应用末端限制性片段长度多态性技术对骨科感染中细菌群落的解析
摘要
研究背景：
感染是骨科临床实践中重点关注的问题之一,无论是假体相关性感染还是诸如开放性骨折、慢性骨髓炎等其它骨科感染,往往具有病原学复杂,抗生素耐药等问题,而对诸如“无菌性假体”松动这样的临床诊断中到底有无细菌的存在更是莫衷一是,争议颇多。临床传统应用的微生物纯培养方法对于这些问题的回答力有未逮。脱胎于坏境工程学科的分子生物学技术末端限制性片段长度多态性(Terminal Restriction Fragment Length Polymorphism, T-RFLP)通过对系统中微生物的标记DNA-16S rDNA—的检测,获取许多长度不同的末端限制性片段(Terminal Restriction Fragment, T-RF)或称分类操作单元(Operational Taxonomic Unit, OTU),从而可以全景式的解析系统中所有细菌群落的结构,这尤其对于检测那些临床传统方法不能培养的细菌意义重大。近年来T-RFLP方法正被逐渐应用于临床病原菌的检测,例如肠道菌群、阴道菌群等,但应用于骨科感染的研究尚未见报道。
研究目的：
1.应用T-RFLP技术分析怀疑感染的骨科标本中细菌群落分布情况；
2.比较T-RFLP技术得到的结果与临床传统纯培养结果的异同；
3.初步评价该技术的优缺点及其应用于临床的前景。
研究方法：
1.实验对象：骨科感染标本(包括假体相关性和非假体相关性),其它科感染标本；
2.细菌检测方法：T-RFLP,临床纯培养；
3.分析方法：i.对T-RFLP自身结果进行聚类分析；ii.对两者的比较则应用克隆文库测序、TAP (T-RFLP Analysis Program)和临床结果电子酶切的方法。
研究结果：
1.收集了59份临床样品,最终14份得到了T-RFLP谱图；
2.聚类分析显示,样品可按照解剖部位分类,每类中末端限制性片段(T-RFs)的分布具有一般性规律：其中膝关节样品6、11、12、17中丰度最高的片段都是80bp、82bp、85bp,其总丰度超过40%；脊椎样品30、47中丰度最高的片段都是479bp,总丰度超过60%；
3.对13号样品构建了克隆文库并测序,Blast分析(Basic Local Alignment Search Tool)序列结果为停乳链球菌,与临床培养结果一致；
4.临床已知培养结果者电子酶切分析得到的T-RFs大多与T-RFLP实测情况相符(为丰度最多的片段,且两者相差在1-3bp)。
结论：
本研究应用末端限制性片段长度多态性技术对骨科感染标本中细菌群落结构的分布进行了全景式的扫描分析,并同临床传统纯培养方法进行了比较,发现：
1.无论有无假体的存在,相同解剖部位的感染中细菌群落的分布都具有很高的相似性；
2.部分临床诊断为“无菌性假体松动”的病例中可能也是有细菌感染存在的,且其菌群分布规律与相同解剖部位感染患者亦相似；
3.通过对各种不同临床来源的样品处理以及与临床结果比对,证实了末端限制性片段长度多态性技术安全、稳定、可靠,具有广阔的临床应用前景；但为进一步提高临床样品的检出率,实验条件尚有待优化。
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.
