Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.     

A novel series of 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing 1,2,3-triazole group: Design,synthesis and antibacterial evaluation

A series of novel 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing the 1,2,3-triazole group were designed, synthesized, and evaluated for their in vitro antibacterial activity. The antibacterial results indicated that most of the target compounds not only increased their activity against resistant bacterial strains, but also partially retained the activity against sensitive bacterial strains compared with clarithromycin. Among them, 13d had the best antibacterial activity against resistant strains, including Streptococcus pneumoniae B1 expressing the ermB gene (16 µg/mL), Streptococcus pneumoniae AB11 expressing the mefA and ermB genes (16 µg/mL) and Streptococcus pyogenes R1 (16 µg/mL), showing >16, 8 and 16-fold higher activity than that of CAM, respectively. Moreover, 13d and 13g exhibited the best antibacterial activity against sensitive bacterial strains, including Staphylococcus aureus ATCC25923 (4 µg/mL) and Bacillus Subtilis ATCC9372 (1 µg/mL). The MBC results showed that the most promising compounds 13d and 13g exhibited antibacterial activity through bacteriostatic mechanism, while the time-kill kinetic experiment revealed bactericidal kinetics of 13g from microscopic point of view. In vitro antibacterial experiments and molecular docking results further confirmed that it was feasible to our initial design strategy by modifying the C-3 and C-11 positions of clarithromycin to increase the activity against resistant bacteria.     

Design,synthesis and biological evaluation of spiropyrimidinetriones oxazolidinone derivatives as antibacterial agents

Gram-positive bacteria are among the most common human pathogens associated with clinical infections which range from mild skin infections to sepsis. Resistance towards existing class of drugs by Gram-positive bacteria including methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (MRSE) and vancomycin resistant enterococci (VRE) is a growing concern. There is an urgent need to discover new antibiotics which are active against resistant strains of Gram positive bacteria. We report herein a novel class of spiropyrimidinetrione oxazolidinone derivatives as novel antibacterial agents. Key step towards the synthesis of title compounds involved the use of tert-amino reaction with [1,5]-hydride shift leading to the new CC bond formation. Compound 30n has demonstrated potent antibacterial activity against a panel of Gram-positive microbial strains including MRSA, MRSE, and LNZ and vancomycin resistant strains of E. faecalis. Further, molecular docking studies suggest that 30n has binding mode similar to that of LNZ in 50S RNA ribosome.     

Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3

Defensins are natural mucosal antimicrobial peptides and their broad spectrum activity against aerobic or facultative anaerobic bacteria has been well investigated. The aim of this study was to systematically examine the antibacterial activity of the small intestinal Paneth cell derived α-defensin HD5 and the major colonic β-defensins HBD-1–3 against strict anaerobic intestinal bacteria. The antibacterial activity was assessed with a flow cytometric assay employing a membrane potential sensitive dye as marker for loss of cell viability. The majority of the tested strains belonging to the dominant anaerobe genera of the gut, Bacteroides and Parabacteroides, were only minimally affected by the constitutively expressed defensins HD5 and HBD-1. The inducible defensin HBD-2 had a limited antibacterial effect, whereas the inducible HBD-3 exhibited potent activity against most strains. The effect of HBD-3 on Bacteroides sp. appeared to be dependent on the presence of oxygen. Bacteroides fragilis strains isolated from blood during bacteremia or from extraintestinal infections were more resistant to HBD-3 than strains from the physiological gut flora. Thus, defensin resistance is not only species- but also strain-specific and may be clinically relevant in the host–bacteria interaction influencing mucosal translocation and systemic infection.     

