Phylogeny and identification of Nocardia species on the basis of multilocus sequence analysis

Abstract

Nocardia species identification is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA and cellular fatty acid analysis to discriminate many species, and the unreliability of biochemical testing. Here, Nocardia species identification was achieved through multilocus sequence analysis (MLSA) of gyrase B of the β subunit of DNA topoisomerase (gyrB), 16S rRNA (16S), subunit A of SecA preprotein translocase (secA1), the 65-kDa heat shock protein (hsp65), and RNA polymerase (rpoB) applied to 190 clinical, 36 type, and 11 reference strains. Phylogenetic analysis resolved 30 sequence clusters with high (>85%) bootstrap support. Since most clusters contained a single type strain and the analysis corroborated current knowledge of Nocardia taxonomy, the sequence clusters were equated with species clusters and MLSA was deemed appropriate for species identification. By comparison, single-locus analysis was inadequate because it failed to resolve species clusters, partly due to the presence of foreign alleles in 22.1% of isolates. While MLSA identified the species of the majority (71.3%) of strains, it also identified clusters that may correspond to new species. The correlation of the identities by MLSA with those determined on the basis of microscopic examination, biochemical testing, and fatty acid analysis was 95%; however, MLSA was more discriminatory. Nocardia cyriacigeorgica (21.58%) and N. farcinica (14.74%) were the most frequently encountered species among clinical isolates. In summary, five-locus MLSA is a reliable method of elucidating taxonomic data to inform Nocardia species identification; however, three-locus (gyrB-16S-secA1) or four-locus (gyrB-16S-secA1-hsp65) MLSA was nearly as reliable, correctly identifying 98.5% and 99.5% of isolates, respectively, and would be more feasible for routine use in a clinical reference microbiology laboratory.

FIG. 1.

An NJ tree constructed from the 2,190-bp concatenated gyrB-16S-secA1-hsp65-rpoB sequences of 237 strains. Strains were colored to represent species clusters inferred from sequence clusters. Each of the 36 type strains is labeled with its species name. Bootstrap values (500 replicates) for nodes leading to sequence clusters are indicated. Branches indicated in boldface were also recovered in the MP tree. The three subgroups of N. cyriacigeorgica are indicated A, B, and C. Also indicated are the six drug-type patterns (I, II, III, IV, V, VI) previously described (3, 4). Bar, 1% nucleotide substitutions.

FIG. 2.

An NJ tree constructed from the 2,190-bp concatenated gyrB-16S-secA-hsp65-rpoB sequences of 237 strains. Strains were colored to represent species identification on the basis of microscopic examination, biochemical testing, and cellular fatty acid analysis. Each of the 36 type strains is labeled with its species name. Bar, 1% nucleotide substitutions.