BACKGROUND
although cystic fibrosis  is fundamentally a genetic disorder, the majority of patients with cf may ultimately succumb to respiratory failure subsequent to chronic bacterial infections  <cit> .
in early childhood, lungs of cf patients are often infected with staphylococcus aureus and haemophilus influenzae, but these organisms are usually outnumbered by pseudomonas aeruginosa as patients become older.
however, s. aureus often persists in the airways of cf patients and the role of s. aureus in the progression of cf patients to respiratory failure is not yet understood whereas infections with p. aeruginosa is considered as one of the main factors for a decline in lung function and mortality  <cit> .
interestingly, both organisms are commonly co-isolated from cf airways  <cit> .
infections with mixed microbial communities are common, although very little is known about the importance and the impact of interspecies interactions  <cit> .
it is now becoming obvious that the different bacteria found in cf airways interact together in several different ways  <cit> .
one possibility is that polymicrobial interactions influence pathogenic processes such as biofilm formation  <cit> .
accordingly, the biofilm lifestyle is now recognized as an integrated and complex polymicrobial community and it is thought that cell-to-cell interspecies signals play a role in the control of this behavior  <cit> .
it has recently been shown that prolonged growth of s. aureus with physiological concentrations of the p. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-n-oxide  selects for a sub-population of slow-growing respiratory deficient s. aureus named small-colony variants   <cit> .
the respiratory deficiency of scvs provides resistance to aminoglycoside antibiotics, which can contribute to microbial persistence during antibiotherapy  <cit> .
furthermore, it has been recently demonstrated that scv selection is a survival strategy of s. aureus against p. aeruginosa  <cit> .
s. aureus scvs are often isolated from chronic infections  <cit> , as in the case of lung infections of cf patients  <cit> .
several studies have shown that s. aureus scvs possess an increased capacity to invade and persist in host cells  <cit> , which is thought to confer the bacterium protection against the immune system and the action of antibiotics  <cit> .
using scvs isolated from cystic fibrosis patients, we have previously demonstrated that the alternative transcription sigma factor b  influences the expression of several virulence factors and is associated with an increased ability to adhere, invade and persist within host cells  <cit> .
furthermore, our more recent results suggest that sigb is involved in the emergence of scvs under aminoglycoside pressure  <cit> , which suggests that the appearance of scvs may be a regulated process influenced by environmental cues.
our current hypothesis is that sigb plays an important role in the establishment of chronic and difficult-to-treat s. aureus infections.
sigb is involved in the response to environmental stresses such as during stationary phase, heat exposure and change in osmotic pressure  <cit> .
moreover, the activity of sigb positively influences the expression of several cell-surface proteins whereas it down-regulates a variety of toxins  <cit> , which suggest an important role for sigb in pathogenesis.
the effect of sigb on virulence gene expression can be direct or indirect, since the genes regulated by sigb also include at least another global regulator of virulence, sara   <cit> .
sara modulates the expression of several virulence factors either by stimulating rnaiii transcription or by pathway independent of the agr  system  <cit> .
in turn, it is proposed that the quorum-sensing agr system controls the transition from colonization to dissemination by up-regulating the expression of several exotoxins and proteolytic enzymes and by repressing the expression of cell-surface proteins involved in colonization  <cit> .
agr  <cit> , sigb  <cit>  and sara  <cit>  are known to influence the formation of biofilms by s. aureus.
at least two different mechanisms of biofilm formation exist in s. aureus  <cit> .
the first mechanism implies the production of the polysaccharide intercellular adhesin , which requires the ica gene cluster, whereas the second mechanism is ica-independent.
with opposite effects, sara and agr are both involved in the ica-independent mechanism of biofilm formation.
sara is thought to be indirectly required for the initial attachment step to biological matrices  <cit> , while agr is controlling the dispersal process of biofilms  <cit> .
recently, lauderdale et al.
<cit>  have shown that sigb is an essential regulator of the ica-independent biofilm formation and suggested that sigb acts upstream of the agr system, allowing the formation of biofilm to be regulated as a function of environmental factors.
noteworthy, biofilms have been linked to chronic infections, especially in the case of those found in the airways of cf patients  <cit> , and an increased formation of biofilms has been associated with the scv phenotype  <cit> .
the aim of this study was to investigate the association between the activity of sigb, the emergence of scvs and biofilm production in s. aureus when exposed to p. aeruginosa hqno.
RESULTS
hqno inhibits the growth of normal strains and provokes the emergence of scvs in s. aureus
fig.
<dig> confirms that hqno suppresses the growth of s. aureus and causes the emergence of scvs.
isolates cf1a-l and cf1d-s are two related strains co-isolated from a cf patient which have a normal and a scv phenotype, respectively .
at a concentration of  <dig> μg/ml, hqno significantly attenuated the growth of cf1a-l  whereas hqno had no apparent effect on the growth of cf1d-s which was already significantly slower than that of cf1a-l in the absence of hqno  .
similar observations were also reproduced with other strains .
fig.
1b shows that an overnight treatment with hqno provokes the emergence of scvs from cf1a-l, as determined by plating the culture on solid medium containing a concentration of gentamicin selective for the scv phenotype.
very little or no scv were detected on gentamicin plates when cultures were not exposed to hqno .
hence, this technique allowed detection and quantification of scvs emerging during the growth of normal bacteria exposed or not to hqno.
this approach was thus used to distinguish the transitory suppression of growth of normal s. aureus by hqno from the emerging slow-growing scvs for which gentamicin resistance and slow growth persist even after removal of hqno.
fig.
