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

Abstract

This study compares the identification, typing, and phylogenetic relationships of the most prevalent clinical Nocardia species in Spain, as determined via sequence analysis of their housekeeping genes gyrB and rpoB, with the results returned by the gold standard 16S rRNA method. gyrB and rpoB analyses identified Nocardia abscessus, N. cyriacigeorgica, N. farcinica, and the N. nova complex, species that together account for more than half of the human nocardiosis cases recorded in Spain. The individual discriminatory power of gyrB and rpoB with respect to intraspecies typing, calculated using the Hunter-Gaston discriminatory index (HGDI), was generally high (HGDI, 0.85 to 1), except for rpoB with respect to N. farcinica (HGDI, 0.71). Phylogenetically, different degrees of intra-and interspecies microheterogeneity were observed for gyrB and rpoB in a group of 119 clinical strains. A single 16S haplotype was obtained for each species, except for the N. nova complex (8 types), while gyrB and rpoB were more polymorphic: N. abscessus had 14 and 18 haplotypes, N. cyriacigeorgica had 17 and 12, N. farcinica had 11 and 5, and the N. nova complex had 26 and 29 haplotypes, respectively. A diversity gradient was therefore seen, with N. farcinica at the bottom followed by N. abscessus and N. cyriacigeorgica in the middle and N. nova complex at the top. The complexity of the N. nova complex is highlighted by its six variations in the GyrB 126 AAAPEH motif. gyrB sequencing (with or without rpoB sequencing) offers a simple means for identifying the most prevalent Nocardia species in Spanish medical laboratories and for determining the intraspecific diversity among their strains. Members of the genus Nocardia are ramified Gram-positive bacilli that normally live in dust, sand, soil, decaying vegetation, and stagnant water (1). To date, nearly 99 Nocardia species have been identified (see NCBI taxonomy for Nocardia, http://www.ncbi.nlm .nih.gov/Taxonomy/Browser/wwwtax.cgi?idϭ1817 and http://www .bacterio.cict.fr/n/nocardia.html), but this number undergoes constant modification. Some one-third of Nocardia species are known human pathogens, and new pathogenic species causing different clinical problems are constantly being discovered (2, 3). Infection occurs mainly via the respiratory tract, later disseminating to other locations, such as the central nervous system. Localized infection is caused by traumatic injury and gives rise to abscesses (4).Partial 16S rRNA gene sequencing is the gold standard for identifying Nocardia spp. (1). 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 th...

AutochthonousNocardia cerradoensisInfection in Humans, Spain, 2011 and 2014

“…Species identification was performed by sequencing 1,188-bp and 401-bp fragments of the 16S rRNA ( 5 ) and hsp65 (65-kDa heat shock protein) ( 6 ) genes, respectively. A total of 3 N. cerradoensis isolates were identified; for these and other infrequently found species, we also analyzed 400-bp and 445-bp fragments of the rpoB (RNA polymerase B) ( 7 ) and secA1 (essential secretory protein SecA1) ( 8 ) genes. We then identified similar gene sequences in GenBank by using blastn (http://www.ncbi.nlm.nih.gov/blast).…”

First report ofNocardia fuscaisolated in humans

“…The identification of the N. fusca isolate was performed by sequencing a 1881 bp fragment of the 16S ribosomal RNA gene,4 of 401 bp of the hsp 65 using the primers described by Telenti et al ,5 of 400 bp of the rpoB 6 and of 445 bp of the secA1 7. The sequences of the four genes of the two isolates showed 100% similarity with the sequences of the N. fusca reference isolates of databases of the NCBI, leBIBI, the ribosomal database project and also with the sequences of the reference strains disposable at the National Center for Emerging and Zoonotic Infectious Disease of Centers for Disease Control and Prevention (Atlanta).…”

gyrB Analysis as a Tool for Identifying Nocardia Species and Exploring Their Phylogeny

“…gyrB has been used on its own in phylogenetic studies of Nocardia (2,3) and in multilocus sequence typing pattern strategies (4) and has allowed new species to be described (5,6). The phylogenetic relationships between very similar species can be difficult to establish when using 16S rRNA-based trees (7).…”

“…However, knowledge of gyrB sequences may help in this respect. Certainly, gyrB shows remarkable variation across Nocardia species and has been used in the identification, typing, and phylogenetic examination of populations of the more common Nocardia species in Spain (i.e., those involved in 67% of clinical cases: N. abscessus, N. cyriacigeorgica, N. farcinica, and N. nova) (3).…”

“…Isolates were grown, their DNA was extracted, sequencing was performed, and phylogenetic trees were constructed as previously described (3). Isolates that were Ն99.0% (for the 606-bp 16S fragment and full 16S gene sequence [8]) and Ն93.5% (for gyrB [2]) similar to those with sequences in the GenBank database were deemed to be of the same species.…”