BACKGROUND
the mutations that lead to the genetic disorder cystic fibrosis  predispose patients to chronic bacterial lung infections, particularly with the opportunist pseudomonas aeruginosa <cit> .
once established, these chronic bacterial infections are virtually impossible to eradicate and lead to a decline in pulmonary function, reduction in quality of life and premature death  <cit> .
during chronic lung infections in cf patients, p. aeruginosa populations accumulate mutations generating considerable population diversity, leading to both genotypic and phenotypic variations  <cit> .
this diversification process can lead to various phenotypic sub-types co-existing in the same population, varying in characteristics such as colony morphology, including mucoid conversion, the inactivation of quorum-sensing  and other virulence-associated traits, hypermutation, loss of the o-antigen components of the lipopolysaccharide, loss of motility, resistance to antibiotics and changes in nutritional requirements  <cit> .
in a previous study, we analysed  <dig> isolates of the liverpool epidemic strain  of p. aeruginosa from  <dig> sputum samples obtained from  <dig> chronically infected adult cf patients  <cit> .
following the characterisation of the isolates for  <dig> traits,  <dig> haplotypes  of the les were identified.
the majority of phenotypic diversity occurred within individual cf patients.
we further showed that this diversity was highly dynamic, with a rapid turnover of subtypes over time.
certain phenotypic changes, such as the evolution of hypermutability and mucoidy, are commonly reported in cf isolates of p. aeruginosa and, therefore, suggest conserved evolutionary pathways of adaptation  <cit> .
the cf lung presents a highly complex environment that is viscous, spatially heterogeneous and compartmentalized.
moreover, it houses a rich microbiota of coexisting species, which may compete for resources or cause p. aeruginosa mortality .
furthermore, the cf lung environment exposes colonising bacteria to physiologically stressful conditions, including host immune responses, oxidative stress and antibiotic treatment  <cit> .
thus it has been hypothesised that phenotypic diversification allows p. aeruginosa to adapt to the hostile environment of the cf lung thereby enabling long-term persistence.
moreover, it has been argued that such diversification leads to either increased or reduced virulence  <cit>  and could therefore be crucial to understanding disease progression and treatment.
while all of these facets of the cf lung environment could potentially play a role in mediating the diversification of p. aeruginosa, it is not possible to disentangle or determine the relative importance of these selective forces in vivo.
a powerful approach to understanding the contribution of particular selective forces to driving bacterial diversification is through experimental evolution, whereby replicate populations are exposed to defined selective conditions in the laboratory.
cf patients are typically subject to extended antibiotic regimes, but the drugs do not necessarily reach the entire lung at inhibitory concentrations  <cit> .
therefore, sub-inhibitory antibiotic exposure could be one factor that promotes p. aeruginosa diversification in the cf lung.
consequently, a better understanding of the responses of p. aeruginosa populations to these sub-inhibitory concentrations of antibiotics in the cf lung would allow clinicians to make better informed choices of antibiotic regimes.
although it is likely that most cf patients acquire p. aeruginosa infections from diverse environmental reservoirs and thus carry their own unrelated strains, several multidrug-resistant “epidemic” strains capable of patient to patient transmission have been identified  <cit> .
the les is the most widespread transmissible strain of p. aeruginosa in the uk  <cit> , and has also been reported in north america  <cit> .
it has been detected in as many as 79% of adult cf patients in a liverpool cf centre  <cit> .
the high prevalence of les in cf patients is a concern, given that chronic les infection has been associated with a greater deterioration in pulmonary function and nutritional state  <cit>  and increased antibiotic resistance  <cit> .
in this study, we analysed p. aeruginosa les populations in an artificial sputum medium  model that mimics cf sputum in terms of composition.
various groups have utilised asm models to study, for example, gene expression patterns and the effects of bacteriophages  <cit> .
p. aeruginosa, when cultured in asm, forms biofilms and diversifies with respect to phenotype, in a manner that resembles behaviour in the cf lung  <cit> .
we hypothesise that exposure to sub-inhibitory concentrations of antibiotics will drive bacterial diversification, possibly through a combination of antibiotic-induced mutagenesis or through the regulation of gene transcription  <cit> .
consequently, the objective of this study was to test the hypothesis that exposure to sub-inhibitory concentrations of antibiotics has a role to play in promoting p. aeruginosa population diversification during growth in an asm model.
