Role of Active Efflux in Antibiotic and Oxidative Stress Induced Aminoglycoside Resistance in Burkholderia vietnamiensis

The Burkholderia cepacia complex (BCC) group of Gram-negative bacteria are highly virulent, opportunistic pathogens in cystic fibrosis (CF) patients, notorious for their multidrug resistance. We previously found that one species within the BCC, B. vietnamiensis (Bv), is uniquely susceptible to aminoglycosides, a class of antimicrobials used in the treatment of respiratory infections in CF, but aminoglycoside resistance is acquired during chronic infection and under tobramycin pressure in vitro. The objectives of this study were to identify Bv aminoglycoside resistance inducing conditions, and to determine the role of active antimicrobial efflux in Bv acquired aminoglycoside resistance. The early, aminoglycoside-susceptible Bv isolate C8395 that was previously reported to acquire aminoglycoside resistance after in vitro tobramycin exposure, also acquired aminoglycoside resistance after repeated exposure to azithromycin, a macrolide antibiotic, and hydrogen peroxide; minimum inhibitory concentrations increased 4- to 16-fold. However, in vitro exposure to ceftazidime or trimethoprim/sulfamethoxazole did not induce aminoglycoside resistance. The aminoglycoside-resistant Bv isolate D0774 that was recovered late in chronic infection expressed the putative amrB efflux system transporter gene at a level ≥8-fold higher than C8395 at late exponential phase. After serial exposure to tobramycin and azithromycin, but not hydrogen peroxide, C8395 expressed amrB to similar levels as D0774, 10- and 9-fold more, respectively. After serial exposure to tobramycin, C8395 also acquired the capacity to accumulate 3 times less [3H]gentamicin at stationary phase (C8395 accumulates 5 times more [3H]gentamicin than D0774; other in vitro exposed isolates are currently being evaluated). These data suggest that in Bv antibiotic and oxidative stress can induce aminoglycoside resistance, and active efflux is involved in acquired aminoglycoside resistance in vivo and in vitro after antibiotic, but not oxidative, exposure.