scholarly journals Pseudomonas aeruginosa Reveals High Intrinsic Resistance to Penem Antibiotics: Penem Resistance Mechanisms and Their Interplay

2001 ◽  
Vol 45 (7) ◽  
pp. 1964-1971 ◽  
Author(s):  
Kiyomi Okamoto ◽  
Naomasa Gotoh ◽  
Takeshi Nishino
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Laboratory Strain ◽  
Resistance Mechanisms ◽  
Efflux System ◽  
Intrinsic Resistance ◽  
Active Efflux ◽  
Membrane Barrier ◽  
Susceptibility Tests ◽  
Almost All

ABSTRACT Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC β-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encodedEscherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC β-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC β-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC β-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC β-lactamase but not from the lower affinity of PBPs for penems.

scholarly journals Pseudomonas aeruginosa – a phenomenon of bacterial resistance

2009 ◽  
Vol 58 (9) ◽  
pp. 1133-1148 ◽  
Author(s):  
Tanya Strateva ◽  
Daniel Yordanov
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Bacterial Resistance ◽  
Antimicrobial Agents ◽  
Constitutive Expression ◽  
Resistance Mechanisms ◽  
Intrinsic Resistance ◽  
Active Efflux ◽  
Class B ◽  
Outer Membrane Permeability

Pseudomonas aeruginosa is one of the leading nosocomial pathogens worldwide. Nosocomial infections caused by this organism are often hard to treat because of both the intrinsic resistance of the species (it has constitutive expression of AmpC β-lactamase and efflux pumps, combined with a low permeability of the outer membrane), and its remarkable ability to acquire further resistance mechanisms to multiple groups of antimicrobial agents, including β-lactams, aminoglycosides and fluoroquinolones. P. aeruginosa represents a phenomenon of bacterial resistance, since practically all known mechanisms of antimicrobial resistance can be seen in it: derepression of chromosomal AmpC cephalosporinase; production of plasmid or integron-mediated β-lactamases from different molecular classes (carbenicillinases and extended-spectrum β-lactamases belonging to class A, class D oxacillinases and class B carbapenem-hydrolysing enzymes); diminished outer membrane permeability (loss of OprD proteins); overexpression of active efflux systems with wide substrate profiles; synthesis of aminoglycoside-modifying enzymes (phosphoryltransferases, acetyltransferases and adenylyltransferases); and structural alterations of topoisomerases II and IV determining quinolone resistance. Worryingly, these mechanisms are often present simultaneously, thereby conferring multiresistant phenotypes. This review describes the known resistance mechanisms in P. aeruginosa to the most frequently administrated antipseudomonal antibiotics: β-lactams, aminoglycosides and fluoroquinolones.

scholarly journals Extrusion of Penem Antibiotics by Multicomponent Efflux Systems MexAB-OprM, MexCD-OprJ, and MexXY-OprM of Pseudomonas aeruginosa

2002 ◽  
Vol 46 (8) ◽  
pp. 2696-2699 ◽  
Author(s):  
Kiyomi Okamoto ◽  
Naomasa Gotoh ◽  
Takeshi Nishino
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Efflux System ◽  
Cell Interior ◽  
Active Efflux ◽  
Membrane Barrier

ABSTRACT The high intrinsic penem resistance of Pseudomonas aeruginosa is due to the interplay among the outer membrane barrier, the active efflux system MexAB-OprM, and AmpC β-lactamase. We studied the roles of two other efflux systems, MexCD-OprJ and MexXY-OprM, in penem resistance by overexpressing each system in an AmpC- and MexAB-OprM-deficient background and found that MexAB-OprM is the most important among the three efflux systems for extrusion of penems from the cell interior.

scholarly journals A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
E. A. Rundell ◽  
N. Commodore ◽  
A. L. Goodman ◽  
B. I. Kazmierczak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Critical Role ◽  
Fitness Cost ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Fitness Advantage ◽  
Membrane Barrier ◽  
Flagellar Assembly ◽  
Cell Envelopes

ABSTRACT The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa’s outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae. This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic. IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

scholarly journals Involvement of an Active Efflux System in the Natural Resistance of Pseudomonas aeruginosa to Aminoglycosides

1999 ◽  
Vol 43 (11) ◽  
pp. 2624-2628 ◽  
Author(s):  
Julio Ramos Aires ◽  
Thilo Köhler ◽  
Hiroshi Nikaido ◽  
Patrick Plésiat
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Regulatory Gene ◽  
Natural Resistance ◽  
Insertion Mutagenesis ◽  
Detectable Effect ◽  
Efflux System ◽  
Intrinsic Resistance ◽  
Cloning And Sequencing ◽  
Active Efflux ◽  
Energy Dependent

ABSTRACT A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn501insertion mutagenesis of Pseudomonas aeruginosa PAO1. Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB. This locus also contained a putative regulatory gene,mexZ, transcribed divergently from the efflux operon. Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host. Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides. Furthermore, this system appeared to function with an outer membrane protein, OprM. While the present paper was being written and reviewed, genes with a sequence identical to our pump genes, mexXY of P. aeruginosa, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations inEscherichia coli hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob. Agents Chemother. 43:415–417, 1999). Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of P. aeruginosato aminoglycosides. Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of themexXY operon in P. aeruginosa had no detectable effect on susceptibility to these agents.

