Search

In the search for new antituberculosis drugs with novel mechanisms of action, we evaluated the antimycobacterial activity of a panel of eight phenolic acids against four pathogenic mycobacterial model species, including Mycobacterium tuberculosis. We demonstrated that salicylic acid (SA), as well as the iodinated derivatives 5-iodo-salicylic acid (5ISA) and 3,5-diiodo-salicylic acid (3,5diISA), displayed promising antitubercular activities. Remarkably, using a genetically encoded mycobacterial intrabacterial pH reporter, we describe for the first time that SA, 5ISA, 3,5diISA, and the anti-inflammatory drug aspirin (ASP) act by disrupting the intrabacterial pH homeostasis of M. tuberculosis in a dose-dependent manner under in vitro conditions mimicking the endolysosomal pH of macrophages. In contrast, the structurally related second-line anti-TB drug 4-aminosalicylic acid (PAS) had no pH-dependent activity and was strongly antagonized by l-methionine supplementation, thereby suggesting distinct modes of action. Finally, we propose that SA, ASP, and its two iodinated derivatives could restrict M. tuberculosis growth in a pH-dependent manner by acidifying the cytosol of the bacilli, therefore making such compounds very attractive for further development of antibacterial agents., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The rise of multi-drug-resistant (MDR) pathogenic bacteria presents a grave challenge to global public health, with antimicrobial resistance ranking as the third leading cause of mortality worldwide. Understanding the mechanisms underlying antibiotic resistance is crucial for developing effective treatments. Efflux pumps, particularly those of the resistance-nodulation-cell division (RND) superfamily, play a significant role in expelling molecules from bacterial cells, contributing to the emergence of multi-drug resistance. These are transmembrane transporters naturally produced by Gram-negative bacteria. This review provides comprehensive insights into the modulation of RND efflux pump expression in bacterial pathogens by numerous and common molecules (bile, biocides, pharmaceuticals, additives, plant extracts, etc.). The interplay between these molecules and efflux pump regulators underscores the complexity of antibiotic resistance mechanisms. The clinical implications of efflux pump induction by non-antibiotic compounds highlight the challenges posed to public health and the urgent need for further investigation. By addressing antibiotic resistance from multiple angles, we can mitigate its impact and preserve the efficacy of antimicrobial therapies.

The development of new and effective antimicrobial compounds is urgent due to the emergence of resistant bacteria. Natural plant flavonoids are known to be effective molecules, but their activity and selectivity have to be increased. Based on previous aurone potency, we designed new aurone derivatives bearing acetamido and amino groups at the position 5 of the A ring and managing various monosubstitutions at the B ring. A series of 31 new aurone derivatives were first evaluated for their antimicrobial activity with five derivatives being the most active (compounds 10 , 12 , 15 , 16 , and 20 ). The evaluation of their cytotoxicity on human cells and of their therapeutic index (TI) showed that compounds 10 and 20 had the highest TI. Finally, screening against a large panel of pathogens confirmed that compounds 10 and 20 possess large spectrum antimicrobial activity, including on bioweapon BSL3 strains, with MIC values as low as 0.78 µM. These results demonstrate that 5-acetamidoaurones are far more active and safer compared with 5-aminoaurones, and that benzyloxy and isopropyl substitutions at the B ring are the most promising strategy in the exploration of new antimicrobial aurones.

Antibiotic resistance has increased the demand for novel treatments against multidrug-resistant microorganisms. In the research literature, 5-fluorouracil (5-FU) was proposed as an alternative due to its intrinsic antibacterial property. However, given its toxicity profile at high doses, its use in antibacterial therapy is dubious. In the quest for improving the efficacy of 5-FU, the present study intends to synthesise 5-FU derivatives and assess their susceptibility and mechanism against pathogenic bacteria. It was found that the compounds having tri-hexylphosphonium substitution on both nitrogen groups of 5-FU (6a, 6b and 6c) had considerable activity against both Gram-positive and Gram-negative bacteria. Among the active compounds, those with an asymmetric linker group 6c were found to have higher antibacterial efficacy. However, no conclusive efflux inhibition activity was found. As elucidated by electron microscopy studies, these self-assembling active phosphonium-based 5-FU derivatives caused considerable septal damage and cytosolic alterations in Staphylococcus aureus cells. In Escherichia coli, these compounds triggered plasmolysis. Interestingly, the minimal inhibitory concentration (MIC) of the most potent 5-FU derivative 6c remained constant, regardless of the bacteria's resistance profile. Further analysis revealed that compound 6c generated significant alterations in membrane permeabilization and depolarization in S. aureus and E. coli cells at the MIC. Compound 6c was found to substantially impede bacterial motility, suggesting its importance in regulating bacterial pathogenicity. Additionally, the nonhaemolytic activity of 6c suggested that it could be a potential therapeutic option for treating multidrug-resistant bacterial infections., (© 2023 The Authors. Archiv der Pharmazie published by Wiley-VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Gram-negative bacteria were reported as a significant cause of infections in both community and nosocomial settings. Considered as one of the greatest threats to public health, the spread of bacteria drug resistance and the lack of effective alternative treatment options remains problematic. Herein, we report a promising strategy to combat Gram-negative resistant strains consisting of the combination of a macrolide antibiotic with a polyaminoisoprenyl adjuvant derivative leading to a significant decrease of antibiotic resistance.