RESULTS
sub-inhibitory antibiotics promote diversification of p. aeruginosa lesb58
the emergence of novel haplotypes was observed in all culture conditions, but the presence of sub-inhibitory concentrations of certain antibiotics significantly increased both the number of novel haplotypes  and the haplotype diversity found within populations  relative to control populations .
however, some antibiotics contributed to this diversity more than others.
diversification was highest in the presence of ceftazidime and colisitin  compared to azithromycin , tobramycin  and meropenem .
furthermore, based on mean antibiotic resistance across the antibiotics tested, the brown-forsythe-levene test of equality of variances between  <dig> groups gave a test statistic of f =  <dig> , p <  <dig> .
exposure to ceftazidime and colistin gave a high variance, and the differences between means are statistically significant .
there was no significant difference in the colony forming unit  values between the populations exposed to antibiotics in asm and in populations exposed to asm alone.
asm appears to generate variation in bacterial numbers among replicates.
haplotype properties differ between different antibiotic exposures
diversification of p. aeruginosa lesb <dig> in asm cultured with and without the various antibiotics was observed only with respect to colony morphology, pyocyanin production and antibiotic susceptibilities .
the culture of lesb <dig> in asm with sub-inhibitory concentrations of ceftazidime and colistin led to diversity in antimicrobial susceptibilities, changes in colony morphology and a loss of pyocyanin production .
lesb <dig> cultured in the presence of these antibiotics, generated more isolates that were outside the normal range of the antibiotic sensitivity profiles of lesb <dig> controls .
in addition, exposure to azithromycin and tobramycin promoted increased cross-resistance to other antibiotics .
there was no variation in the auxotrophic phenotype in the isolates analysed in all experimental and control groups .
the populations exposed to meropenem exhibited no clear phenotypic diversification .
isolates that were characterized as being outside the normal range of antimicrobial susceptibility typically observed in lesb <dig>  included isolates that had either an increased or reduced susceptibility to the antibiotic under test.
asm = artificial sputum medium, lb = luria bertani, caz = ceftazidime, ct = colistin, azt = azithromycin, mem = meropenem and tobi = tobramycin.
furthermore, there was no variation in the carriage of the les prophage  <dig>  les prophage  <dig> and les genomic island - <dig> in the isolates analysed .
none of the tested isolates grown in asm  displayed the hypermutable phenotype.
the only hypermutable isolate detected in this study was generated following growth in luria bertani  for 18 hours .
although diversification occurred with respect to only a few of the phenotypic properties tested, the proportions of the isolates exhibiting these traits varied considerably between treatment groups .
the proportions of these phenotypic changes accounted for the within and between-treatment group variation seen in the numbers of mutant haplotypes .
hierarchical analysis of variance indicated that the majority  of diversity was distributed between isolates within populations, rather than the same traits systematically apportioned between replicate populations or between treatments .
discussion
although it is known that the phenotypic and genotypic characteristics of p. aeruginosa populations within the cf lung fluctuate over time  <cit> , the factors that are responsible for this diversification are not fully understood.
when p. aeruginosa lesb <dig> was grown in asm with and without sub-inhibitory concentrations of antibiotics, we observed differential effects of antibiotics commonly used to treat cf patients on the diversity of lesb <dig> populations in the asm model.
in particular, increased levels of phenotypic diversification occurred in lesb <dig> populations grown in asm when sub-inhibitory concentrations of colistin, ceftazidime and azithromycin were present.
however, extensive diversification of the p. aeruginosa populations was not seen in the presence of sub-inhibitory concentrations of meropenem.
there are a number of mechanisms by which sub-inhibitory concentrations of antibiotics could potentially enhance bacterial diversification.
one potential mechanism could involve the antibiotics inducing mutagenesis within bacterial populations, causing variation and/or promoting the hypermutability phenotype  <cit> .
a second potential mechanism could involve the antibiotics acting as signalling molecules, altering the qs systems within bacterial populations and subsequently promoting social evolution and diversification  <cit> .
antibiotic exposure has been shown to induce mutagenesis by triggering the sos response and thus increasing the expression of error-prone dna polymerases, which could give rise to diversity within bacterial populations  <cit> .
