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Antimicrobial resistance (AMR) is a formidable global health challenge. Multidrug-resistant (MDR) Gram-negative bacterial infections are of primary concern due to diminishing treatment options and high morbidity and mortality. Colistin, a polymyxin family antibiotic, is a last-resort treatment for MDR Gram-negative infections, but its wider use has resulted in escalating resistance. In 2022, using a screening approach, we discovered that a [1,2,5]oxadiazolo[3,4-b]pyrazine (ODP)-containing compound selectively re-sensitized various MDR Gram-negative bacteria to colistin. Initial structure-activity relationship (SAR) studies confirmed that bisanilino ODP compounds are colistin adjuvants with low mammalian toxicity. Herein, we report our extended SAR studies on a wide range of ODP analogs bearing alkyl- or arylalkylamines. Specifically, we discovered two new compounds, 5q and 8g, with potent colistin-potentiating activity and low mammalian toxicity in a wide range of clinically relevant pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Acinetobacter baumannii poses a significant public health threat. Phage-encoded antimicrobial peptides (AMPs) have emerged as promising candidates in the battle against antibiotic-resistant A. baumannii ., Methods: Antimicrobial peptides from the endolysin of A. baumannii bacteriophage were designed from bacteriophage vB_AbaM_PhT2 and vB_AbaAut_ChT04. The peptides' minimum inhibitory concentration (MIC) and the synergistic effect of peptides with outer membrane-permeabilizing agents and colistin were determined. Cytotoxicity effects using HepG2 cell lines were evaluated for 24 h with various concentrations of peptides. Biofilm eradication assay was determined using the MIC concentration of each peptide. Galleria mellonella infection assay of phage-encoded antimicrobial peptides was investigated and recorded daily for 10 days., Results: The current research indicates that three peptides, specifically PE04-1, PE04-1(NH 2 ), and PE04-2, encoded from the endolysin of vB_AbaAut_ChT04 demonstrated significant antimicrobial activity, with minimum inhibitory concentrations (MIC) ranging from 156.25 to 312.5 µg/ml. The peptides showed antimicrobial activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) A. baumannii , Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus , and Bacillus subtilis . We found a strong synergistic effect of three peptides with colistin and citric acid, which showed high inhibition percentages (>90%) and low fractional inhibitory concentration (FIC) indexes. The peptides exhibited a high ability to inhibit biofilm formation against twenty A. baumannii strains, with PE04-2 showing the most potent inhibition (91.92%). The cytotoxicity effects of the peptides on human hepatoma cell lines showed that the concentrations at the MIC level did not affect the cell viability. The peptides improved survival rates in the G. mellonella model, exceeding 80% by day 10., Conclusions/significant Finding: Peptides PE04-1, PE04-1(NH 2 ), and PE04-2 showed sequence similarity to mammalian cathelicidin antimicrobial peptides. They are cationic peptides with a positive charge, exhibiting high hydrophobic ratios and high hydropathy values. The modified PE04-2 was designed by enhancing cationic through amino acid substitutions and shows powerful antibiofilm effects due to its cationic, amphipathic, and hydrophobic properties to destroy biofilm. The peptides improved survival rates in G. mellonella infection models and showed no cytotoxicity effect on human cell lines, ensuring their safety for potential therapeutic applications. In conclusion, this study highlights the antimicrobial ability of phage-encoded peptides against multidrug-resistant A. baumannii . It can be an innovative tool, paving the way for future research to optimize their clinical application., Competing Interests: Sutthirat Sitthisak is an Academic Editor for PeerJ., (© 2024 Rothong et al.)

