Anaerobiosis influences virulence properties of Pseudomonas aeruginosa cystic fibrosis isolates and the interaction with Staphylococcus aureus

The airways of individuals with cystic fibrosis (CF) are abundantly colonised by Staphylococcus aureus and Pseudomonas aeruginosa. Co-infecting hypoxic regions of static mucus within CF airways, together with decreases in pulmonary function, mucus plugging and oxygen consumption by host neutrophils gives rise to regions of anoxia. This study determined the impact of anaerobiosis upon S. aureus-P. aeruginosa interactions in planktonic co-culture and mixed species biofilms in vitro. Whilst anoxia reduced the ability for P. aeruginosa CF isolates to dominate over S. aureus, this occurred in an isolate dependent manner. Investigations into the underlying mechanisms suggest that the anti-staphylococcal compound facilitating P. aeruginosa dominance under normoxia and anoxia is greater than 3 kDa in size and is heat-stable. Not all interspecies interactions studied were antagonistic, as S. aureus exoproducts were shown to restore and enhance P. aeruginosa motility under normoxia and anoxia in an isolate dependent manner. Collectively, this study suggests changes in oxygen availability within regions of the CF lung is likely to influence interspecies interactions and in turn, potentially influence disease progression.

www.nature.com/scientificreports www.nature.com/scientificreports/ comparable to those isolated in single species biofilm. CF isolate 7 however, demonstrated a significant increase in CFU/mL in mixed species biofilm compared to single culture (P < 0.05).
Following growth under anoxia, PAO1 and CF isolates 5 and 6 were no longer able to outcompete S. aureus in mixed species biofilm (Fig. 2c) whilst CF isolate 7 retained its ability to reduce S. aureus viability (P < 0.001). All CF isolates tested were unaffected by the presence of S. aureus under anoxia with P. aeruginosa being enumerated at numbers similar to those in P. aeruginosa monoculture biofilms (Fig. 2d). The data for S. aureus-P. aeruginosa competition in planktonic co-culture and mixed species biofilms is summarised in a table in Supplementary  Table S1. S. aureus-P. aeruginosa competition in planktonic co-culture under normoxia and anoxia. S. aureus and P. aeruginosa were grown statically at 37 °C for 24 h in either single or dual culture, under normoxia or anoxia. At regular intervals, aliquots were taken and plated onto selective agar. Each data point represents the mean ± S.E.M of three independent experiments (N = 3), each performed in triplicate. Statistical differences were determined using a two-way ANOVA with Bonferroni post-hoc, comparing the CI to the RIR at each time point ***P < 0.001.

Impact of oxygen upon anti-staphylococcal exoproducts.
It is evident that anoxia facilitates S. aureus-P. aeruginosa co-existence in an isolate dependent manner, in both planktonic co-culture and mixed species biofilms. To determine how oxygen availability permits bacterial co-existence and why P. aeruginosa CF isolate 7 was still able to reduce S. aureus viability, the production of several known anti-staphylococcal exoproducts in cell-free supernatants of P. aeruginosa were assessed following growth under normoxia and anoxia. This study focused upon the production of proteases, the lysis of S. aureus, the production of the respiratory inhibitor pyocyanin, the siderophore pyoverdine and biosurfactants.
As P. aeruginosa is known to secrete a number of proteases, skimmed milk agar has previously been used to study the total protease activity of P. aeruginosa 50,52 . The diameter of the zones of clearance were measured in millimetres (mm). As shown in Fig. 3a, PAO1 and CF isolates 5 and 7 produced detectable levels of protease under normoxia. Following growth under anoxia, CF isolates 5 and 7 retained their protease activity, whilst this was below the limit of detection for PAO1. CF isolate 6 failed to produce detectable proteases following growth under normoxia and anoxia.
