Search

Antimicrobial resistance has increased rapidly, causing daunting morbidity and mortality rates worldwide. Antimicrobial peptides (AMPs) have emerged as promising alternatives to traditional antibiotics due to their broad range of targets and low tendency to elicit resistance. However, potent antimicrobial activity is often accompanied by excessive cytotoxicity toward host cells, leading to a halt in AMP therapeutic development. Here, we present multivariate analyses that correlate 28 peptide properties to the activity and toxicity of 46 diverse African-derived AMPs and identify the negative lipophilicity of polar residues as an essential physiochemical property for selective antimicrobial activity. Twenty-seven active AMPs are identified, of which the majority are of scorpion or frog origin. Of these, thirteen are novel with no previously reported activities. Principal component analysis and quantitative structure-activity relationships (QSAR) reveal that overall hydrophobicity, lipophilicity, and residue side chain surface area affect the antimicrobial and cytotoxic activity of an AMP. This has been well documented previously, but the present QSAR analysis additionally reveals that a decrease in the lipophilicity, contributed by those amino acids classified as polar, confers selectivity for a peptide to pathogen over mammalian cells. Furthermore, an increase in overall peptide charge aids selectivity toward Gram-negative bacteria and fungi, while selectivity toward Gram-positive bacteria is obtained through an increased number of small lipophilic residues. Finally, a conservative increase in peptide size in terms of sequence length and molecular weight also contributes to improved activity without affecting toxicity. Our findings suggest a novel approach for the rational design or modification of existing AMPs to increase pathogen selectivity and enhance therapeutic potential., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

A new class of amphiphilic molecules, the lipoguanidines, designed as hybrids of guanidine and fatty acid compounds, has been synthesized and developed. The new molecules present both a guanidine polar head and a lipophilic tail that allow them to disrupt bacterial membranes and to sensitize Gram-negative bacteria to the action of the narrow-spectrum antibiotics rifampicin and novobiocin. The lipoguanidine 5g sensitizes Klebsiella pneumonia , Acinetobacter baumannii , Pseudomonas aeruginosa , and Escherichia coli to rifampicin, thereby reducing the antibiotic minimum inhibitory concentrations (MIC) up to 256-fold. Similarly, 5g is able to potentiate novobiocin up to 64-fold, thereby showing a broad spectrum of antibiotic potentiating activity. Toxicity and mechanism studies revealed the potential of 5g to work synergistically with rifampicin through the disruption of bacterial membranes without affecting eukaryotic cells., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Conventional wound infection treatments neither actively promote wound healing nor address the growing problem of antibacterial resistance. Antimicrobial peptides (AMPs) are natural defense molecules, released from host cells, which may be rapidly bactericidal, modulate host-immune responses, and/or act as endogenous mediators for wound healing. However, their routine clinical use has hitherto been hindered due to their instability in the wound environment. Here we describe an electrospun carrier system for topical application of pleurocidin, demonstrating sufficient AMP release from matrices to kill wound-associated pathogens including Acinetobacter baumannii and Pseudomonas aeruginosa. Pleurocidin can be incorporated into polyvinyl alcohol (PVA) fiber matrices, using coaxial electrospinning, without major drug loss with a peptide content of 0.7% w/w predicted sufficient to kill most wound associated species. Pleurocidin retains its activity on release from the electrospun fiber matrix and completely inhibits growth of two strains of A. baumannii (AYE; ATCC 17978) and other ESKAPE pathogens. Inhibition of P. aeruginosa strains (PAO1; NCTC 13437) is, however, matrix weight per volume dependent, with only larger/thicker matrices maintaining complete inhibition. The resulting estimation of pleurocidin release from the matrix reveals high efficiency, facilitating a greater AMP potency. Wound matrices are often applied in parallel or sequentially with the use of standard wound care with biocides, therefore the presence and effect of biocides on pleurocidin potency was tested. It was revealed that combinations displayed additive or modestly synergistic effects depending on the biocide and pathogens which should be considered during the therapy. Taken together, we show that electrospun, pleurocidin-loaded wound matrices have potential to be investigated for wound infection treatment., 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., (Crown Copyright © 2023. Published by Elsevier B.V. All rights reserved.)

