Universal antibiotic tolerance arising from antibiotic-triggered accumulation of redox metabolites

AbstractPseudomonas aeruginosais an opportunistic pathogen that often infects open wounds or patients with cystic fibrosis. Once established,P. aeruginosainfections are notoriously difficult to eradicate. This difficulty is in part due to the ability ofP. aeruginosato tolerate antibiotic treatment at the individual-cell level or through collective behaviors. Here we describe a new mechanism by whichP. aeruginosatolerates antibiotic treatment by modulating its global cellular metabolism. In particular, treatment ofP. aeruginosawith sublethal concentrations of antibiotics covering all major classes promoted accumulation of the redox-sensitive phenazine - pyocyanin (PYO). PYO in turn conferred general tolerance against diverse antibiotics for bothP. aeruginosaand other Gram-negative and Gram-positive bacteria. We show that PYO promotes energy generation to enhance the activity of efflux pumps, leading to enhanced antibiotic tolerance. This property is shared by other redox-active phenazines produced byP. aeruginosa. Our discovery sheds new insights into the physiological functions of phenazines and has implications for designing effective antibiotic treatment protocols.Author SummaryAntibiotic tolerance can facilitate the evolution of resistance, and here we describe a previously unknown mechanism of collective antibiotic tolerance inPseudomonas aeruginosa. In particular,P. aeruginosatreated with sublethal concentrations of antibiotics covering all major classes promotes accumulation of pyocyanin (PYO), an important virulence factor. In turn, PYO confers general tolerance against diverse antibiotics for bothP. aeruginosaand other bacteria. Our discovery is a perfect example of what Nietzsche once said:That which does not kill me makes me stronger.

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PQS Produced by the Pseudomonas aeruginosa Stress Response Repels Swarms Away from Bacteriophage and Antibiotics

Journal of Bacteriology ◽

10.1128/jb.00383-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 7

Author(s):

Jean-Louis Bru ◽

Brandon Rawson ◽

Calvin Trinh ◽

Katrine Whiteson ◽

Nina Molin Høyland-Kroghsbo ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Stress Response ◽

Antibiotic Treatment ◽

Bacterial Pathogen ◽

Warning Signal ◽

Opportunistic Pathogen ◽

Evolutionary Strategy ◽

Swarming Motility ◽

Content Type ◽

Bacteriophage Infection

ABSTRACT We investigate the effect of bacteriophage infection and antibiotic treatment on the coordination of swarming, a collective form of flagellum- and pilus-mediated motility in bacteria. We show that phage infection of the opportunistic bacterial pathogen Pseudomonas aeruginosa abolishes swarming motility in the infected subpopulation and induces the release of the Pseudomonas quinolone signaling molecule PQS, which repulses uninfected subpopulations from approaching the infected area. These mechanisms have the overall effect of limiting the infection to a subpopulation, which promotes the survival of the overall population. Antibiotic treatment of P. aeruginosa elicits the same response, abolishing swarming motility and repulsing approaching swarms away from the antibiotic-treated area through a PQS-dependent mechanism. Swarms are entirely repelled from the zone of antibiotic-treated P. aeruginosa, consistent with a form of antibiotic evasion, and are not repelled by antibiotics alone. PQS has multiple functions, including serving as a quorum-sensing molecule, activating an oxidative stress response, and regulating the release of virulence and host-modifying factors. We show that PQS serves additionally as a stress warning signal that causes the greater population to physically avoid cell stress. The stress response at the collective level observed here in P. aeruginosa is consistent with a mechanism that promotes the survival of bacterial populations. IMPORTANCE We uncover a phage- and antibiotic-induced stress response in the clinically important opportunistic pathogen Pseudomonas aeruginosa. Phage-infected P. aeruginosa subpopulations are isolated from uninfected subpopulations by the production of a stress-induced signal. Activation of the stress response by antibiotics causes P. aeruginosa to physically be repelled from the area containing antibiotics altogether, consistent with a mechanism of antibiotic evasion. The stress response observed here could increase P. aeruginosa resilience against antibiotic treatment and phage therapy in health care settings, as well as provide a simple evolutionary strategy to avoid areas containing stress.

