Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

1982 ◽  
Vol 28 (7) ◽  
pp. 830-840 ◽  
Author(s):  
H. E. Gilleland Jr. ◽  
Linda B. Farley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Resistant Strain ◽  
Cytoplasmic Membrane ◽  
Osmotic Shock ◽  
Membrane Repair ◽  
Permeability Characteristics ◽  
Minimal Inhibitory Concentrations ◽  
Adaptive Resistance ◽  
Resistant Strains

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appeared to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.

scholarly journals Pyoverdine-Mediated Iron Uptake in Pseudomonas aeruginosa: the Tat System Is Required for PvdN but Not for FpvA Transport

2006 ◽  
Vol 188 (9) ◽  
pp. 3317-3323 ◽  
Author(s):  
Romé Voulhoux ◽  
Alain Filloux ◽  
Isabelle J. Schalk
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Iron Uptake ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Iron Release ◽  
Outer Membrane Receptor ◽  
Uptake Pathway ◽  
Uptake Process ◽  
Tat System

ABSTRACT Under iron-limiting conditions, Pseudomonas aeruginosa PAO1 secretes a fluorescent siderophore called pyoverdine (Pvd). After chelating iron, this ferric siderophore is transported back into the cells via the outer membrane receptor FpvA. The Pvd-dependent iron uptake pathway requires several essential genes involved in both the synthesis of Pvd and the uptake of ferric Pvd inside the cell. A previous study describing the global phenotype of a tat-deficient P. aeruginosa strain showed that the defect in Pvd-mediated iron uptake was due to the Tat-dependent export of proteins involved in Pvd biogenesis and ferric Pvd uptake (U. Ochsner, A. Snyder, A. I. Vasil, and M. L. Vasil, Proc. Natl. Acad. Sci. USA 99:8312-8317, 2002). Using biochemical and biophysical tools, we showed that despite its predicted Tat signal sequence, FpvA is correctly located in the outer membrane of a tat mutant and is fully functional for all steps of the iron uptake process (ferric Pvd uptake and recycling of Pvd on FpvA after iron release). However, in the tat mutant, no Pvd was produced. This suggested that a key element in the Pvd biogenesis pathway must be exported to the periplasm by the Tat pathway. We located PvdN, a still unknown but essential component in Pvd biogenesis, at the periplasmic side of the cytoplasmic membrane and showed that its export is Tat dependent. Our results further support the idea that a critical step of the Pvd biogenesis pathway involving PvdN occurs at the periplasmic side of the cytoplasmic membrane.

scholarly journals Mechanism of Enhanced Activity of Liposome-Entrapped Aminoglycosides against Resistant Strains of Pseudomonas aeruginosa

2006 ◽  
Vol 50 (6) ◽  
pp. 2016-2022 ◽  
Author(s):  
Clement Mugabe ◽  
Majed Halwani ◽  
Ali O. Azghani ◽  
Robert M. Lafrenie ◽  
Abdelwahab Omri
Keyword(s):  
Flow Cytometry ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Fusion Rate ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Transmission Electron ◽  
Resistant Strains ◽  
Conventional Antibiotics ◽  
Liposomal Drugs

ABSTRACT Pseudomonas aeruginosa is inherently resistant to most conventional antibiotics. The mechanism of resistance of this bacterium is mainly associated with the low permeability of its outer membrane to these agents. We sought to assess the bactericidal efficacy of liposome-entrapped aminoglycosides against resistant clinical strains of P. aeruginosa and to define the mechanism of liposome-bacterium interactions. Aminoglycosides were incorporated into liposomes, and the bactericidal efficacies of both free and liposomal drugs were evaluated. To define the mechanism of liposome-bacterium interactions, transmission electron microscopy (TEM), flow cytometry, lipid mixing assay, and immunocytochemistry were employed. Encapsulation of aminoglycosides into liposomes significantly increased their antibacterial activity against the resistant strains used in this study (MICs of ≥32 versus ≤8 μg/ml). TEM observations showed that liposomes interact intimately with the outer membrane of P. aeruginosa, leading to the membrane deformation. The flow cytometry and lipid mixing assays confirmed liposome-bacterial membrane fusion, which increased as a function of incubation time. The maximum fusion rate was 54.3% ± 1.5% for an antibiotic-sensitive strain of P. aeruginosa and 57.8% ± 1.9% for a drug-resistant strain. The fusion between liposomes and P. aeruginosa significantly enhanced the antibiotics' penetration into the bacterial cells (3.2 ± 2.3 versus 24.2 ± 6.2 gold particles/bacterium, P ≤ 0.001). Our data suggest that liposome-entrapped antibiotics could successfully resolve infections caused by antibiotic-resistant P. aeruginosa through an enhanced mechanism of drug entry into the bacterial cells.

