Within-Host Evolution of Burkholderia pseudomallei during Chronic Infection of Seven Australasian Cystic Fibrosis Patients

Abstract

Cystic fibrosis (CF) is a genetic disorder characterized by progressive lung function decline. CF patients are at an increased risk of respiratory infections, including those by the environmental bacterium Burkholderia pseudomallei, the causative agent of melioidosis. Here, we compared the genomes of B. pseudomallei isolates collected between ~4 and 55 months apart from seven chronically infected CF patients. Overall, the B. pseudomallei strains showed evolutionary patterns similar to those of other chronic infections, including emergence of antibiotic resistance, genome reduction, and deleterious mutations in genes involved in virulence, metabolism, environmental survival, and cell wall components. We documented the first reported B. pseudomallei hypermutators, which were likely caused by defective MutS. Further, our study identified both known and novel molecular mechanisms conferring resistance to three of the five clinically important antibiotics for melioidosis treatment. Our report highlights the exquisite adaptability of microorganisms to long-term persistence in their environment and the ongoing challenges of antibiotic treatment in eradicating pathogens in the CF lung. Convergent evolution with other CF pathogens hints at a degree of predictability in bacterial evolution in the CF lung and potential targeted eradication of chronic CF infections in the future.

Molecular signatures of non-typeable Haemophilus influenzae lung adaptation in paediatric chronic lung disease

“…Due to the necessary passaging of strains to ensure purity 413 and to obtain sufficient RNA material for sequencing, we cannot discount that these 414 procedures may have influenced the NTHi expression profiles so that they no longer represent 415 their in vivo counterparts. However, as previously demonstrated with other bacterial species, 416 isolates appear to maintain their expression profiles even after microbiological handling and 417 culturing in vitro (63). For future studies, we recommend maximising the number of strains 418 isolated from individual patients together with longitudinal sampling to better understand 419 pathogen adaptation over time.…”

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

“…188 The triplex qPCR assay was tested on eight laboratory-generated Bp82 mutants con- respectively, resulting in death (32). It is becoming clearer that at least some of these 208 cases can be linked to the development of previously unrecognized resistance or 209 decreased susceptibility towards clinically relevant antibiotics (17,22,33,34). Of most 210 concern, antibiotic resistance in B. pseudomallei has now been documented towards 211 almost all antibiotics (17 for methods that can provide rapid detection of emerging antibiotic resistance.…”

“…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.…”