Pseudomonas aeruginosa is intrinsically resistant to many antibiotics due to the impermeability of its outer membrane and to the constitutive expression of efflux pumps. Here, we show that the polyaminoisoprenyl compound NV716 at sub-MIC concentrations re-sensitizes P. aeruginosa to abandoned antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane and increasing antibiotic accumulation inside the bacteria. It also prevents selection of resistance to antibiotics and increases their activity against biofilms. No stable resistance could be selected to NV716-itself after serial passages with subinhibitory concentrations, but the transcriptome of the resulting daughter cells shows an upregulation of genes involved in the synthesis of lipid A and LPS, and a downregulation of quorum sensing-related genes. Accordingly, NV716 also reduces motility, virulence factors production, and biofilm formation. NV716 shows a unique and highly promising profile of activity when used alone or in combination with antibiotics against P. aeruginosa, combining in a single molecule anti-virulence and potentiator effects. Additional work is required to more thoroughly understand the various functions of NV716., (© 2022. The Author(s).)

Potentiators can improve antibiotic activity against difficult-to-treat Gram-negative bacteria like Escherichia coli, Klebsiella pneumoniae or Acinetobacter baumannii. They represent an appealing strategy in view of the paucity of therapeutic alternatives in case of multidrug resistance. Here, we examine the ability of the polyamino-isoprenyl compound NV716 to restore the activity of a series of disused antibiotics (rifampicin, azithromycin, linezolid, fusidic acid, novobiocin, chloramphenicol, and doxycycline, plus ciprofloxacin as an active drug) against these three species in planktonic cultures, but also in infected human monocytes and biofilms and we study its underlying mechanism of action. NV716 considerably reduced the MICs of these antibiotics (2-11 doubling dilutions), the highest synergy being observed with the more lipophilic drugs. This potentiation was related to a strong interaction of NV716 with LPS, ensuing permeabilization of the outer membrane, and leading to an increased accumulation of the antibiotics inside bacteria. Moreover, NV716 increased the relative potency of all drugs against intracellular infection by the same bacteria as well as their maximal efficacy, probably related to an improvement of antibiotic activity against persisters. Lastly, NV716 also enhanced rifampicin activity against biofilms from these three species. All these effects were observed at sub-MIC concentrations of NV716 (and thus unrelated to a bactericidal effect), and in conditions for which no toxicity was evidenced towards eukaryotic cells. Altogether, these data highlight for the first time the potential interest of NV716 as an adjuvant against these Gram-negative pathogens placed in the priority list of WHO for search of new therapies., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Prosthetic implants are widely used in dentistry and orthopedics and, as a result, infections can occur which cause their removal. Therefore, it is essential to propose methods of eradicating the bacteria that remain on the prosthesis during treatment. For this purpose, it is necessary to develop surfaces whose antibacterial activity can be controlled. Herein, we designed innovative and smart phosphonium self-assembled monolayer (SAM) interfaces that can be electrically activated on demand for controlling bacterial contaminations on solid surfaces. Upon electroactivation with a low potential (0.2 V for 60 min., conditions determined through a DOE ), a successful stamping out of Gram-positive and Gram-negative bacterial strains was obtained with SAM-modified titanium surfaces, effectively killing 95% of Staphylococcus aureus and 90% Klebsiella pneumoniae . More importantly, no toxicity towards eukaryotic cells was observed which further enhances the biocompatible character of these novel surfaces for further implementation.

Producing industrially significant compounds with more environmentally friendly represents a challenging task. The large-scale production of an exogenous molecule in a host microfactory can quickly cause toxic effects, forcing the cell to inhibit production to survive. The key point to counter these toxic effects is to promote a gain of tolerance in the host, for instance, by inducing a constant flux of the neo-synthetized compound out of the producing cells. Efflux pumps are membrane proteins that constitute the most powerful mechanism to release molecules out of cells. We propose here a new biological model, Deinococcus geothermalis, organism known for its ability to survive hostile environment; with the aim of coupling the promising industrial potential of this species with that of heterologous efflux pumps to promote engineering tolerance. In this study, clones of D. geothermalis containing various genes encoding chromosomal heterologous efflux pumps were generated. Resistant recombinants were selected using antibiotic susceptibility tests to screen promising candidates. We then developed a method to determine the efflux efficiency of the best candidate, which contains the gene encoding the MdfA of Salmonella enterica serovar Choleraesuis. We observe 1.6 times more compound in the external medium of the hit recombinant than that of the WT at early incubation time. The data presented here will contribute to better understanding of the parameters required for efficient production in D. geothermalis.

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds ( 7 - 23 ) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14 - 16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7 - 23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds ( 10 , 15 ) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% ( 19 ). The 4-phenylbenzylidene derivative ( 15 ) demonstrated significant MDR-reversal "dual action" for β -lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes . 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

