Development of dilipid polymyxins: Investigation on the effect of hydrophobicity through its fatty acyl component

Continuous development of new antibacterial agents is necessary to counter the problem of antimicrobial resistance. Polymyxins are considered as drugs of last resort to combat multidrug-resistant Gram-negative pathogens. Structural optimization of polymyxins requires an in-depth understanding of its structure and how it relates to its antibacterial activity. Herein, the effect of hydrophobicity was explored by adding a secondary fatty acyl component of varying length onto the polymyxin structure at the amine side-chain of l-diaminobutyric acid at position 1, resulting to the development of dilipid polymyxins. The incorporation of an additional lipid was found to confer polymyxin activity against Gram-positive bacteria, to which polymyxins are inherently inactive against. The dilipid polymyxins showed selective antibacterial activity against Pseudomonas aeruginosa. Moreover, dilipid polymyxin 1 that consists of four carbon-long aliphatic lipids displayed the ability to enhance the antibacterial potency of other antibiotics in combination against P. aeruginosa, resembling the adjuvant activity of the well-known outer membrane permeabilizer polymyxin B nonapeptide (PMBN). Interestingly, our data revealed that dilipid polymyxin 1 and PMBN are substrates for the MexAB-OprM efflux system, and therefore are affected by efflux. In contrast, dilipid polymyxin analogs that consist of longer lipids and colistin were not affected by efflux, suggesting that the lipid component of polymyxin plays an important role in resisting active efflux. Our work described herein provides an understanding to the polymyxin structure that may be used to usher the development of enhanced polymyxin analogs.     

Structure–activity studies on polymyxin derivatives carrying three positive charges only reveal a new class of compounds with strong antibacterial activity

Recent years have brought in an increased interest to develop improved polymyxins. The currently used polymyxins, i.e. polymyxin B and colistin (polymyxin E) are pentacationic lipopeptides that possess a cyclic heptapeptide part with three positive charges, a linear “panhandle” part with two positive charges, and a fatty acyl tail. Unfortunately, their clinical use is shadowed by their notable nephrotoxicity. We have previously developed a polymyxin derivative NAB739 which lacks the positive charges in the linear part. This derivative is better tolerated than polymyxin B in cynomolgus monkeys and is, in contrast to polymyxin B, excreted into urine in monkeys and rats. Here we have conducted further structure-activity relationship (SAR) studies on 17 derivatives with three positive charges only. We discovered a remarkably antibacterial class, as exemplified by NAB815, that carries two positive charges only in the cyclic part.     

Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa

The two-component regulatory system PhoP-PhoQ of Pseudomonas aeruginosa regulates resistance to cationic antimicrobial peptides, polymyxin B and aminoglycosides in response to low Mg2+ conditions. We have identified a second two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides. This system responds to limiting Mg2+, and is affected by a phoQ, but not a phoP mutation. Inactivation of the pmrB sensor kinase and pmrA response regulator greatly decreased the expression of the operon encoding pmrA-pmrB while expression of the response regulator pmrA in trans resulted in increased activation suggesting that the pmrA-pmrB operon is autoregulated. Interposon mutants in pmrB, pmrA, or in an intergenic region upstream of pmrA-pmrB exhibited two to 16-fold increased susceptibility to polymyxin B and cationic antimicrobial peptides. The pmrA-pmrB operon was also found to be activated by a number of cationic peptides including polymyxins B and E, cattle indolicidin and synthetic variants as well as LL-37, a component of human innate immunity, whereas peptides with the lowest minimum inhibitory concentrations tended to be the weakest inducers. Additionally, we showed that the putative LPS modification operon, PA3552-PA3559, was also induced by cationic peptides, but its expression was only partially dependent on the PmrA-PmrB system. The discovery that the PmrA-PmrB two-component system regulates resistance to cationic peptides and that both it and the putative LPS modification system are induced by cationic antimicrobial peptides has major implications for the development of these antibiotics as a therapy for P. aeruginosa infections.     

