Molecular identification of Nocardia species using the sodA gene: Identificación molecular de especies de Nocardia utilizando el gen sodA.

Currently for bacterial identification and classification the rrs gene encoding 16S rRNA is used as a reference method for the analysis of strains of the genus Nocardia. However, it does not have enough polymorphism to differentiate them at the species level. This fact makes it necessary to search for molecular targets that can provide better identification. The sodA gene (encoding the enzyme superoxide dismutase) has had good results in identifying species of other Actinomycetes. In this study the sodA gene is proposed for the identification and differentiation at the species level of the genus Nocardia. We used 41 type species of various collections; a 386 bp fragment of the sodA gene was amplified and sequenced, and a phylogenetic analysis was performed comparing the genes rrs (1171 bp), hsp65 (401 bp), secA1 (494 bp), gyrB (1195 bp) and rpoB (401 bp). The sequences were aligned using the Clustal X program. Evolutionary trees according to the neighbour-joining method were created with the programs Phylo_win and MEGA 6. The specific variability of the sodA genus of the genus Nocardia was analysed. A high phylogenetic resolution, significant genetic variability, and specificity and reliability were observed for the differentiation of the isolates at the species level. The polymorphism observed in the sodA gene sequence contains variable regions that allow the discrimination of closely related Nocardia species. The clear specificity, despite its small size, proves to be of great advantage for use in taxonomic studies and clinical diagnosis of the genus Nocardia.

Introduction

Actinomycetes are Gram-positive bacteria, soil saprophytes that play an essential role in the processes of humification and decomposition of organic matter, and are also known for their ability to produce metabolites, such as antibiotics, antitumor agents, immunosuppressive agents and enzymes. Apart from their ecologic and industrial role, members of the genus Nocardia are responsible for infections such as nocardiosis and actinomycetoma [1], [2]. Identification of the various species of Nocardia is no longer possible by standard methods such as conventional biochemical tests. Luckily, identification based on molecular tests is every day more promising.

In recent years the sequencing methodology used the rrs gene that encodes the 16S rRNA as a reference method to identify and classify strains of the genus Nocardia at the species level. However, some studies have reported that this gene does not have enough discriminatory power [3] or multiple copies of the rrs gene are present [4]; thus generating problems in the identification of clinical isolates [5].

Other genes have been used as novel genetic targets for the characterization of Nocardia, such as PCR amplification of a segment of the hsp65 gene [6], [7]; the secA1 gene [8]; the gene gyrB [9]; the rpoB and the intergenic spacer 16S-23S, but they can not solve certain identification problems at the species level; nor can they differentiate between species that are closely related and difficult to separate.

The combined use of different genes makes it possible to refine phylogenetic analysis and provide a molecular basis for accurate identification at the species level. However, the use of these genes in a multiple analysis involves the sequencing of each gene for each isolate, which remains laborious and costly for certain diagnostic laboratories [10].

The amplification and sequencing of the sodA gene (gene encoding the enzyme superoxide dismutase) has been used by Zolg and Philippi-Schulz [11] for species-level identification of Mycobacterium strains.

On this basis, we decided to explore the polymorphism of the sodA gene in the genus Nocardia. The objective of this study was to propose a new molecular target that may be useful for the identification and differentiation of clinical and environmental isolates at the species level of the genus Nocardia as well as to evaluate their efficiency.

Materials and Methods

We used 41 strains of the genus Nocardia from international collections, in which the rrs and hsp65 genes absent in GenBank were sequenced. Sequences of the secA1, rpoB and gyrB genes were obtained completely from GenBank. The sodA gene of all strains was sequenced in our laboratory (Table 1).

