Microbiological characterization of the gut in patients with sudden total deafness

Microbiological characterization of the gut in patients with sudden total deafness | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Microbiological characterization of the gut in patients with sudden total deafness Weiwei Jiang, Ming Yu, Zhengpeng Gong, Zhaolei Ma, Kai Song, Zongying Yang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3893609/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 Objective To investigate a differential analysis of the intestinal flora characteristics of individuals with sudden total deafness and a healthy control population, it will be possible to determine whether there is a link between intestinal flora and sudden total deafness. Methods Fresh stool samples were taken from 10 patients with sudden total deafness and 10 healthy subjects (family members of 10 patients with sudden total deafness) who were hospitalized at Baiyun Hospital of Guizhou Medical University between December 2021 and February 2022. All subjects' stool samples were then analyzed using 16S rRNA sequencing technology. Results We examined the differences in intestinal bacterial diversity between sudden total deafness and healthy normal patients using high-throughput sequencing technologies. The results of sample hierarchical clustering and principal coordinates analysis revealed that the sample flora structure varied between the sudden total deafness and normal control groups; multi-level species difference discriminant analysis and between-group difference analysis revealed that the intestinal flora of the 2 groups in the phylum Aspergillus ( P =0.034), Clostridium ( P = 0.003), with statistically significant differences between the 2 groups, but the relative absorption was similar between the 2 groups. The prevalence of Raoulia spp., Klebsiella spp., Caulobacter spp., Haemophilus spp., and Clostridium spp. was positively connected with sudden total deafness, according to a correlation study.At the genus level, five genera, including Porphyromonas spp., were considerably enriched in the entire deafness group, whereas only Clostridium spp. were significantly enriched in the normal control group, according to an LEfSe multilevel species hierarchy analysis.Four paths existed between the groups, according to PICRUSt functional prediction study.The four routes were considerably different at level 3 according to the results of the PICRUSt functional prediction analysis. Conclusion Patients with sudden total deafness and their families have dramatically different gut flora in terms of composition and functional pathways. Biological sciences/Biochemistry Biological sciences/Biological techniques sudden total deafness gut microbiota 16SrRNA PICRUSt. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Sudden sensorineural hearing loss (SSNHL) is a unilateral or bilateral sensorineural hearing loss with an unexplained hearing loss of at least two adjacent frequencies ≥ 20 dB (Decibel Hearing Level, dBHL) that occurs suddenly within 72 h [1] . In China, the prevalence of SSNHL is rising yearly.Its occurrence seriously disrupts the patients' regular professional and personal lives [2–3] . The occurrence of SSNHL is currently thought to be closely related to a number of factors, including inner ear ischemia, mental and psychological factors, viral infections, tumor lesions, drug intoxication, and autoimmune diseases [4-6] . This is because it is currently unknown what causes and how SSNHL works. There is no established and efficient course of treatment because the disease's mechanism and origin are unknown. Therefore, additional study is urgently required to clarify the precise pathophysiology and offer a foundation for clinical treatment. The intestinal flora refers to the collection of bacteria and other microorganisms that live in harmony with humans in the gastrointestinal tract, including bacteria, fungus, viruses, and archaea [7] . The microbial content of feces is 10 13 ~10 14 /g, of which 70% of microorganisms come from the colon, so the microbial composition of feces can be approximated as the microbial composition of the intestinal mucus layer, thus indirectly reflecting the colitis, The human intestinal flora is made up of about 35,000 different types of bacteria that are distributed throughout the respiratory tract, digestive system, skin surface, and even peripheral circulatory system [8] . In recent years, it has become a popular location for study.The composition of the intestinal microbial population is directly correlated with human health and disease states and is engaged in all areas of body metabolism, immunological illnesses, infectious diseases, and tumor growth and progression. Gut flora has been linked to epilepsy [9–10] , diabetes [11–12] , and Alzheimer's disease [13–14] in studies. Research has demonstrated a relationship between dysbiosis of the gut flora and ear health. By consuming oral gut probiotics, healthy children can lower their risk of otitis media, and children with poor gut health may also be more likely to get sensorineural hearing loss (SNHL) [15] . Hearing loss is linked to type 2 diabetes, diet-induced obesity, high-fat diets, and inflammatory bowel disease (IBD) [16] . Additionally, HFD might cause pathogenic alterations in the intestinal microbiota and have an impact on the intestinal A leaky gut can result from HFD, which may also cause pathological alterations in the intestinal microbiota and compromise the integrity of the intestinal barrier (IB). Chronic systemic inflammation that affects extraintestinal organs might then develop as a result. A systemic inflammatory response brought on by HFD and DIO's effects on the gut flora may compromise the permeability of the blood-vagus barrier (BLB) in the inner ear, causing cochlear inflammation and hearing loss. Our hypothesis was that the dysbiosis of the gut flora may have an impact on the onset and progression of SSNHL based on the aforementioned studies. In order to better understand the onset and progression of SSNHL and to provide a theoretical foundation for developing more effective treatments to aid in the restoration of hearing in patients with the condition, we used 16S rRNA high-throughput sequencing to analyze the gut microbial composition of patients with SSNHL and their families. 1 Material and methods All experimental protocols were approved by Committee of Baiyun Hospital affiliated to Guizhou Medical University,Informed consent has been obtained from all subjects and/or their legal guardians.all methods were performed in accordance with the relevant guidelines and regulations. 1.1 Research Subjects Ten patients were chosen, four men and six females, with an average age of (41±7.52) years, who had been diagnosed with sudden total deafness between December 2021 and February 2022 at Baiyun Hospital, Guizhou Medical University. Ten accompanying family members were chosen (normal control group), consisting of 5 males and 5 females, aged (46±8.13) years, who shared the same food and lifestyle as the patients. Between the two groups, there was no age or gender difference that was statistically significant (P>0.05). 1.2 Inclusion and exclusion criteria Patients with SSNHL who matched the diagnostic standards of the "Guidelines for the Diagnosis and Treatment of Sudden Deafness (2015)" created by the Chinese Medical Association in 2015 [1] were included in the experimental group. Age ranged from 30 to 60, and neither gender was excluded. Patients and their families all shared the same geographic location; none of the patients had recently used probiotic or prebiotic supplements, hormonal or injectable immunizations, or antibacterial or hormonal medications. neither smoking nor drinking has ever been a habit. Family members of patients with sudden total deafness who met the following criteria were included in the control group: never had a sudden deafness episode; lived in the same location as the patient; and had similar food habits.Patients with a clear history of chronic diseases, such as hypertension, diabetes, inflammatory bowel disease, irritable bowel syndrome, and other gastrointestinal diseases; patients and their families who signed an informed consent form; patients who had been treated with antibiotics, probiotics (or prebiotics), hormonal drugs, or had been injected with viral vaccines within 3 months. 1.3 Methodology 1.3.1 Fecal specimen collection Urine was emptied prior to defecation in order to prevent the sample from coming into contact with urine and affecting the test results. Stool specimen collection was based on the principle of immediate collection and storage (preservation immediately after sampling). About 2 g of stool was collected and kept in a sterile stool box after being deeply penetrated by a stool collection tube. Information such as name, label number, and sampling date were recorded when the samples were taken, and then it was promptly stored in liquid nitrogen at -196°C. All subjects' information was comprehensive, and after being verified as accurate, a complete information database was created. 