BACKGROUND
bacteria that form biofilms have been shown to be highly resistant to antimicrobial therapy
<cit>  and contribute to the chronic nature of many bacterial infections in humans because cells in biofilms are highly resistant to antibiotic treatment .
developing effective treatments for biofilm-related infections requires an understanding of the processes that lead biofilms to persist in the face of antimicrobial treatment
<cit> .
there are multiple hypotheses to explain biofilm persistence during antibiotic treatment
<cit> .
physical factors, like diffusion limitation, may prevent antibiotic concentrations from reaching inhibitory or lethal levels within biofilms
<cit> .
however, several studies report biofilm persistence despite substantial antibiotic diffusion .
another hypothesis posits that non-genetic phenotypic heterogeneity, including the plastic expression of phenotypes that are resistant to antibiotics, may occur in biofilms.
for example, resource gradients may lead cells to experience different microenvironments and thus express distinct phenotypes in different parts of a biofilm
<cit> .
in particular, a “persister” phenotype has been hypothesized to arise in biofilms and provide immunity against antibiotics
<cit> .
these persister cells are postulated to have the ability to reconstitute biofilms upon release from antibiotic threat
<cit> .
alternatively, bacteria cultured as biofilms may evolve heritable variation for resistance to antibiotics de novo <cit> .
we know that evolutionary change can occur rapidly within tens to hundreds of generations
<cit> , which are time scales relevant to medical treatment of infectious diseases
<cit> .
furthermore, bacteria in biofilms have huge population sizes so that many new mutations will arise over relatively short time scales.
it is possible that antibiotic resistance might arise in bacterial biofilms through straightforward population genetic processes.
we suggest that, given enough time, variation in antibiotic resistance may arise in biofilms even in the absence of antibiotic selection.
this could happen due to the accumulation of neutral variation or as a result of selection for phenotypes that by happenstance are correlated with antibiotic resistance .
evolutionary change in biofilms is plausible since recent studies have shown tremendous phenotypic variation among cells isolated from biofilms growing in patients
<cit>  and demonstrated heritable variation in traits within experimentally cultured biofilm populations
<cit> .
however, few hypotheses about antibiotic resistance in biofilms invoke evolutionary change as an alternative explanation for biofilm persistence during antibiotic treatment .
evolution could involve mutations that convey resistance to single antibiotics  or to whole suites of antibiotics .
the occurrence of variants resistant to antibiotics may provide an “insurance effect”
<cit>  by creating subpopulations of cells that can survive or even proliferate should the biofilm come under antibiotic assault.
resistant variants could thus facilitate reconstitution of bacterial biofilm populations following cessation of antibiotic treatment.
documenting whether genetic variation for antibiotic resistance arises during the course of biofilm development is an important first step to exploring biofilm persistence in the face of antibiotic treatment.
as we suggest, evolutionary explanations predict that genetic variation in the susceptibility to antibiotics will arise in biofilms, and that the frequency of antibiotic resistant cells will increase through time.
here we demonstrate the rapid evolution of heritable variation for broad-spectrum antibiotic resistance during the course of biofilm development by e. coli.
we tested three hypotheses in this work:  heritable variation for antibiotic resistance evolves during biofilm development;  this variation includes both resistant and susceptible mutants to a range of antibiotics; and  phenotypic variation in biofilms increases through time.
we found evidence for both  and  but not .
variation in antibiotic resistance emerged within  <dig> days of biofilm growth, a time frame that is consistent with many common bacterial infections.
thus, these findings have important implications for the development of treatments for bacteria that form biofilms during infection.
RESULTS
we cultured escherichia coli k <dig> mg <dig> as biofilms in the absence of antibiotics.
briefly, we inoculated e. coli into flow-cells and cultured biofilms using minimal medium with glucose as a sole carbon source.
we sampled biofilm populations at  <dig>   <dig> and  <dig> days and isolated ten bacterial clones from three replicate flow cells at each time point.
using the kirby-bauer disc diffusion method
<cit> , we characterized each bacterial clone for resistance to twelve antibiotics by measuring the diameters of zones of  inhibition  for each clone on each antibiotic .
