Functionally distinct mutations within AcrB underpin antibiotic resistance in different lifestyles

Abstract

Antibiotic resistance is a pressing healthcare challenge and is mediated by various mechanisms, including the active export of drugs via multidrug efflux systems, which prevent drug accumulation within the cell. Here, we studied how Salmonella evolved resistance to two key antibiotics, cefotaxime and azithromycin, when grown planktonically or as a biofilm. Resistance to both drugs emerged in both conditions and was associated with different substitutions within the efflux-associated transporter, AcrB. Azithromycin exposure selected for an R717L substitution, while cefotaxime for Q176K. Additional mutations in ramR or envZ accumulated concurrently with the R717L or Q176K substitutions respectively, resulting in clinical resistance to the selective antibiotics and cross-resistance to other drugs. Structural, genetic, and phenotypic analysis showed the two AcrB substitutions confer their benefits in profoundly different ways. R717L reduces steric barriers associated with transit through the substrate channel 2 of AcrB. Q176K increases binding energy for cefotaxime, improving recognition in the distal binding pocket, resulting in increased efflux efficiency. Finally, we show the R717 substitution is present in isolates recovered around the world.

Introduction

Antibiotics are crucial for modern medicine, but their introduction and use have resulted in the widespread emergence of antibiotic-resistant bacteria. Bacteria can rapidly adapt to changing environments, and exposure to antibiotics selects for genetic traits that confer resistance, promoting the expansion of resistant mutants¹. Several important mechanisms of antibiotic resistance have been described, including enzymatic degradation, target modification or bypass, membrane alterations and changes in efflux activity².

Energy-dependent efflux systems are responsible for the export of toxic compounds from the cell to the environment, are found in all bacteria, and act synergistically with other mechanisms of resistance³. In Gram-negative bacteria, efflux systems are tripartite transmembrane protein complexes that secrete molecules from the periplasm to the exterior of the cell. The ‘Resistance Nodulation cell Division’ (RND) efflux family is the most important for antibiotic export^4,5,6,7, and RND systems have been shown to determine the basal level of susceptibility of cells to many antimicrobials.

Within the RND family, the Enterobacterial AcrAB-TolC is the best characterised tripartite efflux system and is built around the energised inner membrane H⁺/drug-antiporter AcrB⁵. The functional unit of AcrB is a homotrimer, containing three functionally interdependent protomers, cycling consecutively through loose (L), tight (T) and open (O) conformational states during the efflux cycle in a supposedly cooperative fashion^8,9. This allosteric “pumping” allows a drug to be acquired from either periplasmic space or the outer leaflet of the inner membrane and passed out of the cell via a conduit produced by the partner outer membrane factor (OMF) and periplasmic adaptor proteins (PAPs)^4,10,11.

AcrB can export multiple classes of antibiotics, including macrolides, β-lactams, quinolones, rifamycins, tetracyclines, as well as other substrates, including anticancer drugs, bile salts, dyes and solvents^{12,13,14,15,16,17}. This broad substrate specificity is underpinned by the presence of distinct binding pockets within the pump. Drugs of different molecular weights are suggested to be processed in two principal multisite binding pockets, termed the ‘Proximal Binding Pocket’ (PBP) and the ‘Distal Binding Pocket’ (DBP), which have wide specificities and are separated from each other by the so-called gating or switch-loop^{8,18,19,20,21}. High-molecular-weight drugs appear to be predominantly recognised by the PBP, and recent evidence suggests they may be exported directly to the OMF, bypassing the DBP altogether²², whilst low-molecular-weight drugs are thought to be processed predominantly within the DBP^8,19. Access to these multisite binding pockets is governed by at least four distinct substrate channels, each of which also exhibits different substrate specificities^{22,23,24,25,26}. The principal periplasmic drug access channel for polar compounds is proposed to be channel 2 (CH2), preferred by macrolide, rifamycin and tetracycline antibiotics^23,26, while hydrophobic compounds, such as linezolid, phenicols, fluoroquinolones and novobiocin are suggested to be acquired from the outer leaflet of the inner membrane via channel 1 (CH1). Compounds entering via CH1 and CH2 are thought to pass sequentially through both the PBP and DBP, with access to the latter being restricted by the switch-loop. On the other hand, channel 3 (CH3), implicated in the transport of planar aromatic cations (PACs), such as benzalkonium chloride, crystal violet, ethidium bromide, methylene blue, and rhodamine 6G, is suggested to bypass the PBP and the gating loop altogether, allowing direct access to the DBP²⁶. Similarly, membrane-localised carboxylated substrates, such as fusidic acid and hydrophobic β-lactams, access the pump via a groove between the transmembrane helices TM1 and TM2, which forms part of the recently described CH4, again bypassing the PBP, allowing direct access to the DBP²⁵.

