BACKGROUND
haemophilus influenzae is a γ-proteobacterium from within the order the pasteurellacae.
it is an obligate human commensal of the nasopharynx and in most cases it remains as a commensal but some strains can transit from the nasopharynx to other parts of the body and in doing so cause numerous types of disease  <cit> .
there are strain-specific factors that enable pathogenic strains to transit to, and then survive within, different parts of the body, where the stresses of multiple environmental conditions require a breadth of adaptive abilities that permit survival and growth  <cit> .
there are a number of physical parameters that are known to vary between parts of the human host, including: oxygen tension, carbon/energy/nitrogen source, ph and the presence of reactive oxygen and reactive nitrogen species.
defence against these can be directly encoded through detoxification genetic pathways, but also through broader mechanisms for environmental adaptation.
in addition to specific pathways that respond to and deal with each of the damaging physical or chemical stressors present within the various environments the bacteria may encounter, many bacteria have a capacity to switch their lifestyle such that these stresses no longer cause damage to their cell.
one key lifestyle adaptation is the switch from planktonic  cells to a biofilm  <cit> .
this process is based upon numerous features of the bacterial cell including alterations in their metabolism and physiology, the presence and nature of surface structures, and the general physical properties of the bacterial cell.
the process of biofilm formation is defined in stages and each of these has specific features and profiles  <cit> .
put simply, under stressed conditions bacterial cells can switch from a free-living and a rapidly dividing phenotype to an altered metabolic form associated with cell-cell aggregation and attachment to a surface.
there are then early, mid, and late stages for the maturation of a bacterial biofilm.
the particular stresses that induce a change in lifestyle and subsequently the process of biofilm formation are poorly defined for many pathogenic bacteria, however antibiotic usage is certainly one, nutrient starvation and oxidative stress are others  <cit> .
these conditions or signals do seem to be specific for different species.
despite some previous disagreement about the ability of h. influenzae to form a biofilm  <cit> , there is now overwhelming evidence that h. influenzae use biofilm formation for survival within the host and certainly in their colonization of the host  <cit> .
there are elements of h. influenzae which seem to be induced and therefore important for biofilm formation  <cit> .
there are numerous examples of studies that have shown that iron uptake is central to growth within a biofilm  <cit> .
there is a need to further characterise the differences between biofilm-forming and non-biofilm-forming isolates of h. influenzae.
this can be accomplished through a comparison of the genetic and transcriptomic differences between h. influenzae strains that respond to stresses by forming a biofilm, and those that continue to grow under those conditions without forming a biofilm.
changes in ph provides a suitable stressor, being central to its colonisation of different anatomical niches, and identification of the molecular pathways that vary between such isolates would be significant in our understanding of h. influenzae pathogenesis.
h. influenzae strains and isolates display more variation than many other pathogens and underpinning the basis for the strain-specific actors that underlie their biofilm formation .
indeed, coupled to this, there are many features of the h. influenzae physiology  <cit>  and stress response  <cit>  that indicate that this particular host-adapted bacterium has unique molecular mechanisms for survival in the various locations of its host that it can exist.
the ph is known to be elevated in the middle ear, compared to other parts of the body  <cit>  and in this niche there is some evidence that it is ph that induces particular isolates of h. influenzae to form a biofilm  <cit> .
we have assessed the response of different clinical isolates of h. influenzae to changing ph; their growth and biofilm formation.
RESULTS
the growth of different strains of h. influenzae with changing ph
the growth of  <dig> strains  of h. influenzae were assessed over a range of ph values; ph  <dig> ,  <dig>  and  <dig>  as the physiological ph is known to vary among host organs, tissues and niches.
even within a particular body site there can be spatial and temporal changes in ph as a consequence of specific events  <cit> .
despite this uncertainty in the precise nature of the ph value associated with host-pathogen microenvironments, it is clear that there are distinct differences between the primary site of colonization  and the various sites of infection, including the lower respiratory tract, the blood and the middle ear.