1based on sensi-disks from bd.
numbers following letters indicate ug of antibiotic applied to each disk.
we tested whether heritable variation for antibiotic resistance evolved during biofilm development by assessing change in mean zoi for the biofilm-derived clones relative to the ancestor.
we observed the evolution of statistically significant differences in antibiotic resistance in biofilms , with clones that were more sensitive or more resistant appearing independently in each of our replicates .
the data used for the analysis was formatted to create a balanced design matrix.
using a set of nine planned contrasts, we found significant changes in the mean evolved resistance for several antibiotics through time .
many evolved clones also showed increased susceptibility to antibiotics.
although resistant clones were uncommon in our experiments, when they appeared in evolved biofilms, they did so at notable frequencies .
for some combinations of antibiotics and sampling times, multiple samples from evolved biofilms showed higher variability than seen among multiple samples from the ancestral clone .
these findings support hypotheses  <dig> and 2; genetic variation in levels of resistance to antibiotics evolves during biofilm development in the absence of antibiotics, and this variation includes both resistant and sensitive clones.
there was no evidence of increased variation through time in our data .
symbols denote direction of change: ↓ = smaller zoi for latter group, ↑ = larger zoi for latter group.
our results suggest that antibiotic resistance and susceptibility can rapidly evolve in biofilms over relatively short time scales , which begs the question of how these rates of mutation accumulation compare to those in well-mixed liquid cultures that support exponential growth.
such a comparison is difficult because estimating the “mutation rate” in the spatially structured bacterial cells of biofilms is problematic.
mutation rates are almost always calculated and compared on a “per-generation” basis , but rates of bacterial cell division in biofilms vary widely depending on location within the biofilm matrix.
this variation in cellular growth rates is a consequence of nutrient depletion and the creation of strong gradients of substrates, electron acceptors and other resources within the spatially structured environment of biofilms
<cit> .
these gradients cause growth rates to vary tremendously within biofilms, such that cells deep within the biofilm matrix may not divide at all
<cit> .
because of this, mutation frequency cannot be expressed in the same terms, i.e., per generation, as in well-mixed liquid cultures; nor can one calculate a meaningful population-wide average growth rate for cells in biofilms.
one can imagine applying models that account for differential growth in biofilms , and then using current data to calculate mutation rates that can be compared to rates in well-mixed cultures.
however, such calculations require data about mutation rates in in non-growing bacterial cells that is largely lacking, so direct and simple comparisons between biofilms and well-mixed cultures are not possible at this time.
the evolution of antibiotic resistance and susceptibility in bacterial biofilms involves the interaction between mutation, selection, genetic drift, and spatial structure
<cit> .
the data presented here cannot determine the importance of these multiple explanatory factors.
it seems likely that evolution in biofilms typically occurs under conditions contrary to what is typically assumed in standard population genetics theory  and rather involves strong mutational mechanisms typical in bacteria under stress
<cit>  coupled with weak selection .
future work combining spatially explicit models for biofilm growth  with model-based estimates of mutation rates and effect sizes for bacteria  would provide more insight into the details of evolution in biofilms.
CONCLUSIONS
these data show the rapid de novo evolution of heritable variation in antibiotic sensitivity and resistance during e. coli biofilm development.
we suggest that evolutionary processes, whether genetic drift or natural selection, should be considered as a factor to explain the elevated tolerance to antibiotics typically observed in bacterial biofilms.
we do not yet know whether evolution of antibiotic resistance requires high rates of mutation as can arise in biofilms  or can be explained by normal mutation rates in bacteria.
in either case, biofilms quickly evolve high levels of variation in antibiotic resistance.
we hypothesize that rare, highly resistant variants may allow biofilms to regrow following antibiotic treatment.
this mechanism is an important potential explanation for why biofilm populations are, in general, highly resistant to antibiotic treatment.