P. aeruginosa PAO1 has previously been shown to lyse S. aureus 16 . Thus, the ability of P. aeruginosa CF isolates to lyse heat-killed S. aureus was also determined following growth under normoxia and anoxia, a method used previously [53][54][55] . As shown in Fig. 3b, CF isolate 6 was the only isolate unable to significantly lyse heat-killed S.   . Impact of normoxia and anoxia upon the production of anti-staphylococcal exoproducts by P. aeruginosa. (a) Protease production was determined following the addition of P. aeruginosa cell-free supernatants to wells in milk agar, prior to the diameter of the zones of clearance (mm) being measured following 24 h incubation. (b) P. aeruginosa cell-free supernatants were added to heat-killed S. aureus and staphylolytic activity was determined by measuring changes in the OD 595 after 60 min. The control consisted of heat-killed S. aureus with sterile LBN broth only. (c) Pyocyanin was extracted from P. aeruginosa cell-free supernatants using chloroform and 0.2 M hydrochloric acid. Spectrophotometric measurements were taken at OD 520 and multiplied by extinction co-efficient of pyocyanin at this wavelength (17.072) to express pyocyanin in µg/mL. (d) Pyoverdine production was quantified by measuring the relative fluorescence units (RFU) of P. aeruginosa cell-free supernatants following excitation at 460 nm and emission at 490 nm. (e) Surfactant activity was measured using the established drop collapse assay, where cell-free supernatants were serially diluted (1:1) with deionised water containing 0.005% crystal violet to aid visualisation. Surfactant scores are equal to the www.nature.com/scientificreports www.nature.com/scientificreports/ aureus under normoxia (P > 0.05). Following growth under anoxia (Fig. 3b), PAO1 lost its ability to significantly lyse heat-killed S. aureus compared to normoxia (P < 0.001), whilst CF isolates 5 and 7 retained their significant staphylolytic activity under anoxia. CF isolate 6 did not lyse heat-killed S. aureus under anoxia.
The complete cell-free secretomes of P. aeruginosa PAO1 and CF isolates 5, 6 and 7 are shown in Supplementary Tables S2 to S5. Expression levels of elastase and protease LasA following growth under normoxia and anoxia were studied by mass spectrometry (Fig. 4) to determine whether there was any correlation with the protease and staphylolytic activity shown in Fig. 3a,b. As shown in Fig. 4, whilst all of the isolates tested secreted detectable levels of elastase under both environmental conditions, CF isolate 6 was the only isolate which did not produce detectable levels of protease LasA under both normoxia and anoxia. Analysis of the PAO1 cell-free secretome demonstrated a 20.2-fold decrease in the relative abundance of elastase under anoxia, whilst exhibiting a minimal fold change in protease LasA. Conversely, CF isolates 5 and 7 exhibited a 3.0-fold increase and 1.4-fold decrease in elastase respectively, along with a 3.0-fold increase and 1.3-fold decrease in protease LasA. Once more, CF isolate 6 failed to exhibit staphylolytic activity under anaerobiosis (Fig. 4b), with no protease LasA being detected in its secretome (Fig. 4).
The production of the respiratory inhibitor pyocyanin was also determined. Under normoxia, PAO1 and CF isolates 5 and 6 produced varying degrees of pyocyanin following phenol-chloroform extraction, whilst this was below the limit of detection for CF isolate 7 (Fig. 3c). No pyocyanin was detected for any of the P. aeruginosa isolates following growth under anoxia.
As shown in Fig. 3d, P. aeruginosa PAO1 and CF isolate 5 produced pyoverdine under normoxia, with this being significantly reduced under anoxia (P < 0.001). CF isolates 6 and 7 produced minimal levels of pyoverdine, which was unaffected by anoxia. Lastly, PAO1 and all CF isolates tested exhibited varying degrees of biosurfactant activity, with this being the greatest for CF isolate 7 (Fig. 3e). Anoxia reduced the surfactant activity of PAO1 and CF isolate 5, but not for CF isolates 6 and 7. The data shown in Fig. 3 demonstrates that each P. aeruginosa CF isolate produces a varying array of anti-staphylococcal exoproducts under normoxia, with their production being further influenced by anoxia.
Using these data, we sought to greater determine the nature of the P. aeruginosa anti-staphylococcal compound which mediates P. aeruginosa dominance under both normoxia and anoxia. Cell-free supernatants from P. aeruginosa cultures were passed through 3 kDa molecular weight cut-off filters, in an effort to determine the size of the anti-staphylococcal compound. Whilst the 20 kDa protease LasA would be retained within the >3 kDa fraction, smaller virulence factors such as pyocyanin, rhamnolipids and pyoverdine would pass into the <3 kDa fraction. Heat-treatment was used to determine whether the anti-staphylococcal compound was proteinaceous in nature, as boiling would be expected to abolish its anti-staphylococcal activity. Cell-free culture supernatants were subsequently added to log-phase cultures of S. aureus to assess their ability to inhibit S. aureus planktonic growth over 14 h.