Active efflux confers intrinsic resistance to multiple antibiotics in Pseudomonas aeruginosa , including old disused molecules. Beside resistance, intracellular survival is another reason for failure to eradicate bacteria with antibiotics. We evaluated the capacity of polyaminoisoprenyl potentiators (designed as efflux pump inhibitors [EPIs]) NV716 and NV731 compared to PAβN to restore the activity of disused antibiotics (doxycycline, chloramphenicol [substrates for efflux], and rifampin [nonsubstrate]) in comparison with ciprofloxacin against intracellular P. aeruginosa (strains with variable efflux levels) in THP-1 monocytes exposed over 24 h to antibiotics alone (0.003 to 100× MIC) or combined with EPIs. Pharmacodynamic parameters (apparent static concentrations [ C s ] and maximal relative efficacy [ E max ]) were calculated using the Hill equation of concentration-response curves. PAβN and NV731 moderately reduced (0 to 4 doubling dilutions) antibiotic MICs but did not affect their intracellular activity. NV716 markedly reduced (1 to 16 doubling dilutions) the MIC of all antibiotics (substrates or not for efflux; strains expressing efflux or not); it also improved their relative potency and maximal efficacy (i.e., lower C s ; more negative E max ) intracellularly. In parallel, NV716 reduced the persister fraction in stationary cultures when combined with ciprofloxacin. In contrast to PAβN and NV731, which act only as EPIs against extracellular bacteria, NV716 can resensitize P. aeruginosa to antibiotics whether they are substrates or not for efflux, both extracellularly and intracellularly. This suggests a complex mode of action that goes beyond a simple inhibition of efflux to reduce bacterial persistence. NV716 appears to be a useful adjuvant, including to disused antibiotics with low antipseudomonal activity, to improve their activity, including against intracellular P. aeruginosa ., (Copyright © 2021 American Society for Microbiology.)

The growing number of multidrug resistant strains in Tunisia has become a serious health concern contributing to high rate of mortality and morbidity. Since current antibiotics are rapidly becoming ineffective, novel strategies to combat resistance are needed. Recently, we demonstrated that combination of a tetracycline antibiotic with various polyaminoisoprenyl adjuvants can sustain the life span and enhance the activity of these drugs against Pseudomonas aeruginosa reference strain (PA01). In the context of our continuing studies, the effective approach of antibiotic-adjuvant was investigated against a large panel of P. aeruginosa Tunisian clinical strains collected from the Military Hospital of Tunis. In this paper, we demonstrated that the combination of a farnesyl spermine compound 3 used at concentrations ranging from 2.5 to 10 µM, in the presence of doxycycline or minocycline leads to a significant decrease of P. aeruginosa antibiotic resistance.

A series consisting of new polyaminoisoprenyl derivatives were prepared in moderate to good chemical yields varying from 32 to 64% according to two synthetic pathways: (1) using a titanium-reductive amination reaction affording a 50/50 mixture of cis and trans isomers and (2) a direct nucleophilic substitution leading to a stereoselective synthesis of the compounds of interest. These compounds were then successfully evaluated for their in vitro antibiotic enhancer properties against resistant Gram-negative bacteria of four antibiotics belonging to four different families. The mechanism of action against Enterobacter aerogenes of one of the most efficient of these chemosensitizing agents was precisely evaluated by using fluorescent dyes to measure outer-membrane permeability and to determine membrane depolarization. The weak cytotoxicity encountered led us to perform an in vivo experiment dealing with the treatment of mice infected with Salmonella typhimurium and affording preliminary promising results in terms of tolerance and efficiency of the polyaminoisoprenyl derivative 5r /doxycycline combination.

Emergence of antibioresistance is currently a major threat of public health worldwide. Hence there is an urge need of finding new antibacterial material. Herein, we report a simple and eco-friendly method to synthesize homo and heterodicationic ionic liquids based on quaternary phosphonium and ammonium salt. In order to investigate the structure activity relationship (SAR) we measured the MICs of a series of 16 derivatives with structural variations (nature of cations and counter-ions, size of linker and alkyl side chains as well as structural symmetry) over a range of Gram-positive and Gram-negative bacterial strains from the ESKAPE group. Some of the tested structures exhibit high antimicrobial activities (MIC = 0.5 mg/L) and are active over a wide range of bacteria from Gram-positive to Gram-negative. Overall, these results reveal the strong potential of di-cationic derivatives as antibacterial agents and the determination of activities from structural features gives decisive information for future synthesis of such di-cationic structures for biocidal purpose., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The great emergence of multi-resistant bacterial strains and the low renewal of antibiotics molecules are leading human and veterinary medicine to certain therapeutic impasses. Therefore, there is an urgent need to find new therapeutic alternatives including new molecules in the current treatments of infectious diseases. Methionine aminopeptidase (MetAP) is a promising target for developing new antibiotics because it is essential for bacterial survival., Objective: To screen for potential MetAP inhibitors by in silico virtual screening of the ZINC database and evaluate the best potential lead molecules by in vitro studies., Methods: We have considered 200,000 compounds from the ZINC database for virtual screening with FlexX software to identify potential inhibitors against bacterial MetAP. Nine chemical compounds of the top hits predicted were purchased and evaluated in vitro. The antimicrobial activity of each inhibitor of MetAP was tested by the disc-diffusion assay against one Gram-positive (Staphylococcus aureus) and two Gram-negative (Escherichia coli & Pseudomonas aeruginosa) bacteria. Among the studied compounds, compounds ZINC04785369 and ZINC03307916 showed promising antibacterial activity. To further characterize their efficacy, the minimum inhibitory concentration was determined for each compound by the microdilution method which showed significant results., Results: These results suggest compounds ZINC04785369 and ZINC03307916 as promising molecules for developing MetAP inhibitors., Conclusion: Furthermore, they could therefore serve as lead molecules for further chemical modifications to obtain clinically useful antibacterial agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The spreading of multidrug-resistant bacteria and the lack of novel antibiotic molecules leave clinicians and veterinarians with very limited options to treat bacterial infections, especially those caused by Gram-negative pathogens. To reduce the selection of antibiotic resistance mechanisms and their transfer to human pathogens, veterinary pharmaceutical companies have dramatically decreased the number of antibiotics used. Among all the investigated alternate solutions, chemosensitizers, which decrease the amount of the used drugs, appear to be one of the most promising strategies. In this study, we reported that polyamino-isoprenyl derivatives can potentiate florfenicol activity against veterinary sensitive reference strains as well as clinical isolates. These molecules induce inner membrane depolarization and subsequently inhibit efflux pumps by collapsing the proton-motive force (PMF). Considering that Bordetella bronchiseptica rotor flagellum is highly PMF dependent and that flagellar motility represents an important factor involved in colonization, we monitored the swimming and swarming motilities of bacteria and showed a strong inhibition in the presence of the lead selected compound. Taken together, our results suggest that this class of molecules are able to increase treatment efficacy and decrease drug consumption.

