High levels of intrinsic tetracycline resistance in Mycobacterium abscessus is conferred by a tetracycline- modifying monooxygenase [PublishAheadOfPrint]

Tetracyclines have been one of the most successful class of antibiotics. However, its extensive use has led to the emergence of wide-spread drug resistance resulting in discontinuation of use against several bacterial infections. Prominent resistance mechanisms include drug efflux and the use of ribosome protection proteins. Infrequently, tetracyclines can be inactivated by the TetX class of enzymes, also referred to as tetracycline destructases. Low levels of tolerance to tetracycline in M. smegmatis and M. tuberculosis has been previously attributed to the WhiB7- dependent TetV/Tap efflux pump. However, M. abscessus is ~500 fold more resistant to tetracycline as compared to M. smegmatis and M. tuberculosis. In the present study, we show that this high level of resistance to tetracycline and doxycycline in M. abscessus is conferred by a WhiB7 independent tetracycline inactivating monooxygenase, MabTetX (MAB_1496c). Presence of sublethal doses of tetracycline and doxycycline results in >200-fold induction of MabTetX and an isogenic deletion strain is highly sensitive to both antibiotics. Further, purified MabTetX can rapidly monooxygenate both antibiotics. We also demonstrate that expression of MabTetX is repressed by MabTetR_x, by binding to an inverted repeat sequence upstream of MabTetR_x; presence of either antibiotic relieves this repression. Moreover, anhydrotetracycline (ATc) can effectively inhibit MabTetX activity in vitro and decreases the MIC of both tetracycline and doxycycline in vivo. Finally, we show that tigecycline, a third-generation tetracycline, is not only a poor substrate of MabTetX, but is also incapable of inducing the expression of MabTetX. This is the first demonstration of a tetracycline inactivating enzyme in mycobacteria. It a) elucidates the mechanism of tetracycline resistance in M. abscessus, 2) demonstrates the use of an inhibitor that can potentially reclaim the use of tetracycline and doxycycline and 3) identifies two sequential bottlenecks — MabTetX and MabTetR_x — for acquiring resistance to tigecycline thereby reiterating its use against M. abscessus.

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