As shown in Fig. 5, under normoxia, P. aeruginosa PAO1 whole supernatant, >3 kDa and <3 kDa fractions were able to significantly reduce S. aureus growth compared to S. aureus alone (P < 0.001). This anti-staphylococcal activity was not lost following heat-treatment of the fractions. Interestingly, S. aureus antagonism across all the CF clinical isolates tested under normoxia were restricted to the whole supernatant and >3 kDa fractions only, which was unaffected by heat-treatment.
Under anoxia (Fig. 5) significant anti-staphylococcal activity was detected in all but the untreated <3 kDa fraction of PAO1. Once again, the anti-staphylococcal activity of the fractions was unaffected by heat-treatment. The heat-treated whole culture supernatant and both the untreated and heat-treated >3 kDa fractions obtained from CF isolate 5 significantly reduced S. aureus growth. For CF isolate 6, only the >3 kDa fractions significantly reduced S. aureus growth, where once again, heat-treatment failed to abolish this activity. As seen with CF isolate 5, the heat-treated whole culture supernatant and the untreated and heat-treated >3 kDa fractions obtained from CF isolate 7 were the most inhibitory towards S. aureus growth. Collectively, this suggests that the major anti-staphylococcal exoproduct produced by P. aeruginosa is heat-stable and >3 kDa in size.
Proteomic analysis of the cell-free secretomes of PAO1 and CF isolates 5, 6 and 7 (Supplementary Tables S2 to S5) did not identify any obvious lead candidate for the active factor which is likely to mediate P. aeruginosa dominance under normoxia and anoxia. Whilst the mass spectrometry experiments performed were used to generate a proteomic dataset only, it is possible that the extracellular anti-staphylococcal factor is not proteinaceous in nature. Thus, further work is required in order to identify the active anti-staphylococcal factor. Impact of S. aureus exoproducts and anoxia upon P. aeruginosa motility. Lastly, as S. aureus infection precedes P. aeruginosa, we sought to determine the influence of S. aureus exoproducts upon P. aeruginosa swimming and swarming motilities. Both free and surface attached forms of motility are likely to play important roles in airway colonisation and disease progression. The impact of anoxia upon P. aeruginosa motility in the presence and absence of S. aureus exoproducts was also determined.
PAO1 and the CF isolates tested exhibited varying degrees of swimming motility under normoxia (Fig. 6). CF isolate 5 alone exhibited significantly greater swimming motility under normoxia compared to PAO1, CF isolate 6 and CF isolate 7 alone (P < 0.001). P. aeruginosa motility was unaffected by the inclusion of S. aureus exoproducts. Under anoxia, CF isolate 5 alone exhibited the greatest swimming motility compared to PAO1 alone (P < 0.01) and CF isolates 6 and 7 alone (P < 0.05). Compared to normoxia, anoxia reduced the swimming motility of PAO1 reciprocal of the greatest dilution at which there was surfactant activity. Data shown are the mean ± S.E.M. of three independent experiments (N = 3), each performed in triplicate. In panels A, B and D, statistical differences were determined using one-way ANOVA with Tukey's post-hoc test *P < 0.05, **P < 0.01 and ***P < 0.001. www.nature.com/scientificreports www.nature.com/scientificreports/ alone (P < 0.01), CF isolate 5 alone (P < 0.001) and CF isolate 5 in the presence of S. aureus (P < 0.001). PAO1 alone lost its swimming motility under anoxia whilst the addition of S. aureus cell-free supernatant significantly restored the swimming motility of PAO1 (P < 0.001). S. aureus cell-free supernatant also enhanced the swimming motility of CF isolate 7 compared to CF isolate 7 alone (P < 0.01).
Under normoxia, CF isolate 5 alone exhibited the greatest swarming motility compared to PAO1 and CF isolates 6 and 7 alone (P < 0.001) (Fig. 6). The addition of S. aureus exoproducts only enhanced the swarming motility of PAO1 (P < 0.001). Under anoxia, CF isolate 5 exhibited the greatest swarming motility compared to PAO1, including CF isolates 6 and 7 alone (P < 0.001). S. aureus exoproducts did not modulate P. aeruginosa swarming motility under anoxia. Only CF isolate 5 alone exhibited a significant reduction in swarming motility under anoxia compared to normoxia (P < 0.01).