The widespread emergence of antibiotic resistance poses a substantial challenge to global health. While polymyxin E serves as a final option in treatment, its effectiveness is hindered by dose-related toxicity. A crucial strategy for addressing this issue involves incorporating an antibiotic adjuvant to enhance the antibiotic's efficacy and decrease the required dosage. Here, we reported a multifunctional antibacterial compound A33, containing a 2-aminothiazole scaffold. In vitro studies demonstrated that A33 in combination with polymyxin E inhibited the growth of various Gram-negative bacteria, meanwhile with minimum inhibitory concentrations (MICs) of 0.5-4 μg/mL against twenty-three Gram-positive bacteria, including two drug-resistant strains. In vivo studies showed significant efficacy of the combined treatment of A33 and polymyxin E in a mouse infection model. The mice treated with compound A33 (64 mg/kg) and polymyxin E (0.5 mg/kg) in combination had a 100 % survival rate. Mechanistic studies suggested that A33 might exert its synergistic effect by targeting the outer membrane of Gram-negative bacteria. The ADMET data demonstrated that A33 possessed good pharmacokinetic profiles and drug-likeness properties. Overall, the optimized compound A33 assisted polymyxin E in combating various Gram-negative bacteria and exhibited bactericidal effects against drug-resistant Gram-positive bacteria, offering a new potential therapeutic approach for managing mixed bacterial infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Antibiotic resistance has emerged as a pressing global health issue. Polymyxin E, commonly regarded as a clinically important antibiotic, faces limitations due to its dose-dependent toxicity. A crucial strategy to combat this is the development of an antibiotic adjuvant to enhance efficacy while reducing dosage. Screening from our extensive in-house compound library, KJ1071 emerged as a promising adjuvant candidate for polymyxin E. Subsequent structure-activity relationship studies led to the discovery of compound A35, which demonstrated superior synergistic activity. However, its limited aqueous solubility posed challenges for biological experiments. To address this, we carried out a structural derivatization aimed at enhancing its solubility. The amino acid derivative A59 notably improved aqueous solubility to 3.237 mg/mL, a substantial 3237-fold increase compared to compound A35. Moreover, A59 amplified the efficacy of polymyxin E in eradicating a variety of Gram-negative bacteria, including certain clinical strains that are resistant to polymyxin E. The mechanism studies indicated that A59 might target the bacterial membrane. These findings suggest that the discovery of this innovative scaffold could provide critical insights for new adjuvant therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Multidrug-resistant P. aeruginosa (MDR-P. aeruginosa), associated with elevated morbidity, mortality, and readmission rates, presents a formidable challenge to eradication due to its robust resistance to antimicrobial agents and biofilm formation. Herein, self-assembling nanoparticles (NO-PE/PLL NPs) comprised of NO donor-conjugated γ-polyglutamic acid (GSNO-PGA), epsilon-poly-l-lysine (PLL) and colistin were fabricated. The negatively charged NO-PE/PLL NPs exhibited effective penetration through airway mucus, reaching the infection site where GSNO-PGA released NO in response to glutathione within biofilm. PLL worked synergistically with colistin (fractional inhibitory concentration index: 0.281), reducing the minimum inhibitory concentration (MIC) of colistin from 2 to 0.5 μg/mL. Benefiting from this synergistic antibacterial action and NO-mediated biofilm disruption, NO-PE/PLL NPs achieved a 99.99 % eradication rate against MDR-P. aeruginosa biofilms. Additionally, NO-PE/PLL NPs efficiently inhibited endotoxins-stimulated inflammation response. In a chronic pulmonary infection model, NO-PE/PLL NPs displayed the highest eradication efficiency (99.78 %) to infected mice, while having no adverse effects on their major organs or pulmonary functions. These results highlight NO-PE/PLL NPs as a promising therapeutic strategy for treating recalcitrant infections caused by MDR-P. aeruginosa biofilms., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The use of highly potent but very toxic antibiotics such as colistin has become inevitable due to the rise of antimicrobial resistance. We aimed for a chemically-triggered, controlled release of colistin at the infection site to lower its systemic toxicity by harnessing the power of click-to-release reactions. Kinetic experiments with nine tetrazines and three dienophiles demonstrated a fast release via an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene (TCO) and the amine-functionalised tetrazine Tz7. The antibiotic activity of colistin against Escherichia coli was masked by TCO units, but restored upon reaction with d-Ubi-Tz, a tetrazine functionalised with the bacterial binding peptide d-Ubi 29-41 . While standard TCO did not improve toxicity against human proximal tubular kidney HK-2 cells, the installation of an aspartic acid-modified TCO masking group reduced the overall charge of the peptide and entry to the kidney cells, thereby dramatically lowering its toxicity. The analog Col-(TCO-Asp) 1 had favourable pharmacokinetic properties in mice and was successfully activated locally in the lung by d-Ubi-Tz in an in vivo infection model, whereas it remained inactive and non-harmful without the chemical trigger. This study constitutes the first example of a systemically acting two-component antibiotic with improved drug tolerability., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The impact of microbial infections is increasing over time, and it is one of the major reasons for death in both developed and developing countries. colistin is considered as the antibiotic of last choice for infections brought by major multidrug-resistant (MDR), gram-negative bacteria such as Enterobacter species, Acinetobacter species, and Pseudomonas aeruginosa. Existing approaches to diagnose these resistant species are relatively slow and take up to 2 to 3 days. In this work, we propose a novel interdisciplinary method based on Raman spectroscopy and heavy water to identify colistin-resistant microbes. Our hypothesis is based on the fact that resistant bacteria will be metabolically active in the culture medium containing antibiotics and heavy water, and these bacteria will take up deuterium instead of hydrogen to newly synthesized lipids and proteins. This effect will generate a 'C - D' bond-specific Raman spectral marker. Successful identification of this band in the spectral profile can confirm the presence of colistin-resistant bacteria. We have validated the efficacy of this approach in identifying colistin-resistant bacteria spiked in artificial urine and have compared sensitivity at different bacterial concentrations. Overall findings suggest that heavy water can potentially serve as a suitable Raman probe for identifying metabolically active colistin-resistant bacteria via urine under clinically implementable time and can be used in clinical settings after validation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The plasmid-encoded mobile colistin resistance enzyme (MCR) is challenging the clinical efficacy of colistin as a last-resort antibiotic against multidrug-resistant bacteria. This transferase catalyzes the addition of positively charged phosphoethanolamine to lipid A, and its catalytic domain in the periplasm has been elucidated. To date, there are many works on the catalytic domain and function of this enzyme class. However, the roles of unreported soluble or inter-membrane domains remain undefined, which might cause an inaccurate or even incorrect understanding of substrate recognition and binding. In this review, MCR-1 is first compared and analyzed from the perspective of the full-length alpha-fold MCR-1. Specifically, some disputed issues, especially in its architecture and catalytic mechanism are discussed independently. Meanwhile, the structure-based insights into MCRs variants, their evolutions, and the balance between colistin-resistance and survival costs, are also critically analyzed. Importantly, by comparing it with the full-length MCR-1, several potential pockets for drug design have been re-identified. Finally, recent advancements in inhibitors targeting MCR-1 are also in-depth summarized. These details offer a new perspective on MCRs and serve as a valuable foundation for drug development., Competing Interests: Declaration of Competing Interest The author declares no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Purpose: The eradication of bacterial biofilms poses an enormous challenge owing to the inherently low antibiotic susceptibility of the resident microbiota. The complexation of antibiotics with polyphosphate can substantially improve antimicrobial performance., Methods: Nanoparticular complexes of the model drug colistin and polyphosphate (CP-NPs) were developed and characterized in terms of their particle size and morphology, polydispersity index (PDI), zeta potential, and cytotoxicity. Enzyme-triggered monophosphate and colistin release from the CP-NPs was evaluated in the presence of alkaline phosphatase (AP). Subsequently, antimicrobial efficacy was assessed by inhibition experiments on planktonic cultures, as well as time-kill assays on biofilms formed by the model organism Micrococcus luteus ., Results: The CP-NPs exhibited a spherical morphology with particle sizes <200 nm, PDI <0.25, and negative zeta potential. They showed reduced cytotoxicity toward two human cell lines and significantly decreased hemotoxicity compared with native colistin. Release experiments with AP verified the enzymatic cleavage of polyphosphate and subsequent release of monophosphate and colistin from CP-NPs. Although CP-NPs were ineffective against planktonic M. luteus cultures, they showed major activity against bacterial biofilms, outperforming native colistin treatment. Strongly elevated AP levels in the biofilm state were identified as a potential key factor for the observed findings., Conclusion: Accordingly, polyphosphate-based nanocomplexes represent a promising tool to tackle bacterial biofilm., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this study., (© 2024 To et al.)

The growth of (multi)drug resistance in bacteria is among the most urgent global health issues. Monocationic amphiphilic α-hydrazido acid derivatives are structurally simple mimics of antimicrobial peptides (AMPs) with fewer drawbacks. Their mechanism of membrane permeabilization at subtoxic concentrations was found to begin with an initial electrostatic attraction of isolated amphiphile molecules to the phospholipid heads, followed by a rapid insertion of the apolar portions. As the accumulation into the bilayer proceeded, the membrane increased its fluidity and permeability without being subjected to major structural damage. After having ascertained that α-hydrazido acid amphiphiles do not interact with bacterial DNA, they were subjected to synergy evaluation for combinations with conventional antibiotics. Synergy was observed for combinations with tetracycline against sensitive S. aureus and E. coli , as well as with ciprofloxacin and colistin against resistant strains. Additivity with a remarkable recovery in activity of conventional antibiotics (from 2-fold to ≥32-fold) together with largely subtoxic concentrations of α-hydrazido acid derivatives was found for combinations with ciprofloxacin toward susceptible S. aureus and methicillin toward MRSa. However, no potentiation of conventional antibiotics was observed for combinations with linezolid and gentamicin against the corresponding resistant S. aureus and E. coli strains.

Colistin methanesulfonate (CMS) is the less-toxic prodrug of highly nephrotoxic colistin. To develop and understand highly necessary new antibiotic formulations, the hydrolysis of CMS to colistin must be better understood. Herein, with the addition of poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMAA) to CMS, we show that we can follow the hydrolysis kinetics, employing small-angle X-ray scattering (SAXS) through complex coacervation. During this hydrolysis, hydroxy methanesulfonate (HMS) groups from CMS are cleaved, while the newly formed cationic amino groups complex with the anionic charge from the PMAA block. As the hydrolysis of HMS groups is slow, we can follow the complex coacervation process by the gradual formation of complex micelles containing activated antibiotics. Combining mass spectrometry (MS) with SAXS, we quantify the hydrolysis as a function of pH. Upon modeling the kinetic pathways, we found that complexation only happens after complete hydrolysis into colistin and that the process is accelerated under acidic conditions. At pH = 5.0, effective charge switching was identified as the slowest step in the CMS conversion, constituting the rate-limiting step in colistin formation.

Multicomponent therapy combining antibiotics with enhancer molecules known as adjuvants is an emerging strategy to combat antimicrobial resistance. Niclosamide is a clinically relevant anthelmintic drug with potential to be repurposed for its inherent antibacterial activity against Gram-positive bacteria and its ability to potentiate the antibacterial activity of colistin against susceptible and resistant Gram-negative bacteria. Herein, sulfonamide analogs of niclosamide were prepared and found to enhance colistin activity against Gram-negative bacteria. The ability of niclosamide and the new sulfonamide analogs to synergize with bacitracin against vancomycin-resistant Enterococcus faecium was also discovered., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

To combat the ever-growing increase of multidrug-resistant (MDR) bacteria, action must be taken in the development of antibiotic formulations. Colistin, an effective antibiotic, was found to be nephrotoxic and neurotoxic, consequently leading to a ban on its use in the 1980s. A decade later, colistin use was revived and nowadays used as a last-resort treatment against Gram-negative bacterial infections, although highly regulated. If cytotoxicity issues can be resolved, colistin could be an effective option to combat MDR bacteria. Herein, we investigate the complexation of colistin with poly(ethylene oxide)- b -poly(methacrylic acid) (PEO- b -PMAA) block copolymers to form complex coacervate core micelles (C3Ms) to ultimately improve colistin use in therapeutics while maintaining its effectiveness. We show that well-defined and stable micelles can be formed in which the cationic colistin and anionic PMAA form the core while PEO forms a protecting shell. The resulting C3Ms are in a kinetically arrested and stable state, yet they can be made reproducibly using an appropriate experimental protocol. By characterization through dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), we found that the best C3M formulation, based on long-term stability and complexation efficiency, is at charge-matching conditions. This nanoparticle formulation was compared to noncomplexed colistin on its antimicrobial properties, enzymatic degradation, serum protein binding, and cytotoxicity. The studies indicate that the antimicrobial properties and cytotoxicity of the colistin-C3Ms were maintained while protein binding was limited, and enzymatic degradation decreased after complexation. Since colistin-C3Ms were found to have an equal effectivity but with increased cargo protection, such nanoparticles are promising components for the antibiotic formulation toolbox.