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The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

Infection and Immunity ◽

10.1128/iai.00764-16 ◽

2017 ◽

Vol 85 (5) ◽

Cited By ~ 21

Author(s):

Alexandria A. Reinhart ◽

Angela T. Nguyen ◽

Luke K. Brewer ◽

Justin Bevere ◽

Jace W. Jones ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Small Rnas ◽

Iron Homeostasis ◽

Critical Role ◽

Opportunistic Pathogen ◽

Lung Infection ◽

Content Type ◽

The Individual

ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrHIG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrHIG sequence [prrF(ΔHIG)]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.

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RpoS Mutation Leads to Prolonged Biofilm Mode of Growth and a Higher Fitness in Pseudomonas Aeruginosa Biofilms

10.21203/rs.3.rs-488211/v1 ◽

2021 ◽

Author(s):

Xiangke Duan ◽

Yanrong Pan ◽

Zhao Cai ◽

Yumei Liu ◽

Yingdan Zhang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Treatment ◽

Biofilm Formation ◽

Point Mutations ◽

Opportunistic Pathogen ◽

Antimicrobial Treatment ◽

Clinical Isolates ◽

High Dose ◽

Sequencing Analysis ◽

Cyclic Treatment

Abstract BackgroundPseudomonas aeruginosa is a notorious opportunistic pathogen causing various biofilm-related infections. Biofilm formation is a unique microbial strategy that allows P. aeruginosa to survive adverse conditions such as antibiotic treatment and human immune responses. ResultsIn this study, we experimentally evolved P. aeruginosa PAO1 bioﬁlms for cyclic treatment in the presence of high dose of imipenem, and enriched hyperbiofilm mutants within six cycles in two independent lineages. The competition assay showed the evolved hyperbiofilm mutants can outcompete the ancestral strain within biofilm by prolonging the biofilm mode of growth but not in planktonic cultures. Whole-genome sequencing analysis revealed the hyperbiofilm phenotype is caused by point mutations in rpoS gene in all independently evolved mutants and the same mutation was found in P. aeruginosa clinical isolates. We further showed that mutation in rpoS enhanced biofilm formation by prolonging the biofilm mode of growth and elevating the intracellular c-di-GMP level. Mutation in rpoS increased pyocyanin production and virulence in both P. aeruginosa laboratory strains and clinical isolates. ConclusionHere, our study revealed that antibiotic treatment of biofilm-related P. aeruginosa infections might induce a hyperbiofilm phenotype via rpoS mutation, which might partially explain antimicrobial treatment failure of many P. aeruginosa biofilm-related infections.

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Signal Sensing and Transduction Are Conserved between the Periplasmic Sensory Domains of BifA and SagS

mSphere ◽

10.1128/msphere.00442-19 ◽

2019 ◽

Vol 4 (4) ◽

Author(s):