1.3.2 Sample DNA extraction, library construction and, sequencing On all stool samples, DNA extraction and library building were done. To collect the raw sequencing data, sequencing was done using the Illumina Hiseq sequencing platform. To learn more about the intestinal flora and to describe it, bioinformatic data from all samples was evaluated. 1.3.3 Bioinformatics data analysis By double-end sequencing (Paired-end) of this project using the Illumina Hiseq sequencing platform, the raw data in fastq format were sequenced (samples correspond to a pair of sequences S_1.fastq and S_2.fastq). Then paired and spliced into single sequences. The samples corresponding to the sequences were identified by a barcode. The sequences were quality-controlled and filtered with QIIME software to remove chimeras and obtain valid sequences. The raw data in fastq format were sequenced utilizing double-end sequencing (Paired-end) for this project using the Illumina Hiseq sequencing platform (samples correspond to a pair of sequences S_1.fastq and S_2.fastq). the sequences were then linked together and joined. A barcode was used to distinguish the samples that corresponded to the sequences. Using QIIME software, the sequences were quality-controlled and filtered to get rid of chimeras and get legitimate sequences. In order to determine the values of the samples' Alpha diversity and create the appropriate dilution curves, a series of microbial community richness and diversity analyses were carried out at the gene level. R was used to depict the species cumulative box plot analysis. The alpha diversity of the samples was expressed using the Shannon index, and the higher the Shannon value, the more diverse the community. The similarity and differences between several samples in two-dimensional coordinates were displayed using principal coordinate analysis (PCoA) based on the bray-Curtis distance. The principal bacterial species in charge of the variations in the bacterial communities were identified using the similarity percentage method. The units that differed considerably at each taxonomic level were identified using the Lefse analysis. For function prediction, a phylogenetic analysis of the community employing unobserved state reconstruction (PICRUSt) was used. From gene information on OTUs in the Greengene database and for additional untested species, gene function profiles were deduced. 1.4 Statistical methods Software named SPSS 24.0 was utilized for the statistical evaluation. The analysis of categorical variables was conducted using the chi-square test, and the Student's t-test was used to compare the means of the two groups. Standard deviation (SD) was used to describe the measurement data as mean (mean). The association between gut flora and abrupt deafness was examined using Spearman's correlation methodology. P＜0.05 was regarded as a significant value. Correlation coefficients |r| between 0.8 and 1.0 denoted very strong correlation, 0.6 and 0.8, strong correlation, 0.4 and 0.6, moderate correlation, 0.2 and 0.4, weak connection, and 0.0 and 0.2, very weak or no association. 2 Results 2.1 Statistics of sequencing results The sequencing of a total of 10 patients who experienced abrupt total deafness and 10 healthy controls yielded raw sequencing data with reads ranging from 32,445,682 to 50,972,477 samples and a mean of 41,037,118. A non-redundant gene collection was created using de novo assembly, gene structure prediction, and clustering after sequencing results underwent quality check. The Venn diagram of the genes between the two groups is shown in Fig. 1. The Venn diagrams show that there are variations in the fecal sample gene expression between the group experiencing sudden total deafness and the healthy control group. 2.2 Bacterial flora composition analysis To determine the relative abundance, the intestinal flora of each study participant was examined at every level, from phylum to genus. The results showed that the intestinal flora of the sudden total deafness group and the healthy control group contained a total of 10 phyla and 118 genera, with statistically significant differences between the 2 groups for the phyla Methylobacteria (P = 0.034) and Fusobacteria (P = 0.003). However, the phylum Fusobacteria had a low relative abundance in the structure of the intestinal flora; phylum Thick-wal With the exception of the phylum Aspergillus, there was no statistically significant difference in the relative abundance of the dominating phylum between the two groups (P > 0.05). Romboutsia, Lactobacillus, Lactococcus, Escherichia coli, Shigella, Blautia, Streptococcus, and Bifidobacterium were the top 8 bacteria in terms of relative abundance in the sudden total deafness group, and there was no statistically significant difference between the 2 groups (P > 0.05). (Fig. 2B) Alpha diversity analysis Twenty samples were sequenced, and 56 OTUs were identified with sequencing coverage of > 99.97% and a classification level of 97% similarity. The amount of sequencing data was reasonable and could adequately reflect the majority of information on the variety of the flora in the samples, as shown by the community richness index's tendency to be flat for each sample.(Fig. 3). The Shannon, Chao1, and Simpson indices of the alpha diversity analysis did not substantially differ between the two groups of samples (P = 0.871, P = 0.433, and P = 1.000, respectively), showing that the diversity of the flora was identical in both sets of samples. (Table 1) Table 1 Differences in alpha diversity index based on t-test Group Shannon Index Simpson Index Chao1 Index Sudden Total Deafness Group 3.53 ± 0.46 0.90 ± 0.38 911.76 ± 89.39 Normal control group 3.19 ± 1.08 0.78 ± 0.24 785.74 ± 187.28 P -value P = 0.871 P = 1.000 P = 0.433 Beta diversity analysis Samples from the groups with sudden total deafness and the normal control group were clustered independently, according to PCoA (Fig. 4). The combined -diversity analysis revealed that there were differences in the structure of the flora in the 2 sets of samples because the first principal component contributed 17.02% and the second principal component contributed 15.15% to the difference in the sample flora's structure. 2.3 Bacterial flora variation analysis Figure 5A of the LEfSe multilevel species hierarchical analysis revealed that the phylum Anaplasma was significantly enriched in the sudden total deafness group, whereas Clostridium was significantly enriched in the normal control group. At the genus level, 5 genera, including Porphyromonas, were significantly enriched in the sudden total deafness group. Together with the test for the significance of differences between groups, the LDA (LDA threshold > 2.0) (Fig. 5B) revealed that the samples from the two groups did not differ significantly. Clostridium spp, Trichosporon spp, Microbacterium spp, Clostridium faecium spp, Raoul spp, Klebsiella spp, Caulobacter spp, Aminococcus spp, Haemophilus spp, Pasteurella spp, Aspergillus spp, Lactobacillus spp, and 12 other species(p < 0.05) were among those that significantly. 2.4 Correlation analysis between intestinal flora According to Table 2, Spearman's correlation study revealed that Raoulia, Klebsiella, Caulobacter, Haemophilus, and Pachybotrys species were all positively connected with sudden total deafness. Table 2 Analysis of the correlation between microorganisms and sudden total deafness. Microorganisms r p Trichoderma spp. −0.434 0.056 Raoulia spp. 0.926 0.000 Klebsiella spp. 0.712 0.000 Corynebacterium spp. 0.712 0.000 Haemophilus spp. 0.624 0.003 Lactobacillus spp. 0.225 0.339 Genus parabacteroides 0.416 0.068 Phyllococcus spp. 0.503 0.024 2.5 Functional Prediction of gut microbes in the sudden total deafness and control groups Utilizing PICRUSt, the functional prediction was carried out. In the group with sudden total deafness, the penicillin and cephalosporin biosynthesis pathway, the phosphatidylinositol metabolic pathway, the phosphatidylinositol signaling pathway, and the progesterone-mediated biosynthesis pathway were all enriched, whereas the proteasome metabolism, antigen processing and presentation, and aminobenzoic acid degradation were enriched in the control group (Fig. 6). 