This tutorial review focuses on providing a summary of the key techniques used for the characterisation of supramolecular amphiphiles and their self-assembled aggregates; from the understanding of low-level molecular interactions, to materials analysis, use of data to support computer-aided molecular design and finally, the translation of this class of compounds for real world application, specifically within the clinical setting. We highlight the common methodologies used for the study of traditional amphiphiles and build to provide specific examples that enable the study of specialist supramolecular systems. This includes the use of nuclear magnetic resonance spectroscopy, mass spectrometry, X-ray scattering techniques (small- and wide-angle X-ray scattering and single crystal X-ray diffraction), critical aggregation (or micelle) concentration determination methodologies, machine learning, and various microscopy techniques. Furthermore, this review provides guidance for working with supramolecular amphiphiles in in vitro and in vivo settings, as well as the use of accessible software programs, to facilitate screening and selection of druggable molecules. Each section provides: a methodology overview - information that may be derived from the use of the methodology described; a case study - examples for the application of these methodologies; and a summary section - providing methodology specific benefits, limitations and future applications.

We determine the efficacy for three known structurally related, membrane active detergents against multidrug resistant and wild type strains of Pseudomonas aeruginosa . Accessible solution state NMR experiments are used to quantify phospholipid headgroup composition of the microbial membranes and to gain molecular level insight into antimicrobial mode of action.

Antimicrobial resistance is one of the greatest threats to human health. Gram-positive methicillin resistant Staphylococcus aureus (MRSA), in both its planktonic and biofilm form, is of particular concern. Herein we identify the hydrogelation properties for a series of intrinsically fluorescent, structurally related supramolecular self-associating amphiphiles and determine their efficacy against both planktonic and biofilm forms of MRSA. To further explore the potential translation of this hydrogel technology for real-world applications, the toxicity of the amphiphiles was determined against the eukaryotic multicellular model organism, Caenorhabditis elegans . Due to the intrinsic fluorescent nature of these supramolecular amphiphiles, material characterisation of their molecular self-associating properties included; comparative optical density plate reader assays, rheometry and widefield fluorescence microscopy. This enabled determination of amphiphile structure and hydrogel sol dependence on resultant fibre formation.

12-Bis-THA Cl 2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl 2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro , but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regarding their toxicity and which could be more useful if such risks are reduced or eliminated. We modified a bolalipid antiseptic used in throat lozenges to see if it could be made more effective against some of the highest-priority bacteria and less toxic. We found that structural modifications that rendered the lipid more toxic against human cells made it less toxic in infection models and we could effectively treat caterpillars infected with either Mycobacterium tuberculosis, methicillin resistant Staphylococcus aureus, or Acinetobacter baumannii. The study provides a rationale for further adaptation toward diversifying the range of indications in which this class of antimicrobial may be used.

Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of Acinetobacter baumannii burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2023.)

It is urgent to find new antibiotic classes against multidrug-resistant bacteria as the rate of discovery of new classes of antibiotics has been very slow in the last 50 years. Recently, pyrrolobenzodiazepines (PBDs) with a C8-linked aliphatic-heterocycle have been identified as a new broad-spectrum antibiotic class with activity against Gram-negative bacteria. The active imine moiety of the reported lead pyrrolobenzodiazepine compounds was replaced with amide to obtain the non-DNA binding and noncytotoxic dilactam analogues to understand the structure-activity relationship further and improve the safety potential of this class. The synthesised compounds were tested against panels of multidrug-resistant Gram-positive and Gram-negative bacteria, including WHO priority pathogens. Minimum inhibitory concentrations for the dilactam analogues ranged from 4 to 32 mg/L for MDR Gram-positive bacteria, compared to 0.03 to 2 mg/L for the corresponding imine analogues. At the same time, they were found to be inactive against MDR Gram-negative bacteria, with a MIC > 32 mg/L, compared to a MIC of 0.5 to 32 mg/L for imine analogues. A molecular modelling study suggests that the lack of imine functionality also affects the interaction of PBDs with DNA gyrase. This study suggests that the presence of N10-C11 imine moiety is crucial for the broad-spectrum activity of pyrrolobenzodiazepines.

