DNA degradation degree and potential pollution were monitored on the 1% agarose gel, and the purity and integrity of DNA were analyzed. The DNA concentration was accurately quantified with Qubit 4.0 fluorometer.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.2 Construction of standard sequencing library\\u003c/h2\\u003e \\u003cp\\u003eThe DNA samples were randomly broken into fragments with a length of about 350 bp using a Covaris ultrasonic crusher, and the library was constructed through terminal repair, A-tailed addition, splicing, purification, and PCR amplification. After the construction of the library, we used a Qubit 4.0 fluorometer for preliminary quantification, and diluted the library to 2 \\u0026micro; g/ \\u0026micro; l. Subsequently, the length of inserted fragments in the library was detected using an Aglent 2100 biological analyzer.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.3 High-throughput Sequencing\\u003c/h2\\u003e \\u003cp\\u003eAccording to the standard scheme, the Illumina platform (PE150 sequencing method) was used for standardized sequencing to obtain the required DNA sequence.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Data processing\\u003c/h2\\u003e \\u003cp\\u003eWe used FastQC (version v0.11.9) to perform base quality statistics on the original sequencing data, and used R statistical software to visualize the results.Trimmatic(Version 0.39)was used to cut the joint sequence, double-ended low-quality sequence, and double-ended pairing sequence greater than 36 bp was reserved. BMTagger (version 3.102) was used to remove the host genome sequence.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6 Beta Diversity Analysis\\u003c/h2\\u003e \\u003cp\\u003eNon-metric Multi-dimensional Scaling (NMDS) based on Bray-Curtis distancewas used to compare the composition of microbial community among different samples, and the difference of microbial community structure between different samples was evaluated by the distance between points.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003ch2\\u003e3.1 Raw Data and Quality Control\\u003c/h2\\u003e \\u003cp\\u003eThe bacterial and fungal DNA in the EAC secretion of patients with otomycosis was sequenced with high flux, and the sequences obtained from each sample ranged from 29815652 to 87812501. After quality control, the sequence number of fungi varied from 11144 to 1746156, whilethe number of bacteria varied from 418491 to 8968300. The sequence number of bacteria were significantly higher than that of fungi.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2 Community Composition of Bacteria and Fungi\\u003c/h2\\u003e \\u003cp\\u003eAt the phylum level, the difference of bacteria between the two groups was not obvious. Three categories of microorganisms, namely Firmicutes, Actinobacteria and Proteobacteria occupied an absolute dominant position, and their proportions in different samples ranged from 89.9\\u0026ndash;99.8% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAt the phylum level, the fungal community structure of TEG and TRG was significantly different.Ascomycota was the main fungi in TEG, accounting for more than 95% in 8 samples, while Basidiomycota was dominant in TRG, accounting for more than 79% in 3 samples(Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003eAt the genus level of bacteria, Staphylococcus and Bacillus_ A were the most common bacteria in the two groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). For fungi, Aspergillus of Ascomycetes was the main fungus in TEG, of which 7 cases accounted for more than 90%, while the fungi in TFG was mainly Malassezia, of which 2 samples accounted for more than 65% and 1 case accounted even for 97% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAt the bacterial species level, Staphylococcus aureus of Staphylococcus was dominant, while Staphylococcus epidermidis, Staphylococcus capitis and Bacillus_Abombysepticusin Bacillus_ A were also common bacteria (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). For fungi, the most common fungus in TEG was Aspergillus terreus (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB), while the most common fungus in TRG was Malasseziarestricta, of which one case accounted for more than 90%.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAt the same time, in all samples, Staphylococcus aureus and Aspergillus terreus accounted for the largest proportion of bacteria and fungi respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA and \\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3 Analysis of Microbial Community Difference\\u003c/h2\\u003e \\u003cp\\u003eBeta diversity analysis was performed in order to show the difference between the two groups.Non-Metric Multi-Dimensional Scaling(NMDS) based on species level showed that the bacteria in two groups were not completely separated, and there was no significant difference between samples(Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA), which indicated that there was no obvious difference in bacterial community structure inotomycosis, and bacteria were not the main factor causing fungal drug resistance.