1c shows that  <dig> μg of hqno/ml significantly increased the presence of scvs in cultures of the prototypical strains atcc  <dig>  newman and newbould as well as of the other normal strains isolated from cf patients cf03-l, cf07-l and cf1a-l. differences in hqno-mediated scv emergence between strains were not significant, except between atcc  <dig> and newbould .
these results corroborate that hqno generally suppresses the growth of normal s. aureus populations and provokes the emergence of scvs from strains of different origins.
hqno stimulates biofilm production in normal strains but does not alter high biofilm production in scvs
several pairs of related normal and scvs strains were used in order to study the effect of hqno on biofilm production by s. aureus.
fig.
2a shows that scvs produce significantly more biofilm than their normal counterparts.
the use of the strain newbouldhemb  ensured that scvs  are indeed responsible for this increase in biofilm production .
furthermore, as shown in mitchell et al.
<cit> , supplementation of the scv strains cf <dig> and cf <dig> with menadione abolished this phenomenon and thus demonstrated that if there was a reversion of scvs to the normal phenotype, the biofilm production would be greatly reduced.
besides, the presence of hqno at  <dig> μg/ml did stimulate biofilm production in the normal strains .
this observation was statistically significant for the normal strains atcc  <dig>  newman, newbould, cf03-l, cf07-l and cf1a-l whereas hqno had no detectable effect on the already high biofilm production of the scv strains newbouldhemb, cf03-s, cf07-s and cf1d-s .
moreover, cf03-l produced significantly more biofilm than atcc  <dig> and newman in presence of hqno, revealing that the amplitude of the response of normal strains to hqno may individually differs .
interestingly, an overnight exposure to  <dig> μg/ml of hqno resulted in a significant increase in biofilm production  for strain newman, cf03-l and cf1a-l even after sub-culturing strains in hqno-free medium .
this indicates that an exposure of s. aureus to hqno may result in a sustained increase in biofilm production.
overall, these results suggest that hqno increases biofilm production in normal s. aureus strains and that the sustained effect of hqno on biofilm production in subsequent hqno-free medium may result from the relative increase in the sub-population of scvs which are good biofilm producers.
sigb is involved in hqno-mediated emergence of scvs and biofilm production
strains newbould and newbouldΔsigb were used to determine whether sigb is involved in the emergence of scvs and biofilm production under an exposure to hqno.
fig.
3a illustrates the ability of hqno  to favor the emergence of the scv phenotype only in a sigb+ background.
hqno significantly increased the presence of scvs in strain newbould, but not in newbouldΔsigb .
this result was confirmed with strains sh <dig> and 8325- <dig> , which are isogenic strains with a functional and dysfunctional sigb system, respectively  <cit> .
fig.
3c demonstrates that the presence of hqno significantly inhibits the growth of both newbould and newbouldΔsigb .
however, the ability of hqno to increase biofilm formation was observed with strain newbould, but not with newbouldΔsigb .
these results suggest that, even if the inhibition of growth caused by hqno is not influenced by sigb , hqno-mediated emergence of scvs and biofilm production is triggered by a sigb-dependent mechanism .
sigb and agr activities are modulated by an exposure to hqno
fig.
<dig> shows qpcr measurements of the expression of the genes asp <dig>  fnba, hld , hla, sara and gyrb at the exponential growth phase for strains newbould and newbouldΔsigb exposed or not to hqno.
the expression of asp <dig> and fnba was evaluated in order to verify the hypothesis that sigb is activated during hqno exposure.
the gene asp <dig> is a well-known marker for sigb activity as for the gene fnba, although the transcription of the latter is not exclusively influenced by sigb  <cit> .
fig.
4a and 4b show that hqno at  <dig> μg/ml induced sigb activity in strain newbould, as revealed by significant increases of asp <dig> and fnba expression.
the effect of hqno on the expression of asp <dig> and fnba was further confirmed with the sequenced strain newman .
these results suggest that sigb activity is increased by hqno.
the activity of the agr system is known to be reduced in scvs  <cit> .
we have thus hypothesized that hqno exposure would repress the agr quorum-sensing system due to the general suppression of growth toward normal strains  but also due to the overall emergence of the scv sub-population as seen in fig.
<dig>  indeed as expected, fig.
4c shows that exposure of newbould and newbouldΔsigb to hqno significantly repressed the expression of hld .
with the increased in sigb activity and the reduced expression of agr observed under exposure to hqno, it was also justified to measure the expression of the α-hemolysin gene hla which can be influenced by both agr and sigb  <cit> .
hla was only significantly repressed in newbould and not in newbouldΔsigb by the presence of hqno .
furthermore, the expression of hla was, in both exposed and unexposed conditions, significantly increased in newbouldΔsigb in comparison to newbould, which confirms the negative influence of sigb on hla expression  <cit> .
these results show that the expression of hla is reduced by hqno and that the influence of sigb on hla expression under hqno exposure seems to be predominant over the agr system.
the expression level of sara was also measured because of its partial dependency on sigb for expression  <cit> , and its roles in the regulation of virulence factors expression  <cit>  and in biofilm formation  <cit> .
fig.
4e shows that sara expression is significantly induced by hqno in strain newbould but not in newbouldΔsigb.
the specificity of the observed modulations in gene expression was validated by monitoring the impact of hqno on the expression of the housekeeping gene gyrb.
the expression of gyrb was not modulated in the different conditions tested .
these results suggest that hqno induces the expression of sara by a sigb-dependent mechanism.
overall, these results suggest that exposure of s. aureus to hqno reproduces the transcriptional signature found in scvs  <cit>  and stimulates biofilm production by having opposite effects on the activity of sigb  and agr  as well as on the expression of sara .
p. aeruginosa stimulates biofilm formation and increases the activity of sigb of a s. aureus cf isolate
in order to ascertain that the effect of hqno on s. aureus is representative of what may happen when p. aeruginosa and s. aureus are in close proximity during a co-infection, we conducted experiments in which s. aureus was exposed to supernatants from overnight cultures of p. aeruginosa as well as experiments using a double chamber co-culture model.
we used the e. coli strain k <dig> in control experiments to ensure that the observed effect was specific to p. aeruginosa and was not only caused by the close proximity of a gram-negative bacterium or non specific alterations of the growth medium.
we used e. coli because it is known that this bacterium does not produce hqno .
fig.5a shows that p. aeruginosa pao <dig> inhibits the growth of the s. aureus strain cf1a-l whereas this phenomenon was not observed with e. coli k <dig>  the supernatant collected from an overnight culture of pao <dig> significantly inhibited the growth of s. aureus.
this growth inhibition was accompanied by a significant increase in biofilm production .
fig.