3 Discussion Intestinal flora has attracted the attention of researchers both domestically and internationally in recent years, and a large number of studies have demonstrated that the effects of an imbalance in intestinal microbial-host interactions on endocytosis can contribute to extraintestinal disease progression in addition to intestinal inflammation. Studies conducted in the United States and abroad have revealed that issues with the intestinal flora can also cause vascular blood flow problems, ear neuropathy, and sensorineural hearing loss. We posited the possibility that gut flora might affect the development of SSNHL in conjunction with the reading of the literature.In order to test this theory, we carried out high-throughput sequencing of the gut microbiota's 16sRNA in the stools of patients with sudden total deafness and their families. The results revealed differences in genus-level abundance between the sudden total deafness group and the control group, but they did not reveal the precise number of bacteria present. We propose that the sudden total deafness activity is closely related to the bacterial species. Therefore, we explained how the bacterial species changed in both groups. Aspergillus and Clostridium were found in greater abundance in the sudden total deafness group than in the control group, as were 12 genera, including Clostridium spp., Trichosporon spp., Colletotrichum spp., and Clostridium faecalis, at the genus level. At the species level, Lactobacillus and Clostridium spp. were closely related to sudden deafness.Raoulia spp., Klebsiella spp., Corynebacterium spp., Haemophilus spp., and Clostridium perfringens spp. were all substantially and positively connected with abrupt total deafness, according to Spearman's correlation analysis. Patients with abrupt total deafness had significantly higher levels of the phylum Aspergillus, which was statistically significant. Numerous hazardous bacteria, including Escherichia coli, Salmonella spp., Helicobacter pylori, and other well-known genera, are members of the phylum Aspergillus. The phylum Aspergillus contained the genera Raoulia and Klebsiella in the intestinal bacteria of patients with sudden complete deafness in this investigation. All members of the phylum Aspergillus are Gram-negative bacteria, and lipopolysaccharide (LPS) makes up the majority of their outer membranes. The gut is shielded in the intestinal flora of healthy people by an outer mucus layer, anti-inflammatory microbial compounds, and immune system components that have a dual role in generating and reducing inflammation.Pro-inflammatory bacteria in the gut can produce endotoxins, and in people with intestinal flora dysbiosis, a significant rise in Gram-negative bacteria and an increase in lipopolysaccharide (LPS)-producing bacteria can be seen[17,18]. LPS in the intestinal lumen enters the circulation, increasing intestinal permeability [ 19 – 23 ] , and high intestinal permeability can cause toxic substances to leak through the brain-intestinal axis to the brain, where they can According to studies using animal models, hearing loss is correlated with a rise in gram-negative bacteria in the diseased gastrointestinal tract and blood levels of LPS, which binds to the toll-like receptor 4 (TLR4) on immune cellsand activates an inflammatory response [ 24 ] ,increases the permeability of the intestinal barrier, which makes it easier for microorganisms to enter the inner ear and causes the growth of pathogenic bacteria and focal metastasis, further aggravating the inflammatory response. Additionally, it induces the transcription factor NF-B in macrophages and stimulates the release of inflammatory cytokines like interleukin 1 (IL−1) and tumor necrosis factor (TNF) [ 25 , 26 ] . As a result, we speculate that different Aspergillus species may contribute to the onset of SSNHL by influencing intestinal permeability and creating a lot of LPS. The prevalence of Haemophilus influenzae is increased and strongly connected with the occurrence of sudden complete deafness in patients with this condition. Harmes KM [ 27 – 28 ] et al. noted that Haemophilus influenzae was isolated in large numbers from cultures of middle ear fluid from patients with acute otitis media and that Haemophilus influenzae has become one of the major causes of vaccination in patients with pneumococcal disease. Haemophilus influenzae is also a cause of chronic infection and acute otitis media in children, as well as hearing loss. The main reason for the rise in acute otitis media due to the increased use of antibiotics is resistance to beta-lactams and other antibiotics. According to a prior study, Haemophilus colonizes the respiratory tract's epithelial cells through adhesins and induces inflammation that impairs hearing. Adhesins also have supplementary virulence effects, which may also be a factor in the development of SSNHL. These effects include preventing immune clearance, altering pore size to prevent antimicrobial damage, establishing microcolonies, and inducing protein-mediated phase shifts [ 29 ] . According to correlation research, sudden total deafness was also strongly related with the genus Lactococcus. A species of cocci called Lactococcus lamellaris belongs to the Lactobacillus family and order. Lactobacillus, a probiotic commonly found in yogurt and other fermented dairy products, belongs to the thick-walled phylum that produces acetate, lactate, and antimicrobial substances that prevent pathogens from interfering with health. There was a significant increase in the abundance of Lactobacillus at the species level of this study in patients with sudden deafness. Short-chain fatty acids (SCFAs), including acetate, are created by the fermentation of dietary fiber and resistant starch in the colon. These SCFs are involved in host metabolism, immunological response, and gastrointestinal physiology. Prebiotic FOS, as demonstrated by Kondo T et al. [ 30 ] , increased the production of short-chain fatty acids (SCFAs). This resulted in a notable rise in SCFAs in the cecum and a decline in oxidative stress indicators in the serum, as well as better hearing in mice. When histones are deacetylated by the enzyme histone deacetylase (HDAC), they can bind strongly to negatively charged DNA and stop gene transcription. As ligands for HDAC inhibitors, SCFAs, as ligands for HDAC inhibitors, stimulate monocytes and neutrophils by inducing HDAC inhibition, leading to NF-κB inactivation, reducing pro-IL-2, IL-6, tumor necrosis factor -SCFAs can also promote the cytosolic transport of intestinal epithelial cells (IECs), increase the secretion of IgA into the intestinal lumen, increase the content of sIgA in the intestinal mucosa, agglutinate and adhere bacteria to the mucus, prevent direct contact between bacteria and the surface of intestinal epithelial cells, and prevent the invasion and infection of bacteria. It was found that expression of intestinal tight junction proteins was significantly reduced in germ-free mice, leading to increased blood-brain barrier permeability; treatment with complex microorganisms or SCFAs restored the integrity of the blood-brain barrier, for example, treatment of cerebrovascular endothelial cells with propionate attenuated the permeability of exposed lipopolysaccharide (LPS) and increased blood-brain barrier function. In order to minimize generated neuroinflammatory reactions, we predicted that C. lamellar and its related metabolites would shield the blood-brain barrier. Our data showed a positive correlation between lamellococci and the occurrence of sudden total deafness as opposed to a poor correlation, which may have been caused by a connection with a smaller sample size and diverse demographics. In order to better define the gut microbiota in patients with abrupt SSNHL, we will increase the size of our sample. Our research also revealed a rise in Clostridium difficile species abundance in the gut flora of patients with sudden complete deafness. The Clostridium difficile toxin B (TcdB) is the main pathogenic "weapon" of this anaerobic, Gram-positive bacterium, commonly known as Clostridium difficile. The most typical cause of sensorineural hearing loss is damage to the cochlear hair cells. In comparison to the Corti apparatus treated with C. difficile toxin B and gentamicin for 72 hours, Bodmer D [ 31 ] et al. treated the Corti apparatus of the rat cochlea with C. difficile toxin B for 12 hours. C. difficile toxin B treatment significantly reduced the mortality of cochlear hair cells. According to this, C. difficile toxin B may be able to protect auditory hair cells from aminoglycoside toxicity and restore their ability to hear. The usage of Clostridium difficile in SSNHL has not been documented, however it may contribute to hearing loss in drug-induced deafness. According to PICRUSt analysis, sudden total deafness may be closely correlated with the upregulation of certain metabolic pathways, including the phosphatidylinositol metabolic pathway, the phosphatidylinositol signaling pathway, the penicillin and cephalosporin biosynthesis pathway, and progesterone-mediated biosynthesis. An organic osmolyte and volume modulator, inositol. Modifications in the vestibular epithelium and basement membrane's micromechanical coupling due to alterations in the inner ear's osmotic pressure or hair cells may result in pathological changes in sensory transduction. