Polymyxins are often administered as last resort antibiotics for Gram-negative bacterial infections. However, given unwanted side-effects, efficacy and recent reports of resistance against polymyxins, there is an urgency to develop alternatives. This necessitates an understanding of how polymyxins associate with and translocate across the formidable permeability barrier of the Gram-negative bacterial outer membrane. We employ multi-scale molecular simulations to explore the initial association of polymyxin B1 using E. coli outer membrane models that incorporate the latest details of supramolecular lattice networks formed by lipids and native proteins. We show that polymyxin molecules attach to the outer membrane surface, reducing the lateral displacement of proteins and lipopolysaccharides, and polymyxins often associate into large protein-polymyxin aggregates that link individual proteins. Furthermore, we provide atomistic resolution insights into the interaction network between proteins, lipopolysaccharides and polymyxins that lead to the reduced lateral mobility of proteins and lipopolysaccharides in the E. coli outer membrane.

Development of an LC-MS/MS method for quantification of colistin and colistin methanesulfonate in human plasma and its application to stability studies and therapeutic drug monitoring.

Abstract Macrocycles have emerged as important new modalities in drug discovery. In the context of addressing the global threat of antimicrobial resistance, here we used a genetic algorithm as a computational tool to evolve peptide‐peptoid macrocycles to resemble polymyxin B2 (PMB2), a macrocyclic lipopeptide natural product used as last resort antibiotic. Synthesis and testing of 41 PMB2 analogs revealed several peptide‐peptoid macrocycles showing strong, although salt sensitive, activity against Escherichia coli and multidrug‐resistant strains of Pseudomonas aeruginosa, high serum stability, and lower toxicity to kidney cells compared to PMB2. These macrocycles resembled PMB2 in terms of outer membrane permeabilization, inner membrane depolarization, lipopolysaccharide binding, and loss of activity when linearized, but, unlike PMB2, induced aggregation of intracellular contents, an effect was reported for other antimicrobial peptoids. These experiments exemplify a combined computational and experimental approach which might be generally useful to explore the chemical space of macrocyclic peptide natural products.

Macrocycles have emerged as important new modalities in drug discovery. In the context of addressing the global threat of antimicrobial resistance, here we used a genetic algorithm as a computational tool to evolve peptide-peptoid macrocycles to resemble polymyxin B2 (PMB2), a macrocyclic lipopeptide natural product used as last resort antibiotic. Synthesis and testing of forty-one PMB2 analogs revealed several peptide-peptoid macrocycles showing strong, although salt sensitive, activity against Escherichia coli and multidrug-resistant strains of Pseudomonas aeruginosa, high serum stability and lower toxicity to kidney cells compared to PMB2. These macrocycles resembled PMB2 in terms of outer membrane permeabilization, inner membrane depolarization, lipopolysaccharide binding, and loss of activity when linearized, but unlike PMB2 induced aggregation of intracellular contents, an effect reported for other antimicrobial peptoids. These experiments exemplify a combined computational and experimental approach which might be generally useful to explore the chemical space of macrocyclic peptide natural products.

Development and validation of a UPLC-MS/MS method for simultaneous quantification of polymyxins and caspofungin in human plasma for therapeutic drug monitoring.

To facilitate clinical therapeutic drug monitoring (TDM) of polymyxin B (PB) and polymyxin E (PE), we developed and validated a simple LC-MS/MS method for simultaneous determination of PB (including polymyxin B1 (PB1), polymyxin B2 (PB2), polymyxin B3 (PB3) and isoleucine-polymyxin B1 (ile-PB1)) and PE (including polymyxin E1 (PE1) and polymyxin E2 (PE2)) in human plasma. PB or PE was extracted from 20.0μL plasma using a 5% (v/v) formic acid acetonitrile solution and separated on a BEH-C18 column (2.1 × 100 mm, 1.7μm) with a mobile phase consisting of 0.8% formic acid aqueous solution and 0.2% formic acid acetonitrile solution. Gradient elution was performed over 5.5min at a flow rate of 0.250 mL/min. Quantitative analysis was conducted in positive ion scanning mode by electrospray ionization and multiple reaction monitoring. The method validation was conducted based on bioanalytical method validation guidance, including specificity, calibration curve, precision, accuracy, recovery, matrix effect, stability and dilution integrity and all of the results satisfied the requirements. The method was simple, robust and high-throughput and is currently being used to provide a TDM service to enhancing therapeutic efficacy and safety use of the PB and PE.

