Effect of polymyxin B on ion permeability of bacterial membranes

Effect of polymyxin B on the movement of K+ and H+ in polymyxin-sensitive cells of E. coli under different metabolic states has been studied. It was shown that polymyxin B induced the efflux of K+, decreased the efflux of H+ and inhibited the consumption of oxygen in bacterial cells. The effect of antibiotic on ion movement was independent of respiratory conditions. It was suggested that polymyxin B increased ion permeability and destroyed lipid-protein interactions of the respiratory chain simultaneously.     

Lipopolysaccharide as an antibiotic target

Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are amongst the highest priority drug-resistant pathogens, for which new antibiotics are urgently needed. Whilst antibiotic drug development is inherently challenging, this is particularly true for Gram-negative bacteria due to the presence of the outer membrane, a highly selective permeability barrier that prevents the ingress of several classes of antibiotic. This selectivity is largely due to an outer leaflet composed of the glycolipid lipopolysaccharide (LPS), which is essential for the viability of almost all Gram-negative bacteria. This essentiality, coupled with the conservation of the synthetic pathway across species and recent breakthroughs in our understanding of transport and membrane homeostasis has made LPS an attractive target for novel antibiotic drug development. Several different targets have been explored and small molecules developed that show promising activity in vitro. However, these endeavours have met limited success in clinical testing and the polymyxins, discovered more than 70 years ago, remain the only LPS-targeting drugs to enter the clinic thus far. In this review, we will discuss efforts to develop therapeutic inhibitors of LPS synthesis and transport and the reasons for limited success, and explore new developments in understanding polymyxin mode of action and the identification of new analogues with reduced toxicity and enhanced activity.     

Polymyxin E Induces Rapid Paenibacillus polymyxa Death by Damaging Cell Membrane while Ca2+ Can Protect Cells from Damage

Polymyxin E, produced by Paenibacillus polymyxa, is an important antibiotic normally against Gram-negative pathogens. In this study, we found that polymyxin E can kill its producer P. polymyxa, a Gram-positive bacterium, by disrupting its cell membrane. Membrane damage was clearly revealed by detecting the leakage of intracellular molecules. The observation using scanning electron microscopy also supported that polymyxin E can destroy the cell membrane and cause an extensive cell surface alteration. On the other hand, divalent cations can give protection against polymyxin E. Compared with Mg²⁺, Ca²⁺ can more effectively alleviate polymyxin E-induced damage to the cell membrane, thus remarkably increasing the P. polymyxa survival. Our findings would shed light on a not yet described bactericidal mechanism of polymyxin E against Gram-positive bacteria and more importantly the nature of limited fermentation output of polymyxin E from P. polymyxa.     

Effect of polymyxin B on liposomal membranes derived from Escherichia coli lipids

The specificity of the action of polymyxin B was studied using liposomes as a model membrane system. Liposomes prepared from total lipids of Gram-negative bacteria Escherichia coli, a mixture of purified E. coli phosphatidylethanolamine and cardiolipin and a mixture of phosphatidylethanolamine and phosphatidylglycerol, were extremely sensitive to polymyxin while those prepared from lipids of Gram-positive bacteria Streptococcus sanguis, lipids of sheep erythrocyte membranes, mixtures of egg lecithin and negatively charged amphiphatic molecules, were less sensitive to the action of the antibiotic. Chlolesterol was shown to suppress the polymyxin-induced response in liposomes.     

Cell envelope impermeability to daptomycin inPseudomonas aeruginosa andPasteurella multocida

The exclusively gram-positive antibacterial spectrum of the lipopeptide daptomycin (LY146032) suggests that the underlying basis for intrinsic resistance in gram-negative organisms involves envelope impermeability. This study was undertaken to determine whether the outer membranes ofPseudomonas aeruginosa andPasteurella multocida can be rendered permeable to daptomycin by experimental modifications that result in susceptibility of gram-negative bacteria to lipophilic molecules. Turbidimetric growth assays revealed sublethal concentrations of polymyxin B or ethylenediaminetetraacetate (EDTA) sensitized all strains examined to the hydrophobic antibiotic novobiocin. Neither permeabilizer renderedPs. aeruginosa or a hydrophilicPa. multocida variant susceptible to daptomycin; however, polymyxin B sensitized a hydrophobicPa. multocida variant, whereas EDTA did not. Cells cultured with sublethal concentrations of polymyxin B or EDTA retained negatively charged cell surfaces comparable to those of control cells. Growth ofPa. multocida strains in the presence of polymyxin B did not result in modification of cell envelope lipid composition. These findings indicate that the ability of the outer membrane to retard the diffusion of daptomycin does not require normally intact structure, thereby suggesting that the residual negative charge of the cell surface may preclude interaction with the acidic antibiotic owing to electrostatic repulsion.     

