Molecular Methods for Identifying Mycobacteria, Nocardia

诺卡氏菌分离株的研究进展。已经清楚地表明，分子方法对于准确鉴定临床分离株的种级是必要的。我们以前的研究表明，通过扩增16S rRNA和热休克蛋白基因的部分片段，然后对扩增出的扩增产物进行限制性片段长度多态性分析，可以快速、准确地进行鉴定。使用这一程序需要获取所有具有临床意义的所描述物种的模式菌株，以保持RFLP模式数据库的最新；此外，这也是相当劳动密集型的。我们最近对鉴定诺卡氏菌的另一个基因区域secA1的研究结果。已经发表，并表明这个相对较短的区域通常比16S区域更有助于准确识别这些生物。我们现在使用secA1基因区域的测序作为鉴定诺卡氏菌分离株的常规程序；如果结果有任何不明确的地方，我们还会对完整的16S rRNA基因区域进行测序，以获得额外的遗传信息，试图解决这种不确定问题。 我们对诺卡氏菌临床分离株的详细分子分析。已发现其中几个含有多个不同的16S rRNA基因拷贝。对一些分离株的不同基因的克隆和测序表明，没有一个序列与目前描述的任何物种的16S序列相同。在一个案例中，临床分离株与16S基因序列与患者分离株中的一个序列非常相似但不完全相同的分离株之间的DNA-DNA杂交表明，这两个分离株不是同种的。一份总结我们对这种含有多拷贝分离株的研究的报告目前正在准备中。 焦磷酸测序是临床实验室快速鉴定微生物病原菌的一种很有前途的新分子技术，但其对诺卡氏菌的适用性较差。还没有被研究过。利用我们收集的大量具有临床意义的不同物种，我们将在接下来的几个月里调查这一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。他们对抗菌剂的敏感性，可能还有物种内的变异性。然而，由于它们的生长相对较慢，而且在液体介质中倾向于形成块状，而不是更均匀的悬浮液，在解释药敏试验结果时，很难获得实验室内和实验室间的一致性。我们正在开展诺卡氏菌药敏试验的机构间研究，目的是更好地标准化这种试验的性能和解释。 目前，我们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具有临床意义的真菌。大多数霉菌分离物的鉴定仍然几乎完全基于形态。然而，霉菌隔离可能需要一段时间才能形成能够进行识别的结构，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，我们将探索对不同基因区域进行焦磷酸测序的可行性，特别是将不同基因和基因区域用于不同组霉菌的可行性。我们还将研究焦磷酸测序在酵母菌分离鉴定中的应用。在形态上，许多酵母种类彼此非常相似，而且只有少量的表型试验可用于酵母鉴定，这使得表型试验的判别力相对较差。焦磷酸测序可能不仅能提供更准确的酵母菌分离鉴定，而且还能提供更快速的鉴定。

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.