Acacia senegal Budmunchiamines as a Potential Adjuvant for Rejuvenating Phenicol Activities towards Escherichia coli-Resistant Strains.

The continuous emergence of bacterial resistance alters the activities of different antibiotic families and requires appropriate strategies to solve therapeutic impasses. Medicinal plants are an attractive source for researching alternative and original therapeutic molecules. In this study, the fractionation of natural extracts from A. senegal and the determination of antibacterial activities are associated with molecular networking and tandem mass spectrometry (MS/MS) data used to characterize active molecule(s). The activities of the combinations, which included various fractions plus an antibiotic, were investigated using the "chessboard" test. Bio-guided fractionation allowed the authors to obtain individually active or synergistic fractions with chloramphenicol activity. An LC-MS/MS analysis of the fraction of interest and molecular array reorganization showed that most identified compounds are Budmunchiamines (macrocyclic alkaloids). This study describes an interesting source of bioactive secondary metabolites structurally related to Budmunchiamines that are able to rejuvenate a significant chloramphenicol activity in strains that produce an AcrB efflux pump. They will pave the way for researching new active molecules for restoring the activity of antibiotics that are substrates of efflux pumps in enterobacterial-resistant strains.

Contribution of efflux and mutations in fluoroquinolone susceptibility in MDR enterobacterial isolates: a quantitative and molecular study.

OBJECTIVES: The emergence of MDR strains is a public health problem in the management of associated infections. Several resistance mechanisms are present, and antibiotic efflux is often found at the same time as enzyme resistance and/or target mutations. However, in the laboratory routinely, only the latter two are identified and the prevalence of antibiotic expulsion is underestimated, causing a misinterpretation of the bacterial resistance phenotype. The development of a diagnostic system to quantify the efflux routinely would thus improve the management of patients. METHODS: A quantitative technique based on detection of clinically used fluoroquinolones was investigated in Enterobacteriaceae clinical strains with a high or basal efflux activity. The detail of efflux involvement was studied from MIC determination and antibiotic accumulation inside bacteria. WGS was carried out on selected strains to determine the genetic background associated with efflux expression. RESULTS: Only 1 Klebsiella pneumoniae isolate exhibited a lack of efflux whereas 13 isolates had a basal efflux and 8 presented efflux pump overexpression. The antibiotic accumulation evidenced the efficacy of the efflux mechanism in strains, and the contribution of dynamic expulsion versus target mutations in fluoroquinolone susceptibility. CONCLUSIONS: We confirmed that phenylalanine arginine ß-naphthylamide is not a reliable marker of efflux due to the affinity of the AcrB efflux pump for different substrates. We have developed an accumulation test that can be used efficiently on clinical isolates collected by the biological laboratory. The experimental conditions and protocols ensure a robust assay that with improvements in practice, expertise and equipment could be transferred to the hospital laboratory to diagnose the contribution of efflux in Gram-negative bacteria.

Rejuvenating the Activity of Usual Antibiotics on Resistant Gram-Negative Bacteria: Recent Issues and Perspectives.

Antibiotic resistance continues to evolve and spread beyond all boundaries, resulting in an increase in morbidity and mortality for non-curable infectious diseases. Due to the failure of conventional antimicrobial therapy and the lack of introduction of a novel class of antibiotics, novel strategies have recently emerged to combat these multidrug-resistant infectious microorganisms. In this review, we highlight the development of effective antibiotic combinations and of antibiotics with non-antibiotic activity-enhancing compounds to address the widespread emergence of antibiotic-resistant strains.

A framework for dissecting affinities of multidrug efflux transporter AcrB to fluoroquinolones.

Sufficient concentration of antibiotics close to their target is key for antimicrobial action. Among the tools exploited by bacteria to reduce the internal concentration of antibiotics, multidrug efflux pumps stand out for their ability to capture and expel many unrelated compounds out of the cell. Determining the specificities and efflux efficiency of these pumps towards their substrates would provide quantitative insights into the development of antibacterial strategies. In this light, we developed a competition efflux assay on whole cells, that allows measuring the efficacy of extrusion of clinically used quinolones in populations and individual bacteria. Experiments reveal the efficient competitive action of some quinolones that restore an active concentration of other fluoroquinolones. Computational methods show how quinolones interact with the multidrug efflux transporter AcrB. Combining experiments and computations unveils a key molecular mechanism acting in vivo to detoxify bacterial cells. The developed assay can be generalized to the study of other efflux pumps.

Cephalosporin translocation across enterobacterial OmpF and OmpC channels, a filter across the outer membrane.

Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation of structurally related cephalosporins, ceftazidime (CAZ), cefotaxime (CTX) and cefepime (FEP). CAZ is highly active on E. coli producing OmpF (Outer membrane protein F) but less efficient on cells expressing OmpC (Outer membrane protein C), whereas FEP and CTX kill bacteria regardless of the porin expressed. This matches with the different capacity of CAZ and FEP to accumulate into bacterial cells as quantified by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry). Furthermore, porin reconstitution into planar lipid bilayer and zero current assays suggest permeation of ≈1,000 molecules of CAZ per sec and per channel through OmpF versus ≈500 through OmpC. Here, the instant killing is directly correlated to internal drug concentration. We propose that the net negative charge of CAZ represents a key advantage for permeation through OmpF porins that are less cation-selective than OmpC. These data could explain the decreased susceptibility to some cephalosporins of enterobacteria that exclusively express OmpC porins.

An Outer Membrane Vesicle-Based Permeation Assay (OMPA) for Assessing Bacterial Bioavailability.

When searching for new antibiotics against Gram-negative bacterial infections, a better understanding of the permeability across the cell envelope and tools to discriminate high from low bacterial bioavailability compounds are urgently needed. Inspired by the phospholipid vesicle-based permeation assay (PVPA), which is designed to predict non-facilitated permeation across phospholipid membranes, outer membrane vesicles (OMVs) of Escherichia coli either enriched or deficient of porins are employed to coat filter supports for predicting drug uptake across the complex cell envelope. OMVs and the obtained in vitro model are structurally and functionally characterized using cryo-TEM, SEM, CLSM, SAXS, and light scattering techniques. In vitro permeability, obtained from the membrane model for a set of nine antibiotics, correlates with reported in bacterio accumulation data and allows to discriminate high from low accumulating antibiotics. In contrast, the correlation of the same data set generated by liposome-based comparator membranes is poor. This better correlation of the OMV-derived membranes points to the importance of hydrophilic membrane components, such as lipopolysaccharides and porins, since those features are lacking in liposomal comparator membranes. This approach can offer in the future a high throughput screening tool with high predictive capacity or can help to identify compound- and bacteria-specific passive uptake pathways.

Clinical Status of Efflux Resistance Mechanisms in Gram-Negative Bacteria.

Antibiotic efflux is a mechanism that is well-documented in the phenotype of multidrug resistance in bacteria. Efflux is considered as an early facilitating mechanism in the bacterial adaptation face to the concentration of antibiotics at the infectious site, which is involved in the acquirement of complementary efficient mechanisms, such as enzymatic resistance or target mutation. Various efflux pumps have been described in the Gram-negative bacteria most often encountered in infectious diseases and, in healthcare-associated infections. Some are more often involved than others and expel virtually all families of antibiotics and antibacterials. Numerous studies report the contribution of these pumps in resistant strains previously identified from their phenotypes. The authors characterize the pumps involved, the facilitating antibiotics and those mainly concerned by the efflux. However, today no study describes a process for the real-time quantification of efflux in resistant clinical strains. It is currently necessary to have at hospital level a reliable and easy method to quantify the efflux in routine and contribute to a rational choice of antibiotics. This review provides a recent overview of the prevalence of the main efflux pumps observed in clinical practice and provides an idea of the prevalence of this mechanism in the multidrug resistant Gram-negative bacteria. The development of a routine diagnostic tool is now an emergency need for the proper application of current recommendations regarding a rational use of antibiotics.

Toxicity and bacterial anti-motility activities of the hydroethanolic extract of Acacia senegal (L.) Willd (Fabaceae) leaves.

BACKGROUND: Acacia senegal is a plant traditionally used for its various properties, including the treatment of infectious diseases. Recently, our team has demonstrated the ability of the hydroethanolic extract of the leaves to increase the activity of phenicol antibiotics against multi-resistant bacteria. The aim of this work is to determine the toxicological effects of the extract and its capacity to inhibit the bacterial mobility of Gram-negative bacteria, in order to evaluate the level of safety use of this plant. METHODS: The cytotoxicity test was performed using the neutral red absorption method. Acute and sub-acute oral toxicity were conducted on NMRI mice and Wistar rats. The behaviour and adverse effects were recorded during the 14 days of the acute study. For the subacute test, biochemical parameters, food and water consumption, and morphological parameters were determined. The anti-motility activities were evaluated on Pseudomonas aeruginosa PA01 and Escherichia coli AG100, using specific concentrations of Agar as required by the method. RESULTS: HEASG induced inhibition of keratinocytes cell growth with an IC50 of 1302 ± 60 µg/mL. For the acute toxicity study in mice, the single dose of extract of 2000 mg/kg body weight caused no deaths and no behavioural changes were observed; therefore, the median lethal dose (LD50) of HEASG was calculated to 5000 mg/kg body weight. In Wistar rats, no mortality was observed at 250, 500 and 1000 mg/kg/day during the 28-day subacute oral toxicity study. The weights of both females and males increased globally over time, regardless of the batch. No statistically significant differences were registered for organ weights and biochemical parameters, except for chloride for biochemical parameters. Water and food consumption did not change significantly. Furthermore, no macroscopic changes in organ appearance were observed. Regarding anti-motility activity, the extract has reduced the swarming motility of PA01 and AG100 significantly at the concentration of 32 µg/mL (P < 0.001). The extract has reduced the swimming motility (P < 0.01) of PA01 but not AG100. CONCLUSIONS: The results suggest that hydroethanolic extract of A. senegal leaves has significant activity against bacterial motility and relatively low toxicity.