The emergence of multidrug-resistant bacteria and pathogens has created an urgent need for the development of new antibiotics. Herein we report our investigations into the broad-spectrum activity of an easily prepared water-soluble polyaminosterol compound, namely claramine A1, against both drug-sensitive and drug-resistant Gram-negative and Gram-positive bacterial strains. We also report its peculiar mechanism of action, which differs from that of all the other well-known classes of antibiotics, toward Gram-negative and Gram-positive bacteria. Given their low cytotoxicity, this class of compounds based on claramine A1 could constitute an effective response to combat the emergence of multidrug-resistant bacteria and nosocomial diseases., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The gram-negative pathogen Providencia stuartii forms floating communities within which adjacent cells are in apparent contact, before depositing as canonical surface-attached biofilms. Because porins are the most abundant proteins in the outer membrane of gram-negative bacteria, we hypothesized that they could be involved in cell-to-cell contact and undertook a structure-function relationship study on the two porins of P. stuartii , Omp-Pst1 and Omp-Pst2. Our crystal structures reveal that these porins can self-associate through their extracellular loops, forming dimers of trimers (DOTs) that could enable cell-to-cell contact within floating communities. Support for this hypothesis was obtained by studying the porin-dependent aggregation of liposomes and model cells. The observation that facing channels are open in the two porin structures suggests that DOTs could not only promote cell-to-cell contact but also contribute to intercellular communication., Competing Interests: The authors declare no conflict of interest.

We report the synthesis of new mono, di and tri phosphonium ionic liquids and the evaluation of their antibacterial activities on both Gram-positive and Gram-negative bacteria from the ESKAPE-group. Among the molecules synthesized some of them reveal a strong bactericidal activity (MIC = 0.5 mg/L) for Gram-positive bacteria (including resistant strains) comparable to that of standard antibiotics. A comparative Gram positive and Gram negative antibacterial activities shows that the nature of counter-ion has no significant effects. Interestingly, the increase of phosphonium lateral chains (from 4 to 8 carbons) results in a decrease of antibacterial activities. However, the increase of the spacer length has a positive influence on the activity on both Gram-positive and Gram-negative bacteria except for E. aerogenes. Finally, the increased charge density has no effect on the Gram-positive antibacterial activities (MIC between 2 and 4 mg/L) but seems to attenuate (except for P. aeruginosa) the discrimination between Gram-positive and Gram-negative. Overall these results suggest a unique mechanism of action of these triphenylamine-phosphonium ionic liquid derivatives., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Antibiotic resistance is now a worldwide therapeutic problem. Since the beginning of anti-infectious treatment bacteria have rapidly shown an incredible ability to develop and transfer resistance mechanisms. In the last decades, the design variation of pioneer bioactive molecules has strongly improved their activity and the pharmaceutical companies partly won the race against the clock. Since the 1980s, the new classes of antibiotics that emerged were mainly directed to Gram-positive bacteria. Thus, we are now facing to multidrug-resistant Gram-negative bacteria, with no therapeutic options to deal with them. These bacteria are mainly resistant because of their double membrane that conjointly impairs antibiotic accumulation and extrudes these molecules when entered. The main challenge is to allow antibiotics to cross the impermeable envelope and reach their targets. One promising solution would be to associate, in a combination therapy, a usual antibiotic with a non-antibiotic chemosensitizer. Nevertheless, for effective drug discovery, there is a prominent lack of tools required to understand the rules of permeation and accumulation into Gram-negative bacteria. By the use of a multidrug-resistant enterobacteria, we introduce a high-content screening procedure for chemosensitizers discovery by quantitative assessment of drug accumulation, alteration of barriers, and deduction of their activity profile. We assembled and analyzed a control chemicals library to perform the proof of concept. The analysis was based on real-time monitoring of the efflux alteration and measure of the influx increase in the presence of studied compounds in an automatized bio-assay. Then, synergistic activity of compounds with an antibiotic was studied and kinetic data reduction was performed which led to the calculation of a score for each barrier to be altered.

Here we report on translocation of short poly-arginines across the MOMP porin, the major outer membrane protein in the cell wall of Campylobacter jejuni . MOMP was purified to homogeneity from a pathogenic strain of C. jejuni . Its reconstitution in lipid membranes and measuring the ion-current revealed two main distinct populations of protein channels which we interpreted as mono and trimers. Addition of poly-arginines causes concentration and voltage dependent ion-current fluctuations. Increasing the transmembrane potential decreases the residence time of the peptide inside the channel indicating successful translocation. We conclude that poly-arginines can cross the outer membrane of Campylobacter through the MOMP channel.