Bis(N-amidinohydrazones) and N-(amidino)-N′-aryl-bishydrazones: New classes of antibacterial/antifungal agents

The emergence of multidrug-resistant bacterial and fungal strains poses a threat to human health that requires the design and synthesis of new classes of antimicrobial agents. We evaluated bis(N-amidinohydrazones) and N-(amidino)-N′-aryl-bishydrazones for their antibacterial and antifungal activities against panels of Gram-positive/Gram-negative bacteria as well as fungi. We investigated their potential to develop resistance against both bacteria and fungi by a multi-step resistance-selection method, explored their potential to induce the production of reactive oxygen species, and assessed their toxicity. In summary, we found that these compounds exhibited broad-spectrum antibacterial and antifungal activities against most of the tested strains with minimum inhibitory concentration (MIC) values ranging from <0.5 to >500 μM against bacteria and 1.0 to >31.3 μg/mL against fungi; and in most cases, they exhibited either superior or similar antimicrobial activity compared to those of the standard drugs used in the clinic. We also observed minimal emergence of drug resistance to these newly synthesized compounds by bacteria and fungi even after 15 passages, and we found weak to moderate inhibition of the human Ether-à-go-go-related gene (hERG) channel with acceptable IC₅₀ values ranging from 1.12 to 3.29 μM. Overall, these studies show that bis(N-amidinohydrazones) and N-(amidino)-N′-aryl-bishydrazones are potentially promising scaffolds for the discovery of novel antibacterial and antifungal agents.     

New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria

Background

Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs.

Methodology/Principal Findings

Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains.

Conclusions

We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.     

Antibacterial potential of hGlyrichin encoded by a human gene

Emerging multidrug‐resistant (MDR) bacteria are an enormous threat to human life because of their resistance to currently available antibiotics. The genes encoding antibacterial peptides have been studied extensively and are excellent candidates for a new generation of antibiotic drugs to fight MDR bacteria. In contrast to traditional antibiotics, antibacterial peptides, which do not cause drug resistance, have an unparalleled advantage. However, because most antibacterial peptides originate in species other than humans, the hetero‐immunological rejection of antibacterial peptides is a key disadvantage that limits their clinical application. In this study, we identify hGlyrichin as a potential human antibacterial polypeptide. The hGlyrichin polypeptide kills a variety of bacteria including the MDR bacteria methicillin‐resistant Staphylococcus aureus, MDR Pseudomonas aeruginosa, and MDR tubercle bacillus. A 19 amino acid peptide (pCM19) at positions 42–60 of hGlyrichin is crucial for its antibacterial activity. The hGlyrichin polypeptide kills bacteria through the destruction of the bacterial membrane. In addition, all peptides that are homologous to hGlyrichin have antibacterial activity and can penetrate the bacterial membrane. Importantly, hGlyrichin does not cause hemolytic side effects in vitro or in vivo. Therefore, based on the virtues of hGlyrichin, i.e., the absence of hetero‐immunological rejection and hemolytic side effects and the unambiguous efficacy of killing pathogenic MDR bacteria, we propose hGlyrichin as a potential human antibacterial polypeptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A Pipeline for Screening Small Molecules with Growth Inhibitory Activity against Burkholderia cenocepacia

Infections with the bacteria Burkholderia cepacia complex (Bcc) are very difficult to eradicate in cystic fibrosis patients due the intrinsic resistance of Bcc to most available antibiotics and the emergence of multiple antibiotic resistant strains during antibiotic treatment. In this work, we used a whole-cell based assay to screen a diverse collection of small molecules for growth inhibitors of a relevant strain of Bcc, B. cenocepacia K56-2. The primary screen used bacterial growth in 96-well plate format and identified 206 primary actives among 30,259 compounds. From 100 compounds with no previous record of antibacterial activity secondary screening and data mining selected a total of Bce bioactives that were further analyzed. An experimental pipeline, evaluating in vitro antibacterial and antibiofilm activity, toxicity and in vivo antibacterial activity using C. elegans was used for prioritizing compounds with better chances to be further investigated as potential Bcc antibacterial drugs. This high throughput screen, along with the in vitro and in vivo analysis highlights the utility of this experimental method to quickly identify bioactives as a starting point of antibacterial drug discovery.     

Antibacterial activity against β- lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: fractional Inhibitory Concentration Index (FICI) determination

Background

The present study reports the antibacterial capacity of alkaloid compounds in combination with Methicillin and Ampicillin-resistants bacteria isolated from clinical samples. The resistance of different bacteria strains to the current antibacterial agents, their toxicity and the cost of the treatment have led to the development of natural products against the bacteria resistant infections when applied in combination with conventional antimicrobial drugs.

