Stress induced alterations in the outer membrane of Escherichia coli K-12 strain (original) (raw)
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Influence of growth media on Escherichia coli cell composition and ceftazidime susceptibility
Antimicrobial Agents and Chemotherapy, 1991
Cell composition and surface properties of Escherichia coli were modified by using various growth media to investigate the role of yet uncharacterized components in ceftazidime susceptibility. An eightfold dilution of Luria broth was used as the basic growth medium and was supplemented with up to 4% phosphate, 5% glucose, or 12% L-glutamate. Decreases in cephaloridine and ceftazidime susceptibility, of two-and eightfold, respectively, were observed only in the glucose-enriched medium. The outer membrane permeability to ceftazidime and cephaloridine was evaluated by crypticity indices. Indices were unchanged under all growth conditions. Fluorometry of whole cells with 1-N-phenylnaphthylamine showed that glucose does not affect the interaction of this hydrophobic probe with the membranes but showed that elevated concentrations of phosphate or glutamate cause a marked increase in cell hydrophobicity, which, in turn, correlates with an increase in the susceptibility of E. coli to nalidixic acid. Growth in phosphateor glutamate-enriched media caused an augmentation in major phospholipid species and may explain the increased hydrophobicity and susceptibility of E. coli to nalidixic acid. These data showed that E. coli susceptibility to ceftazidime is not influenced by cell surface hydrophobicity and suggested that the contribution of a nonspecific lipophilic diffusion route for entry of ceftazidime into cells is not likely to occur or is distinct from that of more hydrophobic molecules such as nalidixic acid. Finally, the penicillin-binding proteins of the E. coli cells were also investigated. Penicillin-binding protein 8 was only markedly labeled with 125I-penicillin V in inner membranes extracted from cells grown with glucose. Results of this study suggest that the unexpected change in penicillin-binding protein 8 observed in the presence of glucose may be responsible for the increase in MICs of cephaloridine and ceftazidime.
Role of Membrane Architecture in Development of Sensitivity to Cephalosporin Group of Antibiotics
Advances in Life Science and Technology, 2012
Due to efficient adaptation process the spread of resistance among microbial pathogens is always a major problem to therapy. The resistance towards an antibiotic can be either biochemical or genetic. The molecular orientation of bacterial cell wall is responsible in determining drug resistance. The role of cephalosporins for e.g.: ceftriaxone & cefazolin was studied in relation to its toxicity to B. subtilis (Gram positive) & E. coli (Gram negative).In the presence of cefazolin an abnormally high level of protein release was observed when intact cells as well as membrane vesicles. In the presence of cefazolin protein release was observed to been enhanced in contrast to ceftriaxone. The protein export in case of intact bacterial cells was higher than membrane vesicles suggesting the involvement of membrane proteins in drug sensitivity/resistance. The extent of protein released was also found to be modulated when both the cells were subjected to temperature treatment. However, maximum protein export was seen when gram positive& gram-negative cells were subjected to EDTA concentrations. In contrast availability of Mg 2+ ions in the medium resulted in slight fall in protein release indicating stabilization of membrane vesicles as well as bacterial cell wall, which might have resulted in lowered protein export due to involvement of transport system. From this study it can be concluded that outer membrane orientation determines the therapeutic value of cefazolin & ceftriaxone.
Antimicrobial Agents and Chemotherapy, 1979
A new technique has been devised to investigate the penetration of antibiotics through the gram-negative outer membrane; the application here was to study intrinsic resistance of Escherichia coli K-12. Exponential cells in broth were briefly treated with 2.5 mM ethylenediaminetetraacetic acid at 5°C to disrupt the outer membrane penetration barrier, and the response of treated and untreated cells to antibiotics was compared by turbidimetry. A barrier index was derived to describe the ability of 7 beta-lactam and 10 other antibiotics to penetrate the outer membrane of strain Y10. There was correlation between the molecular weight and logio barrier index (r = 0.59, P = 0.01). The envelope mutant D22 (envA) had low barrier indexes for erythromycin, rifampin, ampicillin, and cloxacillin. For the beta-lactams, outer membrane penetration and affinity for inner membrane target site(s) triggering cell lysis were measured as independent components of the overall activity; although penetration and overall activity varied greatly, the affinities of most were within a narrow range.
