Application Terminal Restriction Fragment Length Polymorphism Orthopedic Infections Bacterial Community Analysis

Background:Infecion is one of the most concerned problem in orthopaedic clinical practice. Both implant-associated infections and other orthopaedic infections such as open bone fracture, chronic pyogenic osteomyelitis and so on are often characterized with complex pathogens and antibiotic resistance. The question wheather "aseptic loosening" is truly aeptic is still debatable since traditional cultivation method applied in clinical medicine can’t give the answer. Molecular biology method Terminal Restriction Fragment Length Polymorphism, originated from environmental engineering science can obtain Terminal Restriction Fragments of various lengths or Operational Taxonomic Units by detecting marker DNA, that is16S rDNA for bacteria, of concerned microbe so as to systemically analyze the constitution of bacterial communities. It is of great significance especially for those bacteria can’t be cultivated by traditional method. Although T-RFLP has already been used to detect some clinical pathogens such as intestinal or vaginal mircobe recently, applying it in orthopaedic infection has not been reported so far.Objects:1. Apply T-RFLP method to analyze the distribution of bacterial communites in suspected infection specimens;2. Compare the result of T-RFLP from that of traditional clinical cultivation method;3. Primarily evaluate this technology and its prospect in clinical medicine.Material and methods:1. Subjects:infection specimens from orthopaedic dept.(including both implant-associated and other infections); infection speciemens from other clinical depts.;2. Bacterial detecing methods:T-RFLP, traditional clinical cultivation;3. Analyzing methods: i. Cluster analysis for the result of T-RFLP; ii. Clone library construction and sequencing, TAP (T-RFLP Analysis Program) for T-RFLP results and virtual enzyme cutting for clinical results.Results:.1. Collected59speciemens and got the final T-RFLP results from14of them.2. According to the cluster analysis, specimens could be categorized by their anatomical positions and the distribution of T-RFs in each category had common characters. T-RFs with the length of80bp,82bp,85bp were the richest ones (total richness>40%) in the knee group (NO.6,11,12,17), while T-RF with the length of479bp was the richest one (total richness>60%) in the spinal group (NO.30,47).3. Constructed clone library and made sequencing of speciemne NO.13. Blast analysis (Basic Local Alignment Search Tool) showed the result was Streptococcus Dysgalactiae which coincieded with that of clinical cultivation.4. T-RFs got by virtual enzyme cutting of clinical cultivation results generally coincided with actural experimental results of T-RFLP (be the richest fragments and length differences not more than3bp).Conclusions:In the dissertation we used the method of Terminal Restriction Fragment Length Polymorphism to systemically analyze the constitution of bacterial communities of infectious orthopaedic specimens as well as made comparison with traditional clinical cultivation method.1. Constitution of bacterial communities in the infection of same anatomical position beared great similarity wheather there was implant or not.2. Some cases with the clinical diagnosis of "aseptic implant loosening" might acturally have bacteria infection and the constitution of bacterial communities was also quite similar to that of clinical infection cases in the same anatomical position.3. Verified the safety, stability and reliability of T-RFLP which had a broad clinical prospect by processing specimens of various origins and comparing with conrespongding clinical results. Anyway, the experimental conditions should be further improved so as to elevate its examining-out rate.