Molecular Methods for Identifying Mycobacteria, Nocardiae, and Fungi

正在进行的诺卡菌分离株的研究清楚地表明，分子方法对于临床分离株的准确物种水平鉴定是必要的。我们之前的研究表明，通过扩增16S rRNA和热休克蛋白基因的部分片段，然后对扩增子进行限制性内切片段长度多态性分析，可以获得快速准确的鉴定。我们最近对诺卡菌的另一个基因区secA1的研究结果已经发表，并证明了这个相对较短的区域通常比16S区域更有用，以准确识别这些生物。我们现在使用secA1基因区域测序作为我们鉴定诺卡菌分离物的常规程序；如果结果有任何不明确的地方，我们也会对16S rRNA基因区域进行测序，以获得额外的遗传信息，以试图解决不明确的问题。为了最大限度地提高我们使用secA1基因区域鉴定的可靠性，我们正在获取所有新描述的具有临床意义的诺卡菌种的型菌株。

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. In collaboration with investigators from the Centers for Disease Control and Prevention and the University of Texas Health Center at Tyler, we have published descriptions of two clinically significant species of Nocardia, N. blacklockiae and N. wallacei. 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 are participating in an inter-institutional study of susceptibility testing of Nocardia spp., with the goal of better standardizing the performance and interpretation of such testing. Testing of all isolates at five different centers has been completed, and the data from the study are currently being analyzed. 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. We have found that pyrosequencing, utilizing a hyper-variable internal transcribed spacer region, can reliably identify most common clinical isolates of yeast species, and, while somewhat less precise than conventional cycle sequencing, is less costly. A manuscript summarizing the results of our studies has recently been published.