as an example, the blood can be  <dig> - <dig>  and the middle ear is usually considered to be around ph  <dig>   <cit> .
we assessed ph response of a small set of isolates of h. influenzae that were known to colonise either the blood or the middle ear.
we grew the bacteria  at ph  <dig> ,  <dig>  and  <dig>  and plotted their growth curves  and from this we calculated mean growth rates .
there were no clear patterns, and the observed changes represented only slight variations.
the equivocal differences in growth at different ph levels does not exclude the possibility that the cells are responding differently, such as with an alternative lifestyle .
h. influenzae
isolates grown at different ph
strain
type
ph  <dig>
ph  <dig>
ph  <dig>
*doubling per hour.
the formation of biofilm by h. influenzae as a consequence of changing ph
given that colonization by h. influenzae within various host niches, such as the middle ear, is linked to their induction of a biofilm, and increased ph is characteristic of these environments, we assessed the possibility that biofilm induction is a consequence of increased ph.
it has been previously suggested that for h. influenzae the biofilm formation is induced at ph  <dig>   <cit> .
we assayed for biofilm formation at ph  <dig> ,  <dig>  and  <dig>  .
these screening assays were performed by counting planktonic cells  followed by washing and then releasing the biofilm cells, and counting colonies .
in some strains, such as isolate r <dig>  there was significant induction of biofilm at ph  <dig>  .
other strains, including eagan, did not form biofilm at any ph.
to compare in detail contrasting isolates from this screening of h. influenzae, eagan  and r <dig>  were taken for further analysis , more biological and experimental replicates.
planktonic cell growth was assessed and then biofilm cell numbers were enumerated.
eagan grew equally well at ph  <dig>  and  <dig> , as did r <dig>  but eagan did not form any biofilm at either ph  <dig>  or  <dig>  whereas r <dig> produced a significant biofilm at ph  <dig> , within the context of this assay there was an increase in biofilm formation at ph  <dig>  .
these results are consistent with what is generally accepted and known with regard to h. influenzae pathogenesis; that the capsular strains cope with increased ph by continuing planktonic growth while nthi isolates that colonizes the middle ear switches to a biofilm mode of growth  <cit> .
transcriptional analyses of eagan and r <dig> under different ph
given the definite, growth-style, variations in response to a shift in ph from  <dig>  to  <dig>  between eagan and r <dig>  we were interested in determining the underlying transcriptional differences that varied between eagan and r <dig>  we therefore used rnaseq to analyse the whole cell transcriptome at ph  <dig>  and  <dig>  for both eagan and r <dig> .
the shift from ph  <dig>  to  <dig> , while biologically relevant and certainly impacting bacterial style of growth , is still a subtle change and it was not expected to generate a large set of cellular pathways with changed expression patterns.
genes that were differentially expressed in eagan  revealed predominantly an up-regulation of two gluconate:h+ symporters  and the associated gluconate  metabolic genes  and a potential glycerate kinase  that links into glycolysis.
it is worth noting that these genes/pathways are genetically unlinked, adding to validity of the response.
in addition to the hi1015/gntp symporter, the hi1010- <dig> genes include homologs to a sugar epimerase, aldolase and isomerases that are within the first stages of the pentose phosphate pathway .
the first gene  is a potential 6-phosphogluconate dehydrogenase that generates ribulose-5-phosphate.
this links directly into the ppp and other energy and biosynthetic pathways .
h. influenzae
eagan at ph  <dig>  compared to ph  <dig>
genes up-regulated at ph  <dig>  compared to  <dig>
2
p
<dig>
p
<dig>
fold
p
genes down-regulated at ph  <dig>  compared to  <dig>
<dig>
p
the gntp symporter family of transporters also import h+, as part of the survival response associated with an increased environmental ph .
it is interesting to note that our bioinformatic analyses have identified an operator/promoter upstream of hi <dig>  with a putative deor binding site; hi <dig> is divergent to a deor-like gene.
while not within the scope of this project it is known in other bacteria that deor-like regulators variously control pathways directing sugar metabolism and are connected to the ppp.