As S. aureus exoproducts were shown to modulate the swimming motility of PAO1 and CF isolate 7 under anoxia, including the swarming motility of PAO1 under normoxia (Fig. 6), S. aureus cell-free supernatants were subjected to size fractionation and heat-treatment to determine the size and nature of the effector compound(s). As shown in Fig. 7, all fractions regardless of size and heat-treatment were able to modulate P. aeruginosa motility to the same extent as the whole culture supernatants (Fig. 6).

Discussion
It is now appreciated that mucus plugging, decreases in pulmonary function and the consumption of oxygen by polymicrobial communities and host neutrophils create microenvironments in CF airways which are devoid of oxygen 31,56 . Whilst previous studies have demonstrated anaerobiosis attenuates the growth and virulence of P. aeruginosa laboratory strains and clinical isolates 43,44 , this study aimed to provide novel insights into the impact of anaerobiosis upon the interactions of S. aureus and CF clinical isolates of P. aeruginosa in planktonic co-culture and mixed species biofilms, including the possible mechanisms governing interspecies interactions under both environmental conditions.   WS: Whole supernatant, WSHI: Whole supernatant heat-inactivated, >3: greater than 3 kDa, >3HI: greater than 3 kDa heat-inactivated, <3: less than 3 kDa, <3HI: less than 3 kDa heat-inactivated. Statistical differences were determined using one-way ANOVA with Dunnett's post-hoc (versus S. aureus only control). *P < 0.05, **P < 0.01 ***P < 0.001.
www.nature.com/scientificreports www.nature.com/scientificreports/ Our competition data showed oxygen availability exerts a major role in S. aureus and P. aeruginosa competition in both planktonic co-culture and mixed species biofilms. Under normoxia (Fig. 1), PAO1 and all CF isolates 5, 6 and 7 were all able to outcompete S. aureus in planktonic co-culture at 24 h, without their own growth being adversely affected. This finding is supported by other in vitro studies conducted under normoxia where P. aeruginosa CF isolates were shown to reduce the viability of S. aureus in planktonic culture 25,27,57 . In all planktonic 24 h co-culture experiments, P. aeruginosa was unable to completely kill S. aureus. PAO1 and CF isolates 5 and 7 retained their ability to antagonise S. aureus growth in mixed species biofilms under normoxia, whilst CF isolate 6 did not (Fig. 2). The inability of CF isolate 6 to outcompete S. aureus in mixed species biofilms as in planktonic co-culture (Figs 1 and 2) is potentially explained by previous studies which demonstrate differences in bacterial virulence following growth in planktonic and biofilm culture [58][59][60] .
The introduction of anoxia caused PAO1 and CF isolates 5 and 6 to lose their ability to outcompete S. aureus in both planktonic co-culture and mixed species biofilms (Figs 1 and 2), where S. aureus was enumerated in densities seen in monoculture. These results support those of a previous study which demonstrated anaerobiosis reduced the ability of P. aeruginosa PAO1 to induce zones of clearance upon confluent bacterial lawns of Staphylococcus epidermidis 61 . Furthermore, the culture of P. aeruginosa strains under hypoxia (1% oxygen) has previously been shown to attenuate P. aeruginosa virulence in an in vivo model of infection compared to normoxia through decreased expression of several virulence factors 62 . From these data, it is possible that anoxia is one of several mechanisms facilitating isolate specific S. aureus-P. aeruginosa co-existence in the CF lung. NORMOXIA ANOXIA SWIMMING SWARMING Figure 6. Effect of S. aureus culture supernatant and oxygen upon P. aeruginosa motility. Swimming and swarming motilities of P. aeruginosa isolates were assessed following an overnight incubation on swimming and swarming agar plates. To test the effects of S. aureus culture supernatant upon these two forms of motility, a 1:100 dilution of the culture supernatant was added to plates before the agar set. Data are presented as mean ± S.E.M from three independent experiments each performed in duplicate. Statistical differences were determined using one-way ANOVA with Tukey's post-hoc. **P < 0.01 ***P < 0.001. (2019)   www.nature.com/scientificreports www.nature.com/scientificreports/ Interestingly, CF isolate 7 was the only P. aeruginosa isolate able to reduce S. aureus viability under anoxia, in both planktonic co-culture and mixed species biofilm (Figs 1 and 2). To greater understand this phenomenon, the production of several anti-staphylococcal exoproducts was assessed across isolates, following growth under normoxia and anoxia. The use of molecular weight cut off filters and heat-treatment of the cell-free fractions served to determine the size and nature of the anti-staphylococcal compound likely to be mediate P. aeruginosa dominance under normoxia and anoxia.
