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 ␤ 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.

Molecular identification of clinical Nocardia isolates from India

“…Although MALDI-TOF MS is evolving as an important technique for the identification of Nocardia, further refinement is essential to finally accept this technique for routine identification in the clinical laboratory. It has been proposed that the combination of two or more gene sequences may increase the confidence level of species-specific identification and even the detection of new species (McTaggart et al, 2010). In the present study, we compared the utility of sequencing the most promising genes, hsp65 and the 16S rRNA gene, for identification of 30 Indian clinical isolates of Nocardia spp.…”

“…In spite of the availability of many other molecular methods such as PCR restriction enzyme pattern analysis (Rodríguez-Nava et al, 2006), PCR-RFLP (Conville et al, 2000;Steingrube et al, 1995Steingrube et al, , 1997 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Segawa et al, 2015), gene sequencing remains the best method of identification of Nocardia spp. (McTaggart et al, 2010). Although MALDI-TOF MS is evolving as an important technique for the identification of Nocardia, further refinement is essential to finally accept this technique for routine identification in the clinical laboratory.…”

“…Currently, 16S rRNA gene sequencing is considered a better method for identification of Nocardia to the species level (Borriello et al, 2007), although certain isolates that have been found to have a high sequence similarity (99.8 %) from 16S rRNA sequences are recorded as distinct species by DNA-DNA hybridization (Conville et al, 2004;Rodríguez-Nava et al, 2006). McTaggart et al (2010) applied multilocus sequence analysis to 190 clinical isolates of Nocardia spp. using sequences of 16S rRNA, hsp65, RNA polymerase (rpoB), gyrase B of the b-subunit of DNA topoisomerase (gyrB) and SecA pre-protein translocase-subunit A (secA1) genes and found this technique to be reliable for accurate speciation of Nocardia (McTaggart et al, 2010).…”

“…McTaggart et al (2010) applied multilocus sequence analysis to 190 clinical isolates of Nocardia spp. using sequences of 16S rRNA, hsp65, RNA polymerase (rpoB), gyrase B of the b-subunit of DNA topoisomerase (gyrB) and SecA pre-protein translocase-subunit A (secA1) genes and found this technique to be reliable for accurate speciation of Nocardia (McTaggart et al, 2010). Due to its high discriminatory power and accuracy, multilocus sequence analysis has replaced DNA-DNA hybridization in taxonomy and speciation and for the study of intrageneric relationships (Shneyer, 2007).…”

Identification, Typing, and Phylogenetic Relationships of the Main Clinical Nocardia Species in Spain According to Their gyrB and rpoB Genes

“…However, the conservation of 16S can be an obstacle to distinguishing between closely related species, such as those of the Nocardia nova complex (5,6). To overcome this disadvantage and to establish phylogenetic relationships at the intra-and interspecies levels, other protein-encoding genes have been studied, such as the 65-kDa heat shock protein hsp65 (7), the essential secretory protein secA1 (8), gyrB, which is the ␤-subunit of DNA gyrase and a type II DNA topoisomerase (9), and rpoB, which is the ␤-subunit of DNA-dependent RNA polymerase (RNAP) (6).…”