The rise of antimicrobial resistance remains one of the greatest global health threats facing humanity. Furthermore, the development of novel antibiotics has all but ground to a halt due to a collision of intersectional pressures. Herein we determine the antimicrobial efficacy for 14 structurally related supramolecular self-associating amphiphiles against clinically relevant Gram-positive methicillin resistant Staphylococcus aureus and Gram-negative Escherichia coli . We establish the ability of these agents to selectively target phospholipid membranes of differing compositions, through a combination of computational host:guest complex formation simulations, synthetic vesicle lysis, adhesion and membrane fluidity experiments, alongside our novel 1 H NMR CPMG nanodisc coordination assays, to verify a potential mode of action for this class of compounds and enable the production of evermore effective next-generation antimicrobial agents. Finally, we select a 7-compound subset, showing two lead compounds to exhibit 'druggable' profiles through completion of a variety of in vivo and in vitro DMPK studies., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome, characterized by low levels of lactobacilli and overgrowth of a diverse group of bacteria, associated with higher risk of a variety of infections, surgical complications, cancer, and preterm birth (PTB). Despite the lack of a consistently applicable etiology, Prevotella spp. are often associated with both BV and PTB, and Pr. bivia has known symbiotic relationships with both Peptostreptococcus anaerobius and Gardnerella vaginalis. Higher risk of PTB can also be predicted by a composite of metabolites linked to bacterial metabolism, but their specific bacterial source remains poorly understood. Here, we characterize diversity of metabolic strategies among BV-associated bacteria and lactobacilli and the symbiotic metabolic relationships between Pr. bivia and its partners and show how these influence the availability of metabolites associated with BV/PTB and/or pro- or anti-inflammatory immune responses. We confirm a commensal relationship between Pe. anaerobius and Pr. bivia , refining its mechanism, which sustains a substantial increase in acetate production. In contrast, the relationship between Pr. bivia and G. vaginalis strains, with sequence variant G2, is mutualistic, with outcome dependent on the metabolic strategy of the G. vaginalis strain. Taken together, our data show how knowledge of inter- and intraspecies metabolic diversity and the effects of symbiosis may refine our understanding of the mechanism and approach to risk prediction in BV and/or PTB. IMPORTANCE Bacterial vaginosis (BV) is the most common vaginal infection for women of childbearing age. Although 50% of women with BV do not have any symptoms, it approximately doubles the risk of catching a sexually transmitted infection and also increases the risk of preterm delivery in pregnant women. Recent studies of the vaginal microbiota have suggested that variation between species in the same genus or between strains of the same species explain better or poorer outcomes or at least some coexistence patterns for bacteria of concern. We tested whether such variation is manifested in how vaginal bacteria grow in the laboratory and whether and how they may share nutrients. We then showed that this affected the overall cocktail of chemicals they produce, including bacterially derived chemicals that we have previously shown are linked to a higher risk of preterm delivery.