Purpose: Special consideration is needed when intravenous drugs are administered simultaneously using a Y-site connector. This study aimed to investigate the physical compatibility of colistin with 6 analgesics at concentrations commonly used in clinical practice., Methods: A pharmaceutical preparation of colistin was dissolved according to the manufacturer's instructions and diluted to a concentration of 1.5 mg/mL or 0.67 mg/mL (of colistin base). Simulated administration via Y-site infusion set was performed by mixing 5 mL of colistin solution with an equal volume of a solution of one of 6 intravenous analgesics. Infusion solutions of ibuprofen, ketoprofen, metamizole sodium, morphine sulfate, paracetamol, and tramadol hydrochloride were studied. For each analgesic tested, concentrates for injection were diluted with 2 solvents, resulting in 11 different combinations with each concentration of the colistin solution. The mixtures were visually inspected, and their turbidity was measured directly after mixing and at 3 consecutive time points (30, 60, and 120 minutes). Additionally, the pH of the mixtures was measured after 120 minutes and compared with the pH of the analgesic and the colistin solutions., Results: During visual inspection with the unaided eye, no precipitate formation or gas evolution was observed in any of the tested analgesics except for sodium metamizole, where the yellow color of the solutions was observed. For samples containing the mixture of ibuprofen and colistin, the turbidity measurements revealed the presence of turbidity in the studied mixtures. The greatest change in pH relative to the value immediately after preparation was noted for combinations of ketoprofen and morphine sulfate with the tested antibiotic., Conclusion: Colistin was found to be incompatible with ibuprofen and metamizole sodium formulations. It should also not be combined with morphine sulfate due to the significant differences in the pH value of the preparations. The colistin 0.67 mg/mL and 1.5 mg/mL infusion solutions were physically compatible with ketoprofen, tramadol hydrochloride, and paracetamol., (© American Society of Health-System Pharmacists 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Addressing microbial resistance urgently calls for alternative treatment options. This study investigates the impact of a bimetallic formulation containing colistin, silver, and copper oxide on a pandrug-resistant, highly virulent Pseudomonas aeruginosa (P. aeruginosa) isolate from a cancer patient at the National Cancer Institute, Cairo University, Egypt., Methods: Silver nanoparticles (Ag NPs), copper oxide nanoparticles (CuO NPs), and bimetallic silver-copper oxide nanoparticles (Ag-CuO NPs) were synthesized using gamma rays, combined with colistin (Col), and characterized by various analytical methods. The antimicrobial activity of Col-Ag NPs, Col-CuO NPs, and bimetallic Col-Ag-CuO NPs against P. aeruginosa was evaluated using the agar well diffusion method, and their minimum inhibitory concentration (MIC) was determined using broth microdilution. Virulence factors such as pyocyanin production, swarming motility, and biofilm formation were assessed before and after treatment with bimetallic Col-Ag-CuO NPs. The in vivo efficacy was evaluated using the Galleria mellonella model, and antibacterial mechanism were examined through membrane leakage assay., Results: The optimal synthesis of Ag NPs occurred at a gamma ray dose of 15.0 kGy, with the highest optical density (OD) of 2.4 at 375 nm. Similarly, CuO NPs had an optimal dose of 15.0 kGy, with an OD of 1.5 at 330 nm. Bimetallic Ag-CuO NPs were most potent at 15.0 kGy, yielding an OD of 1.9 at 425 nm. The MIC of colistin was significantly reduced when combined with nanoparticles: 8 µg/mL for colistin alone, 0.046 µg/mL for Col-Ag NPs, and 0.0117 µg/mL for Col-Ag-CuO NPs. Bimetallic Col-Ag-CuO NPs reduced the MIC four-fold compared to Col-Ag NPs. Increasing the sub-inhibitory concentration of bimetallic nanoparticles from 0.29 × 10 -2 to 0.58 × 10 -2 µg/mL reduced P. aeruginosa swarming by 32-64% and twitching motility by 34-97%. At these concentrations, pyocyanin production decreased by 39-58%, and biofilm formation was inhibited by 33-48%. The nanoparticles were non-toxic to Galleria mellonella, showing 100% survival by day 3, similar to the saline-treated group., Conclusions: The synthesis of bimetallic Ag-CuO NPs conjugated with colistin presents a promising alternative treatment for combating the challenging P. aeruginosa pathogen in hospital settings. Further research is needed to explore and elucidate the mechanisms underlying the inhibitory effects of colistin-bimetallic Ag-CuO NPs on microbial persistence and dissemination., (© 2024. The Author(s).)

Antimicrobial peptides (AMPs) are presented as potential scaffolds for antibiotic development due to their desirable qualities including broad-spectrum activity, rapid action, and general lack of susceptibility to current resistance mechanisms. However, they often lose antibacterial activity under physiological conditions and/or display mammalian cell toxicity, which limits their potential use. Identification of AMPs that overcome these barriers will help develop rules for how this antibacterial class can be developed to treat infection. Here we describe the development of our novel synthetic AMP, from discovery through in vivo application. Our evolved AMP, DTr18-dab, has broad-spectrum antibacterial activity and is nonhemolytic. It is active against planktonic bacteria and biofilm, is unaffected by colistin resistance, and importantly is active in both human serum and a Galleria mellonella infection model. Several modifications, including the incorporation of noncanonical amino acids, were used to arrive at this robust sequence. We observed that the impact on antibacterial activity with noncanonical amino acids was dependent on assay conditions and therefore not entirely predictable. Overall, our results demonstrate how a relatively weak lead can be developed into a robust AMP with qualities important for potential therapeutic translation.

Introduction: Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine is a major goal in AML therapy towards developing agents that can be used to treat patients with different 'subtypes' in combination with current chemotherapies. We have previously developed dextrin-colistin conjugates to combat the rise in multi-drug resistant bacterial infections and overcome dose-limiting nephrotoxicity. Recent evidence of colistin's anticancer activity, mediated through inhibition of intracellular lysine-specific histone demethylase 1 (LSD1/KDM1A), suggests that dextrin-colistin conjugates could be used to treat cancer cells, including AML. This study aimed to evaluate whether dextrin conjugation (which reduces in vivo toxicity and prolongs plasma half-life) could enhance colistin's cytotoxic effects in myeloid leukemia cell lines and compare the intracellular uptake and localization of the free and conjugated antibiotic., Results: Our results identified a conjugate (containing 8000 g/mol dextrin with 1 mol% succinoylation) that caused significantly increased toxicity in myeloid leukemia cells, compared to free colistin. Dextrin conjugation altered the mechanism of cell death by colistin, from necrosis to caspase 3/7-dependent apoptosis. In contrast, conjugation via a reversible ester linker, instead of an amide, had no effect on the mechanism of the colistin-induced cell death. Live cell confocal microscopy of fluorescently labelled compounds showed both free and dextrin-conjugated colistins were endocytosed and co-localized in lysosomes, and increasing the degree of modification by succinoylation of dextrin significantly reduced colistin internalization., Discussion: Whilst clinical translation of dextrin-colistin conjugates for the treatment of AML is unlikely due to the potential to promote antimicrobial resistance (AMR) and the relatively high colistin concentrations required for anticancer activity, the ability to potentiate the effectiveness of an anticancer drug by polymer conjugation, while reducing side effects and improving biodistribution of the drug, is very attractive, and this approach warrants further investigation., Competing Interests: Dr Siân Rizzo’s current affiliation is at Department of Haematology, School of Medicine, Cardiff University, Cardiff, UK. Dr Mathieu Varache’s current affiliation is at Advanced BioDesign, 655 All. des Parcs, 69800 Saint-Priest, France. Dr Elaine Ferguson and Prof David Thomas report a patent EP2585114B1 issued. The authors report no other conflicts of interest in this work., (© 2024 Rizzo et al.)