Biofilms are organized communities of bacterial cells that are responsible for the majority of human chronic bacterial infections. Providencia stuartii is a Gram-negative biofilm-forming bacterium involved in high incidence of urinary tract infections in catheterized patients. Yet, the structuration of these biofilms, and their resistance to environmental insults remain poorly understood. Here, we report on planktonic cell growth and biofilm formation by P. stuartii, in conditions that mimic its most common pathophysiological habitat in humans, i.e. the urinary tract. We observed that, in the planktonic state, P. stuartii forms floating communities of cells, prior to attachment to a surface and subsequent adoption of the biofilm phenotype. P. stuartii planktonic and biofilm cells are remarkably resistant to calcium, magnesium and to high concentrations of urea, and show the ability to grow over a wide range of pHs. Experiments conducted on a P. stuartii strain knocked-out for the Omp-Pst2 porin sheds light on the role it plays in the early stages of growth, as well as in the adaptation to high concentration of urea and to varying pH.

Dihydromotuporamine C and its derivatives were evaluated for their in vitro antimicrobial activities and antibiotic enhancement properties against Gram-negative bacteria and clinical isolates. The mechanism of action of one of these derivatives, MOTU-N44, was investigated against Enterobacter aerogenes by using fluorescent dyes to evaluate outer-membrane depolarization and permeabilization. Its efficiency correlated with inhibition of dye transport, thus suggesting that these molecules inhibit drug transporters by de-energization of the efflux pump rather than by direct interaction of the molecule with the pump. This suggests that depowering the efflux pump provides another strategy to address antibiotic resistance., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The Gram-negative organism Campylobacter jejuni is the major cause of food poisoning. Unlike Escherichia coli, which has two major porins, OmpC and OmpF, C. jejuni has one, termed major outer membrane protein (MOMP) through which nutrients and antibiotics transit. We report the 2.1-Å crystal structure of C. jejuni MOMP expressed in E. coli and a lower resolution but otherwise identical structure purified directly from C. jejuni. The 2.1-Å resolution structure of recombinant MOMP showed that although the protein has timeric arrangement similar to OmpC, it is an 18-stranded, not 16-stranded, β-barrel. The structure has identified a Ca 2+ bound at the constriction zone, which is functionally significant as suggested by molecular dynamics and single-channel experiments. The water-filled channel of MOMP has a narrow constriction zone, and single-molecule studies show a monomeric conductivity of 0.7±0.2 nS and a trimeric conductance of 2.2±0.2 nS. The ion neutralizes negative charges at the constriction zone, reducing the transverse electric field and reversing ion selectivity. Modeling of the transit of ciprofloxacin, an antibiotic of choice for treating Campylobacter infection, through the pore of MOMP reveals a trajectory that is dependent upon the presence metal ion., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Peptide deformylase (PDF) is considered an excellent target to develop antibiotics. We have performed an extensive characterization of a new PDF from the pathogen Streptococcus agalactiae, showing properties similar to other known PDFs. S. agalactiae PDF could be used as PDF prototype as it allowed to get complete sets of 3-dimensional, biophysical and kinetic data with virtually any inhibitor compound. Structure-activity relationship analysis with this single reference system allowed us to reveal distinct binding modes for different PDF inhibitors and the key role of a hydrogen bond in potentiating the interaction between ligand and target. We propose this protein as an irreplaceable tool, allowing easy and relevant fine comparisons between series, to design, challenge and validate novel series of inhibitors. As proof-of-concept, we report here the design and synthesis of effective specific bacterial PDF inhibitors of an oxadiazole series with potent antimicrobial activity against a multidrug resistant clinical isolate.

Staphylococcus aureus, a Gram positive coccal bacterium is a major cause of nosocomial infection. We report the synthesis of new triphenylamine phosphonium ionic liquids which are able to self-assemble into multiwall nanoassemblies and to reveal a strong bactericidal activity (MIC=0.5mg/L) for Gram positive bacteria (including resistant strains) comparable to that of standard antibiotics. Time kill, metabolism and fluorescence confocal microscopy studies show a quasi-instantaneously penetration of the nanoassemblies inside the bacteria resulting of a rapid blocking (30min) of their proliferation. As confirmed by rezasurin reduction monitoring, these compounds strongly affect the bacterial metabolism and a Gram positive versus Gram negative selectivity is clearly observed. These fluorescent phosphonium ionic liquid might constitute a useful tool for both translocation studies and to tackle infectious diseases related to the field of implantology., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multidrug resistant bacteria have been a worldwide concern for decades. Though new molecules that effectively target Gram-positive bacteria are currently appearing on the market, a gap remains in the treatment of infections caused by Gram-negative bacteria. Therefore, new strategies must be developed against these pathogens. The aim of this study was to select an antibiotic for which a bacterium is naturally resistant and to use an escort molecule to restore susceptibility, similarly to the model of β-lactam/ β-lactamase inhibitors. High-content screening was performed on the reference strain PA01, allowing the selection of four polyamino-isoprenic compounds that acted synergistically with doxycycline. They were assayed against clinical isolates and Multi-Drug-Resistant strains. One of these compounds was able to decrease the MIC of doxycycline on the reference strain, efflux pump overproducers and clinical isolates of P. aeruginosa, to the susceptibility level. Similar results were obtained using chloramphenicol as the antibiotic. Membrane permeation assays and real-time efflux experiments were used to characterize the mechanism of doxycycline potentiation. The results showed that the selected compound strongly decreases the efficiency of glucose-triggered efflux associated with a slight destabilization of the outer membrane. According to these data, targeting natural resistance may become an interesting way to combat MDR pathogens and could represent an alternative to already devised strategies.