感染是骨科临床实践中重点关注的问题之一,无论是假体相关性感染还是诸如开放性骨折、慢性骨髓炎等其它骨科感染,往往具有病原学复杂,抗生素耐药等问题,而对诸如“无菌性假体”松动这样的临床诊断中到底有无细菌的存在更是莫衷一是,争议颇多。临床传统应用的微生物纯培养方法对于这些问题的回答力有未逮。脱胎于坏境工程学科的分子生物学技术末端限制性片段长度多态性(Terminal Restriction Fragment Length Polymorphism, T-RFLP)通过对系统中微生物的标记DNA-16S rDNA—的检测,获取许多长度不同的末端限制性片段(Terminal Restriction Fragment, T-RF)或称分类操作单元(Operational Taxonomic Unit, OTU),从而可以全景式的解析系统中所有细菌群落的结构,这尤其对于检测那些临床传统方法不能培养的细菌意义重大。近年来T-RFLP方法正被逐渐应用于临床病原菌的检测,例如肠道菌群、阴道菌群等,但应用于骨科感染的研究尚未见报道。
研究目的：
1.应用T-RFLP技术分析怀疑感染的骨科标本中细菌群落分布情况；
2.比较T-RFLP技术得到的结果与临床传统纯培养结果的异同；
3.初步评价该技术的优缺点及其应用于临床的前景。
研究方法：
1.实验对象：骨科感染标本(包括假体相关性和非假体相关性),其它科感染标本；
2.细菌检测方法：T-RFLP,临床纯培养；
3.分析方法：i.对T-RFLP自身结果进行聚类分析；ii.对两者的比较则应用克隆文库测序、TAP (T-RFLP Analysis Program)和临床结果电子酶切的方法。
研究结果：
1.收集了59份临床样品,最终14份得到了T-RFLP谱图；
2.聚类分析显示,样品可按照解剖部位分类,每类中末端限制性片段(T-RFs)的分布具有一般性规律：其中膝关节样品6、11、12、17中丰度最高的片段都是80bp、82bp、85bp,其总丰度超过40%；脊椎样品30、47中丰度最高的片段都是479bp,总丰度超过60%；
3.对13号样品构建了克隆文库并测序,Blast分析(Basic Local Alignment Search Tool)序列结果为停乳链球菌,与临床培养结果一致；
4.临床已知培养结果者电子酶切分析得到的T-RFs大多与T-RFLP实测情况相符(为丰度最多的片段,且两者相差在1-3bp)。
结论：
本研究应用末端限制性片段长度多态性技术对骨科感染标本中细菌群落结构的分布进行了全景式的扫描分析,并同临床传统纯培养方法进行了比较,发现：
1.无论有无假体的存在,相同解剖部位的感染中细菌群落的分布都具有很高的相似性；
2.部分临床诊断为“无菌性假体松动”的病例中可能也是有细菌感染存在的,且其菌群分布规律与相同解剖部位感染患者亦相似；
3.通过对各种不同临床来源的样品处理以及与临床结果比对,证实了末端限制性片段长度多态性技术安全、稳定、可靠,具有广阔的临床应用前景；但为进一步提高临床样品的检出率,实验条件尚有待优化。
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.
引文
1. Hedrick, T.L., J.D. Adams, and R.G Sawyer, Implant-associated infections:an overview. J Long Term Eff Med Implants,2006.16(1):p.83-99.
2. Schierholz, J.M. and J. Beuth, Implant infections:a haven for opportunistic bacteria. J Hosp Infect,2001.49(2):p.87-93.
3. Mack, D., et al., Identification of three essential regulatory gene loci governing expression of Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm formation. Infect Immun,2000.68(7):p. 3799-807.
4. H, O.G.J.H., staphylococcus epidermidis biofilms: importance and implications. J Med. Microbiol.,2001.50(2001):p.1.
5. 刘琼,黄纪坚,林吉忠,等,铜绿假单胞菌致呼吸机相关性肺炎的危险因素和耐药性分析.重庆医学,2002.12(31)：p.1.
6. Schmidt, A.H. and M.F. Swiontkowski, Pathophysiology of infections after internal fixation of fractures. J Am Acad Orthop Surg,2000.8(5):p.285-91.
7. Gristina, A.G., Implant failure and the immuno-incompetent fibro-inflammatory zone. Clin Orthop Relat Res,1994(298):p.106-18.
