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.

Bach Institute of Biochemistry Research Center of Biotechnology of the Russian Academy of Sciences Leninsky Prospect 33 Moscow 119071 Russia

Department of Biology and Biotechnology University of Pavia Via Ferrata 9 27100 Pavia Italy

Department of Medical Microbiology 2nd Faculty of Medicine Charles University and Motol University Hospital 5 Uvalu 84 15400 Prague Czech Republic

Department of Medical Microbiology and Infectious Diseases Rady Faculty of Health Sciences University of Manitoba 727 McDermot Avenue Winnipeg MB R3E 3P5 Canada

Department of Microbiology Faculty of Science University of Manitoba 213 Buller Building Winnipeg MB R3T 2N2 Canada

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