Quinazoline Derivatives Designed as Efflux Pump Inhibitors: Molecular Modeling and Spectroscopic Studies.

Multidrug resistance of bacteria is a worrying concern in the therapeutic field and an alternative method to combat it is designing new efflux pump inhibitors (EPIs). This article presents a molecular study of two quinazoline derivatives, labelled BG1189 and BG1190, proposed as EPIs. In silico approach investigates the pharmacodynamic and pharmacokinetic profile of BG1189 and BG1190 quinazolines. Molecular docking and predicted ADMET features suggest that BG1189 and BG1190 may represent attractive candidates as antimicrobial drugs. UV-Vis absorption spectroscopy was employed to study the time stability of quinazoline solutions in water or in dimethyl sulfoxide (DMSO), in constant environmental conditions, and to determine the influence of usual storage temperature, normal room lighting and laser radiation (photostability) on samples stability. The effects of irradiation on BG1189 and BG1190 molecules were also assessed through Fourier-transform infrared (FTIR) spectroscopy. FTIR spectra showed that laser radiation breaks some chemical bonds affecting the substituents and the quinazoline radical of the compounds.

Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria.

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 ß-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

Chemical Highlights Supporting the Role of Lipid A in Efficient Biological Adaptation of Gram-Negative Bacteria to External Stresses.

The outer membrane (OM) of Gram-negative bacteria provides an efficient barrier against external noxious compounds such as antimicrobial agents. Associated with drug target modification, it contributes to the overall failure of chemotherapy. In the complex OM architecture, Lipid A plays an essential role by anchoring the lipopolysaccharide in the membrane and ensuring the spatial organization between lipids, proteins, and sugars. Currently, the targets of almost all antibiotics are intracellularly located and require translocation across membranes. We report herein an integrated view of Lipid A synthesis, membrane assembly, a structure comparison at the molecular structure level of numerous Gram-negative bacterial species, as well as its recent use as a target for original antibacterial molecules. This review paves the way for a new vision of a key membrane component that acts during bacterial adaptation to environmental stresses and for the development of new weapons against microbial resistance to usual antibiotics.

Acacia senegal Extract Rejuvenates the Activity of Phenicols on Selected Enterobacteriaceae Multi Drug Resistant Strains.

This study reported the phytochemical composition of two hydroethanolic extracts of Acacia senegal and Acacia seyal trees from Burkina Faso and their activities, alone or in combination with selected antibiotics, against multidrug resistant bacteria. High performance thin layer chromatography (HPTLC) method was used for phytochemical screening. Total phenolic and total flavonoid ant tannins in leaves extracts contents were assessed by spectrophotometric method. The minimal inhibitory concentrations (MICs) of plant extracts and antibiotics were determined using the microdilution method and p-iodonitrotetrazolium chloride. Combinations of extracts and antibiotics were studied using checkerboard assays. Screening revealed the presence of phenolic compounds, flavonoids, and tannins in the hydroethanolic extract (HE) of the leaves. The HE of A. seyal showed the highest total phenolic (571.30 ± 6.97 mg GAE/g), total flavonoids (140.41 ± 4.01 mg RTE/g), and tannins (24.72 ± 0.14%, condensed; 35.77 ± 0.19%, hydrolysable tannins). However, the HE of A. senegal showed the lowest total phenolic (69.84 ± 3.54 mg GAE/g), total flavonoids (27.32 ± 0.57 mg RTE/g), and tannins (14.60 ± 0.01%, condensed; 3.09 ± 0.02%, hydrolysable). The MICs for HE and antibiotics were in the range of 2-512 and 0.008-1024 mg/L, respectively. All tested HE presented an MIC greater than 512 mg/L except HE of A. senegal. The lowest MIC value (128 mg/L) was obtained with HE of A. senegal against Klebsiella aerogenes EA298 and Escherichia coli AG100A. Interesting restoring effects on chloramphenicol and florphenicol activity were detected with alcoholic extracts of A. senegal against resistant E. coli and K. aerogenes strains that overproduce AcrAB or FloR pumps. The adjuvant effect of HE of A. senegal suggests that the crude extract of leaves could be a potential source of molecules for improving the susceptibility of bacteria to phenicols antibiotics.