PAO1 has previously been shown to produce LasA, responsible for cleaving the peptidoglycan cell wall of S. aureus 24,63 . Unlike CF isolate 6, PAO1 and CF isolates 5 and 7 were able to lyse heat-killed S. aureus under normoxia (Fig. 3b). PAO1 lost its staphylolytic ability under anoxia however (Fig. 3b), a finding supported by reports of a decrease in the transcription of LasA for PAO1 under anoxia 64 and a reduction in PAO1 elastase B (LasB) production under anoxia 44 . Perhaps the LasA produced by PAO1 following growth under anoxia is below a threshold to exert a considerable affect upon S. aureus viability compared to that produced by CF isolates 5 and 7, although further work is required to determine this. S. aureus has also previously been shown to exhibit an increase in cell wall thickness under anaerobiosis 65 and whilst this was not investigated in this study, this mechanism may further reduce S. aureus susceptibility to P. aeruginosa LasA under anoxia.
Whilst LasA may be advantageous to P. aeruginosa under growth in polymicrobial communities with S. aureus, collectively, LasA mediated lysis of S. aureus alone does not appear to be essential for facilitating P. aeruginosa dominance under normoxia. All CF isolates were able to outcompete S. aureus in planktonic co-culture (Fig. 1), yet CF clinical isolate 6 failed to exhibit detectable staphylolytic activity (Fig. 3b) or secrete detectable protease LasA as determined by mass spectrometry (Fig. 4). LasA also does not appear to be essential for modulating P. aeruginosa dominance under anoxia, as CF isolates 5 and 7 both retained their staphylolytic activity under anoxia (Fig. 3b). Only CF isolate 7 was able to outcompete in planktonic co-culture (Fig. 1) and mixed species biofilm (Fig. 2). Furthermore, whilst whole culture supernatants and >3 kDa fractions from PAO1 and the CF isolates were able to antagonise S. aureus growth, heat-treatment failed to abolish their inhibitory activity (Fig. 5). As staphylolysis is known to be mediated by LasA, boiling such fractions would be expected to denature this 20 kDa protease.
The role of LasA in governing interspecies interactions may also be complicated by previous findings which demonstrate how the growth of S. aureus in the presence of P. aeruginosa LasA and staphylolysin produced by Staphylococcus staphylolyticus, selects for S. aureus colonies which lack a cell wall 66,67 . Whilst the emergence of such colonies in CF airways requires further study, selection of such a phenotype may serve to facilitate S. aureus persistence under both normoxia and anoxia in the presence of P. aeruginosa.
As previously mentioned, pyocyanin inhibits S. aureus oxidative respiration, causing a reduction in growth. PAO1 and CF isolates 5 and 6 produced detectable levels of pyocyanin under normoxia following phenol-chloroform extraction, whilst this was below the limit of detection for CF isolate 7 (Fig. 3c). Despite this, CF isolate 7 was still able to dominate S. aureus under normoxia in planktonic co-culture (Fig. 1) and mixed species biofilms (Fig. 2). Furthermore, pyocyanin was below the limit of detection for all culture supernatants cultured under anoxia following phenol-chloroform extraction (Fig. 3c), yet CF isolate 7 was still able to dominate. Whilst the inability to detect pyocyanin under anoxia may be due to the redox nature of the phenazine 68 , a previous study demonstrated that hypoxia significantly reduced pyocyanin production by P. aeruginosa 69 .