Multidrug resistant (MDR) bacteria are an increasing health problem with the shortage of new active antibiotic agents. Among effective mechanisms that contribute to the spread of MDR Gram-negative bacteria are drug efflux pumps that expel clinically important antibiotic classes out of the cell. Drug pumps are attractive targets to restore the susceptibility toward the expelled antibiotics by impairing their efflux activity. Arylhydantoin derivatives were investigated for their potentiation of activities of selected antibiotics described as efflux substrates in Enterobacter aerogenes expressing or not AcrAB pump. Several compounds increased the bacterial susceptibility toward nalidixic acid, chloramphenicol and sparfloxacin and were further pharmacomodulated to obtain a better activity against the AcrAB producing bacteria.

The Cj1169c-encoded putative protein of Campylobacter was expressed and purified from E. coli after sequence optimization. The purified protein allowed the production of a specific rabbit antiserum that was used to study the protein expression in vitro and its subcellular localization in the bacterial cell and putative interactions with other proteins. This protein is produced in Campylobacter and it clearly localizes into the periplasmic space. The level of protein production depends on factors, including pH, temperature, osmolarity, and growth phase suggesting a role in the Campylobacter environmental adaptation. The cysteine residues present in the sequence are probably involved in disulfide bridges, which may promote covalent interactions with other proteins of the Campylobacter envelope.

A series consisting of ianthelliformisamimes A, B, and C as well as its synthetic analogues was prepared in high chemical yield, from 27 to 91%, using peptide coupling as the key step, and the compounds were evaluated for their in vitro antibiotic enhancer properties against resistant Gram-negative bacteria and clinical isolates. The mechanism of action of one of these derivatives against Pseudomonas aeruginosa when combined with doxycycline was precisely evaluated utilizing bioluminescence to measure ATP efflux and fluorescence to evaluate membrane depolarization.

Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate reagent (BOP) serves as an efficient and versatile coupling reagent for the design and synthesis of new polyamino geranic acid derivatives in moderate to good chemical yields varying from 47% to 83%. These compounds induced a significant decrease of antibiotic resistance in two Gram-negative bacterial MDR strains. Our data suggested that their mechanism of action is closely associated with the inhibition of the efflux pumps., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The role of major porin OmpPst1 of Providencia stuartii in antibiotic susceptibility for two carbapenems is investigated by combining high-resolution conductance measurements, liposome swelling, and microbiological assays. Reconstitution of a single OmpPst1 into a planar lipid bilayer and measuring the ion current, in the presence of imipenem, revealed a concentration-dependent decrease in conductance, whereas meropenem produced well-resolved short ion current blockages. Liposome swelling assays suggested a small flux of imipenem in contrast to a rapid permeation of meropenem. The lower antibiotic susceptibility of P. stuartii to imipenem compared to meropenem correlated well with the decreased level of permeation of the former through the OmpPst1 channel.

Aims: The effects of Thymus maroccanus essential oil (EO) on the integrity of the cell membranes and the permeability of the outer membrane (OM) and inner membrane (IM) of Escherichia coli, Enterobacter aerogenes and Salmonella enterica Typhimurium were investigated., Methods and Results: The bacterial release of intracellular proteins, cytoplasmic β-galactosidase and periplasmic β-lactamase induced by T. maroccanus EO was compared to the membranotropic activity of polymyxin B (PB) known as an effective permeabilizer of the membrane of Gram-negative bacteria. Results showed that T. maroccanus EO increased the permeability of the OM and IM of studied bacteria and induced the release of intracellular proteins into the external medium., Conclusions: The effect of T. maroccanus EO on the outer membrane was comparable to that of PB, and both T. maroccanus EO and PB induce similar levels of β-lactamase release. In addition, it also promoted the release of the cytoplasmic β-galactosidase. Moreover, the lipopolysaccharide molecules and the overexpression of efflux pumps seem to play a crucial role in the level of susceptibility of studied bacteria to the permeabilizing effect of T. maroccanus EO., Significance and Impact of Study: These results demonstrate that T. maroccanus EO can restore antibiotic activity by targeting the two bacterial membranes and would be attractive candidates for developing new adjuvants for combating resistant Gram-negative bacteria., (© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

Campylobacter jejuni is responsible for the major foodborne bacterial enteritis in humans. In contradiction with its fastidious growth requirements, this microaerobic pathogen can survive in aerobic food environments, suggesting that it must employ a variety of protection mechanisms to resist oxidative stress. For the first time, C. jejuni 81-176 inner and outer membrane subproteomes were analyzed separately using two-dimensional protein electrophoresis (2-DE) of oxygen-acclimated cells and microaerobically grown cells. LC-MS/MS analyses successfully identified 42 and 25 spots which exhibited a significantly altered abundance in the IMP-enriched fraction and in the OMP-enriched fraction, respectively, in response to oxidative conditions. These spots corresponded to 38 membrane proteins that could be grouped into different functional classes: (i) transporters, (ii) chaperones, (iii) fatty acid metabolism, (iv) adhesion/virulence and (v) other metabolisms. Some of these proteins were up-regulated at the transcriptional level in oxygen-acclimated cells as confirmed by qRT-PCR. Downstream analyses revealed that adhesion of C. jejuni to inert surfaces and swarming motility were enhanced in oxygen-acclimated cells or paraquat-stressed cells, which could be explained by the higher abundance of membrane proteins involved in adhesion and biofilm formation. The virulence factor CadF, over-expressed in the outer membrane of oxygen-acclimated cells, contributes to the complex process of C. jejuni adhesion to inert surfaces as revealed by a reduction in the capability of C. jejuni 81-176 ΔCadF cells compared to the isogenic strain.Taken together, these data demonstrate that oxygen-enriched conditions promote the over-expression of membrane proteins involved in both the biofilm initiation and virulence of C. jejuni.

