Abstract
Species identification of Nocardia is not straightforward due to rapidly evolving taxonomy, insufficient discriminatory power of conventional phenotypic methods and also of single gene locus analysis including 16S rRNA gene sequencing. Here we evaluated the ability of a 5-locus (16S rRNA, gyrB, secA1, hsp65 and rpoB) multilocus sequence analysis (MLSA) approach as well as that of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in comparison with sequencing of the 5’-end 606 bp partial 16S rRNA gene to provide identification of 25 clinical isolates of Nocardia. The 5’-end 606 bp 16S rRNA gene sequencing successfully assigned 24 of 25 (96%) clinical isolates to species level, namely Nocardia cyriacigeorgica (n = 12, 48%), N. farcinica (n = 9, 36%), N. abscessus (n = 2, 8%) and N. otitidiscaviarum (n = 1, 4%). MLSA showed concordance with 16S rRNA gene sequencing results for the same 24 isolates. However, MLSA was able to identify the remaining isolate as N. wallacei, and clustered N. cyriacigeorgica into three subgroups. None of the clinical isolates were correctly identified to the species level by MALDI-TOF MS analysis using the manufacturer-provided database. A small “in-house” spectral database was established incorporating spectra of five clinical isolates representing the five species identified in this study. After complementation with the “in-house” database, of the remaining 20 isolates, 19 (95%) were correctly identified to species level (score ≥ 2.00) and one (an N. abscessus strain) to genus level (score ≥ 1.70 and < 2.00). In summary, MLSA showed superior discriminatory power compared with the 5’-end 606 bp partial 16S rRNA gene sequencing for species identification of Nocardia. MALDI-TOF MS can provide rapid and accurate identification but is reliant on a robust mass spectra database.
Highlights
Nocardia species are ubiquitous environmental bacteria that cause suppurative infections in humans, including in the lung, central nervous system and skin [1]
Phylogenetic analysis of concatenated gyrB-16S-secA1-hsp65-rpoB sequences obtained from the 25 clinical isolates and 20 Nocardia type strain sequences clearly revealed five major clusters with bootstrap threshold values of 90% (Fig 1)
As strain PUNC020 was more phylogenetically closely related to N. wallacei strain DSM 45136T compared with N. transvalensis strain NRRL B-16037T (Fig 1), the isolate was assigned as N. wallacei
Summary
Nocardia species are ubiquitous environmental bacteria that cause suppurative infections in humans, including in the lung, central nervous system and skin [1]. 16S rRNA gene sequencing is unable to distinguish certain closely related species due to insufficient interspecies gene polymorphisms [6, 8], whilst unable to resolve certain species, e.g. Nocardia nova due to the presence of multiple yet different copies of this gene [9]. To overcome this limitation, other gene polymorphisms have been evaluated, such as those within the β-subunit of the type II DNA topoisomerase gene (gyrB) [10, 11], the subunit A of the SecA preprotein translocase gene (secA1) [12, 13], the 65-kDa heat shock protein gene (hsp65) [14, 15], and the β subunit of DNA-dependent RNA polymerase gene (rpoB) [11]. A multilocus sequence analysis (MLSA) scheme analyzing sequence polymorphisms of multiple Nocardia genes was reported to be accurate for the identification of known Nocardia species but the unveiling of novel species [16]
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