Evolution of Antibiotic Resistance in Biofilm and Planktonic Pseudomonas aeruginosa Populations Exposed to Subinhibitory Levels of Ciprofloxacin

Ahmed, Marwa N.; Porse, Andreas; Sommer, Morten Otto Alexander; Høiby, Niels; Ciofu, Oana

doi:10.1128/aac.00320-18

Cited by 118 publications

(129 citation statements)

References 49 publications

(51 reference statements)

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“…The rise of mutations in LPS biosynthesis genes ( orfKHLN ) and electron transport chain components ( cyoAB ) primarily in biofilm populations indicates that altered binding or cell permeability may have been selected and can indicate a distinct mode of action of TOB. These lifestyle distinctions in resistance traits suggest that the environment may influence the evolutionary dynamics of antimicrobial resistance, in concordance with previous studies (Ahmed et al, 2018b; Santos-Lopez et al, 2019; Trampari et al, 2019). Therefore, while genes like fusA1 represent mechanisms of resistance that are robust across a wide range of species, environments, and host conditions, the prevailing mode of bacterial growth is likely crucial in attempting to predict the evolution of antimicrobial resistance.…”

Section: Discussionsupporting

confidence: 91%

“…If we acknowledge that most forecasting efforts rely on history to anticipate the future, the explosive growth of whole-genome sequencing (WGS) sets the stage to resolve evolutionary phenomena in action and suggest the next selected path. Among the best examples, bacterial populations exposed to strong selection like antibiotics and analyzed by WGS are likely to identify gene regions that produce resistance (Ahmed et al, 2018a; Cooper, 2018; Feng et al, 2016; Palmer and Kishony, 2013). Repeated instances of the same antibiotic selection may enrich the same types of mutations and ultimately enable some measure of predictability (Ibacache-Quiroga et al, 2018; Wong et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…Yet predicting evolution may be hampered when antibiotic selection produces species or environment-specific outcomes. Evolution experiments in antibiotics have demonstrated that subjecting different bacterial strains to the same antibiotic treatment regime (Gifford et al, 2018; Vogwill et al, 2014, 2016), or the same strains to different environments (Ahmed et al, 2018a; Santos-Lopez et al, 2019) can select for different drug resistance levels as well as molecular targets. We can test the extent of predictability of evolved levels and causes of drug resistance by studying the evolution of resistance in different environments and across different species that inherently have different genetic backgrounds.…”

Section: Introductionmentioning

confidence: 99%

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Parallel evolution of tobramycin resistance across species and environments

Scribner

Santos-López

Marshall

et al. 2019

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words)13 An important problem in evolution is identifying the genetic basis of how different species adapt 14 to similar environments. Understanding how various bacterial pathogens evolve in response to 15 antimicrobial treatment is a pressing example of this problem, where discovery of molecular 16 parallelism could lead to clinically useful predictions. Evolution experiments with pathogens in 17 environments containing antibiotics combined with periodic whole population genome 18 sequencing can be used to characterize the evolutionary dynamics of the pathways to 19 antimicrobial resistance. We separately propagated two clinically relevant Gram-negative 20 pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii, in increasing 21 concentrations of tobramycin in two different environments each: planktonic and biofilm. 22Independent of the pathogen, populations adapted to tobramycin selection by parallel evolution 23 of mutations in fusA1, encoding elongation factor G, and ptsP, encoding phosphoenolpyruvate 24 phosphotransferase. As neither gene is a direct target of this aminoglycoside, both are relatively 25 novel and underreported causes of resistance. Additionally, both species acquired antibiotic-26 associated mutations that were more prevalent in the biofilm lifestyle than planktonic, in electron 27 transport chain components in A. baumannii and LPS biosynthesis enzymes in P. aeruginosa 28 populations. Using existing databases, we discovered both fusA1 and ptsP mutations to be 29 prevalent in antibiotic resistant clinical isolates. Additionally, we report site-specific parallelism of 30 fusA1 mutations that extend across several bacterial phyla. This study suggests that strong 31 selective pressures such as antibiotic treatment may result in high levels of predictability in 32 molecular targets of evolution despite differences between organisms' genetic background and 33 environment.The notion that evolution can be forecasted at the level of phenotype, gene, or even 36 amino acid is no longer a fantasy in the post-genomic era (Lässig et al., 2017). If we 37 acknowledge that most forecasting efforts rely on history to anticipate the future, the explosive 38 growth of whole-genome sequencing (WGS) sets the stage to resolve evolutionary phenomena 39 in action and suggest the next selected path. Among the best examples, bacterial populations 40 exposed to strong selection like antibiotics and analyzed by WGS are likely to identify gene 41 regions that produce resistance (Ahmed et al., 2018a; Cooper, 2018; Feng et al., 2016; Palmer 42 and Kishony, 2013). Repeated instances of the same antibiotic selection may enrich the same 43 types of mutations and ultimately enable some measure of predictability (Ibacache-Quiroga et 44 al., 2018; Wong et al., 2012). For instance, we can be confident that exposure of many bacteria 45 to high doses of fluoroquinolones like ciprofloxacin may select for substitutions in residues 83 or 46 87 of the drug target, DNA gyrase A (Fàbrega et al., 2009; Wong and Kassen, 2011).47 Furt...