In Gram-negative bacteria, the envelope is a sophisticated barrier protecting the cell against external toxic compounds. Membrane transporters, e.g., porins or efflux pumps, are main filters regulating the internal accumulation of various hydrophilic molecules. Regarding bacterial susceptibility towards antibacterial agents, membrane permeability is part of the early bacterial defense. The bacterium manages the translocation process, influx and efflux, to control the intracellular concentration of various molecules. Antibiotics and biocides are substrates of these mechanisms and the continuing emergence of multidrug resistant isolates is a growing worldwide health concern. Different strategies could be proposed to bypass the bacterial membrane barrier, comprising influx and efflux mechanisms, in order to restore the activity of antibiotics against resistant bacteria., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The present study assessed the antimicrobial activities of various natural products belonging to the terpenoids, alkaloids and phenolics against a collection of Gram-negative multidrug-resistant (MDR) bacteria. The results demonstrated that most of the compounds were extruded by bacterial efflux pumps. In the presence of the efflux pump inhibitor phenylalanine arginine β-naphthylamide (PAβN), the activities of laurentixanthone B (xanthone), plumbagin (naphthoquinone), 4-hydroxylonchocarpin (flavonoid) and MAB3 (coumarin) increased significantly against all studied MDR bacteria. Laurentixanthone B, 4-hydroxylonchocarpin and MAB3 contained the same pharmacophoric moiety as plumbagin. This study indicates that the AcrAB-TolC (Enterobacteriaceae) and MexAB-OprM (Pseudomonas aeruginosa) efflux pumps are involved in resistance of Gram-negative bacteria to most of the natural products., (Copyright © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The activities of two naturally occurring compounds, isobavachalcone and diospyrone, against documented strains and multidrug-resistant (MDR) Gram-negative bacterial isolates were evaluated. The results indicated that the two compounds exhibited intrinsic antibacterial activity against several Gram-negative bacteria, and their activities were significantly improved in the presence of an efflux pump inhibitor (MIC values decreased to below 10 microg/ml). In addition, the activities of isobavachalcone and diospyrone against various strains exhibiting deletions of the major efflux pump components (AcrAB, TolC) were significantly increased. The overall results indicate that isobavachalcone and diospyrone could be candidates for the development of new drugs against MDR strains and that their use in combination with efflux pump inhibitors reinforces their activity.

Antibiotic resistance mechanisms reported in Gram-negative bacteria are a worldwide health problem. The continuous dissemination of multi-drug resistant (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In MDR bacteria the over-expression of efflux pumps expel structurally-unrelated antibiotics decreasing their intracellular concentration. It is necessary to clearly define the molecular and genetic bases of the efflux pump in order to combat this mechanism. The characterization of efflux pumps, from genetic to structural studies, allows the definition of a new, original antiresistance bullet, the efflux pump inhibitor (EPI). This new class of antibacterial molecules may act conjointly to the usual antibiotic in order to restore its activity. Several families of EPIs have been now reported and described. The use of these EPIs promotes a significant increase of susceptibility to one or more antibiotics in strains or clinical isolates which were initially resistant. These EPIs may target different efflux targets, (i) the expression of genes that induces MDR, the transporters that pump the antibiotic out of bacterium, (ii) the assembly of membrane transporter complex involved in drug efflux, (iii) the energy involved in this active transport, (iv) the inhibition of the flux of molecules inside the efflux channel by competition or blocking (via steric hindrances). With the recent thorough characterization of the efflux pump AcrB at its structural and physiological level including the identification of drug affinity sites and kinetic parameters for some antibiotics, it is now possible to rationally develop an improved new generation of EPIs.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Infection generally occurs after ingestion of contaminated poultry products, usually conserved at low temperatures. The mechanisms promoting survival of C. jejuni in the cold remain poorly understood despite several investigations. The present study provides insight into the survival mechanism by establishing the involvement of polynucleotide phosphorylase (PNPase), a 3'-5' exoribonuclease with multiple biological functions in cold survival. The role of PNPase was demonstrated genetically using strains with altered pnp genes (which encode PNPase) created in C. jejuni F38011 and C. jejuni 81-76 backgrounds. Survival assays carried out at low temperatures (4 and 10 degrees C) revealed a difference of 3 log CFU/ml between the wild-type and the pnp deletion (Deltapnp) strains. This did not result from a general requirement for PNPase because survival rates of the strains were similar at higher growth temperatures (37 or 42 degrees C). trans-Complementation with plasmid pNH04 carrying the pnp gene under the control of its natural promoter restored the cold survival phenotype to the pnp deletion strains (at 4 and 10 degrees C) but not to the same level as the wild type. In this study we demonstrate the role of PNPase in low-temperature survival of C. jejuni and therefore attribute a novel biological function to PNPase directly related to human health.