Whilst AcrB helps determine the intrinsic level of susceptibility to many drugs, it can also confer resistance when overexpressed due to mutations in the regulatory circuits controlling its production^27,28. Changes within AcrB itself that alter the export of specific antibiotics can also be selected by antibiotic exposure^{3,6,29,30,31,32}. For example, substitutions M78I and P319L were shown to confer decreased susceptibility to multiple antimicrobial substrates³³, and substitution G288D has been linked to increased tolerance against ciprofloxacin²⁹. These examples demonstrate how selection can favour strains with mutant AcrB proteins altering substrate recognition or export efficiency, as well as mutations in regulators which control pump expression.

Despite the benefits provided, the selection of resistance can have impacts on the fitness of a bacterium, and the fate of any resistance mutation that occurs within a population will depend on how permissive it is for the organism’s lifestyle³⁴. Efflux pumps contribute to various important cellular functions, including those relevant to infection. Relationships between efflux pump function and the ability to form biofilms have been established in multiple species³⁵, and loss of pump function commonly compromises virulence³⁶. Life within a biofilm is common for bacteria and is an important determinant of many infections, as biofilms are also, by nature, highly tolerant of antibiotics³⁷.

In this work, we used an evolution model to study how subinhibitory concentrations of two clinically important antibiotics, cefotaxime (Cef) and azithromycin (Azi), representing two major structural classes of antibiotics, cephalosporins and macrolides respectively, selected for resistance mechanisms in Salmonella, in both biofilm and planktonic conditions. We found that both antibiotics selected for unique substitutions within AcrB. We confirmed these substitutions affect antibiotic susceptibility and identified their prevalence in the real world of these mutant acrB alleles. Using structural and computational approaches supported by genetic and phenotypic analysis, we demonstrate how these two distinct substitutions within AcrB facilitate drug translocation through the efflux conduit of the pump in fundamentally different ways.

Results

Cefotaxime and azithromycin both select for substitutions within AcrB

To investigate the adaptation of Salmonella to clinically important antibiotics, we used representatives of two antibiotic families amongst the drugs of choice for the treatment of salmonellosis: cefotaxime, a third-generation cephalosporin, and azithromycin, a second-generation macrolide. We repeatedly exposed independent planktonic and biofilm lineages of S. Typhimurium 14028S to concentrations of azithromycin and cefotaxime that restricted planktonic growth rates by ~50% (10 and 0.062 μg/ml, respectively) for 17 passage cycles (each lasting 72 h). Estimation of the number of generations each population went through (based on calculating log2 × the dilution factor of cells in each condition by the number of passages) gave ~170 for planktonic conditions, ~264 for cefotaxime-exposed biofilms, ~289 for azithromycin-exposed biofilms and ~317 for control biofilms. The number of generations was higher for biofilms than planktonic conditions as we used a bead-based evolution model³⁸, where the dilution factor of cells which occurs when new, sterile beads are colonised, is higher than the dilution in planktonic cultures.