Method

The antibacterial assays in this study were performed by using inhibition zone diameters, MIC, MBC methods, the time-kill assay and the Fractional Inhibitory Concentration Index (FICI) determination. On the whole, fifteen Gram-positive bacterial strains (MRSA/ARSA) were used. Negative control was prepared using discs impregnated with 10 % DMSO in water and commercially available Methicillin and Ampicillin from Alkom Laboratories LTD were used as positive reference standards for all bacterial strains.

Results

We noticed that the highest activities were founded with the combination of alkaloid compounds and conventional antibiotics against all bacteria strains. Then, results showed that after 7 h exposition there was no viable microorganism in the initial inoculums.

Conclusion

The results of this study showed that alkaloid compounds in combination with conventional antibiotics (Methicillin, Ampicillin) exhibited antimicrobial effects against microorganisms tested. These results validate the ethno-botanical use of Cienfuegosia digitata Cav. (Malvaceae) in Burkina Faso. Moreover, this study demonstrates the potential of this herbaceous as a source of antibacterial agent that could be effectively used for future health care purposes.     

Preliminary selection study of potential probiotic bacteria from aquacultural area in Tunisia

In order to test the ability to produce antibacterial substances within marine bacteria, prior to select potential probiotics for use in shellfish farming, we targeted a large collection of bacterial isolates (132 strains), brought from the clamRuditapes decussatus and 37 reference strains. First, we proceeded to their biochemical identification and the screening of antibiotic resistance profiles. Else, we investigated their inhibitory activityin vitro against several fish and shellfish pathogens, using two methods: the double-layer agar and the direct simultaneous antagonism methods. The results showed high frequencies of inhibitory producing bacteria (IPB) within the isolates. These bacteria (25%) were aerobic mesophylic bacteria belonging to various bacterial groups: 33.7% oxidase-positive Gram-negative bacteria, 7.4%Enterobacteriaceae and 28% lactic acid bacteria. Besides this group, nine strains produced strong inhibition effect. These bacteria belonged to:Aeromonas hydrophila, Aeromonas sobria, Pseudomonas cepacia, Vibrio sp,Serratia liquefaciens andLactobacillus rhamnosus. They were active against pathogenic bacteria belonging to the genera:Aeromonas, Pseudomonas andVibrio. These potential probiotics were submitted to further investigations prior to their introduction in larval shellfish farming.     

Antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian medicinal plant Eremophila neglecta

Aims: To determine the antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian plant Eremophila neglecta. Methods and Results: Antimicrobial activities of serrulatane compounds 8,19‐dihydroxyserrulat‐14‐ene ( 1 ) and 8‐hydroxyserrulat‐14‐en‐19‐oic acid ( 2 ) were tested against Gram‐negative and Gram‐positive bacteria including human and veterinary pathogens and some multidrug‐resistant isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the compounds were determined by broth microdilution assay. Both compounds exhibited antibacterial activity against all Gram‐positive test strains. They showed antimycobacterial activity against isolates of Mycobacterium fortuitum and Mycobacterium chelonae. Of the five Gram‐negative bacteria tested, only Moraxella catarrhalis showed susceptibility to the compounds. Cytotoxic activities were tested in the Vero cell line. Compound 1 showed more activity than 2 in both antibacterial and cytotoxicity assays with cytotoxicity at concentrations similar to the MBC. Conclusions: Serrulatane compounds showed significant activity against medically important bacteria, with 1 exhibiting stronger antibacterial activity. However, they also displayed toxicity to mammalian cells. Significance and Impact of the Study: Serrulatanes are of interest as novel antibacterial compounds for use in biomedical applications; this study reports data obtained with a range of bacterial strains and mammalian cells, essential for assessing the capabilities and limitations of potential applicability of these compounds.     