Polymyxins as Novel and Safe Mucosal Adjuvants to Induce Humoral Immune Responses in Mice

There is currently an urgent need to develop safe and effective adjuvants for enhancing vaccine-induced antigen-specific immune responses. We demonstrate here that intranasal immunization with clinically used polypeptide antibiotics, polymyxin B (PMB) and colistin (CL), along with ovalbumin (OVA), increases OVA-specific humoral immune responses in a dose-dependently manner at both mucosal and systemic compartments. Enhanced immunity by boosting was found to persist during 8 months of observation. Moreover, mice intranasally immunized with OVA plus various doses of PMB or CL showed neither inflammatory responses in the nasal cavity and olfactory bulbs nor renal damages, compared to those given OVA alone. These data suggest that polymyxins may serve as novel and safe mucosal adjuvants to induce humoral immune responses. The polymyxin adjuvanticity was found to be independent of endotoxins liberated by its bactericidal activity, as indicated by similar enhancing effects of PMB in lipopolysaccharide (LPS)-hyporesponsive and LPS-susceptible mice. However, despite the presence of preexisting anti-PMB antibodies, we observed no reduction in the adjuvant function of polymyxins when they were given intranasally. Furthermore, the titers of OVA-specific Abs in mice intranasally immunized with OVA plus PMB or CL were significantly higher than those in mice administered with polymyxin analogues, such as polymyxin B nonapeptide and colistin methanesulfonate. The levels of released β-hexosaminidase and histamine in mast cell culture supernatants stimulated by PMB or CL were also significantly higher than those stimulated by their analogues. These results suggest that both the hydrophobic carbon chain and hydrophilic cationic cyclic peptide contribute to the mucosal adjuvanticity of PMB and CL.     

Effect of polymyxins on Bacillus polymyxa sporogenesis

Bacillus polymyxa var. Ross. producing polymyxin M and Bacillus polymyxa 153 producing polymyxin B form spores during submerged cultivation when the rate of biosynthesis of antibiotic peptides is low and when the production of antibiotics is over. However, sporogenesis is stimulated if polymyxins are added at the early stage of cultural growth. Inhibition of the synthesis of antibiotics suppresses the formation of spores. Substances other than polymyxins do not exhibit such a specific effect on sporogenesis. The fact that the culture requires endogenous polymyxins which are most effective in the period prior to the appearance of spores in the culture suggests the regulatory action of these peptides at the stage between vegetative growth and spore formation in Bacillus polymyxa.     

Farm animals and aquaculture: significant reservoirs of mobile colistin resistance genes

Colistin resistance has attracted substantial attention after colistin was considered as a last-resort drug for the treatment of infections caused by carbapenem-resistant and/or multidrug-resistant (MDR) Gram-negative bacteria in clinical settings. However, with the discovery of highly mobile colistin resistance (mcr) genes, colistin resistance has become an increasingly urgent issue worldwide. Despite many reviews, which summarized the prevalence, mechanisms, and structures of these genes in bacteria of human and animal origin, studies on the prevalence of mobile colistin resistance genes in aquaculture and their transmission between animals and humans remain scarce. Herein, we review recent reports on the prevalence of colistin resistance genes in animals, especially wildlife and aquaculture, and their possibility of transmission to humans via the food chain. This review also gives some insights into the routine surveillance, changing policy and replacement of polymyxins by polymyxin derivatives, molecular inhibitors, and traditional Chinese medicine to tackle colistin resistance.     

The activity of polymyxins against dense populations of Escherichia coli.

