Molecular Methods for Identifying Mycobacteria, Nocardiae, and Fungi

鉴定分枝杆菌、诺卡氏菌和真菌的分子方法

• 批准号: 7593103
• 负责人: Frank G Witebsky
• 金额: $ 6.5万
• 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/7593103
• 关键词: Clinical; Cloning; Collection; Data; Diagnostic; Discrimination; Futility; Genes; Genetic; Genus Mycobacterium; Goals; Growth; Heat shock proteins; Laboratories; Liquid substance; Manuscripts; Methods; Molds; Molecular; Morphology; Names; Nocardia; Numbers; Organism; Patients; Performance; Predisposition; Preparation; Procedures; Published Comment; Publishing; Reporting; Restriction Fragment Length Polymorphism Analysis; Ribosomal RNA; Speed; Structure; Suspension substance; Suspensions; Techniques; Test Result; Testing; Time; Yeasts; antimicrobial drug; base; clinically significant; fungus; microbial; pathogen; rRNA Genes; rapid technique; species difference

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. 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. To maximize the reliability of our identifications using the secA1 gene region, we are acquiring the type strains for all newly-described Nocardia species of clinical significance. 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 has recently been published, and we have commented on the futility of assigning different species names to isolates with such subtle differences among them that most diagnostic laboratories would be unable to distinguish one from another. We have also recently published our results of a study of Nocardia cyriacigeorgica, which has been considered by some to be a newly-recognized pathogen; however, we have shown that it is in fact a relatively common clinical isolate, and is only new in that it has now been formally described and named. Pyrosequencing is a promising new molecular technique for the rapid identification of microbial pathogens in clinical laboratories. Using our large collection of different species of clinical significance, we have investigated the utility of this procedure for identification of Nocardia species, and have found that the technique cannot accurately identify all of the species that are currently considered to be clinically significant. 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 participating in the organization of an inter-institutional study of susceptibility testing of Nocardia spp., with the goal of better standardizing the performance and interpretation of such testing; we hope to begin the actual testing of isolates in the next few months. We are not planning any additional studies of rapidly growing mycobacteria at present. We are, however, 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 have also been 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 studies thus far indicate that the procedure, utilizing the region spanning internal transcribed spacer genes 1 and 2, can provide not only more accurate identifications of yeast isolates, but more rapid ones as well. Our studies thus far have indicated that at least 38 species of yeast can be reliably identified utilizing this procedure. A manuscript summarizing our results with yeast isolates is in preparation.

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。 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。 为了最大限度地提高我们使用secA 1基因区域进行鉴定的可靠性，我们正在获取所有新描述的具有临床意义的诺卡氏菌属的模式菌株。 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。 最近发表了一份报告，总结了我们对这种含有多拷贝的分离株的一些研究，我们评论了为分离株分配不同的物种名称是徒劳的，这些分离株之间存在细微差异，大多数诊断实验室无法区分它们。 我们最近还发表了我们对Nocardia cyriacigeorgica的研究结果，这被一些人认为是一种新认识的病原体;然而，我们已经表明，它实际上是一种相对常见的临床分离株，并且只是新的，因为它现在已经被正式描述和命名。 焦磷酸测序技术是一种很有前途的新的分子生物学技术，可用于临床实验室快速鉴定微生物病原体。 使用我们收集的大量具有临床意义的不同菌种，我们研究了该方法用于鉴定诺卡氏菌属的实用性，发现该技术无法准确鉴定目前认为具有临床意义的所有菌种。 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。 我们参与组织了一项机构间诺卡氏菌敏感性试验研究，为了更好地标准化这种测试的性能和解释，我们希望在接下来的几个月里开始对分离株的实际测试。 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，我们正在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。 我们也一直在研究焦磷酸测序鉴定酵母菌株的效用。 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。 焦磷酸测序的研究表明，该程序，利用跨区域的内部转录间隔区基因1和2，不仅可以提供更准确的鉴定酵母菌株，但更快速的。 迄今为止，我们的研究表明，至少有38种酵母可以可靠地确定利用这一程序。 一份总结我们用酵母菌分离物所得结果的手稿正在准备中。