An Intertwined Network of Regulation Controls Membrane Permeability Including Drug Influx and Efflux in Enterobacteriaceae.

The transport of small molecules across membranes is a pivotal step for controlling the drug concentration into the bacterial cell and it efficiently contributes to the antibiotic susceptibility in Enterobacteriaceae. Two types of membrane transports, passive and active, usually represented by porins and efflux pumps, are involved in this process. Importantly, the expression of these transporters and channels are modulated by an armamentarium of tangled regulatory systems. Among them, Helix-turn-Helix (HTH) family regulators (including the AraC/XylS family) and the two-component systems (TCS) play a key role in bacterial adaptation to environmental stresses and can manage a decrease of porin expression associated with an increase of efflux transporters expression. In the present review, we highlight some recent genetic and functional studies that have substantially contributed to our better understanding of the sophisticated mechanisms controlling the transport of small solutes (antibiotics) across the membrane of Enterobacteriaceae. This information is discussed, taking into account the worrying context of clinical antibiotic resistance and fitness of bacterial pathogens. The localization and relevance of mutations identified in the respective regulation cascades in clinical resistant strains are discussed. The possible way to bypass the membrane/transport barriers is described in the perspective of developing new therapeutic targets to combat bacterial resistance.

The challenge of intracellular antibiotic accumulation, a function of fluoroquinolone influx versus bacterial efflux.

With the spreading of antibiotic resistance, the translocation of antibiotics through bacterial envelopes is crucial for their antibacterial activity. In Gram-negative bacteria, the interplay between membrane permeability and drug efflux pumps must be investigated as a whole. Here, we quantified the intracellular accumulation of a series of fluoroquinolones in population and in individual cells of Escherichia coli according to the expression of the AcrB efflux transporter. Computational results supported the accumulation levels measured experimentally and highlighted how fluoroquinolones side chains interact with specific residues of the distal pocket of the AcrB tight monomer during recognition and binding steps.

Complex Response of the CpxAR Two-Component System to ß-Lactams on Antibiotic Resistance and Envelope Homeostasis in Enterobacteriaceae.

The Cpx stress response is widespread among Enterobacteriaceae We previously reported a mutation in cpxA in a multidrug-resistant strain of Klebsiella aerogenes isolated from a patient treated with imipenem. This mutation yields a single-amino-acid substitution (Y144N) located in the periplasmic sensor domain of CpxA. In this work, we sought to characterize this mutation in Escherichia coli by using genetic and biochemical approaches. Here, we show that cpxAY144N is an activated allele that confers resistance to ß-lactams and aminoglycosides in a CpxR-dependent manner, by regulating the expression of the OmpF porin and the AcrD efflux pump, respectively. We also demonstrate the effect of the intimate interconnection between the Cpx system and peptidoglycan integrity on the expression of an exogenous AmpC ß-lactamase by using imipenem as a cell wall-active antibiotic or by inactivating penicillin-binding proteins. Moreover, our data indicate that the Y144N substitution abrogates the interaction between CpxA and CpxP and increases phosphotransfer activity on CpxR. Because the addition of a strong AmpC inducer such as imipenem is known to cause abnormal accumulation of muropeptides (disaccharide-pentapeptide and N-acetylglucosamyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamy-meso-diaminopimelic-acid-d-alanyl-d-alanine) in the periplasmic space, we propose these molecules activate the Cpx system by displacing CpxP from the sensor domain of CpxA. Altogether, these data could explain why large perturbations to peptidoglycans caused by imipenem lead to mutational activation of the Cpx system and bacterial adaptation through multidrug resistance. These results also validate the Cpx system, in particular, the interaction between CpxA and CpxP, as a promising therapeutic target.

Synthesis and Biological Evaluation of Four New Ricinoleic Acid-Derived 1-O-alkylglycerols.