The activities of polymyxin B sulphate, colistin (polymyxin E) sulphate and their sulphomethyl derivatives were compared by continuous turbidimetric monitoring of dense cultures of an Escherichia coli strain exposed to these agents. Judged by the concentration of antibiotic which caused a rapid fall in opacity of the culture, polymyxin B sulphate and colistin sulphate had similar activities, but the sulphomethyl compounds differed considerably: sulphomyxin sodium induced lysis of the culture at a concentration four times that of the parent compound, whereas colistin sulphomethate sodium induced a delayed fall in opacity consistent with recruitment of activity as the inactive sulphomethyl derivative was broken down to the parent compound. Durign overnight incubation, regrowth of cultures which had initially succumbed to polymyxin action occurred, apparently due to the selection of phenotypically resistant variants from within the population. In this way cultures could easily be adapted to growth in concentrations of antibiotic well above the conventionally-determined minimum inhibitory concentration. The comparative ease of adaptation was in the order: colistin sulphomethate greater than sulphomyxin greater than colistin sulphate greater than polymyxin B sulphate.     

Novel polymyxin derivatives are less cytotoxic than polymyxin B to renal proximal tubular cells

The emergence of very multiresistant Gram-negative bacterial strains has reinstated polymyxins (polymyxin B, colistin), pentacationic lipopeptides, in the therapy, in spite of their nephrotoxicity. Extensive tubular reabsorption concentrates polymyxin in proximal tubular cells. The novel polymyxin derivatives NAB739, NAB7061 and NAB741 have their cyclic part identical to that of polymyxin B, but their side chain consists of uncharged octanoyl-threonyl-d-serinyl, octanoyl-threonyl-aminobutyryl, and acetyl-threonyl-D-serinyl respectively. In this study, we compared the toxicities of NAB739, NAB7061 and NAB741 with that of polymyxin B by using the porcine renal proximal tubular cell line LLC-PK1 electroporated or incubated with the selected compound. Both the ability to cause cell necrosis (quantified as the leakage of lactate dehydrogenase) and the ability to cause apoptosis (as quantified by counting apoptotic nuclei) were assessed. In electroporated cells, polymyxin B induced total (>85%) necrosis of the cells at 0.016 mM, whereas an approx. 8-fold concentration of NAB739 and NAB7961 and an approx. 32-fold concentration of NAB741 was required for the same effect. In cells treated without electroporation (incubated), polymyxin B elicited a marked degree (approx. 50%) of necrosis at 0.5mM, whereas the NAB compounds were inert even at 1mM. Neither polymyxin B nor the NAB compounds induced apoptosis.     

Synergism between Chlorhexidine and Polymyxins against Pseudomonas aeruginosa

Chlorhexidine acts synergistically with the polymyxins B and E causing increased and rapid release of cell contents from Pseudomonas aeruginosa. While the polymyxin target molecule is phospholipid, evidence is provided for the proposal that the primary target for Chlorhexidine action is a protein component of the cytoplasmic membrane.     

Performance of different methods for testing polymyxin B: comparison of broth microdilution,agar dilution and MIC test strip in mcr-1 positive and negative Escherichia coli

Antimicrobial susceptibility testing with the last-resort antibiotics polymyxins (polymyxin B and colistin) is associated with several methodological issues. Currently, broth microdilution (BMD) is recommended for colistin and polymyxin B. BMD is laborious and the utility of alternative methods needs to be evaluated for polymyxin B susceptibility testing. In this study, using BMD as a reference method, the performance of agar dilution (AD) and MIC test strips (MTS) were evaluated in polymyxin B susceptibility testing. BMD, AD and MTS were used to determine MICs of 193 clinical isolates of Escherichia coli. Seventy-nine were positive for the polymyxin resistance gene mcr-1. Method performances were evaluated based on pair-wise agreements with the reference method (BMD) and statistical testing. AD and MTS showed an unacceptable number of very major errors (VMEs) compared with BMD, 9·3 and 10·7%, respectively. The essential agreement (EA) was low for AD (49·7%), but high for MTS (97·8%). However, statistical testing showed that MTS tended to yield a one-step lower MIC (P < 0·01) compared with BMD. The discordances observed with MTS and AD in comparison with BMD for polymyxin B susceptibility testing for E. coli suggest their inapplicability in routine testing. A large number of isolates clustered around the susceptibility breakpoint (2–4 mg l⁻¹) and several mcr-1 positive isolates (17%) were determined as susceptible with BMD. A screening breakpoint for mcr-1 of 2 mg l⁻¹ should also be considered.     