The pharmacodynamic profile of antimicrobial peptides (AMPs) and their in vivo synergy are two factors that are thought to restrict resistance evolution and ensure their conservation. The frog Rana temporaria secretes a family of closely related AMPs, temporins A-L, as an effective chemical dermal defense. The antibacterial potency of temporin L has been shown to increase synergistically in combination with both temporins B and A, but this is modest. Here we show that the less potent temporin B enhances the cooperativity of the in vitro antibacterial activity of the more potent temporin L against EMRSA-15 and that this may be associated with an altered interaction with the bacterial plasma membrane, a feature critical for the antibacterial activity of most AMPs. Addition of buforin II, a histone H2A fragment, can further increase the cooperativity. Molecular dynamics simulations indicate temporins B and L readily form hetero-oligomers in models of Gram-positive bacterial plasma membranes. Patch-clamp studies show transmembrane ion conductance is triggered with lower amounts of both peptides and more quickly when used in combination, but conductance is of a lower amplitude and pores are smaller. Temporin B may therefore act by forming temporin L/B hetero-oligomers that are more effective than temporin L homo-oligomers at bacterial killing and/or by reducing the probability of the latter forming until a threshold concentration is reached. Exploration of the mechanism of synergy between AMPs isolated from the same organism may therefore yield antibiotic combinations with advantageous pharmacodynamic properties.

Herein, we report a series of di-anionic supramolecular self-associating amphiphiles (SSAs). We elucidate the antimicrobial properties of these SSAs against both methicillin resistant Staphylococcus aureus and Escherichia coli . In addition, we show this class of compound to form both intra- and intermolecular hydrogen bonded macrocyclic structures in the solid state.

Reliable antimicrobial susceptibility testing is essential in informing both clinical antibiotic therapy decisions and the development of new antibiotics. Mammalian cell culture media have been proposed as an alternative to bacteriological media, potentially representing some critical aspects of the infection environment more accurately. Here, we use a combination of NMR metabolomics and electron microscopy to investigate the response of Escherichia coli and Pseudomonas aeruginosa to growth in differing rich media to determine whether and how this determines metabolic strategies, the composition of the cell wall, and consequently susceptibility to membrane active antimicrobials including colistin and tobramycin. The NMR metabolomic approach is first validated by characterizing the expected E. coli acid stress response to fermentation and the accompanying changes in the cell wall composition, when cultured in glucose rich mammalian cell culture media. Glucose is not a major carbon source for P. aeruginosa but is associated with a response to osmotic stress and a modest increase in colistin tolerance. Growth of P. aeruginosa in a range of bacteriological media is supported by consumption of formate, an important electron donor in anaerobic respiration. In mammalian cell culture media, however, the overall metabolic strategy of P. aeruginosa is instead dependent on consumption of glutamine and lactate. Formate doping of mammalian cell culture media does not alter the overall metabolic strategy but is associated with polyamine catabolism, remodelling of both inner and outer membranes, and a modest sensitization of P. aeruginosa PAO1 to colistin. Further, in a panel of P. aeruginosa isolates an increase between 2- and 3-fold in sensitivity to tobramycin is achieved through doping with other organic acids, notably propionate which also similarly enhances the activity of colistin. Organic acids are therefore capable of nonspecifically influencing the potency of membrane active antimicrobials.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.

It is urgent to find new antibiotic classes with activity against multidrug-resistant (MDR) Gram-negative pathogens as the pipeline of antibiotics is essentially empty. Modified pyrrolobenzodiazepines with a C8-linked aliphatic heterocycle provide a new class of broad-spectrum antibacterial agents with activity against MDR Gram-negative bacteria, including WHO priority pathogens. The structure-activity relationship established that the third ring was particularly important for Gram-negative activity. Minimum inhibitory concentrations for the lead compounds ranged from 0.125 to 2 mg/L for MDR Gram-negative, excluding Pseudomonas aeruginosa , and between 0.03 and 1 mg/L for MDR Gram-positive species. The lead compounds were rapidly bactericidal with >5 log reduction in viable count within 4 h for Acinetobacter baumannii and Klebsiella pneumoniae . The lead compound inhibited DNA gyrase in gel-based assays, with an IC 50 of 3.16 ± 1.36 mg/L. This study provides a new chemical scaffold for developing novel broad-spectrum antibiotics which can help replenish the pipeline of antibiotics.

