Use of 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis for analysis of the microfloras of healing and nonhealing chronic venous leg ulcers
Use of 16S ribosomal DNA PCR and denaturing gradient gel electrophoresis for analysis of the microfloras of healing and nonhealing chronic venous leg ulcers
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DOI：
10.1128/jcm.42.8.3549-3557.2004
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发表时间：
2004-08-01
影响因子：
9.4
通讯作者：
Thomas, DW
中科院分区：
文献类型：
--
作者：
Davies, CE;Hill, KE;Thomas, DW
通过结合培养分析和PCR扩增16SrRNA基因产物的变性梯度凝胶电泳（DGGE），比较了8例愈合和10例未愈合的慢性下肢静脉溃疡的细菌菌群。微生物区系的培养分析显示，两种伤口类型中的大多数携带需氧菌葡萄球菌和假单胞菌。(89 80%）。DGGE分析的基础上选择的16S核糖体DNA的测序允许的菌株未检测到的文化手段的鉴定。相当有趣的是，发现超过40%的序列代表的生物体不是从伤口中培养出来的。DGGE图谱还显示，所有的伤口具有一个明显的共同带，确定为假单胞菌属测序。该PCR信号的强度表明，细菌负荷的非愈合伤口的假单胞菌愈合伤口相比，高得多，它可能被大大低估了文化分析。因此，本研究表明，DGGE可以提供有关慢性伤口菌群的有价值的额外信息，这些信息仅从文化分析中是不明显的。
The bacterial microfloras of 8 healing and 10 nonhealing chronic venous leg ulcers were compared by using a combination of cultural analysis and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene products. Cultural analysis of the microflora revealed that the majority of both wound types carried the aerobes Staphylococcus and Pseudomonas spp. (89 and 80%, respectively). Sequencing of 16S ribosomal DNAs selected on the basis of DGGE profiling allowed the identification of strains not detected by cultural means. Of considerable interest was the finding that more than 40% of the sequences represented organisms not cultured from the wound from which they were amplified. DGGE profiles also revealed that all of the wounds possessed one apparently common band, identified by sequencing as Pseudomonas sp. The intensity of this PCR signal suggested that the bacterial load of nonhealing wounds was much higher for pseudomonads compared to healing wounds and that it may have been significantly underestimated by cultural analysis. Hence, the present study shows that DGGE could give valuable additional information about chronic wound microflora that is not apparent from cultural analysis alone.