Proximal binding pocket Arg717 substitutions in Escherichia coli AcrB cause clinically relevant divergencies in resistance profiles

Abstract

Multidrug resistance (MDR) in bacteria can be caused by the over-expression of multidrug efflux pumps belonging to the Resistance-Nodulation-Division (RND) superfamily of proteins. These intrinsic or acquired pumps can export a wide range of antibiotics. Recently, amino acid substitutions within these pumps have been observed in resistant clinical strains. Among others, two of these worrying gain-of-function mutations are R717L and R717Q in the proximal binding pocket of efflux pump AcrB (AcrB-Sa) found in azithromycin-resistant Salmonella enterica spp. We investigated the ramifications of these (and other) mutations in phylogenetically closely related AcrB from Escherichia coli (AcrB-Ec). We found that AcrB-Ec harboring Arg717 substitutions were significantly more effective in exporting all tested macrolides, with an up to 8-fold increase in the minimum inhibitory concentration (MIC) values (from 16 to 128 g/mL for azithromycin). Interestingly, gain-of-function was also seen for fluoroquinolones (2-fold higher MICs), while there was a consistent loss-of-function for the export of novobiocin and {beta}-lactam cloxacillin (2-fold lower MICs), pointing to a protein adaptation, which simultaneously partly compromised the efflux ability of other compounds with different molecular properties. Disk diffusion susceptibility testing corroborated the findings, as the R717Q and R717L mutant strains had significantly smaller inhibition zones for macrolides and fluoroquinolones and a larger inhibition zone for novobiocin, compared to the wild type. The spread and independent emergences of these potent efflux pump mutations highlight the necessity of control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors. ImportanceThe over-expression of multidrug efflux pumps is a major cause of multidrug-resistant bacteria. The emergence of gain-of-function mutations in these pumps is additionally worrying, as these render last-resort antibiotic treatment options ineffective. The Arg717-mutations in related Salmonella Typhi and Paratyphi A AcrB caused azithromycin-resistant strains, with resistance levels past breakpoints. This is worrying, as azithromycin is a last-resort antibiotic after fluoroquinolones to treat typhoid and paratyphoid fever. Our findings that the same substitutions in closely related Escherichia coli AcrB cause an increase in MICs for both fluoroquinolones and macrolides is worrying as it shows the effect of one amino acid substitution on a multitude of treatment options. Additionally, the finding that the substitutions negatively impact the export of other drugs, such as {beta}-lactam cloxacillin, suggests that treatment with multiple antibiotics may mitigate resistance and improve treatment. Our findings also imply the urge to develop novel antibiotics and inhibitors.