5c shows that when s. aureus cf1a-l was co-cultured with pao <dig> for  <dig> h, significantly more scvs were recovered than that seen when the co-culture was done with e. coli k <dig>  of striking interest, the co-cultivation of s. aureus cf1a-l with p. aeruginosa pao <dig> specifically and significantly increased the expression of asp <dig>  these results confirm that p. aeruginosa has the potential to specifically inhibit the growth, stimulate biofilm production, favor the emergence of the scv phenotype and increase the activity of sigb in non-scv s. aureus strains.
hqno from p. aeruginosa stimulates s. aureus biofilm production by a sigb-dependent mechanism
we used the pqsa and pqsl mutants derived from p. aeruginosa pa <dig> to further confirm the specific effect of hqno on biofilm production by s. aureus.
the pqsa mutant does not produce any 4-hydroxy-2-alkylquinolines  at all  <cit> , whereas the pqsl mutant is specifically altered in hqno biosynthesis  <cit> .
thus, we have used both pqsa and pqsl mutants in order to distinguish the global impact of all p. aeruginosa haqs from the specific impact of hqno on biofilm production by s. aureus.
fig.
6a shows that the growth of the pqsa and pqsl mutants is not impaired compared to that of the parental strain pa <dig>  thus excluding variations in supernatant composition caused by differences in growth rates among strains.
fig.
6b shows that the supernatant from an overnight culture of p. aeruginosa pa <dig> stimulates biofilm production by s. aureus cf1a-l in comparison to the supernatant from the pqsl mutant .
the effect of different doses of supernatants from overnight cultures of p. aeruginosa pa <dig>  the pqsa mutant, the pqsl mutant or e. coli k <dig> on biofilm production by s. aureus cf1a-l is shown in fig.
6c.
while supernatants from both mutants significantly induced less biofilm production in comparison to pa <dig>  this attenuated effect was more pronounced for the pqsa mutant  than the pqsl mutant.
this result can be explained by the fact that other haqs secreted by p. aeruginosa, although less potent than hqno, can also have a growth-inhibitory activity against s. aureus  <cit> .
noteworthy, all three strains of p. aeruginosa stimulated biofilm production in comparison to e. coli, suggesting that other p. aeruginosa exoproducts can indeed stimulate biofilm production by s. aureus.
fig.
6d confirms that hqno from the supernatant of strain pa <dig> stimulates biofilm production by a sigb-dependent mechanism.
the increase in biofilm production observed when s. aureus newbould is in contact with the supernatant from pa <dig> is significantly higher than that seen with supernatants from the pqsa and pqsl mutants.
surprisingly, both mutants did not significantly stimulate biofilm production by newbould as that observed for cf1a-l, suggesting that differences between s. aureus strains may exist in respect to their response to the presence of non-hqno exoproducts.
as expected, biofilm production by newbouldΔsigb in contact with supernatants from the three p. aeruginosa strains was significantly inferior to that observed using the pa <dig> supernatants with strain newbould.
moreover, supernatants from pa <dig> generally did not significantly stimulate biofilm production by newbouldΔsigb in comparison to supernatants from pqsa and pqsl mutants, which confirms that sigb is involved in hqno-mediated s. aureus biofilm production.
overall, the results of this section support the hypothesis that hqno from p. aeruginosa stimulates biofilm production by s. aureus through a sigb-dependent mechanism.
discussion
we found that the p. aeruginosa exoproduct hqno increases the production of biofilm by s. aureus.
the effects on biofilm production, as well as on growth, were only seen on normal strains whereas the already high biofilm formation and slow growth rate of scvs were not altered by the presence of hqno.
it is known that hqno specifically inhibits electron transport in gram-positive bacteria  <cit>  and its immediate action toward normal strains is likely to reduce atp production and growth to a similar level to that observed in scvs.
interestingly, our results also indicate that hqno provokes a sustained stimulatory effect on the production of biofilms by s. aureus.
we indeed found that a pre-treatement of s. aureus with hqno still led to a subsequent increase in biofilm formation even after hqno removal.
this sustained effect is probably associated with the increased proportion of the sub-population of scvs resulting from hqno exposure.
an exposure of s. aureus to hqno may thus, in addition to its immediate effect, favor the emergence of scvs having a long-term impact on biofilm formation.
aminoglycosides are also known to favor the emergence of scvs  <cit>  and are often used in cf patient care  <cit> .
interestingly, a synergistic effect between hqno and tobramycin for the formation of s. aureus scvs was previously observed by hoffmann et al.
