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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 β-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.

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.

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.

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 was 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 our 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 future a high throughput screening tool with high predictive capacity or can help to identify compound- and bacteria-specific passive uptake pathways. This article is protected by copyright. All rights reserved.

International audience; Recent reports predict increases in harmful cyanobacteria in water systems worldwide due to climatic and environmental changes, which would compromise water quality and public health. Among abiotic changes, higher salinities are expected to promote the growth of certain harmful species, such as Planktothrix agardhii, which forms blooms in brackish waters. Since P. agardhii is a common producer of cyanotoxin, we investigated the growth and tolerance of this species when exposed in vitro to a range of salinities, while assessing variations in its microcystin diversity and production in batch cultures during a time-frame experiment spanning 18 days. The study revealed salt acclimation of the brackish P. agardhii, which continued to produce microcystins in salty cultures, while maintaining its growth capacity at low to medium NaCl (ranging from 0 to 7.5 g l−1). With higher concentrations (10 to 15 g l−1) significantly less growth occurred, corresponding to the shortening of cyanobacterial filaments, which nevertheless maintained their metabolic functions, as revealed by the high intensity of chlorophyll auto-fluorescence and persistent microcystin production. These findings showed that moderate to high salt levels do not inhibit microcystin production by P. agardhii, at least for several weeks. This raises questions concerning the persistence of harmful cyanobacteria in shallow water systems more exposed to evaporation and consequently to an increase in salinity in the future, as predicted by various climate models.

Structure of bacterial envelope is one of the major factors contributing to Gram negative bacterial resistance. To develop new agents that target the bacterial membranes, we synthesized, by analogy with our previous peptide conjugates, new amphiphilic 3',4',6-trinaphthylmethylene neamines functionalized at position 5 through a short spacer by a chelating group, tris(2-pyridylmethyl)amine (TPA) and di-(picolyl)amine (DPA) and tetraazacyclotetradecane (Cyclam). ESI+ mass spectrometry analyses showed that neither Zn(II)(NeaDPA) nor Cu(II)(NeaCyclam) were stable in the Mueller Hinton (MH) medium used for antibacterial assays. In contrast Zn(NeaTPA) was stable in the MH medium. Interestingly, in MH, the free ligand NeaTPA was found bound to zinc, the zinc salt being the most abundant salt in this medium. Thus, the antibacterial activities of all compounds were evaluated as free ligands against E. coli strains, wild type AG100 and E. aerogenes EA289 (a clinical MDR strain that overexpresses AcrAB-TolC efflux pump), as well as AG100A an AcrAB- E. coli strain and EA298 a TolC- derivative. NeaCyclam and Zn(NeaTPA) were by far the most efficient compounds active against resistant isolate EA289 with MICs in the range 16-4 and 4 μM, respectively, while usual antibiotics such as β-lactams and phenicols were inactive (MICs > 128) and ciprofloxacin was at 64 μM. Zn(NeaTPA) and NeaCyclam were shown to target and permeabilize the outer membrane of EA289 by promoting the cleavage of nitrocefin by periplasmic β-lactamase. Moreover, all the neamine conjugates were able to block the efflux of 1,2'-dinaphthylamine in EA289, by acting on the efflux transporter located in the inner membrane. These membranotropic properties contribute to explain the activities of these neamine conjugates toward the MDR EA289 strain.

International audience; 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.

A maltotriose-conjugate can deliver molecules into the cytoplasmic space of Gram-negative bacteria by parasitizing the maltose uptake pathway., Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin–fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of the Escherichia coli maltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathway via the activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.