it is possible that ceftazidime induced mutagenesis in the lesb <dig> populations through the induction of the sos response.
it has been suggested that this increase in mutagenesis is an adaptive strategy that favours the acquisition of antimicrobial resistance and survival in harsh environments  <cit> .
however, it has been argued that the generation of genetic variants within the cf lung does not require the sos response, and that starvation and oxidative stress caused by antibiotic exposure can promote diversity within p. aeruginosa biofilms  <cit> .
the hypermutable phenotype occurs as a consequence of defects in error avoidance or dna repair genes, typically termed anti-mutator genes  <cit> .
it has been suggested that hypermutability, promoted by extrinsic and intrinsic factors, is the driver of p. aeruginosa adaptation and survival in the cf lung  <cit> .
although phenotypic diversification of lesb <dig> was observed following culture in asm, especially when sub-inhibitory concentrations of colisitin, ceftazidime or azithromycin were present, no hypermutable isolates were detected.
in our previous study using les isolates from multiple cf patients, we found hypermutable sub-types but only at low frequency  <cit> .
in this study we found no evidence that hypermutability was driving this diversification and adaptation process.
this supports work by ciofu et al.
<cit>  who found that the hypermutability phenotype was not essential for the acquisition of mucoidy and loss of qs.
other studies have also suggested that spontaneous mutation and mutator strains are not required to produce genetic variants in populations of p. aeruginosa within the cf lung  <cit> .
it has been shown that sub-inhibitory concentrations of antibiotics can act as signalling molecules that regulate bacterial gene transcription, physiology and virulence  <cit> .
in particular, tobramycin, colistin and azithromycin at sub-inhibitory concentrations have been shown to modulate the qs networks in bacterial populations  <cit> .
these antibiotics are commonly used to treat cf patients and, therefore, the signalling activities of these antibiotics could increase bacterial fitness for survival in the harsh environment of the cf lung  <cit> , suggesting that the classical view of antibiotics acting only to reduce bacterial fitness and virulence is not always the case.
in the current study, across all the asm cultures, no single dominant phenotypic variant emerged.
some patterns in the diversification process were evident.
for example, isolates lacking the pyocyanin production phenotype occurred following culture in asm with ceftazidime or colistin.
however this was only evident in two out of the three biological replicates , highlighting the variability between replicates.
a previous study by cummins et al.
<cit>  has shown that sub-inhibitory concentrations of colisitin actually increases pyocyanin production.
pyocyanin production is regulated by qs, which relies upon small diffusible signal molecules interacting with transcriptional activators to couple gene expression with cell population density.
although qs-deficiency is a common feature amongst p. aeruginosa cf isolates  <cit> , qs regulates a number of factors of relevance to cf, including pyocyanin and lasa production  <cit> .
our previous studies suggested that les populations in cf comprise a mixture of qs-positive and qs-deficient bacteria  <cit> , which is what we have observed in this study in asm.
the qs-deficient populations could benefit at the cost of qs-positive populations.
the main phenotypic variations involved changes in colony morphology, pyocyanin production and antimicrobial susceptibilities.
a high diversity in the antimicrobial susceptibility profiles of cf isolates within adult sputum samples has been demonstrated previously  <cit> , highlighting the limitations of performing antimicrobial susceptibility tests on a single isolate from a cf patient sputum sample.
it was also shown that using one antibiotic could lead to enhanced resistance to a different, unrelated antibiotic  <cit> .
a similar pattern was observed in this study, when exposure to one antibiotic altered the antibiotic susceptibility profiles to unrelated antibiotics.
in particular, exposure to azithromycin, tobramycin or ceftazidime led to an increase in resistance to tazobactam/piperacillin.
this could have serious clinical consequences for the cf patient, in terms of the generation of antimicrobial resistant p. aeruginosa populations, because cf patients are regularly exposed to a number of different antibiotics.
in our study, the presence of meropenem had a weaker effect on diversification compared to the other antibiotics, despite having a similar mechanism of action to ceftazidime.
however, it is possible that cell death was occurring in these populations, since the numbers of cells obtained following culture were generally lower.
this is despite the meropenem concentration in asm being 8-fold less than the minimum inhibitory concentration of this antibiotic.
therefore, the apparent reduction in diversity could be attributed to the populations exhibiting cell death.
this suggests that there may be a clinical advantage to using some antibiotics  rather than others.
it would also be interesting to analyse combinations of two antibiotics, since it is often the case that dual therapy is used clinically.
the identification of individual mutations within the lesb <dig> populations to explain the changes in individual phenotypic traits would have been beyond the scope of this work.