8. Tang, L. and J.W. Eaton, Inflammatory responses to biomaterials. Am J Clin Pathol,1995.103(4):p.466-71.
9. Sanderson, P.J., Infection in orthopaedic implants. J Hosp Infect,1991.18 Suppl A:p.367-75.
10. Mousa, H.A., Infection following orthopaedic implants and bone surgery. East Mediterr Health J,2001.7(4-5):p.738-43.
11. Nelson, C.L., et al., Is aseptic loosening truly aseptic? Clin Orthop Relat Res, 2005(437):p.25-30.
12. 王化芬,王晓军,于茜,骨科感染病原菌谱与抗菌谱3年报告.中华矫形外科杂志,2007.15(4)：p.3.
13. 李金钟,谢正平,相青珍,段雄波,慢性化脓性骨髓炎病原菌分布特点及耐药性分析.河北中西医结合杂志,1997.6(5)：p.3.
14. Sia, l.G. and E.F. Berbari, Infection and musculoskeletal conditions: Osteomyelitis. Best Pract Res Clin Rheumatol,2006.20(6):p.1065-81.
15. 王洪媛,管华诗,江晓路,微生物生态学中分子生物学方法及T-RFLP技 术研究.中国生物工程杂志,2004.24(8)：p.5.
16. Sekiguchi, Y., et al., Phylogenetic diversity of mesophilic and thermophilic granular sludges determined by 16S rRNA gene analysis. Microbiology,1998. 144 (Pt 9):p.2655-65.
17. von Wintzingerode, F., U.B. Gobel, and E. Stackebrandt, Determination of microbial diversity in environmental samples:pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev,1997.21(3):p.213-29.
18. Fogel, G.B., et al., Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population. Microb Ecol,1999.38(2):p.93-113.
19. Lueders, T. and M.W. Friedrich, Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl Environ Microbiol,2003.69(1):p. 320-6.
20. Thies, F.L., W. Konig, and B. Konig, Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprinting. J Med Microbiol,2007.56(Pt 6):p.755-61.
21. Hayashi, H., et al., Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. J Med Microbiol.2005. 54(Pt 11):p.1093-101.
22. Christensen, J.E., et al., Rapid molecular diagnosis of lactobacillus bacteremia by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene. Clin Med Res,2004.2(1):p.37-45.
23. 袁三青,薛.高.,汪卫东,马延和,T-RFLP技术分析油藏微生物多样性.微生物学报,2007.47(2)：p.5.
24. Siala, M., et al., Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing. Arthritis Res Ther,2008.10(2):p. R40.
25. Siala, M., et al., Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis. Arthritis Res Ther,2009.11(4):p. R102.
1. Hedrick, T.L., J.D. Adams, and R.G. Sawyer, Implant-associated infections:an overview. J Long Term Eff Med Implants,2006.16(1):p.83-99.
2. Schierholz, J.M. and J. Beuth, Implant infections:a haven for opportunistic bacteria. J Hosp Infect,2001.49(2):p.87-93.
3. Mack, D., et al., Identification of three essential regulatory gene loci governing expression of Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm formation. Infect Immun,2000.68(7):p. 3799-807.
4. H, O.G.J.H., staphylococcus epidermidis biofilms:importance and implications. J Med. Microbiol.,2001.50(2001):p.1.
5. 刘琼,黄纪坚,林吉忠,等,铜绿假单胞菌致呼吸机相关性肺炎的危险因素和耐药性分析.重庆医学,2002.12(31)：p.1.
6. Schmidt, A.H. and M.F. Swiontkowski, Pathophysiology of infections after internal fixation of fractures. J Am Acad Orthop Surg,2000.8(5):p.285-91.
7. Gristina, A.G., Implant failure and the immuno-incompetent fibro-inflammatory zone. Clin Orthop Relat Res,1994(298):p.106-18.