Background In Gram-negative bacteria, passing through the double membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibiotic activity. Spectrofluorimetry has been developed to follow fluoroquinolone accumulation inside bacteria using intrinsic bacterial fluorescence as an internal standard. However, adaptation for non-fluorescent antibiotics is needed; quantitative methods based on MS offer the possibility of expanding the detection range obtained by spectrofluorimetry. Objectives To validate, with spectrofluorimetry, the use of MS to measure antibiotic accumulation in cells and to determine the relationship between antibiotic concentrations and the amount of intrabacterial accumulation in different efflux backgrounds on the same batch of molecules. Methods Spectrofluorimetry was performed in parallel with MS on the same samples to measure the ciprofloxacin and fleroxacin accumulation in cells expressing various efflux pump levels. A microplate protocol was set up to determine the antibiotic accumulation as a function of external antibiotic concentrations. Results A correlation existed between the data obtained with spectrofluorimetry and MS, whatever the efflux pump or tested antibiotic. The results highlighted different dynamics of uptake between ciprofloxacin and fleroxacin as well as the relationship between the level of efflux activity and antibiotic accumulation. Conclusions We have developed a microplate protocol and cross-validated two complementary methods: spectrofluorimetry, which contains a reliable internal standard; and MS, which allows detection of low antibiotic amounts. These assays allow study of the dose effect and the efflux impact on the intrabacterial accumulation of antibiotics.

International audience; The efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.

Klebsiella pneumoniae, an Enterobacteriaceae that mostly causes hospital-acquired infections, belongs to the recently published WHO’s list of antibiotic-resistant pathogens that pose the greatest threat to human health. Indeed, K. pneumoniae is the enterobacterial species most concerned by both resistance to extended-spectrum cephalosporins, due to extended-spectrum β-lactamase (ESBL) production, and resistance to carbapenems, i.e. the β-lactams with the broadest activity. Carbapenem resistance is related not only to carbapenemase production, but also the production of ESBL or AmpC and the loss of general porins. Here, we characterized the mechanisms that deprived a urinary ESBL-producing, porin-deficient K. pneumoniae isolate, isolated 13 days after the end of a 40-day course of imipenem treatment, of its carbapenem resistance. These mechanisms were observed in two in-vivo derivatives of this isolate and consisted of mutations in genes encoding molecules that participate in the downregulation of the synthesis of PhoE, a porin specialized in phosphate transport. We obtained three new derivatives from one of the two original derivatives, following in-vitro antibiotic pressure, in which the carbapenem resistance was restored because of mutations in genes encoding molecules that participate in the upregulation of PhoE synthesis. Thus, we uncovered novel mechanisms of carbapenem resistance/susceptibility switching in K. pneumoniae.

International audience; To understand the antibiotic resistance in Gram-negative bacteria, a key point is to investigate antibiotic accumulation which is defined by influx and efflux. Several methods exist to evaluate the membrane permeability and efflux pump activity but they present some disadvantages and limitations. An optimized spectrofluorimetric method using the intrinsic tryptophan fluorescence as internal standard as well as a complementary microfluorimetric assay following the time-course accumulation in intact individual cells have been developed. Comparing the latter population and single cell approaches can lead to the understanding of the phenotypic heterogeneity within a population. The two methodologies lead to the determination of parameters, concentration, accumulation rates, localization that contribute to emerging concepts (RTC2T, SICAR) with the aim to identify and detail the antibiotic chemotypes that are involved in influx/efflux.