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) to the second messenger's inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (DG), and in the guinea pig corti organ, the InsP3 second messenger system is associated with muscarinic cholinergic receptors and purinergic receptors, affecting hearing in guinea pigs. Purinergic receptor activation and ATP release from the cochlear epithelium also have a significant impact on the cochlea's physiology. Purinergic P2X and P2Y receptors are widely distributed in the inner and outer hair cells as well as in the Corti apparatus supporting cells, and they both have an impact on the development of hearing by changing the sensitivity of the receptor cells to K +. Additionally, purinergic signaling through purinergic receptors can affect hearing sensitivity and cochlear blood flow in noise-induced hearing impairment, which is consistent with the findings of our study. Accordingly, we hypothesize that the phosphatidylinositol metabolic pathway and phosphatidylinositol signaling pathway may be one of the pathogenic mechanisms of SSNHL. Our study has several limitations. First, this is a correlational study, and there is no proof that abnormally high levels of specific gut flora are the direct cause of SSNHL, even though we employed 16S rRNA PICRUSt functional prediction to examine potential pathways of the effect of gut flora on sudden complete deafness. Therefore, the next step will be to increase the sample size and further describe the pathophysiological mechanisms underlying SSNHL utilizing bigger sample size studies based on proteomics, metabolomics, and macroeconomics. Further research on this mechanism will be done in the following study because, in the previous study, the group with sudden total deafness was significantly enriched in five genera, including Porphyromonas, at the genus level, while the normal control group was only significantly enriched in Clostridium. Conclusion In conclusion, patients with sudden total deafness and healthy controls have significantly different gut microbial compositions. Potential risk factors for abrupt deafness include Raoulia species, Klebsiella species, Haemophilus species, Clostridium species, Lactobacillus species, and the phosphatidylinositol signaling and metabolic pathways. Overall, the distinctive alterations in the gut flora and metabolic pathways found in people who have experienced sudden total deafness offer new research areas for the pathophysiology and treatment of these conditions. Declarations Author Contribution WWJ and ZYY caried out most of the experiments and drafted the manuscript.KS、ZLM and XLZ did parts of the experiments and performed the statistical analysis;MY and ZPG conceived of hte study and revised the manuscript. All authors reviewed the manuscript. References Editorial Board of Chinese Journal of Otolaryngology, Head and Neck Surgery, Chinese Medical Association, Division of Otolaryngology, Head and Neck Surgery. Guidelines for the diagnosis and treatment of sudden deafness (2015) [J]. 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Prevalence of antimicrobial-resistant pathogens in middle ear fluid: a multinational study of 917 children with acute otitis media. Antimicrob Agents Chemother. 1998 Mar;42(3):589-95. Osman KL, Jefferies JM, Woelk CH, Cleary DW, Clarke SC. The adhesins of non-typeable Haemophilus influenza. Expert Rev Anti Infect Ther. 2018 Mar. 16(3):187-196. Kondo T, Saigo S, Ugawa S, Kato M, Yoshikawa Y, Miyoshi N, Tanabe K. Prebiotic effect of fructooligosaccharides on the inner ear of DBA/2 J mice with early-onset progressive hearing loss. J Nutr Biochem. 2020 Jan;75:108247. Bodmer D, Brors D, Pak K, Gloddek B, Ryan A. Rescue of auditory hair cells from aminoglycoside toxicity by Clostridium difficile toxin B, an inhibitor Hear Res. 2002 Oct;172(1-2):81-6. Additional Declarations No competing interests reported. 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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-3893609","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":270427650,"identity":"4726c75b-adc9-4f31-a5f1-fd45f8de9e03","order_by":0,"name":"Weiwei Jiang","email":"","orcid":"","institution":"Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Jiang","suffix":""},{"id":270427651,"identity":"29b5eb17-ac23-4364-afa0-d610f76b6368","order_by":1,"name":"Ming Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYJCCAwwM/+XsjzcfOPDhB/FamI0ZzhxLPDizh3iLmBMbbuQYH+ZgI0KtwY3sxMMFv9gYGxtyPhxm4GGQ5xc7QEhL7obDM/t4mJkZzm44XGDBYDhzdgIRWnh7JNjYGHs3HJ7Bw5BgcJs4LQY8PMw8Dw7zsBGrhedHgoQEGw8DcVokz7wF2tJwwMCAh80AGMgShP3Cdzx382eePwfqN8g/fvzhww8beX5pAloUDgAJxjY4XwK/chCQbwCRfwgrHAWjYBSMghEMACXJTf3yl6fVAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Guizhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ming","middleName":"","lastName":"Yu","suffix":""},{"id":270427652,"identity":"f1698604-ce27-4802-8d07-e7ab1efdfb95","order_by":2,"name":"Zhengpeng Gong","email":"","orcid":"","institution":"Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhengpeng","middleName":"","lastName":"Gong","suffix":""},{"id":270427653,"identity":"d7d431a3-fcba-447e-a709-c5c67465d6a8","order_by":3,"name":"Zhaolei Ma","email":"","orcid":"","institution":"Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhaolei","middleName":"","lastName":"Ma","suffix":""},{"id":270427654,"identity":"2cae4aba-4433-477f-a281-d83dbe03300b","order_by":4,"name":"Kai Song","email":"","orcid":"","institution":"Affiliated Hospital of Guizhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Song","suffix":""},{"id":270427655,"identity":"12b26e3f-3dae-4e93-8c95-c13b49ca0a35","order_by":5,"name":"Zongying Yang","email":"","orcid":"","institution":"Guizhou Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zongying","middleName":"","lastName":"Yang","suffix":""},{"id":270427656,"identity":"0b8bb590-fbbe-4b00-87f1-e26f9175b70a","order_by":6,"name":"Xianli Zhang","email":"","orcid":"","institution":"guizhou","correspondingAuthor":false,"prefix":"","firstName":"Xianli","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-01-24 09:29:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3893609/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3893609/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50670442,"identity":"82aedb39-6dbd-4822-be60-2ad040e9cc7d","added_by":"auto","created_at":"2024-02-05 14:35:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11210,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of gene expression in fecal samples from the sudden total deafness group and normal control group\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3893609/v1/53f7bcc4d83e1c4f05588f94.jpg"},{"id":50670443,"identity":"0ebd505f-e891-4503-82e6-5b21b48931dc","added_by":"auto","created_at":"2024-02-05 14:35:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56001,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance composition of sudden total deafness and normal control groups at the phylum and genus levels\u003c/p\u003e\n\u003cp\u003eA. Relative abundance of flora at the phylum level; B. Relative abundance of flora at the genus level.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3893609/v1/634a3cc986c8d353e0144ebb.jpg"},{"id":50671573,"identity":"5b03b00a-934b-40e3-ab27-9b9501a9281e","added_by":"auto","created_at":"2024-02-05 14:43:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27438,"visible":true,"origin":"","legend":"\u003cp\u003eDilution curve of α-diversity index\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3893609/v1/6a3b1f47a0f0cf2106b99652.jpg"},{"id":50671572,"identity":"1a422c62-ba6b-4dad-8c7d-7f5ba83b59ef","added_by":"auto","created_at":"2024-02-05 14:43:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33778,"visible":true,"origin":"","legend":"\u003cp\u003eSample distance PCoA results based on colony calculation\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3893609/v1/adc387af4fbbfad66e0f4c5e.jpg"},{"id":50670446,"identity":"ee2c5334-e218-498f-9cf5-3a149bd5dab5","added_by":"auto","created_at":"2024-02-05 14:35:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":97694,"visible":true,"origin":"","legend":"\u003cp\u003eLEFSe analysis of the difference in flora in the sudden total deafness group and normal control group\u003c/p\u003e\n\u003cp\u003eA. LEfSe multilevel species hierarchical analysis; B. LDA.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3893609/v1/2e6f0c26b61faa7642da176f.jpg"},{"id":50670445,"identity":"e44d892d-4714-43f5-ad0f-e3c5e7527d55","added_by":"auto","created_at":"2024-02-05 14:35:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":66963,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential analysis of the third level of classification of the KEGG metabolic pathway\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3893609/v1/25d8dfae3f871571e2c787ba.jpg"},{"id":57750239,"identity":"9e68c995-baa9-42bf-96c2-aa9b4c22942a","added_by":"auto","created_at":"2024-06-05 06:54:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":688155,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3893609/v1/b059b14c-53bf-4a6a-b793-535eb1fca43b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Microbiological characterization of the gut in patients with sudden total deafness","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSudden sensorineural hearing loss (SSNHL) is a unilateral or bilateral sensorineural hearing loss with an unexplained hearing loss of at least two adjacent frequencies \u0026ge; 20 dB (Decibel Hearing Level, dBHL) that occurs suddenly within 72 h\u003csup\u003e[1]\u0026nbsp;\u003c/sup\u003e. In China, the prevalence of SSNHL is rising yearly.Its occurrence seriously disrupts the patients\u0026apos; regular professional and personal lives \u003csup\u003e[2\u0026ndash;3]\u003c/sup\u003e. The occurrence of SSNHL is currently thought to be closely related to a number of factors, including inner ear ischemia, mental and psychological factors, viral infections, tumor lesions, drug intoxication, and autoimmune diseases\u003csup\u003e[4-6]\u003c/sup\u003e. This is because it is currently unknown what causes and how SSNHL works. There is no established and efficient course of treatment because the disease\u0026apos;s mechanism and origin are unknown. Therefore, additional study is urgently required to clarify the precise pathophysiology and offer a foundation for clinical treatment.