A series of novel substituted 1-O-alkylglycerols (AKGs) containing methoxy (8), gem-difluoro (9), azide (10) and hydroxy (11) group at 12 position in the alkyl chain were synthesized from commercially available ricinoleic acid (12). The structures of these new synthesized AKGs were established by NMR experiments as well as from the HRMS and elementary analysis data. The antimicrobial activities of the studied AKGs 8-11 were evaluated, respectively, and all compounds exhibited antimicrobial activity to different extents alone and also when combined with some commonly used antibiotics (gentamicin, tetracycline, ciprofloxacin and ampicillin). AKG 11 was viewed as a lead compound for this series as it exhibited significantly higher antimicrobial activity than compounds 8-10.

A simple phenotypic test for detecting the contribution of outer membrane permeability to carbapenem resistance.

Introduction. The worldwide emergence of carbapenem resistance in Gram-negative bacteria makes the development of simple tests mandatory to identify antimicrobial resistance mechanisms. Enzymatic and membrane barriers are the prominent resistance mechanisms described in these bacteria. Several tests are currently used to detect carbapenemase activities.Aim. However, a simple test for the identification of membrane-associated mechanisms of resistance is not yet available and this mechanism is often inferred after the exclusion of a carbapenemase in carbapenem-resistant Gram-negative bacteria.Methodology. Different media (liquid and solid) containing a membrane permeabilizer were tested to identify the existence of a membrane barrier. Here, polymyxin B nonapeptide (PMBN) was selected to bypass the role of impermeability in clinical carbapenem-resistant Enterobacteriaceae, including Escherichia coli, Enterobacter cloacae , Klebsiella pneumoniae and Klebsiella aerogenes isolates. In parallel, the expression of porins (OmpC and OmpF types) was checked in the various bacterial strains in order to search for a correlation between the restoration of susceptibility and the expression of porin.Results. Using a large number of clinical isolates, PMBN associated with a carbapenem allowed us to detect porin-deficient isolates with a sensitivity ranging from 89 to 93 % and a specificity ranging from 86 to 100 %.Conclusion. This paves the way for a diagnostic assay allowing the detection of this membrane-associated mechanism of resistance in Enterobacteriaceae.

Porins and small-molecule translocation across the outer membrane of Gram-negative bacteria.

Gram-negative bacteria and their complex cell envelope, which comprises an outer membrane and an inner membrane, are an important and attractive system for studying the translocation of small molecules across biological membranes. In the outer membrane of Enterobacteriaceae, trimeric porins control the cellular uptake of small molecules, including nutrients and antibacterial agents. The relatively slow porin-mediated passive uptake across the outer membrane and active efflux via efflux pumps in the inner membrane creates a permeability barrier. The synergistic action of outer membrane permeability, efflux pump activities and enzymatic degradation efficiently reduces the intracellular concentrations of small molecules and contributes to the emergence of antibiotic resistance. In this Review, we discuss recent advances in our understanding of the molecular and functional roles of general porins in small-molecule translocation in Enterobacteriaceae and consider the crucial contribution of porins in antibiotic resistance.

Enterobacter spp.: Update on Taxonomy, Clinical Aspects, and Emerging Antimicrobial Resistance.

The genus Enterobacter is a member of the ESKAPE group, which contains the major resistant bacterial pathogens. First described in 1960, this group member has proven to be more complex as a result of the exponential evolution of phenotypic and genotypic methods. Today, 22 species belong to the Enterobacter genus. These species are described in the environment and have been reported as opportunistic pathogens in plants, animals, and humans. The pathogenicity/virulence of this bacterium remains rather unclear due to the limited amount of work performed to date in this field. In contrast, its resistance against antibacterial agents has been extensively studied. In the face of antibiotic treatment, it is able to manage different mechanisms of resistance via various local and global regulator genes and the modulation of the expression of different proteins, including enzymes (ß-lactamases, etc.) or membrane transporters, such as porins and efflux pumps. During various hospital outbreaks, the Enterobacter aerogenes and E. cloacae complex exhibited a multidrug-resistant phenotype, which has stimulated questions about the role of cascade regulation in the emergence of these well-adapted clones.

Fluoroquinolone-derived fluorescent probes for studies of bacterial penetration and efflux.

Fluorescent probes derived from the fluoroquinolone antibiotic ciprofloxacin were synthesised using a Cu(i)-catalysed azide-alkyne cycloaddition (CuAAC) to link a ciprofloxacin azide derivative with alkyne-substituted green and blue fluorophores. The azide (2) and fluorophore (3 and 4) derivatives retained antimicrobial activity against Gram-positive and Gram-negative bacteria. The use of confocal fluorescent microscopy showed intracellular penetration, which was substantially enhanced in the presence of carbonyl cyanide 3-chlorophenylhydrazone as an efflux pump inhibitor in Escherichia coli.