Molecular & Phenotypic Methods for Identifying Bacteria

对快速生长的分枝杆菌和诺卡菌的部分基因组进行聚合酶链反应（PCR）扩增，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。与基于生化测试的常规鉴定需要一个月或更长时间相比，使用该技术可以在生物分离后几天内获得许多物种水平的分离物的鉴定。此外，与生物化学测试相比，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）进行了研究，发现了临床意义重大的诺卡菌分离物，这些分离物既属于很少被认为是病原体的诺卡菌（如退伍诺卡菌），也属于迄今为止未被描述的诺卡菌。我们最近描述了新物种克氏诺卡菌，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工作。我们与Richard J. Wallace, Jr.博士的研究小组合作，对他们收集的分离株的异质性程度进行了调查，这些分离株以前被确定属于诺卡迪亚新星，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。我们已经描述了几个有趣的诺卡菌分离株，我们发现每个细胞具有几个不同的16S rRNA基因拷贝。在我们分析的一些诺卡菌分离株中，我们发现了两个在碱基对组成上不同的16S rDNA基因拷贝。迄今为止，在4个不同的分离株中，我们发现了4种不同的16S基因分布模式。事实上，这四个人属于同一物种已经通过DNA-DNA杂交得到证实。我们对这些分离株的研究结果总结已经发表，对其他含有多种不同16S rDNA基因的分离株的研究正在进行中。需要进一步的工作来确定物种内和物种间这些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、致病性和抗菌药物敏感性方面的物种特异性差异。各需氧放线菌对抗菌药物的敏感性存在差异。临床实验室标准协会最近发布了一项标准，其中包含用于这些生物体的敏感性测试的程序的建议。然而，用某些药物-有机体组合来解释终点仍然很困难，我们希望在不久的将来开展机构间合作，以评估目前推荐的建议的可重复性，以及可能需要进行一些程序更改。

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.