An efficient and practical one-pot synthesis of isoindolines from readily available starting materials was achieved under mild conditions by implementing an isoindole umpolung strategy. A variety of isoindolines were prepared with good to excellent yields. Biological screens of these identified compounds demonstrated that they are potent potentiators of colistin for multi-drug resistant Acinetobacter baumannii .

The single-component colistin E2, with superior antibacterial activity and lower toxicity, was being developed as the latest generation of polymyxin drugs. However, colistin E2 has not been tested quantitatively in biological matrices. In this study, based on the quantitative detection of colistin methanesulphonate (CMS) and colistin by Zhao et al., 15 N-labeled colistin E2 was used as an internal standard (IS) for a more accurate quantitative detection of CMS E2 in human plasma. A rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay method was developed for determination of CMS E2 and colistin E2 in human plasma. After pretreatment of plasma samples by 96-well SPE Supra-Clean Weak Cation Exchange (WCX) plate, the formed colistin E2 was detected and quantified by UHPLC-MS/MS system. All plasma lots were found to be free of interferences with the analyte. The matrix has no effect on the quantitation of the analyte. No significant effect of the carryover was observed. The dilution integrity was demonstrated in plasma samples without the loss of accuracy and precision. The lower limit of quantification (LLOQ) was 0.0300 mg/L for colistin E2 in plasma with accuracy (relative error, 5.1-12.7%) and precision (relative standard deviation,  - 5.7-9.3%). Stability of CMS E2 and colistin E2 was demonstrated in biological samples before and during sample treatment, and in the extract. Furthermore, this method was successfully applied to the analysis of plasma samples obtained from Chinese healthy volunteers receiving a single intravenous CMS E2 dose of 5 mg/kg. In conclusion, the detection method was characterized by speed and high accuracy, which laid a solid foundation for the subsequent development of CMS E2 drug., (© 2023. The Author(s).)

Polymyxins, a last resort antibiotic, target the outer membrane of pathogens and are used to address the increasing prevalence of multidrug-resistant Gram-negative bacteria. The plasmid-encoded enzyme MCR-1 confers polymyxin resistance to bacteria by modifying the outer membrane. Transferable resistance to polymyxins is a major concern; therefore, MCR-1 is an important drug target. In this review, we discuss recent structural and mechanistic aspects of MCR-1 function, its variants and homologs, and how they are relevant to polymyxin resistance. Specifically, we discuss work on polymyxin-mediated disruption of the outer and inner membranes, computational studies on the catalytic mechanism of MCR-1, mutagenesis and structural analysis concerning residues important for substrate binding in MCR-1, and finally, advancements in inhibitors targeting MCR-1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Owing to the emergence of antibiotic resistance, the polymyxin colistin has been recently revived to treat acute, multidrug-resistant Gram-negative bacterial infections. Positively charged colistin binds to negatively charged lipids and damages the outer membrane of Gram-negative bacteria. However, the MCR-1 protein, encoded by the mobile colistin resistance (mcr) gene, is involved in bacterial colistin resistance by catalysing phosphoethanolamine (PEA) transfer onto lipid A, neutralising its negative charge, and thereby reducing its interaction with colistin. Our preliminary results showed that treatment with a reference pyrazolone compound significantly reduced colistin minimal inhibitory concentrations in Escherichia coli expressing mcr-1 mediated colistin resistance (Hanpaibool et al. in ACS Omega, 2023). A docking-MD combination was used in an ensemble-based docking approach to identify further pyrazolone compounds as candidate MCR-1 inhibitors. Docking simulations revealed that 13/28 of the pyrazolone compounds tested are predicted to have lower binding free energies than the reference compound. Four of these were chosen for in vitro testing, with the results demonstrating that all the compounds tested could lower colistin MICs in an E. coli strain carrying the mcr-1 gene. Docking of pyrazolones into the MCR-1 active site reveals residues that are implicated in ligand-protein interactions, particularly E246, T285, H395, H466, and H478, which are located in the MCR-1 active site and which participate in interactions with MCR-1 in ≥ 8/10 of the lowest energy complexes. This study establishes pyrazolone-induced colistin susceptibility in E. coli carrying the mcr-1 gene, providing a method for the development of novel treatments against colistin-resistant bacteria., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Colistin (polymyxin E) is a group of cationic antimicrobial cyclic peptides and is recognized as a last-resort defense against lethal infections with carbapenem-resistant pathogens. In addition to the plasmid-borne mobilized phosphoethanolamine (PEA) transferases, the functional expression of lipid A-modifying enzymes encoded on chromosomes has been attributed to intrinsic bacterial colistin resistance. However, the mechanisms of colistin resistance in Riemerella anatipestifer remain unknown. Herein, the GE296&#95;RS09715 gene-encoded Lipid A PEA transferases (RaEptA) was identified in R. anatipestifer. Genetic and structural analyses revealed that the amino acid sequence of RaEptA shared 26.6%-33.1% similarities with the family of Lipid A PEA transferases (EptA) and MCR-like proteins and have defined 12 residues that contribute to the formation of phosphatidylethanolamine (PE)-recognizable cavities. Comparative analyses of colistin resistance in RA-LZ01 and RA-LZ01ΔRaEptA showed the level of colistin has fallen from 96 μg mL -1 down to 24 ~ 32 μg mL -1 . Site-directed mutagenesis assay of the PE-binding cavity and expression of the mutants reveals that K309-rRaEptA can remodel the surface of Escherichia coli and rendering it resistant to colistin, suggesting this point-mutation of P309K is necessary for EptA-mediated lipid A modification. Moreover, the virulence of RA-LZ01ΔRaEptA was attenuated compared with RA-LZ01 both in vivo and vitro. Taken together, the results represent the RaEptA involved in the colistin resistance and pathogenicity, and the P309K mutation might alter bacterial adaptation and increase the spread of colistin resistance from R. anatipestifer to other gram-negative bacteria. The findings of this study suggest another scenario for the spread of colistin resistance genes and should be considered by a wide audience., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Polymyxins (polymyxin B and colistin) are lipopeptide antibiotics used as a last-line treatment for life-threatening multidrug-resistant (MDR) Gram-negative bacterial infections. Unfortunately, their clinical use has been affected by dose-limiting toxicity and increasing resistance. Structure-activity (SAR) and structure-toxicity (STR) relationships are paramount for the development of safer polymyxins, albeit very little is known about the role of the conserved position 10 threonine (Thr) residue in the polymyxin core scaffold. Here, we synthesized 30 novel analogues of polymyxin B 1 modified explicitly at position 10 and examined the antimicrobial activity against Gram-negative bacteria and in vivo toxicity and performed molecular dynamics simulations with bacterial outer membranes. For the first time, this study revealed the stereochemical requirements and role of the β-hydroxy side chain in promoting the correctly folded conformation of the polymyxin that drives outer membrane penetration and antibacterial activity. These findings provide essential information for developing safer and more efficacious new-generation polymyxin antibiotics.