also, the bioinformatics analyses indicate that the hi1010- <dig> genes are on a single transcriptional unit, forming an operon.
traditionally high concentrations of glucose are thought to be oxidized extracellularly by membrane-bound dehydrogenases.
whereas under low glucose conditions, oxidized glucose is imported and phosphorylated within the cell to 6-phosphogluconate  <cit>  which feeds into either the entner-dourdooff  pathway or the ppp  <cit>  for energy.
in addition, gluconate can act as an exogenous carbon source and therefore be taken up as a direct mode of growth.
it has been shown in some contexts that such metabolism is related to bacterial growth in the host-pathogen environment, such as with escherichia coli colonization of the mouse large intestine  <cit>  where gluconate is also important in the growth and pathogenesis of other pathogens  <cit> .
some bacteria possess multiple gluconate uptake systems  <cit> , such as those characterized in e. coli, where there are four  <cit> .
not all of these are necessarily primary gluconate transporters, with some acting on other sugar acids that are able to be utilized by the same permeases.
at least one of these has been shown to be likely to preferentially import fructuronate and not gluconate  <cit> .
in e. coli and other bacteria these transporters are regulated through different transcriptional pathways controlled by sugar-utilizing systems and signals; such as the sensing of the presence of gluconate by gntr, or as in a camp-dependent catabolite repression system/s, by the global transcriptional regulator crp  <cit> .
there is an emerging consensus that the regulation and role of these sugar acid metabolic systems is broader than originally thought.
recently it has been shown that in e. coli, the hexuronate utilizing pathways are regulated by a complex interplay of regulatory systems including induction under osmotic stress conditions  <cit> .
what is clear from our results is that there are two homologous gluconate transport systems in h. influenzae eagan and that both are upregulated at ph  <dig> .
the media used throughout our studies was rich in glucose and other carbon and energy sources .
it is worth noting that there are other genes responsible for these steps in the ppp in the genomes of h. influenzae, however these genes are not physically linked on an operon as with hi1010- <dig>  the indication is that in the eagan strain the hi1010- <dig> operon is uniquely regulated based on ph and it feeds into the ppp functioning under increased ph.
the duplication of genes for steps in the ppp is not unusual, there are homologs of these h. influenzae genes  in several bacteria that have a similar duplication.
in pectobacterium carotovorum the homologs to hi1011- <dig> are vguabcd and these function in gluconate metabolism and have an as yet uncharacterized role in the pathogenesis of this plant pathogen  <cit> .
interestingly, the sugar acid metabolism pathways can also feed into cell wall composition or modifications.
glucuronic acid can be incorporated into the inner core of the lipopolysaccharide  in e. coli and these potential modifications are a response to environmental stresses, specifically those associated with envelope stress, such as ph, and this response is controlled by several regulatory pathways  <cit> .
we demonstrated that as the ph increases to  <dig> , the eagan isolate induced two gluconate permeases, one being part of an operon with gluconate metabolism genes, these likely providing the proteins and enzymes linked into energy production  but also potentially providing other cellular alterations for coping with the stress .
in contrast, the nthi r <dig> isolate did not induce the hi1010- <dig> operon at ph  <dig> .
consistent with this isolate inducing its biofilm formation at ph  <dig> , it induced various, genetically unlinked iron acquisition genes .
in multiple bacterial species iron acquisition pathways have been linked to the development of the biofilm lifestyle; such that if these pathways are removed or iron is unavailable it depletes their biofilm-forming ability  <cit> .
likewise in studies on nthi biofilm formation and biofilm maturation, the iron uptake has been shown to be essential  <cit> .
it should be noted that in our comparative analyses of r <dig> and eagan at ph  <dig>  we showed that eagan did not form significant amounts of biofilm.
as a comparison of their profile of growth pathways at ph  <dig>  and then for r <dig> at  <dig>  , the transcriptional switch in the planktonic r <dig> cells at ph  <dig>  compared to  <dig>  is an indication of their response to this environmental condition and mechanisms that predispose the cells to biofilm formation as well as allowing a direct comparison to the eagan planktonic cells at ph  <dig> .