The rise in multi-drug resistant Gram-negative bacterial infections has led to an increased need for 'last-resort' antibiotics such as polymyxins. However, the emergence of polymyxin-resistant strains threatens to bring about a post-antibiotic era. Thus, there is a need to develop new polymyxin-based antibiotics, but a lack of knowledge of the mechanism of action of polymyxins hinders such efforts. It has recently been suggested that polymyxins induce cell lysis of the Gram-negative bacterial inner membrane (IM) by targeting trace amounts of lipopolysaccharide (LPS) localized there. We use multiscale molecular dynamics (MD) including long-timescale coarse-grained (CG) and all-atom (AA) simulations to investigate the interactions of polymyxin B1 (PMB1) with bacterial IM models containing phospholipids (PLs), small quantities of LPS, and IM proteins. LPS was observed to (transiently) phase separate from PLs at multiple LPS concentrations, and associate with proteins in the IM. PMB1 spontaneously inserted into the IM and localized at the LPS-PL interface, where it cross-linked lipid headgroups via hydrogen bonds, sampling a wide range of interfacial environments. In the presence of membrane proteins, a small number of PMB1 molecules formed interactions with them, in a manner that was modulated by local LPS molecules. Electroporation-driven translocation of PMB1 via water-filled pores was favored at the protein-PL interface, supporting the 'destabilizing' role proteins may have within the IM. Overall, this in-depth characterization of PMB1 modes of interaction reveals how small amounts of mislocalized LPS may play a role in pre-lytic targeting and provides insights that may facilitate rational improvement of polymyxin-based antibiotics.

Introduction: Polymyxin B is widely used to treat infections caused by multidrug-resistant Gram-negative bacteria. However, the pharmacokinetic study data of PB in the elderly are scarce. Herein, a simple method to measure the concentration of PB in human plasma was developed and validated by high performance liquid chromatography-tandem mass spectrometry, and it was applied to a PK study in the elderly. Methods: PB was extracted from human plasma by a rapid protein-precipitation method using 0.1% formic acid in methanol and then separated on an ultimate AQ-C18 column using linear gradient elution with a 0.5-mL/min flow rate. Subsequently, PB was detected using a mass spectrometer operated in positive-ion and multiple-reaction-monitoring modes. Results: The lower limits of quantification of the method for Polymyxin B1 and Polymyxin B2 were 1.00 and 0.10μg/mL, respectively. The linear ranges for PB1 and PB2 were 1.00-20.02 and 0.10-2.04μg/mL, respectively. Patients receiving a 75-mg maintenance dose every 12h had AUCss, 24h, and Css, av values of 117.70 ± 37.03μg h/mL and 4.14 ± 1.74μg/mL, respectively. For patients receiving a 100mg maintenance dose, these values were 152.73 ± 70.09μg h/mL and 5.43 ± 2.85μg/mL, respectively. Conclusion: The validated HPLC-MS/MS method was successfully applied to a study on the pharmacokinetics of PB in elderly patients infected with multidrug-resistant Gram-negative bacteria. Both two dose strategies in this study would have a excessive PB exposure in the elderly patients then the therapeutic window recommended by guidelines.

Combinatorial metabolic engineering of Bacillus subtilis for de novo production of polymyxin B

Extremophiles are known to produce antibiotics. This study aimed to isolate and purify polypeptide antibiotics from Pria Laot Sabang (PLS) 76, a bacterium isolated from an undersea fumarole. The antibiotic production was done by cultivating PLS 76 in a 2.5% TSB liquid medium and incubated at 70°C, 150 rpm for 112 hours. Ninhydrin assay on the crude supernatant after centrifugation indicated that the PLS 76 produced a polypeptide antibiotic. The supernatant was partitioned using methanol. The antibiotic activity was tested using the Kirby-Bauer method, and the inhibition zone was about 12 mm against Escherichia coli and Staphylococcus aureus. The methanol fraction was purified further by Preparative Thin Layer Chromatography and produced several fractions. Fractions with the same Rf were pooled and subjected to the ninhydrin assay. Fraction A, which gave a positive result after the ninhydrin assay, showed an inhibition zone of only 7 mm against E. coli and S. aureus. The Quadrupole Time-of-Flight (QTOF) Mass Spectrometry data indicated that the antibiotic's structure was similar to Polymyxin B2, an antibiotic of a polypeptide group.

In order to investigate the potential mechanisms of probiotic-fermented coconut water in treating enteritis, this study conducted a comprehensive analysis of the effects of probiotic intervention on the recovery from Dextran Sodium Sulfate-induced acute enteritis in Wenchang chicks. The analysis encompassed the assessment of growth performance, serum indicators, intestinal tissue structure, and metagenomic and metabolomic profiles of cecal contents in 60 Wenchang chicks subjected to intervention. This approach aimed to elucidate the impact of probiotic intervention on the recovery process from acute enteritis at both the genetic and metabolic levels in the avian model. The results revealed that intervention with Saccharomyces cerevisiae Y301 improved the growth rate of chicks. and intervention with Lactiplantibacillus plantarum MS2c regulated the glycerophospholipid metabolism pathway and reshaped the gut microbiota structure in modeling chicks with acute enteritis, reducing the abundance of potentially pathogenic bacteria from the Alistipes and increasing the abundance of potentially beneficial species from the Christensenellaceae. This intervention resulted in the production of specific gut metabolites, including Gentamicin C and polymyxin B2, recognized for their therapeutic effects on acute enteritis. The combined intervention of S. cerevisiae Y301 and L. plantarum MS2c not only enhanced growth performance but also mitigated intestinal wall damage and increased the abundance of gut metabolites such as gentamicin C and polymyxin B2, thereby mitigating symptoms of enteritis. Furthermore, this combined intervention reduced the levels of serum immune markers, including IL-10, IL-6, TNF-α, IFN-γ, and D-lactic acid, thus mitigating intestinal epithelial cell damage and promoting acute enteritis recovery. This study provides crucial insights into the mechanisms of action of probiotics and probiotic-fermented coconut water in acute enteritis recovery, offering new perspectives for sustainable farming practices for Wenchang chicken.