CONCLUSIONS
this study suggests that the exposure to sub-inhibitory concentrations of certain antibiotics can drive phenotypic diversification of p. aeruginosa populations in the asm model.
this may help to explain the observed diversification of p. aeruginosa in natural cf lung infections, although other factors such as the host immune response, other members of the microflora, or bacteriophages may also contribute.
understanding p. aeruginosa diversification in the cf lung could alter the way we control these infections, in particular during the early stages.
diversification of the p. aeruginosa populations in the cf lung, and the emergence of phenotypes such as mucoidy, are signs of adaptation leading to a chronic infection state.
diversification may also lead to enhanced antimicrobial resistance.
antibiotics that do not cause extensive diversification might be utilised to prevent diversification, and possibly slow down the development of a chronic infection state.
therefore, being able to delay, control or possibly reduce diversification could be advantageous for the cf patient.
this could also be achieved by using antibiotics that permeate the lung and the bacterial biofilms better to achieve inhibitory concentrations, but it could also be important to choose antibiotics that do not promote diversification.
hence a better understanding of the differential effects of various antibiotics on diversification of p. aeruginosa populations could provide valuable information to help clinicians choose the best antibiotics for cf patients.

 the mutations that lead to the genetic disorder cystic fibrosis predispose patients to chronic bacterial lung infections, particularly with the opportunist pseudomonas aeruginosa. once established, these chronic bacterial infections are virtually impossible to eradicate and lead to a decline in pulmonary function, reduction in quality of life and premature death. during chronic lung infections in cf patients, p. aeruginosa populations accumulate mutations generating considerable population diversity, leading to both genotypic and phenotypic variations. this diversification process can lead to various phenotypic sub-types co-existing in the same population, varying in characteristics such as colony morphology, including mucoid conversion, the inactivation of quorum-sensing and other virulence-associated traits, hypermutation, loss of the o-antigen components of the lipopolysaccharide, loss of motility, resistance to antibiotics and changes in nutritional requirements. in a previous study, we analysed isolates of the liverpool epidemic strain of p. aeruginosa from sputum samples obtained from chronically infected adult cf patients. following the characterisation of the isolates for traits, haplotypes of the les were identified. the majority of phenotypic diversity occurred within individual cf patients. we further showed that this diversity was highly dynamic, with a rapid turnover of subtypes over time. certain phenotypic changes, such as the evolution of hypermutability and mucoidy, are commonly reported in cf isolates of p. aeruginosa and, therefore, suggest conserved evolutionary pathways of adaptation. the cf lung presents a highly complex environment that is viscous, spatially heterogeneous and compartmentalized. moreover, it houses a rich microbiota of coexisting species, which may compete for resources or cause p. aeruginosa mortality. furthermore, the cf lung environment exposes colonising bacteria to physiologically stressful conditions, including host immune responses, oxidative stress and antibiotic treatment. thus it has been hypothesised that phenotypic diversification allows p. aeruginosa to adapt to the hostile environment of the cf lung thereby enabling long-term persistence. moreover, it has been argued that such diversification leads to either increased or reduced virulence and could therefore be crucial to understanding disease progression and treatment. while all of these facets of the cf lung environment could potentially play a role in mediating the diversification of p. aeruginosa, it is not possible to disentangle or determine the relative importance of these selective forces in vivo. a powerful approach to understanding the contribution of particular selective forces to driving bacterial diversification is through experimental evolution, whereby replicate populations are exposed to defined selective conditions in the laboratory. cf patients are typically subject to extended antibiotic regimes, but the drugs do not necessarily reach the entire lung at inhibitory concentrations. therefore, sub-inhibitory antibiotic exposure could be one factor that promotes p. aeruginosa diversification in the cf lung. consequently, a better understanding of the responses of p. aeruginosa populations to these sub-inhibitory concentrations of antibiotics in the cf lung would allow clinicians to make better informed choices of antibiotic regimes. although it is likely that most cf patients acquire p. aeruginosa infections from diverse environmental reservoirs and thus carry their own unrelated strains, several multidrug-resistant “epidemic” strains capable of patient to patient transmission have been identified. the les is the most widespread transmissible strain of p. aeruginosa in the uk, and has also