the r <dig> cells at ph  <dig>  that are in the biofilm were therefore excluded from our comparison; these by definition would be greatly different  and not a clear comparison to the non-biofilm forming eagan cells.
it was not our aim to compare planktonic against biofilm cell but the response to increased ph, conditions we know shift the r <dig> cells to biofilm-forming state.
it is worth noting that there were iron-associated genes up-regulated in eagan at ph  <dig>  but not to the extent observed in r <dig>
h. influenzae
r <dig> at ph  <dig>  compared to ph  <dig>
genes up-regulated at ph  <dig>  compared to  <dig>
<dig>
p
<dig>
p
<dig>
genes down-regulated at ph  <dig>  compared to  <dig>
<dig>
p
CONCLUSIONS
h. influenzae can adapt to the physical and chemical properties that exist in different anatomical niches .
various strains of this pathogen adapt to these niches differently, such growing rapidly and planktonically or alternatively by forming a biofilm.
the different niches are known to vary in a range of properties, the ph being one of these that subtly but significantly shifts from about neutral in the blood to ph  <dig>  in the middle ear  <cit> .
the ph does not remain constant within a niche and even in the blood there can various reasons for the ph to shift.
while blood ph is tightly regulated at around ph  <dig> , there are other parts of the body encountered by h. influenzae as a result of systemic infection starting in the blood that can include conditions that do reach ph  <dig> .
a capsular isolate taken from the blood would therefore need to be able to exist in the ph range of  <dig> - <dig>  but in this lifestyle it is rarely associated with a biofilm.
a nthi isolate from the middle ear  would predominantly encounter ph  <dig>  and its processes of colonization would occur at this ph .
in this niche as part of its colonization, the bacterial cell would form a biofilm.
indeed some studies have shown that biofilm is induced in the middle ear as a very likely consequence of the increased ph   <cit> .
the type iv pili genes are more likely to be highly regulated in the biofilm cells themselves and not the planktonic cells we analysed.
not all h. influenzae isolates respond to the changes in physical and chemical properties between the niches that h. influenzae can occupy with the same capacity or in the same manner.
we show that h. influenzae isolates respond differently to the subtle and yet physiologically relevant changes in ph from  <dig>  to  <dig> .
these changes are slight in regards to the observed growth rates but the changes are underpinned by lifestyle changes, such as modes of growth or biofilm formation.
a capsular isolate , continues to grow, with variation from ph  <dig>  to  <dig>  and does not form a biofilm while a nthi isolate known to colonize the middle ear, does form a biofilm at ph  <dig> .
this is consistent with the established knowledge that the middle ear is more basic in its ph than the nasopharynx or the blood and nthi colonize the middle ear by biofilm formation.
while it was not unexpected that the nthi isolate induced its iron-uptake pathways during its growth at ph  <dig>  as it cells become predisposed to forming a biofilm, it was a novel finding that the eagan strain induced gluconate:h+ uptake and sugar acid/gluconate metabolic genes.
this pathway was not induced in the biofilm-forming r <dig> cells.
this obviously provides a pathway for growth, through the link from gluconate to the ed and ppp energy production pathways, while at the same time providing a mechanism for maintaining ph homeostasis .
our study has therefore identified clear differences between a capsular isolate and a nthi isolate in their response to a relevant ph shift; these differences seem likely to be the basis for their mode of growth and survival within a specific niche.