Table 1

Accession numbers for six genes of different strains of Nocardia obtained from GenBank

No.	Species	Strain	Gene
			rrs	hsp65	secA1	rpoB	gyrB	sodA
1	N. abscessus	DSM 44432T	DQ659895	AY544983	DQ360260	JN215593	AB447398	EPVa
2	N. amamiensis	DSM 45066T	AB275164	JN041700	JN041937	JN215567	JN041226	JX519285b
3	N. anaemiae	DSM 44821T	JF797304	JN041769	JN042006	JN215636	AB447400	KX944462b
4	N. aobensis	DSM 44805T	JF797305	JN041852	JN042089	JN215719	AB447401	EPV
5	N. araoensis	DSM 44729T	JF797306	AY903637b	EU178745	DQ085145	AB450768	DQ085169b
6	N. arthritidis	DSM 44731T	DQ659896	AY903633b	JN041946	JN215576	AB450769	DQ085166b
7	N. asiatica	DSM 44668T	DQ659897	AY903631b	DQ360263	JN215591	AB450770	DQ085165b
8	N. asteroides	ATCC 19247T	DQ659898	AY756513b	DQ360267	JN215563	AB450771	DQ085146b
9	N. beijingensis	JCM10666T	DQ659901	AY756515b	JN041942	JN215572	AB450772	DQ085147b
10	N. brasiliensis	ATCC 19296T	AF430038	AY756516b	DQ360269	JN215639	AB450773	DQ085148b
11	N. brevicatena	ATCC 15333T	AF430040	AY756517b	DQ360270	JN215692	AB450774	EPV
12	N. carnea	DSM 43397T	AF430035	AY756518b	DQ360271	JN215702	AB450782	EPV
13	N. cerradoensis	DSM 44546T	NR_028704	AY756519b	JN042082	JN215712	AB450777	EPV
14	N. coubleae	DSM 44960T	DQ265689b	DQ250024b	JN041930	JN215560	JN041219	EPV
15	N. cyriacigeorgica	DSM 44484T	AF430027	AY756522b	DQ360272	JN215664	AB450784	EF408035b
16	N.exalbida	DSM 44883T	JF797308	JN041715	GU584191	JN215582	AB447397	EPV
17	N. farcinica	DSM 43665T	AF430033	AY756523b	DQ360274	DQ085117	AB014169	JX519286b
18	N. flavorosea	JCM3332T	AF430048	AY756524b	JN042071	JN215701	AB450787	EPV
19	N. fluminea	DSM 44489T	AF430053	AY756525b	JN041926	JN215556	AB450788	DQ085150b
20	N. gamkensis	DSM 44956T	DQ235272	JN041716	JN041953	JN215583	JN041242	JX519284b
21	N. higoensis	DSM 44732T	AB108778	AY903634b	EU178747	DQ085142	AB450789	DQ085167b
22	N. ignorata	DSM 44496T	DQ659907	AY756526b	JN041928	JN215558	AB450790	DQ085151b
23	N. inohanensis	DSM 44667T	AB092560	AY903625b	DQ360276	DQ085133	AB450791	DQ085159b
24	N. mexicana	CIP 108295T	JF797310	AY903624b	GU584192	DQ085132	GQ496104	EPV
25	N. neocaledoniensis	DSM 44717T	AY282603	AY903628b	JN041932	JN215562	AB450795	DQ085162b
26	N. niigatensis	DSM 44670T	DQ659910	AY903629b	DQ360278	DQ085137	AB450796	DQ085163b
27	N. nova	CIP 104777T	AY756555b	AY756527b	DQ360279	JN215752	AB450797	EPV
28	N. otitidiscaviarum	ATCC 14629T	AF430067	AY756528b	DQ360280	JN215616	AB450798	EPV
29	N. paucivorans	DSM 44386T	AF179865	AY756529b	DQ360281	JN215691	AB450799	EPV
30	N. pneumoniae	DSM 44730T	JF797313	AY903636b	EU178749	JN215569	AB450801	EPV
31	N. pseudobrasiliensis	DSM 44290T	DQ659914	AY756530b	DQ360282	JN215625	AB450802	DQ085152b
32	N. puris	DSM 44599T	JF797314	AY903632b	EU178750	DQ085140	AB450804	EPV
33	N. seriolae	DSM 44129T	AF430039	AY756533b	DQ360284	DQ085125	AB450805	DQ085154b
34	N. shimofusensis	DSM 44733T	AB108775	AY903635b	EU178751	DQ085143	AB450806	DQ085168b
35	N. sienata	DSM 44766T	NR_024825	JN041831	DQ360285	JN215698	AB450807	EPV
36	N. thailandica	DSM 44808T	AB126874	JN041686	EU178752	JN215553	AB450811	EPV
37	N. transvalensis	DSM 43405T	AF430047	AY756535b	DQ360287	JN215628	AB450812	EPV
38	N. vaccinii	DSM 43285T	AF430045	AY756537b	DQ366276	DQ085129	AB450814	EPV
39	N. veterana	DSM 44445T	AF430055	AY756538b	DQ360288	JN215706	AB450816	EPV
40	N. vinacea	JCM 10988T	DQ659919	AY756539b	DQ360289	JN215634	AB450817	DQ085158b
41	N. yamanashiensis	DSM 44669T	AB092561	AY903630b	DQ360290	DQ085138	AB450819	DQ085164b

