Loss of Methyltransferase Function and Increased Efflux Activity Leads to Doxycycline Resistance in Burkholderia pseudomallei.

The soil-dwelling bacterium is the causative agent of the potentially fatal disease melioidosis. The lack of a vaccine toward means that melioidosis treatment relies on prolonged antibiotic therapy, which can last up to 6 months in duration or longer. Due to intrinsic resistance, few antibiotics are effective against The lengthy treatment regimen required increases the likelihood of resistance development, with subsequent potentially fatal relapse. Doxycycline (DOX) has historically played an important role in the eradication phase of melioidosis treatment. Both primary and acquired DOX resistances have been documented in ; however, the molecular mechanisms underpinning DOX resistance have remained elusive. Here, we identify and functionally characterize the molecular mechanisms conferring acquired DOX resistance in an isogenic pair. Two synergistic mechanisms were identified. The first mutation occurred in a putative -adenosyl-l-methionine-dependent methyltransferase (encoded by ), which we propose leads to altered ribosomal methylation, thereby decreasing DOX binding efficiency. The second mutation altered the function of the efflux pump repressor gene, , resulting in increased expression of the resistance-nodulation-division efflux pump, AmrAB-OprA. Our findings highlight the diverse mechanisms by which can become resistant to antibiotics used in melioidosis therapy and the need for resistance monitoring during treatment regimens, especially in patients with prolonged or recrudesced positive cultures for .