Microbial Primer: Multidrug efflux pumps.

Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell. While involved in a variety of biological roles, they are primarily known for their contribution to antibiotic resistance by limiting the intracellular accumulation of antimicrobial compounds within bacteria. These transporters are often overexpressed in clinical isolates, leading to multidrug-resistant phenotypes. Efflux pumps are classified into several families based on their structure and understanding the characteristics of each family is important for the development of novel therapies to restore antibiotic potency.

E. coli ST11 (O157:H7) does not encode a functional AcrF efflux pump.

Escherichia coli is a facultative anaerobe found in a wide range of environments. Commonly described as the laboratory workhorse, E. coli is one of the best characterized bacterial species to date, however much of our understanding comes from studies involving the laboratory strain E. coli K-12. Resistance-nodulation-division efflux pumps are found in Gram-negative bacteria and can export a diverse range of substrates, including antibiotics. E. coli K-12 has six RND pumps; AcrB, AcrD, AcrF, CusA, MdtBC and MdtF, and it is frequently reported that all E. coli strains possess these six pumps. However, this is not true of E. coli ST11, a lineage of E. coli, which is primarily composed of the highly virulent important human pathogen, E. coli O157:H7. Here we show that acrF is absent from the pangenome of ST11 and that this lineage of E. coli has a highly conserved insertion within the acrF gene, which when translated encodes 13 amino acids and two stop codons. This insertion was found to be present in 97.59 % of 1787 ST11 genome assemblies. Non-function of AcrF in ST11 was confirmed in the laboratory as complementation with acrF from ST11 was unable to restore AcrF function in E. coli K-12 substr. MG1655 ΔacrB ΔacrF. This shows that the complement of RND efflux pumps present in laboratory bacterial strains may not reflect the situation in virulent strains of bacterial pathogens.

EnvR is a potent repressor of acrAB transcription in Salmonella.

BACKGROUND: Resistance nodulation division (RND) family efflux pumps, including the major pump AcrAB-TolC, are important mediators of intrinsic and evolved antibiotic resistance. Expression of these pumps is carefully controlled by a network of regulators that respond to different environmental cues. EnvR is a TetR family transcriptional regulator encoded upstream of the RND efflux pump acrEF. METHODS: Binding of EnvR protein upstream of acrAB was determined by electrophoretic mobility shift assays and the phenotypic consequence of envR overexpression on antimicrobial susceptibility, biofilm motility and invasion of eukaryotic cells in vitro was measured. Additionally, the global transcriptome of clinical Salmonella isolates overexpressing envR was determined by RNA-Seq. RESULTS: EnvR bound to the promoter region upstream of the genes coding for the major efflux pump AcrAB in Salmonella, inhibiting transcription and preventing production of AcrAB protein. The phenotype conferred by overexpression of envR mimicked deletion of acrB as it conferred multidrug susceptibility, decreased motility and decreased invasion into intestinal cells in vitro. Importantly, we demonstrate the clinical relevance of this regulatory mechanism because RNA-Seq revealed that a drug-susceptible clinical isolate of Salmonella had low acrB expression even though expression of its major regulator RamA was very high; this was caused by very high EnvR expression. CONCLUSIONS: In summary, we show that EnvR is a potent repressor of acrAB transcription in Salmonella, and can override binding by RamA so preventing MDR to clinically useful drugs. Finding novel tools to increase EnvR expression may form the basis of a new way to prevent or treat MDR infections.

Efflux pumps mediate changes to fundamental bacterial physiology via membrane potential.

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. Efflux pump inactivation 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 Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting 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 hyperpolarization. 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.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants.

Molecular mechanisms of antibiotic resistance revisited.

Antibiotic resistance is a global health emergency, with resistance detected to all antibiotics currently in clinical use and only a few novel drugs in the pipeline. Understanding the molecular mechanisms that bacteria use to resist the action of antimicrobials is critical to recognize global patterns of resistance and to improve the use of current drugs, as well as for the design of new drugs less susceptible to resistance development and novel strategies to combat resistance. In this Review, we explore recent advances in understanding how resistance genes contribute to the biology of the host, new structural details of relevant molecular events underpinning resistance, the identification of new resistance gene families and the interactions between different resistance mechanisms. Finally, we discuss how we can use this information to develop the next generation of antimicrobial therapies.

Characteristics of antimicrobial peptide OaBac5mini and its bactericidal mechanism against Escherichia coli.

Introduction: Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac5mini is an active fragment of the sheep-derived OaBac5 belonging to the PrAMPs family. Methods: In this study, the antibacterial activity of OaBac5mini was investigated by testing the MICs against different stains of E. coli and S. aureus as well as the time-kill curve. The bactericidal mechanism was explored by determining the effect of OaBac5mini on the cell membrane. The stability and biosafety were also evaluated. Results: The susceptibility test demonstrated that OaBac5mini showed potent antibacterial activity against the multidrug-resistant (MDR) E. coli isolates. It is noticeable that the absence of inner membrane protein SbmA in E. coli ATCC 25922 caused the MIC of OaBac5mini to increase 4-fold, implying OaBac5mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac5mini against E. coli ATCC 25922 was not remarkably affected by the serum salts except for CaCl2 at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < 20 µg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac5mini at the concentration of 200 µg/mL (8 × MICs) could effectively kill E. coli ATCC 25922 after co-incubation for 12 h. In addition, OaBac5mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J2). Bioinformatic analysis indicated that OaBac5mini is a linear peptide with 8 net positive charges. Furthermore, OaBac5mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of E. coli ATCC25922. Conclusion: OaBac5mini is a stable and potent PrAMP that kills E. coli by two different modes of action - inhibiting intracellular target(s) and damaging cell membrane.