<cit> .
it is thus possible that the administration of aminoglycosides to cf patients co-infected with both s. aureus and p. aeruginosa further increases the formation of biofilm by s. aureus.
besides, it is well known that the abnormal function of the cf transmembrane conductance regulator  protein in cf patients has profound consequences on the airway physiology and it will be of great interest to determine whether other parameters related to the cf airways influence the emergence of scvs and the production of biofilms by s. aureus.
the expression of virulence factors in s. aureus is indeed controlled by diverse and complex regulatory networks in a time- and environment-dependent manner, being influenced for example by ionic forces, ph and o <dig>  <cit> .
consequently, it is likely that s. aureus specifically responds to the particular environment of cf airways.
whether this response is sigb-dependent and will lead to the emergence of scvs and biofilm production remains to be determined.
naturally-occurring mutations altering the activity of virulence regulators in s. aureus have been previously reported  <cit> .
our results suggest that the inactivation of sigb will importantly influence the outcome of the hqno-mediated interaction between p. aeruginosa and s. aureus.
we are currently studying s. aureus isolates from cf patients co-infected with p. aeruginosa which are not influenced by the presence of p. aeruginosa.
this, in addition to the observation that differences between s. aureus strains exist relative to their response to haqs , suggest that s. aureus strains isolated from cf patients may adapt or evolve toward a long-term coexistence with p. aeruginosa.
whether this involves mutations in sigb or any other genes encoding regulators is now under investigation and will greatly help to understand the dynamic behavior and the adaptation of s. aureus in response to the cf airway environment as well as to the presence of p. aeruginosa.
the effect of hqno on the regulators sara, agr and sigb suggests that several virulence factors should be influenced by the presence of hqno.
a concomitant activation of sigb and repression of agr should result in the up-regulation of a variety of cell-surface proteins  involved in adhesion to host tissues, and in the repression of several exotoxins  and proteolytic enzymes  <cit> .
it is then tempting to speculate that the presence of hqno will prevent s. aureus from disseminating and will rather favor tissue colonization, biofilm production and invasion of host cells.
it has indeed been suggested that s. aureus fnbps mediates cellular invasion  <cit>  whereas the capacity of the bacterium to remain intracellular is helped by the repression of hla  <cit> .
accordingly, we showed that an exposure of s. aureus to hqno up-regulates the expression of fnba and represses the expression of hla.
however, whether or not hqno and p. aeruginosa increase the invasion of host cells by s. aureus remains to be confirmed.
interestingly, o'neil et al.
<cit>  have recently demonstrated that the fnbps are also involved in the ica-independent mechanism of biofilm formation.
it is thus possible that fnbps are directly responsible for the observed hqno-mediated sigb-dependent increase in biofilm production and, more specifically, fnbpa which is under the control of sigb for expression  <cit> .
as such, the fnbps would represent the main effectors for both biofilm formation and cellular invasion in s. aureus scvs.
hqno may be one of several bacterial exoproducts influencing s. aureus during polymicrobial infections.
our results and those of machan et al.
<cit>  suggest that other haqs may also affect s. aureus, although not as efficiently as hqno.
moreover, it is known that other p. aeruginosa exoproducts such as pyocyanin have an inhibitory activity against the electron transport chain of s. aureus  <cit> .
loss of pyocyanin production has been associated with mutations in the pqsa-e genes  <cit> , which may provide an additional explanation for the different effects of the pqsa and pqsl mutants we have observed on the growth  and biofilm formation of s. aureus .
furthermore, qazi et al.
<cit>  found that an n-acyl-homoserine-lactone from p. aeruginosa antagonizes quorum sensing and virulence gene expression in s. aureus.
more precisely, it was shown that the 3-oxo-c12-hsl interacts with the cytoplasmic membrane of s. aureus and down-regulates both sara and agr expression.
although we also observed here a down-regulation of agr, the hqno-mediated up-regulation of sara suggests further complexity in the response of s. aureus to p. aeruginosa exoproducts.
it is possible that the outcome of the s. aureus-p. aeruginosa interaction is dependent on the amount and the types of exoproducts secreted by the specific strain of p. aeruginosa interacting with s. aureus.
it should be kept in mind that the diversity of diffusible bacterial signals and their role in interspecies communication are just beginning to be appreciated  <cit>  and that potentially numerous other bacterial species may be interacting with s. aureus, especially during infectious diseases.
it is then likely that s. aureus interacts with other bacterial genus than pseudomonas during infection of the airways of cf patients.
as an example, the cf pathogen burkholderia cepacia also produces n-acylhomoserine lactones  <cit>  and some burkholderia species are able to synthesize haq analogues  <cit> .
nevertheless, the observation that p. aeruginosa favors the emergence of scvs and biofilm production by s. aureus is likely to have a significant clinical impact.
the clinical consequences may actually surpass the previously anticipated formation of aminoglycoside-resistant scvs by hoffman et al.
<cit> .
persistence of bacteria in chronic infections has been associated with biofilm production  <cit>  and biofilms are known to confer protection from host defenses and antibiotic treatments at large  <cit> .
in the cystic fibrosis context, where obstructive infections worsen the health prognosis of patients, the clinical significance of biofilm production by normal s. aureus and scv strains will need to be further investigated.
CONCLUSIONS
this study strongly supports the hypothesis that p. aeruginosa influences the pathogenicity of s. aureus by producing hqno, which favors the acquisition of the scv phenotype through the activation of the stress- and colonization-related s. aureus alternative sigma factor b. although several p. aeruginosa exoproducts may potentially influence s. aureus, our observations with pure hqno were confirmed and supported by experiments using whole supernatants from two p. aeruginosa strains as well as mutants unable to produce hqno.
considering that biofilms and scvs are both suspected to play a role in chronic infections of cf airways, the observation that p. aeruginosa increases the emergence of scvs and biofilm formation by s. aureus may influence the patient health prognosis.
new therapeutic strategies should aim at preventing interspecies interactions and the development of specific phenotypes such as biofilm-producing scvs in order to reduce the likelihood of chronic infections.