Purpose: Tobramycin shows synergistic antibacterial activity with colistin and can reduce the toxic effects of colistin. The purpose of this study is to prepare pulmonary powder formulations containing both colistin and tobramycin and to assess their in vitro aerosol performance and storage stability., Methods: The dry powder formulations were manufactured using a lab-scale spray dryer. In vitro aerosol performance was measured using a Next Generation Impactor. The storage stability of the dry powder formulations was measured at 22°C and two relative humidity levels - 20 and 55%. Colistin composition on the particle surface was measured using X-ray photoelectron spectroscopy., Results: Two combination formulations, with 1:1 and 1:5 molar ratios of colistin and tobramycin, showed fine particle fractions (FPF) of 85%, which was significantly higher than that of the spray dried tobramycin (45%). FPF of the tobramycin formulation increased significantly when stored for four weeks at both 20% and 55% RH. In contrast, FPF values of both combination formulations and spray dried colistin remained stable at both humidity levels. Particle surface of each combination was significantly enriched in colistin molecules; 1:5 combination showed 77% by wt. colistin., Conclusions: The superior aerosol performance and aerosolization stability of 1:1 and 1:5 combination formulations of colistin and tobramycin could be attributed to enrichment of colistin on the co-spray dried particle surface. The observed powder properties may be the result of a surfactant-like assembly of these colistin molecules during spray drying, thus forming a hydrophobic particle surface., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Colistin is a potent antibiotic but its severe side effects including nephrotoxicity and neurotoxicity are the roadblock for their wide use in clinics. To solve this problem, we synthesized a new prodrug, mannose-maltose-colistin conjugate, termed MMCC that can reversibly mask the five amines of colistin that are primarily responsible for the toxicity. The deliberated design of disulfide-based self-immolative linker warranted the reversibly release of the pristine amines of colistin on demand without sacrificing antimicrobial efficacy. Once MMCC was delivered in cells, reducing agents cleaves the disulfide bond and release the pristine amines. The targeting ligands of maltose and mannose were grafted on colistin conjugate for targeting delivery of colistin to bacteria and macrophages, respectively. Taken together, MMCC as a new class of antimicrobial biomaterials, demonstrates its great potential for the treatment of intracellular bacterial infections., (© 2022 Wiley Periodicals LLC.)

The development of nanotechnology-based antibiotic delivery systems (nanoantibiotics) is an important challenge in the effort to combat microbial multidrug resistance. These systems have improved biopharmaceutical characteristics by increasing local bioavailability and reducing systemic toxicity and the number and frequency of drug side effects. Conjugation of low -molecular -weight antibacterial agents with natural polysaccharides is an effective strategy for developing optimal targeted delivery systems with programmed release and reduced cytotoxicity. This study describes the synthesis of conjugates of colistin (CT) and hyaluronic acid (HA) using carbodiimide chemistry to conjugate the amino groups of CT with the carboxyl groups of HA. The obtained polysaccharide carriers had a degree of substitution (DS) with CT molecules of 3-10 %, and the CT content was 129-377 μg/mg. The size of the fabricated particles was 300-600 nm; in addition, there were conjugates in the form of single macromolecules (30-50 nm). The ζ-potential of developed systems was about -20 mV. In vitro release studies at pH 7.4 and pH 5.2 showed slow hydrolysis of amide bonds, with a CT release of 1-5 % after 24 h. The conjugates retained antimicrobial activity depending on the DS: at DS 8 %, the minimum inhibitory concentration (MIC) of the conjugate corresponded to the MIC of free CT. The resulting systems also reduced CT nephrotoxicity by 20-50 %. These new conjugates of CT with HA are promising for the development of nanodrugs for safe and effective antimicrobial therapy., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Colistin (polymyxin E) and polymyxin B have been used as last-resort agents for treating infections caused by multidrug-resistant Gram-negative bacteria. However, their efficacy has been challenged by the emergence of the mobile colistin resistance gene mcr-1 , which encodes a transmembrane phosphoethanolamine (PEA) transferase enzyme, MCR-1. The enzyme catalyzes the transfer of the cationic PEA moiety of phosphatidylethanolamine (PE) to lipid A, thereby neutralizing the negative charge of lipid A and blocking the binding of positively charged polymyxins. This study aims to facilitate understanding of the mechanism of the MCR-1 enzyme by investigating its active-site sequence requirements. For this purpose, 23 active-site residues of MCR-1 protein were randomized by constructing single-codon randomization libraries. The libraries were individually selected for supporting Escherichia coli cell growth in the presence of colistin or polymyxin B. Deep sequencing of the polymyxin-resistant clones revealed that wild-type residues predominates at 17 active-site residue positions, indicating these residues play critical roles in MCR-1 function. These residues include Zn 2+ -chelating residues as well as residues that may form a hydrogen bond network with the PEA moiety or make hydrophobic interactions with the acyl chains of PE. Any mutations at these residues significantly decrease polymyxin resistance levels and the PEA transferase activity of the MCR-1 enzyme. Therefore, deep sequencing of the randomization libraries of MCR-1 enzyme identifies active-site residues that are essential for its polymyxin resistance function. Thus, these residues may be utilized as targets to develop inhibitors to circumvent MCR-1-mediated polymyxin resistance. IMPORTANCE Polymyxin antibiotics are used as last-line antibiotics in treating infections caused by multidrug-resistant pathogens. However, widespread use of polymyxins has led to the emergence of resistance. Although multiple mechanisms for resistance exist, that due to mcr-1 is a particular concern, as it can be readily transferred among bacterial pathogens. The mcr-1 gene encodes a transmembrane phosphoethanolamine (PEA) transferase that modifies lipid A to block the binding of polymyxin antibiotics. We utilized random mutagenesis coupled with next-generation sequencing to determine the amino acid sequence requirements of 23 residues in and near the active site of MCR-1. We show that the enzyme has stringent sequence requirements, with 75% of the residues examined being essential for function. Coupled with the finding that these residues are largely conserved among PEA enzymes, the results suggest inhibitors that bind near these sites will broadly inhibit MCR-1 and other enzymes of this class.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) poses a major threat to human health worldwide. Combination therapies of antibiotics with different mechanisms have been recommended in literatures. This study assessed in vitro antibacterial activities and synergistic activities of ceftazidime/avibactam alone and in combinations against KPC-Kp. In total, 70 isolates from 2 hospitals in Beijing were examined in our study. By using the agar dilution method and broth dilution method, we determined the minimum inhibitory concentration (MIC) of candidate antibiotics. Ceftazidime/avibactam demonstrated promising susceptibility against KPC-Kp (97.14%). Synergistic activities testing was achieved by checkerboard method and found ceftazidime/avibactam-amikacin displayed synergism in 90% isolates. Ceftazidime/avibactam-colistin displayed partial synergistic in 43% isolates, and ceftazidime/avibactam-tigecycline displayed indifference in 67% isolates. In time-kill assays, antibiotics at 1-fold MIC were mixed with bacteria at 1 × 105 CFU/ml and Mueller-Hinton broth (MHB). Combinations of ceftazidime/avibactam with amikacin and tigecycline displayed better antibacterial effects than single drug. Ceftazidime/avibactam-colistin combination did not exhibit better effect than single drug. In KPC-Kp infections, susceptibility testing suggested that ceftazidime/avibactam may be considered as first-line choice. However, monotherapy is often inadequate in infection management. Thus, our study revealed that combination therapy including ceftazidime/avibactam colistin and ceftazidime/avibactam tigecycline may benefit than monotherapy in KPC-Kp treatment. Further pharmacokinetic/pharmacodynamic and mutant prevention concentration studies should be performed to optimize multidrug-regimens., Competing Interests: The authors have declared that no competing interests exist.

