scholarly journals The Genomic Basis of Rapid Adaptation to Antibiotic Combination Therapy in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Camilo Barbosa ◽  
Niels Mahrt ◽  
Julia Bunk ◽  
Matthias Graßer ◽  
Philip Rosenstiel ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Combination Therapy ◽  
Bacterial Resistance ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Opportunistic Pathogen ◽  
Regulatory Function ◽  
Central Target ◽  
Resistance Evolution ◽  
Intergenic Regions

Abstract Combination therapy is a common antibiotic treatment strategy that aims at minimizing the risk of resistance evolution in several infectious diseases. Nonetheless, evidence supporting its efficacy against the nosocomial opportunistic pathogen Pseudomonas aeruginosa remains elusive. Identification of the possible evolutionary paths to resistance in multidrug environments can help to explain treatment outcome. For this purpose, we here performed whole-genome sequencing of 127 previously evolved populations of P. aeruginosa adapted to sublethal doses of distinct antibiotic combinations and corresponding single-drug treatments, and experimentally characterized several of the identified variants. We found that alterations in the regulation of efflux pumps are the most favored mechanism of resistance, regardless of the environment. Unexpectedly, we repeatedly identified intergenic variants in the adapted populations, often with no additional mutations and usually associated with genes involved in efflux pump expression, possibly indicating a regulatory function of the intergenic regions. The experimental analysis of these variants demonstrated that the intergenic changes caused similar increases in resistance against single and multidrug treatments as those seen for efflux regulatory gene mutants. Surprisingly, we could find no substantial fitness costs for a majority of these variants, most likely enhancing their competitiveness toward sensitive cells, even in antibiotic-free environments. We conclude that the regulation of efflux is a central target of antibiotic-mediated selection in P. aeruginosa and that, importantly, changes in intergenic regions may represent a usually neglected alternative process underlying bacterial resistance evolution, which clearly deserves further attention in the future.

scholarly journals Pseudomonas aeruginosa Polynucleotide Phosphorylase Controls Tolerance to Aminoglycoside Antibiotics by Regulating the MexXY Multidrug Efflux Pump

2020 ◽  
Author(s):  
Zheng Fan ◽  
Xiaolei Pan ◽  
Dan Wang ◽  
Ronghao Chen ◽  
Tongtong Fu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Efflux Pump ◽  
Opportunistic Pathogen ◽  
Aminoglycoside Antibiotics ◽  
Polynucleotide Phosphorylase ◽  
Multidrug Efflux ◽  
Intrinsic Resistance ◽  
Multidrug Efflux Pump ◽  
Fluoroquinolone Antibiotics

Pseudomonas aeruginosa is an opportunistic pathogen that shows high intrinsic resistance to a variety of antibiotics. The MexX-MexY-OprM efflux pump plays an important role in the bacterial resistance to aminoglycoside antibiotics. Polynucleotide phosphorylase (PNPase) is a highly conserved exonuclease that plays important roles in RNA processing and bacterial response to environmental stresses. Previously, we demonstrated that PNPase controls the tolerance to fluoroquinolone antibiotics by influencing the production of pyocin in P. aeruginosa. In this study, we found that mutation of the PNPase coding gene (pnp) in P. aeruginosa increases the bacterial tolerance to aminoglycoside antibiotics. We further demonstrate that upregulation of the mexXY genes is responsible for the increased tolerance in the pnp mutant. Furthermore, our experimental results revealed that PNPase controls translation of the armZ mRNA through its 5′ untranslated region (5′-UTR). ArmZ had previously been shown to positively regulate the expression of mexXY. Therefore, our results revealed a novel role of PNPase in the regulation of armZ and subsequently the MexXY efflux pump.

Pseudomonas aeruginosa Oligoribonuclease Controls Susceptibility to Polymyxin B by Regulating Pel Exopolysaccharide Production

2022 ◽  
Author(s):  
Baopeng Yang ◽  
Yujun Jiang ◽  
Yongxin Jin ◽  
Fang Bai ◽  
Zhihui Cheng ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Defense Mechanism ◽  
Extracellular Polysaccharide ◽  
Opportunistic Pathogen ◽  
Polymyxin B ◽  
Guanosine Monophosphate ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Bacterial Survival

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa . Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa .

scholarly journals ANTIBACTERIAL RESISTANCE PATTERN OF PSEUDOMONAS AERUGINOSA ISOLATED FROM CLINICAL SAMPLES AT A GENERAL HOSPITAL IN PADANG, WEST SUMATRA, INDONESIA

2017 ◽  
Vol 10 (8) ◽  
pp. 158
Author(s):  
Rustini Rustini ◽  
Jamsari Jamsari ◽  
Marlina Marlina ◽  
Nasrul Zubir ◽  
Yori Yuliandra
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Clinical Sample ◽  
Opportunistic Pathogen ◽  
Positive Control ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Resistance Pattern ◽  
Tract Infections ◽  
Almost All