Proteins involved in phenazine biosynthesis were only detected in the secretomes of P. aeruginosa PAO1 and P. aeruginosa CF isolate 5. As shown in Supplementary Table S2, P. aeruginosa PAO1 exhibited a 10.4-fold decrease in phenazine biosynthesis protein PhzB 1 and a 12.3-fold decrease in phenazine biosynthesis protein PhzB 2 respectively under anoxia. Additionally, there was a 21.0-fold decrease in Phenazine biosynthesis protein PhzB 1 for P. aeruginosa CF isolate 5 under anoxia (Supplementary Table S3). These data support the phenotypic data relating to pyocyanin production shown in Fig. 3c.
Size exclusion and heat-treatment of the P. aeruginosa cell-free supernatants also demonstrated that S. aureus antagonism was restricted to the >3 kDa fraction and was not abolished following heat-treatment (Fig. 5). However, it is expected that pyocyanin would be present in the <3 kDa fraction and that heat-treatment would abolish its activity.
Pyoverdine production by P. aeruginosa is known to reduce S. aureus growth, and enhance P. aeruginosa virulence 27,29,70 . As shown in Fig. 3d, PAO1 and CF isolate 5 produced large amounts of pyoverdine. Furthermore, anoxia was shown to significantly reduce pyoverdine production by PAO1 and CF isolate 5 compared to normoxia, a finding also reported following the growth of P. aeruginosa under hypoxia 69 . Despite CF isolates 6 and 7 producing minimal amounts of pyoverdine under normoxia (Fig. 3d), all P. aeruginosa CF isolates were able to outcompete S. aureus in planktonic co-culture (Fig. 1). Additionally, CF isolate 7 was able to outcompete S. aureus under anoxia despite producing minimal amounts of pyoverdine. As the antimicrobial activity of P. aeruginosa cell-free supernatants was restricted to the >3 kDa fractions (Fig. 5), it is unlikely that siderophore production in isolation drives S. aureus antagonism. However, we recognise the importance of pyoverdine in governing interspecies interactions may be underestimated in this study as iron-depleted growth media was not used to culture P. aeruginosa.
The surfactant activity of P. aeruginosa rhamnolipids has been previously shown to target S. aureus and reduce its viability [71][72][73] . Our data demonstrated that PAO1 and the CF P. aeruginosa isolates tested harboured biosurfactant activity under normoxia, with this being the greatest for CF isolate 7 (Fig. 3e). Whilst CF isolate 7 retained its high surfactant score under anoxia, size exclusion studies demonstrated that the anti-staphylococcal activity for all isolates was restricted to the >3 kDa fraction under both normoxia and anoxia (Fig. 5) and yet it is expected that the rhamnolipids would be retained within the <3 kDa fractions.
Collectively, it is reasonable to suggest from the results of this study that the anti-staphylococcal compound which facilitates P. aeruginosa dominance under both normoxia and anoxia is heat-stable, >3 kDa in size and is likely not one of the anti-staphylococcal factors studied. An interesting observation in Fig. 5  www.nature.com/scientificreports www.nature.com/scientificreports/ heat-treated whole supernatant and >3 kDa fractions for most P. aeruginosa CF isolates under normoxia and anoxia appeared to more greatly reduce S. aureus growth compared to those which had not been heat treated. It is reasonable that heating may either increase the activity of the anti-staphylococcal factor, or alternatively lead to the loss of a factor within the supernatant which may otherwise protect S. aureus directly or indirectly. We also recognise that a limitation of this study is that the absolute concentration of the P. aeruginosa exoproducts in cell-free culture supernatants was not determined and it may be that some of the virulence factors were at concentrations too low to exert an antagonistic effect upon S. aureus. P. aeruginosa is known to employ several other virulence properties which were not characterised in this study and may contribute to the differences seen in bacterial competition. This includes the respiratory inhibitor hydrogen cyanide 74 . However, its role in governing S. aureus-P. aeruginosa interactions is likely to be minimal in this study as it would be found in the <3 kDa fraction, and yet these fractions did not exert considerable anti-staphylococcal activity upon exposure to planktonic S. aureus under normoxia (Fig. 5). Furthermore, previous research has demonstrated that hydrogen cyanide is rapidly inactivated under strict anoxia 75 and thus its ability to reduce S. aureus viability under anoxia is considered minimal.