Study of Alterations of Bacterial Membrane Proteins involved in ?-Lactam Sensitivity
Journal of Biology Agriculture and Healthcare, 2012
The selective nature of organisms to prevailing stress-β-lactam antibiotics is obviously concerned with the architecture of outer membrane. Bacillus subtilis Gram-positive bacteria advocates variation in achieving cellular morphogenetic dimensions. In the present study more protonated species of β-lactam i.e. Ceftriaxone proved to be more effective in contrast to cefazolin as far as sensitivity of Bacillus subtilis is concerned. The MIC 50 for ceftriaxone (CT) was observed to be 1.5 ppm in contrast to first generation β-lactam antibiotic i.e. cefazolin with MIC 50 18 ppm concentration. The relative sensitivity of bacterial cells to both the cephalosporins was more positively inclined to third generation species. The role of modified cephalosporin was observed under various physiological situations under laboratory conditions such as pH, Temp, chelating agent and Mg 2+ ion concentrations. The sensitivity of Bacillus subtilis to β-lactam was more potentiated at acidic pH than at alkaline pH. At alkaline pH only 16% inhibition of growth of Bacillus subtilis in Dye's minimal media at 37±1 0 C was seen in contrast to 60% inhibition of growth in the presence of CZ. The growth was enhanced / potentiated in the presence of chelating agent-EDTA at 0.25 mM concentration in combination with MIC 50 of CT and CZ 84% inhibition of growth was recorded in the presence of CT (MIC 50) in contrast to 82% inhibition in the presence of CZ (MIC 50). The growth inhibition with 0.25 mM EDTA alone was also seen to be 82%.
Antimicrobial Agents and Chemotherapy, 1991
The relative binding affinities of the extended-spectrum cephalosporins cefepime, cefpirome, and cefaclidine for the penicillin-binding proteins (PBPs) of Escherichia coli K-12 and Pseudomonas aeruginosa SC8329 were determined. Affinities were calculated from competition experiments between these antibiotics and [3H]benzylpenicillin in isolated membrane preparations. The concentrations which reduced binding to a PBP by 50% (IC50s) were determined. For E. coli, all three antibiotics displayed good PBP 3 binding (IC50s of 0.5 ,ug/ml or less), and MICs roughly correlated with these values. Cefepime had a greater than 20-fold-lower IC50 for PBP 2 of E. coli than the other antibiotics. For P. aeruginosa, all of the antibiotics bound poorly (>25 ,ug/ml) to PBP 2 but showed excellent pseudomonal (<0.0025 ,ig/ml) PBP 3 binding. No correlations were seen between IC50s and MICs for P. aeruginosa. Despite differences in PBP binding, cefepime, cefpirome, and cefaclidine all displayed similar bactericidal activity for E. coli K-12 over the initial 3 h after antibiotic addition. All three caused E. coli to form filaments at values close to the MICs. In addition, cefepime induced "bleb" formation along the filaments at concentrations >10x the MIC.
Transport of Cephalosporin Antibiotics Across the Outer Membrane
Biophysical Journal, 2010
Outer membrane channels in Gram-negative bacteria are implicated in the influx of the latest generation of cephalosporins. We have measured the interaction strengths of ceftriaxone, cefpirome and ceftazidime in the two most abundant outer membrane porins of Escherichia coli, OmpF and OmpC, by both ion current fluctuations through single protein channels and fluorescence quenching. Statistical analysis of individual antibiotic entry events in membrane-incorporated porins yielded the kinetic rates and the equilibrium binding constant of each antibiotic-porin pair. Affinity constants were independently obtained by measuring the static quenching of inherent tryptophan fluorescence in the porins in the presence of the antibiotics. Through an empirical inner filter effect correction we have succeeded in measuring the chemical interaction of these strongly absorbing antibiotics, and obtained a qualitative agreement with conductance measurements. The interaction of all three antibiotics is smaller for OmpC than OmpF, and in the case of each porin the interaction strength series ceftriaxone > cefpirome > ceftazidime is maintained.
