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

Abstract

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.

Keywords: Nocardia; hsp65; polymorphism; rrs; sodA.

Fig. 1

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.

Fig. 2

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.

Fig. 3

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.

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.

Fig 1

Árbol filogenético de especies de Nocardia (sodA). Distribución filogenética del gen sodA (386 pb) de las 41 cepas tipo de Nocardia analizadas en este estudio. Usando el método de neighbor-joining, el modelo de dos parámetros de Kimura y un bootstrap de 1000. Se indicaron solo los valores de significancia de bootstrap mayores de 50% (programas: Clustal X, Phylo_win)

Fig 2

Árbol filogenético de especies de Nocardia (hsp65). Distribución filogenética del gen hsp65 (401 pb) de las 41 cepas tipo de Nocardia analizadas en este estudio. Usando el método de neighbor-joining, el modelo de dos parámetros de Kimura y un bootstrap de 1000. Se indicaron solo los valores de significancia de bootstrap mayores de 50% (programas: Clustal X, Phylo_win).

Fig 3

Árbol filogenético de especies de Nocardia (rrs). Distribución filogenética del gen rrs (1171 pb) de las 41 cepas tipo de Nocardia analizadas en este estudio. Usando el método de neighbor-joining, el modelo de dos parámetros de Kimura y un bootstrap de 1000. Se indicaron solo los valores de significancia de bootstrap mayores de 50% (programas: Clustal X, Phylo_win).

Fig 4

Árbol filogenético de especies de Nocardia (secA1). Distribución filogenética del gen secA1 (494 pb) de las 41 cepas tipo de Nocardia analizadas en este estudio. Usando el método de neighbor-joining, el modelo de dos parámetros de Kimura y un bootstrap de 1000. Se indicaron solo los valores de significancia de bootstrap mayores de 50% (programas: Clustal X, Phylo_win)

Fig 5

Árbol filogenético de especies de Nocardia (gyrB). Distribución filogenética del gen gyrB (1195 pb) de las 41 cepas tipo de Nocardia analizadas en este estudio. Usando el método de neighbor-joining, el modelo de dos parámetros de Kimura y un bootstrap de 1000. Se indicaron solo los valores de significancia de bootstrap mayores de 50% (programas: Clustal X, Phylo_win)

Fig 6

Árbol filogenético de especies de Nocardia (rpoB). Distribución filogenética del gen rpoB(401 pb) de las 41 cepas tipo de Nocardia analizadas en este estudio. Usando el método de neighbor-joining, el modelo de dos parámetros de Kimura y un bootstrap de 1000. Se indicaron solo los valores de significancia de bootstrap mayores de 50% (programas: Clustal X, Phylo_win)