Page 1 of 32 Diabetes Analysis of the Composition and Functions of the Microbiome in Diabetic Foot Osteomyelitis based on 16S rRNA and Metagenome Sequencing Technology Zou Mengchen1*; Cai Yulan2*; Hu Ping3*; Cao Yin1; Luo Xiangrong1; Fan Xinzhao1; Zhang Bao4; Wu Xianbo4; Jiang Nan5; Lin Qingrong5; Zhou Hao6; Xue Yaoming1; Gao Fang1# 1Department of Endocrinology and Metabolism, Nanfang Hospital, Southern Medical University, Guangzhou, China 2Department of Endocrinology, Affiliated Hospital of Zunyi Medical College, Zunyi, China 3Department of Geriatric Medicine, Xiaogan Central Hospital, Xiaogan, China 4School of Public Health and Tropic Medicine, Southern Medical University, Guangzhou, China 5Department of Orthopaedics & Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou, China 6Department of Hospital Infection Management of Nanfang Hospital, Southern Medical University, Guangzhou, China *Zou mengchen, Cai yulan and Hu ping contributed equally to this work. Running title: Microbiome of Diabetic Foot Osteomyelitis Word count: 3915 Figures/Tables Count: 4Figures / 3 Tables References: 26 Diabetes Publish Ahead of Print, published online August 14, 2020 Diabetes Page 2 of 32 Keywords: diabetic foot osteomyelitis; microbiome; 16S rRNA sequencing; metagenome sequencing #Corresponding author: Gao Fang, E-mail: [email protected], Tel: 13006871226 Page 3 of 32 Diabetes ABSTRACT Metagenome sequencing has not been used in infected bone specimens. This study aimed to analyze the microbiome and its functions. This prospective observational study explored the microbiome and its functions of DFO (group DM) and posttraumatic foot osteomyelitis (PFO) (group NDM) based on 16S rRNA sequencing and metagenome sequencing technologies. Spearman analysis was used to explore the correlation between dominant species and clinical indicators of patients with DFO. High-throughput sequencing showed that all the specimens were polymicrobial. The microbial diversity was significantly higher in DM than in NDM. Firmicutes, Prevotellaceae, and Prevotella were the most abundant microbes in DM. The most abundant microbes in NDM were Proteobacteria, Halomonadaceae, and Halomonas. Prevotella denticola, Prevotella jejuni and Prevotella fusca had positive correlation with the duration of diabetic foot infection (DFI_d). Proteus vulgaris was positively correlated with the infection index, while Bacteroides fragilis was negatively correlated. The microbial functional genes were more abundant in DM than in NDM. Metagenome sequencing is feasible for the analysis of the microbiome in infected bone specimens. G- bacteria and anaerobes are dominant in DFO. Clinical trials registration: NCT04240964, ClinicalTrials.gov Diabetes Page 4 of 32 Diabetes-related lower-extremity complications are a large and growing contributor to the disability burden worldwide (1). Diabetic foot osteomyelitis (DFO) is the most severe stage of DF. More than 20% of patients with severe diabetic foot infection (DFI) and 50%-60% of patients with moderate DFI develop DFO. The minor amputation rate of patients with DFO reaches 59.4% (2). Five year mortality for patients undergoing minor amputations is 46.2%. Long-term survival was worse in patients who underwent a major amputation with a 5-year mortality of 65.6%. Which exceeds the reported 5-year overall mortality rate of cancer by 31.0%. DFO is common, complicated and costly. In 2017, the direct cost of diabetes care was $237 billion, and more than one-third of the direct cost was attributed to the lower extremities (3). In recent years, the importance of antibiotics in DFO treatment has gradually been recognized, but the application of antibiotics is limited due to the limitations and one-sidedness of microbial identification technology (4). Fast and comprehensive analysis of the microbiome in wounds is a prerequisite for reducing the amputation rate and mortality. Although culture methods have a long history and have been widely used, shortcomings remain (e.g. time consumption and low sensitivity) (5). These deficiencies have hindered a correct understanding of the microbiome and made it difficult to meet the needs of clinical application and the development of modern biological research. Consequently, technological updates are urgently needed in the field of microbial research. High-throughput sequencing technology does not rely on culture and can be used to examine the microbiome and its functions in wounds quickly and comprehensively, providing a new direction for the development of microbial research. The most widely used high-throughput sequencing technology for identifying bacteria is 16S rRNA sequencing. Page 5 of 32 Diabetes Among its advantages, 16S rRNA sequencing does not require culture and is simple, fast, low-cost