Molecular & Phenotypic Methods for Identifying Bacteria

分子

• 批准号: 7215823
• 负责人: Frank G Witebsky
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7215823
• 关键词: Mycobacterium; Nocardiaceae; bacterial genetics; phenotype; polymerase chain reaction; restriction fragment length polymorphism

Polymerase chain reaction (PCR) amplification of portions of the genome of both rapidly growing mycobacteria and nocardiae, followed by restriction fragment length polymorphism (RFLP) analysis of the amplification products, has proven to be a useful technique in the diagnostic laboratory. Identification of many isolates to the species level can be obtained within a few days of organism isolation using this technique, as compared with the month or more required for conventional identification based on biochemical testing. In addition, these molecular procedures allow better discrimination among species and subspecies than is possible with biochemical testing, and facilitate the detection of hitherto undescribed species. Our work with two different areas of the Nocardia genome (a portion of the gene for 16S ribosomal RNA and a portion of the gene for the heat-shock protein) has resulted in our detecting clinically significant isolates belonging both to species of Nocardia that have rarely been recognized as pathogens (such as Nocardia veterana), and to species that have been hitherto undescribed. We have recently described the new species Nocardia kruckzakiae, and work is ongoing with a number of other clinical isolates that probably belong to additional as yet undescribed species. We have collaborated with the research group of Dr. Richard J. Wallace, Jr., in an investigation of the extent of hererogeneity in the isolates in their collection that had been previously identified as belonging to the species Nocardia nova, and have found that a significant fraction of the isolates actually belong to other newly described or as yet undescribed species. We have characterized several interesting Nocardia isolates we have found that possess several different 16S rRNA gene copies per cell. In some Nocardia nova isolates that we have analyzed, we have found two 16S rDNA gene copies, which differ from each other in base pair composition. To date, in four different isolates, we have found four different patterns of 16S gene distribution. The fact that all four belong to the same species has been confirmed using DNA-DNA hybridization. A summary of our findings with these isolates has been published, and work with other isolates containing multiple different 16S rDNA genes is ongoing. Much further work is needed to determine the taxonomic and physiological significance of these genetic differences within and between species, as well as to assess such possible species-specific differences in geographic distribution, pathogenicity, and antimicrobial susceptibility as may exist. There is variablity among aerobic actinomycete isolates in their susceptibility to antimicrobial agents. The Clinical Laboratory Standards Institute has recently published a standard which contains recommendations for the procedures to be used in the susceptibility testing of these organisms. However interpreting the endpoint with certain drug-organism combinations remains difficult, and we hope to embark in the near future on an inter-institutional collaboration to assess the reproducibility of the currently recommended recommendations and the possible need for some procedural alterations.

聚合酶链反应（PCR）扩增快速生长的分枝杆菌和诺卡氏菌基因组的一部分，然后对扩增产物进行限制性片段长度多态性（RFLP）分析，已被证明是诊断实验室中有用的技术。与基于生化检测的常规鉴定所需的一个月或更长时间相比，使用该技术可以在微生物分离的几天内获得许多分离株的种水平鉴定。此外，这些分子程序允许更好的物种和亚种之间的区别比可能与生化检测，并促进迄今未描述的物种的检测。我们对诺卡氏菌基因组的两个不同区域（16 S核糖体RNA基因的一部分和热休克蛋白基因的一部分）的研究导致我们检测到临床上显著的分离株，这些分离株既属于很少被认为是病原体的诺卡氏菌（如兽医诺卡氏菌），也属于迄今未被描述的物种。我们最近描述了一个新的种克氏诺卡氏菌，并正在进行一些其他的临床分离株，可能属于其他尚未描述的物种。我们与小理查德·J·华莱士博士的研究小组合作，在对他们收集的先前被鉴定为属于诺卡氏菌种的分离物中的异源性程度的调查中，发现相当大部分的分离物实际上属于其他新描述的或尚未描述的种。我们已经确定了几个有趣的诺卡氏菌分离株，我们发现每个细胞具有几个不同的16 S rRNA基因拷贝。在我们分析的一些诺卡氏菌分离株中，我们发现了两个16 S rDNA基因拷贝，它们在碱基对组成上彼此不同。到目前为止，在四个不同的分离物中，我们发现了四种不同的16 S基因分布模式。使用DNA-DNA杂交证实了这四种都属于同一物种的事实。我们对这些分离株的研究结果的总结已经发表，对含有多种不同16 S rDNA基因的其他分离株的研究正在进行中。需要进一步的工作，以确定这些遗传差异的分类学和生理学意义的物种内和物种之间，以及评估这种可能存在的地理分布，致病性和抗菌药物敏感性的物种特异性差异。不同的好氧放线菌对抗菌药物的敏感性存在差异。临床实验室标准研究所最近发布了一项标准，其中包含对这些微生物敏感性试验中使用的程序的建议。然而，解释某些药物-微生物组合的终点仍然很困难，我们希望在不久的将来开始进行机构间合作，以评估目前推荐的建议的可重复性和可能需要的一些程序改变。