 although cystic fibrosis is fundamentally a genetic disorder, the majority of patients with cf may ultimately succumb to respiratory failure subsequent to chronic bacterial infections. in early childhood, lungs of cf patients are often infected with staphylococcus aureus and haemophilus influenzae, but these organisms are usually outnumbered by pseudomonas aeruginosa as patients become older. however, s. aureus often persists in the airways of cf patients and the role of s. aureus in the progression of cf patients to respiratory failure is not yet understood whereas infections with p. aeruginosa is considered as one of the main factors for a decline in lung function and mortality. interestingly, both organisms are commonly co-isolated from cf airways. infections with mixed microbial communities are common, although very little is known about the importance and the impact of interspecies interactions. it is now becoming obvious that the different bacteria found in cf airways interact together in several different ways. one possibility is that polymicrobial interactions influence pathogenic processes such as biofilm formation. accordingly, the biofilm lifestyle is now recognized as an integrated and complex polymicrobial community and it is thought that cell-to-cell interspecies signals play a role in the control of this behavior. it has recently been shown that prolonged growth of s. aureus with physiological concentrations of the p. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-n-oxide selects for a sub-population of slow-growing respiratory deficient s. aureus named small-colony variants. the respiratory deficiency of scvs provides resistance to aminoglycoside antibiotics, which can contribute to microbial persistence during antibiotherapy. furthermore, it has been recently demonstrated that scv selection is a survival strategy of s. aureus against p. aeruginosa. s. aureus scvs are often isolated from chronic infections, as in the case of lung infections of cf patients. several studies have shown that s. aureus scvs possess an increased capacity to invade and persist in host cells, which is thought to confer the bacterium protection against the immune system and the action of antibiotics. using scvs isolated from cystic fibrosis patients, we have previously demonstrated that the alternative transcription sigma factor b influences the expression of several virulence factors and is associated with an increased ability to adhere, invade and persist within host cells. furthermore, our more recent results suggest that sigb is involved in the emergence of scvs under aminoglycoside pressure, which suggests that the appearance of scvs may be a regulated process influenced by environmental cues. our current hypothesis is that sigb plays an important role in the establishment of chronic and difficult-to-treat s. aureus infections. sigb is involved in the response to environmental stresses such as during stationary phase, heat exposure and change in osmotic pressure. moreover, the activity of sigb positively influences the expression of several cell-surface proteins whereas it down-regulates a variety of toxins, which suggest an important role for sigb in pathogenesis. the effect of sigb on virulence gene expression can be direct or indirect, since the genes regulated by sigb also include at least another global regulator of virulence, sara. sara modulates the expression of several virulence factors either by stimulating rnaiii transcription or by pathway independent of the agr system. in turn, it is proposed that the quorum-sensing agr system controls the transition from colonization to dissemination by up-regulating the expression of several exotoxins and proteolytic enzymes and by repressing the expression of cell-surface proteins involved in colonization. agr, sigb and sara are known to influence the formation of biofilms by s. aureus. at least two different mechanisms of biofilm formation exist in s. aureus. the first mechanism implies the production of the polysaccharide intercellular adhesin, which requires the ica gene cluster, whereas the second mechanism is ica-independent. with opposite effects, sara and agr are both involved in the ica-independent mechanism of biofilm formation. sara is thought to be indirectly required for the initial attachment step to biological matrices, while agr is controlling the dispersal process of biofilms. recently, lauderdale et al. have shown that sigb is an essential regulator of the ica-independent biofilm formation and suggested that sigb acts upstream of the agr system, allowing the formation of biofilm to be regulated as a function of environmental factors. noteworthy, biofilms have been linked to chronic infections, especially in the case of those found in the airways of cf patients, and an increased formation of biofilms has been associated with the scv phenotype. the aim of this study was to investigate the association between the activity of sigb, the emergence of scvs and biofilm production in s. aureus when exposed to p. aeruginosa hqno.  hqno inhibits the growth of normal strains and provokes the emergence of scvs in s. aureus fig. confirms that hqno suppresses the growth of s. aureus and causes the emergence of scvs. isolates cf1a-l and cf1d-s are two related strains co-isolated from a cf patient which have a normal and a scv phenotype, respectively. at a concentration of μg/ml, hqno significantly attenuated the growth of cf1a-l whereas hqno had no apparent effect on the growth of cf1d-s which was already significantly slower than that of cf1a-l in the absence of hqno. similar observations were also reproduced with other strains. fig. 1b shows that an overnight treatment with hqno provokes the emergence of scvs from cf1a-l, as determined by plating the culture on solid medium containing a concentration of gentamicin selective for the scv phenotype. very little or no scv were detected on gentamicin plates when cultures were not exposed to hqno. hence, this technique allowed detection and quantification of scvs emerging during the growth of normal bacteria exposed or not to hqno. this approach was thus used to distinguish the transitory suppression of growth of normal s. aureus by hqno from the emerging slow-growing scvs for which gentamicin resistance and slow growth persist even after removal of hqno. fig. 1c shows that μg of hqno/ml significantly increased the presence of scvs in cultures of the prototypical strains atcc newman and newbould as well as of the other normal strains isolated from cf patients cf03-l, cf07-l and cf1a-l. differences in hqno-mediated scv emergence between strains were not significant, except between atcc and newbould. these results corroborate that hqno generally suppresses the growth of normal s. aureus populations and provokes the emergence of scvs from strains of different origins. hqno stimulates biofilm production in normal strains but does not alter high biofilm production in scvs several pairs of related normal and scvs strains were used in order to study the effect of hqno on biofilm production by s. aureus. fig. 2a shows that scvs produce significantly more biofilm than their normal counterparts. the use