been reported in north america. it has been detected in as many as 79% of adult cf patients in a liverpool cf centre. the high prevalence of les in cf patients is a concern, given that chronic les infection has been associated with a greater deterioration in pulmonary function and nutritional state and increased antibiotic resistance. in this study, we analysed p. aeruginosa les populations in an artificial sputum medium model that mimics cf sputum in terms of composition. various groups have utilised asm models to study, for example, gene expression patterns and the effects of bacteriophages. p. aeruginosa, when cultured in asm, forms biofilms and diversifies with respect to phenotype, in a manner that resembles behaviour in the cf lung. we hypothesise that exposure to sub-inhibitory concentrations of antibiotics will drive bacterial diversification, possibly through a combination of antibiotic-induced mutagenesis or through the regulation of gene transcription. consequently, the objective of this study was to test the hypothesis that exposure to sub-inhibitory concentrations of antibiotics has a role to play in promoting p. aeruginosa population diversification during growth in an asm model.  sub-inhibitory antibiotics promote diversification of p. aeruginosa lesb58 the emergence of novel haplotypes was observed in all culture conditions, but the presence of sub-inhibitory concentrations of certain antibiotics significantly increased both the number of novel haplotypes and the haplotype diversity found within populations relative to control populations. however, some antibiotics contributed to this diversity more than others. diversification was highest in the presence of ceftazidime and colisitin compared to azithromycin, tobramycin and meropenem. furthermore, based on mean antibiotic resistance across the antibiotics tested, the brown-forsythe-levene test of equality of variances between groups gave a test statistic of f = , p < . exposure to ceftazidime and colistin gave a high variance, and the differences between means are statistically significant. there was no significant difference in the colony forming unit values between the populations exposed to antibiotics in asm and in populations exposed to asm alone. asm appears to generate variation in bacterial numbers among replicates. haplotype properties differ between different antibiotic exposures diversification of p. aeruginosa lesb in asm cultured with and without the various antibiotics was observed only with respect to colony morphology, pyocyanin production and antibiotic susceptibilities. the culture of lesb in asm with sub-inhibitory concentrations of ceftazidime and colistin led to diversity in antimicrobial susceptibilities, changes in colony morphology and a loss of pyocyanin production. lesb cultured in the presence of these antibiotics, generated more isolates that were outside the normal range of the antibiotic sensitivity profiles of lesb controls. in addition, exposure to azithromycin and tobramycin promoted increased cross-resistance to other antibiotics. there was no variation in the auxotrophic phenotype in the isolates analysed in all experimental and control groups. the populations exposed to meropenem exhibited no clear phenotypic diversification. isolates that were characterized as being outside the normal range of antimicrobial susceptibility typically observed in lesb included isolates that had either an increased or reduced susceptibility to the antibiotic under test. asm = artificial sputum medium, lb = luria bertani, caz = ceftazidime, ct = colistin, azt = azithromycin, mem = meropenem and tobi = tobramycin. furthermore, there was no variation in the carriage of the les prophage les prophage and les genomic island - in the isolates analysed. none of the tested isolates grown in asm displayed the hypermutable phenotype. the only hypermutable isolate detected in this study was generated following growth in luria bertani for 18 hours. although diversification occurred with respect to only a few of the phenotypic properties tested, the proportions of the isolates exhibiting these traits varied considerably between treatment groups. the proportions of these phenotypic changes accounted for the within and between-treatment group variation seen in the numbers of mutant haplotypes. hierarchical analysis of variance indicated that the majority of diversity was distributed between isolates within populations, rather than the same traits systematically apportioned between replicate populations or between treatments. discussion although it is known that the phenotypic and genotypic characteristics of p. aeruginosa populations within the cf lung fluctuate over time, the factors that are responsible for this diversification are not fully understood. when p. aeruginosa lesb was grown in asm with and without sub-inhibitory concentrations of antibiotics, we observed differential effects of antibiotics commonly used to treat cf patients on the diversity of lesb populations in the asm model. in particular, increased levels of phenotypic diversification occurred in lesb populations grown in asm when sub-inhibitory concentrations of colistin, ceftazidime and azithromycin were present. however, extensive diversification of the p. aeruginosa populations was not seen in the presence