Jozef Dingemans ◽

Rebecca E. Al-Feghali ◽

Holger Sondermann ◽

Karin Sauer

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Structural Similarity ◽

Opportunistic Pathogen ◽

Sequence Conservation ◽

Sensor Kinase ◽

Antibiotic Tolerance ◽

Bacterial Cells ◽

Chronic Infections ◽

Content Type

ABSTRACT The hybrid sensor kinase SagS of Pseudomonas aeruginosa plays a key role in the transition from the planktonic to the biofilm mode of growth. Recently, we have shown that distinct sets of residues in its periplasmic HmsP sensory domain are involved in the regulation of biofilm formation or antibiotic tolerance. Interestingly, the HmsP domain of the phosphodiesterase BifA shows great predicted structural similarity to that of SagS, despite moderate sequence conservation and only a number of residues involved in SagS signaling being conserved between both proteins. Based on this observation, we hypothesized that BifA and SagS may use similar mechanisms to sense and transduce signals perceived at their periplasmic HmsP domains and, therefore, may be interchangeable. To test this hypothesis, we constructed SagS hybrids in which the HmsP domain of SagS was replaced by that of BifA (and vice versa) or by the DISMED2 sensory domain of NicD. The SagS-BifA hybrid restored attachment and biofilm formation by the ΔbifA mutant. Likewise, while the NicD-SagS hybrid was nonfunctional, the BifA-SagS hybrid partially restored pathways leading to biofilm formation and antibiotic tolerance in a ΔsagS mutant background. Furthermore, alanine substitution of key residues previously associated with the biofilm formation and antibiotic tolerance pathways of SagS impaired signal transduction by the BifA-SagS hybrid in a similar way to SagS. In conclusion, our data indicate that the nature of the sensory domain is important for proper functionality of the cytoplasmic effector domains and that signal sensing and transduction are likely conserved in SagS and BifA. IMPORTANCE Biofilms have been associated with more than 60% of all recalcitrant and chronic infections and can render bacterial cells up to a thousand times more resistant to antibiotics than planktonic cells. Although it is known that the transition from the planktonic to the biofilm mode of growth involves two-component regulatory systems, increased c-di-GMP levels, and quorum sensing systems among others, the exact signaling events that lead to biofilm formation remain unknown. In the opportunistic pathogen Pseudomonas aeruginosa, the hybrid sensor kinase SagS regulates biofilm formation and antibiotic tolerance through two independent pathways via distinct residues in its periplasmic sensory domain. Interestingly, the sensory domains of SagS and BifA show great predicted structural similarity despite moderate sequence conservation. Here we show that the sensory domains of BifA and SagS are functionally interchangeable and that they use a similar mechanism of signal sensing and transduction, which broadens our understanding of how bacteria perceive and transduce signals when transitioning to the biofilm mode of growth.

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Parallel evolutionary paths to produce more than one Pseudomonas aeruginosa biofilm phenotype

npj Biofilms and Microbiomes ◽

10.1038/s41522-019-0113-6 ◽

2020 ◽

Vol 6 (1) ◽

Cited By ~ 6

Author(s):

Janne G. Thöming ◽

Jürgen Tomasch ◽

Matthias Preusse ◽

Michal Koska ◽

Nora Grahl ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Parallel Evolution ◽

Opportunistic Pathogen ◽

Clinical Isolates ◽

Transcriptional Responses ◽

Regulatory Pathways ◽

Extracellular Matrix Components ◽

The Individual ◽

Biofilm Development ◽

General Response

AbstractStudying parallel evolution of similar traits in independent within-species lineages provides an opportunity to address evolutionary predictability of molecular changes underlying adaptation. In this study, we monitored biofilm forming capabilities, motility, and virulence phenotypes of a plethora of phylogenetically diverse clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa. We also recorded biofilm-specific and planktonic transcriptional responses. We found that P. aeruginosa isolates could be stratified based on the production of distinct organismal traits. Three major biofilm phenotypes, which shared motility and virulence phenotypes, were produced repeatedly in several isolates, indicating that the phenotypes evolved via parallel or convergent evolution. Of note, while we found a restricted general response to the biofilm environment, the individual groups of biofilm phenotypes reproduced biofilm transcriptional profiles that included the expression of well-known biofilm features, such as surface adhesive structures and extracellular matrix components. Our results provide insights into distinct ways to make a biofilm and indicate that genetic adaptations can modulate multiple pathways for biofilm development that are followed by several independent clinical isolates. Uncovering core regulatory pathways that drive biofilm-associated growth and tolerance towards environmental stressors promises to give clues to host and environmental interactions and could provide useful targets for new clinical interventions.

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rpoS-mutation variants are selected in Pseudomonas aeruginosa biofilms under imipenem pressure

Cell & Bioscience ◽

10.1186/s13578-021-00655-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Xiangke Duan ◽

