The high persister phenotype ofPseudomonas aeruginosais associated with increased fitness and persistence in cystic fibrosis airways

AbstractDespite intensive antibiotic treatment of cystic fibrosis (CF) patients,Pseudomonas aeruginosaoften persists in patient airways for decades, and can do so without the development of antibiotic resistance. Using a high-throughput screening assay of survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of high-persister variants (Hip) in a large patient cohort. In a screen of 467 longitudinal clinical isolates ofP. aeruginosafrom 40 CF patients, we classified 25.7% as Hip. Hip were identified in 26 patients, but only a few bacterial lineages were dominated by Hip. Instead, the emergence of Hip increased over time, suggesting that CF airways treated with ciprofloxacin select for Hip with an increased fitness in this environment. We generally observed diverse genetic changes in the Hip isolate population (as many co-occurring routes to increased fitness exist), but interestingly elevated mutation counts in the RpoN gene of 18 Hip isolates suggest that this sigma factor plays a role in shaping levels of antibiotic tolerance. To probe the impact of the Hip phenotype in a CF-similar environment, we tested the fitness properties of otherwise genotypically and phenotypically similar low-persister (Lop) and Hip isolates in co-culture using a specialized flow-cell biofilm system mimicking pharmacokinetic/-dynamic antibiotic dosing. Hip survived ciprofloxacin treatment far better than Lop isolates. The results of this investigation provide novel insights into persister dynamics and fitness contributions to survival in the CF lung, and show that the Hip phenotype of antibiotic susceptible bacteria plays an important role in long-term infections.SignificanceAntibiotic resistance is emphasized as a rapidly increasing health threat, but antibiotic tolerance via the occurrence of persister cells in antibiotic-treated bacterial populations is clinically and publicly neglected. In 40 CF patients representing a well-established human infection model – long-term lung infections byPseudomonas aeruginosa– we show the emergence and accumulation of persister variants in a clinical population heavily reliant on antibiotic therapy. We observe that the high-persister (Hip) phenotype is independent of resistance and likely the consequence of numerous genetic alterations, complicating surveillance and inhibition in the clinic. Furthermore, we find Hip are selected for over time, survive better than ‘normal’ bacteria, and can outcompete them in CF-similar conditions, ultimately affecting 65% of patients in an early disease cohort.

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Adaptation to an amoeba host leads to Pseudomonas aeruginosa isolates with attenuated virulence

Applied and Environmental Microbiology ◽

10.1128/aem.02322-21 ◽

2022 ◽

Author(s):

Wai Leong ◽

Wee Han Poh ◽

Jonathan Williams ◽

Carla Lutz ◽

M. Mozammel Hoque ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Late Stage ◽

Early Stage ◽

Opportunistic Pathogen ◽

Phenotypic Traits ◽

Nucleotide Polymorphisms ◽

Chronic Infections ◽

The Impact

The opportunistic pathogen Pseudomonas aeruginosa , is ubiquitous in the environment, and in humans is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when co-incubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans , was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and non-adapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoeba as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoeba resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. Importance Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by bacterial predators, e.g. protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examine the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa . We show that long term co-incubation with protozoa resulted in mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures amongst host cell types as well as similar adaptation strategies.

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Long-term coexistence of Pseudomonas aeruginosa and Staphylococcus aureus using an in vitro cystic fibrosis model

Future Microbiology ◽

10.2217/fmb-2021-0025 ◽

2021 ◽

Author(s):

Rosana Monteiro ◽

Andreia Patrícia Magalhães ◽

Maria Olivia Pereira ◽

Ana Margarida Sousa

Keyword(s):

Cystic Fibrosis ◽

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Physicochemical Characteristics ◽

Antibiotic Tolerance ◽

Virulence Potential ◽

Cystic Fibrosis Airway ◽

Lung Colonization

Aim: To investigate the role of pre-established Staphylococcus aureus on Pseudomonas aeruginosa adaptation and antibiotic tolerance. Materials & methods: Bacteria were cultured mimicking the sequential pattern of lung colonization and exposure to ciprofloxacin. Results: In the absence of ciprofloxacin exposure, S. aureus and P. aeruginosa coexisted supported by the physicochemical characteristics of the artificial sputum medium. S. aureus had no role in P. aeruginosa tolerance against ciprofloxacin and did not select P. aeruginosa small-colony variants during antibiotic treatment. rhlR and psqE were downregulated after the contact with S. aureus indicating that P. aeruginosa attenuated its virulence potential. Conclusion: P. aeruginosa and S. aureus can cohabit in cystic fibrosis airway environment for long-term without significant impact on P. aeruginosa adaptation and antibiotic tolerance.