of sub-inhibitory concentrations of meropenem. there are a number of mechanisms by which sub-inhibitory concentrations of antibiotics could potentially enhance bacterial diversification. one potential mechanism could involve the antibiotics inducing mutagenesis within bacterial populations, causing variation and/or promoting the hypermutability phenotype. a second potential mechanism could involve the antibiotics acting as signalling molecules, altering the qs systems within bacterial populations and subsequently promoting social evolution and diversification. antibiotic exposure has been shown to induce mutagenesis by triggering the sos response and thus increasing the expression of error-prone dna polymerases, which could give rise to diversity within bacterial populations. it is possible that ceftazidime induced mutagenesis in the lesb populations through the induction of the sos response. it has been suggested that this increase in mutagenesis is an adaptive strategy that favours the acquisition of antimicrobial resistance and survival in harsh environments. however, it has been argued that the generation of genetic variants within the cf lung does not require the sos response, and that starvation and oxidative stress caused by antibiotic exposure can promote diversity within p. aeruginosa biofilms. the hypermutable phenotype occurs as a consequence of defects in error avoidance or dna repair genes, typically termed anti-mutator genes. it has been suggested that hypermutability, promoted by extrinsic and intrinsic factors, is the driver of p. aeruginosa adaptation and survival in the cf lung. although phenotypic diversification of lesb was observed following culture in asm, especially when sub-inhibitory concentrations of colisitin, ceftazidime or azithromycin were present, no hypermutable isolates were detected. in our previous study using les isolates from multiple cf patients, we found hypermutable sub-types but only at low frequency. in this study we found no evidence that hypermutability was driving this diversification and adaptation process. this supports work by ciofu et al. who found that the hypermutability phenotype was not essential for the acquisition of mucoidy and loss of qs. other studies have also suggested that spontaneous mutation and mutator strains are not required to produce genetic variants in populations of p. aeruginosa within the cf lung. it has been shown that sub-inhibitory concentrations of antibiotics can act as signalling molecules that regulate bacterial gene transcription, physiology and virulence. in particular, tobramycin, colistin and azithromycin at sub-inhibitory concentrations have been shown to modulate the qs networks in bacterial populations. these antibiotics are commonly used to treat cf patients and, therefore, the signalling activities of these antibiotics could increase bacterial fitness for survival in the harsh environment of the cf lung, suggesting that the classical view of antibiotics acting only to reduce bacterial fitness and virulence is not always the case. in the current study, across all the asm cultures, no single dominant phenotypic variant emerged. some patterns in the diversification process were evident. for example, isolates lacking the pyocyanin production phenotype occurred following culture in asm with ceftazidime or colistin. however this was only evident in two out of the three biological replicates, highlighting the variability between replicates. a previous study by cummins et al. has shown that sub-inhibitory concentrations of colisitin actually increases pyocyanin production. pyocyanin production is regulated by qs, which relies upon small diffusible signal molecules interacting with transcriptional activators to couple gene expression with cell population density. although qs-deficiency is a common feature amongst p. aeruginosa cf isolates, qs regulates a number of factors of relevance to cf, including pyocyanin and lasa production. our previous studies suggested that les populations in cf comprise a mixture of qs-positive and qs-deficient bacteria, which is what we have observed in this study in asm. the qs-deficient populations could benefit at the cost of qs-positive populations. the main phenotypic variations involved changes in colony morphology, pyocyanin production and antimicrobial susceptibilities. a high diversity in the antimicrobial susceptibility profiles of cf isolates within adult sputum samples has been demonstrated previously, highlighting the limitations of performing antimicrobial susceptibility tests on a single isolate from a cf patient sputum sample. it was also shown that using one antibiotic could lead to enhanced resistance to a different, unrelated antibiotic. a similar pattern was observed in this study, when exposure to one antibiotic altered the antibiotic susceptibility profiles to unrelated antibiotics. in particular, exposure to azithromycin, tobramycin or ceftazidime led to an increase in resistance to tazobactam/piperacillin. this could have serious clinical consequences for the cf patient, in terms of the generation of antimicrobial resistant p. aeruginosa populations, because cf patients are regularly exposed to a number of different antibiotics. in our study, the presence of meropenem had a weaker effect on diversification