The Nocardial aph(2″) Gene Confers Tobramycin and Gentamicin Resistance and Is an Effective Positive Selection Marker in Mycobacteria and Nocardia

Abstract

The current study aimed to evaluate the feasibility of using the aminoglycoside 2″-O-phosphotransferase aph(2″) gene as a positive selection marker in N. asteroides, M. smegmatis, M. abscessus and M. tuberculosis. The aph(2″) gene, known to confer resistance to tobramycin, was PCR amplified from N. farcinica and cloned into two plasmid vectors, pMSG383 and pDB151, harboring hygromycin and zeocin selection markers, respectively. The recombinant plasmids were transformed into the target microorganisms, and selectability was assessed against varying concentrations of tobramycin and using an E-test against gentamicin. The results indicated that the aph(2″) gene is a useful selection marker in Mycobacteria and Nocardia against tobramycin, with a good selectability at 2.5–10 µg/mL for M. smegmatis mc2-155 and N. asteroides ATCC 19,247, and 60–160 µg/mL for M. abscessus ATCC 19,977 and M. tuberculosis H37Ra. The minimum inhibitory concentration (MIC) of gentamicin for recombinant N. asteroides, M. smegmatis and M. abscessus was >256 µg/mL, whereas respective MIC in wild-type strains was 0.125 µg/mL, 0.38 µg/mL and 8 µg/mL, respectively. These findings demonstrate the potential of aph(2″) as a positive selection marker for genetic manipulation processes in Mycobacteria and Nocardia, thus facilitating their research and improving the efficiency of biotechnology applications. Conclusions: the aph(2″) gene is a useful, new selection marker for genetic manipulation of Nocardia and various Mycobacteria.