Objective: To analyze the characteristics of pathogenic microorganisms in the infected bone tissues in patients with diabetic foot osteomyelitis (DFO) using 16S rRNA high-throughput sequencing to facilitate rapid and accurate detection of pathogens and effective infection control.
Methods: Between 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.
Results: 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. 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 (67.16% vs 50.00%) in the samples. 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.
Conclusions: 16S rRNA high-throughput sequencing is capable of demonstrating the diversity and abundance of microflora in DFO bone tissues, where diverse and uniformly distributed pathogens can be detected with a discrete distribution of the dominant genera, most of which are gram-negative. Compared with routine culture method, 16S rRNA sequencing allows more convenient and accurate identification of the pathogens (especially gram-negative bacteria and anaerobes), and can be useful in clinical decision on appropriate treatment of DFO.
目的: 利用16s rRNA高通量测序技术分析糖尿病足骨髓炎（DFO）感染骨组织中病原微生物特点，为临床感染病原菌的鉴定及治疗提供快速、准确的方法。
方法: 收取2016年9月~2017年4月于本科室住院的16例DFO患者清创术中获取的感染骨标本，分别利用16s rRNA高通量测序技术和血培养分析仪进行病原微生物的鉴定，分析16s rRNA测序结果中DFO的菌群特点，并与培养结果相比较。
结果: 16s rRNA测序显示DFO骨组织病原微生物多样性较大，分布较为均匀，共获得优势菌属20种，占所有菌属的87.00%，其中Prevotella是丰度最大的菌属。两种鉴定方法结果均显示DFO病原菌以革兰氏阴性菌为主。与培养法相比，16s rRNA测序阳性率较高（100% vs 88.24%），平均每个样本病原菌数较多（12.56 vs 1.50），革兰氏阴性菌所占的比例较高（67.16% vs 50.00%）。此外，16s rRNA测序结果覆盖了除Escherichia coli、Serratia marcescens及Enterobacter cloaca外的所有培养病原菌结果，甚至有高达13种菌属不存在于培养结果，其中Anaerococcus、Veillonella、Bacteroides、Fusobacterium、Porphyromonas、Finegoldia、Prevotella、Peptostreptococcus、Parvimonas、Peptoniphilus和Bulleidia均为专性厌氧菌或严格厌氧菌，而培养结果中并无厌氧菌的出现。但培养结果显示，DFO中多重耐药菌的比例高达58.33%。
结论: 16s rRNA高通量测序能较好展示DFO骨组织中菌群微生态的多样性及丰度特点。DFO骨组织病原微生物多样性大，分布均匀，但优势菌属分布离散，以革兰氏阴性菌为主。与培养法相比，16s rRNA测序对病原菌的鉴定简单而准确，尤其在对革兰氏阴性菌和厌氧菌鉴定方面有显著的优势，可快速而准确地指导DFO感染治疗。