Release of rhodanese from Pseudomonas aeruginosa by cold shock and its localization within the cell

1979 ◽  
Vol 25 (3) ◽  
pp. 340-351 ◽  
Author(s):  
R. W. Ryan ◽  
M. P. Gourlie ◽  
R. C. Tilton
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Outer Membrane ◽  
Electron Micrographs ◽  
Cold Shock ◽  
Cytoplasmic Membrane ◽  
Lactic Dehydrogenase ◽  
Periplasmic Space ◽  
Whole Cells ◽  
Periplasmic Enzyme

Whole cells of Pseudomonas aeruginosa possess rhodanese activity. The enzyme can be released by rapidly resuspending the cells in cold Tris–HCl buffer. Approximately 95% of the rhodanese activity is released by cold shock. Release of the enzyme can be inhibited either by preincubating the cells with Mg2+ or by incorporating Mg2+ into the shocking buffer. The effect of Mg2+ can be reversed by washing the cells twice with buffer prior to cold shock. While rhodanese can be released from P. aeruginosa by cold shock, lactic dehydrogenase, a cytoplasmic enzyme, remains within the cell. Diazo-7-amino-1,3-naphthalenedisulfonic acid, a compound which does not penetrate the cytoplasmic membrane, completely inactivated rhodanese and alkaline phosphatase, a periplasmic enzyme, whereas lactic dehydrogenase retained its full activity. These data suggest that rhodanese in P. aeruginosa, like alkaline phosphatase, is located distal to the cytoplasmic membrane in the periplasmic space. Electron micrographs also show that portions of the lipopolysaccharide outer membrane are shed from the cell during cold shock, while cells preincubated with Mg2+ did not release segments of their outer membrane.

scholarly journals Art-175 Is a Highly Efficient Antibacterial against Multidrug-Resistant Strains and Persisters of Pseudomonas aeruginosa

2014 ◽  
Vol 58 (7) ◽  
pp. 3774-3784 ◽  
Author(s):  
Yves Briers ◽  
Maarten Walmagh ◽  
Barbara Grymonprez ◽  
Manfred Biebl ◽  
Jean-Paul Pirnay ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Time Lapse ◽  
Bactericidal Effect ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Chronic Infections ◽  
Content Type ◽  
Resistant Strains

ABSTRACTArtilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and killPseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (∼5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect againstP. aeruginosapersisters (up to >4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections.

scholarly journals In vitro characterization of aminoglycoside adaptive resistance in Pseudomonas aeruginosa.

1996 ◽  
Vol 40 (6) ◽  
pp. 1387-1393 ◽  
Author(s):  
J A Karlowsky ◽  
M H Saunders ◽  
G A Harding ◽  
D J Hoban ◽  
G G Zhanel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Cytoplasmic Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proton Motive Force ◽  
Motive Force ◽  
Adaptive Resistance ◽  
Resistant Cells

Aminoglycoside adaptive resistance was characterized in one reference strain and four clinical isolates of Pseudomonas aeruginosa. Adaptive resistance was initiated with a 2-h gentamicin or tobramycin exposure at the MIC. Each P. aeruginosa strain demonstrated an adaptive-resistance period of between 8 and 12 h when tested with both aminoglycosides. Aminoglycoside adaptive resistance was shown to correlate with a decrease in [3H] gentamicin accumulation and a small (5%) but significant (P < 0.05) reduction in proton motive force. The mean generation time of P. aeruginosa during peak levels of adaptive resistance (i.e., maximum reductions in aminoglycoside killing) was not significantly different from that of control organisms (P < 0.05). No changes in outer membrane protein or lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles were noted when control, adaptively resistant, and postadaptively resistant cells were compared. Cytoplasmic membrane profiles of adaptively resistant cells, however, demonstrated several band changes when compared with control and postadaptively resistant cells. We conclude that the decrease in aminoglycoside accumulation associated with adaptive resistance in P. aeruginosa may be, in part, a function of reductions in proton motive force and/or cytoplasmic membrane protein changes. However, the importance of these changes requires further investigation.

scholarly journals Correlation between Resistance of Pseudomonas aeruginosa to Quaternary Ammonium Compounds and Expression of Outer Membrane Protein OprR

2003 ◽  
Vol 47 (7) ◽  
pp. 2093-2099 ◽  
Author(s):  
Atsushi Tabata ◽  
Hideaki Nagamune ◽  
Takuya Maeda ◽  
Keiji Murakami ◽  
Yoichiro Miyake ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Quaternary Ammonium ◽  
Resistant Strain ◽  
Quaternary Ammonium Compounds ◽  
Cross Resistance ◽  
Knockout Mutant ◽  
Ammonium Compounds