ATCC, American Type Culture Collection; CIP, Collection de l’Institut Pasteur; DSM, Deutsche Sammlung von Mikroorganismen; JCM, Japan Collection of Microorganisms.

EPV indicates sequences in process of validation in GenBank.

Sequenced by our laboratory.

Methods of DNA extraction

DNA extraction from type strains was performed with the commercial kit UltraClean Microbial DNA isolation kit Mobio, previously reported for bacteria of the order Actinomycetales by Soddell et al. [12]; with the exception of some species such as N. nova, N. anaemiae and N. puris, where the Chelex resin method was used because of problems of concentration and purity of DNA obtained with other methods.

Chelex resin

A bacterial suspension was prepared in an Eppendorf tube containing a dozen 1 mm glass beads and 250 μL of sterile ultrapure water which was homogenized for 5 minutes in a vortex, and 60 μL of Resin Chelex (InstaGene Matrix; 6% w/v Chelex Resin; Bio-Rad, Hercules, CA, USA) which was previously agitated. The tubes were placed on a heat plate at 100°C for 25 minutes. It was then centrifuged at 10 000g for 5 minutes. Supernatant (200 μL) containing the DNA was taken and transferred to a sterile tube, then frozen at −20°C for storage.

UltraClean Microbial DNA isolation Kit Mobio

A total of 300 μL of the MicroBead solution were added to a MicroBead tube. Once the beads were wetted, the bacterial suspension was added to the tube. Next 50 μL of the MD1 solution was added and the tubes were vortexed at the maximum rate for 10 minutes. The tubes were centrifuged at 10 000g for 30 seconds. The supernatant was then transferred to a tube containing 100 μL of the MD2 solution, vortexed and allowed to incubate at 4°C for 15 minutes, after which the tubes were centrifuged at 10 000g for 1 minute. After this step, 200 μL of the supernatant was transferred to a tube containing 450 μL of the MD3 solution and vortexed for 2 minutes. Immediately 650 μL was transferred to a clean tube with filtration column, centrifuged at 10 000g for 30 seconds, the filtrate discarded and 300 μL of MD4 solution added to the filtration column, which was then centrifuged at 10 000g per 30 seconds. The filtrate column was placed in a 2.0 mL clean microtube, and 35 μL of the MD5 solution was added to the centre of the filter column (where it was allowed to incubate for 5 minutes at room temperature), then centrifuged at 10 000g for 30 seconds. The filtration column was discarded; the DNA was extracted and recovered in the microtube, then stored at −20°C until use.

Amplification and sequencing

Gene rrs (1171 bp)

Sequencing of the rrs gene (16SrRNA) was performed using the following primers: SQ1 (5′-AGAGTTTGATCMTGGCTCAG-3′), SQ2 (5′-AAACTCAAAGRATTGACGGG-3′), SQ3 (5′-CCCGTCAATYCTTTGAGTTT-3′), SQ4 (5′-CGTGCCAGCAGCCGCG-3′), SQ5 (5′-CGCGGCTGCTGGCACG-3′) and SQ6 (5′-CGGTGTGTACAAGGCCC-3′) (0.2 μM) according to the Sanger method adapted by the DYE terminator sequencing kit (Amersham Biosciences, Uppsala, Sweden). The nucleotide sequences were determined with an automated sequencer ABI 377 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions by the Biofidal (Vaux-en-Velin, France).

