Rresistance-nodulation-division (RND) efflux pumps in Gram-negative bacteria remove multiple, structurally distinct classes of antimicrobials from inside bacterial cells therefore directly contributing to multidrug resistance. There is also emerging evidence that many other mechanisms of antibiotic resistance rely on the intrinsic resistance conferred by RND efflux. In addition to their role in antibiotic resistance, new information has become available about the natural role of RND pumps including their established role in virulence of many Gram-negative organisms. This review also discusses the recent advances in understanding the regulation and structure of RND efflux pumps.

There are currently no clinically available inhibitors of metallo-β-lactamases (MBLs), enzymes that hydrolyze β-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3–7.2 μM; Ki values of 0.03–1.5 μM). Minimum inhibitory concentration assays demonstrated potentiation of β-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N–C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.

Gram-negative infections are increasingly difficult to treat because of their impermeable outer membranes (OM) and efflux pumps which maintain a low intracellular accumulation of antibiotics within cells. Historically, measurement of accumulation of drugs or dyes within Gram-negative cells has concentrated on analyzing whole bacterial populations. Here, we have developed a method to measure the intracellular accumulation of ethidium bromide, a fluorescent DNA intercalating dye, in single cells using flow cytometry. Bacterial cells were stained with SYTOTM 84 to easily separate cells from background cell debris. Ethidium bromide fluorescence was then measured within the SYTOTM 84 positive population to measure accumulation. In S. Typhimurium SL1344, ethidium bromide accumulation was low, however, in a number of efflux mutants, accumulation of ethidium bromide increased more than twofold, comparable to previous whole population analysis of accumulation. We demonstrate simultaneous measurement of ethidium bromide accumulation and GFP allowing quantification of gene expression or other facets of phenotype in single cells. In addition, we show here that this assay can be adapted for use with efflux inhibitors, with both Gram-negative and Gram-positive bacteria, and with other fluorescent substrates with different fluorescence spectra.

This study shows that efflux is important for maintaining low intracellular accumulation only in actively growing cells and that envelope permeability is the predominant factor in stationary-phase cells. This conclusion means that (i) antibiotics with intracellular targets may be less effective in complex infections with nongrowing or slow-growing bacteria, where intracellular accumulation may be low; (ii) efflux inhibitors may be successful in potentiating the activity of existing antibiotics, but potentially only for bacterial infections where cells are actively growing; and (iii) the remodeling of the cell envelope prior to stationary phase could provide novel drug targets.

Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Inactivation of efflux pumps is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella enterica serovar Typhimurium is drastically altered during slower growth leading to stationary phase cells having a re-modelled, less permeable, envelope that prevents antibiotics entering the cell. Here, we investigated the effects of knocking out the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild type strain SL1344, and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarisation. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella, with likely impacts on multiple phenotypes.