Effects of polymyxin B sulfate and polymyxin B nonapeptide on growth and permeability of the yeast Saccharomyces cerevisiae

Summary Polymyxin B, a toxic, membrane-affecting antibiotic, can be rendered harmless to yeast cells by enzymatic removal of its fatty acyl moiety. The remaining cyclic peptide portion, polymyxin B nonapeptide, has no significant effect on growth and viability but it drastically reduces mating efficiency. In addition, the cyclic peptide enhances sensitivity of cells to several drugs, presumably by increasing membrane permeability. Mutants resistant to polymyxin B are simultaneously less responsive to the combination of the nonapeptide and the drugs. This indicates that the peptide portion of polymyxin B is the moiety responsible for the permeability changes. The resistance is inherited as a simple recessive trait. The mutation has been mapped to chromosome XV of Saccharomyces cerevisiae.     

The Effect of the Extracellular Bacteriolytic Enzymes of Lysobacter sp. on Gram-Negative Bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells was altered by treating them with polymyxin B.     

Polymyxin B-phosphatidylglycerol interactions. A monolayer (π, ΔV) study

Through a monolayer investigation (π, ΔV), it is shown that the cationic antibiotic polymyxin B (or E) strongly interacts with films of acidic lipids, namely the didodecanoyl- and dihexadecanoylphosphatidylglycerol. The zwitterionic dihexadecanoylphosphatidylcholine was an unsuitable substrate. Interactions occurred at and above a polymyxin B concentration in the subphase of 2.5 · 10^?7 M, bringing about a considerable increase of both π and ΔV. These interactions proceeded in two steps, as revealed by a biphasic change of ΔV with time. They were independent of the film molecular packing (fluid or gel states) and of the initial film pressure.Since it was possible to monitor the relative number of polymyxin B and didodecanoyl- or dihexadecanoylphosphatidylglycerol molecules in the monolayer, it is demonstrated that, at saturation, one polymyxin B molecule is bound to five phosphatidylglycerol molecules, a result which accounts for an exact neutralization of the charges.From competition experiments, it is shown that Na⁺ is ineffective in removing polymyxin B from the interface. Ca²⁺ appeared to be a stronger competitor but no complete antibiotic desorption was observed even at a Ca²⁺ concentration of 100 mM.As a working hypothesis, the antibiotic/lipid ($\text{1}\text{5}$) system was assumed to constitute by itself one molecular species. The mixing of the polymyxin B/didodecanoylphosphatidylglycerol ($\text{1}\text{5}$) system with an excess of lipid molecules in the monolayer was found to be ideal both in terms of π and ΔV. With dihexadecanoylphosphatidylglycerol, a small condensing effect could be detected only at intermediate surface pressures, in a region where the lipid phase transition occurred.The molecular area of polymyxin B interacting with didodecanoylphosphatidylglycerol can be calculated to be 1.23 ± 0.05 nm². It is proposed that the whole antibiotic molecule penetrates the film, the five bound lipid molecules being distributed around the peptide structure, at given positions imposed by the five 2,4-diaminobutyric acid residues.     

Assessment of synergistic antibacterial activity of combined biosurfactants revealed by bacterial cell envelop damage

Besides potential surface activity and some beneficial physical properties, biosurfactants express antibacterial activity. Bacterial cell membrane disrupting ability of rhamnolipid produced by Pseudomonas aeruginosa C2 and a lipopeptide type biosurfactant, BS15 produced by Bacillus stratosphericus A15 was examined against Staphylococcus aureus ATCC 25923 and Escherichia coli K8813. Broth dilution technique was followed to examine minimum inhibitory concentration (MIC) of both the biosurfactants. The combined effect of rhamnolipid and BS15 against S. aureus and E. coli showed synergistic activity by expressing fractional inhibitory concentration (FIC) index of 0.43 and 0.5. Survival curve of both the bacteria showed bactericidal activity after treating with biosurfactants at their MIC obtained from FIC index study as it killed > 90% of initial population. The lesser value of MIC than minimum bactericidal concentration (MBC) of the biosurfactants also supported their bactericidal activity against both the bacteria. Membrane permeability against both the bacteria was supported by amplifying protein release, increasing of cell surface hydrophobicity, withholding capacity of crystal violet dye and leakage of intracellular materials. Finally cell membrane disruption was confirmed by scanning electron microscopy (SEM). All these experiments expressed synergism and effective bactericidal activity of the combination of rhamnolipid and BS15 by enhancing the bacterial cell membrane permeability. Such effect of the combination of rhamnolipid and BS15 could make them promising alternatives to traditional antibiotic in near future.     

