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 126AAAPEH 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.
INTRODUCTION
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?id=1817 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 β-subunit of DNA gyrase and a type II DNA topoisomerase (9), and rpoB, which is the β-subunit of DNA-dependent RNA polymerase (RNAP) (6).
gyrB promotes negative supercoiling in the bacterial chromosome during DNA replication, while rpoB is involved in transcription. These protein-encoding genes offer advantages over RNA-encoding genes as molecular markers; as housekeeping genes, they are less susceptible to horizontal gene transfer and can be analyzed at the nucleotide and amino acid levels to determine phylogenetic relationships (10).
The majority of phylogenetic studies on Nocardia spp. have involved just one strain that is representative of each species, taking into account the combinations of many genes (6, 11). No studies, however, have been performed that have examined large numbers of clinical strains per species.
The aims of the present work were to (i) compare a sequence analysis of gyrB and rpoB against partial 16 rRNA gene sequencing (the gold standard) for identifying, typing, and determining the phylogenetic relationships between clinical strains of N. abscessus, N. cyriacigeorgica, N. farcinica, and the N. nova complex (collectively the most prevalent clinical Nocardia species in Spain), and (ii) to gain insight into the intraspecific diversity of these strains via the analysis of these housekeeping genes.
MATERIALS AND METHODS
Nocardia strains.
A total of 119 strains of Nocardia spp., belonging to the four species most commonly collected in Spain, N. abscessus, N. cyriacigeorgica, N. farcinica, and the N. nova complex, were isolated from clinical samples submitted for identification to the Spanish National Center of Microbiology (Majadahonda, Madrid, Spain) between 2006 and 2010. Isolates were grown in heart infusion Columbia agar supplemented with 5% (vol/vol) sheep blood and buffered charcoal-yeast extract (BCYE) for 48 to 72 h at 37°C under aerobic conditions.
16S, gyrB, and rpoB sequencing.
DNA was extracted by the boiling method. Amplification was performed using PuReTaq Ready-To-Go PCR beads (Amersham Biosciences, Buchinghamshire, United Kingdom) under the following conditions: 30 cycles at 95°C for 1 min, primer annealing (5 pM) at 55°C for 1 min, and extension at 72°C for 1.5 min. The primers used for 16S amplification and sequencing were 5′-GCTTAACACATGCAAGTCG-3′ and 5′-GAATTCCAGTCTCCCCTG-3′ (8), for gyrB they were 5′-GAGGTCGTCATGACCCAGCTGCA-3′ and 5′-GTCTTGGTCTGGCCCTCGAACTG-3′ (9), and for rpoB they were 5′-CGACCACTTCGGCAACCG-3′ and 5′-TCGATCGGGCACATCCGG-3′ (12). The amplification products were electrophoresed and purified using ExoSAP-IT reagent (GE Healthcare, NJ, USA) and sequenced by capillary electrophoresis in an ABI Prism 3100 apparatus (Applied Biosystems, Foster City, CA, USA).
16S, gyrB, and rpoB analyses.
Sequences were assembled using SeqMan software (DNAStar, Inc., Madison, WI). The sequence lengths were adjusted to match the length of the shortest sequence of each species and aligned using the ClustalW algorithm (see http://www.ebi.ac.uk/Tools/clustalw2/index.html). The Hunter-Gaston discrimination index (HGDI) (13), single nucleotide polymorphisms (SNPs), haplotype numbers, and other variables (Table 1) were assessed using DnaSP software (14). A phylogenetic assessment of each species was undertaken using MEGA 4.1 software (15). Phylogenetic trees were constructed using the neighbor-joining (16), maximum-parsimony (17), and maximum-likelihood methods (18), with bootstrap analyses based on 1,000 resamplings. Branches corresponding to partitions that were reproduced in <50% of bootstrap replicates were collapsed. The evolutionary distances between the nucleotide and amino acid sequences of the rpoB and gyrB genes were determined using the Kimura 2-parameter model and the Poisson correction model (19). N. farcinica strain DSM 43665T (GenBank accession no. NC_006361) was used as an outgroup (see http://nocardia.nih.go.jp/), except in N. farcinica analyses, in which N. abscessus strain DSM 44432T (GenBank accession no. JN041489 for 16S, AB447398 for gyrB, and JN215593 for rpoB) was employed.