Objectives: Pseudomonas aeruginosa is an opportunistic pathogen that has an innate resistance to some antibiotics. This bacterium is one of the mostcommon causes of nosocomial infections that include surgical wound infections, burns, and urinary tract infections. The bacteria have been reportedlyresistant to many antibiotics and have developed multidrug resistance (MDR). The objective of the study was to determine the resistance pattern ofP. aeruginosa isolated from clinical samples of patients against some major antibiotics.Methods: Isolates of P. aeruginosa were obtained from clinical sample of urine, sputum, swabs, pus, feces, and blood and cultured in cetrimide agar. P.aeruginosa ATCC 27853 was used as a positive control. The antibacterial susceptibility testing was conducted against 13 antibiotics: Ceftazidime, cefotaxime,ceftriaxone, cefoperazone, ciprofloxacin, levofloxacin, ofloxacin, gentamicin, amikacin, piperacillin, ticarcillin, meropenem, and imipenem. The examinationwas carried out using agar diffusion method of Kirby-Bauer and following the standards from Clinical and Laboratory Standards Institute (CLSI).Results: The results showed that bacterial resistance was established against all tested antibiotics. The highest number of resistance was shownagainst ceftriaxone (44.21%), whereas the most susceptibility was exhibited against amikacin (only 9.47% of resistance). MDR P. aeruginosa (MDRPA)was detected on almost all clinical samples tested, except the feces. The sample with the highest percentage of MDRPA was the pus.Conclusion: The study concludes that the most effective antibiotic against P. aeruginosa is amikacin (91.51%), whereas the most resistance is exhibited to ceftriaxone (43.16%).

scholarly journals Effect of MexXY Overexpression on Ceftobiprole Susceptibility in Pseudomonas aeruginosa

2009 ◽  
Vol 53 (7) ◽  
pp. 2785-2790 ◽  
Author(s):  
Ellen Z. Baum ◽  
Steven M. Crespo-Carbone ◽  
Brian J. Morrow ◽  
Todd A. Davies ◽  
Barbara D. Foleno ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Broad Spectrum ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Resistance Mechanism ◽  
Common Mechanism ◽  
Spontaneous Mutant ◽  
Minimal Effect ◽  
Strain Pao1

ABSTRACT Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus broad-spectrum cephalosporin, has activity (MIC for 50% of strains tested, ≤4 μg/ml) against many Pseudomonas aeruginosa strains. A common mechanism of P. aeruginosa resistance to β-lactams, including cefepime and ceftazidime, is efflux via increased expression of Mex pumps, especially MexAB. MexXY has differential substrate specificity, recognizing cefepime but not ceftazidime. In ceftobiprole clinical studies, paired isolates of P. aeruginosa from four subjects demonstrated ceftobiprole MICs of 2 to 4 μg/ml at baseline but 16 μg/ml posttreatment, unrelated to β-lactamase levels. Within each pair, the level of mexXY RNA, but not mexAB, mexCD, and mexEF, increased by an average of 50-fold from baseline to posttreatment isolates. Sequencing of the negative regulatory gene mexZ indicated that each posttreatment isolate contained a mutation not present at baseline. mexXY expression as a primary ceftobiprole and cefepime resistance mechanism was further examined in isogenic pairs by using cloned mexXY and mexZ. Expression of cloned mexXY in strain PAO1 or in a baseline isolate increased the ceftobiprole MIC to that for the posttreatment isolate. In contrast, in posttreatment isolates, lowering mexXY expression via introduction of cloned mexZ decreased the ceftobiprole MIC to that for the baseline isolates. Similar changes were observed for cefepime. A spontaneous mutant selectively overexpressing mexXY displayed a fourfold elevation in its ceftobiprole MIC, while overexpression of mexAB, -CD, and -EF had a minimal effect. These data indicate that ceftobiprole, like cefepime, is an atypical β-lactam that is a substrate for the MexXY efflux pump in P. aeruginosa.

scholarly journals Role of RgsA in oxidative stress resistance in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Shuyi Hou ◽  
Jiaqing Zhang ◽  
Xiaobo Ma ◽  
Qiang Hong ◽  
Lili Fang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Regulatory Function ◽  
Rna Molecules ◽  
Protein Mass ◽  
Protein Levels ◽  
Protein Mass Spectrometry ◽  
Small Regulatory Rnas