P. aeruginosa also produces the respiratory inhibitor HQNO, which is known to reduce S. aureus viability 19,27,76 . Its production is regulated by Pseudomonas quinolone signal (PQS) 77 . PQS production is known to be suppressed under anoxia 61,78 . As shown in Supplementary Table S5, PQS proteins were only detected in the secretome of P. aeruginosa CF isolate 7. Whilst there was a 9.4-fold decrease in PqsB under anoxia, there was a 4.5-fold increase in PqsC under anoxia. Thus, further work is required to determine the role of HQNO in governing S. aureus-P. aeruginosa interspecies interactions.
It is also reasonable to suggest that the ability of P. aeruginosa to dominate under both normoxia and anoxia is not due to the secretion of a single anti-staphylococcal factor, but is due to the production of many 20,27,[79][80][81] . A previous study has reported that deletion of a single virulence property has been shown to reduce P. aeruginosa antagonism towards S. aureus 82 . The production of alginate by P. aeruginosa has been reported to facilitate S. aureus-P. aeruginosa co-existence within the CF lung, with the overproduction of alginate inhibiting the synthesis secretion of pyoverdine, HQNO and rhamnolipids 82 . However, as none of the P. aeruginosa CF isolates used in this study were mucoid, alginate is unlikely to play a role in facilitating interspecies co-existence.
Finally, we also show how not all S. aureus-P. aeruginosa interactions studied are antagonistic. Whilst S. aureus has previously been shown to enhance P. aeruginosa antibiotic resistance 83,84 , we have demonstrated that S. aureus exoproducts were able to restore and enhance P. aeruginosa swimming and swarming motility in an isolate dependent manner under normoxia and anoxia (Fig. 6).
Previous research has demonstrated that the intracellular accumulation of the signalling molecule bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) favours P. aeruginosa biofilm formation 85 through the production of exopolysaccharide, whilst repressing flagella motility. Conversely, intracellular decreases in c-di-GMP inhibits exopolysaccharide production and promotes flagella mediated motility 86,87 . These effects of intracellular c-di-GMP are mediated through its ability to bind and modulate the transcriptional regulator FleQ 88,89 . It is possible that one or more S. aureus exoproducts within the cell-free supernatant may lower the intracellular concentration c-di-GMP in P. aeruginosa, promoting motility (Fig. 6). Additional work is required as the modulating ability of S. aureus culture supernatant upon P. aeruginosa motility was not seen in all isolates and could not be restricted to a single fraction (Fig. 7).
Twitching motility in the presence and absence of S. aureus exoproducts was also investigated but in our hands no twitching motility was detected in any strains. As anoxia was also shown to influence P. aeruginosa motility, the presence of S. aureus and changes in oxygen availability are both likely to influence P. aeruginosa colonisation and dissemination into microenvironments within the CF lung.

Conclusion
This study provides novel findings concerning the impact of anoxia upon interactions between S. aureus and CF isolates of P. aeruginosa in mixed planktonic culture and mixed species biofilms. We hypothesise from our data that anoxia is one of several potential mechanisms facilitating S. aureus-P. aeruginosa co-existence in the CF lung, with co-infection having been associated with poor clinical outcomes and a worsening of pulmonary function in CF 20,90 . However, not all P. aeruginosa CF isolates lose their ability to antagonise S. aureus growth under anoxia. Further work is required to determine the nature of the major anti-staphylococcal compound which facilitates P. aeruginosa dominance under both normoxia and anoxia, although this study provides evidence that the factor is likely to be heat-stable and >3 kDa in size. The ability of select P. aeruginosa CF isolates to retain their virulence and motility under anoxia is likely to influence disease progression differently across the CF population. The ability of S. aureus exoproducts to further modulate P. aeruginosa motility in an isolate dependent manner, reinforces the complex interspecies interactions known occur within the CF lung.
As co-isolated strains of S. aureus and P. aeruginosa are shown to be less antagonistic than those from mono-infected patients 82 , a longitudinal study of select S. aureus and P. aeruginosa co-isolates could be performed. This would allow interspecies interactions and the pathoadaptive mechanisms that occur overtime in both S. aureus and P. aeruginosa to be studied in greater detail and allow comparisons to be made under different environmental conditions. It is hoped that this study encourages interspecies interactions of other key CF pathogens to be determined under anoxia, an environmental condition shown to be important in modulating bacterial virulence and interspecies interactions.