SSAs are a class of supramolecular self-associating amphiphilic salt, the anionic component of which contains a covalently bound hydrogen bond donor-acceptor motif. This results in a monomeric unit which can adopt multiple hydrogen bonding modes simultaneously. Previous investigations have shown examples of SSAs to act as antimicrobial agents against clinically relevant methicillin-resistant Staphylococcus aureus (MRSA). Herein, we report an intrinsically fluorescent SSA which can self-associate producing dimers, spherical aggregates and hydrogels dependent on solvent environment, while retaining antimicrobial activity against both model Gram-positive (MRSA) and Gram-negative (Escherichia coli) bacteria. Finally, we demonstrate the SSA supramolecular hydrogel to tolerate the inclusion of the antibiotic ampicillin, leading to the enhanced inhibition of growth with both model bacteria, and derive initial molecular structure-physicochemical property-antimicrobial activity relationships.

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

To understand the potential utility of novel nitroreductase (NR)-activated prodrugs, NR enzyme activity was assessed in clinical Klebsiella pneumoniae isolates using a NR-activated fluorescent probe. NR activity was constant throughout the bacterial growth cycle, but individual K. pneumoniae isolates exhibited a wide range of NR activity levels. The genes of major NR enzymes (nfsA and nfnB) showed a number of sequence variants. Aside from a C-terminal extension of NfnB, which may be responsible for lower NR activity in specific isolates, the genetic differences did not explain the variation in activity. Analysis of important clinical strains (ST11, ST258, ST14 and ST101) showed significant variation in NR activity between isolates within the same sequence type despite conservation of nfsA/nfnB sequences. Addition of methyl viologen (MV), a known activator of soxRS, caused a significant increase in NR activity for all strains, with proportionally larger increases in activity seen for strains with low uninduced NR levels. Real-time PCR on selected strains following exposure to MV showed upregulation of soxS (15-32-fold) and nfsA (5-22-fold) in all strains tested. Expression of nfnB was upregulated 2-5-fold in 4/6 strains tested. High levels of NR activity in the absence of MV activation correlated with nitrofurantoin susceptibility. These data provide evidence that NR gene mutations and regulatory pathways influence NR activity in K. pneumoniae isolates and this is likely to impact treatment efficacy with novel nitro-containing drugs or prodrugs., (Copyright © 2019. Published by Elsevier B.V.)

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.

The Prestwick library was screened for antibacterial activity or "antibiotic resistance breaker" (ARB) potential against four species of Gram-negative pathogens. Discounting known antibacterials, the screen identified very few ARB hits, which were strain/drug specific. These ARB hits included antimetabolites (zidovudine, floxuridine, didanosine, and gemcitabine), anthracyclines (daunorubicin, mitoxantrone, and epirubicin), and psychoactive drugs (gabapentin, fluspirilene, and oxethazaine). These findings suggest that there are few approved drugs that could be directly repositioned as adjunct antibacterials, and these will need robust testing to validate efficacy., (© Crown copyright 2019.)

Antimicrobial peptides (AMPs) are a potential source of new molecules to counter the increase in antimicrobial resistant infections but a better understanding of their properties is required to understand their native function and for effective translation as therapeutics. Details of the mechanism of their interaction with the bacterial plasma membrane are desired since damage or penetration of this structure is considered essential for AMPs activity. Relatively modest modifications to AMPs primary sequence can induce substantial changes in potency and/or spectrum of activity but, hitherto, have not been predicted to substantially alter the mechanism of interaction with the bacterial plasma membrane. Here we use a combination of molecular dynamics simulations, circular dichroism, solid-state NMR and patch clamp to investigate the extent to which temporin B and its analogues can be distinguished both in vitro and in silico on the basis of their interactions with model membranes. Enhancing the hydrophobicity of the N-terminus and cationicity of the C-terminus in temporin B improves its membrane activity and potency against both Gram-negative and Gram-positive bacteria. In contrast, enhancing the cationicity of the N-terminus abrogates its ability to trigger channel conductance and renders it ineffective against Gram-positive bacteria while nevertheless enhancing its potency against Escherichia coli. Our findings suggest even closely related AMPs may target the same bacterium with fundamentally differing mechanisms of action.