Nosocomial infections, caused by bacterial contamination of medical devices and implants, are a serious healthcare concern. We demonstrate here, the use of fluorous-cured protein nanofilm coatings for generating antimicrobial surfaces. In this approach, bacteria-repelling films are created by heat-curing proteins in fluorous media. These films are then loaded with antibiotics, with release controlled via electrostatic interactions between therapeutic and protein film building blocks to provide bactericidal surfaces. This film fabrication process is additive-free, biocompatible, biodegradable, and can be used to provide antimicrobial coatings for both three-dimensional (2D) and 3D objects for use in indwelling devices.

Nanotechnology-based modification of known antimicrobial agents is a rational and straightforward way to improve their safety and effectiveness. The aim of this study was to develop colistin (CT)-loaded polymeric carriers based on hyaluronic acid (HA) for potential application as antimicrobial agents against multi-resistant gram-negative microorganisms (including ESKAPE pathogens). CT-containing particles were obtained via a polyelectrolyte interaction between protonated CT amino groups and HA carboxyl groups (the CT-HA complex formation constant [logK CT-HA ] was about 5.0). The resulting polyelectrolyte complexes had a size of 210-250 nm and a negative charge (ζ-potential -19 mV), with encapsulation and loading efficiencies of 100% and 20%, respectively. The developed CT delivery systems were characterized by modified release (45% and 85% of CT released in 15 and 60 min, respectively) compared to pure CT (100% CT released in 15 min). In vitro tests showed that the encapsulation of CT in polymer particles did not reduce its pharmacological activity; the minimum inhibitory concentrations of both encapsulated CT and pure CT were 1 μg/mL (against Pseudomonas aeruginosa)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In order to render potent, but toxic antibiotics more selective, we have explored a novel conjugation strategy that includes drug accumulation followed by infection-triggered release of the drug. Bacterial targeting was achieved using a modified fragment of the human antimicrobial peptide ubiquicidin, as demonstrated by fluorophore-tagged variants. To limit the release of the effector colistin only to infection-related situations, we introduced a linker that was cleaved by neutrophil elastase (NE), an enzyme secreted by neutrophil granulocytes at infection sites. The linker carried an optimized sequence of amino acids that was required to assure sufficient cleavage efficiency. The antibacterial activity of five regioisomeric conjugates prepared by total synthesis was masked, but was released upon exposure to recombinant NE when the linker was attached to amino acids at the 1- or the 3-position of colistin. A proof-of-concept was achieved in co-cultures of primary human neutrophils and Escherichia coli that induced the secretion of NE, the release of free colistin, and an antibacterial efficacy that was equal to that of free colistin., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Colistin is widely used in the treatment of nosocomial infections caused by carbapenem-resistant gram-negative bacilli (CR-GNB). Colistin-induced nephrotoxicity is one of the major adverse reactions during colistin treatment. Comparisons of colistin-induced nephrotoxicity between different formulations of colistin are rarely reported., Methods: In this retrospective cohort study, we enrolled intensive care unit-admitted patients if they had culture isolates of CR-GNB and underwent intravenous treatment with colistin. The occurrence of acute kidney injury (AKI) during intravenous treatment with colistin was recorded. The occurrence of colistin-induced nephrotoxicity was compared between two formulations of colistin, Locolin®, and Colimycin®. Treatment outcomes associated with the occurrence of colistin-induced nephrotoxicity were also investigated., Results: Among 195 patients, 95 who were treated with Locolin® and 100 who were treated with Colimycin® were included for analysis. Patients treated with Locolin® had a higher rate of occurrence of stage 2 (46.3% vs. 32%, p = 0.040) and stage 3 (29.5% vs. 13%, p = 0.005) AKI than did those treated with Colimycin®. In multivariate analysis, the presence of septic shock (adjusted odds ratio [aOR] 2.17, 95% confidence interval [CI] 1.10-4.26) and inappropriate colistin dosage (aOR 2.52, 95% CI 1.00-6.33) were clinical factors associated with colistin-induced nephrotoxicity. Treatment with Colimycin® was an independent factor associated with a lower risk of colistin-induced nephrotoxicity (aOR 0.37, 95% CI 0.18-0.77). The mortality rate was comparable between patients with and without colistin-induced nephrotoxicity., Conclusions: The risk of colistin-induced nephrotoxicity significantly varied in different formulations of colistin in critically ill patients. Colistin-induced nephrotoxicity was not associated with increased mortality rate., (© 2021. The Author(s).)

Current cystic fibrosis (CF) treatment strategies are primarily focused on oral/inhaled anti-inflammatories and antibiotics, resulting in a considerable treatment burden for CF patients. Therefore, combination treatments consisting of anti-inflammatories with antibiotics could reduce the CF treatment burden. However, there is an imperative need to understand the potential drug-drug interactions of these combination treatments to determine their efficacy. Thus, this study aimed to determine the interactions of the anti-inflammatory agent Ibuprofen with each of the CF-approved inhaled antibiotics (Tobramycin, Colistin and its prodrug colistimethate sodium/Tadim) and anti-bacterial and anti-inflammatory efficacy. Chemical interactions of the Ibuprofen:antibiotic combinations were elucidated using High-Resolution Mass-Spectrometry (HRMS) and 1 H NMR. HRMS showed pairing of Ibuprofen and Tobramycin, further confirmed by 1 H NMR whilst no pairing was observed for either Ibuprofen:Colistin or Ibuprofen:Tadim combinations. The anti-bacterial activity of the combinations against Pseudomonas aeruginosa showed that neither paired nor non-paired Ibuprofen:antibiotic therapies altered the anti-bacterial activity. The anti-inflammatory efficacy of the combination therapies was next determined at two different concentrations (Low and High) using in vitro models of NuLi-1 (healthy) and CuFi-1 (CF) cell lines. Differential response in the anti-inflammatory efficacy of Ibuprofen:Tobramycin combination was observed between the two concentrations due to changes in the structural conformation of the paired Ibuprofen:Tobramycin complex at High concentration, confirmed by 1 H NMR. In contrast, the non-pairing of the Ibuprofen:Colistin and Ibuprofen:Tadim combinations showed a significant decrease in IL-8 secretion at both the concentrations. Importantly, all antibiotics alone showed anti-inflammatory properties, highlighting the inherent anti-inflammatory properties of these antibiotics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The increase in the use of antimicrobials such as colistin for the treatment of infectious diseases has led to the appearance of Aeromonas strains resistant to this drug. However, resistance to colistin not only occurs in the clinical area but has also been determined in Aeromonas isolates from the environment or animals, which has been determined by the detection of mcr genes that confer a resistance mechanism to colistin. The variants mcr-1 , mcr-3 , and mcr-5 have been detected in the genus Aeromonas in animal, environmental, and human fluids samples. In this article, an overview of the resistance to colistin in Aeromonas is shown, as well as the generalities of this molecule and the recommended methods to determine colistin resistance to be used in some of the genus Aeromonas .

Attachment of polysaccharide carriers is increasingly being used to achieve precision delivery and improved effectiveness of protein and peptide drugs. Although it is clear that their clinical effectiveness relies on the purity and integrity of the conjugate in storage, as well as following administration, instability of polysaccharide-based conjugates can reduce the protective efficacy of the polymer, which may adversely affect the bioactive's potency. As a model, these studies used dextrin-colistin conjugates, with varying degrees of polymer modification (1, 2.5 and 7.5 mol% succinoylation) to assess the effect of storage temperature (- 20, 4, 21 and 37 °C) and duration (up to 12 months) on saccharide and colistin release and antimicrobial activity. Estimation of the proportion of saccharide release (by comparison of area under the curve from size exclusion chromatograms) was more pronounced at higher temperatures (up to 3 and 35% at - 20 °C and 37 °C, respectively after 12 months), however, repeated freeze-thaw did not produce any measurable release of saccharides, while addition of amylase (20, 100, 500 IU/L) caused rapid release of saccharides (> 70% total within 24 h). At all temperatures, conjugates containing the lowest degree of succinoylation released the highest proportion of free colistin, which increased with storage temperature, however no trend in saccharide release was observed. Despite the clear physical effects of prolonged storage, antimicrobial activity of all samples was only altered after storage at 37 °C for 12 months (> threefold decreased activity). These results demonstrate significant release of saccharides from dextrin-colistin conjugates during prolonged storage in buffered solution, especially at elevated temperature, which, in most cases, did not affect antimicrobial activity. These findings provide vital information about the structure-activity relationship of dextrin-colistin conjugates, prior to full-scale commercial development, which can subsequently be applied to other polysaccharide-protein and -peptide conjugates.