AbstractPseudomonas aeruginosa is an extremely common opportunistic pathogen in clinical practice. Patients with metabolic disorders, hematologic diseases, malignancies, who have undergone surgery or who have received certain treatments are susceptible to this bacterium. In addition, P. aeruginosa is a multidrug-resistant that tends to form biofilms and is refractory to treatment. Small regulatory RNAs are RNA molecules that are 40–500 nucleotides long, possess regulatory function, are ubiquitous in bacteria, and are also known as small RNA (sRNA). sRNAs play important regulatory roles in various vital life processes in diverse bacteria and their quantity and diversity of regulatory functions exceeds that of proteins. In this study, we showed that deletion of the sRNA RgsA decreases the growth rate and ability to resist different concentrations and durations of peroxide in P. aeruginosa. These decreases occur not only in the planktonic state, but also in the biofilm state. Finally, protein mass spectrometry was employed to understand changes in the entire protein spectrum. The results presented herein provide a description of the role of RgsA in the life activities of P. aeruginosa at the molecular, phenotypic, and protein levels.

scholarly journals Constitutive Activation of MexT by Amino Acid Substitutions Results in MexEF-OprN Overproduction in Clinical Isolates ofPseudomonas aeruginosa

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Paulo Juarez ◽  
Isabelle Broutin ◽  
Christophe Bordi ◽  
Patrick Plésiat ◽  
Catherine Llanes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Content Type ◽  
Constitutive Activation ◽  
Multidrug Efflux System

ABSTRACTWhen overproduced, the multidrug efflux system MexEF-OprN increases the resistance ofPseudomonas aeruginosato fluoroquinolones, chloramphenicol, and trimethoprim. In this work, we demonstrate that gain-of-function mutations in the regulatory genemexTresult in oligomerization of the LysR regulator MexT, constitutive upregulation of the efflux pump, and increased resistance in clinical isolates.

scholarly journals Bacterial biodiversity drives the evolution of CRISPR-based phage resistance in Pseudomonas aeruginosa

2019 ◽  
Author(s):  
Ellinor O Alseth ◽  
Elizabeth Pursey ◽  
Adela M Luján ◽  
Isobel McLeod ◽  
Clare Rollie ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Dna Sequences ◽  
Bacterial Species ◽  
Opportunistic Pathogen ◽  
Phage Resistance ◽  
Natural Environments ◽  
Human Pathogens ◽  
Resistance Evolution ◽  
Trade Offs

Approximately half of all bacterial species encode CRISPR-Cas adaptive immune systems1, which provide immunological memory by inserting short DNA sequences from phage and other parasitic DNA elements into CRISPR loci on the host genome2. Whereas CRISPR loci evolve rapidly in natural environments3, bacterial species typically evolve phage resistance by the mutation or loss of phage receptors under laboratory conditions4,5. Here, we report how this discrepancy may in part be explained by differences in the biotic complexity of in vitro and natural environments6,7. Specifically, using the opportunistic pathogen Pseudomonas aeruginosa and its phage DMS3vir, we show that coexistence with other human pathogens amplifies the fitness trade-offs associated with phage receptor mutation, and therefore tips the balance in favour of CRISPR-based resistance evolution. We also demonstrate that this has important knock-on effects for P. aeruginosa virulence, which became attenuated only if the bacteria evolved surface-based resistance. Our data reveal that the biotic complexity of microbial communities in natural environments is an important driver of the evolution of CRISPR-Cas adaptive immunity, with key implications for bacterial fitness and virulence.

scholarly journals The small RNAs PA2952.1 and PrrH as regulators of virulence, motility and iron metabolism in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Shannon R. Coleman ◽  
Manjeet Bains ◽  
Maren L. Smith ◽  
Victor Spicer ◽  
Ying Lao ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Differential Expression ◽  
Small Rnas ◽  
Efflux Pump ◽  
Iron Acquisition ◽  
Opportunistic Pathogen ◽  
Multidrug Resistant ◽  
Rna Seq ◽  
Swimming Motility ◽  
A Genome