scholarly journals Contribution of the MexX-MexY-OprM Efflux System to Intrinsic Resistance in Pseudomonas aeruginosa

2000 ◽  
Vol 44 (9) ◽  
pp. 2242-2246 ◽  
Author(s):  
Nobuhisa Masuda ◽  
Eiko Sakagawa ◽  
Satoshi Ohya ◽  
Naomasa Gotoh ◽  
Hideto Tsujimoto ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laboratory Strain ◽  
Immunoblot Analysis ◽  
Rabbit Serum ◽  
Deficient Mutant ◽  
Efflux System ◽  
Intrinsic Resistance ◽  
Strain Pao1 ◽  
Isogenic Mutants ◽  
Increased Susceptibility

ABSTRACT To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in Pseudomonas aeruginosa, we constructed a series of isogenic mutants lacking mexXY and/ormexAB and/or oprM from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility. Introduction of an OprM expression plasmid decreased the susceptibility of themexAB-oprM-deficient-/mexXY-maintaining mutant, yet caused no change in the susceptibility of amexAB-oprM- and mexXY-deficient double mutant. Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent. These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

scholarly journals Phenotypic Variation of Tobramycin and Ofloxacin Resistance of Pseudomonas aeruginosa by Repeated Exhibition

2020 ◽  
pp. 1-9
Author(s):  
Andry Maharo Andrianarivelo ◽  
Christian Emmanuel Mahavy ◽  
Blandine Andrianarisoa ◽  
Tsiry Rasamiravaka
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phenotypic Variation ◽  
Culture Medium ◽  
Wild Strain ◽  
Resistance Mechanisms ◽  
Luria Bertani ◽  
University Hospital ◽  
Buffer System ◽  
Visual Evaluation ◽  
Almost All

Pseudomonas aeruginosa has the ability to resist almost all available antibiotics by rapidly accumulating multiple resistance mechanisms and thus lead to a therapeutic impasse and higher mortality in infected patients. The objective of this study was to assess the phenotypic variation in resistance to tobramycin and ofloxacin from Pseudomonas aeruginosa by repeated exhibition after determination of the minimum inhibitory concentration. This is a prospective and descriptive study carried out in the Laboratory of Microbiology of Fundamental and Applied Biochemistry (Faculty of Sciences, Antananarivo) during the month of January 2020. The strains studied were the virulent wild strain of Pseudomonas aeruginosa PAO1 supplied by the Laboratory and two clinical strains of Pseudomonas aeruginosa from the Microbiology Laboratory of the Joseph Ravoahangy Andrianavalona University Hospital Center, Antananarivo. The strains of P. aeruginosa were cultured in the liquid culture medium (which is Luria Bertani, added with a buffer system of 3- (N-morpholino) propanesulfonic acid (LB-MOPS) which will stabilize the pH and a solid culture medium which is Columbia agar. Repeated exhibition to Tobramycin and Ofloxacin from these strains have been made. The MIC is determined by a visual evaluation of the turbidity of the various wells of the microplate. The MIC value of Pseudomonas aeruginosa with tobramycin and ofloxacin is very variable for the initial MIC until the 5th generation after repeated exhibition. More Pseudomonas aeruginosa is exposed to an antibiotic many times, the more it develops resistance to this antibiotic, even being sensitive at the start. That is to say, clinically, the dose prescribed for the antibiotic has been greatly exceeded if Pseudomonas aeruginosa is repeatedly exposed to the same antibiotic.

scholarly journals Multidrug efflux in intrinsic resistance to trimethoprim and sulfamethoxazole in Pseudomonas aeruginosa.

1996 ◽  
Vol 40 (10) ◽  
pp. 2288-2290 ◽  
Author(s):  
T Köhler ◽  
M Kok ◽  
M Michea-Hamzehpour ◽  
P Plesiat ◽  
N Gotoh ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane Proteins ◽  
Multiple Drug ◽  
Drug Efflux ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Intrinsic Resistance ◽  
Wild Type Parent ◽  
Multidrug Efflux System

Pseudomonas aeruginosa possesses at least two multiple drug efflux systems which are defined by the outer membrane proteins OprM and OprJ. We have found that mutants overexpressing OprM were two- and eightfold more resistant than their wild-type parent to sulfamethoxazole (SMX) and trimethoprim (TMP), respectively. For OprJ-overproducing strains, MICs of TMP increased fourfold but those of SMX were unchanged. Strains overexpressing OprM, but not those overexpressing OprJ, became hypersusceptible to TMP and SMX when oprM was inactivated. The wild-type antibiotic profile could be restored in an oprM mutant by transcomplementation with the cloned oprM gene. These results demonstrate that the mexABoprM multidrug efflux system is mainly responsible for the intrinsic resistance of P. aeruginosa to TMP and SMX.

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