According to the recommendations of Rodríguez-Nava et al. [7]; the sequences were aligned using the Clustal X program [13]. The Phylo_win program [14] and MEGA 6 were used to infer the evolutionary trees according to the neighbour-joining method [15] and Kimura’s two-parameter model [16]. The robustness of the tree was performed with a bootstrap of 1000 replicates.

Gene hsp65 (401 bp)

A fragment of the hsp65 gene encoding the 65 kDa heat shock protein was amplified and sequenced with the following primers: TB11, 5′-ACCAACGATGGTGTGTCCAT-3′ and TB12 5′-CTTGTCGAACCGCATACCCT-3′ (0.6 μM) [17]. The amplification was carried out in Ready-to-Go PCR beads (GE Healthcare UK, Little Chalfont, UK) in a final volume of 25 μL (2.5 U of Taq polymerase, 10 mM Tris-HCl (pH 9) and 50 mM KCl, 1.5 mM MgCl2 and 200 μM of each desoxynucleoside triphosphate) with 10 μL of the DNA extracted by the Chelex method. After initial denaturation at 94°C for 5 minutes, the reaction mixture was run for 35 denaturation cycles at 94°C for 60 seconds, the primers were aligned at 55°C for 60 seconds and the extension was carried out at 72°C for 60 seconds, followed by a postextension extension at 72°C for 5 minutes.

The nucleotide sequence, alignment and analysis of the sequences and the elaboration of the phylogenetic trees were performed under the same conditions described above for the rrs gene.

Gene sodA (386 bp)

A 440 bp fragment of the sodA gene was amplified and sequenced with primers SodV1 (5′-CAC CAY WSC AAG CAC CA-3′) and SodV2 (5′-CCT TAG CGT TCT GGT ACT G-3′) where Y = C or T, W = A or T and S = C or G (0.6 μM) (V. Rodríguez-Nava, personal communication). Amplification was performed using both primers in PCR tubes (illustra puReTaq Ready-to-Go PCR Beads; GE Healthcare Biosciences, Piscataway, NJ, USA) under the same conditions as the rrs gene. Amplification was carried out in a thermocycler (PTC-100; MJ Research, Boston, MA, USA). The amplification run included an initial denaturation step of 5 minutes at 94°C, followed by 35 cycles (94°C for 60 seconds, 55°C for 60 seconds and 72°C for 60 seconds) and a final step of 10 minutes at 72°C. The sequences obtained were verified by DNA sequencing in both directions. The nucleotide sequence, alignment, sequence analysis and elaboration of phylogenetic trees was performed under the same conditions described above for the rrs gene).

Phylogenetic analysis

For the comparative phylogenetic analysis, the sodA gene as well as the rrs (1171 bp), hsp65 (401 bp), secA1 (494 bp), gyrB (1195 bp) and rpoB (401 bp) genes of the 41 Nocardia species studied were used. Several newly described species of the genus Nocardia were not included in this study because they were not available at the time we performed the phylogenetic analysis of the sequences.

Results

Phylogenetic analysis of sequences of sodA gene of 41 type strains

The sodA gene was successfully amplified in the 41 type species, and phylogenetic analysis of the amplicon sequences showed 15 nodes with bootstrap values of ≥90% representing 38.46%, allowing a robust tree to be constructed. The percentage of minimum similarity found was 79.8% and had a maximum of 100%, which shows a high variability among species. The 386 bp fragment of the sodA gene presented variable regions, with segments of 4 and 5 bp, thus showing an interspecies variability of the sodA gene with the potential to be used as a molecular marker (Table 2).