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFor fungi, there were significant differences between the two groups. The sample points of TEG and TRG were gathered respectively and separated significantly, indicating that there were significant differences in the fungal community structure between the two groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB). Samples of TRG were resistant to terbinafine because of their different fungal community structures.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eEarly identification of pathogens is essential for the diagnosis and treatment of otomycosis. In general, detection of pathogens of otomycosis mainly depends on traditional culture-based methods and modern sequencing technology. However,culture-negative strains or resident funguses could be hardly detected by regular culture conditions\\u003csup\\u003e\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e\\u003c/sup\\u003e. Some scholars had proved that thepositive culture rate of the agents causing fungal infections was 50%, especially when Malassezia species or Mucorales were highly suspected\\u003csup\\u003e\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e\\u003c/sup\\u003e. It had been reported thateight causative fungal genera were identified by ITS sequencing while five fungal genera were identified by culture in fungal keratitis. Moreover, Gu et al. identified the fungal community of otomycosis patients in Nanjing, Jiangsu Province, China, through ITS, and successfully detected some uncommon funguses such as Sagenomella and Cladosporium\\u003csup\\u003e\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e\\u003c/sup\\u003e. However, the limitation of ITS is that it is not suitable for the marking of species within the genus due to the small differences in the interval because of the evolutionary order and variation of fungi.\\u003c/p\\u003e \\u003cp\\u003eMNGS is highly sensitive and informative, which possesses the ability to detect mixed infections of bacteria, fungi and virus. Therefore, it has the advantage of congenital thickening for diseases caused by multiple pathogens.In this work, fungi and bacterial communities in otomycosis were identified simultaneously by mNGS. Most researchers from different countries indicatedthat A. nigerserved as the most commonotomycosis agent\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR23 CR24\\\" citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u003c/sup\\u003e. However, through mNGS, we found that the most common fungus in otomycosis was Aspergillus terreus, which was consistent with the study of Zhang et al.\\u003csup\\u003e\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e in Hangzhou, China. This indicates that the fungal community of otomycosis may vary in different region. In western China, Aspergillus tubingensiswas the most common fungi, followed by Aspergillus fumigatus and Aspergillus terreus\\u003csup\\u003e\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e.Studies in Iran had found that Aspergillus flavus was the most common species of otomycosis, followed by Aspergillus tubingensis and Aspergillus niger\\u003csup\\u003e\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u003c/sup\\u003e, while Aspergillus awamori was considered to be the most common fungi causing otomycosis in studies in southern Hungary\\u003csup\\u003e\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u003c/sup\\u003e. In India, Aspergillus niger and Aspergillus fumigatus were dominant in otomycosis\\u003csup\\u003e\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e\\u003c/sup\\u003e.Candida albicans had also been reported as a common pathogen of otomycosis, which mainly occurred in patients with abnormal immune function\\u003csup\\u003e\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e\\u003c/sup\\u003e. To date, however, the bacterial community in otomycosis has been studied sparingly. In the present study, we found through mNGS that the most common bacteria in otomycosis patients was Staphylococcus aureus.At the same time, staphylococcus aureus is the main pathogen of bacterial external otitis, which can proliferate and infect the human body, leading to inflammation when human immunity is reduced and the environment of the ear canal