Table 1.
Characteristics of the four most prevalent species of Nocardia isolated from clinical samples in Spain between 2006 and 2010
| Nocardia species (no. of strains) | Genes (bp)a | No. of haplotypes (HGDI, S2, SD)b | No. of SNPs (divergence rate)c | SNPs per strain (avg, mode) |
|---|---|---|---|---|
| N. abscessus (29) | 16S rRNA (409) | 1 | 0 | 0 |
| gyrB (653) | 14 (0.889, 0.002, 0.046) | 24 (0.0–1.7) | 0–9 (3, 2) | |
| rpoB (354) | 18 (0.948, 0.00058, 0.024) | 65 (0.0–16) | 6–49 (42, 44) | |
| N. cyriacigeorgica (30) | 16S rRNA (514) | 1 | 0 | 0 |
| gyrB (727) | 17 (0.94, 0.00055, 0.023) | 77 (0.0–5.6) | 0–38 (28, 36/38) | |
| rpoB (354) | 12 (0.88, 0.00135, 0.037) | 24 (0.0–4.4) | 4–15 (9, 13) | |
| N. farcinica (31) | 16S rRNA (507) | 1 | 0 | 0 |
| gyrB (739) | 11 (0.854, 0.0014, 0.038) | 11 (0.0–0.8) | 0–6 (3, 2/4) | |
| rpoB (351) | 5 (0.716, 0.001, 0.036) | 9 (0.0–1.4) | 4–6 (4, 4) | |
| N. nova (29) | 16S rRNA (529) | 8 (0.672, 0.00739, 0.086) | 8 (0.0–1.1) | 0–4 (1, 2) |
| gyrB (698) | 26 (0.985, 0.0003, 0.017) | 51 (0.0–5.2) | 0–36 (22, 34) | |
| rpoB (285) | 29 (1.0, 0.00008, 0.009) | 49 (0.7–12.6) | 16–44 (32, 31) |
Analyzed size in number of base pairs.
HGDI, Hunter-Gaston discriminatory index; S2, variance.
SNP, single nucleotide polymorphism; the divergence rate is expressed as a percentage among strains of each group.
Species assignation and assignment of detected polymorphisms.
The 16S, gyrB, and rpoB fragments sequenced for each Nocardia strain were compared to sequences in the GenBank database and identified using BLAST (version 2.2.10; see http://www.ncbi.nlm.nih.gov/BLAST) and the Bioinformatics Bacteria Identification (BIBI) version 0.2 software (see http://umr5558-sud-str1.univ-lyon1.fr/lebibi/lebibi.cgi). A similarity score of ≥99.0% between the 16S rRNA sequence and database sequence(s) was deemed to indicate that strains belonged to the same species (20). Species assignment was confirmed via the greatest similarity with respect to the 16S, gyrB, and rpoB sequences. To detect polymorphisms and their genome positions, the following type strains were used: N. abscessus DSM 44432T (GenBank accession no. JN041489 for 16S, AB447398 for gyrB, and JN215593 for rpoB), N. cyriacigeorgica DSM 44484T (GenBank accession no. GQ376180 for 16S, GQ496121 for gyrB, and JN215664 for rpoB), N. farcinica DSM 43665T (GenBank accession no. GQ217499 for 16S, GQ496115 for gyrB, and DQ085117 for rpoB), and N. nova DSM 44481T (GenBank accession no. GQ376190 for 16S, GQ496102 for gyrB, and JN215754 for rpoB).
RESULTS
Between 2006 and 2010, 698 clinical strains of Nocardia spp. were submitted to our center for identification by 16S analysis. The most prevalent Nocardia spp. responsible for severe clinical conditions were N. abscessus/N. asiatica (15.9%), N. cyriacigeorgica (24.5%), N. farcinica (13.6%), and the N. nova complex (13.0%). For each species, some 30 strains of different geographical origins and clinical backgrounds were selected (see Table S1 in the supplemental material).