\u003c/p\u003e\n\u003cp\u003eThe intestinal flora refers to the collection of bacteria and other microorganisms that live in harmony with humans in the gastrointestinal tract, including bacteria, fungus, viruses, and archaea\u003csup\u003e[7]\u003c/sup\u003e. \u0026nbsp;The microbial content of feces is 10\u003csup\u003e13\u003c/sup\u003e ~10\u003csup\u003e14\u003c/sup\u003e /g, of which 70% of microorganisms come from the colon, so the microbial composition of feces can be approximated as the microbial composition of the intestinal mucus layer, thus indirectly reflecting the colitis, The human intestinal flora is made up of about 35,000 different types of bacteria that are distributed throughout the respiratory tract, digestive system, skin surface, and even peripheral circulatory system\u003csup\u003e[8]\u003c/sup\u003e. In recent years, it has become a popular location for study.The composition of the intestinal microbial population is directly correlated with human health and disease states and is engaged in all areas of body metabolism, immunological illnesses, infectious diseases, and tumor growth and progression. Gut flora has been linked to epilepsy\u003csup\u003e[9\u0026ndash;10]\u003c/sup\u003e, diabetes\u003csup\u003e[11\u0026ndash;12]\u003c/sup\u003e, and Alzheimer\u0026apos;s disease\u003csup\u003e[13\u0026ndash;14]\u003c/sup\u003e in studies. Research has demonstrated a relationship between dysbiosis of the gut flora and ear health. By consuming oral gut probiotics, healthy children can lower their risk of otitis media, and children with poor gut health may also be more likely to get sensorineural hearing loss (SNHL)\u003csup\u003e[15]\u003c/sup\u003e. Hearing loss is linked to type 2 diabetes, diet-induced obesity, high-fat diets, and inflammatory bowel disease (IBD)\u003csup\u003e[16]\u003c/sup\u003e. Additionally, HFD might cause pathogenic alterations in the intestinal microbiota and have an impact on the intestinal A leaky gut can result from HFD, which may also cause pathological alterations in the intestinal microbiota and compromise the integrity of the intestinal barrier (IB). Chronic systemic inflammation that affects extraintestinal organs might then develop as a result. A systemic inflammatory response brought on by HFD and DIO\u0026apos;s effects on the gut flora may compromise the permeability of the blood-vagus barrier (BLB) in the inner ear, causing cochlear inflammation and hearing loss.\u003c/p\u003e\n\u003cp\u003eOur hypothesis was that the dysbiosis of the gut flora may have an impact on the onset and progression of SSNHL based on the aforementioned studies. In order to better understand the onset and progression of SSNHL and to provide a theoretical foundation for developing more effective treatments to aid in the restoration of hearing in patients with the condition, we used 16S rRNA high-throughput sequencing to analyze the gut microbial composition of patients with SSNHL and their families.\u003c/p\u003e"},{"header":"1 Material and methods","content":"\u003cp\u003eAll experimental protocols were approved by Committee of Baiyun Hospital affiliated to Guizhou Medical University,Informed consent has been obtained from all subjects and/or their legal guardians.all methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e1.1 Research Subjects\u003c/p\u003e\n\u003cp\u003eTen patients were chosen, four men and six females, with an average age of (41\u0026plusmn;7.52) years, who had been diagnosed with sudden total deafness between December 2021 and February 2022 at Baiyun Hospital, Guizhou Medical University. Ten accompanying family members were chosen (normal control group), consisting of 5 males and 5 females, aged (46\u0026plusmn;8.13) years, who shared the same food and lifestyle as the patients. Between the two groups, there was no age or gender difference that was statistically significant (P\u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003e1.2 Inclusion and exclusion criteria\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePatients with SSNHL who matched the diagnostic standards of the \u0026quot;Guidelines for the Diagnosis and Treatment of Sudden Deafness (2015)\u0026quot; created by the Chinese Medical Association in 2015\u003csup\u003e[1]\u003c/sup\u003e were included in the experimental group. Age ranged from 30 to 60, and neither gender was excluded. Patients and their families all shared the same geographic location; none of the patients had recently used probiotic or prebiotic supplements, hormonal or injectable immunizations, or antibacterial or hormonal medications. neither smoking nor drinking has ever been a habit. Family members of patients with sudden total deafness who met the following criteria were included in the control group: never had a sudden deafness episode; lived in the same location as the patient; and had similar food habits.Patients with a clear history of chronic diseases, such as hypertension, diabetes, inflammatory bowel disease, irritable bowel syndrome, and other gastrointestinal diseases; patients and their families who signed an informed consent form; patients who had been treated with antibiotics, probiotics (or prebiotics), hormonal drugs, or had been injected with viral vaccines within 3 months.\u003c/p\u003e\n\u003cp\u003e1.3 Methodology\u003c/p\u003e\n\u003cp\u003e1.3.1 Fecal specimen collection\u003c/p\u003e\n\u003cp\u003eUrine was emptied prior to defecation in order to prevent the sample from coming into contact with urine and affecting the test results. Stool specimen collection was based on the principle of immediate collection and storage (preservation immediately after sampling). About 2 g of stool was collected and kept in a sterile stool box after being deeply penetrated by a stool collection tube. Information such as name, label number, and sampling date were recorded when the samples were taken, and then it was promptly stored in liquid nitrogen at -196\u0026deg;C. All subjects\u0026apos; information was comprehensive, and after being verified as accurate, a complete information database was created.\u003c/p\u003e\n\u003cp\u003e1.3.2 Sample DNA extraction, library construction and, sequencing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn all stool samples, DNA extraction and library building were done. To collect the raw sequencing data, sequencing was done using the Illumina Hiseq sequencing platform. To learn more about the intestinal flora and to describe it, bioinformatic data from all samples was evaluated.\u003c/p\u003e\n\u003cp\u003e1.3.3 Bioinformatics data analysis\u003c/p\u003e\n\u003cp\u003eBy double-end sequencing (Paired-end) of this project using the Illumina Hiseq sequencing platform, the raw data in fastq format were sequenced (samples correspond to a pair of sequences S_1.fastq and S_2.fastq). Then paired and spliced into single sequences. The samples corresponding to the sequences were identified by a barcode. The sequences were quality-controlled and filtered with QIIME software to remove chimeras and obtain valid sequences. The raw data in fastq format were sequenced utilizing double-end sequencing (Paired-end) for this project using the Illumina Hiseq sequencing platform (samples correspond to a pair of sequences S_1.fastq and S_2.fastq). the sequences were then linked together and joined. A barcode was used to distinguish the samples that corresponded to the sequences. Using QIIME software, the sequences were quality-controlled and filtered to get rid of chimeras and get legitimate sequences. In order to determine the values of the samples\u0026apos; Alpha diversity and create the appropriate dilution curves, a series of microbial community richness and diversity analyses were carried out at the gene level. R was used to depict the species cumulative box plot analysis. The alpha diversity of the samples was expressed using the Shannon index, and the higher the Shannon value, the more diverse the community. The similarity and differences between several samples in two-dimensional coordinates were displayed using principal coordinate analysis (PCoA) based on the bray-Curtis distance. The principal bacterial species in charge of the variations in the bacterial communities were identified using the similarity percentage method. The units that differed considerably at each taxonomic level were identified using the Lefse analysis. For function prediction, a phylogenetic analysis of the community employing unobserved state reconstruction (PICRUSt) was used. From gene information on OTUs in the Greengene database and for additional untested species, gene function profiles were deduced.\u003c/p\u003e\n\u003cp\u003e1.4 Statistical methods\u003c/p\u003e\n\u003cp\u003eSoftware named SPSS 24.0 was utilized for the statistical evaluation. The analysis of categorical variables was conducted using the chi-square test, and the Student\u0026apos;s t-test was used to compare the means of the two groups. Standard deviation (SD) was used to describe the measurement data as mean (mean). The association between gut flora and abrupt deafness was examined using Spearman\u0026apos;s correlation methodology. P＜0.05 was regarded as a significant value. Correlation coefficients |r| between 0.8 and 1.0 denoted very strong correlation, 0.6 and 0.8, strong correlation, 0.4 and 0.6, moderate correlation, 0.2 and 0.4, weak connection, and 0.0 and 0.2, very weak or no association.