Polymyxin E production by P. amylolyticus

AIMS: To investigate antibiotic production by bacteria isolated from the hindgut of Tipula abdominalis, the aquatic crane fly. METHODS AND RESULTS: A group of five isolates with 99.1% 16S rRNA sequence similarity to Paenibacillus amylolyticus were identified as antibacterial producers using the cross-streak method against both Gram-positive and Gram-negative bacteria. For one isolate, P. amylolyticus C27, biochemical tests were performed to confirm 16S rRNA identification and the antibacterials were purified using chromatographic methods. Postsource decay (PSD) mass spectroscopy (MS) was used to identify the antimicrobials, which were found to be polymyxins E(1) and E(2). Investigation of the remaining four isolates using PSD MS revealed they all produce polymyxins E(1) and E(2) as well. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Although variants of the polymyxin antibiotics are known to be produced by several species within the Paenibacillus genus, this first investigation of antibacterial production by bacteria isolated from the hindgut of T. abdominalis describes a novel source for polymyxin E production as well as the first report of antibiotic production by P. amylolyticus.     

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.     

分子模拟在多黏菌素药理机制研究中的应用

多黏菌素是一种膜靶向的脂肽类抗生素，是临床上治疗革兰氏阴性多重耐药菌感染的最后一道防线。通过与脂多糖相互作用，多黏菌素破坏细菌外膜结构并导致细菌死亡。然而，受限于生物化学和结构生物学手段对细胞膜-药物相互作用的表征能力，目前对多黏菌素药理机制的认识还不充分，从而限制了新一代多黏菌素药物的设计和开发。为此，本文总结了近年来利用分子动力学方法对细胞膜系统与多黏菌素相互作用的研究进展，为深入理解多黏菌素药理机制与细胞膜系统的内在联系，加快新型抗生素药物研发提供新思路。     

Susceptibility to Polymyxin B of Penicillin G-induced Proteus mirabilis L Forms and Spheroplasts

A polymyxin B-resistant strain of Proteus mirabilis was converted into L forms and spheroplasts in the presence of penicillin G. This treatment caused a 400-fold increase in polymyxin B susceptibility. The acquired susceptibility was in the range of the natural susceptibility reported for susceptible gram-negative bacteria ( approximately 1 mug/ml). The high susceptibility to polymyxin B was lost as soon as the spheroplasts and L forms were allowed to reconvert into the bacillary form in penicillin-free media. This behavior is strong evidence that the natural resistance of Proteus strains to polymyxins is due to the impermeability of the outer cell wall structures to these antibiotic substances.     

Interaction of phosphorylase kinase with polymyxins

Nonactivated rabbit skeletal muscle phosphorylase kinase is inhibited by the polymyxins A, B, D and E when assayed at pH 8.6. Polymyxin B is the most effective inhibitor, causing 50% inhibition at 0.3 mM. Following the effect of polymyxin B on the kinase activity toward troponin, no inhibition was observed. In contrast, polymyxin B was found to greatly stimulate the autophosphorylation of phosphorylase kinase. About 10 mol of phosphate per tetramer (alpha beta nu delta) were incorporated in presence of polymyxin B (full autophosphorylation). This incorporation was about 6-fold higher than that observed without polymyxin. The stimulation of autophosphorylation by polymyxin B was accompanied with enhancement of the rate of autoactivation at pH 6.8.     

Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions

Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]¹polymyxin B₃. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]¹polymyxin B₃ for LPS was comparable to polymyxin B and colistin, and considerably greater (K_d < 1 μM) than for whole cells (K_d ∼ 6–12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]¹polymyxin B₃ to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]¹polymyxin B₃–LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]¹polymyxin B₃–LPS complex due to steric hindrance from the dansyl[Lys]¹ fluorophore; this corresponded with diminished antibacterial activity (MIC ?16 μg/mL). Dansyl[Lys]¹polymyxin B₃ may prove useful as a screening tool for drug development.     