The effectiveness of several cationic disinfectants as well as colistin and polymyxin B were assessed under different growth conditions against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains. These conditions included different media (MH1, MH2, TSB and LB) and plate material (polypropylene and polystyrene). Results showed that Minimum inhibitory and bactericidal concentrations (MIC/MBC) values of colistin and polymyxin B were significantly lower on polypropylene plates when compared to polystyrene plates regardless of media used. There were also differences in MIC/MBC values to certain biocides e.g. chlorhexidine and octenidine particularly for S. aureus and E. coli strains, with polypropylene again showing lower values. Other biocides appear to be mostly unaffected by plate type. Whether biocide efficacy was altered by media composition was organism dependent with S. aureus and E. coli more affected than P. aeruginosa. Lower MIC values were more commonly associated with MH2 media and higher MIC values with TSB media for both polypropylene and polystyrene plates, although there were exceptions. Results obtained for standard strains were, in general, indicative for other S. aureus, E. coli and P. aeruginosa strains tested. This study demonstrates the importance of media composition and plate material on biocide effectiveness and highlights the need for optimized disinfectant testing methods., Significance and Impact of the Study: There are an increasing number of reports of bacterial strains that are multi-drug resistant. The use of biocides as part of infection control is crucial in helping to combat the spread of these particular strains. Unlike for antibiotics, there are few standardized measuring techniques to understand if an isolate has become more resistant to biocides. This study demonstrates the importance of media composition and plate material on variation and reporting of susceptibility of several bacterial species to specific cationic biocides. It is a useful comparison study to highlight the need to standardize biocide susceptibility testing., (© 2018 Crown copyright. © 2018 The Society for Applied Microbiology.)

Antimicrobial resistance has become a major global concern. Development of novel antimicrobial agents for the treatment of infections caused by multidrug resistant (MDR) pathogens is an urgent priority. Pyrrolobenzodiazepines (PBDs) are a promising class of antibacterial agents initially discovered and isolated from natural sources. Recently, C8-linked PBD biaryl conjugates have been shown to be active against some MDR Gram-positive strains. To explore the role of building block orientations on antibacterial activity and obtain structure activity relationship (SAR) information, four novel structures were synthesized in which the building blocks of previously reported compounds were inverted, and their antibacterial activity was studied. The compounds showed minimum inhibitory concentrations (MICs) in the range of 0.125-32 μg/mL against MDR Gram-positive strains with a bactericidal mode of action. The results showed that a single inversion of amide bonds reduces the activity while the double inversion restores the activity against MDR pathogens. All inverted compounds did not stabilize DNA and lacked eukaryotic toxicity. The compounds inhibit DNA gyrase in vitro, and the most potent compound was equally active against both wild-type and mutant DNA gyrase in a biochemical assay. The observed activity of the compounds against methicillin resistant S. aureus (MRSA) strains with equivalent gyrase mutations is consistent with gyrase inhibition being the mechanism of action in vivo, although this has not been definitively confirmed in whole cells. This conclusion is supported by a molecular modeling study showing interaction of the compounds with wild-type and mutant gyrases. This study provides important SAR information about this new class of antibacterial agents.

Proteus mirabilis forms extensive crystalline biofilms on indwelling urethral catheters that block urine flow and lead to serious clinical complications. The Bcr/CflA efflux system has previously been identified as important for development of P. mirabilis crystalline biofilms, highlighting the potential for efflux pump inhibitors (EPIs) to control catheter blockage. Here we evaluate the potential for drugs already used in human medicine (fluoxetine and thioridazine) to act as EPIs in P. mirabilis, and control crystalline biofilm formation. Both fluoxetine and thioridazine inhibited efflux in P. mirabilis, and molecular modelling predicted both drugs interact strongly with the biofilm-associated Bcr/CflA efflux system. Both EPIs were also found to significantly reduce the rate of P. mirabilis crystalline biofilm formation on catheters, and increase the time taken for catheters to block. Swimming and swarming motilies in P. mirabilis were also significantly reduced by both EPIs. The impact of these drugs on catheter biofilm formation by other uropathogens (Escherichia coli, Pseudomonas aeruginosa) was also explored, and thioridazine was shown to also inhibit biofilm formation in these species. Therefore, repurposing of existing drugs with EPI activity could be a promising approach to control catheter blockage, or biofilm formation on other medical devices.