changes\\u003csup\\u003e\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eCurrently, the treatment of otomycosis currently relies mainly on topical use of antifungal drugs. Terbinafine is more effective against partial Aspergillus species in vitro than itraconazole or amphotericin B\\u003csup\\u003e\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e\\u003c/sup\\u003e. Meanwhile, terbinafine is considered to be an effective drug in the treatment of otomycosis\\u003csup\\u003e\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. It has been reported that the cure rate of Terbinafine in the treatment of otomycosis reaches 100%\\u003csup\\u003e32\\u003c/sup\\u003e, while, in this study, 14 of the 17 patients treated with Terbinafine have been cured, with a cure rate of 82.4%, which might be related to the increasingly severe fungal resistance at present. It is worth noting that Aspergillus is the main cause of cured cases in our study, beingconsistent with the above report\\u003csup\\u003e\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u003c/sup\\u003e. Some Malassezia strains have been reported to be resistant to terbinafine\\u003csup\\u003e\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u003c/sup\\u003e. We found that the fungal community dominated by Malassezia has resistance to terbinafine, and the precise mechanism of resistance needs to be further studied.\\u003c/p\\u003e \\u003cp\\u003eIn summary, the present study identifies by means of mNGSthat the most common fungal species among patients with otomycosis in the city of Jinan, Shandong Province, of the People\\u0026rsquo;s Republic of China is Aspergillus terreus, and the most common bacterial species is Staphylococcus aureus. Fungi dominated by Malassezia are more resistant to terbinafine than fungi dominated by Aspergillus. Findings from this work have certain guiding significance for the clinical treatment of otomycosis with terbinafine. The specific mechanism underpinning drug resistance needs to be further researched.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u0026nbsp;\\u003c/strong\\u003eThis work was supported by the National Natural Science Foundation of China (No. 82071039) and the National Natural Science Foundation of China (No. 82101215\\u0026nbsp;）.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u0026nbsp;\\u003c/strong\\u003eThe authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions\\u0026nbsp;\\u003c/strong\\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Shuai Xu, Xin Zhang, Jianfeng Li and Zhaoyan Yu. The first draft of the manuscript was written by Shuai Xu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical Approval\\u0026nbsp;\\u003c/strong\\u003eThis research was approved by the Biomedical Research Ethics Committee of Shandong Provincial Hospital.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eGupta A K , Venkataraman M . Antifungal resistance in Superficial mycoses[J]. Journal of Dermatological Treatment, 2021:1-25.\\u003c/li\\u003e\\n\\u003cli\\u003eHazarika D, Jahan N, Sharma A. Changing Trend of Superficial Mycoses with Increasing Nondermatophyte Mold Infection: A Clinicomycological Study at a Tertiary Referral Center in Assam. Indian journal of dermatology. 2019;64:261-5.\\u003c/li\\u003e\\n\\u003cli\\u003eBrown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. Hidden killers: human fungal infections. Science translational medicine. 2012;4:165rv13.\\u003c/li\\u003e\\n\\u003cli\\u003eLee Y, Puumala E, Robbins N, Cowen LE. Antifungal Drug Resistance: Molecular Mechanisms in Candida albicans and Beyond. 2021;121:3390-411.\\u003c/li\\u003e\\n\\u003cli\\u003eNowrozi H, Arabi FD, Mehraban HG, Tavakoli A, GhooshchiG. Mycological and clinical study of otomycosis in Tehran,Iran. Bull Environ Pharmacol Life Sci. 2014;3(2):29\\u0026ndash;31.\\u003c/li\\u003e\\n\\u003cli\\u003eKoltsidopoulos P, Skoulakis C. Otomycosis With Tympanic Membrane Perforation: A Review of the Literature. Ear, nose, \\u0026amp; throat journal. 2020;99:518-21.\\u003c/li\\u003e\\n\\u003cli\\u003eHagiwara S, Tamura T, Satoh K, Kamewada H, Nakano M, Shinden S, et al. The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi, Japan. Mycopathologia. 2019;184:13-21.\\u003c/li\\u003e\\n\\u003cli\\u003eKiakojuri K, Mahdavi Omran S, Roodgari S, Taghizadeh Armaki M, Hedayati MT, Shokohi T, et al. Molecular Identification and Antifungal Susceptibility of Yeasts and Molds Isolated from Patients with Otomycosis. 2021;186:245-57.\\u003c/li\\u003e\\n\\u003cli\\u003eKamali Sarwestani Z, Hashemi SJ, Rezaie S, Gerami Shoar M, Mahmoudi S, Elahi M, et al. Species identification and in vitro antifungal susceptibility testing of Aspergillus section Nigri strains isolated from otomycosis patients. Journal de mycologie medicale. 