Tangled bank of experimentally evolved Burkholderia biofilms reflects selection during chronic infections

Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH 03824.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2012; 110(3). DOI: 10.1073/pnas.1207025110
Source: PubMed

ABSTRACT How diversity evolves and persists in biofilms is essential for understanding much of microbial life, including the uncertain dynamics of chronic infections. We developed a biofilm model enabling long-term selection for daily adherence to and dispersal from a plastic bead in a test tube. Focusing on a pathogen of the cystic fibrosis lung, Burkholderia cenocepacia, we sequenced clones and metagenomes to unravel the mutations and evolutionary forces responsible for adaptation and diversification of a single biofilm community during 1,050 generations of selection. The mutational patterns revealed recurrent evolution of biofilm specialists from generalist types and multiple adaptive alleles at relatively few loci. Fitness assays also demonstrated strong interference competition among contending mutants that preserved genetic diversity. Metagenomes from five other independently evolved biofilm lineages revealed extraordinary mutational parallelism that outlined common routes of adaptation, a subset of which was found, surprisingly, in a planktonic population. These mutations in turn were surprisingly well represented among mutations that evolved in cystic fibrosis isolates of both Burkholderia and Pseudomonas. These convergent pathways included altered metabolism of cyclic diguanosine monophosphate, polysaccharide production, tricarboxylic acid cycle enzymes, global transcription, and iron scavenging. Evolution in chronic infections therefore may be driven by mutations in relatively few pathways also favored during laboratory selection, creating hope that experimental evolution may illuminate the ecology and selective dynamics of chronic infections and improve treatment strategies.

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Available from: Vaughn S Cooper, Aug 28, 2015
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    • "SD = 2.8cm, t = -8.96, P < 0.0001), and obviously produced more biofilm, given copious production on the tube at the airliquid interface (as shown in (Traverse et al., 2013)). "
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    ABSTRACT: A common phenotype within bacterial biofilms is the small, “wrinkly” colony, which may associate with worse prognoses from biofilm-associated infections. The mechanisms that produce these variants in Burkholderia are undefined. Here we report the mutational and ecological causes of wrinkly (W) colonies that evolved during experimental biofilm evolution of B. cenocepacia. Mutations clustered in a homologous pathway to the Pseudomonas wsp operon but with a distinct terminal signaling mechanism, and their parallel evolution suggested that they inhabited an equivalent biofilm niche. We tested this hypothesis of niche complementarity by measuring effects of substituting different W variants in the same evolved biofilm community. Despite phenotypic differences among W mutants growing alone, fitness of reconstituted mixed biofilms did not differ significantly. In conclusion, the evolution of small-colony variants in Burkholderia biofilms appears to be driven by an ecological opportunity that generates strong selection for constitutive wsp mutants to inhabit a common niche.
    Genomics 09/2014; 104(6). DOI:10.1016/j.ygeno.2014.09.007 · 2.79 Impact Factor
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    • "A number of studies have examined adaptation of BCC bacteria to various in-vitro growth environments [45]–[49]. However, there are currently little data on adaptive strategies of BCC bacteria to chronic pulmonary infection in CF. "
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    ABSTRACT: Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.
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    • "Elevated intracellular levels of c-di-GMP through expression of the E. coli DGC protein YedQ in B. cenocepacia resulted in the formation of wrinkled colonies on solid medium, robust pellicles at the air–liquid interface of static liquid cultures and increased biofilm formation in flow-cells. Wrinkled colony morphology has been found to be highly correlated with increased biofilm formation ability of various bacteria (Rainey and Travisano, 1998; Spiers et al., 2002; 2003; Friedman and Kolter, 2004a,b), including B. cenocepacia (Fazli et al., 2011; 2013; Traverse et al., 2013). A genetic screen for mutants that were unable to form wrinkled colonies in response to high intracellular c-di-GMP levels led to the identification of the Crp/Fnr superfamily transcription factor BCAM1349 (Fazli et al., 2011). "
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    ABSTRACT: In the present review we describe and compare the molecular mechanisms that are involved in biofilm formation by Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Burkholderia cenocepacia. Our current knowledge suggests that biofilm formation is regulated by cyclic diguanosine-5'-monophosphate (c-di-GMP), small RNAs (sRNA) and quorum sensing (QS) in all these bacterial species. The systems that employ c-di-GMP as a second messenger regulate the production of exopolysaccharides and surface proteins which functions as extracellular matrix components in the biofilms formed by the bacteria. The systems that make use of sRNAs appear to regulate the production of exopolysaccharide biofilm matrix material in all these species. In the pseudomonads QS regulates the production of extracellular DNA, lectins and biosurfactants which all play a role in biofilm formation. In B. cenocepacia QS regulates the expression of a large surface protein, lectins and extracellular DNA that all function as biofilm matrix components. Although the three regulatory systems all regulate the production of factors used for biofilm formation, the molecular mechanisms involved in transducing the signals into expression of the biofilm matrix components differ between the species. Under the conditions tested, exopolysaccharides appears to be the most important biofilm matrix components for P. aeruginosa, whereas large surface proteins appears to be the most important biofilm matrix components for P. putida, P. fluorescens, and B. cenocepacia.
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