International audience; A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to understand membrane permeation and intracellular concentration of antibiotics in clinical isolates: passing the membrane barrier to reach the threshold concentration inside the bacterial periplasm or cytoplasm is the pivotal step of antibacterial activity. Ceftazidime (CAZ) is a key molecule of the combination therapy for treating resistant bacteria. We designed and synthesized different fluorescent CAZ derivatives (CAZ*, CAZ**) to dissect the early step of translocation-accumulation across bacterial membrane. Their activities were determined on E. coli strains and on selected clinical isolates overexpressing ß-lactamases. The accumulation of CAZ* and CAZ** were determined by microspectrofluorimetry and epifluorimetry. The derivatives were properly translocated to the periplasmic space when we permeabilize the outer membrane barrier. The periplasmic location of CAZ** was related to a significant antibacterial activity and with the outer membrane permeability. This study demonstrated the correlation between periplasmic accumulation and antibiotic activity. We also validated the method for approaching ß-lactam permeation relative to membrane permeability and paved the way for an original matrix for determining " Structure Intracellular Accumulation Activity Relationship " for the development of new therapeutic candidates. With the misuse and overuse of the various antibiotic families and the scarcity of new drugs available, multid-rug resistance is now a major bacterial threat to healthcare worldwide 1–4. A major concern discussed in various studies is the intracellular concentration of the antibiotic molecule 5–7. In Gram-negative bacteria, the challenge for a drug is to permeate the outer membrane (OM) that protects the cell against external attacks 8, 9. This membrane plays a key role in controlling the diffusion of external toxic molecules and the antibiotics must translocate through the outer membrane to reach the periplasmic target or additionally pass the inner membrane to reach the cytoplasmic target 10, 11. During their translocation, the molecules can use various hydrophilic channels such as porins that form trimeric hydrophilic pores present in the OM 7, 8. Today a key challenge for hospital microbiologists is to bypass the resistance in a Gram-negative strain that can produce carbapenemase and/or ß-lactamase 12, 13. Recent studies proposed an alternative way by using a combination of ceftazidime (CAZ) + ß-lactamase inhibitor 14–16. Regarding this strategy, it is urgently required to dissect the relationships between the role of membrane and enzymatic barrier and the antibacterial activity of CAZ as recently reported 17. Presently, various groups have studied the binding of ß-lactams to penicillin binding proteins (PBP) using fluorophore-conjugated-penicillin, termed Bocillin 18, 19 , fluorescent cephalosporin C 20 or fluorescent meropenem 21. Despite these various efforts to develop labeled antibiotic probes, no key information has been obtained regarding the role of porins, penetration rate, and the effect of ß-lactamase on periplasmic concentrations when addressing the choice of combination of clinically-used cephalosporin and ß-lactamase

Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed “Structure Intracellular Concentration Activity Relationship” (SICAR) concept.

International audience; Planktothrix agardhii (P. agardhii), a filamentous cyanobacterium commonly found in freshwater environments, has also been reported in brackish areas. In order to test the hypothesis of an acclimation of P. agardhii to its environmental salinity, we monitored growth and morphometry of P. agardhii brackish and freshwater strains under various salinities (0 to 15 psu). Despite differences in their response under increasing salinities (0–15 psu), the brackish strains show: (i) growth less gradually inhibited by increasing salinities than the freshwater strains; (ii) growth nearly optimal in the salinity of their natural environment (iii) a salt tolerance range wider (0 to [12.5–15 psu]) than that of freshwater P. agardhii (0 to [7.5–10 psu]). Our findings suggest that this tolerance may be due to an acclimation of the strains to their environment by effective salt extrusion and compatible solutes accumulation. This salt acclimation, just as the population response to various salinities, is well reflected by the filaments morphometry. This study highlights the ability of potentially harmful cyanobacteria to acclimate to salinity in a salinization context.

International audience; The cyanobacterium, Microcystis aeruginosa, is able to proliferate in a wide range of freshwater ecosystems and to produce many secondary metabolites that are a threat to human and animal health. The dynamic of this production and more globally the metabolism of this species is still poorly known. A DNA microarray based on the genome of M. aeruginosa PCC 7806 was constructed and used to study the dynamics of gene expression in this cyanobacterium during the light/dark cycle, because light is a critical factor for this species, like for other photosynthetic microorganisms. This first application of transcriptomics to a Microcystis species has revealed that more than 25% of the genes displayed significant changes in their transcript abundance during the light/dark cycle and in particular during the dark/light transition. The metabolism of M. aeruginosa is compartmentalized between the light period, during which carbon uptake, photosynthesis and the reductive pentose phosphate pathway lead to the synthesis of glycogen, and the dark period, during which glycogen degradation, the oxidative pentose phosphate pathway, the TCA branched pathway and ammonium uptake promote amino acid biosynthesis. We also show that the biosynthesis of secondary metabolites, such as microcystins, aeruginosin and cyanopeptolin, occur essentially during the light period, suggesting that these metabolites may interact with the diurnal part of the central metabolism.