\u003c/p\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e2.1 Statistics of sequencing results\u003c/h2\u003e\n \u003cp\u003eThe sequencing of a total of 10 patients who experienced abrupt total deafness and 10 healthy controls yielded raw sequencing data with reads ranging from 32,445,682 to 50,972,477 samples and a mean of 41,037,118. A non-redundant gene collection was created using de novo assembly, gene structure prediction, and clustering after sequencing results underwent quality check. The Venn diagram of the genes between the two groups is shown in Fig.\u0026nbsp;1. The Venn diagrams show that there are variations in the fecal sample gene expression between the group experiencing sudden total deafness and the healthy control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e2.2 Bacterial flora composition analysis\u003c/h2\u003e\n \u003cp\u003eTo determine the relative abundance, the intestinal flora of each study participant was examined at every level, from phylum to genus. The results showed that the intestinal flora of the sudden total deafness group and the healthy control group contained a total of 10 phyla and 118 genera, with statistically significant differences between the 2 groups for the phyla Methylobacteria (P\u0026thinsp;=\u0026thinsp;0.034) and Fusobacteria (P\u0026thinsp;=\u0026thinsp;0.003). However, the phylum Fusobacteria had a low relative abundance in the structure of the intestinal flora; phylum Thick-wal With the exception of the phylum Aspergillus, there was no statistically significant difference in the relative abundance of the dominating phylum between the two groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Romboutsia, Lactobacillus, Lactococcus, Escherichia coli, Shigella, Blautia, Streptococcus, and Bifidobacterium were the top 8 bacteria in terms of relative abundance in the sudden total deafness group, and there was no statistically significant difference between the 2 groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). (Fig. 2B)\u003c/p\u003e\n \u003cp\u003eAlpha diversity analysis\u003c/p\u003e\n \u003cp\u003eTwenty samples were sequenced, and 56 OTUs were identified with sequencing coverage of \u0026gt;\u0026thinsp;99.97% and a classification level of 97% similarity. The amount of sequencing data was reasonable and could adequately reflect the majority of information on the variety of the flora in the samples, as shown by the community richness index\u0026apos;s tendency to be flat for each sample.(Fig.\u0026nbsp;3).\u003c/p\u003e\n \u003cp\u003eThe Shannon, Chao1, and Simpson indices of the alpha diversity analysis did not substantially differ between the two groups of samples (P\u0026thinsp;=\u0026thinsp;0.871, P\u0026thinsp;=\u0026thinsp;0.433, and P\u0026thinsp;=\u0026thinsp;1.000, respectively), showing that the diversity of the flora was identical in both sets of samples. (Table\u0026nbsp;1)\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eDifferences in alpha diversity index based on t-test\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eShannon Index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSimpson Index\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChao1 Index\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSudden Total Deafness Group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e911.76\u0026thinsp;\u0026plusmn;\u0026thinsp;89.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal control group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e785.74\u0026thinsp;\u0026plusmn;\u0026thinsp;187.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.871\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.433\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eBeta diversity analysis\u003c/p\u003e\n \u003cp\u003eSamples from the groups with sudden total deafness and the normal control group were clustered independently, according to PCoA (Fig.\u0026nbsp;4). The combined -diversity analysis revealed that there were differences in the structure of the flora in the 2 sets of samples because the first principal component contributed 17.02% and the second principal component contributed 15.15% to the difference in the sample flora\u0026apos;s structure.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e2.3 Bacterial flora variation analysis\u003c/h2\u003e\n \u003cp\u003eFigure 5A of the LEfSe multilevel species hierarchical analysis revealed that the phylum Anaplasma was significantly enriched in the sudden total deafness group, whereas Clostridium was significantly enriched in the normal control group. At the genus level, 5 genera, including Porphyromonas, were significantly enriched in the sudden total deafness group. Together with the test for the significance of differences between groups, the LDA (LDA threshold\u0026thinsp;\u0026gt;\u0026thinsp;2.0) (Fig. 5B) revealed that the samples from the two groups did not differ significantly. Clostridium spp, Trichosporon spp, Microbacterium spp, Clostridium faecium spp, Raoul spp, Klebsiella spp, Caulobacter spp, Aminococcus spp, Haemophilus spp, Pasteurella spp, Aspergillus spp, Lactobacillus spp, and 12 other species(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were among those that significantly.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e2.4 Correlation analysis between intestinal flora\u003c/h2\u003e\n \u003cp\u003eAccording to Table\u0026nbsp;2, Spearman\u0026apos;s correlation study revealed that Raoulia, Klebsiella, Caulobacter, Haemophilus, and Pachybotrys species were all positively connected with sudden total deafness.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAnalysis of the correlation between microorganisms and sudden total deafness.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMicroorganisms\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003er\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTrichoderma spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus;0.434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRaoulia spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.926\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKlebsiella spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCorynebacterium spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHaemophilus spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.003\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLactobacillus spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.225\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.339\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGenus parabacteroides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.068\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhyllococcus spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.503\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.024\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e2.5 Functional Prediction of gut microbes in the sudden total deafness and control groups\u003c/h2\u003e\n \u003cp\u003eUtilizing PICRUSt, the functional prediction was carried out. In the group with sudden total deafness, the penicillin and cephalosporin biosynthesis pathway, the phosphatidylinositol metabolic pathway, the phosphatidylinositol signaling pathway, and the progesterone-mediated biosynthesis pathway were all enriched, whereas the proteasome metabolism, antigen processing and presentation, and aminobenzoic acid degradation were enriched in the control group (Fig.\u0026nbsp;6).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eIntestinal flora has attracted the attention of researchers both domestically and internationally in recent years, and a large number of studies have demonstrated that the effects of an imbalance in intestinal microbial-host interactions on endocytosis can contribute to extraintestinal disease progression in addition to intestinal inflammation. Studies conducted in the United States and abroad have revealed that issues with the intestinal flora can also cause vascular blood flow problems, ear neuropathy, and sensorineural hearing loss. We posited the possibility that gut flora might affect the development of SSNHL in conjunction with the reading of the literature.In order to test this theory, we carried out high-throughput sequencing of the gut microbiota's 16sRNA in the stools of patients with sudden total deafness and their families. The results revealed differences in genus-level abundance between the sudden total deafness group and the control group, but they did not reveal the precise number of bacteria present. We propose that the sudden total deafness activity is closely related to the bacterial species. Therefore, we explained how the bacterial species changed in both groups. Aspergillus and Clostridium were found in greater abundance in the sudden total deafness group than in the control group, as were 12 genera, including Clostridium spp., Trichosporon spp., Colletotrichum spp., and Clostridium faecalis, at the genus level. At the species level, Lactobacillus and Clostridium spp. were closely related to sudden deafness.Raoulia spp., Klebsiella spp., Corynebacterium spp., Haemophilus spp., and Clostridium perfringens spp. were all substantially and positively connected with abrupt total deafness, according to Spearman's correlation analysis.