8. Tang, L. and J.W. Eaton, Inflammatory responses to biomaterials. Am J Clin Pathol,1995.103(4):p.466-71.
9. Sanderson, P.J., Infection in orthopaedic implants. J Hosp Infect,1991.18 Suppl A:p.367-75.
10. Mousa, H.A., Infection following orthopaedic implants and bone surgery. East Mediterr Health J,2001.7(4-5):p.738-43.
11. Nelson, C.L., et al., Is aseptic loosening truly aseptic? Clin Orthop Relat Res, 2005(437):p.25-30.
12.王化芬,王晓军,于茜,骨科感染病原菌谱与抗菌谱3年报告.中华矫形外科杂志,2007.15(4)：p.3.
13. 李金钟,谢正平,相青珍,段雄波,慢性化脓性骨髓炎病原菌分布特点及耐药性分析.河北中西医结合杂志,1997.6(5)：p.3.
14. Sia, I.G. and E.F. Berbari, Infection and musculoskeletal conditions: Osteomyelitis. Best Pract Res Clin Rheumatol,2006.20(6):p.1065-81
2. Schierholz, J.M. and J. Beuth, Implant infections:a haven for opportunistic bacteria. J Hosp Infect,2001.49(2):p.87-93.
3. Mack, D., et al., Identification of three essential regulatory gene loci governing expression of Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm formation. Infect Immun,2000.68(7):p. 3799-807.
4. H, O.G.J.H., staphylococcus epidermidis biofilms: importance and implications. J Med. Microbiol.,2001.50(2001):p.1.
5. 刘琼,黄纪坚,林吉忠,等,铜绿假单胞菌致呼吸机相关性肺炎的危险因素和耐药性分析.重庆医学,2002.12(31)：p.1.
6. Schmidt, A.H. and M.F. Swiontkowski, Pathophysiology of infections after internal fixation of fractures. J Am Acad Orthop Surg,2000.8(5):p.285-91.
7. Gristina, A.G., Implant failure and the immuno-incompetent fibro-inflammatory zone. Clin Orthop Relat Res,1994(298):p.106-18.
8. Tang, L. and J.W. Eaton, Inflammatory responses to biomaterials. Am J Clin Pathol,1995.103(4):p.466-71.
9. Sanderson, P.J., Infection in orthopaedic implants. J Hosp Infect,1991.18 Suppl A:p.367-75.
10. Mousa, H.A., Infection following orthopaedic implants and bone surgery. East Mediterr Health J,2001.7(4-5):p.738-43.
11. Nelson, C.L., et al., Is aseptic loosening truly aseptic? Clin Orthop Relat Res, 2005(437):p.25-30.
12. 王化芬,王晓军,于茜,骨科感染病原菌谱与抗菌谱3年报告.中华矫形外科杂志,2007.15(4)：p.3.
13. 李金钟,谢正平,相青珍,段雄波,慢性化脓性骨髓炎病原菌分布特点及耐药性分析.河北中西医结合杂志,1997.6(5)：p.3.
14. Sia, l.G. and E.F. Berbari, Infection and musculoskeletal conditions: Osteomyelitis. Best Pract Res Clin Rheumatol,2006.20(6):p.1065-81.
15. 王洪媛,管华诗,江晓路,微生物生态学中分子生物学方法及T-RFLP技 术研究.中国生物工程杂志,2004.24(8)：p.5.
16. Sekiguchi, Y., et al., Phylogenetic diversity of mesophilic and thermophilic granular sludges determined by 16S rRNA gene analysis. Microbiology,1998. 144 (Pt 9):p.2655-65.
17. von Wintzingerode, F., U.B. Gobel, and E. Stackebrandt, Determination of microbial diversity in environmental samples:pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev,1997.21(3):p.213-29.
18. Fogel, G.B., et al., Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population. Microb Ecol,1999.38(2):p.93-113.
19. Lueders, T. and M.W. Friedrich, Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts. Appl Environ Microbiol,2003.69(1):p. 320-6.
20. Thies, F.L., W. Konig, and B. Konig, Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprinting. J Med Microbiol,2007.56(Pt 6):p.755-61.
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