Table 1 shows the corresponding HGDI and SNP variables (number, arithmetic mean and mode, etc.) for the 16S, gyrB, and rpoB genes for each species.
Analysis of 16S polymorphisms.
Twenty-nine N. abscessus strains shared a 16S rRNA haplotype that was also identical to that of N. abscessus strain DSM 44432T (GenBank accession no. JN041489) and N. asiatica DSM 44668T (GenBank accession no. DQ659897). This full similarity between N. abscessus and N. asiatica is a consequence of the fact that the current 16S fragment used in routine clinical testing does not include the area of difference (which extends from the middle of the gene to the 3′-end).
Thirty N. cyriacigeorgica strains had a common 16S haplotype, which was also that of N. cyriacigeorgica DSM 44484T (GenBank accession no. GQ376180). All 31 N. farcinica strains had the same 16S haplotype, which was identical to that of N. farcinica DSM 43665T (GenBank accession no. GQ217499). Eight 16S haplotypes were found in 29 N. nova complex strains with respect to the reference strain N. nova DSM 44481T (GenBank accession no. GQ376190) (similarity, ≥99%).
Analysis of gyrB polymorphisms.
Table 2 shows the identified GyrB amino acid substitutions, their types, and frequencies, along with the HGDI values and the percent divergence between the studied strains. Figure S1 in the supplemental material shows the inferred phylogenetic tree for the 4 studied species. The topologies of the trees produced by the neighbor-joining, maximum-parsimony, and maximum-likelihood methods were almost identical (data not shown).
Table 2.
GyrB amino acid polymorphisms identified in the N. abscessus (n = 29), N. cyriacigeorgica (n = 30), and N. nova (n = 29) strains
| Species |
Amino acid (nucleotides) at the indicated positions (1–4)a |
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N. farcinica DSM 43665b | 160 | 161 | 164 | 181 | 183 | 185 | 187 | 188 | 233 | 237 | 238 | 240 | 257 | 258 | 259 | 260 | 262 | 265 | 292 | 349 |
| N. abscessus strainsc | ||||||||||||||||||||
| DSM 44432b (n = 11) | 10 Glu (GAG) | 38 Lys (AAG) | 87 Asp (GAC) | 109 Ala (GCC) | ||||||||||||||||
| Mutatedb (n = 18) | Lys (AAG) | Gln (CAG) | Glu (GAA) | Ser (TCC) | ||||||||||||||||
| Frequency (%) | 3.4 | 3.4 | 58.6 | 10.3 | ||||||||||||||||
| N. cyriacigeorgica strainsd | ||||||||||||||||||||
| DSM 44484c (n = 8) | 29 Val (GTC) | 32 Ala (GCC) | 49 Ile (ATC) | 53 Pro (CCG) | 55 Thr (ACG) | 106 Thr (ACC) | 108 Val (GTC) | 125 Ala (GCC) | 126 Glu (GAG) | 127 Ala (GCG) | 128 Gln (CAG) | 130 Thr (ACC) | ||||||||
| Mutatedc (n = 22) | Ile (ATC) | Asp (GAC) | Val (GTT) | Ala (GCG) | Ser (TCC), Lys (AAG) | Ala (GCC) | Ile (ATC) | Glu (GAG) | Gln (CAG) | Thr (ACG) | Pro (CCG), Ala(GCG) | Ala (GCC) | ||||||||
| Frequency (%) | 3.3 | 3.3 | 3.3 | 43.3 | 36.7 6.6 | 6.6 | 20.0 | 23.3 | 23.3 | 23.3 | 66.6 3.3 | 50.0 | ||||||||
| N. nova strainse | ||||||||||||||||||||
| DSM 44481d (n = 8) | 51 Gly (GGT) | 101 Glu (GAG) | 105 Asp (GAT) | 126 Ala (GCG) | 129′ | 131 Hys (CAG) | 157 Pro (CCG) | 215 Val (GTG) | ||||||||||||
| Mutatedd (n = 21) | Asp (GAC), Ser (AGC) | Gln (CAG) | Glu (GAG) | Thr (ACC), Ser (AGC), Asn (AAC) | Pro (CCG) | Pro (CCG), Arg (CGG) | Ala (GCG) | Phe (TTC) | ||||||||||||
| Frequency (%) | 3.4, 3.4 | 62.0 | 62.0 | 44.8, 10.34, 3.4 | 65.5 | 62, 3.4 | 68.9 | 3.4 | ||||||||||||
Amino acid polymorphism positions (modified nucleotides are underlined) with respect to the complete genome sequence of reference strain N. farcinica DSM 43665T (accession no. NC_006361), N. abscessus DSM 44432T (accession no. AB447398), N. cyriacigeorgica DSM 44484T (accession no. GQ496121), and N. nova DSM 44481T (accession no. GQ496102).