and widely applied. However, 16S rRNA sequencing provides only partial bacterial genome information. Even after full optimization, it is impossible to obtain detailed information at the species level, and it is difficult to analyze the functions of microorganisms in detail. Compared with 16S rRNA sequencing, metagenome sequencing is a revolutionary method that can examine all DNA in specimens without the use of culture or PCR amplification. Metagenome sequencing can provide sufficient and accurate species information, thus compensating for the shortcomings of the above two methods (6). However, there have been no studies detecting microorganisms in infected bone specimens based on metagenome sequencing. We therefore conducted a prospective study with the goal of better observing the microbiome and its functions in wounds of DFO based on cultivation, 16S rRNA sequencing and metagenome sequencing. Research Design and Methods Population No study has analyzed the differences and similarities between the microorganisms in wounds of DFO and posttraumatic foot osteomyelitis (PFO). Over a one-year period, we prospectively enrolled 28 consecutive patients aged over 18 years who presented with osteomyelitis at Nanfang Hospital from September 2017 to September 2018. The patients were divided into two groups, namely, the group DM (DFO, 17 patients) and the group NDM (PFO, 11 patients), according to Diabetes Page 6 of 32 whether or not they had diabetes mellitus. Osteomyelitis was suspected based on the patient's clinical manifestations, laboratory tests, and imaging examination: 1) clinical manifestations: bone exposure (especially if the area was >2 cm2) or probe-to-bone test was positive, erythema and hardening of the toes (sausage-like toes) occurred; 2) laboratory tests: erythrocyte sedimentation rate (ESR) >70 mm/h; C-reactive protein (CRP), procalcitonin (PCT) ,white blood cell (WBC) levels increased and microorganisms could be cultivated from specimens; 3) Radiographs examination: Radiographs showed loss of cortical bone, accompanied by bone erosion or demineralization, focal trabecular morphology loss or loss of bone marrow radio permeability; 4) MRI examination: MRI images showed T1-weighted low-focus signals, T2-weighted high-focus signals, and high bone marrow short-term inversion recovery (STIR) sequence signals (4). Patients were included if osteomyelitis was diagnosed, microorganisms could be cultivated from specimens, and the infected bone was exposed. Furthermore, the patients exhibited no obvious skin lesions on the wound surface or surroundings, and they were in good health and can withstand debridement. The patients and/or his/her family were informed and agreed to participate in the study. The patients were ≥18 years old. Patients were excluded if they had an immune system that could not tolerate debridement or had taken immunosuppressants within 3 months before admission. The study was designed and implemented in accordance with the Helsinki declaration (2013), approved by the ethics committee of Nanfang Hospital (NFEC-2017-013), and registered on the Clinical Trials website (NCT04240964). Informed consent was obtained from all patients. Page 7 of 32 Diabetes Specimen collection After the wounds were rinsed with sterile saline and hydrogen peroxide solution, 17 bone specimens were collected in the clinic setting from patients with DFO who required debridement to manage their osteomyelitis after removal of necrotic tissue from the surface to avoid soft tissue or sinus tract contamination, which was not predictive of the presence of pathogen with sufficient accuracy (9). And after the wounds were rinsed with sterile saline and iodophor solution, 11 bone specimens were collected in an operating room from patients with PFO routinely. We obtained bone specimens and divided them into two parts under sterile conditions. One part was used for routine microbial culture, and the other was used for high-throughput sequencing. 16S rRNA sequencing Genomic DNA was extracted using a DNA extraction kit (YiRui, ShenZhen, China) according to the manufacturer's instructions. The extracted DNA was quantified and quality controlled by 1% agarose gel electrophoresis (JS-power 300, PeiQin, Shanghai, China). Then, we amplified the V3-V4 variable region of the 16S rRNA gene for sequencing using forward and reverse fusion primers (341F: 5’-CCTAYGGGRBGCASCAG-3’ and 806R: 5’-GGACTACNNGGGTATCTAAT-3’). PCR was performed in a total volume of 20 μl containing 4 μl of 10x PCR buffer, 2 μl of 2.5 mM dNTPs, 0.8 μl of forward primer (5 M), 0.8 μl of reverse primer (5 M), 0.4 μl of FastPfu polymerase, template DNA (10 μl) and H2O (2 μl). PCR amplification was conducted under the following conditions: initial denaturation at