The Effect of 2-Thiocyanatopyridine Derivative 11026103 on Burkholderia Cenocepacia: Resistance Mechanisms and Systemic Impact

Abstract

Bacteria of the Burkholderia cepacia complex (Bcc) are associated with significant decline of lung functions in cystic fibrosis patients. Bcc infections are virtually impossible to eradicate due to their irresponsiveness to antibiotics. The 2-thiocyanatopyridine derivative 11026103 is a novel, synthetic compound active against Burkholderia cenocepacia. To characterize mechanisms of resistance to 11026103, B. cenocepacia was subjected to chemical mutagenesis, followed by whole genome sequencing. Parallel mutations in resistant isolates were localized in a regulatory protein of the efflux system Resistance-Nodulation-Division (RND)-9 (BCAM1948), RNA polymerase sigma factor (BCAL2462) and its cognate putative anti-sigma factor (BCAL2461). Transcriptomic analysis identified positive regulation of a major facilitator superfamily (MFS) efflux system BCAL1510-1512 by BCAL2462. Artificial overexpression of both efflux systems increased resistance to the compound. The effect of 11026103 on B. cenocepacia was analyzed by RNA-Seq and a competitive fitness assay utilizing an essential gene knockdown mutant library. 11026103 exerted a pleiotropic effect on transcription including profound downregulation of cluster of orthologous groups (COG) category “Translation, ribosomal structure, and biogenesis”. The competitive fitness assay identified many genes which modulated susceptibility to 11026103. In summary, 11026103 exerts a pleiotropic cellular response in B. cenocepacia which can be prevented by efflux system-mediated export.

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

“…To pro le genome-wide contributions to antibiotic susceptibility and resistance, we turned to transposon mutagenesis and Illumina sequencing to measure mutant abundance during antibiotic challenges. Previously, our lab has constructed transposon mutant libraries in K56-2, a multidrug-resistant ET12 epidemic lineage clinical isolate 26 , to identify the essential genome 27 and characterise targets and mechanisms of action for antimicrobials [28][29][30] . To leverage advances in sequencing and bioinformatic capabilities, we modi ed our Tn5 transposon to contain a random 20 bp barcode (Supplementary Fig.…”

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

“…Previously, our lab has constructed transposon mutant libraries in K56-2, a multidrug-resistant ET12 epidemic lineage clinical isolate (Darling et al 1998), to identify the essential genome (Gislason et al 2017b) and characterise targets and mechanisms of action for antimicrobials (Gislason et al 2017a; Hogan et al 2018; Nunvar et al 2019). To leverage advances in sequencing and bioinformatic capabilities, we modified our Tn 5 transposon to contain a random 20 bp barcode (Figure S1D), greatly facilitating tracking mutant abundance in many conditions by Illumina sequencing (Wetmore et al 2015).…”

“…To profile genome-wide contributions to antibiotic susceptibility and resistance, we turned to transposon mutagenesis and Illumina sequencing to measure mutant abundance during antibiotic challenges. Previously, our lab has constructed transposon mutant libraries in K56-2, a multidrugresistant ET12 epidemic lineage clinical isolate [26], to identify the essential genome [27] and characterise targets and mechanisms of action for antimicrobials [28][29][30]. To leverage advances in sequencing and bioinformatic capabilities, we modified our Tn5 transposon to contain a random 20 bp barcode (Figure S1D), greatly facilitating the tracking of mutant abundance in many conditions by Illumina sequencing [21].…”

Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens

“…Noteworthy, when the conserved RND-4 efflux pump is missing or inactivated, overexpression of the RND-9 system can compensate for its function. For example, in a B. cenocepacia RND-4 deletion strain, mutations in a gene (bcam1948) encoding a transcriptional repressor of the RND-9 operon were demonstrated to confer resistance to a new antitubercular thiopyridine compound whose antimicrobial activity was previously demonstrated to be impaired by RND-4 mediated extrusion [130,133]. Interestingly, mutations in the same regulator confer resistance to a 2,1,3-benzothiadiazol-5-yl family compound and to multiple antibiotics (chloramphenicol, ciprofloxacin, levofloxacin, norfloxacin, sparfloxacin and nalidixic acid) [134].…”