Biofilm dispersal cells of a cystic fibrosis<i>Pseudomonas aeruginosa</i>isolate exhibit variability in functional traits likely to contribute to persistent infection

Woo, Jerry K. K.; Webb, Jeremy S.; Kirov, SM; Kjelleberg, Staffan; Rice, Scott A.

doi:10.1111/j.1574-695x.2012.01006.x

Cited by 35 publications

(43 citation statements)

References 62 publications

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“…Both clinical isolates and the dispersal populations of laboratory-grown P. aeruginosa biofilms are known to exhibit high numbers of heritable phenotypic variants (8,9), indicating withinpopulation genetic diversification during biofilm growth. Genetic diversification during biofilm development has been proposed as a mechanism underlying P. aeruginosa's propensity for swift adaptation to antibiotics.…”

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confidence: 99%

Strain-specific parallel evolution drives short-term diversification during Pseudomonas aeruginosa biofilm formation

McElroy

Hui

Woo

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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113

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Generation of genetic diversity is a prerequisite for bacterial evolution and adaptation. Short-term diversification and selection within populations is, however, largely uncharacterised, as existing studies typically focus on fixed substitutions. Here, we use whole-genome deep-sequencing to capture the spectrum of mutations arising during biofilm development for two Pseudomonas aeruginosa strains. This approach identified single nucleotide variants with frequencies from 0.5% to 98.0% and showed that the clinical strain 18A exhibits greater genetic diversification than the type strain PA01, despite its lower per base mutation rate. Mutations were found to be strain specific: the mucoid strain 18A experienced mutations in alginate production genes and a c-di-GMP regulator gene; while PA01 acquired mutations in PilT and PilY1, possibly in response to a rapid expansion of a lytic Pf4 bacteriophage, which may use type IV pili for infection. The Pf4 population diversified with an evolutionary rate of 2.43 × 10 −3 substitutions per site per day, which is comparable to single-stranded RNA viruses. Extensive within-strain parallel evolution, often involving identical nucleotides, was also observed indicating that mutation supply is not limiting, which was contrasted by an almost complete lack of noncoding and synonymous mutations. Taken together, these results suggest that the majority of the P. aeruginosa genome is constrained by negative selection, with strong positive selection acting on an accessory subset of genes that facilitate adaptation to the biofilm lifecycle. Long-term bacterial evolution is known to proceed via few, nonsynonymous, positively selected mutations, and here we show that similar dynamics govern shortterm, within-population bacterial diversification.dispersal | prophage | haplotypes D iversifying selection within bacterial populations underpins a range of ecological and clinical phenomena, for example niche adaptation (1-3) and antibiotic resistance (4). During diversifying selection, a single population explores multiple fitness peaks, resulting in subpopulations with different adaptive mutations. This kind of within-population genetic diversity can provide the raw material on which long-term evolution acts. However, diversifying selection of bacterial populations is still poorly understood, because previous experimental evolution and epidemiological studies typically have focused on fixed substitutions at the end point of evolution (5), ignoring short-term or withinpopulation effects.Laboratory-grown Pseudomonas aeruginosa biofilms provide an ideal model for investigating within-population diversification. The bacterium P. aeruginosa is a widespread, Gram-negative generalist and can have either a planktonic, motile lifestyle or exist as a biofilm (i.e., a surface-attached cells embedded within an extracellular polymeric matrix). P. aeruginosa has been the focus of extensive research, because of both its status as a model organism and its ability to form opportunistic, chronic, often lethal biof...

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confidence: 99%

Strain-specific parallel evolution drives short-term diversification during Pseudomonas aeruginosa biofilm formation

McElroy

Hui

Woo

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

113

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“…These results and those of the previous experiment in ASMDMϩ indicate that, given the structural similarity of phenazines, GSH is able to react structurally with more than one type. This is important, given the typical heterogeneity of phenotypes encountered in vivo (10,11).…”

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confidence: 99%

“…Biofilms that form in CF sputum are unique in their structure, existing as intercellular aggregates near the air-surface interface of sputum (8), rather than typical "mushroom-shaped" microcolonies (9). Populations within these persistent biofilms undergo substantial within-host evolution over time, with seeding dispersion from biofilms resulting in phenotypically divergent strains throughout the CF lung and the emergence of antibiotic-resistant strains (10,11).…”

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confidence: 99%

Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome

Klare

Das

Ibugo

et al. 2016

Antimicrob Agents Chemother

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bPseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM؉). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies.

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“…A second model consists of induced biofilms, in which bacteria collected from the supernatant of naive biofilms are used as a starting inoculum. This model may better take into account the adaptative process mediated by the quorum sensing molecules that takes place during biofilm maturation (27,28) and which was already well demonstrated to take place with clinical isolates form other bacterial species (29,30). The models have been tested with antibiotics representative of the 3 main classes of antibiotics active against S. pneumoniae, namely, amoxicillin (for ␤-lactams), clarithromycin (for macrolides), and levofloxacin and moxifloxacin (for fluoroquinolones).…”

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Antibiotic Activity against Naive and Induced Streptococcus pneumoniae Biofilms in an In Vitro Pharmacodynamic Model

Vandevelde

Tulkens

2014

Antimicrob Agents Chemother

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Biofilms play a role in the pathogenicity of pneumococcal infections. A pharmacodynamic in vitro model of biofilm was developed that allows characterization of the activity of antibiotics against viability and biomass by using in parallel capsulated (ATCC 49619) and noncapsulated (R6) reference strains. Naive biofilms were obtained by incubating fresh planktonic cultures for 2 to 11 days in 96-well polystyrene plates. Induced biofilms were obtained using planktonic bacteria collected from the supernatant of 6-day-old naive biofilms. Biomass production was more rapid and intense in the induced model, but the levels were similar for both strains. Full concentration responses fitting sigmoidal regressions allowed calculation of maximal efficacies and relative potencies of drugs. All antibiotics tested (amoxicillin, clarithromycin, solithromycin, levofloxacin, and moxifloxacin) were more effective against young naive biofilms than against old or induced biofilms, except macrolides/ketolides, which were as effective at reducing viability in 2-day-old naive biofilms and in 11-day-old induced biofilms of R6. Macrolides/ketolides, however, were less potent than fluoroquinolones against R6 (approximately 5-to 20-fold-higher concentrations needed to reduction viability of 20%). However, at concentrations obtainable in epithelial lining fluid, the viabilities of mature or induced biofilms were reduced 15 to 45% (amoxicillin), 17 to 44% (macrolides/ketolides), and 12 to 64% (fluoroquinolones), and biomasses were reduced 5 to 45% (amoxicillin), 5 to 60% (macrolides/ketolides), and 10 to 76% (fluoroquinolones), with solithromycin and moxifloxacin being the most effective and the most potent agents (due to lower MICs) in their respective classes. This study allowed the ranking of antibiotics with respect to their potential effectiveness in biofilm-related infections, underlining the need to search for still more effective options.

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Biofilm dispersal cells of a cystic fibrosisPseudomonas aeruginosaisolate exhibit variability in functional traits likely to contribute to persistent infection

Cited by 35 publications

References 62 publications

Strain-specific parallel evolution drives short-term diversification during Pseudomonas aeruginosa biofilm formation

Strain-specific parallel evolution drives short-term diversification during Pseudomonas aeruginosa biofilm formation

Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome

Antibiotic Activity against Naive and Induced Streptococcus pneumoniae Biofilms in an In Vitro Pharmacodynamic Model

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