Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and plasmid profile of soil and clinical isolates of Nocardia

The aim of this study was to develop a polymerase chain reaction-restrictio n fragment length polymorphism (PCR-RFLP) assay for generic and species-spe cific differentiation of Nocardia from other morphologically similar bacter ial pathogens. To examine the utility of the PCR-RFLP approach in species i dentification, Genomic DNA was prepared from 40 soil isolates, 10 clinical isolates and 8 reference strains of Nocardia. A set of oligonucleotide prim ers was designed from the consensus sequence of the highly conserved groEL gene that encodes the 65-kDa heat shock protein (hsp 65). The primers selec tively amplified 422 bp DNA from the genomic DNA of all Nocardia species an d isolates. The digestion of the amplicons with the restriction enzyme MspI produced DNA fragments that could differentiate between different Nocardia species regardless of their origin. Additionally, the RFLP patterns obtain ed with restriction enzymes MspI and BsaHI resulted in the differentiation of six Nocardia species which were earlier identified by biochemical tests. Apart from soil isolates of N. asteroides, which had shown some degree of genotypic polymorphism with BsaHI, the remaining taxa yielded mon consisten t results. Our results on the isolation of plasmids indicated that their oc currence is not a consistent feature in Nocardia species. It is neither rel ated to the source of origin (clinical versus saprobic), nor to virulence, antimicrobial resistance or species specificity.