All the N. farcinica strains harbored the same GyrB as N. farcinica DSM 43665T (accession no. NC_006361).
No. of GyrB types, 5; HGDI, 0.94; divergence, 0.5 to 1.4%.
No. of GyrB types, 9; HGDI, 0.94; divergence, 0.4 to 4.3%.
No. of GyrB types, 11; HGDI, 0.81; divergence, 0.4 to 2.6%.
(i) N. abscessus strains.
To confirm the above identifications that were made using the partial 16S sequence, several reference strains were used in gyrB analysis: N. abscessus DSM 44432T (GenBank accession no. AB447398) and N. asiatica DSM 44668T (GenBank accession no. GQ217495). All 29 studied strains showed a gyrB sequence that was similar (98.3 to 99.8%) to that of the reference strain N. abscessus DSM 44332T. None showed the sequence seen in N. asiatica DSM 44668T (similarity, 94.9 to 95.8%), which lacks three nucleotides possessed by N. abscessus DSM 44432T (109Ala [GCC]-110Ala [GCG] is modified to 109Ala [GCG]). The 14 gyrB haplotypes found fell into one SNP cluster group (SCG) along with N. abscessus DSM 44332T, and the gyrB haplotype of N. asiatica DSM 44668T lay outside this cluster (Table 1; see also Fig. S1 in the supplemental material). A total of 24 SNPs were detected.
The GyrB protein of all 29 strains harbored 109Ala-110Ala. Eighteen strains showed four amino acid substitutions with respect to the GyrB protein of N. abscessus DSM 44432T (accession no. BAH10710). The most common change was the appearance of 87Glu (seen as the only substitution in 13 strains but appearing in 58.6% of the strains in some combination with other changes). 10Lys, 38Gln, and 109Ser appeared in one, one, and three strains, respectively.
(ii) N. cyriacigeorgica strains.
Seventeen gyrB haplotypes were identified for N. cyriacigeorgica DSM 44484T (GenBank accession no. GQ496121) (similarity values, 95.6 to 99.9%), which fell into two SCGs. Seventy-seven SNPs were detected (Table 1; see also Fig. S1 in the supplemental material).
In comparison with the N. cyriacigeorgica DSM 44484T (GenBank accession no. ACV89678) GyrB reference sequence, 22 strains (73.3%) showed a total of 14 amino acid replacements in 12 locations, resulting in 9 GyrB protein types (Table 2). The amino acid changes accumulated in individual strains ranged from 1 to 7.
(iii) N. farcinica strains.
Eleven gyrB haplotypes were identified for N. farcinica DSM 43665T (GenBank accession no. GQ217499) (similarity, 99.2 to 99.9%) (Table 1), all of which fell into one SCG. Eleven SNPs were detected, all of which were silent.
(iv) N. nova complex strains.