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that undergoes swarming motility in response to semisolid conditions with amino acids as a nitrogen source. With a genome encoding hundreds of potential intergenic small RNAs (sRNAs), P. aeruginosa can easily adapt to different conditions and stresses. We previously identified 20 sRNAs that were differentially expressed under swarming conditions. Here, these sRNA were overexpressed in strain PAO1 and subjected to an array of phenotypic screens. Overexpression of PrrH resulted in decreased swimming motility; while a ΔprrH mutant had decreased cytotoxicity and increased pyoverdine production. Overexpression of the previously uncharacterized PA2952.1 resulted in decreased swarming and swimming motilities, increased gentamicin and tobramycin resistance under swarming conditions, and increased trimethoprim susceptibility. RNA-Seq and proteomics were performed on the wildtype overexpressing PA2952.1 cf. the empty vector control under swarming conditions, and revealed the differential expression (FC ≥ ±1.5) of 784 genes and the differential abundance (FC ≥ ±1.25) of 59 proteins. Amongst these were found 73 transcriptional regulators, two-component systems and sigma and anti-sigma factors. Downstream effectors included downregulated pili and flagellar genes, the upregulated efflux pump MexGHI-OpmD, and the upregulated arn operon. Genes involved in iron and zinc uptake were generally upregulated, and certain pyoverdine genes were upregulated. Overall, the sRNAs PA2952.1 and PrrH appeared to be involved in regulating virulence-related programs in P. aeruginosa, including iron acquisition and motility. IMPORTANCE Due to the rising incidence of multidrug-resistant strains and the difficulty of eliminating P. aeruginosa infections, it is important to understand the regulatory mechanisms that allow this bacterium to adapt to and thrive under a variety of conditions. Small RNAs (sRNAs) are one regulatory mechanism that allow bacteria to change the amount of protein synthesized. In this study, we overexpressed 20 different sRNAs in order to investigate how this might affect different bacterial behaviours. We found that one of the sRNAs, PrrH, played a role in swimming motility and virulence phenotypes, indicating a potentially important role in clinical infections. Another sRNA, PA2952.1, affected other clinically relevant phenotypes, including motility and antibiotic resistance. RNA-Seq and proteomics of the strain overexpressing PA2952.1 revealed the differential expression of 784 genes and 59 proteins, with a total of 73 regulatory factors. This substantial dysregulation indicates an important role for the sRNA PA2952.1.

scholarly journals Pseudomonas aeruginosa adapts to octenidine via a combination of efflux and membrane remodelling

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lucy J. Bock ◽  
Philip M. Ferguson ◽  
Maria Clarke ◽  
Vichayanee Pumpitakkul ◽  
Matthew E. Wand ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Fold Increase ◽  
Opportunistic Pathogen ◽  
Major Facilitator Superfamily ◽  
Membrane Composition ◽  
Base Pair Deletion ◽  
Family Gene ◽  
Major Facilitator

AbstractPseudomonas aeruginosa is an opportunistic pathogen capable of stably adapting to the antiseptic octenidine by an unknown mechanism. Here we characterise this adaptation, both in the laboratory and a simulated clinical setting, and identify a novel antiseptic resistance mechanism. In both settings, 2 to 4-fold increase in octenidine tolerance was associated with stable mutations and a specific 12 base pair deletion in a putative Tet-repressor family gene (smvR), associated with a constitutive increase in expression of the Major Facilitator Superfamily (MFS) efflux pump SmvA. Adaptation to higher octenidine concentrations led to additional stable mutations, most frequently in phosphatidylserine synthase pssA and occasionally in phosphatidylglycerophosphate synthase pgsA genes, resulting in octenidine tolerance 16- to 256-fold higher than parental strains. Metabolic changes were consistent with mitigation of oxidative stress and altered plasma membrane composition and order. Mutations in SmvAR and phospholipid synthases enable higher level, synergistic tolerance of octenidine.

scholarly journals Discovery and Characterization of Chemical Compounds That Inhibit the Function of Aspartyl-tRNA Synthetase from Pseudomonas aeruginosa

2017 ◽  
Vol 23 (3) ◽  
pp. 294-301 ◽  
Author(s):  
Araceli Corona ◽  
Stephanie O. Palmer ◽  
Regina Zamacona ◽  
Benjamin Mendez ◽  
Frank B. Dean ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aspartic Acid ◽  
Cell Cultures ◽  
Human Cell ◽  
Efflux Pump ◽  
Chemical Compounds ◽  
Opportunistic Pathogen ◽  
Trna Synthetase ◽  
Bacterial Strains ◽  
Human Cell Cultures

Pseudomonas aeruginosa, an opportunistic pathogen, is highly susceptible to developing resistance to multiple antibiotics. The gene encoding aspartyl-tRNA synthetase (AspRS) from P. aeruginosa was cloned and the resulting protein characterized. AspRS was kinetically evaluated, and the KM values for aspartic acid, ATP, and tRNA were 170, 495, and 0.5 μM, respectively. AspRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 1690 chemical compounds, resulting in the identification of two inhibitory compounds, BT02A02 and BT02C05. The minimum inhibitory concentrations (MICs) were determined against nine clinically relevant bacterial strains, including efflux pump mutant and hypersensitive strains of P. aeruginosa. The compounds displayed broad-spectrum antibacterial activity and inhibited growth of the efflux and hypersensitive strains with MICs of 16 μg/mL. Growth of wild-type strains were unaffected, indicating that efflux was likely responsible for this lack of activity. BT02A02 did not inhibit growth of human cell cultures at any concentration. However, BT02C05 did inhibit human cell cultures with a cytotoxicity concentration (CC50) of 61.6 μg/mL. The compounds did not compete with either aspartic acid or ATP for binding AspRS, indicating that the mechanism of action of the compound occurs outside the active site of aminoacylation.

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