Abstract
Chromobacterium violaceum is an environmental Gram-negative bacterium that causes infections in humans. Treatment of C. violaceum infections is difficult and little is known about the mechanisms of antibiotic resistance in this bacterium. In this work, we identified mutations in the MarR family transcription factor EmrR and in the protein GyrA as key determinants of quinolone resistance in C. violaceum, and we defined EmrR as a repressor of the MFS-type efflux pump EmrCAB. Null deletion of emrR caused increased resistance to nalidixic acid, but not to other quinolones or antibiotics of different classes. Moreover, the ΔemrR mutant showed decreased production of the purple pigment violacein. Importantly, we isolated C. violaceum spontaneous nalidixic acid-resistant mutants with a point mutation in the DNA-binding domain of EmrR (R92H), with antibiotic resistance profile similar to that of the ΔemrR mutant. Other spontaneous mutants with high MIC values for nalidixic acid and increased resistance to fluoroquinolones presented point mutations in the gene gyrA. Using DNA microarray, Northern blot and EMSA assays, we demonstrated that EmrR represses directly a few dozen genes, including the emrCAB operon and other genes related to transport, oxidative stress and virulence. This EmrR repression on emrCAB was relieved by salicylate. Although mutation of the C. violaceum emrCAB operon had no effect in antibiotic susceptibility or violacein production, deletion of emrCAB in an emrR mutant background restored antibiotic susceptibility and violacein production in the ΔemrR mutant. Using a biosensor reporter strain, we demonstrated that the lack of pigment production in ΔemrR correlates with the accumulation of quorum-sensing molecules in the cell supernatant of this mutant strain. Therefore, our data revealed that overexpression of the efflux pump EmrCAB via mutation and/or derepression of EmrR confers quinolone resistance and alters quorum-sensing signaling in C. violaceum, and that point mutation in emrR can contribute to emergence of antibiotic resistance in bacteria.
Highlights
Antibiotic resistance is a global public health problem with high impact on the treatment of bacterial infections, as many multidrug-resistant (MDR) strains have evolved in clinically relevant pathogens (Davies and Davies, 2010; Rossolini et al, 2014)
Antibiogram tests with 24 antibiotics (Supplementary Figure S1) and determination of MIC with eight antibiotics (Supplementary Table S3) revealed that the emrR mutant showed increased resistance to nalidixic acid, a quinolone (a 13-mm decrease in the halo and a fourfold increase in the MIC)
A phenotype of decreased violacein production, verified for the emrR mutant in LB liquid cultures, was complemented (Figure 2B). These results indicate that EmrR controls antibiotic resistance and pigment production in C. violaceum
Summary
Antibiotic resistance is a global public health problem with high impact on the treatment of bacterial infections, as many multidrug-resistant (MDR) strains have evolved in clinically relevant pathogens (Davies and Davies, 2010; Rossolini et al, 2014). Antibiotic-resistant strains arise by mutations in transcription factors that regulate the genes involved in antibiotic resistance. Members of the multiple antibiotic resistance regulator (MarR) family of transcription factors are involved in several biological processes in bacteria, including oxidative stress, virulence, and antibiotic resistance; most of them act as transcription repressors (Deochand and Grove, 2017). The first described member of this family, the marR gene of Escherichia coli, represses the marRAB operon, involved in resistance to multiple antibiotics (George and Levy, 1983; Seoane and Levy, 1995). Other MarR family transcription factors have been described regulating efflux pumps that contribute to antibiotic resistance, such as MexR in Pseudomonas aeruginosa (Chen et al, 2008), MepR in Staphylococcus aureus (Birukou et al, 2013), MarR in Mycobacterium smegmatis (Zhang et al, 2014), and EmrR in E. coli (Lomovskaya and Lewis, 1992). A similar mechanism has been described controlling the EmrCABsm efflux pump in Stenotrophomonas maltophilia (Huang et al, 2013), it is still unclear whether antibiotic resistance can emerge as a consequence of point mutation in the emrR gene, and other genes regulated by EmrR have not been identified
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