Polymerase chain reaction (PCR) amplification of portions of the genome of both rapidly growing mycobacteria and nocardiae, followed by restriction fragment length polymorphism (RFLP) analysis of the amplification products, has proven to be a useful technique in the diagnostic laboratory. Identification of many isolates to the species level can be obtained within a few days of organism isolation using this technique, as compared with the month or more required for conventional identification based on biochemical testing. In addition, these molecular procedures allow better discrimination among species and subspecies than is possible with biochemical testing, and facilitate the detection of hitherto undescribed species. Our work with two different areas of the Nocardia genome (a portion of the gene for 16S ribosomal RNA and a portion of the gene for the heat-shock protein) has resulted in our detecting clinically significant isolates belonging both to species of Nocardia that have rarely been recognized as pathogens (such as Nocardia veterana), and to species that have been hitherto undescribed. We have recently described the new species Nocardia kruckzakiae, and work is ongoing with a number of other clinical isolates that probably belong to additional as yet undescribed species. We have collaborated with the research group of Dr. Richard J. Wallace, Jr., in an investigation of the extent of hererogeneity in the isolates in their collection that had been previously identified as belonging to the species Nocardia nova, and have found that a significant fraction of the isolates actually belong to other newly described or as yet undescribed species. We have characterized several interesting Nocardia isolates we have found that possess several different 16S rRNA gene copies per cell. In some Nocardia nova isolates that we have analyzed, we have found two 16S rDNA gene copies, which differ from each other in base pair composition. To date, in four different isolates, we have found four different patterns of 16S gene distribution. The fact that all four belong to the same species has been confirmed using DNA-DNA hybridization. A summary of our findings with these isolates has been published, and work with other isolates containing multiple different 16S rDNA genes is ongoing. Much further work is needed to determine the taxonomic and physiological significance of these genetic differences within and between species, as well as to assess such possible species-specific differences in geographic distribution, pathogenicity, and antimicrobial susceptibility as may exist. There is variablity among aerobic actinomycete isolates in their susceptibility to antimicrobial agents. The Clinical Laboratory Standards Institute has recently published a standard which contains recommendations for the procedures to be used in the susceptibility testing of these organisms. However interpreting the endpoint with certain drug-organism combinations remains difficult, and we hope to embark in the near future on an inter-institutional collaboration to assess the reproducibility of the currently recommended recommendations and the possible need for some procedural alterations.

Agency
National Institute of Health (NIH)
Institute
Clinical Center (CLC)
Type
Intramural Research (Z01)
Project #
1Z01CL010247-11
Application #
7215823
Study Section
(DLM)
Project Start
Project End
Budget Start
Budget End
Support Year
11
Fiscal Year
2005
Total Cost
Indirect Cost
Name
Clinical Center
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Palmore, Tara N; Shea, Yvonne R; Conville, Patricia S et al. (2009) ""Mycobacterium tilburgii,"" a newly described, uncultivated opportunistic pathogen. J Clin Microbiol 47:1585-7
Montero, C I; Shea, Y R; Jones, P A et al. (2008) Evaluation of Pyrosequencing technology for the identification of clinically relevant non-dematiaceous yeasts and related species. Eur J Clin Microbiol Infect Dis 27:821-30
Conville, Patricia S; Brown, June M; Steigerwalt, Arnold G et al. (2008) Nocardia wallacei sp. nov. and Nocardia blacklockiae sp. nov., human pathogens and members of the ""Nocardia transvalensis Complex"". J Clin Microbiol 46:1178-84
Conville, Patricia S; Witebsky, Frank G (2007) Organisms designated as Nocardia asteroides drug pattern type VI are members of the species Nocardia cyriacigeorgica. J Clin Microbiol 45:2257-9
Conville, Patricia S; Witebsky, Frank G (2007) Analysis of multiple differing copies of the 16S rRNA gene in five clinical isolates and three type strains of Nocardia species and implications for species assignment. J Clin Microbiol 45:1146-51
Conville, Patricia S; Zelazny, Adrian M; Witebsky, Frank G (2006) Analysis of secA1 gene sequences for identification of Nocardia species. J Clin Microbiol 44:2760-6
Conville, Patricia S; Witebsky, Frank G (2005) Multiple copies of the 16S rRNA gene in Nocardia nova isolates and implications for sequence-based identification procedures. J Clin Microbiol 43:2881-5
Conville, Patricia S; Brown, June M; Steigerwalt, Arnold G et al. (2004) Nocardia kruczakiae sp. nov., a pathogen in immunocompromised patients and a member of the ""N. nova complex"". J Clin Microbiol 42:5139-45
Cloud, Joann L; Conville, Patricia S; Croft, Ann et al. (2004) Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database. J Clin Microbiol 42:578-84
Conville, Patricia S; Brown, June M; Steigerwalt, Arnold G et al. (2003) Nocardia veterana as a pathogen in North American patients. J Clin Microbiol 41:2560-8

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