2018;28:279-84.\\u003c/li\\u003e\\n\\u003cli\\u003eAboutalebian S, Mahmoudi S, Mirhendi H, Okhovat A, Abtahi H, Chabavizadeh J. Molecular epidemiology of otomycosis in Isfahan revealed a large diversity in causative agents. Journal of medical microbiology. 2019;68:918-23.\\u003c/li\\u003e\\n\\u003cli\\u003eZhang S, Jin M, Hu S, Zhang Y, Zhou G. Administration of 1% topical voriconazole drops was effective and safe in the treatment of refractory otomycosis without tympanic membrane perforation. The Annals of otology, rhinology, and laryngology. 2021;130:273-9.\\u003c/li\\u003e\\n\\u003cli\\u003eJia X, Liang Q, Chi F, Cao W. Otomycosis in Shanghai: aetiology, clinical features and therapy. Mycoses. 2012;55:404-9.\\u003c/li\\u003e\\n\\u003cli\\u003eAli K, Hamed MA, Hassan H, Esmail A, Sheneef A. Identification of Fungal Pathogens in Otomycosis and Their Drug Sensitivity: Our Experience. International archives of otorhinolaryngology. 2018;22:400-3.\\u003c/li\\u003e\\n\\u003cli\\u003eCampbell CK, Johnson EM, Warnock DW. Identification of Pathogenic Fungi: Identification of Pathogenic Fungi; 2013.\\u003c/li\\u003e\\n\\u003cli\\u003eAnwar K, Gohar MS. Otomycosis; clinical features, predisposing factors and treatment implications. Pakistan journal of medical sciences. 2014;30:564-7.\\u003c/li\\u003e\\n\\u003cli\\u003eBarati B, Okhovvat SA, Goljanian A, Omrani MR. Otomycosis in central iran: a clinical and mycological study. Iranian Red Crescent medical journal. 2011;13:873-6.\\u003c/li\\u003e\\n\\u003cli\\u003ePrasad SC, Kotigadde S, Shekhar M, Thada ND, Prabhu P, T DS, et al. Primary otomycosis in the Indian subcontinent: predisposing factors, microbiology, and classification. International journal of microbiology. 2014;2014:636493.\\u003c/li\\u003e\\n\\u003cli\\u003eVennewald I, Klemm E. Otomycosis: Diagnosis and treatment. Clinics in dermatology. 2010;28:202-11.\\u003c/li\\u003e\\n\\u003cli\\u003eSzigeti G, Kocsub\\u0026eacute; S, D\\u0026oacute;czi I, Bereczki L, V\\u0026aacute;gv\\u0026ouml;lgyi C, Varga J. Molecular identification and antifungal susceptibilities of black Aspergillus isolates from otomycosis cases in Hungary. Mycopathologia. 2012;174:143-7.\\u003c/li\\u003e\\n\\u003cli\\u003eAboutalebian S, Ahmadikia K, Fakhim H, Chabavizadeh J, Okhovat A, Nikaeen M, et al. Direct detection and identification of the most common bacteria and fungi causing otitis externa by a stepwise multiplex PCR.Frontiers in Cellular and Infection Microbiology. 2021;11: 210. \\u003c/li\\u003e\\n\\u003cli\\u003eGu X, Cheng X, Zhang J, She W. Identification of the Fungal Community in Otomycosis by Internal Transcribed Spacer Sequencing. Frontiers in microbiology. 2022;13:820423.\\u003c/li\\u003e\\n\\u003cli\\u003eMoharram\\u0026sup1; AM, Ahmed\\u0026sup2; HE, Nasr\\u0026sup1; SA-M. Otomycosis in Assiut, Egypt. J Basic Appl Mycol (Egypt). 2013;4:1\\u0026ndash;11.\\u003c/li\\u003e\\n\\u003cli\\u003eجابر H. In Vitro Assessment Of Antifungal Potential Of Apple Cider Vinegar And Acetic Acid Versus Fluconazole In Clinical Isolates Of Otomycosis. 2011.\\u003c/li\\u003e\\n\\u003cli\\u003eGarc\\u0026iacute;a-Agudo L, Aznar-Mar\\u0026iacute;n P, Gal\\u0026aacute;n-S\\u0026aacute;nchez F, Garc\\u0026iacute;a-Martos P, Mar\\u0026iacute;n-Casanova P, Rodr\\u0026iacute;guez-Iglesias M. Otomycosis due to filamentous fungi. Mycopathologia. 2011;172:307-10.\\u003c/li\\u003e\\n\\u003cli\\u003eDeğerli K, Ecemiş T, G\\u0026uuml;nhan K, Başkesen T, Kal E. [Agents of otomycosis in Manisa region, Turkey, 1995-2011]. Mikrobiyoloji bulteni. 2012;46:79-84.\\u003c/li\\u003e\\n\\u003cli\\u003eZhang L, Wang X, Houbraken J, Mei H, Liao W, Hasimu H, et al. Molecular Identification and In Vitro Antifungal Susceptibility of Aspergillus Isolates Recovered from Otomycosis Patients in Western China. 2020;185:527-35.\\u003c/li\\u003e\\n\\u003cli\\u003eMofatteh MR, Naseripour Yazdi Z, Yousefi M, Namaei MH. Comparison of the recovery rate of otomycosis using betadine and clotrimazole topical treatment. Brazilian journal of otorhinolaryngology. 2018;84:404-9.\\u003c/li\\u003e\\n\\u003cli\\u003eViswanatha B, Sumatha D, Vijayashree MS. Otomycosis in immunocompetent and immunocompromised patients: comparative study and literature review. Ear, nose, \\u0026amp; throat journal. 2012;91:114-21.\\u003c/li\\u003e\\n\\u003cli\\u003eLi Q, Jiang HQ. [Clinical diagnosis and treatment progress of acute otitis externa]. Lin chuang er bi yan hou tou jing wai ke za zhi = Journal of clinical otorhinolaryngology, head, and neck surgery. 2016;30:1164-7.\\u003c/li\\u003e\\n\\u003cli\\u003eMoore CB, Walls CM, Denning DW. In vitro activities of terbinafine against Aspergillus species in comparison with those of itraconazole and amphotericin B. Antimicrobial agents and chemotherapy. 