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Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Parallel evolution of tobramycin resistance across species and environments

Scribner

Santos-López

Marshall

et al. 2019

Preprint

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“…Ten of the mutants had mutations in pilin-encoding pil genes ( Table 1, Table S1). The relationship between pil mutations and ciprofloxacin resistance is not clear, although very recently other researchers also reported an association between pil mutations and ciprofloxacin resistance (36). Eleven of the mutants had a mutation in a gene PA3491 that to the best of our knowledge has not previously been associated with antibiotic resistance.…”

Section: Ciprofloxacin-selected Mutantsmentioning

confidence: 83%

A large-scale comparison shows that genetic changes causing antibiotic resistance in experimentally evolvedPseudomonas aeruginosapredict those in naturally evolved bacteria

Wardell¹,

Rehman²,

Martin³

et al. 2019

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Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have acquired mutations that make them antibiotic resistant, making treatment more difficult. To identify resistance-associated mutations we experimentally evolved the antibiotic sensitive strain P. aeruginosa PAO1 to become resistant to three widely used anti-pseudomonal antibiotics, ciprofloxacin, meropenem and tobramycin. Mutants were able to tolerate up to 2048-fold higher concentrations of antibiotic than strain PAO1. Genome sequences were determined for thirteen mutants for each antibiotic. Each mutant had between 2 and 8 mutations. There were at least 8 genes mutated in more than one mutant per antibiotic, demonstrating the complexity of the genetic basis of resistance. Additionally, large deletions of up to 479kb arose in multiple meropenem resistant mutants. For all three antibiotics mutations arose in genes known to be associated with resistance, but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in experimentallyevolved mutants we analysed the corresponding genes in 457 isolates of P. aeruginosa from patients with cystic fibrosis or bronchiectasis as well as 172 isolates from the general environment. Many of the genes identified through experimental evolution had changes predicted to be function-altering in clinical isolates but not in isolates from the general environment, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. These findings expand understanding of the genetic basis of antibiotic resistance in P. aeruginosa as well as demonstrating the validity of experimental evolution in identifying clinically-relevant resistance-associated mutations.' ImportanceThe rise in antibiotic resistant bacteria represents an impending global health crisis. As such, understanding the genetic mechanisms underpinning this resistance can be a crucial piece of the puzzle to combatting it. The importance of this research is that by experimentally evolving P. aeruginosa to three clinically relevant antibiotics, we have generated a catalogue of genes that can contribute to resistance in vitro. We show that many (but not all) of these genes are clinically relevant, by identifying variants in clinical isolates of P. aeruginosa. This research furthers our understanding of the genetics leading to resistance in P. aeruginosa and provides tangible evidence that these genes can play a role clinically, potentially leading to new druggable targets or inform therapies.

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“…1–3 Furthermore, the mechanical integrity and structure of the bacterial biofilm conferred by the matrix gives rise to stable microenvironments that contribute to phenotypic antibiotic tolerance. 4–5 Thus, both the chemical and the structural properties of the biofilm matrix contribute to bacterial tolerance of harsh environments.…”

Section: Introductionmentioning

confidence: 99%

Specific Disruption of EstablishedP. aeruginosaBiofilms Using Polymer-Attacking Enzymes

Kovach

Fleming

Rumbaugh

et al. 2019

Preprint

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12Biofilms are communities of bacteria embedded in an extracellular matrix of self-13 produced polymeric substances. This polymer matrix lends the bacteria protection against 14 a wide array of chemical and mechanical stresses that they may experience in their 15 environment, which might be a location in the human body in the case of a biofilm 16 infection, or a surface immersed in fluid in an industrial setting. Breaking down the matrix 17 network renders biofilms more susceptible to physical disruption and to treatments. 18 Different species of bacteria, and different strains within the same species, produce 19 different types of matrix polymersthis suggests that targeting specific polymers for 20 disruption may be more effective than non-specific approaches to disrupting biofilm 21 matrices. In this study, we treated Pseudomonas aeruginosa biofilms with enzymes that 22 are specific to different matrix polymers. We used bulk rheology to measure the resulting 23 approaches to eradicating biofilms in environmental or industrial settings may need to be 40 very different from effective treatments of infection. 41 42 and the importance of a specific polymer type varies with biofilm-forming species and 47 strain. For biofilm infections, the matrix protects the inhabitant bacteria chemically by 48 inhibiting the diffusion of antibiotics into the biofilm and by binding to antibacterial 49 chemicals produced by the host immune response. 1-3 Furthermore, the mechanical 50 integrity and structure of the bacterial biofilm conferred by the matrix gives rise to stable 51 microenvironments that contribute to phenotypic antibiotic tolerance. 4-5 Thus, both the 52 chemical and the structural properties of the biofilm matrix contribute to bacterial 53 tolerance of harsh environments. 54Many common interventions in P. aeruginosa infections, like antibiotic treatment, 55 are far more successful if the bacteria are in a planktonic state rather than in a biofilm. 6

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Evolution of Antibiotic Resistance in Biofilm and Planktonic Pseudomonas aeruginosa Populations Exposed to Subinhibitory Levels of Ciprofloxacin

Cited by 118 publications

References 49 publications

Parallel evolution of tobramycin resistance across species and environments

Parallel evolution of tobramycin resistance across species and environments

A large-scale comparison shows that genetic changes causing antibiotic resistance in experimentally evolvedPseudomonas aeruginosapredict those in naturally evolved bacteria

Specific Disruption of EstablishedP. aeruginosaBiofilms Using Polymer-Attacking Enzymes

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