\u003c/p\u003e \u003cp\u003ePatients with abrupt total deafness had significantly higher levels of the phylum Aspergillus, which was statistically significant. Numerous hazardous bacteria, including Escherichia coli, Salmonella spp., Helicobacter pylori, and other well-known genera, are members of the phylum Aspergillus. The phylum Aspergillus contained the genera Raoulia and Klebsiella in the intestinal bacteria of patients with sudden complete deafness in this investigation. All members of the phylum Aspergillus are Gram-negative bacteria, and lipopolysaccharide (LPS) makes up the majority of their outer membranes. The gut is shielded in the intestinal flora of healthy people by an outer mucus layer, anti-inflammatory microbial compounds, and immune system components that have a dual role in generating and reducing inflammation.Pro-inflammatory bacteria in the gut can produce endotoxins, and in people with intestinal flora dysbiosis, a significant rise in Gram-negative bacteria and an increase in lipopolysaccharide (LPS)-producing bacteria can be seen[17,18]. LPS in the intestinal lumen enters the circulation, increasing intestinal permeability\u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e–\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, and high intestinal permeability can cause toxic substances to leak through the brain-intestinal axis to the brain, where they can According to studies using animal models, hearing loss is correlated with a rise in gram-negative bacteria in the diseased gastrointestinal tract and blood levels of LPS, which binds to the toll-like receptor 4 (TLR4) on immune cellsand activates an inflammatory response\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e ,increases the permeability of the intestinal barrier, which makes it easier for microorganisms to enter the inner ear and causes the growth of pathogenic bacteria and focal metastasis, further aggravating the inflammatory response. Additionally, it induces the transcription factor NF-B in macrophages and stimulates the release of inflammatory cytokines like interleukin 1 (IL−1) and tumor necrosis factor (TNF) \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. As a result, we speculate that different Aspergillus species may contribute to the onset of SSNHL by influencing intestinal permeability and creating a lot of LPS.\u003c/p\u003e \u003cp\u003eThe prevalence of Haemophilus influenzae is increased and strongly connected with the occurrence of sudden complete deafness in patients with this condition. Harmes KM\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e–\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e et al. noted that Haemophilus influenzae was isolated in large numbers from cultures of middle ear fluid from patients with acute otitis media and that Haemophilus influenzae has become one of the major causes of vaccination in patients with pneumococcal disease. Haemophilus influenzae is also a cause of chronic infection and acute otitis media in children, as well as hearing loss. The main reason for the rise in acute otitis media due to the increased use of antibiotics is resistance to beta-lactams and other antibiotics. According to a prior study, Haemophilus colonizes the respiratory tract's epithelial cells through adhesins and induces inflammation that impairs hearing. Adhesins also have supplementary virulence effects, which may also be a factor in the development of SSNHL. These effects include preventing immune clearance, altering pore size to prevent antimicrobial damage, establishing microcolonies, and inducing protein-mediated phase shifts\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to correlation research, sudden total deafness was also strongly related with the genus Lactococcus. A species of cocci called Lactococcus lamellaris belongs to the Lactobacillus family and order. Lactobacillus, a probiotic commonly found in yogurt and other fermented dairy products, belongs to the thick-walled phylum that produces acetate, lactate, and antimicrobial substances that prevent pathogens from interfering with health. There was a significant increase in the abundance of Lactobacillus at the species level of this study in patients with sudden deafness. Short-chain fatty acids (SCFAs), including acetate, are created by the fermentation of dietary fiber and resistant starch in the colon. These SCFs are involved in host metabolism, immunological response, and gastrointestinal physiology. Prebiotic FOS, as demonstrated by Kondo T et al. \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, increased the production of short-chain fatty acids (SCFAs). This resulted in a notable rise in SCFAs in the cecum and a decline in oxidative stress indicators in the serum, as well as better hearing in mice. When histones are deacetylated by the enzyme histone deacetylase (HDAC), they can bind strongly to negatively charged DNA and stop gene transcription. As ligands for HDAC inhibitors, SCFAs, as ligands for HDAC inhibitors, stimulate monocytes and neutrophils by inducing HDAC inhibition, leading to NF-κB inactivation, reducing pro-IL-2, IL-6, tumor necrosis factor -SCFAs can also promote the cytosolic transport of intestinal epithelial cells (IECs), increase the secretion of IgA into the intestinal lumen, increase the content of sIgA in the intestinal mucosa, agglutinate and adhere bacteria to the mucus, prevent direct contact between bacteria and the surface of intestinal epithelial cells, and prevent the invasion and infection of bacteria. It was found that expression of intestinal tight junction proteins was significantly reduced in germ-free mice, leading to increased blood-brain barrier permeability; treatment with complex microorganisms or SCFAs restored the integrity of the blood-brain barrier, for example, treatment of cerebrovascular endothelial cells with propionate attenuated the permeability of exposed lipopolysaccharide (LPS) and increased blood-brain barrier function. In order to minimize generated neuroinflammatory reactions, we predicted that C. lamellar and its related metabolites would shield the blood-brain barrier. Our data showed a positive correlation between lamellococci and the occurrence of sudden total deafness as opposed to a poor correlation, which may have been caused by a connection with a smaller sample size and diverse demographics. In order to better define the gut microbiota in patients with abrupt SSNHL, we will increase the size of our sample.\u003c/p\u003e \u003cp\u003eOur research also revealed a rise in Clostridium difficile species abundance in the gut flora of patients with sudden complete deafness. The Clostridium difficile toxin B (TcdB) is the main pathogenic \"weapon\" of this anaerobic, Gram-positive bacterium, commonly known as Clostridium difficile. The most typical cause of sensorineural hearing loss is damage to the cochlear hair cells. In comparison to the Corti apparatus treated with C. difficile toxin B and gentamicin for 72 hours, Bodmer D\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e et al. treated the Corti apparatus of the rat cochlea with C. difficile toxin B for 12 hours. C. difficile toxin B treatment significantly reduced the mortality of cochlear hair cells. According to this, C. difficile toxin B may be able to protect auditory hair cells from aminoglycoside toxicity and restore their ability to hear. The usage of Clostridium difficile in SSNHL has not been documented, however it may contribute to hearing loss in drug-induced deafness.\u003c/p\u003e \u003cp\u003e According to PICRUSt analysis, sudden total deafness may be closely correlated with the upregulation of certain metabolic pathways, including the phosphatidylinositol metabolic pathway, the phosphatidylinositol signaling pathway, the penicillin and cephalosporin biosynthesis pathway, and progesterone-mediated biosynthesis.\u003c/p\u003e \u003cp\u003eAn organic osmolyte and volume modulator, inositol. Modifications in the vestibular epithelium and basement membrane's micromechanical coupling due to alterations in the inner ear's osmotic pressure or hair cells may result in pathological changes in sensory transduction. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) to the second messenger's inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol (DG), and in the guinea pig corti organ, the InsP3 second messenger system is associated with muscarinic cholinergic receptors and purinergic receptors, affecting hearing in guinea pigs. Purinergic receptor activation and ATP release from the cochlear epithelium also have a significant impact on the cochlea's physiology. Purinergic P2X and P2Y receptors are widely distributed in the inner and outer hair cells as well as in the Corti apparatus supporting cells, and they both have an impact on the development of hearing by changing the sensitivity of the receptor cells to K +. Additionally, purinergic signaling through purinergic receptors can affect hearing sensitivity and cochlear blood flow in noise-induced hearing impairment, which is consistent with the findings of our study. Accordingly, we hypothesize that the phosphatidylinositol metabolic pathway and phosphatidylinositol signaling pathway may be one of the pathogenic mechanisms of SSNHL.