Preliminary screening for antibacterial and antimycobacterial activity of actinomycetes from less explored ecosystems

Actinomycetes from less explored ecosystems were screened for antibacterial and antimycobacterial activity. Crude bioactive compounds were produced by growing these strains by shake flask fermentation using soybean meal medium. Culture supernatant and mycelia were extracted with ethyl acetate and methanol, respectively. Antibacterial activity of crude extracts was tested by disc diffusion method against gram positive and gram negative bacteria. Actinomycete strains D10, D5, NEK5, ANS2, M104 and R2 showed prominent activity. Culture filtrates and crude extracts were tested against standard strain Mycobacterium tuberculosis H₃₇Rv and drug sensitive and drug resistant clinical isolates of M. tuberculosis by luciferase reporter phage (LRP) assay. Considerable variation was observed in antimycobacterial activity between actinomycete culture filtrates and solvent extracts. Actinomycete strains viz., D10, D5 (desert), CSA14 (forest), CA33 (alkaline soil), NEK5 (Neem plant), MSU, ANS2, R2 and M104 (marine) screened in the present study were found to be highly potent showing good antibacterial and antimycobacterial activity. Five of them such as A3, CSA1, EE9, ANS5 and R9 were exclusively active against M. tuberculosis. Secretary products of actinomycetes of rare ecosystems are meant to antagonize organisms in their respective environments. These are likely to be novel antimycobacterial compounds as they unknown to human pathogens.     

Propolis as a novel antibacterial agent

Propolis (bee glue) is a bee glue, sticky resinous material released from various plant sources such as bud exudates, flowers, and leaves modified by bee secretions and wax propolis is composed of resins, waxes, polyphenols, polysaccharides, volatile materials, and secondary metabolites that are responsible for various bioactivity such as antibacterial, anti-angiogenic, antiulcer, anti-inflammatory, antioxidant, and anti-viral activities. The physico-chemical characteristics and the natural properties of various kinds of propolis have been studied for the past decade. Novel active anti-microbial compounds have been identified in propolis. Those compounds positively modulated the antimicrobial resistance of multidrug resistant bacteria. Published research has indicated that propolis and its derivatives has many natural antimicrobial compounds with a broad spectrum against different types of bacteria and that it enhanced the efficacy of conventional antibiotics. Besides, the combination of propolis with other compounds such as honey has been studied whereby, such combinations have a synergistic effect against bacterial strains such as Escherichia coli and Staphylococcus aureus. The activity of propolis is very much dependent on seasonal and regional factors, and Middle Eastern propolis have shown best antibacterial efficacy. Propolis and its main flavonoids ingredients should not be overlooked and should be evaluated in clinical trials to better elucidate their potential application in various fields of medicine. Clinical antibacterial potential and its use in new drugs of biotechnological products should be conducted. This review aims at highlighting some of the recent scientific findings associated with the antibacterial properties of propolis and its components.     

Potentiation of the activity of β-lactam antibiotics by farnesol and its derivatives

Farnesol, a sesquiterpene alcohol, potentiates the activity of β-lactam antibiotics against antibiotic-resistant bacteria. We document that farnesol and two synthetic derivatives (compounds 2 and 6) have poor antibacterial activities of their own, but they potentiate the activities of ampicillin and oxacillin against Staphylococcus aureus strains (including methicillin-resistant S. aureus). These compounds attenuate the rate of growth of bacteria, which has to be taken into account in assessment of the potentiation effect.     

Substituted 1,6-diphenylnaphthalenes as FtsZ-targeting antibacterial agents

Bacterial cell division occurs in conjunction with the formation of a cytokinetic Z-ring structure comprised of FtsZ subunits. Agents that disrupt Z-ring formation have the potential, through this unique mechanism, to be effective against several of the newly emerging multidrug-resistant strains of infectious bacteria. Several 1-phenylbenzo[c]phenanthridines exhibit notable antibacterial activity. Based upon their structural similarity to these compounds, a distinct series of substituted 1,6-diphenylnaphthalenes were synthesized and evaluated for antibacterial activity against Staphylococcus aureus and Enterococcus faecalis. In addition, the effect of select 1,6-diphenylnaphthalenes on the polymerization dynamics of S. aureus FtsZ and mammalian β-tubulin was also assessed. The presence of a basic functional group or a quaternary ammonium substituent on the 6-phenylnaphthalene was required for significant antibacterial activity. Diphenylnaphthalene derivatives that were active as antibiotics, did exert a pronounced effect on bacterial FtsZ polymerization and do not appear to cross-react with mammalian tubulin to any significant degree.     