Investigating the action of the microalgal pigment marennine on Vibrio splendidus by in vivo 2H and 31P solid-state NMR

This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo ²H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(?) bacteria membranes. We also investigated the effect of marennine using ³¹P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis.     

Effects of Polymyxin B on Mammalian Urinary Bladder

Previous reports have demonstrated that large cationic polypeptides (of molecular mass 5,000 daltons or greater) cause an increase in the apical membrane conductance of the rabbit urinary bladder epithelium. This report investigates the effects of the small cationic molecule polymyxin B (PX: a 1,400 dalton antibiotic) on the permeability of the rabbit urinary bladder. The addition of micromolar concentrations of polymyxin B to the luminal solution of the rabbit urinary bladder resulted in an increase in the transepithelial conductance of the bladder. The magnitude of the increase in the conductance was dependent upon the concentration of PX, and the polarity and magnitude of the apical membrane potential. As the apical membrane potential was made more cell interior negative, the larger was the increase in the membrane conductance. This voltage-dependent increase in conductance was an exponential function of the applied voltage, with a negligible increase in conductance occurring when the membrane potential was cell interior positive. Upon changing the membrane voltage from cell interior positive to negative, there was a delay before there was a measurable change in the membrane conductance. The longer the apical membrane was exposed to PX, the more poorly reversible was its effect on the transepithelial conductance, suggesting a toxic effect of PX on this epithelium. Received: 9 May 1996/Revised: 17 July 1996     

Biosynthesis of polymyxin by Bacillus polymyxa. I. The status of the biosynthetic multienzyme complex during active antibiotic synthesis and sporulation

The involvement of membrane fractions of Bacillus polymyxa in the early stages of the biosynthesis of the peptide antibiotic polymyxin, was established by (a) incorporation of the precursor amino acid in an acid precipitable form in the absence of protein synthesis, (b) presence of all the component amino acid-activating enzymes, and (c) association of bioassayable polymyxin, in the purified membrane fraction. Polymyxin negative mutants that were also blocked at stage 0 of sporulation were shown to be defective in one or more of their membrane-bound amino acid-activating enzymes. A strong correlation between sporulation and antibiotic production had been indicated by the isolation of these mutants which are revertible simultaneously to Ab⁺ and Spo⁺ traits. During the onset of the rapid phase of the elaboration of polymyxin, a delocalization of one of the membrane-bound enzymes, 2,4-diaminobutyric acid-activating enzyme, to the soluble fraction was observed. Concomitant with this change, the levels of the intracellular protein-bound and free polymyxin was increased in the soluble fraction.     

Mechanism of Antimycin A Resistance in Bacillus subtilis

The site of action of antimycin A is known to lie between cytochrome b and c in the respiratory chain of mammalian cells. But in general, bacteria, even those which have cytochromes similar to those of mammalian cells such as Bacillus subtilis, are naturally resistant to this antibiotic.

The mechanism of this natural resistance is studied using a strain of B. subtilis. Succinoxidase activity of the intact cells of this bacterium showed very low sensitivity to the antibiotic, but on disruption of the cells, the sensitivity increased 7.5 times. Moreover, the activity of the intact cells could be sensitized by treatment with cationic detergent. In addition to the permeability barrier suggested by the above results, it was found that the electron transport system of this bacterium contained antimycin A insensitive by-path.     

Influence of Pseudomonas aeruginosa pvdQ Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required and up-regulated during swarming motility, which is triggered by high cell densities. As high density bacterial populations also display elevated antibiotics resistance, studies have demonstrated swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study was to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduces PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a twofold to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.     

Chemical Communication of Antibiotic Resistance by a Highly Resistant Subpopulation of Bacterial Cells

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.     

Phosphorylation of methyl-alpha-D-glucopyranoside in polymyxin B-treated Salmonella typhimurium

Phosphoenolpyruvate-dependent phosphorylation of methyl-alpha-d-glucopyranoside in Salmonella typhimurium is increased by the membrane active polypeptide antibiotic polymyxin B whereas active transport ability is abolished.     