The lack of novel antibiotics for more than a decade has placed increased pressure on existing therapies to combat the emergence of multidrug-resistant (MDR) bacterial pathogens. This study evaluated the Galleria mellonella insect model in determining the efficacy of available antibiotics against planktonic and biofilm infections of MDR Pseudomonas aeruginosa and Klebsiella pneumoniae strains in comparison with in vitro minimum inhibitory concentration (MIC) determination. In general, in vitro analysis agreed with the G. mellonella studies, and susceptibility in Galleria identified different drug resistance mechanisms. However, the carbapenems tested appeared to perform better in vivo than in vitro, with meropenem and imipenem able to clear K. pneumoniae and P. aeruginosa infections with strains that had bla(NDM-1) and bla(VIM) carbapenemases. This study also established an implant model in G. mellonella to allow testing of antibiotic efficacy against biofilm-derived infections. A reduction in antibiotic efficacy of amikacin against K. pneumoniae and P. aeruginosa biofilms was observed compared with a planktonic infection. Ciprofloxacin was found to be less effective at clearing a P. aeruginosa biofilm infection compared with a planktonic infection, but no statistical difference was seen between K. pneumoniae biofilm and planktonic infections treated with this antibiotic (P>0.05). This study provides important information regarding the suitability of Galleria as a model for antibiotic efficacy testing both against planktonic and biofilm-derived MDR infections., (Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.)

Bfl-1 is a pro-survival Bcl-2 family member overexpressed in a subset of chemoresistant tumours, including melanoma. Here, we characterised the expression and regulation of Bfl-1 in normal and malignant melanocytes and determined its role in protecting these cells from chemotherapy-induced apoptosis. Bfl-1 was mitochondrially resident in both resting and apoptotic cells and experienced regulation by the proteasome and NFκB pathways. siRNA-mediated knockdown enhanced sensitivity towards various relevant drug treatments, with forced overexpression of Bfl-1 protective. These findings identify Bfl-1 as a contributor towards therapeutic resistance in melanoma cells and support the use of NFκB inhibitors alongside current treatment strategies., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chronic myeloid leukemia is the first disease in which the potential of molecular targeted therapy with tyrosine kinase inhibitors (TKIs) was realized. Despite this success, a proportion of patients, particularly with advanced disease, are, or become, resistant to this treatment. Overcoming resistance and uncovering the underlying mechanisms is vital for further improvement of clinical outcomes. Here we report the identification, development, and characterization of a novel chronic myeloid leukemia cell line carrying the additional chromosomal aberration t(3;12)(q26;p13) resulting in expression of the TEL/MDS1/EVI1 fusion protein, which is resistant to TKIs. Resistance to TKIs was overcome by the co-administration of the BH3-mimetic, ABT-737. In addition, application of EVI1-specific small interfering RNA decreased expression of the TEL/MDS1/EVI1 fusion, reduced resistance to imatinib, and increased sensitivity to ABT-737. Subsequent studies revealed a role for the BH3-only protein BAD, probably via a phosphoinositide 3-kinase/AKT-dependent pathway, as pharmacological inhibition of AKT could also resensitize cells to death from TKIs. These findings indicate a novel pathway of TKI resistance regulated by EVI1 proteins and provide a promising means for overcoming resistance in chronic myeloid leukemia and other hematological malignancies displaying EVI1 overexpression., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)