 bacteria that form biofilms have been shown to be highly resistant to antimicrobial therapy and contribute to the chronic nature of many bacterial infections in humans because cells in biofilms are highly resistant to antibiotic treatment. developing effective treatments for biofilm-related infections requires an understanding of the processes that lead biofilms to persist in the face of antimicrobial treatment. there are multiple hypotheses to explain biofilm persistence during antibiotic treatment. physical factors, like diffusion limitation, may prevent antibiotic concentrations from reaching inhibitory or lethal levels within biofilms. however, several studies report biofilm persistence despite substantial antibiotic diffusion. another hypothesis posits that non-genetic phenotypic heterogeneity, including the plastic expression of phenotypes that are resistant to antibiotics, may occur in biofilms. for example, resource gradients may lead cells to experience different microenvironments and thus express distinct phenotypes in different parts of a biofilm. in particular, a “persister” phenotype has been hypothesized to arise in biofilms and provide immunity against antibiotics. these persister cells are postulated to have the ability to reconstitute biofilms upon release from antibiotic threat. alternatively, bacteria cultured as biofilms may evolve heritable variation for resistance to antibiotics de novo. we know that evolutionary change can occur rapidly within tens to hundreds of generations, which are time scales relevant to medical treatment of infectious diseases. furthermore, bacteria in biofilms have huge population sizes so that many new mutations will arise over relatively short time scales. it is possible that antibiotic resistance might arise in bacterial biofilms through straightforward population genetic processes. we suggest that, given enough time, variation in antibiotic resistance may arise in biofilms even in the absence of antibiotic selection. this could happen due to the accumulation of neutral variation or as a result of selection for phenotypes that by happenstance are correlated with antibiotic resistance. evolutionary change in biofilms is plausible since recent studies have shown tremendous phenotypic variation among cells isolated from biofilms growing in patients and demonstrated heritable variation in traits within experimentally cultured biofilm populations. however, few hypotheses about antibiotic resistance in biofilms invoke evolutionary change as an alternative explanation for biofilm persistence during antibiotic treatment. evolution could involve mutations that convey resistance to single antibiotics or to whole suites of antibiotics. the occurrence of variants resistant to antibiotics may provide an “insurance effect” by creating subpopulations of cells that can survive or even proliferate should the biofilm come under antibiotic assault. resistant variants could thus facilitate reconstitution of bacterial biofilm populations following cessation of antibiotic treatment. documenting whether genetic variation for antibiotic resistance arises during the course of biofilm development is an important first step to exploring biofilm persistence in the face of antibiotic treatment. as we suggest, evolutionary explanations predict that genetic variation in the susceptibility to antibiotics will arise in biofilms, and that the frequency of antibiotic resistant cells will increase through time. here we demonstrate the rapid evolution of heritable variation for broad-spectrum antibiotic resistance during the course of biofilm development by e. coli. we tested three hypotheses in this work: heritable variation for antibiotic resistance evolves during biofilm development; this variation includes both resistant and susceptible mutants to a range of antibiotics; and phenotypic variation in biofilms increases through time. we found evidence for both and but not. variation in antibiotic resistance emerged within days of biofilm growth, a time frame that is consistent with many common bacterial infections. thus, these findings have important implications for the development of treatments for bacteria that form biofilms during infection.  we cultured escherichia coli k mg as biofilms in the absence of antibiotics. briefly, we inoculated e. coli into flow-cells and cultured biofilms using minimal medium with glucose as a sole carbon source. we sampled biofilm populations at and days and isolated ten bacterial clones from three replicate flow cells at each time point. using the kirby-bauer disc diffusion method, we characterized each bacterial clone for resistance to twelve antibiotics by measuring the diameters of zones of inhibition for each clone on each antibiotic. 1based on sensi-disks from bd. numbers following letters indicate ug of antibiotic applied to each disk. we tested whether heritable variation for antibiotic resistance evolved during biofilm development by assessing change in mean zoi for the biofilm-derived clones relative to the ancestor. we observed the evolution of statistically significant differences in antibiotic resistance in biofilms, with clones that were more sensitive or more resistant appearing independently in each of our replicates. the data used for the analysis was formatted to create a balanced design matrix. using a set of nine planned contrasts, we found significant changes in the mean evolved resistance for several antibiotics through time. many evolved clones also showed increased susceptibility to antibiotics. although resistant clones were uncommon in our experiments, when they appeared in evolved biofilms, they did so at notable frequencies. for