In gyrB analysis, several reference sequences were taken into account due to the closeness of the N. nova complex members (21): N. nova DSM 44481T (GenBank accession no. GQ496102), N. aobensis DSM 44805T (GenBank accession no. JN041378), N. cerradoensis DSM 44546T (GenBank accession no. GQ496123), N. kruczakiae DSM 44877T (GenBank accession no. AB450793), N. elegans DSM 44890T (GenBank accession no. AB450785), and N. africana DSM 44491T (GenBank accession no. JN041368). Twenty-six gyrB haplotypes were found with respect to N. nova DSM 44481T (GenBank accession no. GQ496102) (83.1 to 99.9%), which fell into 2 SCGs. Fifty-one gyrB SNPs were detected. Almost 59% of the strains harbored ≥22 SNPs, with a maximum of 36 SNPs in a strain.
The GyrB protein of 21 strains showed a 129Pro insertion compared to the reference strain N. nova DSM 44481T (GenBank accession no. ACV89659) and 10 of the other studied strains. This difference thus divided them into two separate clades. Eleven GyrB protein types were seen. Eight amino acid changes were detected with respect to the sequence of the N. nova DSM 44481T protein (Table 2). The substitutions accumulated per strain ranged from 0 to 7 (mode, 6). The combination 101Gln-105Glu-129′Pro-131Pro/Arg-157Ala was seen in 17 strains with the 129Pro insertion, and 129′Pro-131Pro/Arg-157Ala was seen in two strains. The triple amino acid association 129′Pro-131Pro-157Ala was found in the N. aobensis, N. cerradoensis, and N. kruczakiae reference strains. In addition, the 126AAAPEH motif was seen in three GyrB protein types (possessed in 10 strains). However, in the strains with the 129Pro insertion (underlined below), this motif was changed to AAAPPEP in 1 GyrB protein type (two strains), to SAAPPEP in 2 GyrB protein types (3 strains) (SAAPPEP was also seen in the N. aobensis DSM 44805T and N. kruczakiae DSM 44877T reference strains), to TAAPPEP in 3 GyrB protein types (12 strains) (similar to the motif TATPPEP seen in the N. cerradoensis DSM 44546T reference strain), to TAAPPER in one GyrB protein type (one strain), and finally to NAAPPEP in 1 GyrB protein type (one strain) (see Fig. S3 in the supplemental material). Strains with the 129′Pro insertion and GyrB changes showed a range of similarity scores of 16S with respect to the N. nova DSM 44481T of 99.2% to 100%. These percentage scores decreased (∼1 to 2 percentage points) when the comparison was done with respect to the N. aobensis, N. cerradoensis, and N. kruczakiae reference strains.
Analysis of rpoB polymorphisms.
Figure S2 in the supplemental material shows the phylogenetic trees produced according to the neighbor-joining method that were based on the rpoB gene.
(i) N. abscessus strains.
Eighteen haplotypes were found for the 29 studied strains with respect to the rpoB gene of the reference strain N. abscessus DSM 44432T (GenBank accession no. JN215593) (similarity range, 84.0 to 99.7%). Sixty-five SNPs were detected. Thirty-three nucleotides were changed in >85% of the strains; these SNPs divided the 29 strains into two clades, with 26 strains in the larger clade. Nineteen of these changes resulted in 15 RpoB protein types (Table 3).
Table 3.