An evolving family of mobile colistin resistance (MCR) enzymes is threatening public health. However, the molecular mechanism by which the MCR enzyme as a rare member of lipid A-phosphoethanolamine (PEA) transferases gains the ability to confer phenotypic colistin resistance remains enigmatic. Here, we report an unusual example that genetic duplication and amplification produce a functional variant (Ah762) of MCR-3 in certain Aeromonas species. The lipid A-binding cavity of Ah762 is functionally defined. Intriguingly, we locate a hinge linker of Ah762 (termed Linker 59) that determines the MCR. Genetic and biochemical characterization reveals that Linker 59 behaves as a facilitator to render inactive MCR variants to regain the ability of colistin resistance. Along with molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC) suggests that this facilitator guarantees the formation of substrate phosphatidylethanolamine (PE)-accessible pocket within MCR-3-like enzymes. Therefore, our finding defines an MCR-3 inside facilitator for colistin resistance., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyses phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calculations on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via transfer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an additional Zn2+. The calculations suggest the involvement of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addition. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases.

Purposes: To evaluate the effects of component contents in different colistin methanesulfonate (CMS) formulas on their clinical pharmacokinetics of the prodrug CMS and the formed colistin., Methods: Two CMS formulas (CTTQ and Parkedale) were investigated in a single dose, randomized, open-label, crossover study conducted in 18 healthy Chinese subjects. Both CMS formulas met the requirements of European Pharmacopoeia 9.2 with 12.1% difference in the two major active components (CMS A and CMS B). The PK parameters after a single intravenous infusion of CMS at 2.5 mg/kg were calculated and the steady-state plasma colistin concentrations (C ss,avg ) following multiple dosing, once every 12 h for 7 days, were simulated with the non-compartment model., Results: The systemic exposure (AUC 0-inf ) of CMS were 59.49 ± 5.90 h·μg/mL and 51.09 ± 4.70 h·μg/mL, and the AUC 0-inf of colistin were 15.39 ± 2.63 h·μg/mL and 12.36 ± 2.10 h·μg/mL for CTTQ and Parkedale, respectively. The ratios (90% CI) of geometric mean of AUC 0-inf of CTTQ to Parkedale were 116.38% (112.95%, 119.91%) and 124.49% (120.76%, 128.35%) for CMS and colistin, respectively. The predicted C ss,avg (95% CI) were 0.92 (0.85, 0.99) μg/mL and 0.74 (0.69, 0.79) μg/mL for CTTQ and Parkedale, respectively., Conclusion: The difference in component content in the two CMS formulas had a significant (P < 0.001) impact on the systemic exposure of colistin in human, thus, warranted essential considerations in clinical applications.

Bacterial resistance has become a major global concern, affecting about 500, 000 individuals in 22 countries. Thus, it is clear that Gram-negative bacteria have been receiving more attention in this scenario. These bacteria perform several resistance mechanisms, such as modifying lipid A from lipopolysaccharides as a product of the mcr-1 gene expression. This gene was initially identified in animals; however, it quickly spread to humans, spreading to 70 countries. Mcr-1 gene attributes resistance to polymyxin B and colistin, which are drugs established as the last alternative to combat Enterobacteriaceae bacteria. Notwithstanding the prevalence and lack of antibiotic therapies for such bacteria, this article aimed to compile information about natural compounds against the resistance attributed by this gene, including the activity of isolated colistin or its associations with other antibiotics. Among the studies that evaluated colistin's synergistic action with other compounds, azidothymidine and isoalantholactone stood out. On the other hand, the paenipeptin 1 analog showed satisfactory activities when associated with other antibiotics. Besides, it is worth mentioning that molecular docking results between ostole and eugenol toward phosphoethanolamine transferase MCR-1 revealed that these compounds could interact with critical amino acid residues for the catalytic action of this enzyme. Based on this, natural agents' role is evident against infections caused by mcr-1-positive bacteria, directly contributing to the development of new effective pharmacotherapies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Treatment of patients affected by severe burns is challenging, especially due to the high risk of Pseudomonas infection. In the present work, we have generated a novel model of bioartificial human dermis substitute by tissue engineering to treat infected wounds using fibrin-agarose biomaterials functionalized with nanostructured lipid carriers (NLCs) loaded with two anti-Pseudomonas antibiotics: sodium colistimethate (SCM) and amikacin (AMK)., Results: Results show that the novel tissue-like substitutes have strong antibacterial effect on Pseudomonas cultures, directly proportional to the NLC concentration. Free DNA quantification, WST-1 and Caspase 7 immunohistochemical assays in the functionalized dermis substitute demonstrated that neither cell viability nor cell proliferation were affected by functionalization in most study groups. Furthermore, immunohistochemistry for PCNA and KI67 and histochemistry for collagen and proteoglycans revealed that cells proliferated and were metabolically active in the functionalized tissue with no differences with controls. When functionalized tissues were biomechanically characterized, we found that NLCs were able to improve some of the major biomechanical properties of these artificial tissues, although this strongly depended on the type and concentration of NLCs., Conclusions: These results suggest that functionalization of fibrin-agarose human dermal substitutes with antibiotic-loaded NLCs is able to improve the antibacterial and biomechanical properties of these substitutes with no detectable side effects. This opens the door to future clinical use of functionalized tissues.

Purpose: This study aims to understand the impact of spray drying nozzles on particle surface composition and aerosol stability., Methods: The combination formulations of colistin and azithromycin were formulated by 2-fluid nozzle (2 N) or 3-fluid (3 N) spray drying in a molar ratio of 1:1. A 3-factor, 2-level (2 3 ) factorial design was selected to investigate effects of flow rate, inlet temperature and feed concentration on yield of spray drying and the performance of the spray dried formulations for the 3 N., Results: FPF values for the 2 N formulation (72.9 ± 1.9% for azithromycin & 73.4 ± 0.8% for colistin) were higher than those for the 3 N formulation (56.5 ± 3.8% for azithromycin & 55.1 ± 1.6% for colistin) when stored at 20% RH for 1 day, which could be attributed to smaller physical size for the 2 N. There was no change in FPF for both drugs in the 2 N formulation after storage at 75% RH for 90 days; however, there was a slight increase in FPF for colistin in the 3 N formulation at the same storage conditions. Surface enrichment of hydrophobic azithromycin was measured by X-ray photoelectron spectroscopy for both 2 N and 3 N formulations and interactions were studied using FTIR., Conclusions: The 3-fluid nozzle provides flexibility in choosing different solvents and has the capability to spray dry at higher feed solid concentrations. This study highlights the impact of hydrophobic azithromycin enrichment on particle surface irrespective of the nozzle type, on the prevention of moisture-induced deterioration of FPF for hygroscopic colistin.