of the strain newbouldhemb ensured that scvs are indeed responsible for this increase in biofilm production. furthermore, as shown in mitchell et al., supplementation of the scv strains cf and cf with menadione abolished this phenomenon and thus demonstrated that if there was a reversion of scvs to the normal phenotype, the biofilm production would be greatly reduced. besides, the presence of hqno at μg/ml did stimulate biofilm production in the normal strains. this observation was statistically significant for the normal strains atcc newman, newbould, cf03-l, cf07-l and cf1a-l whereas hqno had no detectable effect on the already high biofilm production of the scv strains newbouldhemb, cf03-s, cf07-s and cf1d-s. moreover, cf03-l produced significantly more biofilm than atcc and newman in presence of hqno, revealing that the amplitude of the response of normal strains to hqno may individually differs. interestingly, an overnight exposure to μg/ml of hqno resulted in a significant increase in biofilm production for strain newman, cf03-l and cf1a-l even after sub-culturing strains in hqno-free medium. this indicates that an exposure of s. aureus to hqno may result in a sustained increase in biofilm production. overall, these results suggest that hqno increases biofilm production in normal s. aureus strains and that the sustained effect of hqno on biofilm production in subsequent hqno-free medium may result from the relative increase in the sub-population of scvs which are good biofilm producers. sigb is involved in hqno-mediated emergence of scvs and biofilm production strains newbould and newbouldΔsigb were used to determine whether sigb is involved in the emergence of scvs and biofilm production under an exposure to hqno. fig. 3a illustrates the ability of hqno to favor the emergence of the scv phenotype only in a sigb+ background. hqno significantly increased the presence of scvs in strain newbould, but not in newbouldΔsigb. this result was confirmed with strains sh and 8325-, which are isogenic strains with a functional and dysfunctional sigb system, respectively. fig. 3c demonstrates that the presence of hqno significantly inhibits the growth of both newbould and newbouldΔsigb. however, the ability of hqno to increase biofilm formation was observed with strain newbould, but not with newbouldΔsigb. these results suggest that, even if the inhibition of growth caused by hqno is not influenced by sigb, hqno-mediated emergence of scvs and biofilm production is triggered by a sigb-dependent mechanism. sigb and agr activities are modulated by an exposure to hqno fig. shows qpcr measurements of the expression of the genes asp fnba, hld, hla, sara and gyrb at the exponential growth phase for strains newbould and newbouldΔsigb exposed or not to hqno. the expression of asp and fnba was evaluated in order to verify the hypothesis that sigb is activated during hqno exposure. the gene asp is a well-known marker for sigb activity as for the gene fnba, although the transcription of the latter is not exclusively influenced by sigb. fig. 4a and 4b show that hqno at μg/ml induced sigb activity in strain newbould, as revealed by significant increases of asp and fnba expression. the effect of hqno on the expression of asp and fnba was further confirmed with the sequenced strain newman. these results suggest that sigb activity is increased by hqno. the activity of the agr system is known to be reduced in scvs. we have thus hypothesized that hqno exposure would repress the agr quorum-sensing system due to the general suppression of growth toward normal strains but also due to the overall emergence of the scv sub-population as seen in fig. indeed as expected, fig. 4c shows that exposure of newbould and newbouldΔsigb to hqno significantly repressed the expression of hld. with the increased in sigb activity and the reduced expression of agr observed under exposure to hqno, it was also justified to measure the expression of the α-hemolysin gene hla which can be influenced by both agr and sigb. hla was only significantly repressed in newbould and not in newbouldΔsigb by the presence of hqno. furthermore, the expression of hla was, in both exposed and unexposed conditions, significantly increased in newbouldΔsigb in comparison to newbould, which confirms the negative influence of sigb on hla expression. these results show that the expression of hla is reduced by hqno and that the influence of sigb on hla expression under hqno exposure seems to be predominant over the agr system. the expression level of sara was also measured because of its partial dependency on sigb for expression, and its roles in the regulation of virulence factors expression and in biofilm formation. fig. 4e shows that sara expression is significantly induced by hqno in strain newbould but not in newbouldΔsigb. the specificity of the observed modulations in gene expression was validated by monitoring the impact of hqno on the expression of the housekeeping gene gyrb. the expression of gyrb was not modulated in the different conditions tested. these results suggest that hqno induces the expression of sara by a sigb-dependent mechanism. overall, these results suggest that exposure of s. aureus to hqno reproduces the transcriptional signature found in scvs and stimulates biofilm production by having opposite effects on the activity of sigb and agr as well as on the expression of sara. p. aeruginosa stimulates biofilm formation and increases the activity of sigb of a s. aureus cf isolate in order to ascertain that the effect of hqno on s. aureus is representative of what may happen when p. aeruginosa and s. aureus are in close proximity during a co-infection, we conducted experiments in which s. aureus was exposed to supernatants from overnight cultures of p. aeruginosa as well as experiments using a double chamber co-culture model. we used the e. coli strain k in control experiments to ensure that the observed effect was specific to p. aeruginosa and was not only caused by the close proximity of a gram-negative bacterium or non specific alterations of the growth medium. we used e. coli because it is known that this bacterium does not produce hqno. fig.5a shows that p. aeruginosa pao inhibits the growth of the s. aureus strain cf1a-l whereas this phenomenon was not observed with e. coli k the supernatant collected from an overnight culture of pao significantly inhibited the growth of s. aureus. this growth inhibition was accompanied by a significant increase in biofilm production. fig. 5c shows that when s. aureus cf1a-l was co-cultured with pao for h, significantly more scvs were recovered than that seen when the co-culture was done with e. coli k of striking interest, the co-cultivation of s. aureus cf1a-l with p. aeruginosa pao specifically and significantly increased the expression of asp these results confirm that p. aeruginosa has the potential to specifically inhibit the growth, stimulate biofilm production, favor the emergence of the scv phenotype and increase the activity of sigb in non-scv s. aureus strains. hqno from p. aeruginosa stimulates s. aureus biofilm production by a sigb-dependent mechanism we used the pqsa and pqsl mutants derived