scholarly journals Genetic Determinants Involved in the Susceptibility of Pseudomonas aeruginosa to β-Lactam Antibiotics

2010 ◽  
Vol 54 (10) ◽  
pp. 4159-4167 ◽  
Author(s):  
Carolina Alvarez-Ortega ◽  
Irith Wiegand ◽  
Jorge Olivares ◽  
Robert E. W. Hancock ◽  
José Luis Martínez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Detailed Analysis ◽  
Barrier Function ◽  
Mechanisms Of Action ◽  
Mutant Library ◽  
Genetic Determinants ◽  
Intrinsic Resistance ◽  
Resistance Phenotype ◽  
Mixed Phenotype ◽  
Alginate Biosynthesis

ABSTRACT The resistome of P. aeruginosa for three β-lactam antibiotics, namely, ceftazidime, imipenem, and meropenem, was deciphered by screening a comprehensive PA14 mutant library for mutants with increased or reduced susceptibility to these antimicrobials. Confirmation of the phenotypes of all selected mutants was performed by Etest. Of the total of 78 confirmed mutants, 41 demonstrated a reduced susceptibility phenotype and 37 a supersusceptibility (i.e., altered intrinsic resistance) phenotype, with 6 mutants demonstrating a mixed phenotype, depending on the antibiotic. Only three mutants demonstrated reduced (PA0908) or increased (glnK and ftsK) susceptibility to all three antibiotics. Overall, the mutant profiles of susceptibility suggested distinct mechanisms of action and resistance for the three antibiotics despite their similar structures. More detailed analysis indicated important roles for novel and known β-lactamase regulatory genes, for genes with likely involvement in barrier function, and for a range of regulators of alginate biosynthesis.

scholarly journals Complex Ciprofloxacin Resistome Revealed by Screening a Pseudomonas aeruginosa Mutant Library for Altered Susceptibility

2008 ◽  
Vol 52 (12) ◽  
pp. 4486-4491 ◽  
Author(s):  
Elena B. M. Breidenstein ◽  
Bhavjinder K. Khaira ◽  
Irith Wiegand ◽  
Joerg Overhage ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mutant Library ◽  
Intrinsic Resistance ◽  
Adaptive Resistance ◽  
Broth Dilution

ABSTRACT Pseudomonas aeruginosa offers substantial therapeutic challenges due to its high intrinsic resistance to many antibiotics and its propensity to develop mutational and/or adaptive resistance. The PA14 comprehensive mutant library was screened for mutants exhibiting either two- to eightfold increased susceptibilities (revealing genes involved in intrinsic resistance) or decreased susceptibilities (mutational resistance) to the fluoroquinolone ciprofloxacin. Thirty-five and 79 mutants with increased and decreased susceptibilities, respectively, were identified, as confirmed by broth dilution.

scholarly journals Genomewide Identification of Genetic Determinants of Antimicrobial Drug Resistance in Pseudomonas aeruginosa

2009 ◽  
Vol 53 (6) ◽  
pp. 2522-2531 ◽  
Author(s):  
Andreas Dötsch ◽  
Tanja Becker ◽  
Claudia Pommerenke ◽  
Zofia Magnowska ◽  
Lothar Jänsch ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Resistance ◽  
Cost Of Care ◽  
Antimicrobial Drug ◽  
Effective Therapy ◽  
Gram Negative Bacteria ◽  
Genetic Determinants ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Antimicrobial Drug Resistance

ABSTRACT The emergence of antimicrobial drug resistance is of enormous public concern due to the increased risk of delayed treatment of infections, the increased length of hospital stays, the substantial increase in the cost of care, and the high risk of fatal outcomes. A prerequisite for the development of effective therapy alternatives is a detailed understanding of the diversity of bacterial mechanisms that underlie drug resistance, especially for problematic gram-negative bacteria such as Pseudomonas aeruginosa. This pathogen has impressive chromosomally encoded mechanisms of intrinsic resistance, as well as the potential to mutate, gaining resistance to current antibiotics. In this study we have screened the comprehensive nonredundant Harvard PA14 library for P. aeruginosa mutants that exhibited either increased or decreased resistance against 19 antibiotics commonly used in the clinic. This approach identified several genes whose inactivation sensitized the bacteria to a broad spectrum of different antimicrobials and uncovered novel genetic determinants of resistance to various classes of antibiotics. Knowledge of the enhancement of bacterial susceptibility to existing antibiotics and of novel resistance markers or modifiers of resistance expression may lay the foundation for effective therapy alternatives and will be the basis for the development of new strategies in the control of problematic multiresistant gram-negative bacteria.

scholarly journals Evaluation of InSeq to Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization

2018 ◽  
Author(s):  
Ramamoorthy Sivakumar ◽  
Jothi Ranjani ◽  
Udayakumar S. Vishnu ◽  
Sathyanarayanan Jayashree ◽  
Gabriel L. Lozano ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Root Colonization ◽  
Physiological Responses ◽  
Deletion Mutants ◽  
Mutant Library ◽  
Amino Acid Catabolism ◽  
Wild Type ◽  
Genetic Determinants ◽  
Corn Root ◽  
Corn Roots

AbstractThe reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the TnSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5-to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P. aeruginosa PGPR2 for root colonization.

scholarly journals High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials

2020 ◽  
Vol 367 (22) ◽  
Author(s):  
Chris Coward ◽  
Gopujara Dharmalingham ◽  
Omar Abdulle ◽  
Tim Avis ◽  
Stephan Beisken ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Selective Advantage ◽  
Mechanisms Of Action ◽  
Over Expression ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Established Technique ◽  
Bacterial Transposon

ABSTRACT The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon–phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

scholarly journals Genetic determinants of Pseudomonas aeruginosa biofilm establishment

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 431-441 ◽  
Author(s):  
Mathias Müsken ◽  
Stefano Di Fiore ◽  
Andreas Dötsch ◽  
Rainer Fischer ◽  
Susanne Häussler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Growth Conditions ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Laser ◽  
Repair System ◽  
Ph Homeostasis ◽  
Genetic Determinants ◽  
Complex Process ◽  
Genes Encoding

The establishment of bacterial biofilms on surfaces is a complex process that requires various factors for each consecutive developmental step. Here we report the screening of the comprehensive Harvard Pseudomonas aeruginosa PA14 mutant library for mutants exhibiting an altered biofilm phenotype. We analysed the capability of all mutants to form biofilms at the bottom of a 96-well plate by the use of an automated confocal laser-scanning microscope and found 394 and 285 genetic determinants of reduced and enhanced biofilm production, respectively. Overall, 67 % of the identified mutants were affected within genes encoding hypothetical proteins, indicating that novel developmental pathways are likely to be dissected in the future. Nevertheless, a common theme that emerged from the analysis of the genes with a predicted function is that the establishment of a biofilm requires regulatory components that are involved in survival under microaerophilic growth conditions, arginine metabolism, alkyl-quinolone signalling, pH homeostasis and the DNA repair system.

Genetic determinants involved in the biodegradation of naphthalene and phenanthrene in Pseudomonas aeruginosa PAO1

2014 ◽  
Vol 22 (9) ◽  
pp. 6743-6755 ◽  
Author(s):  
Jing Qi ◽  
Bobo Wang ◽  
Jing Li ◽  
Huanhuan Ning ◽  
Yingjuan Wang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genetic Determinants ◽  
Pseudomonas Aeruginosa Pao1

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.

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.

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