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.

聚合酶链式反应 (PCR) 扩增快速生长的分枝杆菌和诺卡氏菌的基因组部分，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的一项有用技术。使用该技术在生物体分离后几天内即可对许多分离株进行物种水平的鉴定，而基于生化测试的常规鉴定则需要一个月或更长时间。此外，与生化测试相比，这些分子程序可以更好地区分物种和亚种，并有助于检测迄今未描述的物种。我们对诺卡氏菌基因组的两个不同区域（16S 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了属于诺卡氏菌属物种的具有临床意义的分离株，这些分离株很少出现在诺卡氏菌属物种中。被认为是病原体（例如退伍诺卡氏菌），以及迄今为止尚未被描述的物种。我们最近描述了新种克氏诺卡氏菌，并且正在对可能属于其他尚未描述的物种的许多其他临床分离株进行研究。我们与 Richard J. Wallace, Jr. 博士的研究小组合作，调查了他们收集的分离株的异质性程度，这些分离株先前被鉴定为属于新诺卡氏菌种，并发现很大一部分分离株实际上属于其他新描述或尚未描述的物种。我们对几种有趣的诺卡氏菌分离株进行了表征，我们发现它们每个细胞拥有多个不同的 16S rRNA 基因拷贝。在我们分析的一些新诺卡氏菌分离株中，我们发现了两个 16S rDNA 基因拷贝，它们的碱基对组成彼此不同。迄今为止，在四个不同的分离株中，我们发现了四种不同的 16S 基因分布模式。 DNA-DNA 杂交已证实这四种病毒属于同一物种。我们对这些分离株的研究结果摘要已经发表，并且对含有多种不同 16S rDNA 基因的其他分离株的研究正在进行中。需要做更多的工作来确定物种内和物种间遗传差异的分类学和生理学意义，并评估可能存在的地理分布、致病性和抗菌敏感性方面的物种特异性差异。需氧放线菌分离株对抗菌剂的敏感性存在差异。临床实验室标准研究所最近发​​布了一项标准，其中包含对这些生物体敏感性测试中使用的程序的建议。然而，用某些药物-生物体组合来解释终点仍然很困难，我们希望在不久的将来开始机构间合作，以评估当前推荐建议的可重复性以及可能需要进行一些程序改变。