Christensen Jens Jørgen, Andresen Keld, Justesen Tage, Kemp Michael
National Reference Laboratory for Identification of Bacteria, Statens Serum Institut, Copenhagen, Denmark.
APMIS. 2005 Sep;113(9):621-8. doi: 10.1111/j.1600-0463.2005.apm_224.x.
Diagnostic tools for identification of bacteria have developed dramatically in the last decade. Sequencing of genes coding for rRNA has led to revolutionary insights into the phylogeny and taxonomy of bacteria, and to new demands on the service provided by national reference laboratories for identification of bacteria. At the Danish Reference Laboratory for Identification of Bacteria, partial 16S rDNA sequencing has been used since 2001 to identify "difficult" strains submitted for taxonomic elucidation. Experiences relating to phenotypic as well as 16S rDNA sequencing of the first 175 strains examined are presented. Approximately 2/3 of the strains were Gram-positive and 1/3 Gram-negative. One fifth of the strains were anaerobic, while 4/5 were either facultatively anaerobic or aerobic. Methodological agreement was seen for most strains at species and/or genus level. Methodological disagreement was relatively rare. In 1/6 of the strains valuable information was obtained from sequencing results, while for some strains identification was based primarily on the phenotypic results. Only a few strains could not be clearly identified by either method. A very large number of strains representing taxons ranging from facultatively anaerobic to aerobic and anaerobic species and genera, Gram-positive as well as Gram-negative, were successfully examined. Of the submitted strains many have only rarely been encountered as human pathogens. Thus, genotypic identification may result in recognition of hitherto seldom recognized or unrecognized bacteria as human pathogens, which will lead to a better understanding of the nature of human infections. It is self evident that we should focus on slowly growing, fastidious or 'difficult' organisms when using sequencing for national reference purposes. Short sequences (450-650 base pairs) seem sufficient for most identifications. Molecular bacterial identification is a powerful tool for national reference laboratories, enhancing both the speed and validity of examinations performed.
在过去十年中，用于鉴定细菌的诊断工具取得了巨大发展。对编码rRNA的基因进行测序，为细菌的系统发育和分类学带来了革命性的见解，也对国家参考实验室提供的细菌鉴定服务提出了新的要求。自2001年以来，丹麦细菌鉴定参考实验室一直使用部分16S rDNA测序来鉴定提交进行分类学阐明的“疑难”菌株。本文介绍了对首批175株菌株进行表型分析以及16S rDNA测序的相关经验。大约2/3的菌株为革兰氏阳性菌，1/3为革兰氏阴性菌。五分之一的菌株为厌氧菌，而4/5为兼性厌氧菌或需氧菌。在物种和/或属水平上，大多数菌株的方法学一致性良好。方法学上的分歧相对较少。在1/6的菌株中，从测序结果中获得了有价值的信息，而对于一些菌株，鉴定主要基于表型结果。只有少数菌株无法通过任何一种方法得到明确鉴定。成功检测了大量代表从兼性厌氧菌到需氧菌和厌氧菌的物种和属的菌株，包括革兰氏阳性菌和革兰氏阴性菌。在提交的菌株中，许多作为人类病原体很少被发现。因此，基因型鉴定可能会导致将迄今很少被认识或未被认识的细菌识别为人类病原体，这将有助于更好地理解人类感染的本质。不言而喻，在将测序用于国家参考目的时，我们应该关注生长缓慢、苛求或“疑难”的微生物。短序列（450 - 650个碱基对）似乎足以满足大多数鉴定需求。分子细菌鉴定是国家参考实验室的有力工具，可提高检测的速度和准确性。