\u003c/p\u003e \u003cp\u003eOur study has several limitations. First, this is a correlational study, and there is no proof that abnormally high levels of specific gut flora are the direct cause of SSNHL, even though we employed 16S rRNA PICRUSt functional prediction to examine potential pathways of the effect of gut flora on sudden complete deafness. Therefore, the next step will be to increase the sample size and further describe the pathophysiological mechanisms underlying SSNHL utilizing bigger sample size studies based on proteomics, metabolomics, and macroeconomics. Further research on this mechanism will be done in the following study because, in the previous study, the group with sudden total deafness was significantly enriched in five genera, including Porphyromonas, at the genus level, while the normal control group was only significantly enriched in Clostridium.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, patients with sudden total deafness and healthy controls have significantly different gut microbial compositions. Potential risk factors for abrupt deafness include Raoulia species, Klebsiella species, Haemophilus species, Clostridium species, Lactobacillus species, and the phosphatidylinositol signaling and metabolic pathways. Overall, the distinctive alterations in the gut flora and metabolic pathways found in people who have experienced sudden total deafness offer new research areas for the pathophysiology and treatment of these conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWWJ and ZYY caried out most of the experiments and drafted the manuscript.KS、ZLM and XLZ did parts of the experiments and performed the statistical analysis;MY and ZPG conceived of hte study and revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEditorial Board of Chinese Journal of Otolaryngology, Head and Neck Surgery, Chinese Medical Association, Division of Otolaryngology, Head and Neck Surgery. Guidelines for the diagnosis and treatment of sudden deafness (2015) [J]. Chinese Journal of Otolaryngology-Head and Neck Surgery,2015,50(6):443-447.\u003c/li\u003e\n\u003cli\u003eStachler, R. J., Chandrasekhar, S. S., Archer, S. M., Rosenfeld, R. M., \u0026amp; Schwartz, S. R. (2011). Clinical Practice Guideline: Sudden Hearing Loss. otolaryngology-Head and Neck Surgery, 145(2_suppl), P28-P28.\u003c/li\u003e\n\u003cli\u003eHerrera M, Berrocal JRG, Arum\u0026iacute; AG, et al. Update on consensus on diagnosis and treatment of idiopathic sudden sensorineural hearing loss [J]. Acta Otorrinolar ingologica (English Edition), 2019, 70 (5): 290-300.\u003c/li\u003e\n\u003cli\u003eWang D, Wang A, Jia Y. Treatment and research progress of sudden deafness[J] . China Medicine Finger,2019,17(16):30-31.\u003c/li\u003e\n\u003cli\u003eYOUNG YH. A contemporary review of the causes and differential diagnosis of sudden sensorineural hearing loss [J]. Int J Audiol,2020,590(4):243-253.\u003c/li\u003e\n\u003cli\u003eLIN RJ, KRALL R, WESTERBERG B D, et al. 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EBioMedicine. 2020 Jan. 51:102590.\u003c/li\u003e\n\u003cli\u003eIatcu CO, Steen A, Covasa M. Gut Microbiota and Complications of Type-2 Diabetes. Nutrients. 2021 Dec 30;14(1):166.\u003c/li\u003e\n\u003cli\u003eJiang C, Li G, Huang P, Liu Z, Zhao B. The Gut Microbiota and Alzheimer\u0026apos;s Disease. J Alzheimers Dis. 2017;58(1):1-15.\u003c/li\u003e\n\u003cli\u003eQuigley EMM. Microbiota-Brain-Gut Axis and Neurodegenerative Diseases. Curr Neurol Neurosci Rep. 2017 Oct 17;17(12):94. \u003c/li\u003e\n\u003cli\u003eAngelucci F, Cechova K, Amlerova J, Hort J. Antibiotics, gut microbiota, and Alzheimer\u0026apos;s disease. J Neuroinflammation. 2019 May 22;16(1):108.\u003c/li\u003e\n\u003cli\u003eGurung M, Li Z, You H, Rodrigues R, Jump DB, Morgun A, Shulzhenko N. Role of gut microbiota in type 2 diabetes pathophysiology. EBioMedicine. 2020 Jan. 51:102590.\u003c/li\u003e\n\u003cli\u003eSalguero, M.V.; Al-Obaide, M.A.; Singh, R.; Siepmann, T.; Vasylyeva, T.L. 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Diabetes 2008 , 57, 1470-1481.\u003c/li\u003e\n\u003cli\u003eGhanim, H.; Abuaysheh, S.; Sia, C.L.; Korzeniewski, K.; Chaudhuri, A.; Fernandez-Real, J.M.; Dandona, P. Increase in plasma endotoxin concentrations and the expression of Toll-like receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high- Diabetes Care 2009, 32, 2281-2287.\u003c/li\u003e\n\u003cli\u003eCaesar, R.; Reigstad, C.S.; Backhed, H.K.; Reinhardt, C.; Ketonen, M.; Lunden, G.O.; Cani, P.D.; Backhed, F. Gut-derived lipopolysaccharide augments adipose macrophage accumulation but is not essential for impaired glucose or insulin tolerance in mice. Gut 2012, 61, 1701-1707. \u003c/li\u003e\n\u003cli\u003ePendyala, S.; Neff, L.M.; Suarez-Farinas, M.; Holt, P.R. Diet-induced weight loss reduces colorectal inflammation: Implications for colorectal Am. J. Clin. Nutr. 2011, 93, 234-242.\u003c/li\u003e\n\u003cli\u003eJanssen, A.W.; Kersten, S. 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Prevalence of antimicrobial-resistant pathogens in middle ear fluid: a multinational study of 917 children with acute otitis media. Antimicrob Agents Chemother. 1998 Mar;42(3):589-95. \u003c/li\u003e\n\u003cli\u003eOsman KL, Jefferies JM, Woelk CH, Cleary DW, Clarke SC. The adhesins of non-typeable Haemophilus influenza. Expert Rev Anti Infect Ther. 2018 Mar. 16(3):187-196. \u003c/li\u003e\n\u003cli\u003eKondo T, Saigo S, Ugawa S, Kato M, Yoshikawa Y, Miyoshi N, Tanabe K. Prebiotic effect of fructooligosaccharides on the inner ear of DBA/2 J mice with early-onset progressive hearing loss. J Nutr Biochem. 2020 Jan;75:108247.\u003c/li\u003e\n\u003cli\u003eBodmer D, Brors D, Pak K, Gloddek B, Ryan A. Rescue of auditory hair cells from aminoglycoside toxicity by Clostridium difficile toxin B, an inhibitor Hear Res. 2002 Oct;172(1-2):81-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"sudden total deafness, gut microbiota, 16SrRNA, PICRUSt.","lastPublishedDoi":"10.21203/rs.3.rs-3893609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3893609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective To \u003c/strong\u003einvestigate a differential analysis of the intestinal flora characteristics of individuals with sudden total deafness and a healthy control population, it will be possible to determine whether there is a link between intestinal flora and sudden total deafness.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eFresh stool samples were taken from 10 patients with sudden total deafness and 10 healthy subjects (family members of 10 patients with sudden total deafness) who were hospitalized at Baiyun Hospital of Guizhou Medical University between December 2021 and February 2022. All subjects' stool samples were then analyzed using 16S rRNA sequencing technology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eWe examined the differences in intestinal bacterial diversity between sudden total deafness and healthy normal patients using high-throughput sequencing technologies. The results of sample hierarchical clustering and principal coordinates analysis revealed that the sample flora structure varied between the sudden total deafness and normal control groups; multi-level species difference discriminant analysis and between-group difference analysis revealed that the intestinal flora of the 2 groups in the phylum Aspergillus (\u003cem\u003eP\u003c/em\u003e=0.034), Clostridium (\u003cem\u003eP\u003c/em\u003e= 0.003), with statistically significant differences between the 2 groups, but the relative absorption was similar between the 2 groups. The prevalence of Raoulia spp., Klebsiella spp., Caulobacter spp., Haemophilus spp., and Clostridium spp. was positively connected with sudden total deafness, according to a correlation study.At the genus level, five genera, including Porphyromonas spp., were considerably enriched in the entire deafness group, whereas only Clostridium spp. were significantly enriched in the normal control group, according to an LEfSe multilevel species hierarchy analysis.Four paths existed between the groups, according to PICRUSt functional prediction study.The four routes were considerably different at level 3 according to the results of the PICRUSt functional prediction analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003ePatients with sudden total deafness and their families have dramatically different gut flora in terms of composition and functional pathways.\u003c/p\u003e","manuscriptTitle":"Microbiological characterization of the gut in patients with sudden total deafness","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-05 14:35:12","doi":"10.21203/rs.3.rs-3893609/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":"8678c28b-8f5a-40d3-b2d6-fdf1e2f3cd10","owner":[],"postedDate":"February 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28504972,"name":"Biological sciences/Biochemistry"},{"id":28504973,"name":"Biological sciences/Biological techniques"}],"tags":[],"updatedAt":"2024-06-05T06:54:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-05 14:35:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3893609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3893609","identity":"rs-3893609","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}