Molecular Methods for Identifying Mycobacteria, Nocardia

诺卡氏菌属分离株的正在进行的研究。已经清楚地表明，分子方法是必要的，准确的物种水平的鉴定临床分离株。我们以前的研究表明，快速和准确的鉴定，可以通过扩增的16 S rRNA和热休克蛋白基因的部分，然后通过限制性片段长度多态性分析得到的扩增子。使用这种方法需要获得所有描述的具有临床意义的物种的模式菌株，以保持RFLP模式的数据库是最新的;此外，它也是相当劳动密集型的。我们最近的研究结果的另一个基因区域，secA 1，用于鉴定诺卡氏菌。已经发表，并且已经证明，这个相对短的区域通常比16 S区域对于准确鉴定这些生物体更有用。我们现在使用secA 1基因区的测序作为我们鉴定诺卡氏菌分离株的常规程序;如果结果以任何方式不明确，我们还对完整的16 S rRNA基因区进行测序，以获得额外的遗传信息，试图解决不明确的问题。 我们对诺卡氏菌临床分离株进行了详细的分子分析。已经揭示了几个含有16 S rRNA基因的许多不同拷贝。一些分离株的不同基因的克隆和测序显示，没有一个序列与目前描述的任何物种的16 S序列相同。在一种情况下，临床分离株和分离株之间的DNA-DNA杂交与患者分离株中的序列之一非常相似，但不相同，证明这两种分离株不是同种的。目前正在编写一份报告，总结我们对这种含有多拷贝的分离株的一些研究。 焦磷酸测序是一种很有前途的新的分子生物学技术，用于临床实验室快速鉴定微生物病原体，但其适用性诺卡氏菌属。尚未被研究。利用我们大量收集的具有临床意义的不同物种，我们将在未来几个月内研究这种程序在此类鉴定中的实用性。 诺卡氏菌属之间存在种间差异。对抗菌剂的敏感性，以及可能的种内变异性。然而，由于其生长相对缓慢，并且在液体介质中倾向于形成团块，而不是更均匀的悬浮液，因此在解释敏感性试验结果时难以获得实验室内和实验室间的一致性。我们正在开展一项机构间诺卡氏菌属敏感性试验研究，目的是更好地标准化这种测试的性能和解释。 我们目前没有计划对快速生长的分枝杆菌进行任何额外的研究。然而，我们将应用类似于我们对诺卡氏菌和快速生长菌所使用的技术来研究临床上重要的真菌。大多数霉菌分离物的鉴定仍然几乎完全基于形态学。然而，霉菌分离物可能需要一些时间来形成允许进行鉴定的结构，并且在某些情况下，这种结构永远不会形成。为了提高霉菌鉴定的速度和准确性，我们将探索使用不同基因区域的焦磷酸测序的可行性，特别是将研究使用不同基因和基因区域用于不同霉菌组的可行性。我们还将研究焦磷酸测序用于鉴定酵母分离株的实用性。在形态学上，许多酵母物种彼此非常相似，并且只有少量的表型测试可用于酵母鉴定的事实使得通过表型测试进行区分相对不可靠。焦磷酸测序不仅可以提供更准确的酵母分离株鉴定，而且可以提供更快速的鉴定。

Ongoing studies of isolates of Nocardia spp. have clearly demonstrated that molecular methods are necessary for the accurate species-level identification of clinical isolates. Our previous studies showed that rapid and accurate identifications could be obtained by amplification of portions of the 16S rRNA and heat-shock protein genes, followed by restriction fragment length polymorphism analysis of the resulting amplicons. The use of this procedure requires the acquisition of the type strain of all described species of clinical significance in order to keep the database of RFLP patterns current; additionally it is also quite labor-intensive. Results of our recent studies of another gene region, secA1, for the identification of Nocardia spp. have been published, and have demonstrated that this relatively short region is generally even more useful than the 16S region for accurate identification of these organisms. We now use sequencing of the secA1 gene region as our routine procedure for identification of Nocardia isolates; if the results are in any way ambiguous, we also sequence the full 16S rRNA gene region to obtain additional genetic information to try to resolve the ambiguity . Our detailed molecular analyses of clinical isolates of Nocardia spp. have revealed that several contain a number of differing copies of the 16S rRNA gene. Cloning and sequencing of the different genes has for some isolates revealed that none of the sequences is identical to the 16S sequence for any currently described species. In one case, DNA-DNA hybridization between the clinical isolate and an isolate with a 16S gene sequence very similar, but not identical, to one of the sequences in the patient isolate demonstrated that the two isolates were not conspecific. A report summarizing some of our studies with such multiple-copy-containing isolates is currently in preparation. Pyrosequencing is a promising new molecular technique for the rapid identification of microbial pathogens in clinical laboratories, but its applicability to Nocardia spp. has not yet been studies. Using our large collection of different species of clinical significance, we will be investigating the utility of this procedure for such identifications over the next several months. There are inter-species differences among Nocardia spp. in their susceptibility to antimicrobial agents, and probably intra-species variability as well. However, because of their relatively slow growth and their tendency to form clumps, rather than more homogeneous suspensions, in liquid media, there have been difficulties in obtaining intra- and inter-laboratory concordance in interpretation of susceptibility testing results. We are embarking on an inter-institutional study of susceptibility testing of Nocardia spp., with the goal of better standardizing the performance and interpretation of such testing. We are not planning any additional studies of rapidly growing mycobacteria at present. We will, however, be applying techniques similar to those we have used with both Nocardia and rapid growers to the study of clinically significant fungi. The identification of most mold isolates is still based almost entirely on morphology. However, it may take some time for a mold isolate to develop the structures that will allow an identification to be made, and in some cases such structures never develop. In an attempt to enhance both the speed and accuracy of mold identification, we will be exploring the feasibility of using pyrosequencing of various gene regions, and will in particular investigate the feasibility of using different genes and gene regions for different groups of molds. We will also be investigating the utility of pyrosequencing for the identification of yeast isolates. Morphologically, many yeast species are quite similar to one another, and the fact that only a small number of phenotypic tests are available for yeast identification makes discrimination by phenotypic testing relatively unreliable. Pyrosequencing may be able to provide not only more accurate identifications of yeast isolates, but more rapid ones as well.