Molecular & Phenotypic Methods for Identifying Bacteria

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部分基因组，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一项有用的技术。使用这项技术可以在生物体分离后的几天内对许多菌株进行物种水平的鉴定，而基于生化测试的常规鉴定需要数月或更长时间。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)的工作导致我们发现了具有临床意义的分离株，它们既属于很少被认为是病原体的诺卡氏菌物种(如兽医诺卡氏菌)，也属于迄今未被描述的物种。我们最近描述了新种克氏诺卡氏菌，与其他一些临床分离株的工作正在进行中，这些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，对他们收集的先前被鉴定为新诺卡氏菌的分离物中的遗传性程度进行了调查，并发现很大一部分分离物实际上属于其他新描述的或尚未描述的物种。我们已经鉴定了几个有趣的诺卡氏菌分离株，我们发现每个细胞都有几个不同的16S 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16S rDNA基因拷贝，它们的碱基对组成不同。到目前为止，我们在四个不同的分离株中发现了四种不同的16S基因分布模式。通过DNA-DNA杂交证实了这四个物种属于同一物种的事实。我们对这些分离物的发现摘要已经发表，与其他含有多个不同16S rDNA基因的分离物的工作正在进行中。需要做更多的工作来确定这些种内和种间遗传差异的分类学和生理学意义，以及评估可能存在的地理分布、致病性和抗菌素敏感性方面可能存在的物种特定差异。不同的好氧放线菌分离株对抗菌药物的敏感性不同。临床实验室标准研究所最近公布了一项标准，其中就这些微生物的敏感性测试程序提出了建议。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进行机构间合作，以评估目前建议的建议的可重复性，以及可能需要进行一些程序上的修改。

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.