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. 2000 Jan;38(1):158-64.
doi: 10.1128/JCM.38.1.158-164.2000.

Identification of nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene

P S Conville  1 , S H Fischer, C P Cartwright, F G Witebsky
Affiliations

Identification of nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene

P S Conville et al. J Clin Microbiol. 2000 Jan.

Abstract

Identification of clinical isolates of Nocardia to the species level is important for defining the spectrum of disease produced by each species and for predicting antimicrobial susceptibility. We evaluated the usefulness of PCR amplification of a portion of the Nocardia 16S rRNA gene and subsequent restriction endonuclease analysis (REA) for species identification. Unique restriction fragment length polymorphism (RFLP) patterns were found for Nocardia sp. type strains (except for the N. asteroides type strain) and representative isolates of the drug pattern types of Nocardia asteroides (except for N. asteroides drug pattern type IV, which gave inconsistent amplification). A variant RFLP pattern for Nocardia nova was also observed. Twenty-eight clinical isolates were evaluated both by traditional biochemical identification and by amplification and REA of portions of the 16S rRNA gene and the 65-kDa heat shock protein (HSP) gene. There was complete agreement among the three methods on identification of 24 of these isolates. One isolate gave a 16S rRNA RFLP pattern consistent with the biochemical identification but was not identifiable by its HSP gene RFLP patterns. Three isolates gave 16S rRNA RFLP patterns which were inconsistent with the identification obtained by both biochemical tests and HSP gene RFLP; sequence analysis suggested that two of these isolates may belong to undefined species. The PCR and REA technique described appears useful both for the identification of clinical isolates of Nocardia and for the detection of new or unusual species.

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Figures

FIG. 1
FIG. 1
Bio-Rad-derived RFLP patterns of various reference strains of Nocardia species. Lanes 1, 6, and 16, base pair ladder, with lengths indicated at left. (A) HinP1I digests. Lane 2, N. transvalensis and N. asteroides drug pattern type II; lane 3, N. farcinica; lane 4, N. asteroides type strain, N. asteroides drug pattern type I, N. brasiliensis, N. nova variant, N. asteroides drug pattern type VI, N. nova, and N. pseudobrasiliensis; lane 5, N. otitidiscaviarum. (B) DpnII digests. Lane 7, N. asteroides drug pattern type II; lane 8, N. asteroides drug pattern type VI and N. otitidiscaviarum; lane 9, N. pseudobrasiliensis; lane 10, N. asteroides type strain, N. asteroides drug pattern type I, N. brasiliensis, N. nova variant, N. transvalensis, and N. farcinica; lane 11, N. nova. (C) SphI and BstEII digests. Lane 12, N. brasiliensis (SphI); lane 13, N. asteroides type strain, N. asteroides drug pattern type I, and N. nova variant (SphI); lane 14, N. asteroides drug pattern type I (BstEII); lane 15, N. nova variant (BstEII).
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