from p. aeruginosa pa to further confirm the specific effect of hqno on biofilm production by s. aureus. the pqsa mutant does not produce any 4-hydroxy-2-alkylquinolines at all, whereas the pqsl mutant is specifically altered in hqno biosynthesis. thus, we have used both pqsa and pqsl mutants in order to distinguish the global impact of all p. aeruginosa haqs from the specific impact of hqno on biofilm production by s. aureus. fig. 6a shows that the growth of the pqsa and pqsl mutants is not impaired compared to that of the parental strain pa thus excluding variations in supernatant composition caused by differences in growth rates among strains. fig. 6b shows that the supernatant from an overnight culture of p. aeruginosa pa stimulates biofilm production by s. aureus cf1a-l in comparison to the supernatant from the pqsl mutant. the effect of different doses of supernatants from overnight cultures of p. aeruginosa pa the pqsa mutant, the pqsl mutant or e. coli k on biofilm production by s. aureus cf1a-l is shown in fig. 6c. while supernatants from both mutants significantly induced less biofilm production in comparison to pa this attenuated effect was more pronounced for the pqsa mutant than the pqsl mutant. this result can be explained by the fact that other haqs secreted by p. aeruginosa, although less potent than hqno, can also have a growth-inhibitory activity against s. aureus. noteworthy, all three strains of p. aeruginosa stimulated biofilm production in comparison to e. coli, suggesting that other p. aeruginosa exoproducts can indeed stimulate biofilm production by s. aureus. fig. 6d confirms that hqno from the supernatant of strain pa stimulates biofilm production by a sigb-dependent mechanism. the increase in biofilm production observed when s. aureus newbould is in contact with the supernatant from pa is significantly higher than that seen with supernatants from the pqsa and pqsl mutants. surprisingly, both mutants did not significantly stimulate biofilm production by newbould as that observed for cf1a-l, suggesting that differences between s. aureus strains may exist in respect to their response to the presence of non-hqno exoproducts. as expected, biofilm production by newbouldΔsigb in contact with supernatants from the three p. aeruginosa strains was significantly inferior to that observed using the pa supernatants with strain newbould. moreover, supernatants from pa generally did not significantly stimulate biofilm production by newbouldΔsigb in comparison to supernatants from pqsa and pqsl mutants, which confirms that sigb is involved in hqno-mediated s. aureus biofilm production. overall, the results of this section support the hypothesis that hqno from p. aeruginosa stimulates biofilm production by s. aureus through a sigb-dependent mechanism. discussion we found that the p. aeruginosa exoproduct hqno increases the production of biofilm by s. aureus. the effects on biofilm production, as well as on growth, were only seen on normal strains whereas the already high biofilm formation and slow growth rate of scvs were not altered by the presence of hqno. it is known that hqno specifically inhibits electron transport in gram-positive bacteria and its immediate action toward normal strains is likely to reduce atp production and growth to a similar level to that observed in scvs. interestingly, our results also indicate that hqno provokes a sustained stimulatory effect on the production of biofilms by s. aureus. we indeed found that a pre-treatement of s. aureus with hqno still led to a subsequent increase in biofilm formation even after hqno removal. this sustained effect is probably associated with the increased proportion of the sub-population of scvs resulting from hqno exposure. an exposure of s. aureus to hqno may thus, in addition to its immediate effect, favor the emergence of scvs having a long-term impact on biofilm formation. aminoglycosides are also known to favor the emergence of scvs and are often used in cf patient care. interestingly, a synergistic effect between hqno and tobramycin for the formation of s. aureus scvs was previously observed by hoffmann et al.. it is thus possible that the administration of aminoglycosides to cf patients co-infected with both s. aureus and p. aeruginosa further increases the formation of biofilm by s. aureus. besides, it is well known that the abnormal function of the cf transmembrane conductance regulator protein in cf patients has profound consequences on the airway physiology and it will be of great interest to determine whether other parameters related to the cf airways influence the emergence of scvs and the production of biofilms by s. aureus. the expression of virulence factors in s. aureus is indeed controlled by diverse and complex regulatory networks in a time- and environment-dependent manner, being influenced for example by ionic forces, ph and o. consequently, it is likely that s. aureus specifically responds to the particular environment of cf airways. whether this response is sigb-dependent and will lead to the emergence of scvs and biofilm production remains to be determined. naturally-occurring mutations altering the activity of virulence regulators in s. aureus have been previously reported. our results suggest that the inactivation of sigb will importantly influence the outcome of the hqno-mediated interaction between p. aeruginosa and s. aureus. we are currently studying s. aureus isolates from cf patients co-infected with p. aeruginosa which are not influenced by the presence of p. aeruginosa. this, in addition to the observation that differences between s. aureus strains exist relative to their response to haqs, suggest that s. aureus strains isolated from cf patients may adapt or evolve toward a long-term coexistence with p. aeruginosa. whether this involves mutations in sigb or any other genes encoding regulators is now under investigation and will greatly help to understand the dynamic behavior and the adaptation of s. aureus in response to the cf airway environment as well as to the presence of p. aeruginosa. the effect of hqno on the regulators sara, agr and sigb suggests that several virulence factors should be influenced by the presence of hqno. a concomitant activation of sigb and repression of agr should result in the up-regulation of a variety of cell-surface proteins involved in adhesion to host tissues, and in the repression of several exotoxins and proteolytic enzymes. it is then tempting to speculate that the presence of hqno will prevent s. aureus from disseminating and will rather favor tissue colonization, biofilm production and invasion of host cells. it has indeed been suggested that s. aureus fnbps mediates cellular invasion whereas the capacity of the bacterium to remain intracellular is helped by the repression of hla. accordingly, we showed that an exposure of s. aureus to hqno up-regulates the expression of fnba and represses the expression of hla. however, whether or not hqno and p. aeruginosa increase the invasion of host cells by s. aureus remains to be confirmed. interestingly, o'neil et al. have recently demonstrated that the fnbps are also involved in the ica-independent mechanism of