compared to the other antibiotics, despite having a similar mechanism of action to ceftazidime. however, it is possible that cell death was occurring in these populations, since the numbers of cells obtained following culture were generally lower. this is despite the meropenem concentration in asm being 8-fold less than the minimum inhibitory concentration of this antibiotic. therefore, the apparent reduction in diversity could be attributed to the populations exhibiting cell death. this suggests that there may be a clinical advantage to using some antibiotics rather than others. it would also be interesting to analyse combinations of two antibiotics, since it is often the case that dual therapy is used clinically. the identification of individual mutations within the lesb populations to explain the changes in individual phenotypic traits would have been beyond the scope of this work.  this study suggests that the exposure to sub-inhibitory concentrations of certain antibiotics can drive phenotypic diversification of p. aeruginosa populations in the asm model. this may help to explain the observed diversification of p. aeruginosa in natural cf lung infections, although other factors such as the host immune response, other members of the microflora, or bacteriophages may also contribute. understanding p. aeruginosa diversification in the cf lung could alter the way we control these infections, in particular during the early stages. diversification of the p. aeruginosa populations in the cf lung, and the emergence of phenotypes such as mucoidy, are signs of adaptation leading to a chronic infection state. diversification may also lead to enhanced antimicrobial resistance. antibiotics that do not cause extensive diversification might be utilised to prevent diversification, and possibly slow down the development of a chronic infection state. therefore, being able to delay, control or possibly reduce diversification could be advantageous for the cf patient. this could also be achieved by using antibiotics that permeate the lung and the bacterial biofilms better to achieve inhibitory concentrations, but it could also be important to choose antibiotics that do not promote diversification. hence a better understanding of the differential effects of various antibiotics on diversification of p. aeruginosa populations could provide valuable information to help clinicians choose the best antibiotics for cf patients. 
however, because cystic fibrosis lungs represent ecologically complex and hostile environments, the selective forces driving this diversification in vivo remain unclear. we took an experimental evolution approach to test the hypothesis that sub-inhibitory antibiotics can drive diversification of p. aeruginosa populations. our study indicates that certain antibiotics can promote population diversification when present in sub-inhibitory concentrations. replicate populations of p. aeruginosa lesb were cultured for seven days in artificial sputum medium with and without sub-inhibitory concentrations of various clinically relevant antibiotics. alterations in colony morphology and pyocyanin production were observed following exposure to ceftazidime and colistin only. hence, the choice of antibiotic may have previously unforeseen implications for the development of p. aeruginosa infections in the lungs of cystic fibrosis patients. divergence occurred due to alterations in antimicrobial susceptibility profiles following exposure to azithromycin, ceftazidime and colistin. we then characterised diversification with respect to phenotypic and genotypic characteristics.
 pseudomonas aeruginosa populations within the cystic fibrosis lung exhibit extensive phenotypic and genetic diversification. the resultant population diversity is thought to be crucial to the persistence of infection and may underpin the progression of disease. however, because cystic fibrosis lungs represent ecologically complex and hostile environments, the selective forces driving this diversification in vivo remain unclear. we took an experimental evolution approach to test the hypothesis that sub-inhibitory antibiotics can drive diversification of p. aeruginosa populations. replicate populations of p. aeruginosa lesb were cultured for seven days in artificial sputum medium with and without sub-inhibitory concentrations of various clinically relevant antibiotics. we then characterised diversification with respect to phenotypic and genotypic characteristics.  we observed that higher population diversity evolved in the presence of azithromycin, ceftazidime or colistin relative to antibiotic-free controls. divergence occurred due to alterations in antimicrobial susceptibility profiles following exposure to azithromycin, ceftazidime and colistin. alterations in colony morphology and pyocyanin production were observed following exposure to ceftazidime and colistin only. diversification was not observed in the presence of meropenem.  our study indicates that certain antibiotics can promote population diversification when present in sub-inhibitory concentrations. hence, the choice of antibiotic may have previously unforeseen implications for the development of p. aeruginosa infections in the lungs of cystic fibrosis patients. antibioticscystic fibrosispopulation biologypseudomonas aeruginosa 