Table 2

Phylogenetic analysis of different genes used

Characteristic	rrs	hsp65	secA1	gyrB	rpoB	sodA
Phylogenetic resolution (%)	23.07	23.68	30.7	38.48	20.51	38.48
Gene size (bp)	1171	401	494	1195	401	386
Divergence in sequences (bp)	145 (12.38%)	114 (28.41%)	178 (36.03%)	530 (44.35%)	151 (37.65%)	168 (43.52%)
Total no. of nodes	39	38	39	39	39	39
No. of nodes ≥90%	9	9	12	15	8	15

Polymorphism analysis of different genes

The evolutionary analysis based on distance matrices allowed us to evaluate the interspecies polymorphism for each molecular marker used (rrs, hsp65, secA1, gyrB, rpoB and sodA). We found that the average percentage similarity for the rrs gene was 97.1%, corresponding to 145 nucleotides of difference between species at the interspecies level, whereas for the sodA gene it was 89.9%, corresponding to 168 nucleotides of difference. The variability of substitutions of the sequences of the sodA gene was much greater than those of the rrs gene (Table 2).

With the analysis of the sequences performed for the sodA gene, 15 nodes (38.48%) were found in the phylogenetic tree (bootstrap values of ≥90%), while for the other genes evaluated, nine nodes (23.07%) were obtained for the rrs gene, nine nodes (23.68%) for hsp65, 12 nodes (30.7%) for secA1 and eight nodes (20.51%) for rpoB; for gyrB, we obtained 15 nodes and a tree robustness of 38.48%.

These results indicate that the sodA gene analysed on a 386 bp fragment compared to the standard rrs identification gene (1171 bp) presents a high resolution and polymorphism as a molecular marker, and presents a variability equivalent to the gyrB gene, with the difference that the gyrB gene corresponds to a bigger fragment of 1195 bp.

As already mentioned, the phylogeny of the genus Nocardia presents certain difficulties, which are reflected in several pairs of species that are hard to differentiate: N. coubleae and N. ignorata; and N. bevicatena and N. paucivorans.

In order to make the phylogenetic analyses more clear and reproducible, all the trees were rooted, as far as possible, with bacteria of the same species. Species of the genus Mycobacterium were used.

Discussion

Sequencing of the rrs gene has been used as the reference method for the identification of Nocardia isolates [18], [19], [20], [21]. However, several studies have revealed the lack of polymorphism of the rrs gene to discriminate between certain closely related species [22]. In our study, the analysis of sequences with the sodA gene revealed an important interspecies polymorphism for the 41 species of Nocardia analysed. It was found that the sequences have a variability of 43.52%, and that contains conserved and other highly variable regions in a small fragment of only 386 bp. It was also observed that it presents the highest phylogenetic resolution (38.48%) compared to the genes already reported, including the reference gene, which presented a lower value (23.07%), except for the gyrB gene, with a resolution of 38.48% but with a large size, resulting in a complex analysis and expensive sequencing.

The phylogenetic distribution found for the Nocardia species obtained with the sodA gene was confirmed by the phylogenetic distribution of the rrs gene, confirming the distribution and position of the associations relative to each species within the tree. The clusters formed between the different species are also present in the other genes (hsp65, secA1, gyrB and rpoB) (Fig. 4, Fig. 5, Fig. 6).

Fig. 4

Phylogenetic tree of Nocardia species (secA1). Phylogenetic distribution of secA1 gene (494 bp) of 41 Nocardia type strains analysed in this study using neighbour-joining method, Kimura’s two-parameter model and bootstrap of 1000. Only values of bootstrap significance greater than 50% (Clustal X, Phylo_win) were reported.

Fig. 5

Phylogenetic tree of Nocardia species (gyrB). Phylogenetic distribution of gyrB gene (1195 bp) of 41 Nocardia type strains analysed in this study using neighbour-joining method, Kimura’s two-parameter model and bootstrap of 1000. Only values of bootstrap significance greater than 50% (Clustal X, Phylo_win) were reported.

Fig. 6

Phylogenetic tree of Nocardia species (rpoB). Phylogenetic distribution of rpoB gene (401 bp) of 41 strains of Nocardia analysed in this study using neighbour-joining method, Kimura’s two-parameter model and bootstrap of 1000. Only values of bootstrap significance greater than 50% (Clustal X, Phylo_win) were reported.