RpoB amino acid polymorphisms identified in the N. abscessus (n = 29) and N. nova (n = 29) strains
| Species | RpoB amino acid (nucleotides) at the indicated positions (1–6)a |
|||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| N. farcinica DSM 43665 | 357 | 358 | 361 | 365 | 367 | 376 | 378 | 405 | 413 | 414 | 420 | 423 | 425 | 427 | 429 | 430 | 434 | 435 | 438 | 451 | 456 | 464 | 465 | 471 |
| N. abscessus strainsb | ||||||||||||||||||||||||
| DSM 44432 (n = 11)c | 3 Leu (CTC) | 3 Asp (GAC) | 3 Gly (GGC) | 13 Ile (ATC) | 42 Ile (ATC) | 50 Ala (GCG) | 60 Leu (CTG) | 62 Gln (CAG) | 64 Met (ATG) | 66 Gln (CAG) | 67 Asn (AAC) | 71 Ser (TCG) | 72 Gly (GGC) | 75 Hys (CAC) | 88 Ser (TCC) | 101 Pro (CCG) | 102 Ser (TCG) | 108 Cys (TGC) | ||||||
| Mutated (n = 18) | Ile (ATC) | Ala (GCC) | Arg (CGC) | Met (ATG) | Met (ATG) | Gly (GGG) | Met (ATG) | Val (GTG), Glu (GAG) | Leu (CTG) | Glu (GAG) | Arg (CGC) | Ala (GCC) | Ser (AGC) | Gln (CAG) | Thr (ACC) | Tyr (TAC) | Thr (ACC) | Ala (GCC) | ||||||
| Frequency (%) | 3.4 | 3.4 | 3.4 | 10.3 | 89.6 | 86.2 | 96.5 | 86.2, 3.4 | 86.2 | 89.6 | 89.6 | 89.6 | 89.6 | 86.2 | 89.6 | 89.6 | 10.3 | 6.8 | ||||||
| N. nova strainsd | ||||||||||||||||||||||||
| DSM 44481 (n = 0) | 2 Leu (CTG) | 4 Thr (ACG) | 13 Leu (CTG) | 15 Val (GTC) | 51 Ile (ATC) | 57 Thr (ACC) | 64 Met (ATG) | 66 Gln (CAG) | 67 Asn (AAC) | 71 Ser (TCG) | 72 Gly (GGT) | 75 Hys (CAC) | 88 Ser (TCC) | 93 Gly (GGC) | ||||||||||
| Mutated(n = 29) | Val (GTC), Gly (GGC) | Pro (CCC) | Ile (ATC), Met (ATG) | Ile (ATC) | Val (GTC) | Ser (ACT) | Leu (CTG) | Glu (GAA) | Arg (CGC), Ser (AGC), Hys (CAC), Cys (TGC) | Ala (GCG) | Ser (AGT) | Asn (AAC) | Cys (TGC), Thr (ACC) | Ala (GCT) | ||||||||||
| Frequency (%) | 93.1, 6.9 | 10.3 | 93.1, 6.9 | 100 | 96.5 | 34.5 | 41.4 | 24.1 | 24.1, 20.7, 3.4, 3.4 | 80.7 | 89.6 | 27.6 | 10.4, 3.4 | 6.8 | ||||||||||
Amino acid polymorphism positions (modified nucleotides are underlined) with respect to the complete genome sequence of reference strain N. farcinica DSM 43665T (accession no. NC_006361), N. abscessus DSM 44432T (accession no. JN215593), and N. nova DMS 44481T (accession no. JN215754). All the N. cyriacigeorgica strains harbored the same RpoB as N. cyriacigeorgica DSM 44484T (accession no. JN215664).
No. of RpoB types, 15; HGDI, 0.93; divergence, 0.9 to 12.6%.
A fragment of RpoB was not indicated in the GenBank-deposited N. abscessus DSM 44432T (accession no. JN215593) data; here, a consensus sequence is described.
No. of RpoB types, 22; HGDI, 0.97; divergence, 1.1 to 11.1%.
(ii) N. cyriacigeorgica strains.
Twelve rpoB haplotypes were identified for N. cyriacigeorgica DSM 44484T (GenBank accession no. DQ085116) (95.6 to 99.7% similarity). Twenty-four SNPs were identified (Table 1; see also Fig. S2 in the supplemental material). All these SNPs were synonymous, i.e., they did not affect the amino acid sequence of the RpoB protein.
(iii) N. farcinica strains.
Five rpoB haplotypes were detected with respect to the reference strain N. farcinica DSM 43665T (GenBank accession no. DQ085117) (96.5 to 99.7% similarity). Nine SNPs were seen. A common RpoB protein type was seen for the entire population.
(iv) N. nova complex strains.
Each strain showed a different rpoB haplotype compared to the reference strain N. nova DMS 44481T (GenBank accession no. JN215754) (87.4 to 99.3% similarity). Forty-nine SNPs were detected. Twenty amino acid changes in 14 locations led to 22 RpoB protein types (Table 3).
Nucleotide sequence accession numbers.
