Molecular Methods for Identifying Mycobacteria, Nocardiae, and Fungi

Clinical Center

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Abstract

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.[unreadable] [unreadable] 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.[unreadable] [unreadable] 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. [unreadable] [unreadable] 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.[unreadable] [unreadable] 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.[unreadable] [unreadable] 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.