2001;45:1882-5.\\u003c/li\\u003e\\n\\u003cli\\u003eKurnatowski P, Filipiak A. Otomycosis: prevalence, clinical symptoms, therapeutic procedure. Mycoses. 2001;44:472-9.\\u003c/li\\u003e\\n\\u003cli\\u003eYang TH, Young YH. Eradicating Otomycosis with Terbinafine Solution: Basic and Clinical Investigation. Audiology \\u0026amp; neuro-otology. 2019;24:183-90.\\u003c/li\\u003e\\n\\u003cli\\u003eGupta AK, Kohli Y, Li A, Faergemann J, Summerbell RC. In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine. The British journal of dermatology. 2000;142:758-65.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"mycopathologia\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"myco\",\"sideBox\":\"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)\",\"snPcode\":\"11046\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11046/3\",\"title\":\"Mycopathologia\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Metagenomic Next Generation Sequencing, Otomycosis, Microbial community, Terbinafine resistance\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-2749783/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-2749783/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe present study was designed to identify the microbial community as well as to analyze its diversity bymeans of metagenomic Next Generation Sequencing(mNGS)in 17 patients with otomycosis treated with terbinafinein the Department of Otolaryngology of Shandong Provincial Hospital from June 2021 to June 2022, so as to evaluate the relationship between microbial community and terbinafine resistance. Those 17 patients were divided into two groups, i.e., Terbinafine Effective Group (TEG, n\\u0026thinsp;=\\u0026thinsp;14 cases) and Terbinafine Resistance Group(TRG, n\\u0026thinsp;=\\u0026thinsp;3 cases) according to the therapy effect, whose microbial community of secretion of external auditory canal (EAC)was identified using mNGS. We found that the sequence of bacteria was significantly more than that of fungi and, whereas, the difference between the two groups of bacteria was not significant.There were significant differences in fungal community between the two groups. Aspergillus was the main pathogenic fungus of TEG patients while Malassezia was a dominant fungus in TRG patients. In conclusion, the results from this work indicate that Aspergillus terreusis the main pathogenic fungus in this cohortof otomycosis patients and MNGS sequencing can offer comprehensive information about the microbial community of otomycosis. The fungus community dominated by Malassezia is more likely to be resistant to terbinafine, which provides certain guidance for clinical treatment of otomycosis with terbinafine.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Identification of Microbial Community in Otomycosis by mNGS: Potential implication for treatment of this disorder with terbinafine\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-04-04 16:27:46\",\"doi\":\"10.21203/rs.3.rs-2749783/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2023-04-06T18:20:32+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2023-04-01T09:52:13+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"Mycopathologia\",\"date\":\"2023-03-29T16:49:28+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2023-03-29T16:04:04+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Mycopathologia\",\"date\":\"2023-03-29T00:45:28+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"mycopathologia\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"myco\",\"sideBox\":\"Learn more about [Mycopathologia](https://www.springer.com/journal/11046)\",\"snPcode\":\"11046\",\"submissionUrl\":\"https://submission.nature.com/new-submission/11046/3\",\"title\":\"Mycopathologia\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"afd3a028-8b5a-46ef-8274-9fa634c1eeab\",\"owner\":[],\"postedDate\":\"April 4th, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-09-25T15:06:29+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-2749783\",\"link\":\"https://doi.org/10.1007/s11046-023-00791-5\",\"journal\":{\"identity\":\"mycopathologia\",\"isVorOnly\":false,\"title\":\"Mycopathologia\"},\"publishedOn\":\"2023-09-19 15:01:37\",\"publishedOnDateReadable\":\"September 19th, 2023\"},\"versionCreatedAt\":\"2023-04-04 16:27:46\",\"video\":\"\",\"vorDoi\":\"10.1007/s11046-023-00791-5\",\"vorDoiUrl\":\"https://doi.org/10.1007/s11046-023-00791-5\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-2749783\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-2749783\",\"identity\":\"rs-2749783\",\"version\":[\"v1\"]},\"buildId\":\"rHA-KDH7Qsr4HCuvH75dn\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}