The effects of polymyxin B (PMB) on the Escherichia coli outer (OM) and cytoplasmic membrane (CM) permeabilities were studied by monitoring the fluxes of tetraphenylphosphonium, phenyldicarbaundecaborane, and K ؉ and H ؉ ions. At concentrations between 2 and 20 g/ml, PMB increased the OM permeability to lipophilic compounds and induced a leakage of K ؉ from the cytosol and an accumulation of lipophilic anions in the cellular membranes but did not cause the depolarization of the CM. At higher concentrations, PMB depolarized the CM, forming ion-permeable pores in the cell envelope. The permeability characteristics of PMB-induced pores mimic those of bacteriophage-and/or bacteriocin-induced channels. However, the bactericidal effect of PMB took place at concentrations below 20 g/ml, indicating that this effect is not caused by pore formation. Under conditions of increased ionic strength, PMB made the OM permeable to lipophilic compounds and decreased the K ؉ gradient but was not able to depolarize the cells. The OM-permeabilizing effect of PMB can be diminished by increasing the concentration of Mg 2؉. The major new findings of this work are as follows: (i) the OM-permeabilizing action of PMB was dissected from its depolarizing effect on the CM, (ii) the PMB-induced ion-permeable pores in bacterial envelope were registered, and (iii) the pore formation and depolarization of the CM are not obligatory for the bactericidal action of PMB and dissipation of the K ؉ gradient on the CM.Lipid bilayers usually are quite permeable to lipophilic compounds (15,41,44). However, the outer membrane (OM) of gram-negative bacteria forms a rather effective permeability barrier against various lipophilic substances, including antibiotics. At least 10-to 100-fold slower rates of lipophilic compound permeation through the OM bilayer compared to those through the cytoplasmic membrane (CM) are observed because of the highly charged lipopolysaccharide (LPS)-formed outer monolayer and the stabilization of this layer by divalent cations (20, 39). These compounds also cannot traverse the porins, the narrow channels for inorganic ions and small hydrophilic nutrients (19,38).Polymyxin B (PMB) is a decapeptide antibiotic characterized by a heptapeptide ring containing four 2,4-diaminobutyric acids. An additional peptide chain covalently bound to the ␥-amino group carries an aliphatic chain attached to the peptide through an amide bond. The molecule carries five positively charged residues of diaminobutyric acid (52). Due to its molecular mass (about 1,200 Da), charge, and amphiphilicity, PMB should be excluded by the OM. However, several polycationic compounds, including PMB, are known to penetrate the OM using pathways other than porins. Although the detailed mechanism of the OM permeability barrier disruption remains undetermined, complex formation by PMB with LPS is expected to be the first stage (26,50,56,60). Being bulkier than the inorganic divalent cations that it displaces, PMB changes the packing order of LPS and increases the permeabi...

Soil is one of the biggest reservoirs of microbial diversity, yet the processes that define the community dynamics are not fully understood. Apart from soil management being vital for agricultural purposes, it is also considered a favorable environment for the evolution and development of antimicrobial resistance, which is due to its high complexity and ongoing competition between the microorganisms. Different approaches to agricultural production might have specific outcomes for soil microbial community composition and antibiotic resistance phenotype. Therefore in this study we aimed to compare the soil microbiota and its resistome in conventional and organic farming systems that are continually influenced by the different treatment (inorganic fertilizers and pesticides vs. organic manure and no chemical pest management). The comparison of the soil microbial communities revealed no major differences among the main phyla of bacteria between the two farming styles with similar soil structure and pH. Only small differences between the lower taxa could be observed indicating that the soil community is stable, with minor shifts in composition being able to handle the different styles of treatment and fertilization. It is still unclear what level of intensity can change microbial composition but current conventional farming in Central Europe demonstrates acceptable level of intensity for soil bacterial communities. When the resistome of the soils was assessed by screening the total soil DNA for clinically relevant and soil-derived antibiotic resistance genes, a low variety of resistance determinants was detected (resistance to β-lactams, aminoglycosides, tetracycline, erythromycin, and rifampicin) with no clear preference for the soil farming type. The same soil samples were also used to isolate antibiotic resistant cultivable bacteria, which were predominated by highly resistant isolates of Pseudomonas, Stenotrophomonas, Sphingobacterium and Chryseobacterium genera. The resistance of these isolates was largely dependent on the efflux mechanisms, the soil Pseudomonas spp. relying mostly on RND, while Stenotrophomonas spp. and Chryseobacterium spp. on RND and ABC transporters.

Bacteriophages 6 and 13 are related enveloped double-stranded RNA viruses that infect gram-negative Pseudomonas syringae cells. 6 uses a pilus as a receptor, and 13 attaches to the host lipopolysaccharide. We compared the entry-related events of these two viruses, including receptor binding, envelope fusion, peptidoglycan penetration, and passage through the plasma membrane. The infection-related events are dependent on the multiplicity of infection in the case of 13 but not with 6. A temporal increase of host outer membrane permeability to lipophilic ions was observed from 1.5 to 4 min postinfection in both virus infections. This enhanced permeability period coincided with the fast dilution of octadecyl rhodamine B-labeled virus-associated lipid molecules. This result is in agreement with membrane fusion, and the presence of temporal virus-derived membrane patches on the outer membrane. Similar to 6, 13 contains a thermosensitive lytic enzyme involved in peptidoglycan penetration. The phage entry also caused a limited depolarization of the plasma membrane. Inhibition of host respiration considerably decreased the efficiency of irreversible virus binding and membrane fusion. An active role of cell energy metabolism in restoring the infection-induced defects in the cell envelope was also observed.

Although several details of the photosensitization mechanisms involved in the photosensitized inactivation of bacteria have been elucidated, there are relatively few data on the morphological alterations induced on the bacterial cell structure during photosensitization. In this work we analysed the photodynamic action of the tetra-cationic photosensitizer tetrakis(N-ethylpyridinium-4-yl)porphyrin tetratosylate (TN-Et-PyP) on the integrity and selected functions of E. coli KMY1 cell membranes, in an effort to combine electron microscopy data with enzymatic assays and electrochemistry measurements. Using low concentrations of photosensitizer, damage is inflicted to the outer membrane and results in a higher permeability of the membrane to fairly small molecules such as deoxycholate; however, larger molecules such as periplasmic alkaline phosphatase are not released or are released after their extensive inactivation, as we could not register any enzyme activity outside the cells. Increasing the TN-Et-PyP concentration correlates with the inactivation of the respiratory chain, drop in plasma membrane voltage, the release of compounds with absorption band at 260 nm, and a decrease in intracellular enzyme β-galactosidase activity, though this activity has not been noticed to increase outside the cells, suggesting that enzyme inactivation probably occurs in inner cell districts.

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We have analysed the photodynamic action of the cationic photosensitiser tetrakis(N-ethylpyridinium-4-yl)porphyrin tetratosylate (TN-Et-PyP) on the integrity and functions of E. coli AN 180 cell membranes. Photosensitisation of bacterial cells with photobactericidal concentrations of TN-Et-PyP resulted in abolition of plasma membrane (PM) voltage (∆Ψ), dissipation of transmembrane K + gradient and inhibition of the respiratory activity of bacteria contemporary with enhanced accumulation of the lipophilic anion phenyldicarbaundecaborane (PCB -). Substantial impairment of cell PM functions suggests that the E. coli cell envelope, mainly at the level of PM, is a critical target of phototoxic action of TN-Et-PyP.

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