Isolation and characteristics of polymyxin-resistant mutants of Shigella flexneri

Some characteristics of S. flexneri 2a mutants resistant to various concentrations of polymyxin M were studied. The data indicate that mutations resulting in low (50 microgram/ml) and high (300 microgram/ml) levels of the antibiotic resistance were determined by different genes. Polymyxin resistance led to changed permeability of the outer membrane with respect to detergents and some antibiotics, such as aminoglycosides, penicillins, chloramphenicol and amphotericin B but did not change sensitivity of the strains to some bacteriophages, except phage PI. Mutants resistant to 50 microgram/ml of polymyxin M preserved their ability to induce keratoconjunctivitis in guinea pigs. Part of the strains resistant to 300 microgram/ml of the antibiotic lost this property. No correlation between the polymyxin M resistance level, loss of the pathogenic properties and toxicity of the bacterial cells was found. It was confirmed that though inactivation of endotoxin by polymyxins is associated with their capacity for interaction with lipid A, this component does not participate in development of resistance to these antibiotics.     

Antibiotic Susceptibilities of Serratia marcescens and Enterobacter liquefaciens

Production of 5'-nucleotides by Serratia marcescens and Enterobacter liquefaciens correlates with deoxyribonuclease production, indicating the close relationship between these two organisms. To determine further relationships, susceptibilities of 279 strains of the tribe Klebsielleae were determined by the high-potency disc method, agar-dilution method, or both, by using 14 antibiotics. Ninety-seven per cent of S. marcescens (201 of 207 strains) and 100% of E. liquefaciens (17 strains) had minimum inhibitory concentration (MIC) of 100 mug/ml or greater with colistin and polymyxin B. With these two antibiotics, 93% of other Enterobacter species (28 strains) had MIC values of less than 1.6 mug/ml, and 100% of Klebsiella (27 strains) had MIC values less than 1.6 mug/ml. Consistent patterns were not noted with the other antibiotics tested, but the results with colistin and polymyxin B provide additional evidence of the close relationship of S. marcescens and E. liquefaciens.     

Draft Genome Sequence of Paenibacillus polymyxa OSY-DF, Which Coproduces a Lantibiotic, Paenibacillin, and Polymyxin E1

Paenibacillus polymyxa OSY-DF is a Gram-positive rod-shaped bacterium isolated from a fermented vegetable food. This bacterial strain displays potent antimicrobial activities against Gram-positive and Gram-negative pathogenic bacteria, attributed to the production of the lantibiotic paenibacillin and the colistin peptide polymyxin E1. Here we report the draft genome sequence of Paenibacillus polymyxa OSY-DF.     

Interaction of antibacterial peptides spanning the carboxy-terminal region of human beta-defensins 1-3 with phospholipids at the air-water interface and inner membrane of E. coli

Synthetic peptides Phd1-3 spanning the cationic carboxy-terminal region of human beta-defensins HBD-1-3 have been shown to have antibacterial activity. Gross morphological changes were seen in E. coli cells treated with these peptides. In this paper, we have studied the surface-active properties of peptides Phd1-3 and their interactions with different phospholipids using Langmuir-Blodgett monolayers. Compression isotherms and increase in pressure on insertion of peptides into lipid monolayers at different initial pressures indicate the affinity of these peptides for negatively charged lipids. Phd3 inserted less effectively into monolayers as compared to Phd1 and Phd2. The peptides differed in their ability to permeabilize the inner membrane of E. coli, with Phd3 being least effective. It is likely that the peptides kill Gram-negative bacteria by more than one mechanism. When hydrophobicity and net charge favor insertion into lipid membranes, then membrane permeabilization could be the primary event in the killing of bacteria. In cases where membrane insertion does not occur, interaction with phospholipid interface induces highly selective stress that leads to stasis and cell death, as proposed for polymyxin B and bactenecin.