Biochemical identification and molecular characterization (PCR-RFLP) of Nocardia isolates from sputum.

BACKGROUND

Nocardia identification has been based on biochemical and morphological characteristics. However, molecular biology techniques allow a better characterization of species and biotypes that are related to invasive diseases.

METHODS

Twelve isolates of Nocardia spp. were obtained from sputum of patients with tuberculosis under retreatment. Identification was done based on morphological characteristics, biochemical tests (casein, tyrosine, xanthine, gelatin, and urea) and molecular biology techniques (PCR-RFLP) using restriction enzymes MspI, HinfI, BsaHI, HaeIII and BstEII.

RESULTS

Biochemical tests identified the 12 isolates as Nocardia asteroides. PCR-RFLP technique identified nine isolates to species and biotype level: five as N. asteroides type II, two as N. asteroides type VI, and two as N. asteroides type I. The remaining three isolates were identified as follows: one to species level as N. farcinica and two at genus level as Nocardia sp.

CONCLUSIONS

Significant statistical differences between the use of traditional techniques and PCR-RFLP were not found at genus level, but there were important differences at species and biotype level. Biochemical tests identified correctly the actinomycete isolates as belonging to Nocardia genus, but at N. asteroides complex level were not able to discern among their different species. PCR-RFLP is a rapid, non-expensive, and reliable method that allows to discriminate the N. asteroides complex species, identifying biotypes related to invasive disease. Our results suggest that the hospital environment was not a contamination source.