Phylogenetic tree of Nocardia species (sodA). Phylogenetic distribution of sodA gene (386 bp) of 41 type Nocardia strains analysed in this study using neighbour-joining method, Kimura’s two-parameter model and bootstrap of 1000. Only values of bootstrap significance greater than 50% (Clustal X, Phylo_win) were reported.

Phylogenetic tree of Nocardia species (hsp65). Phylogenetic distribution of hsp65 gene (401 bp) of 41 Nocardia type strains analysed in this study using neighbour-joining method, Kimura’s two-parameter model and bootstrap of 1000. Only values of bootstrap significance greater than 50% (Clustal X, Phylo_win) were reported.

Phylogenetic tree of Nocardia species (rrs). Phylogenetic distribution of rrs gene (1171 bp) of 41 strains of Nocardia analysed in this study using neighbour-joining method, Kimura’s two-parameter model and bootstrap of 1000. Only values of bootstrap significance greater than 50% (Clustal X, Phylo_win) were reported.

Conclusions

The specific variability of the sodA gene of the genus Nocardia was analysed. The gene proposed in this work as a molecular genetic marker presented a high phylogenetic resolution, an important genetic variability, and specificity and reliability for the differentiation of isolates at the species level. The polymorphism observed in the sodA gene sequence contains variable regions that enable discrimination of closely related Nocardia species.

We observed a clear specificity of the sodA gene, which we think will prove to be of great advantage for use in taxonomic studies; it could also be used for clinical diagnoses of the genus Nocardia.

Conflict of Interest

None declared.

Tabla 1

Números de acceso para 6 genes de las diferentes cepas tipo de Nocardia obtenidos del GenBank

