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: 9.67). 12/2012; 110(3). DOI: 10.1073/pnas.1207025110
Source: PubMed


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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    • "Seven W mutants with different genotypes and evolutionary histories were each used to found several replicate populations that were passaged under planktonic conditions for 3 days and screened for colony variants. Of these W ancestors, five had previously evolved during biofilm selection for 32–64 generations and acquired single wsp mutations (Cooper et al., 2014), one evolved for 315 generations and acquired at least one secondary mutation in rpoC, and one W ancestor evolved for 1050 generations and acquired at least eight mutations (Traverse et al., 2013). Twenty mutants with S morphology from these different ancestors were collected and labeled 'E' for arising from an Early W ancestor, 'I' for arising from the Intermediate W ancestor, or 'L' for the Late ancestor. "
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    ABSTRACT: Experimental evolution paired with modern sequencing can be a powerful approach to identify the mechanisms by which bacteria adapt to discrete environmental conditions found in nature or during infections. We used this approach to identify mechanisms enabling biofilm specialists of the opportunistic respiratory pathogen Burkholderia cenocepacia to regain planktonic fitness. Seven mutants producing wrinkly (W) small-colony variants by mutations in the wrinkly-spreader operon (wsp) cluster, but with varying duration of biofilm adaptation, served as ancestors of this experiment. Following planktonic growth, each W ancestor produced smooth (S) mutants with distinct fitness effects across planktonic, biofilm, and dispersal-phase environments. The causes of the S phenotype traced to mutations in three gene clusters: wsp, Bcen2424_1436, an uncharacterized two-component transcriptional regulator which appears to be critical for wsp signaling, and a cohort of genes involved in polysaccharide synthesis. The genetic pathway from W to S also associated with evolutionary history in the biofilm environment. W mutants isolated from long-term biofilm selection usually produced S types via secondary wsp mutations, whereas S types evolved from less adapted W ancestors by a wider scope of mutations. These different genetic pathways to suppress the W phenotype suggest that prolonged biofilm adaptation limits routes to subsequent planktonic adaptation, despite common initial mechanisms of biofilm adaptation. More generally, experimental evolution can be used as a nuanced screen for gain-of-function mutations in multiple conditions that illustrate tensions that bacteria may face in changing environments or hosts.
    Frontiers in Genetics 02/2015; 6:18. DOI:10.3389/fgene.2015.00018
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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 · 2.28 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.
    PLoS ONE 09/2014; 9(9):e106428. DOI:10.1371/journal.pone.0106428 · 3.23 Impact Factor
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