Analysis Of Pathogenic Microbes Of Diabetic Foot Osteomyelitis And Comparison Of Two Bone Biopsy Methods Based On 16S RRNA Sequencing

BackgroundDiabetic foot osteomyelitis(DFO)is a serious chronic complication of diabetes.It is mainly caused by superficial soft tissue infection of diabetic foot ulcers spreading to bone or bone marrow tissue.Diabetic foot osteomyelitis often involves diabetic peripheral neuropathy,peripheral vascular disease,joint deformity,infection and so on.However,infection is the most important factor leading to the occurrence and development of osteomyelitis.In recent years,with the increase of the number of diabetic patients,the incidence of diabetic foot osteomyelitis is also increasing.The high admission rate,amputation rate and mortality rate caused by diabetic foot osteomyelitis always threaten the physical and mental health of diabetic patients,and consume huge social resources.Advances in early diagnosis and treatment of diabetic foot osteomyelitis are valuable to shorting the hospitalization day,reducing amputation rate and mortality.The gold standard for diagnosis of diabetic foot osteomyelitis is bone biopsy at the infected area.Microbiological analysis of bone biopsy is of great significance for the diagnosis and treatment of osteomyelitis.At present,microbiological identification based on culture has some limitations,which has relatively low accuracy,and needs further study to make the value of guiding anti-infection therapy clear.16S rRNA gene sequencing,an important tool for comprehensive understanding of the microbial community,can quickly and accurately identify pathogenic microorganisms.It has potential value for the clinical analysis of diabetic foot osteomyelitis.Both IDSA and IWGDF recommend surgical bone biopsy as the primary bone biopsy method,followed by percutaneous puncture biopsy.Actually,not all patients with diabetic foot osteomyelitis need or can be treated with surgery,and percutaneous bone biopsy as an invasive examination may not be easy to be accepted by patients,so the debridement may become a more popular method of bone biopsy.Up to now,16S rRNA gene sequencing has not been widely used in clinical studies,and the value of microbiological identification for diabetic foot osteomyelitis is not clear;the research on debridement bone biopsy is limited,and the accuracy rate is not known.So,it is necessary to further explore the clinical feasibility and accuracy of microbiological analysis of diabetic foot osteomyelitis bone tissue using 16S rRNA gene sequencing and debridement bone biopsy in the diagnosis of diabetic foot osteomyelitis,improving the level of diagnosis of osteomyelitis.Chapter1 Pathogen analysis in patients with diabetic foot osteomyelitis using 16S rRNA high-throughput sequencingObjectivesTo analyze the characteristics of pathogenic microorganisms in the infected bone tissues in patients with diabetic foot osteomyelitis using 16S rRNA high-throughput sequencing to facilitate rapid and accurate detection of pathogens and effective infection control.MethodsBetween September,2016 and April,2017,16 patients with DFO were admitted in our department and infected bone specimens were obtained during debridement.The pathogenic microorganisms in the specimens were identified using both 16S rRNA high-throughput sequencing and automatic blood culture analyzer,and the characteristics of the microflora were analyzed based on 16S rRNA sequencing data in comparison with the results of blood culture.Results1.The results of 16S rRNA sequencing showed that bone tissues of DFO contained diverse and uniformly distributed pathogenic organisms,among which 20(87%)dominant genera were identified with Prevotella as the most abundant pathogen.Both 16S rRNA sequencing and routine culture results suggested the domination of gram-negative bacteria among the pathogens in DFO bone tissues.2.16S rRNA sequencing,compared with routine culture,yielded a higher positivity rate(100%vs 88.24%)and detected a greater average number of pathogens(12.56 vs 1.50)and a higher proportion of gram-negative bacteria(60.70%vs 50.00%)in the samples.3.16S rRNA sequencing detected nearly all the pathogens identified by routine culture except for Escherichia coli,Serratia marcescens and Enterobacter cloaca,and identified 13 genera that failed to be detected by routine culture,including the obligate or strict anaerobes Anaerococcus,Veillonella,Bacteroides,Fusobacterium,Porphyromonas,Finegoldia,Prevotella,Peptostreptococcus,Parvimonas,Peptoniphilus and Bulleidia.Routine culture did not detect any anaerobes in the samples but identified multidrug-resistant strains in as many as 58.33%of the pathogens.Conclusions4.The results of 16S rRNA high-throughput sequencing demonstrated that the microbes of the diabetic foot osteomyelitis had large diversity and uniform distribution.But the dominant genera were dispered and dominanted by gram-negative bacteria.5.Compared with traditional culture,16S rRNA sequencing was more convenient and accurate in pathogens identification,especially in gram-negative bacteria and anaerobes.6.Antibiotics,mainly for gram-nagative bacteria and anaerobes,should be empirically used as anti-infection treatment for patients with diabetic foot osteomyelitis.Chapter2 Comparison of microbiological results of two bone sampling methods in diabetic foot osteomyelitis:debridement versus operationObjectivesThe purpose of this study is to compare the microbiological results of debridement bone samples with those of corresponding results of operation bone samples for patients with diabetic foot osteomyelitis,using 16S rRNA high-throughput sequencing approach,and provide a more simple and easier method for clinical bone biopsy.MethodsWe prospectively recruited 24 diabetic foot patients with osteomyelitis from September,2016 to April,2017,and finally obtained 9 bone samples during surgery(Ds-group)and 16 bone samples from debridement(Dd-group).We compared the differences between debridement and operation procedures based on microbiological study of bone samples via 16S rRNA sequencing,using Ion PGMTM System Sequencer.Results1.The differences of sexes,ages,diabetic duration,WBC,N,PCT,CRP,ALB,CCr,BUN,Cys-c between Ds-group and Dd-group were not significant.Ds-group displayed longer foot ulcers infection duration(P<0.01)but lower glycosylated hemoglobin level(P<0.05)than Dd-group.2.There were obvious differences in microbes between Ds-group and Dd-group.Microbes in Ds-group among samples were more similar,and the distribution of dominant bacteria were narrower.On the contrary,pathogens in Dd-group were more different and the distribution were more dispersed.3.Ds-group was dominant by Proteobacteria,and Dd-group was dominant by Firmicutes and Bacteroidetes.In genus,Halomonas was the most dominant one in Ds-group and Prevotella was the most dominant one in Dd-group.4.Genera,such as Anaerococcus、Dialister、Halomonas、Nesterenkonia、Pseudomonas,were significantly different between Ds-group and Dd-group(P<0.05).The abundance of Anaerococcus、Dialister、Pseudomonas in Ds-group were significantly higher than Dd-group,while the abundance of Halomonas and Nesterenkonia were significantly lower.However,there were no significant difference in positive rates and average number of dominant genera between the two group(P>0.05).Conclusions1.Compared with surgical bone biopsy,debridement bone biopsy might affect the abundance of pathogenic microbes,but would not affect the diversity and positive rates of dominant genera.2.Collecting bone fragments during debridement can be ued as a substitution for bone biopsy in non-sugical diabetic foot osteomyelitis patients with exposed bone,and assist the diagnosis and treatment of osteomyelitis.