1. Methods
Acknowledgements
The Hamel Center for Undergraduate Research:
Summer Undergraduate Research Fellowship
!
National Institutes of Health: NIH1R01GM110444
!
David Morejon, Devon O’Rourke and Meghan Hartwick
!
Dr. Jessica Bolker, Ms. Heather Botelle and Mrs. Evelyn Brown
!
My wonderful family and friends
Experimental Adaptation of a Clinical Cystic Fibrosis Isolate to
Biofilm Conditions
Sarah Kremer, Sean Buskirk, Kenneth Flynn, Vaughn Cooper
Department of Molecular, Cellular and Biomedical Sciences
University of New Hampshire
Contacts: Sarahck17@gmail.com and Vaughn.Cooper@unh.edu
ResultsIntroduction
Chronic biofilm-associated infections are the major cause
of morbidity and mortality in patients with Cystic Fibrosis
(CF). The complex biofilm structure protects bacterial cells
from chemotherapeutic (i.e. antibiotics) and mechanical
treatments (i.e. chest percussion) which allows infections to
persist for many months to years and bacterial populations
to evolve and diversify. This dynamic motivates our study of
long term evolution in biofilms. Burkholderia cenocepacia
strain H111 was isolated from a CF lung infection and
evolved in a laboratory biofilm model to study the adaptation
and diversification of populations derived from a clone. We
hypothesized that the clinical H111 strain would adapt and
diversify by similar mechanisms as the previously studied
environmental strain B. cenocepacia HI2424 (Poltak, 2011).
~20 Generations ~200 Generations
Collected 8 colony variant
isolates over 225 generations.
The isolates are phenotypically
and genotypically distinct from
the ancestor.
Clonal
population
Wildtype H111
Continued
for ~225
generations,
32 transfers
1.Experimental
evolution
2.Measure phenotypic
differences from the
ancestor
We used fitness
competition assays to
compare the isolates’
reproductive success
to the ancestor
Evolved Isolate Wildtype
ancestor
Measure of fitness
Biofilm production assays
were used to quantify surface
associated biomass
3.Genomic DNA sequencing
Extracted gDNA from evolved isolates
Illumina
sequencing
Analysis and
mutation calls
Discussion
! The clonal population adapted to the selective
biofilm environment and diversified phenotypically
and genotypically.
! Mutations in phosphoenolpyruvate carboxylase also
explained planktonic adaptation by HI2424,
indicating convergent evolution
! The third chromosome is nonessential and does not
significantly affect biofilm production, but the
absence has a detrimental fitness effect in a
planktonic environment.
! The evolutionary trajectory of environmental and
pathogenic microbe phenotype is loosely
predictable.
! In contrast to prior work, the colony variants were
adapted to planktonic environment rather than
biofilm conditions.
Mutant Isolate:
evolution
environment
Type of
mutation Region affected
1: Biofilm
Complete deletion
3 bp insertion
Third chromosome
Glycerol uptake facilitator protein
2: Biofilm SNP Glycerol kinase
3: Biofilm
15 bp deletion
SNP
Integrase
Phosphoenolpyruvate carboxylase
4: Biofilm - Not sequenced
5: Planktonic*
29 bp deletion
47,000 bp deletion
Glycerol kinase
Prophage
6: Planktonic*
29 bp deletion
47,000 bp deletion
Glycerol kinase
Prophage
7: Biofilm
SNP
SNP
Intergenic, upstream from CzcD protein
Phosphoenolpyruvate carboxylase
8: Planktonic - Not sequenced
Table 1: Illumina sequencing followed by Breseq analysis allowed us to call eleven mutations across six isolates, including
the loss of the third chromosome in isolate one. Phosphoenolpyruvate carboxylase also became mutated in the HI2424
evolution, implying convergent evolution. *5 and 6 are distinct, but were isolated from the same population at the same time point.
Biofilm production by all isolates declined
*Significance reported at the p<0.05 level, isolates that are significantly different from the wild type ancestor are starred
Figure 2: Significant differences in the productivity of the same isolate in different environments shows that biofilm
formation is condition specific. The decreased productivity of the isolates selected for biofilm formation suggests
that other traits may be under stronger selection, such as adaptation to the media.
- Agnoli, K., Frauenknecht, C., Freitag, R., Schwager, S., Jenul, C., Vergunst, A., … Eberl, L. (2014). The third replicon of members of the Burkholderia cepacia complex, plasmid pC3, plays a role
in stress tolerance. Applied and Environmental Microbiology, 80(4), 1340–1348. doi:10.1128/AEM.03330-13
-­‐
Cooper,
V.
S.,
Staples,
R.
K.,
Traverse,
C.
C.,
&
Crystal,
N.
(2014).
Parallel
evolu?on
of
small
colony
variants
in
Burkholderia
cenocepacia
biofilms.
Genomics.
doi:10.1016/j.ygeno.2014.09.007
- Poltak, S. R., & Cooper, V. S. (2011). Ecological succession in long-term experimentally evolved biofilms produces synergistic communities. The ISME Journal, 5(3), 369–78. doi:10.1038/ismej.
2010.136
- Traverse, C. C., Mayo-smith, L. M., Poltak, S. R., & Cooper, V. S. (2012). Tangled bank of experimentally evolved Burkholderia bio films reflects selection during chronic infections, 110(3). doi:
10.1073/pnas.1207025110/-/DCSupplemental.www.pnas.org/cgi/doi/10.1073/pnas.1207025110
6
replicate
biofilm
popula?ons
were
the
experimental
group;
6
planktonic
popula?ons
were
the
control.
*
*
*
*
Figure 1: Relative fitness was measured in a 24 hour competition assay. Four biofilm isolates experienced fitness
gains in both planktonic and biofilm environments. Isolates 5 and 6 adapted to the planktonic environment at the
cost of biofilm fitness, likely due to identical 29 and 47,000 base pair deletions. In isolate 1, the loss of
chromosome 3 only had a significant effect in the planktonic environment. Biofilm isolate 4 had the greatest gain
in both biofilm and planktonic fitness, suggesting an advantageous mutation.
Mutants exhibit altered levels of fitness
*
*
*
*
*
*