 haemophilus influenzae is a γ-proteobacterium from within the order the pasteurellacae. it is an obligate human commensal of the nasopharynx and in most cases it remains as a commensal but some strains can transit from the nasopharynx to other parts of the body and in doing so cause numerous types of disease. there are strain-specific factors that enable pathogenic strains to transit to, and then survive within, different parts of the body, where the stresses of multiple environmental conditions require a breadth of adaptive abilities that permit survival and growth. there are a number of physical parameters that are known to vary between parts of the human host, including: oxygen tension, carbon/energy/nitrogen source, ph and the presence of reactive oxygen and reactive nitrogen species. defence against these can be directly encoded through detoxification genetic pathways, but also through broader mechanisms for environmental adaptation. in addition to specific pathways that respond to and deal with each of the damaging physical or chemical stressors present within the various environments the bacteria may encounter, many bacteria have a capacity to switch their lifestyle such that these stresses no longer cause damage to their cell. one key lifestyle adaptation is the switch from planktonic cells to a biofilm. this process is based upon numerous features of the bacterial cell including alterations in their metabolism and physiology, the presence and nature of surface structures, and the general physical properties of the bacterial cell. the process of biofilm formation is defined in stages and each of these has specific features and profiles. put simply, under stressed conditions bacterial cells can switch from a free-living and a rapidly dividing phenotype to an altered metabolic form associated with cell-cell aggregation and attachment to a surface. there are then early, mid, and late stages for the maturation of a bacterial biofilm. the particular stresses that induce a change in lifestyle and subsequently the process of biofilm formation are poorly defined for many pathogenic bacteria, however antibiotic usage is certainly one, nutrient starvation and oxidative stress are others. these conditions or signals do seem to be specific for different species. despite some previous disagreement about the ability of h. influenzae to form a biofilm, there is now overwhelming evidence that h. influenzae use biofilm formation for survival within the host and certainly in their colonization of the host. there are elements of h. influenzae which seem to be induced and therefore important for biofilm formation. there are numerous examples of studies that have shown that iron uptake is central to growth within a biofilm. there is a need to further characterise the differences between biofilm-forming and non-biofilm-forming isolates of h. influenzae. this can be accomplished through a comparison of the genetic and transcriptomic differences between h. influenzae strains that respond to stresses by forming a biofilm, and those that continue to grow under those conditions without forming a biofilm. changes in ph provides a suitable stressor, being central to its colonisation of different anatomical niches, and identification of the molecular pathways that vary between such isolates would be significant in our understanding of h. influenzae pathogenesis. h. influenzae strains and isolates display more variation than many other pathogens and underpinning the basis for the strain-specific actors that underlie their biofilm formation. indeed, coupled to this, there are many features of the h. influenzae physiology and stress response that indicate that this particular host-adapted bacterium has unique molecular mechanisms for survival in the various locations of its host that it can exist. the ph is known to be elevated in the middle ear, compared to other parts of the body and in this niche there is some evidence that it is ph that induces particular isolates of h. influenzae to form a biofilm. we have assessed the response of different clinical isolates of h. influenzae to changing ph; their growth and biofilm formation.  the growth of different strains of h. influenzae with changing ph the growth of strains of h. influenzae were assessed over a range of ph values; ph , and as the physiological ph is known to vary among host organs, tissues and niches. even within a particular body site there can be spatial and temporal changes in ph as a consequence of specific events. despite this uncertainty in the precise nature of the ph value associated with host-pathogen microenvironments, it is clear that there are distinct differences between the primary site of colonization and the various sites of infection, including the lower respiratory tract, the blood and the middle ear. as an example, the blood can be - and the middle ear is usually considered to be around ph . we assessed ph response of a small set of isolates of h. influenzae that were known to colonise either the blood or the middle ear. we grew the bacteria at ph , and and plotted their growth curves and from this we calculated mean growth rates. there were no clear patterns, and the observed changes represented only slight variations. the equivocal differences in growth at different ph levels does not exclude the possibility that the cells are responding differently, such as with an alternative lifestyle. h. influenzae isolates grown at different ph strain type ph  ph  ph  *doubling per hour. the formation of biofilm by h. influenzae as a consequence of changing ph given that colonization by h. influenzae within various host niches, such as the middle ear, is linked to their induction of a biofilm, and