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

Abstract

The soil-dwelling bacterium Burkholderia pseudomallei is the causative agent of the potentially fatal disease melioidosis. The lack of a vaccine toward B. pseudomallei 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 B. pseudomallei. 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 B. pseudomallei; 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 B. pseudomallei pair. Two synergistic mechanisms were identified. The first mutation occurred in a putative S-adenosyl-L-methionine-dependent methyltransferase (encoded by BPSL3085), 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, amrR, resulting in increased expression of the resistance-nodulation-division efflux pump, AmrAB-OprA. Our findings highlight the diverse mechanisms by which B. pseudomallei 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 B. pseudomallei.

Development and validation of a triplex qPCR assay to detect efflux pump-mediated antibiotic resistance inBurkholderia pseudomallei

“…Despite 218 culture-based methods being accurate, they have lengthy turn-around-times, and do 219 not determine the genetic basis of resistance, both of which are important considera-220 tions when determining optimal therapeutic strategies. Nucleic-acid based detection 221 methods such as real-time PCR have the potential to circumvent many shortcomings 222 of traditional culture-based methods, offering more rapid turn-around times and de-223 termination of the underlying resistance mechanism, which is essential for identifying 224 potential cross-resistance mechanisms (33,36) or stepwise progression towards high- 225 level resistance development (23,37,38). Information generated from such assays has 226 the potential to inform optimal treatment strategies for melioidosis patients in near 227 real-time and reduce the number of cases where undiagnosed antibiotic resistance 228 emerges during treatment (39).…”

“…229 RND efflux pump upregulation is common in B. pseudomallei strains that exhibit 230 decreased susceptibility towards clinically relevant antibiotics. This mechanism can 231 either by itself, or in concert with additional mutations, cause resistance towards DOX, 232 SXT, and MEM (17,22,23,24), three invaluable antibiotics in melioidosis treatment 233 regimens. Therefore, the main goal of this study was to design, optimize and validate a 234 triplex qPCR probe-based assay targeting the AmrAB-OprA, BpeAB-OprB, and BpeEF- 235 OprC RND efflux pumps in B. pseudomallei (40).…”

“…In 239 contrast, probe-based PCR methods can simultaneously identify multiple targets in a 240 single assay while being efficient, affordable, sensitive and specific. 241 Given the large number of mutations that can cause RND efflux pump upregu-242 lation (17,22,23,28,43), we opted for a multiplex probe-based assay format that 243 identifies increased gene expression in favor of designing individual assays targeting 244 each regulatory mutation, the latter of which would be both impractical (due to the 245 large number of assays required) and prone to false negatives (due to the high likeli-246 hood of missing novel variants). In addition, detecting RNA expression ensures that 247 efflux pump upregulation is identified without requiring a thorough understanding 248 of the underlying regulatory networks.…”

“…258 The mechanisms conferring DOX and SXT resistance, and decreased MEM sus-259 ceptibility, have recently been unraveled in B. pseudomallei. Importantly, all resistant 260 strains analyzed to date have upregulated efflux pump expression (17,22,23,24 folM/ptr1, are required for the shift towards high-level DOX and SXT resistance (23,24). 268 The two-step nature of these mechanisms means that the triplex qPCR assay alone 269 cannot be used to definitively determine if a strain is resistant to DOX or SXT.…”

“…DOX. The triplex assay accurately detected efflux pump upregulation between isogenic 23 pairs, which corresponded with decreased antibiotic susceptibility. We further verified 24 assay performance on eight laboratory-generated B. pseudomallei mutants encoding 25 efflux pump regulator mutations.…”