Background: Resistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets. Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can also extrude antibiotics, obtain energy from the hydrolysis of ATP. Because E. coli must pass through two pH distinct environments of the gastrointestinal system of the host, it must be able to extrude toxic agents at very acidic and at near neutral pH (bile salts in duodenum and colon for example). The herein described study examines the effect of pH on the extrusion of ethidium bromide (EB)., Methodology/principal Findings: E. coli AG100 and its tetracycline induced progeny AG100(TET) that over-expresses the acrAB efflux pump were evaluated for their ability to extrude EB at pH 5 and 8, by our recently developed semi-automated fluorometric method. At pH 5 the organism extrudes EB without the need for metabolic energy (glucose), whereas at pH 8 extrusion of EB is dependent upon metabolic energy. Phe-Arg beta-naphtylamide (PAbetaN), a commonly assumed inhibitor of RND efflux pumps has no effect on the extrusion of EB as others claim. However, it does cause accumulation of EB. Competition between EB and PAbetaN was demonstrated and suggested that PAbetaN was preferentially extruded. A K(m) representing competition between PAbetaN and EB has been calculated., Conclusions/significance: The results suggest that E. coli has two general efflux systems (not to be confused with a distinct efflux pump) that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ABC transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host.

The essential oil of Helichrysum italicum significantly reduces the multidrug resistance of Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Combinations of the two most active fractions of the essential oil with each other or with phenylalanine arginine beta-naphthylamide yield synergistic activity. Geraniol, a component of one fraction, significantly increased the efficacy of beta-lactams, quinolones, and chloramphenicol.

Background: Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. beta-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression., Methodology/principal Findings: Here influx of beta-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single beta-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of beta-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility., Conclusions/significance: We propose the idea of a molecular "passport" that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections.

In Enterobacteriaceae, membrane permeability is a key in the level of susceptibility to antibiotics. Modification of the bacterial envelope by decreasing the porin production or increasing the expression of efflux pump systems has been reported. These phenomena are frequently associated with other resistance mechanisms such as alteration of antibiotics or modification of the drug targets, in various clinical isolates showing a Multi Drug Resistant phenotype (MDR). In Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Salmonella enterica several genes and external factors are involved in the emergence of MDR isolates. These bacterial isolates exhibit a noticeable reduction of functional porins per cell due to a decrease, a complete shutdown of synthesis, or the expression of an altered porin and a high expression of efflux systems (e.g. overexpression of the pump). The combined action of these mechanisms during an infection confers a significant decrease in bacterial sensitivity to antibiotherapy ensuring dissemination and colonization of the patient and favours the acquisition of additional mechanisms of resistance. MarA and ramA are involved in a complex regulation cascade controlling membrane permeability and actively participate in the triggering of the MDR phenotype. Mutations in regulator genes have been shown to induce the overproduction of efflux and the down-regulation of porin synthesis. In addition, various compounds such as salicylate, imipenem or chloramphenicol are able to activate the MDR response. This phenomenon has been observed both in vitro during culture of bacteria in the presence of drugs and in vivo during antibiotic treatment of infected patients. These effectors activate the expression of specific global regulators, marA, ramA, or target other genes located downstream in the regulation cascade.

The thermophilic Campylobacters are enteropathogenic for humans. We recently showed that Omp50 is a Campylobacter species-specific porin produced in Campylobacter jejuni and Campylobacter lari but not in Campylobacter coli. In the present study, we investigated regulation of the omp50 gene and found that its expression in C. jejuni was temperature-dependent, but independent of growth phase or medium viscosity. The use of RT-PCR and omp50::lacZ fusions showed that growth temperature control occurred at the transcriptional level. The promoter and the coding sequence were cloned in an Escherichia coli-Campylobacter shuttle plasmid and transferred to E. coli and to a C. jejuni Omp50-deficient strain. Regulation of omp50 gene expression by growth temperature was observed in the recombinant C. jejuni strain, but not in E. coli. The same regulation was also observed in wild-type C. lari strains and in a C. coli strain supplemented by the plasmid, suggesting that omp50 expression is controlled by a mechanism conserved among Campylobacter species.

The essential oil of wild Daucus carota L. obtained from aerial parts at the end of the flowering stage (DCEO) was reported as antimicrobial against the human enteropathogen Campylobacter jejuni. The aim of the present study was to extend this analysis to other Campylobacter species and to identify the active compounds of the essential oil, subjected to GC, GC-MS, and (13)C NMR analysis. A minimum inhibitory concentration assay was used to quantify the antimicrobial activity of DCEO and the major components, isolated on column chromatography. Growth of all the C. jejuni, Campylobacter coli, and Campylobacter lari strains tested, including one multidrug resistant C. jejuni, was inhibited to the same extent by DCEO. Molecules that were responsible for the antibacterial activity were identified as (E)-methylisoeugenol and elemicin. Moreover, the use of structural analogues of these compounds allowed us to identify important features that may account for the activity.

Campylobacter is the most common cause of bacterial gastroenteritis in the world. Poultry is the main reservoir of human infections. The widespread use of antibiotics in agriculture and veterinary medicine has resulted in the emergence of an increasing number of antibiotic-resistant Campylobacter strains that can be transmitted to humans through the food chain. Of particular concern to public health is the prevalence of resistance to macrolides and fluoroquinolones that are used in the treatment of life-threatening campylobacteriosis. The CmeABC efflux system has been shown to contribute to the intrinsic and acquired resistance to these antibiotics. In addition, by mediating resistance to bile, it is essential for colonization of the chicken gut in vivo. Inhibition of CmeABC may provide an effective means of reversing antibiotic resistance and decreasing the transmission of Campylobacter via the food chain. This would positively impact on public health by decreasing the morbidity, mortality and increased healthcare costs associated with the treatment of antibiotic-resistant Campylobacter.