Direction of aminoacylated transfer RNAs into antibiotic synthesis and peptidoglycan-mediated antibiotic resistance

Prokaryotic aminoacylated-transfer RNAs often need to be efficiently segregated between translation and other cellular biosynthetic pathways. Many clinically relevant bacteria, including Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa direct some aminoacylated-tRNA species into peptidoglycan biosynthesis and/or membrane phospholipid modification. Subsequent indirect peptidoglycan cross-linkage or change in membrane permeability is often a prerequisite for high-level antibiotic resistance. In Streptomycetes, aminoacylated-tRNA species are used for antibiotic synthesis as well as antibiotic resistance. The direction of coding aminoacylated-tRNA molecules away from translation and into antibiotic resistance and synthesis pathways are discussed in this review.     

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria

The effect of the extracellular bacteriolytic enzymes of Lysobacter sp. on gram-negative bacteria was studied. These enzymes were found to be able to hydrolyze the peptidoglycan that was isolated from the gram-negative bacteria, the hydrolysis being completely inhibited by the cell wall lipopolysaccharide of these bacteria. The native cells of the gram-negative bacteria became susceptible to the bacteriolytic enzymes after the permeability of the outer membrane of the cells had been altered by treating them with polymyxin B.     

Oxidative stress alters neuronal RNA- and protein-synthesis: Implications for neural viability

Recent studies have demonstrated that impaired protein synthesis occurs in several neurodegenerative conditions associated with oxidative stress. Studies have also demonstrated that administration of oxidative stressors is sufficient to impair different and discrete regulatory aspects of protein synthesis in neural cells, with the majority of these studies focused on the effects of oxidative stressors towards initiation factors. Currently, little is known with regards to oxidative stress effects on the rates of RNA- and protein-synthesis, or the relationship between oxidant-induced impairments in RNA-/protein-synthesis to subsequent neuron death. In the present study, we demonstrate that administration of an oxidative stressor (hydrogen peroxide) induces a significant and time-dependent decrease in both RNA- and protein-synthesis in primary neurons and neural SH-SY5Y cells. Increases in RNA oxidation and disruption of ribosome complexes were selectively observed following the longer durations of oxidant exposure. Significant correlations between the loss of RNA- and protein-synthesis and the amount of oxidant-induced neuron death were also observed. Interestingly, the addition of a protein synthesis inhibitor (cycloheximide) did not significantly alter the amount of neuron death induced by the oxidative stressor. These data demonstrate that oxidant exposure promotes a time-dependent decrease in both RNA- and protein-synthesis in neurons, and demonstrate a role for elevations in RNA oxidation and ribosome dysfunction as potential mediators of impaired protein synthesis. These data also suggest that there is a complex relationship between the ability of oxidative stressors to modulate RNA- and protein-synthesis, and the ability of oxidative stressors to ultimately induce neuron death.     

Oxidative stress alters neuronal RNA- and protein-synthesis: Implications for neural viability

Recent studies have demonstrated that impaired protein synthesis occurs in several neurodegenerative conditions associated with oxidative stress. Studies have also demonstrated that administration of oxidative stressors is sufficient to impair different and discrete regulatory aspects of protein synthesis in neural cells, with the majority of these studies focused on the effects of oxidative stressors towards initiation factors. Currently, little is known with regards to oxidative stress effects on the rates of RNA- and protein-synthesis, or the relationship between oxidant-induced impairments in RNA-/protein-synthesis to subsequent neuron death. In the present study, we demonstrate that administration of an oxidative stressor (hydrogen peroxide) induces a significant and time-dependent decrease in both RNA- and protein-synthesis in primary neurons and neural SH-SY5Y cells. Increases in RNA oxidation and disruption of ribosome complexes were selectively observed following the longer durations of oxidant exposure. Significant correlations between the loss of RNA- and protein-synthesis and the amount of oxidant-induced neuron death were also observed. Interestingly, the addition of a protein synthesis inhibitor (cycloheximide) did not significantly alter the amount of neuron death induced by the oxidative stressor. These data demonstrate that oxidant exposure promotes a time-dependent decrease in both RNA- and protein-synthesis in neurons, and demonstrate a role for elevations in RNA oxidation and ribosome dysfunction as potential mediators of impaired protein synthesis. These data also suggest that there is a complex relationship between the ability of oxidative stressors to modulate RNA- and protein-synthesis, and the ability of oxidative stressors to ultimately induce neuron death.