	Especie		Números de Acceso
			Gen
		Clave	rrs	hsp65	secA1	rpoB	gyrB	sodA
1	N. abscessus	DSM 44432T	DQ659895	AY544983	DQ360260	JN215593	AB447398	EPV
2	N. amamiensis	DSM 45066 T	AB275164	JN041700	JN041937	JN215567	JN041226	JX519285
3	N. anaemiae	DSM 44821 T	JF797304	JN041769	JN042006	JN215636	AB447400	KX944462
4	N. aobensis	DSM 44805 T	JF797305	JN041852	JN042089	JN215719	AB447401	EPV
5	N. araoensis	DSM 44729 T	JF797306	AY903637	EU178745	DQ085145	AB450768	DQ085169
6	N. arthritidis	DSM 44731 T	DQ659896	AY903633	JN041946	JN215576	AB450769	DQ085166
7	N. asiatica	DSM 44668 T	DQ659897	AY903631	DQ360263	JN215591	AB450770	DQ085165
8	N. asteroides	ATCC 19247 T	DQ659898	AY756513	DQ360267	JN215563	AB450771	DQ085146
9	N. beijingensis	JCM 10666 T	DQ659901	AY756515	JN041942	JN215572	AB450772	DQ085147
10	N. brasiliensis	ATCC 19296 T	AF430038	AY756516	DQ360269	JN215639	AB450773	DQ085148
11	N. brevicatena	ATCC 15333 T	AF430040	AY756517	DQ360270	JN215692	AB450774	EPV
12	N. carnea	DSM 43397 T	AF430035	AY756518	DQ360271	JN215702	AB450782	EPV
13	N. cerradoensis	DSM 44546 T	NR_028704	AY756519	JN042082	JN215712	AB450777	EPV
14	N. coubleae	DSM 44960 T	DQ265689	DQ250024	JN041930	JN215560	JN041219	EPV
15	N. cyriacigeorgica	DSM 44484 T	AF430027	AY756522	DQ360272	JN215664	AB450784	EF408035
16	N.exalbida	DSM 44883 T	JF797308	JN041715	GU584191	JN215582	AB447397	EPV
17	N. farcinica	DSM 43665 T	AF430033	AY756523	DQ360274	DQ085117	AB014169	JX519286
18	N. flavorosea	JCM 3332 T	AF430048	AY756524	JN042071	JN215701	AB450787	EPV
19	N. fluminea	DSM 44489 T	AF430053	AY756525	JN041926	JN215556	AB450788	DQ085150
20	N. gamkensis	DSM 44956 T	DQ235272	JN041716	JN041953	JN215583	JN041242	JX519284
21	N. higoensis	DSM 44732 T	AB108778	AY903634	EU178747	DQ085142	AB450789	DQ085167
22	N. ignorata	DSM 44496 T	DQ659907	AY756526	JN041928	JN215558	AB450790	DQ085151
23	N. inohanensis	DSM 44667 T	AB092560	AY903625	DQ360276	DQ085133	AB450791	DQ085159
24	N. mexicana	CIP 108295 T	JF797310	AY903624	GU584192	DQ085132	GQ496104	EPV
25	N. neocaledoniensis	DSM 44717 T	AY282603	AY903628	JN041932	JN215562	AB450795	DQ085162
26	N. niigatensis	DSM 44670 T	DQ659910	AY903629	DQ360278	DQ085137	AB450796	DQ085163
27	N. nova	CIP 104777 T	AY756555	AY756527	DQ360279	JN215752	AB450797	EPV
28	N. otitidiscaviarum	ATCC 14629 T	AF430067	AY756528	DQ360280	JN215616	AB450798	EPV
29	N. paucivorans	DSM 44386 T	AF179865	AY756529	DQ360281	JN215691	AB450799	EPV
30	N. pneumoniae	DSM 44730 T	JF797313	AY903636	EU178749	JN215569	AB450801	EPV
31	N. pseudobrasiliensis	DSM 44290 T	DQ659914	AY756530	DQ360282	JN215625	AB450802	DQ085152
32	N. puris	DSM 44599 T	JF797314	AY903632	EU178750	DQ085140	AB450804	EPV
33	N. seriolae	DSM 44129 T	AF430039	AY756533	DQ360284	DQ085125	AB450805	DQ085154
34	N. shimofusensis	DSM 44733 T	AB108775	AY903635	EU178751	DQ085143	AB450806	DQ085168
35	N. sienata	DSM 44766 T	NR_024825	JN041831	DQ360285	JN215698	AB450807	EPV
36	N. thailandica	DSM 44808 T	AB126874	JN041686	EU178752	JN215553	AB450811	EPV
37	N. transvalensis	DSM 43405 T	AF430047	AY756535	DQ360287	JN215628	AB450812	EPV
38	N. vaccinii	DSM 43285 T	AF430045	AY756537	DQ366276	DQ085129	AB450814	EPV
39	N. veterana	DSM 44445 T	AF430055	AY756538	DQ360288	JN215706	AB450816	EPV
40	N. vinacea	JCM 10988 T	DQ659919	AY756539	DQ360289	JN215634	AB450817	DQ085158
41	N. yamanashiensis	DSM 44669 T	AB092561	AY903630	DQ360290	DQ085138	AB450819	DQ085164

*Todas las claves en “negritas” son cepas secuenciadas por nuestro laboratorio.*EPV: secuencias en proceso de validación en GenBank.

DSM: Deutsche Sammlung von Mikroorganismen, JCM: Japan Collection of Microorganisms; ATCC: American Type Culture Collection; CIP: Collecction de lÍnstitut Pasteur

Tabla 2

“Análisis filogenético de los diferentes genes utilizados”

Gen	rrs	hsp65	secA1	gyrB	rpoB	sodA
Resolución filogenética	23.07%	23.68%	30.7%	38.48%	20.51%	38.48%
Tamaño del gen	1171pb	401pb	494pb	1195pb	401pb	386pb
pb divergentes en las secuencias	145 (12.38%)	114 (28.41%)	178 (36.03%)	530 (44.35%)	151 (37.65%)	168 (43.52%)
Nodos totales	39	38	39	39	39	39
Nodos ≥90%	9	9	12	15	8	15