Yanrong Pan ◽

Zhao Cai ◽

Yumei Liu ◽

Yingdan Zhang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Treatment ◽

Point Mutations ◽

Opportunistic Pathogen ◽

Antimicrobial Treatment ◽

High Dose ◽

Sequencing Analysis ◽

Adverse Conditions ◽

Diguanylate Cyclases ◽

Cyclic Treatment

Abstract Background Pseudomonas aeruginosa is a notorious opportunistic pathogen causing various types of biofilm-related infections. Biofilm formation is a unique microbial strategy that allows P. aeruginosa to survive adverse conditions such as antibiotic treatment and human immune clearance. Results In this study, we experimentally evolved P. aeruginosa PAO1 biofilms for cyclic treatment in the presence of high dose of imipenem, and enriched hyperbiofilm mutants within six cycles in two independent lineages. The competition assay showed that the evolved hyperbiofilm mutants can outcompete the ancestral strain within biofilms but not in planktonic cultures. Whole-genome sequencing analysis revealed the hyperbiofilm phenotype is caused by point mutations in rpoS gene in all independently evolved mutants and the same mutation was found in P. aeruginosa clinical isolates. We further showed that mutation in rpoS gene increased the intracellular c-di-GMP level by turning on the expression of the diguanylate cyclases. Mutation in rpoS increased pyocyanin production and virulence in hyperbiofilm variants. Conclusion Here, our study revealed that antibiotic treatment of biofilm-related P. aeruginosa infections might induce a hyperbiofilm phenotype via rpoS mutation, which might partially explain antimicrobial treatment failure of many P. aeruginosa biofilm-related infections.

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Suspended multiwalled, acid-functionalized carbon nanotubes promote aggregation of the opportunistic pathogen Pseudomonas aeruginosa

10.1101/2020.04.29.068973 ◽

2020 ◽

Author(s):

Kristin Kovach ◽

Indu Venu Sabaraya ◽

Parth Patel ◽

Mary Jo Kirisits ◽

Navid B. Saleh ◽

...

Keyword(s):

Carbon Nanotubes ◽

Pseudomonas Aeruginosa ◽

Functional Materials ◽

Opportunistic Pathogen ◽

Antibiotic Tolerance ◽

Aqueous Systems ◽

Low Concentrations ◽

Animal Pathogens ◽

High Concentrations ◽

Bacterial Aggregates

AbstractThe increasing prevalence of carbon nanotubes (CNTs) as components of new functional materials has the unintended consequence of causing increases in CNT concentrations in aqueous environments. Aqueous systems are reservoirs for bacteria, including human and animal pathogens, that can form biofilms. At high concentrations, CNTs have been shown to display biocidal effects; however, at low concentrations, the interaction between CNTs and bacteria is more complicated, and antimicrobial action is highly dependent upon the properties of the CNTs in suspension. Here, impact of low concentrations of multiwalled CNTs (MWCNTs) on the biofilm-forming opportunistic human pathogen Pseudomonas aeruginosa is studied. Using phase contrast and confocal microscopy, flow cytometry, and antibiotic tolerance assays, it is found that sub-lethal concentrations (2 mg/L) of MWCNTs promote aggregation of P. aeruginosa into multicellular clusters. However, the antibiotic tolerance of these “young” bacterial-CNT aggregates is similar to that of CNT-free cultures. Overall, our results indicate that the co-occurrence of MWCNTs and P. aeruginosa in aqueous systems, which promotes the increased number and size of bacterial aggregates, could increase the dose to which humans or animals are exposed.

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The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin

Microbiology ◽

10.1099/mic.0.031971-0 ◽

2010 ◽

Vol 156 (3) ◽

pp. 678-686 ◽

Cited By ~ 53

Author(s):

Tiffany Vinckx ◽

Qing Wei ◽

Sandra Matthijs ◽

Pierre Cornelis

Keyword(s):