ABSTRACT The adaptation mechanism of Pseudomonas aeruginosa ATCC 10145 to quaternary ammonium compounds (QACs) was investigated. A P. aeruginosa strain with adapted resistance to QACs was developed by a standard broth dilution method. It was revealed that P. aeruginosa exhibited remarkable resistance to N-dodecylpyridinium iodide (P-12), whose structure is similar to that of a common disinfectant, cetylpyridinium chloride. Adapted resistance to benzalkonium chloride (BAC), which is commonly used as a disinfectant, was also observed in P. aeruginosa. Moreover, the P-12-resistant strain exhibited cross-resistance to BAC. Analysis of the outer membrane protein of the P-12-resistant strain by two-dimensional polyacrylamide gel electrophoresis showed a significant increase in the level of expression of a protein (named OprR) whose molecular mass was approximately 26 kDa. The actual function of OprR is not yet clear; however, OprR was expected to be an outer membrane-associated protein with homology to lipoproteins of other bacterial species, according to a search of the National Center for Biotechnology Information website with the BLAST program by use of the N-terminal sequence of OprR. A correlation between the level of expression of OprR and the level of resistance of P. aeruginosa to QACs was observed by using a PA2800 gene knockout mutant derived from the P-12-resistant strain. The knockout mutant recovered susceptibility not only to P-12 but also to BAC. These results suggested that OprR significantly participated in the adaptation of P. aeruginosa to QACs, such as P-12 and BAC.

scholarly journals Evolution toward maximum transport capacity of the Ttg2 ABC system in Pseudomonas aeruginosa

2019 ◽  
Author(s):  
Daniel Yero ◽  
Lionel Costenaro ◽  
Oscar Conchillo-Solé ◽  
Mireia Díaz-Lobo ◽  
Adrià Mayo ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Gene Knockout ◽  
Multidrug Resistant ◽  
Crystallographic Structure ◽  
Intrinsic Resistance ◽  
Head Groups ◽  
Resistant Strains ◽  
System Thought

AbstractIn Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding component of an ABC transport system thought to be involved in maintaining the asymmetry of the outer membrane. The crystallographic structure of Ttg2D at 2.5Å resolution reveals that this protein can bind two diacyl phospholipids. Native and denaturing mass spectrometry experiments confirm that Ttg2D binds two phospholipid molecules, which may have different head groups. Analysis of the available structures of Ttg2D orthologs allowed us to classify this protein family as a novel substrate-binding protein fold and to venture the evolutionary events that differentiated the orthologs binding one or two phospholipids. In addition, gene knockout experiments in P. aeruginosa PAO1 and multidrug-resistant strains show that disruption of this system leads to outer membrane permeabilization. This demonstrates the role of this system in low-level intrinsic resistance against certain antibiotics that use a lipid-mediated pathway to permeate through membranes.

scholarly journals In Vitro Alginate Polymerization and the Functional Role of Alg8 in Alginate Production by Pseudomonas aeruginosa

2006 ◽  
Vol 72 (1) ◽  
pp. 298-305 ◽  
Author(s):  
Uwe Remminghorst ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Outer Membrane Proteins ◽  
Polymerase Activity ◽  
Protein Fusion ◽  
Alginate Production ◽  
Alginate Biosynthesis

ABSTRACT An enzymatic in vitro alginate polymerization assay was developed by using 14C-labeled GDP-mannuronic acid as a substrate and subcellular fractions of alginate overproducing Pseudomonas aeruginosa FRD1 as a polymerase source. The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting that cytoplasmic and outer membrane proteins constitute the functional alginate polymerase complex. Accordingly, no alginate polymerase activity was detected using cytoplasmic membrane or outer membrane proteins, respectively. To determine the requirement of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic alg8 knockout mutants of alginate-overproducing P. aeruginosa FRD1 and P. aeruginosa PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was restored by introducing only the alg8 gene. Surprisingly, this resulted in significant alginate overproduction of the complemented P. aeruginosa Δalg8 mutants compared to nonmutated strains, suggesting that Alg8 is the bottleneck in alginate biosynthesis. 1H-NMR analysis of alginate isolated from these complemented mutants showed that the degree of acetylation increased from 4.7 to 9.3% and the guluronic acid content was reduced from 38 to 19%. Protein topology prediction indicated that Alg8 is a membrane protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus. Subcellular fractionation suggested that the highest specific PhoA activity of Alg8-PhoA is present in the cytoplasmic membrane. A structural model of Alg8 based on the structure of SpsA from Bacillus subtilis was developed.

scholarly journals Efflux-Mediated Resistance to Tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1

2003 ◽  
Vol 47 (3) ◽  
pp. 972-978 ◽  
Author(s):  
Charles R. Dean ◽  
Melissa A. Visalli ◽  
Steven J. Projan ◽  
Phaik-Eng Sum ◽  
Patricia A. Bradford
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Repressor Gene ◽  
Mutant Strains ◽  
Resistant Strains ◽  
Resistant Mutants ◽  
Outer Membrane Channel ◽  
Increased Susceptibility

ABSTRACT Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 μg/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 μg/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 μg/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines.

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