Sodium colistimethate (SCM) and amikacin (AMK) are among the few antibiotics effective against resistant P. aeruginosa, K. pneumoniae and A. baumannii; however, their toxicity severely limits their use. Enclosing antibiotics into nanostructured lipid carriers (NLC) might decrease drug toxicity and improve antibiotic disposition. In this work, SCM or AMK was loaded into different NLC formulations, through high pressure homogenization, and their in vitro and in vivo effectiveness was analyzed. The encapsulation process did not reduce drug effectiveness since in vitro SCM-NLC and AMK-NLC drug activity was equal to that of the free drugs. As cryoprotectant, trehalose showed better properties than dextran. Instead, positive chitosan coating was discarded due to its limited cost-efficiency. Finally, the in vivo study in acute pneumonia model revealed that intraperitoneal administration was superior to the intramuscular route and confirmed that (-) SCM-NLC with trehalose, was the most suitable formulation against an extensively drug-resistant A. baumannii strain., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The prevalence of extensively and pandrug-resistant strains of Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB&#95;AbaP&#95;PMK34). This phage shows fast adsorption and lacks virulence genes; nonetheless, its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8-kb genome (50 open reading frames), encoding an endolysin (LysMK34) with potent muralytic activity (1,499.9 ± 131 U/μM), a typical mesophilic thermal stability up to 55°C, and a broad pH activity range (4 to 10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. The addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are resensitized in both Mueller-Hinton broth and 50% human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic. IMPORTANCE A. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last-resort antibiotic, and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure-dependent intrinsic antibacterial activity, providing new insights in the mode of action of lysins with intrinsic activity against Gram-negative bacteria., (Copyright © 2020 American Society for Microbiology.)

Previous studies have shown that combining colistin (Col), a cationic polypeptide antibiotic, with ivacaftor (Iva), a cystic fibrosis (CF) drug, could achieve synergistic antibacterial effects against Pseudomonas aeruginosa . The purpose of this study was to develop dry powder inhaler formulations for co-delivery of Col and Iva, aiming to treat CF and lung infection simultaneously. In order to improve solubility and dissolution for the water-insoluble Iva, Iva was encapsulated into bovine serum albumin (BSA) nanoparticles (Iva-BSA-NPs). Inhalable composite microparticles of Iva-BSA-NPs were produced by spray-freeze-drying using water-soluble Col as the matrix material and l-leucine as an aerosol enhancer. The optimal formulation showed an irregularly shaped morphology with fine particle fraction (FPF) values of 73.8 ± 5.2% for Col and 80.9 ± 4.1% for Iva. Correlations between " D × ρ tapped " and FPF were established for both Iva and Col. The amorphous solubility of Iva is 66 times higher than the crystalline solubility in the buffer. Iva-BSA-NPs were amorphous and remained in the amorphous state after spray-freeze-drying, as examined by powder X-ray diffraction. In vitro dissolution profiles of the selected DPI formulation indicated that Col and Iva were almost completely released within 3 h, which was substantially faster regarding Iva release than the jet-milled physical mixture of the two drugs. In summary, this study developed a novel inhalable nanocomposite microparticle using a synergistic water-soluble drug as the matrix material, which achieved reduced use of excipients for high-dose medications, improved dissolution rate for the water-insoluble drug, and superior aerosol performance.

Drug-resistant bacteria are rapidly becoming a significant problem across the globe. One element that factors into this crisis is the role played by bacterial biofilms in the recalcitrance of some infections to the effects of conventional antibiotics. Bacteria within a biofilm are highly tolerant of both antibiotic treatment and host immune responses. Biofilms are implicated in many chronic infections, including tuberculosis, in which they can act as bacterial reservoirs, requiring an arduous antibiotic regimen to eradicate the infection. A separate, compounding problem is that antibiotics once seen as last-resort drugs, such as the polymyxin colistin, are now seeing more frequent usage as resistance to front-line drugs in Gram-negative bacteria becomes more prevalent. The increased use of such antibiotics inevitably leads to an increased frequency of resistance. Drugs that inhibit biofilms and/or act as adjuvants to overcome resistance to existing antibiotics will potentially be an important component of future approaches to antibacterial treatment. We have previously demonstrated that analogues of the meridianin natural product family possess adjuvant and antibiofilm activities. In this study, we explore structural variation of the lead molecule from previous studies, and identify compounds showing both improved biofilm inhibition potency and synergy with colistin., (© 2020 Wiley-VCH GmbH.)

Naturally occurring linear polyamines are known to enable bacteria to be resistant to cationic membrane active peptides. To understand this protective mechanism, molecular dynamics simulations are employed to probe their effect on a model bacterial outer membrane. Being protonated at physiological pH, the amine groups of the polyamine engage in favorable electrostatic interactions with the negatively charged phosphate groups of the membrane. Additionally, the amine groups form large number of hydrogen bonds with the phosphate groups. At high concentrations, these hydrogen bonds and the electrostatic network can non-covalently crosslink the lipid A molecules, resulting in stabilization of the outer membrane against membrane active antibiotics such as colistin and polymyxin B. Moreover, large polyamine molecules (e.g., spermidine) have a stronger stabilization effect than small polyamine molecules (e.g., ethylene diamine). The atomistic insights provide useful guidance for the design of next generation membrane active amine-rich antibiotics, especially to tackle the growing threat of multi-drug resistance of Gram negative bacteria., 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. Chandra S Verma is founder director of SiNOPSEE Therapeutics; Roger Beuerman is founder of SINSA Labs, this work has no conflict with the two companies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Antibiotic resistance is a growing global challenge to public health. Polymyxin is considered to be the last-resort antibiotic against most gram-negative bacteria. Recently, discoveries of a plasmid-mediated, transferable mobilized polymyxin resistance gene (mcr-1) in many countries have heralded the increased threat of the imminent emergence of pan-drug-resistant super bacteria. MCR-1 is an inner membrane protein that enables bacteria to develop resistance to polymyxin by transferring phosphoethanolamine to lipid A. However, the mechanism associated with polymyxin resistance has yet to be elucidated, and few drugs exist to address this issue. Here, we review our current understanding regarding MCR-1 and small molecule inhibitors to provide a detailed enzymatic mechanism of MCR-1 and the associated implications for drug design.

A novel UHPLC-MS/MS method for the determination of polypeptide antibiotic residues in animal muscle, milk, and eggs was developed and validated. Bacitracin A, colistin A, colistin B, polymyxin B1, and polymyxin B2 were extracted from the samples with a mixture of acetonitrile/water/ammonia solution 25%, 80/10/10 ( v / v / v ), and put through further evaporation, reconstitution, and filtration steps. The chromatographic separation was performed on a C18 column in gradient elution mode. Mass spectral acquisitions were performed in selective multiple reaction monitoring mode by a triple quadrupole mass spectrometer. The method was validated according to the criteria of Commission Decision 2002/657/EC. The method quantifies polypeptides in a linear range from 10 to 1000 μg kg -1 , where the lowest concentration on the calibration curve refers to the limit of quantification (LOQ). The recoveries ranged from 70 to 99%, the repeatability was below 13%, and within-laboratory reproducibility was lower than 15%. The decision limit (CCα) and detection capability (CCβ) values were calculated, and ruggedness and stability studies were performed, to fulfill the criteria for confirmatory methods. Moreover, the developed method may also be used for screening purposes by its labor efficiency.

MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the "last-resort" antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.

Gram-negative bacteria are a significant cause of infections acquired in both hospital and community settings, resulting in a high mortality rate worldwide. Currently, a Gram-negative infection is diagnosed by symptom evaluation and is treated with empiric antibiotics which target both Gram-negative and Gram-positive bacteria. A rapid and simple diagnostic method would enable immediate and targeted treatment, while dramatically reducing antibiotic overuse. Herein, we introduce a method utilizing a fluorescent derivative of colistin (COL-FL), that can directly label the Gram-negative cell wall of live bacteria and universally detect the targets within 10 min. By using the COL-FL assay, we achieved the differential labeling of various Gram-negative pathogens related to hospital-acquired infections, which could be subsequently detected via spectrofluorometry and microscopy. Further, we determined that our method can be used for complex samples, such as combinations of multiple bacterial types; bacteria in the presence of mammalian cells; and bacteria with serum components. This assay can be integrated into a simple diagnostic platform for rapid screening tests and the stratification of Gram-negative bacterial infections in the clinic.