increased ph is characteristic of these environments, we assessed the possibility that biofilm induction is a consequence of increased ph. it has been previously suggested that for h. influenzae the biofilm formation is induced at ph . we assayed for biofilm formation at ph , and. these screening assays were performed by counting planktonic cells followed by washing and then releasing the biofilm cells, and counting colonies. in some strains, such as isolate r there was significant induction of biofilm at ph . other strains, including eagan, did not form biofilm at any ph. to compare in detail contrasting isolates from this screening of h. influenzae, eagan and r were taken for further analysis, more biological and experimental replicates. planktonic cell growth was assessed and then biofilm cell numbers were enumerated. eagan grew equally well at ph  and, as did r but eagan did not form any biofilm at either ph  or whereas r produced a significant biofilm at ph , within the context of this assay there was an increase in biofilm formation at ph . these results are consistent with what is generally accepted and known with regard to h. influenzae pathogenesis; that the capsular strains cope with increased ph by continuing planktonic growth while nthi isolates that colonizes the middle ear switches to a biofilm mode of growth. transcriptional analyses of eagan and r under different ph given the definite, growth-style, variations in response to a shift in ph from to between eagan and r we were interested in determining the underlying transcriptional differences that varied between eagan and r we therefore used rnaseq to analyse the whole cell transcriptome at ph  and for both eagan and r. the shift from ph  to, while biologically relevant and certainly impacting bacterial style of growth, is still a subtle change and it was not expected to generate a large set of cellular pathways with changed expression patterns. genes that were differentially expressed in eagan revealed predominantly an up-regulation of two gluconate:h+ symporters and the associated gluconate metabolic genes and a potential glycerate kinase that links into glycolysis. it is worth noting that these genes/pathways are genetically unlinked, adding to validity of the response. in addition to the hi1015/gntp symporter, the hi1010- genes include homologs to a sugar epimerase, aldolase and isomerases that are within the first stages of the pentose phosphate pathway. the first gene is a potential 6-phosphogluconate dehydrogenase that generates ribulose-5-phosphate. this links directly into the ppp and other energy and biosynthetic pathways. h. influenzae eagan at ph  compared to ph  genes up-regulated at ph  compared to 2 p p fold p genes down-regulated at ph  compared to p the gntp symporter family of transporters also import h+, as part of the survival response associated with an increased environmental ph. it is interesting to note that our bioinformatic analyses have identified an operator/promoter upstream of hi with a putative deor binding site; hi is divergent to a deor-like gene. while not within the scope of this project it is known in other bacteria that deor-like regulators variously control pathways directing sugar metabolism and are connected to the ppp. also, the bioinformatics analyses indicate that the hi1010- genes are on a single transcriptional unit, forming an operon. traditionally high concentrations of glucose are thought to be oxidized extracellularly by membrane-bound dehydrogenases. whereas under low glucose conditions, oxidized glucose is imported and phosphorylated within the cell to 6-phosphogluconate which feeds into either the entner-dourdooff pathway or the ppp for energy. in addition, gluconate can act as an exogenous carbon source and therefore be taken up as a direct mode of growth. it has been shown in some contexts that such metabolism is related to bacterial growth in the host-pathogen environment, such as with escherichia coli colonization of the mouse large intestine where gluconate is also important in the growth and pathogenesis of other pathogens. some bacteria possess multiple gluconate uptake systems, such as those characterized in e. coli, where there are four. not all of these are necessarily primary gluconate transporters, with some acting on other sugar acids that are able to be utilized by the same permeases. at least one of these has been shown to be likely to preferentially import fructuronate and not gluconate. in e. coli and other bacteria these transporters are regulated through different transcriptional pathways controlled by sugar-utilizing systems and signals; such as the sensing of the presence of gluconate by gntr, or as in a camp-dependent catabolite repression system/s, by the global transcriptional regulator crp. there is an emerging consensus that the regulation and role of these sugar acid metabolic systems is broader than originally thought. recently it has been shown that in e. coli, the hexuronate utilizing pathways are regulated by a complex interplay of regulatory systems including induction under osmotic stress conditions. what is clear from our results is that there are two homologous gluconate transport systems in h. influenzae eagan and that both are upregulated at ph . the media used throughout our studies was rich in glucose and other carbon and energy sources. it is worth noting that there are other genes responsible for these steps in the ppp in the genomes of h. influenzae, however these genes are not physically linked on an operon as with hi1010- the indication is that in the eagan strain the hi1010- operon is uniquely regulated based on ph and it feeds into the ppp functioning under increased ph. the duplication