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Small molecules with antibiofilm, antivirulence and antibiotic synergy activities againstPseudomonas aeruginosa

10.1101/067074 ◽

2016 ◽

Cited By ~ 1

Author(s):

Erik van Tilburg Bernardes ◽

Laetitia Charron-Mazenod ◽

David Reading ◽

Shauna L. Reckseidler-Zenteno ◽

Shawn Lewenza

Keyword(s):

Cystic Fibrosis ◽

Extracellular Matrix ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Small Molecules ◽

Biofilm Formation ◽

Chronic Infections ◽

Term Survival ◽

Lung Infections

AbstractBiofilm formation is a universal bacterial strategy for long-term survival in nature and during infections. Biofilms are dense microbial communities enmeshed within a polymeric extracellular matrix that protects bacteria from antibiotic exposure and the immune system and thus contribute to chronic infections.Pseudomonas aeruginosais an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in Cystic Fibrosis (CF) patients. The extracellular matrix ofP. aeruginosabiofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and non-mucoid isolates ofP. aeruginosaproduces the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation and immune evasion. Given the central importance of the Pel and Psl EPS in biofilm structure, they are attractive targets for novel anti-infective compounds. In this study we used a high throughput gene expression screen to identify compounds that repress expression ofpelandpslgenes as measured by transcriptionalluxfusions. Testing of thepel/pslrepressors demonstrated an antibiofilm activity against microplate and flow chamber biofilms formed by wild type and hyperbiofilm forming strains. To determine the potential role of EPS in virulence, mutants inpel/pslwere shown to have reduced virulence in the feeding behavior and slow killing virulence assays inCaenorhabditis elegans. The antibiofilm molecules also reducedP. aeruginosaPAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds were synergistic in killingP. aeruginosabiofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronicP. aeruginosainfections.Author summaryBacteria use the strategy of growing as a biofilm to promote long-term survival and therefore to cause chronic infections. One of the best examples isPseudomonas aeruginosaand the chronic lung infections in individuals with Cystic Fibrosis (CF). Biofilms are generally a dense community of bacteria enmeshed in an extracellular matrix that protects bacteria from numerous environmental stresses, including antibiotics and the immune system. In this study we developed an approach to identifyP. aeruginosabiofilm inhibitors by repressing the production of the matrix exopolysaccharide (EPS) polymers. Bacteria treated with compounds and then fed to the nematode also had showed reduced virulence by promoting nematode survival. To tackle the problem of biofilm tolerance of antibiotics, the compounds identified here also had the beneficial property of increasing the biofilm sensitivity to different classes of antibiotics. The compounds disarm bacteria but they do not kill or limit growth like antibiotics. We provide further support that disarmingP. aeruginosamay be a critical anti-infective strategy that limits the development of antibiotic resistance, and provides a new way for treating chronic infections.

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Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

The ISME Journal ◽

10.1038/s41396-021-01157-9 ◽

2021 ◽

Author(s):

Jules D. P. Valentin ◽

Hervé Straub ◽

Franziska Pietsch ◽

Marion Lemare ◽

Christian H. Ahrens ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Structural Development ◽

Antibiotic Tolerance ◽

Intrinsic Resistance ◽

Knock Out ◽

Initial Adhesion ◽

Flagellar Proteins ◽

Biofilm Development ◽

The Impact

AbstractPseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.

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P101 The impact of transmissible strains of Pseudomonas aeruginosa on the long-term clinical outcomes of adults with cystic fibrosis

Journal of Cystic Fibrosis ◽

10.1016/s1569-1993(19)30395-9 ◽

2019 ◽

Vol 18 ◽

pp. S86

Author(s):

E.F. Nash ◽

K.J. Hardy ◽

R.G. Edgar ◽

E. Justice ◽

R. Rashid ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Clinical Outcomes ◽

The Impact

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Hypoxia Increases Antibiotic Resistance in Pseudomonas aeruginosa through Altering the Composition of Multidrug Efflux Pumps

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05574-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 2114-2118 ◽

Cited By ~ 56

Author(s):

Bettina Schaible ◽

Cormac T. Taylor ◽

Kirsten Schaffer

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Content Type ◽

Hypoxic Conditions ◽

Multidrug Efflux Pumps ◽

The Impact

ABSTRACTAntibiotic resistance is a significant and developing problem in general medical practice and a common clinical complication in cystic fibrosis patients infected withPseudomonas aeruginosa. Such infections occur within hypoxic mucous deposits in the cystic fibrosis lung; however, little is known about how the hypoxic microenvironment influences pathogen behavior. Here we investigated the impact of hypoxia on antibiotic resistance inP. aeruginosa. The MICs of a selection of antibiotics were determined forP. aeruginosagrown under either normoxic or hypoxic conditions. The expression of mRNAs for resistance-nodulation-cell division (RND) multidrug efflux pump linker proteins was determined by real-time PCR, and multidrug efflux pump activity was inhibited using Phe-Arg β-naphthylamide dihydrochloride. The MIC values of a subset of clinically importantP. aeruginosaantibiotics were higher for bacteria incubated under hypoxia than under normoxia. Furthermore, hypoxia altered the stoichiometry of multidrug efflux pump linker protein subtype expression, and pharmacologic inhibition of these pumps reversed hypoxia-induced antibiotic resistance. We hypothesize that hypoxia increases multidrug resistance inP. aeruginosaby shifting multidrug efflux pump linker protein expression toward a dominance of MexEF-OprN. Thus, microenvironmental hypoxia may contribute significantly to the development of antibiotic resistance inP. aeruginosainfecting cystic fibrosis patients.