The new variants of 16S, gyrB, and rpoB sequences of each studied species were assigned the GenBank accession no. KC662119 to KC662126 and KC631324 to KC631385.
DISCUSSION
In Spain, the estimated incidence of nocardial infection in the population as a whole is 0.55/100,000 (22), although solid organ recipients, people with chronic lung disease, diabetes mellitus, or an immunodeficiency, and those undergoing corticosteroid treatment are more susceptible. Over the last 10 years, the main etiological agents of human nocardiosis in the country have been N. abscessus (prevalence, 13.8%), N. cyriacigeorgica (25.5%), N. farcinica (12.5%), and N. nova complex (14.5%) (figures were obtained from 1,024 Nocardia isolates from across the country). When these rates are compared with those reported in studies that examined >90 isolates, differences in the prevalences of the four species can be seen; e.g., in Guipuzcoa (northern Spain), the prevalences of the four species are 12.4, 15.0, 23.1, and 29.6 (23), in Belgium, 6.5, 14.0, 44.1, and 21.5 (24), in Taiwan, 2.0, 16.0, 8.0, and 5.3 (25), and in the United States, 5.6, 13.2, 14.0, and 28.0, respectively (26).
The classification of species and subgroups of the genus Nocardia has traditionally been based on the evaluation of growth characteristics, antibiotic resistance, and biochemical testing (1). However, these methods are laborious and time-consuming (6 to 8 weeks). Molecular methods provide crucial insights into identification, epidemiology, and intraspecies variability. 16S analysis is the gold standard and allows for rapid identification, but the conservation of the studied sequence means that it cannot be used to study intrageneric relationships. Therefore, other targets need to be explored (11). Typing techniques, such as pulsed-field gel electrophoresis and amplified fragment length polymorphism, have been shown to be unsatisfactory for distinguishing between N. farcinica strains involved in outbreaks (27).
Housekeeping genes, such as secA1, hsp65, rpoB, and gyrB (2, 7–9), may be better molecular markers than 16S for such analyses. When secA1 was examined, several disagreements with 16S-based identifications were seen for very closely related species (e.g., N. abscessus and N. asiatica) (8). hsp65 shows better discriminatory power at the species level but not as much as gyrB and rpoB (6, 9). Even in species with nearly identical 16S sequences, rpoB and gyrB polymorphisms are highly discriminatory (6) and show close correlations with the identification results of DNA-DNA hybridization tests (11). gyrB has even been shown to be useful in the description of new Nocardia species (2, 3).
In two recent multilocus sequence typing studies involving reference strains for each species examined, five and 14 housekeeping genes were tested as markers of diversity in Nocardia (6, 11). However, if smaller numbers of housekeeping genes could be used in identification procedures, these analyses could be simpler and cheaper. Certainly, the analyses of gyrB and rpoB appear to identify the strains of the four main Nocardia species, type them at the inter- and intraspecies levels, and provide information on nocardial community diversity, a relevant event in clinical settings (28).
Because of the sequenced size and the criterion of 99% sequence similarity, the results of 16S analysis were not conclusive for N. abscessus and the N. nova complex strains. The study of the whole-gene 16S may distinguish N. abscessus from N. asiatica and allow for a more accurate species assignment of the N. nova complex. Nocardia species were previously clustered based on gyrB sequence similarity values of ≥93.5% (9). Here, the lower scores for the similarities of each studied species were as follows: N. abscessus, 98.3%; N. cyriacigeorgica, 94.4%; N. farcinica, 99.2%; and N. nova complex, 94.8% (higher values than the one previously proposed). In the case of rpoB, the recommended limit for a wide number of bacterial genera was ≥94.0% (29). N. cyriacigeorgica and N. farcinica strains showed higher minimum similarity scores, 95.6% and 98.6%, respectively. Meanwhile, N. abscessus and the N. nova complex displayed values lower than the suggested one (84.0% and 87.4%). The resulting scores are with respect to the considered type reference strains. Taking into account these criteria for the three genes, 65 strains simultaneously fulfilled them: 3 N. abscessus strains, 30 N. cyriacigeorgica strains, 31 N. farcinica strains, and 1 N. nova strain. Meanwhile, 54 strains fulfilled the 16S and gyrB breakpoints: 26 N. abscessus strains and 28 N. nova complex strains. Because of this dual behavior, gyrB and rpoB are complements of 16S identification, each with their usefulness and limits.