of genes for steps in the ppp is not unusual, there are homologs of these h. influenzae genes in several bacteria that have a similar duplication. in pectobacterium carotovorum the homologs to hi1011- are vguabcd and these function in gluconate metabolism and have an as yet uncharacterized role in the pathogenesis of this plant pathogen. interestingly, the sugar acid metabolism pathways can also feed into cell wall composition or modifications. glucuronic acid can be incorporated into the inner core of the lipopolysaccharide in e. coli and these potential modifications are a response to environmental stresses, specifically those associated with envelope stress, such as ph, and this response is controlled by several regulatory pathways. we demonstrated that as the ph increases to, the eagan isolate induced two gluconate permeases, one being part of an operon with gluconate metabolism genes, these likely providing the proteins and enzymes linked into energy production but also potentially providing other cellular alterations for coping with the stress. in contrast, the nthi r isolate did not induce the hi1010- operon at ph . consistent with this isolate inducing its biofilm formation at ph , it induced various, genetically unlinked iron acquisition genes. in multiple bacterial species iron acquisition pathways have been linked to the development of the biofilm lifestyle; such that if these pathways are removed or iron is unavailable it depletes their biofilm-forming ability. likewise in studies on nthi biofilm formation and biofilm maturation, the iron uptake has been shown to be essential. it should be noted that in our comparative analyses of r and eagan at ph  we showed that eagan did not form significant amounts of biofilm. as a comparison of their profile of growth pathways at ph  and then for r at, the transcriptional switch in the planktonic r cells at ph  compared to is an indication of their response to this environmental condition and mechanisms that predispose the cells to biofilm formation as well as allowing a direct comparison to the eagan planktonic cells at ph . the r cells at ph  that are in the biofilm were therefore excluded from our comparison; these by definition would be greatly different and not a clear comparison to the non-biofilm forming eagan cells. it was not our aim to compare planktonic against biofilm cell but the response to increased ph, conditions we know shift the r cells to biofilm-forming state. it is worth noting that there were iron-associated genes up-regulated in eagan at ph  but not to the extent observed in r h. influenzae r at ph  compared to ph  genes up-regulated at ph  compared to p p genes down-regulated at ph  compared to p  h. influenzae can adapt to the physical and chemical properties that exist in different anatomical niches. various strains of this pathogen adapt to these niches differently, such growing rapidly and planktonically or alternatively by forming a biofilm. the different niches are known to vary in a range of properties, the ph being one of these that subtly but significantly shifts from about neutral in the blood to ph  in the middle ear. the ph does not remain constant within a niche and even in the blood there can various reasons for the ph to shift. while blood ph is tightly regulated at around ph , there are other parts of the body encountered by h. influenzae as a result of systemic infection starting in the blood that can include conditions that do reach ph . a capsular isolate taken from the blood would therefore need to be able to exist in the ph range of - but in this lifestyle it is rarely associated with a biofilm. a nthi isolate from the middle ear would predominantly encounter ph  and its processes of colonization would occur at this ph. in this niche as part of its colonization, the bacterial cell would form a biofilm. indeed some studies have shown that biofilm is induced in the middle ear as a very likely consequence of the increased ph. the type iv pili genes are more likely to be highly regulated in the biofilm cells themselves and not the planktonic cells we analysed. not all h. influenzae isolates respond to the changes in physical and chemical properties between the niches that h. influenzae can occupy with the same capacity or in the same manner. we show that h. influenzae isolates respond differently to the subtle and yet physiologically relevant changes in ph from to. these changes are slight in regards to the observed growth rates but the changes are underpinned by lifestyle changes, such as modes of growth or biofilm formation. a capsular isolate, continues to grow, with variation from ph  to and does not form a biofilm while a nthi isolate known to colonize the middle ear, does form a biofilm at ph . this is consistent with the established knowledge that the middle ear is more basic in its ph than the nasopharynx or the blood and nthi colonize the middle ear by biofilm formation. while it was not unexpected that the nthi isolate induced its iron-uptake pathways during its growth at ph  as it cells become predisposed to forming a biofilm, it was a novel finding that the eagan strain induced gluconate:h+ uptake and sugar acid/gluconate metabolic genes. this pathway was not induced in the biofilm-forming r cells. this obviously provides a pathway for growth, through the link from gluconate to the ed and ppp energy production pathways, while at the same time providing a mechanism for maintaining ph homeostasis. our study has therefore identified clear differences between a capsular isolate and a nthi isolate in their response to a relevant ph shift; these differences seem likely to be the basis for their mode of growth and survival within a specific niche. 