biofilm formation. it is thus possible that fnbps are directly responsible for the observed hqno-mediated sigb-dependent increase in biofilm production and, more specifically, fnbpa which is under the control of sigb for expression. as such, the fnbps would represent the main effectors for both biofilm formation and cellular invasion in s. aureus scvs. hqno may be one of several bacterial exoproducts influencing s. aureus during polymicrobial infections. our results and those of machan et al. suggest that other haqs may also affect s. aureus, although not as efficiently as hqno. moreover, it is known that other p. aeruginosa exoproducts such as pyocyanin have an inhibitory activity against the electron transport chain of s. aureus. loss of pyocyanin production has been associated with mutations in the pqsa-e genes, which may provide an additional explanation for the different effects of the pqsa and pqsl mutants we have observed on the growth and biofilm formation of s. aureus. furthermore, qazi et al. found that an n-acyl-homoserine-lactone from p. aeruginosa antagonizes quorum sensing and virulence gene expression in s. aureus. more precisely, it was shown that the 3-oxo-c12-hsl interacts with the cytoplasmic membrane of s. aureus and down-regulates both sara and agr expression. although we also observed here a down-regulation of agr, the hqno-mediated up-regulation of sara suggests further complexity in the response of s. aureus to p. aeruginosa exoproducts. it is possible that the outcome of the s. aureus-p. aeruginosa interaction is dependent on the amount and the types of exoproducts secreted by the specific strain of p. aeruginosa interacting with s. aureus. it should be kept in mind that the diversity of diffusible bacterial signals and their role in interspecies communication are just beginning to be appreciated and that potentially numerous other bacterial species may be interacting with s. aureus, especially during infectious diseases. it is then likely that s. aureus interacts with other bacterial genus than pseudomonas during infection of the airways of cf patients. as an example, the cf pathogen burkholderia cepacia also produces n-acylhomoserine lactones and some burkholderia species are able to synthesize haq analogues. nevertheless, the observation that p. aeruginosa favors the emergence of scvs and biofilm production by s. aureus is likely to have a significant clinical impact. the clinical consequences may actually surpass the previously anticipated formation of aminoglycoside-resistant scvs by hoffman et al.. persistence of bacteria in chronic infections has been associated with biofilm production and biofilms are known to confer protection from host defenses and antibiotic treatments at large. in the cystic fibrosis context, where obstructive infections worsen the health prognosis of patients, the clinical significance of biofilm production by normal s. aureus and scv strains will need to be further investigated.  this study strongly supports the hypothesis that p. aeruginosa influences the pathogenicity of s. aureus by producing hqno, which favors the acquisition of the scv phenotype through the activation of the stress- and colonization-related s. aureus alternative sigma factor b. although several p. aeruginosa exoproducts may potentially influence s. aureus, our observations with pure hqno were confirmed and supported by experiments using whole supernatants from two p. aeruginosa strains as well as mutants unable to produce hqno. considering that biofilms and scvs are both suspected to play a role in chronic infections of cf airways, the observation that p. aeruginosa increases the emergence of scvs and biofilm formation by s. aureus may influence the patient health prognosis. new therapeutic strategies should aim at preventing interspecies interactions and the development of specific phenotypes such as biofilm-producing scvs in order to reduce the likelihood of chronic infections. 
experiments using culture supernatants from p. aeruginosa pao and a double chamber co-culture model confirmed that p. aeruginosa stimulates biofilm formation and activates sigb in a s. aureus strain isolated from a cf patient. the presence of s. aureus scvs as well as biofilms have both been associated with chronic infections in cf. it was previously shown that the p. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-n-oxide suppresses the growth of s. aureus and provokes the emergence of small-colony variants. furthermore, the supernatant from p. aeruginosa mutants unable to produce hqno induced the production of biofilms by s. aureus to a lesser extent than the wild-type strain only in a s. aureus sigb-functional background. these results suggest that s. aureus responds to hqno from p. aeruginosa by forming scvs and biofilms through sigb activation, a phenomenon that may contribute to the establishment of chronic infections in cf patients. the emergence of scvs and biofilm production under hqno exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor b. staphylococcus aureus and pseudomonas aeruginosa are often found together in the airways of cystic fibrosis patients. we demonstrated that hqno stimulates s. aureus to form a biofilm in association with the formation of scvs. analysis of gene expression revealed that exposure of a prototypical s. aureus strain to hqno activates sigb, which was leading to an increase in the expression of the fibronectin-binding protein a and the biofilm-associated sara genes.
 staphylococcus aureus and pseudomonas aeruginosa are often found together in the airways of cystic fibrosis patients. it was previously shown that the p. aeruginosa exoproduct 4-hydroxy-2-heptylquinoline-n-oxide suppresses the growth of s. aureus and provokes the emergence of small-colony variants. the presence of s. aureus scvs as well as biofilms have both been associated with chronic infections in cf.  we demonstrated that hqno stimulates s. aureus to form a biofilm in association with the formation of scvs. the emergence of scvs and biofilm production under hqno exposure was shown to be dependent on the activity of the stress- and colonization-related alternative sigma factor b. analysis of gene expression revealed that exposure of a prototypical s. aureus strain to hqno activates sigb, which was leading to an increase in the expression of the fibronectin-binding protein a and the biofilm-associated sara genes. conversely, the quorum sensing accessory gene regulator system and the α-hemolysin gene were repressed by hqno. experiments using culture supernatants from p. aeruginosa pao and a double chamber co-culture model confirmed that p. aeruginosa stimulates biofilm formation and activates sigb in a s. aureus strain isolated from a cf patient. furthermore, the supernatant from p. aeruginosa mutants unable to produce hqno induced the production of biofilms by s. aureus to a lesser extent than the wild-type strain only in a s. aureus sigb-functional background.  these results suggest that s. aureus responds to hqno from p. aeruginosa by forming scvs and biofilms through sigb activation, a phenomenon that may contribute to the establishment of chronic infections in cf patients. 