Enhancement of polymyxin B1 production by an artificial microbial consortium of Paenibacillus polymyxa and recombinant Corynebacterium glutamicum producing precursor amino acids

Simple and robust LC–MS/MS method for quantification of colistin methanesulfonate and colistin in human plasma for therapeutic drug monitoring

Kiwifruit bacterial canker (KBC) caused by Pseudomonas syringae pv. actinidiae (Psa) is the main limiting factor in the kiwifruit industry. This study aimed to identify bacterial strains with antagonistic activity against Psa, analyze antagonistically active substances, and provide a new basis for the biological control of KBC. A total of 142 microorganisms were isolated from the rhizosphere soil of asymptomatic kiwifruit. Among them, an antagonistic bacterial strain was identified as Paenibacillus polymyxa YLC1 by 16S rRNA sequencing. KBC control by strain YLC1 (85.4%) was comparable to copper hydroxide treatment (81.8%) under laboratory conditions and field testing. Active substances of strain YLC1 were identified by genetic sequence analysis using antiSMASH. The six biosynthetic active compound gene clusters that were identified as encoding ester peptide synthesis, such as polymyxins. An active fraction was purified and identified as polymyxin B1 using chromatography, hydrogen nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry. In addition, polymyxin B1 was also found significantly to suppressed the expression of T3SS-related genes but did not affect the growth of Psa at low concentrations. In this study, a biocontrol strain P. polymyxa YLC1 obtained from kiwifruit rhizosphere soil exhibited excellent control effects on KBC in vitro and in field tests. Its active compound was identified as polymyxin B1, which inhibits a variety of pathogenic bacteria. We conclude that P. polymyxa YLC1 is a biocontrol strain with excellent prospects for development and application. This article is protected by copyright. All rights reserved.

Dynamic covalent nano-networks comprising antibiotics and polyphenols orchestrate bacterial drug resistance reversal and inflammation alleviation

Polymyxin B (PB) is a dose-dependent drug used to treat multidrug-resistantgram-negative bacteria, for which a suitable method is needed to determine clinical samples. A simple, economical, and efficient high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method was developed and validated for polymyxin B1 (PB1), polymyxin B1-Ile (PB1-I), polymyxin B2 (PB2), and polymyxin B3 (PB3) in human plasma. Chromatographic column was Waters BEH C18 column (2.1 × 50 mm, 1.7 μm). Phase A was water with 0.2% formic acid (FA), and phase B was acetonitrile containing 0.2% FA. The elution method is gradient elutio. The total analysis time was 5 min. The pretreatment method involved protein precipitation using acetonitrile containing 0.2% trifluoroacetic acid and 0.1% FA as the precipitant. The recovery rate was 92–99%. The total quantity of PB1 and PB1-I was measured in the linear range of 100–8000 ng/mL. Simultaneously, the total amounts of PB2 and PB3 were measured in the linear range of 11.9–948.5 ng/mL. This validated method was successfully applied to the pharmacokinetics of PB in critically ill patients.

Polymyxin B is a "last-line-of-defense" antibiotic approved in the 1960s. However, the population pharmacokinetics (PK) of its four main components has not been reported in infected mice. We aimed to determine the PK of polymyxin B1, B1-Ile, B2, and B3 in a murine bloodstream and lung infection model of Acinetobacter baumannii and develop humanized dosage regimens. A linear 1-compartment model, plus an epithelial lining fluid (ELF) compartment for the lung model, best described the PK. Clearance and volume of distribution were similar among the four components. The bioavailability fractions were 72.6% for polymyxin B1, 12.0% for B1-Ile, 11.5% for B2, and 3.81% for B3 for the lung model and were similar for the bloodstream model. While the volume of distribution was comparable between both models (17.3 mL for the lung and ~27 mL for the bloodstream model), clearance was considerably smaller for the lung (2.85 mL/h) compared to that of the bloodstream model (5.59 mL/h). The total drug exposure (AUC) in ELF was high due to the saturable binding of polymyxin B presumably to bacterial lipopolysaccharides. However, the modeled unbound AUC in ELF was ~16.7% compared to the total drug AUC in plasma. The long elimination half-life (~4 h) of polymyxin B enabled humanized dosage regimens with every 12 h dosing in mice. Daily doses that optimally matched the range of drug concentrations observed in patients were 21 mg/kg for the bloodstream and 13 mg/kg for the lung model. These dosage regimens and population PK models support translational studies for polymyxin B at clinically relevant drug exposures.

Determination of polymyxin B in human plasma and epithelial lining fluid using LC-MS/MS and its clinical application in therapeutic drug monitoring

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 B1 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.

Interactions of polymyxin B1 with the gram-negative inner membrane: A simulation study.