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The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.

Antimicrobial resistance (AMR) is a global problem hindering treatment of bacterial infections, rendering many aspects of modern medicine less effective. AMR genes (ARGs) are frequently located on plasmids, which are self-replicating elements of DNA. They are often transmissible between bacteria, and some have spread globally. Novel strategies to combat AMR are needed, and plasmid curing and anti-plasmid approaches could reduce ARG prevalence, and sensitise bacteria to antibiotics. We discuss the use of curing agents as laboratory tools including chemicals (e.g. detergents and intercalating agents), drugs used in medicine including ascorbic acid, psychotropic drugs (e.g. chlorpromazine), antibiotics (e.g. aminocoumarins, quinolones and rifampicin) and plant-derived compounds. Novel strategies are examined; these include conjugation inhibitors (e.g. TraE inhibitors, linoleic, oleic, 2-hexadecynoic and tanzawaic acids), systems designed around plasmid incompatibility, phages and CRISPR/Cas-based approaches. Currently, there is a general lack of in vivo curing options. This review highlights this important shortfall, which if filled could provide a promising mechanism to reduce ARG prevalence in humans and animals. Plasmid curing mechanisms which are not suitable for in vivo use could still prove important for reducing the global burden of AMR, as high levels of ARGs exist in the environment.

Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly understood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regulators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlorpromazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by interfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors. IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) superfamily are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; however, several issues have thus far hindered all efforts at designing new efflux inhibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibitors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds.

Background: The aim of our study is to describe a fast molecular method, able to distinguish and quantize the two different genotypes (652 and JP2) of an important periodontal pathogen: Actinobacillus actinomycetemcomitans. The two genotypes show differences in the expression of an important pathogenic factor: the leukotoxin (ltx). In order to evidence this, we performed a real time PCR procedure on the ltx operon, able to recognize Aa clinical isolates with different leukotoxic potentials.

Antimicrobial-resistant (AMR) infections pose a serious risk to human and animal health. A major factor contributing to this global crisis is the sharing of resistance genes between different bacteria via plasmids. The WHO lists Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae, producing extended-spectrum β-lactamases (ESBL) and carbapenemases as “critical” priorities for new drug development. These resistance genes are most often shared via plasmid transfer. However, finding methods to prevent resistance gene sharing has been hampered by the lack of screening systems for medium-/high-throughput approaches. Here, we have used an ESBL-producing plasmid, pCT, and a carbapenemase-producing plasmid, pKpQIL, in two different Gram-negative bacteria, E. coli and K. pneumoniae. Using these critical resistance-pathogen combinations, we developed an assay using fluorescent proteins, flow cytometry, and confocal microscopy to assess plasmid transmission inhibition within bacterial populations in a medium-throughput manner. Three compounds with some reports of antiplasmid properties were tested; chlorpromazine reduced transmission of both plasmids and linoleic acid reduced transmission of pCT. We screened the Prestwick library of over 1,200 FDA-approved drugs/compounds. From this, we found two nucleoside analogue drugs used to treat HIV, abacavir and azidothymidine (AZT), which reduced plasmid transmission (AZT, e.g., at 0.25 μg/ml reduced pCT transmission in E. coli by 83.3% and pKpQIL transmission in K. pneumoniae by 80.8% compared to untreated controls). Plasmid transmission was reduced by concentrations of the drugs which are below peak serum concentrations and are achievable in the gastrointestinal tract. These drugs could be used to decolonize humans, animals, or the environment from AMR plasmids. IMPORTANCE More and more bacterial infections are becoming resistant to antibiotics. This has made treatment of many infections very difficult. One of the reasons this is such a large problem is that bacteria are able to share their genetic material with other bacteria, and these shared genes often include resistance to a variety of antibiotics, including some of our drugs of last resort. We are addressing this problem by using a fluorescence-based system to search for drugs that will stop bacteria from sharing resistance genes. We uncovered a new role for two drugs used to treat HIV and show that they are able to prevent the sharing of two different types of resistance genes in two unique bacterial strains. This work lays the foundation for future work to reduce the prevalence of resistant infections.

Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors. IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.

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