GyrB analysis was, however, very helpful in this respect. The GyrB of the strains from N. abscessus possessed 109Ala-110Ala, as did the N. abscessus reference strains, while the reference strains of N. asiatica possessed 109Ala. 16S analysis was sufficient to identify the N. nova complex. The gyrB gene of the N. nova complex strains, however, showed great variability, as revealed by the different motifs seen in the corresponding GyrB proteins. Similar heterogeneity was previously reported in the concatenated gyrB-16S-secA1-hsp65-rpoB phylogenetic tree constructed in multilocus sequence analysis studies of species grouped in the N. nova complex (6).
Unfortunately, 16S analysis is unable to distinguish between the species within certain genera. gyrB and rpoB have also been used as identification tools, but the genetic similarity values at which identities are confirmed vary depending on the species and the target gene (30, 31). To make identifications based on gyrB or rpoB, it is necessary to analyze the sequences from large populations of the same species (30). Owing to the faster evolution of these genes, the ranges of similarity are wider than those reported for 16S (31) (in the present case, 83.4 to 99.2 for gyrB and 84.0 to 98.6 for rpoB). To date, no consensus divergence breakpoint has been fully established for the majority of bacterial species.
Typing was successful using just one of the housekeeping genes. For gyrB and rpoB, the HGDI values ranged from 0.88 to 1 in all species, except for N. farcinica (HGDI, 0.85 and 0.72 for gyrB and rpoB, respectively). One gene of these two was usually better, depending on the species. The abilities of gyrB and rpoB to discriminate between strains of the most prevalent species are important in confirming person-to-person transmission and for identifying new and recurrent infections. Thus, gyrB and rpoB are promising genes for use in molecular typing studies. The large number of gyrB and rpoB SNPs detected, both synonymous and nonsynonymous, provided insight into the diversity of these clinical strains (with the exception of N. farcinica).
After describing the genetic diversities of gyrB and rpoB in populations made up of different Nocardia species (7, 10, 12), the second step was to assess the intraspecies variation in a large number of strains. To our knowledge, this is the first work to explore this in a single-species scenario. Three levels of intraspecies diversity were seen: low level, which was shown by the N. farcinica strains with their 11 gyrB and 4 rpoB haplotypes, and single GyrB and RpoB protein types; mid-level, which was shown by the N. abscessus and N. cyriacigeorgica strains, whose large numbers of SNPs resulted in 14 gyrB and 18 rpoB and 17 gyrB and 12 rpoB haplotypes, respectively, along with 5 GyrB and 15 RpoB protein types among the N. abscessus strains and 9 GyrB and 1 RpoB protein types among the N. cyriacigeorgica strains; and high level, which was shown by the N. nova complex strains with their 26 gyrB and 29 rpoB haplotypes and 11 GyrB and 22 RpoB protein types. The absence of the 129Pro insertion differentiates N. nova sensu stricto from other members of the N. nova complex.
N. farcinica, with its few and always monomorphic SNPs, might belong to a younger lineage than the other species. In the N. nova strains identified as such by 16S analysis, variations in the GyrB motif 126AAAPEH suggest different sublineages of recent appearance.
In conclusion, the present work shows that analysis of the gyrB or rpoB genes offers a rapid and relatively cheap means of studying strains of clinically important Nocardia species, in combination with 16S analysis.
Supplementary Material
ACKNOWLEDGMENTS
This work was funded by a grant to N.G. from the Instituto de Salud Carlos III (MPY-1446/11).
We are grateful to the CNM Biopolymers Unit for assistance in sequencing, to Adrian Burton for linguistic assistance in the preparation of the manuscript, and to the laboratories that submitted the Nocardia strains to our center for identification.
Footnotes
Published ahead of print 21 August 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00515-13.
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