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Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing

Scientific Reports ◽

10.1038/s41598-021-85100-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Syed A. K. Shifat Ahmed ◽

Michelle Rudden ◽

Sabrina M. Elias ◽

Thomas J. Smyth ◽

Roger Marchant ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Genomic Islands ◽

Clinical Strain ◽

Whole Genome ◽

Synonymous Mutations ◽

Wide Range

AbstractPseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI’s) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

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The Impact of an Efflux Pump Inhibitor on the Activity of Free and Liposomal Antibiotics against Pseudomonas aeruginosa

Pharmaceutics ◽

10.3390/pharmaceutics13040577 ◽

2021 ◽

Vol 13 (4) ◽

pp. 577

Author(s):

Douweh Leyla Gbian ◽

Abdelwahab Omri

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Antimicrobial Activities ◽

Dilution Method ◽

Efflux Pump Inhibitor ◽

Signal Production ◽

Lung Infections ◽

The Impact ◽

Microbroth Dilution

The eradication of Pseudomonas aeruginosa in cystic fibrosis patients has become continuously difficult due to its increased resistance to treatments. This study assessed the efficacy of free and liposomal gentamicin and erythromycin, combined with Phenylalanine arginine beta-naphthylamide (PABN), a broad-spectrum efflux pump inhibitor, against P. aeruginosa isolates. Liposomes were prepared and characterized for their sizes and encapsulation efficiencies. The antimicrobial activities of formulations were determined by the microbroth dilution method. Their activity on P. aeruginosa biofilms was assessed, and the effect of sub-inhibitory concentrations on bacterial virulence factors, quorum sensing (QS) signals and bacterial motility was also evaluated. The average diameters of liposomes were 562.67 ± 33.74 nm for gentamicin and 3086.35 ± 553.95 nm for erythromycin, with encapsulation efficiencies of 13.89 ± 1.54% and 51.58 ± 2.84%, respectively. Liposomes and PABN combinations potentiated antibiotics by reducing minimum inhibitory and bactericidal concentrations by 4–32 fold overall. The formulations significantly inhibited biofilm formation and differentially attenuated virulence factor production as well as motility. Unexpectedly, QS signal production was not affected by treatments. Taken together, the results indicate that PABN shows potential as an adjuvant of liposomal macrolides and aminoglycosides in the management of lung infections in cystic fibrosis patients.

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Locking down the Impact of New Zealand’s COVID-19 Alert Level Changes on Pets

Animals ◽

10.3390/ani11030758 ◽

2021 ◽

Vol 11 (3) ◽

pp. 758

Author(s):

Fiona Esam ◽

Rachel Forrest ◽

Natalie Waran

Keyword(s):

New Zealand ◽

Separation Anxiety ◽

National Surveys ◽

Normal Life ◽

Pet Owners ◽

Level 1 ◽

The Impact ◽

Better Than

The influence of the COVID-19 pandemic on human-pet interactions within New Zealand, particularly during lockdown, was investigated via two national surveys. In Survey 1, pet owners (n = 686) responded during the final week of the five-week Alert Level 4 lockdown (highest level of restrictions—April 2020), and survey 2 involved 498 respondents during July 2020 whilst at Alert Level 1 (lowest level of restrictions). During the lockdown, 54.7% of owners felt that their pets’ wellbeing was better than usual, while only 7.4% felt that it was worse. Most respondents (84.0%) could list at least one benefit of lockdown for their pets, and they noted pets were engaged with more play (61.7%) and exercise (49.7%) than pre-lockdown. Many respondents (40.3%) expressed that they were concerned about their pet’s wellbeing after lockdown, with pets missing company/attention and separation anxiety being major themes. In Survey 2, 27.9% of respondents reported that they continued to engage in increased rates of play with their pets after lockdown, however, the higher levels of pet exercise were not maintained. Just over one-third (35.9%) of owners took steps to prepare their pets to transition out of lockdown. The results indicate that pets may have enjoyed improved welfare during lockdown due to the possibility of increased human-pet interaction. The steps taken by owners to prepare animals for a return to normal life may enhance pet wellbeing long-term if maintained.

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