some combinations of antibiotics and sampling times, multiple samples from evolved biofilms showed higher variability than seen among multiple samples from the ancestral clone. these findings support hypotheses and 2; genetic variation in levels of resistance to antibiotics evolves during biofilm development in the absence of antibiotics, and this variation includes both resistant and sensitive clones. there was no evidence of increased variation through time in our data. symbols denote direction of change: ↓ = smaller zoi for latter group, ↑ = larger zoi for latter group. our results suggest that antibiotic resistance and susceptibility can rapidly evolve in biofilms over relatively short time scales, which begs the question of how these rates of mutation accumulation compare to those in well-mixed liquid cultures that support exponential growth. such a comparison is difficult because estimating the “mutation rate” in the spatially structured bacterial cells of biofilms is problematic. mutation rates are almost always calculated and compared on a “per-generation” basis, but rates of bacterial cell division in biofilms vary widely depending on location within the biofilm matrix. this variation in cellular growth rates is a consequence of nutrient depletion and the creation of strong gradients of substrates, electron acceptors and other resources within the spatially structured environment of biofilms. these gradients cause growth rates to vary tremendously within biofilms, such that cells deep within the biofilm matrix may not divide at all. because of this, mutation frequency cannot be expressed in the same terms, i.e., per generation, as in well-mixed liquid cultures; nor can one calculate a meaningful population-wide average growth rate for cells in biofilms. one can imagine applying models that account for differential growth in biofilms, and then using current data to calculate mutation rates that can be compared to rates in well-mixed cultures. however, such calculations require data about mutation rates in in non-growing bacterial cells that is largely lacking, so direct and simple comparisons between biofilms and well-mixed cultures are not possible at this time. the evolution of antibiotic resistance and susceptibility in bacterial biofilms involves the interaction between mutation, selection, genetic drift, and spatial structure. the data presented here cannot determine the importance of these multiple explanatory factors. it seems likely that evolution in biofilms typically occurs under conditions contrary to what is typically assumed in standard population genetics theory and rather involves strong mutational mechanisms typical in bacteria under stress coupled with weak selection. future work combining spatially explicit models for biofilm growth with model-based estimates of mutation rates and effect sizes for bacteria would provide more insight into the details of evolution in biofilms.  these data show the rapid de novo evolution of heritable variation in antibiotic sensitivity and resistance during e. coli biofilm development. we suggest that evolutionary processes, whether genetic drift or natural selection, should be considered as a factor to explain the elevated tolerance to antibiotics typically observed in bacterial biofilms. we do not yet know whether evolution of antibiotic resistance requires high rates of mutation as can arise in biofilms or can be explained by normal mutation rates in bacteria. in either case, biofilms quickly evolve high levels of variation in antibiotic resistance. we hypothesize that rare, highly resistant variants may allow biofilms to regrow following antibiotic treatment. this mechanism is an important potential explanation for why biofilm populations are, in general, highly resistant to antibiotic treatment. 
using escherichia coli biofilms, we demonstrate that heritable variation for broad-spectrum antibiotic resistance can arise and accumulate rapidly during biofilm development, even in the absence of antibiotic selection. our results demonstrate the rapid de novo evolution of heritable variation in antibiotic sensitivity and resistance during e. coli biofilm development. we suggest that evolutionary processes, whether genetic drift or natural selection, should be considered as a factor to explain the elevated tolerance to antibiotics typically observed in bacterial biofilms. explanations for bacterial biofilm persistence during antibiotic treatment typically depend on non-genetic mechanisms, and rarely consider the contribution of evolutionary processes. this could be an under-appreciated mechanism that accounts why biofilm populations are, in general, highly resistant to antibiotic treatment.
 explanations for bacterial biofilm persistence during antibiotic treatment typically depend on non-genetic mechanisms, and rarely consider the contribution of evolutionary processes.  using escherichia coli biofilms, we demonstrate that heritable variation for broad-spectrum antibiotic resistance can arise and accumulate rapidly during biofilm development, even in the absence of antibiotic selection.  our results demonstrate the rapid de novo evolution of heritable variation in antibiotic sensitivity and resistance during e. coli biofilm development. we suggest that evolutionary processes, whether genetic drift or natural selection, should be considered as a factor to explain the elevated tolerance to antibiotics typically observed in bacterial biofilms. this could be an under-appreciated mechanism that accounts why biofilm populations are, in general, highly resistant to antibiotic treatment. evolutionantibiotic resistancebacterial biofilmsmutationsdiversity 