a non-typeable h. influenzae, isolated from the middle ear, induced biofilm formation at ph , and at this ph it induced a series of iron acquisition genes, consistent with previous studies linking iron homeostasis to biofilm lifestyle. strain-specific factors allow some strains to migrate to particular anatomical niches, such as the middle ear, bronchi or blood, and induce disease by surviving within the conditions present at these sites in the body. this is exemplified by the contrast between the biofilm-forming strains found in middle ear infections and those isolates that survive within the blood and are rarely associated with biofilm development. the ph is a property that is different from the middle ear compared to other sites that h. influenzae can colonize and infect. screening a group of h. influenzae strains revealed only slight variations in their growth across a range of ph conditions. it is established that h. influenzae colonization and in some cases survival is highly dependent on their ability to form a biofilm. different strains of h. influenzae cope with changes in environmental factors using strain-specific mechanisms. the transcriptional response to increasing ph by h. influenzae varies between strains, and ph is linked to pathways that allow strains to either continue free-living growth or induction of a biofilm. we showed that a biofilm-forming isolate induced iron metabolism pathways, whereas a strain that does not form biofilm at increasing ph induced mechanisms for growth and ph homeostasis based on sugar acid transport. biofilm formation is a key trait in the development of chronic infection by certain isolates.
 haemophilus influenzae colonizes the nasopharynx as a commensal. strain-specific factors allow some strains to migrate to particular anatomical niches, such as the middle ear, bronchi or blood, and induce disease by surviving within the conditions present at these sites in the body. it is established that h. influenzae colonization and in some cases survival is highly dependent on their ability to form a biofilm. biofilm formation is a key trait in the development of chronic infection by certain isolates. this is exemplified by the contrast between the biofilm-forming strains found in middle ear infections and those isolates that survive within the blood and are rarely associated with biofilm development.  screening a group of h. influenzae strains revealed only slight variations in their growth across a range of ph conditions. however, some isolates responded to a ph of by the formation of a biofilm. while the type b capsular blood isolate eagan did not form a biofilm and grew at the same rate regardless of ph  -, transcriptomic analyses demonstrated that at ph  it uniquely induced a gluconate-uptake and metabolism pathway, which concurrently imports h+. a non-typeable h. influenzae, isolated from the middle ear, induced biofilm formation at ph , and at this ph it induced a series of iron acquisition genes, consistent with previous studies linking iron homeostasis to biofilm lifestyle.  different strains of h. influenzae cope with changes in environmental factors using strain-specific mechanisms. these pathways define the scope and mode of niche-survival for an isolate. the ph is a property that is different from the middle ear compared to other sites that h. influenzae can colonize and infect. the transcriptional response to increasing ph by h. influenzae varies between strains, and ph is linked to pathways that allow strains to either continue free-living growth or induction of a biofilm. we showed that a biofilm-forming isolate induced iron metabolism pathways, whereas a strain that does not form biofilm at increasing ph induced mechanisms for growth and ph homeostasis based on sugar acid transport. biofilmh. influenzaeph stresstranscriptomics 