African Journal of Biotechnology, 2009
The study was conducted with the objective of examining the outer membrane proteins and their involvement during the transport of-lactams in multidrug resistant Escherichia coli isolated from extra-intestinal infections. Also, the response of gram negative bacterial biomembrane alteration was studied using extended spectrum-Lactamase (ESBL) positive strains of E.coli in the presence and absence of stress. Outer membrane protein analysis revealed that multidrug resistance was mediated by loss of porins in the ESBL isolates of E. coli. Parallel to this, the level of outer membrane proteins decreased dramatically with increased antibiotic stress in the isolates. In non-ESBL isolates of E. coli, porins seems to have no influence in co-modulation of resistance.
European Journal …, 1989
A pair of strains of Pseudomonas aeruginosa (3-Pre : cefsulodin-sensitive, inducible /?-lactamase ; and 3-Post: cefsulodin-resistant, elevated fl-lactamase, derived from 3-Pre by subculture in the presence of cefsulodin) were taken as representative of the class of bacteria resistant to third-generation cephalosporins due to elevated synthesis of the normally inducible, chromosomally encoded j-lactamase. These two strains were used to differentiate between 'trapping' and 'hydrolytic' mechanisms of cefsulodin resistance by (a) measuring the outer-membrane permeabilities to cefsulodin, (b) measuring the kinetics of cefsulodin hydrolysis and the stoichiometry of cefsulodin trapping by the periplasmic b-lactamase, and (c) comparing the predictions of the trapping and hydrolysis hypotheses with the minimum inhibitory concentrations (MIC) of cefsulodin. The MIC of cefsulodin for strains 3-Pre and 3-Post were 2.35 pM (1.25 pg ml-') and 37.6 pM (20.0 pg ml-') respectively. The permeability parameter for cefsulodin of the outer membrane of the resistant strain was 0.0034 cm3 min-' mg dry mass-', so the flux of cefsulodin across its outer membrane at the MIC was calculated to be 0.120 nmol min-' mg dry mass-'. Hydrolysis of cefsulodin by the p-lactamase in the periplasm occurred at a rate of 0.1 18 nmol min-' mg dry mass-' which can thus account for resistance by matching the above rate of inflow. Trapping by the P-lactamase, even with a 1 : 1 stoichiometry, would require the enzyme to be synthesized at 5.0 pg protein min-' mg dry mass-' or about 40% of the dry mass/generation. We conclude that hydrolysis, but not trapping, adequately explains the resistance to cefsulodin in P. aeruginosa 3-Post. A similar calculation for latamoxef resistance, using data taken from the literature. led to the same conclusion. Resistance to third-generation cephalosporins [l] in gramnegative bacteria possessing inducible class-C [2] P-lactamases can occur by mutation to continuous elevated synthesis of the enzyme [3-91. The mechanism of this form of resistance has been the subject of much debate, because in general these p-lactamases possess low or undetectable hydrolytic activity against such compounds [8,10-131. Then and Angehrn [3] proposed that the mechanism of resistance was one of trapping (i.e. binding only) of the antibiotic by the elevated periplasm-located enzyme. This proposal has been supported by Pechere and Levesque [14] and by Sanders [S, 101 and, in specific situations, by Livermore [15] and Frkre and Joris [16]; but its general validity has been questioned by several authors [ll, 13, 17, 181 who support a conventional hydrolytic mechanism of resistance.