Antibacterial activities of Groebke–Blackburn–Bienaymé-derived imidazo[1,2-a]pyridin-3-amines

We sought to explore the imidazo[1,2-a]pyridin-3-amines for TLR7 (or 8)-modulatory activities. This chemotype, readily accessed via the Groebke–Blackburn–Bienaymé multi-component reaction, resulted in compounds that were TLR7/8-inactive, but exhibited bacteriostatic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). To investigate the mechanism of antibacterial activity of this new chemotype, a resistant strain of S. aureus was generated by serially passaging the organism in escalating doses of the most active analogue. A comparison of minimum inhibitory concentrations (MICs) of known bacteriostatic agents in wild-type and resistant strains indicates a novel mechanism of action. Structure–activity relationship studies have led to the identification of positions on the scaffold for additional structural modifications that should allow for the introduction of probes designed to examine cognate binding partners and molecular targets, while not significantly compromising antibacterial potency.     

Mode of action investigation for the antibacterial cationic anthraquinone analogs

Reported previously by our group, we have developed a novel class of antibacterial cationic anthraquinone analogs with superb potency (MIC <1μg/mL) against Gram positive (G+) pathogens including Methicillin-resistant Staphylococcus aureus (MRSA). However, most of these compounds only manifest modest antibacterial activity against Gram negative (G-) bacteria. Further investigation on the antibacterial mode of action using fluorogenic dyes reveals that these compounds exert two different modes of action that account for the difference in their antibacterial profile. It was found that most of the compounds exert their antibacterial activity by disrupting the redox processes of bacteria. At high concentration, these compounds can also act as membrane disrupting agents. This information can help to design new therapeutics against various bacteria.     

Antibacterial activity of 3-methylbenzo[d]thiazol-methylquinolinium derivatives and study of their action mechanism

The increasing incidence of multidrug resistant bacterial infection renders an urgent need for the development of new antibiotics. To develop small molecules disturbing FtsZ activity has been recognized as promising approach to search for antibacterial of high potency systematically. Herein, a series of novel quinolinium derivatives were synthesized and their antibacterial activities were investigated. The compounds show strong antibacterial activities against different bacteria strains including MRSA, VRE and NDM-1 Escherichia coli. Among these derivatives, a compound bearing a 4-fluorophenyl group (A2) exhibited a superior antibacterial activity and its MICs to the drug-resistant strains are found lower than those of methicillin and vancomycin. The biological results suggest that these quinolinium derivatives can disrupt the GTPase activity and dynamic assembly of FtsZ, and thus inhibit bacterial cell division and then cause bacterial cell death. These compounds deserve further evaluation for the development of new antibacterial agents targeting FtsZ.     

Design, synthesis and structure-activity relationship studies of novel indazole analogues as DNA gyrase inhibitors with Gram-positive antibacterial activity

In this study, we report the design, synthesis and structure-activity relationships of novel indazole derivatives as DNA gyrase inhibitors with Gram-positive antibacterial activity. Our results show that selected compounds from this series exhibit potent antibacterial activity against Gram-positive bacteria including multi-drug resistant strains that is methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE).     

Distribution and antimicrobial multiresistance in Gram-negative bacteria isolated from Turkish sea bass (Dicentrarchus labrax L., 1781) farm

In this study, the frequencies of 111 antibiotic-resistant Gram-negative bacteria obtained from cultured sea bass were investigated. All the strains were identified and tested whether they were resistive against ten different antibiotics or not. The results showed that most of the bacteria were resistant to trimethoprim-sulphamethoxazole, cephalotin, tetracycline and streptomycin. It was found multiple antibiotic resistance (MAR) index values ranging from 0.3–0.8 for the bacteria isolated from gill. A large number ofPseudomanas putida (25.2%),Moellerella wisconsensis (18%) andPseudomonas fluorescens (10.81%) were identified. Also, strains ofRalstonia pickettii (9%),Vibrio fluvialis (8.1%),Pantoea sp. (7.2%) andErwinia sp. (5.4%) were found. This study suggested that Turkish sea bass farms have antibacterial multiresistance bacteria and may play as a reservoirs response for disease pathogen bacteria.