Oxidative Stress ◽

Pseudomonas Aeruginosa ◽

Protective Action ◽

Opportunistic Pathogen ◽

Single Copy ◽

Protective Role ◽

Phenotypic Change ◽

Wild Type ◽

Redox Active ◽

Pyocyanin Production

The LysR-type transcriptional regulator (LTTR) OxyR orchestrates the defence of the opportunistic pathogen Pseudomonas aeruginosa against reactive oxygen species. In previous work we also demonstrated that OxyR is needed for the utilization of the ferrisiderophore pyoverdine, stressing the importance of this regulator. Here, we show that an oxyR mutant is unable to swarm on agar plates, probably as a consequence of absence of production of rhamnolipid surfactant molecules. Another obvious phenotypic change was the increased production of the phenazine redox-active molecule pyocyanin in the oxyR mutant. As already described, the oxyR mutant could not grow in LB medium, unless high numbers of cells (>108 ml−1) were inoculated. However, its growth in Pseudomonas P agar (King's A), a medium inducing pyocyanin production, was like that of the wild-type, suggesting a protective action of this redox-active phenazine compound. This was confirmed by the restoration of the capacity to grow in LB medium upon addition of pure pyocyanin. Although both rhamnolipid and pyocyanin production are controlled by quorum sensing, no obvious changes were observed in the production of N-acylhomoserine lactones or the Pseudomonas quinolone signal (PQS). Complementation of rhamnolipid production and motility, and restoration of normal pyocyanin levels, was only possible when the oxyR gene was in single copy, while pyocyanin levels were increased when oxyR was present in a multicopy vector. Conversely, plating efficiency was increased only when the oxyR gene was present in multicopy, but not when in single copy in the chromosome, due to lower expression of oxyR compared with the wild-type, suggesting that some phenotypes are differently affected in function to the levels of OxyR molecules in the cell. Analysis of transcripts of oxidative stress-response enzymes showed a strong decrease of katB, ahpC and ahpB expression in the oxyR mutant grown in LB, but this was not the case when the mutant was grown on P agar, suggesting that the OxyR dependency for the transcription of these genes is not total.

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Filamentous Bacteriophages and the Competitive Interaction between Pseudomonas aeruginosa Strains under Antibiotic Treatment: a Modeling Study

mSystems ◽

10.1128/msystems.00193-21 ◽

2021 ◽

Author(s):

Julie D. Pourtois ◽

Michael J. Kratochvil ◽

Qingquan Chen ◽

Naomi L. Haddock ◽

Elizabeth B. Burgener ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Treatment ◽

Bacterial Pathogenesis ◽

Competitive Interaction ◽

Antibiotic Tolerance ◽

Filamentous Bacteriophages ◽

Bacterial Fitness ◽

The Impact ◽

Modeling Study ◽

Filamentous Phages

Filamentous phages are a frontier in bacterial pathogenesis, but the impact of these phages on bacterial fitness is unclear. In particular, Pf phages produced by Pa promote antibiotic tolerance but are metabolically expensive to produce, suggesting that competing pressures may influence the prevalence of Pf+ versus Pf− strains of Pa in different settings.

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Antibiotic treatment of Pseudomonas aeruginosa biofilms stimulates expression of the magnesium transporter gene mgtE

Microbiology ◽

10.1099/mic.0.070144-0 ◽

2014 ◽

Vol 160 (1) ◽

pp. 165-178 ◽

Cited By ~ 12

Author(s):

Carly V. Redelman ◽

Shubham Chakravarty ◽

Gregory G. Anderson

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Treatment ◽

Type Iii Secretion ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Transporter Protein ◽

Magnesium Transporter ◽

High Concentrations ◽

Serious Disease

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with the capacity to cause serious disease, including chronic biofilm infections in the lungs of cystic fibrosis (CF) patients. These infections are treated with high concentrations of antibiotics. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified, including the magnesium transporter protein MgtE. We have recently found that isogenic deletion of mgtE leads to increased cytotoxicity through effects on the type III secretion system. To explore the role of the CF lung environment in MgtE activity, we investigated mgtE transcriptional regulation following antibiotic treatment. Utilizing quantitative real-time-PCR, we have demonstrated an increase in mgtE transcript levels following antibiotic treatment with most of the 12 antibiotics tested. To begin to determine the regulatory network governing mgtE expression, we screened a transposon-mutant library of P. aeruginosa to look for mutants with potentially altered mgtE activity, using cytotoxicity as a readout. In this screen, we observed that AlgR, which regulates production of the biofilm polysaccharide alginate, alters MgtE-mediated cytotoxicity. This cross-talk between MgtE and AlgR suggests that AlgR is involved in linking external inducing signals (e.g. antibiotics) to mgtE transcription and downstream virulence and biofilm activities. Analysing such interactions may lead to a better understanding of how the CF lung environment shapes P. aeruginosa biofilm infections.

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