Materials and Methods
Bacterial strains and growth conditions. All bacterial strains used in this study are listed in Table 1, with Table 2 describing their colony morphotype following growth upon LB agar. Eight clinical isolates of P. aeruginosa were cultured from paediatric CF sputum samples from Birmingham Children's Hospital, UK between 1990-1999. All bacterial strains were stored at −80 °C in 50% (v/v) glycerol/water. Bacteria were streaked from frozen stocks to Luria Bertani (LB) agar plates (Fisher Scientific, UK) and incubated at 37 °C for 48 h. Strains were identified using selective agar, Gram staining and biochemical testing.
All assays were carried out using standardised bacterial inoculum. Single well isolated colonies of S. aureus or P. aeruginosa were routinely inoculated into 10 mL of sterile LBN broth (LB broth supplemented with 1% (w/v) potassium nitrate) (Fisher Scientific, UK) and grown at 37 °C under static conditions of normoxia or anoxia (min-iMACS anaerobic workstation, Don Whitley Scientific, UK) for approximately 16 h.
Cross-Streak assay on solid agar. Overnight cultures of S. aureus and P. aeruginosa grown separately under normoxia or anoxia were pelleted, resuspended in fresh broth and adjusted to an OD 470 of 1.0. A sterile cotton swab was immersed in a given P. aeruginosa standardised culture and streaked horizontally across the surface of a LB agar plate. After air drying for 20 min, a sterile cotton swab was immersed in the adjusted S. aureus culture and cross-streaked vertically across the surface of the agar plate. Plates were then incubated either under normoxia or anoxia at 37 °C for 18 h, prior to being visually inspected for growth inhibition.
Mono-culture and co-culture planktonic growth curves. All growth curve experiments were conducted in 250 mL conical flasks containing 50 mL of LBN broth at 37 °C under static conditions. Overnight cultures of S. aureus and P. aeruginosa CF isolates grown under normoxia or anoxia were pelleted, resuspended in fresh medium and adjusted to an OD 470 of 1.0. For co-culture growth curves, the bacteria were inoculated at an equal ratio (1:1, S. aureus to P. aeruginosa) and incubated under static conditions at 37 °C for 24 h. Samples were taken at regular intervals, serially diluted in sterile PBS (Fisher, UK) and 20 µL spots plated onto Pseudomonas isolation agar (PIA) (Fisher Scientific, UK) and mannitol salt agar (MSA) (Fisher Scientific, UK) to allow species differentiation. The plates were incubated for approximately18 h, prior to enumerating the colony forming units (CFU/mL).
The competition index (CI) and Relative Increase Ratio (RIR) were calculated. The RIR was calculated on single growth curve data using the P. aeruginosa-S. aureus ratio at a given time point, divided by the same ratio at time point 0 h (inoculum). The same ratio was used to calculate the CI, although this used data from the mixed culture. A CI that differs statistically from the RIR indicates competition between the two organisms. This method was adapted from Macho et al. 91 .
Mono-culture and co-culture biofilm formation. Overnight cultures of S. aureus and P. aeruginosa grown under normoxia or anoxia were centrifuged and adjusted to an OD 470 of 1.0. Cultures were diluted tenfold and 100 µL added to the central wells of a 96-well tissue culture treated flat bottom plate, either individually or in a 1:1 ratio for an hour under static conditions, at 37 °C. An equal volume of broth was added to the individual culture to compensate for any dilution effect. After 60 min, the well contents were aspirated and replaced with fresh LBN broth. Plates were incubated for a further 24 h at 37 °C under static conditions. Following this, biofilms were washed twice using 200 µL of PBS, detached using 100 µL of 0.25% trypsin-EDTA (Fisher Scientific, UK), collected, vortexed for 70 s, serially diluted and plated onto PIA (Fisher, UK) and MSA (Fisher Scientific, UK). The plates were incubated for approximately18 h, prior to enumerating the colony forming units (CFU/mL). preparation of P. aeruginosa cell-free culture supernatant. Overnight cultures of P. aeruginosa grown under normoxia or anoxia were centrifuged at 4,000 x g for 10 min at 4 °C. Each supernatant was sterile filtered with a low-binding 0.22 µm polyethersulfone membrane filter (Corning, USA) and stored at −20 °C until needed. To confirm sterility after each preparation, a small volume of supernatant was streaked onto LB agar plates and incubated for approximately 20 h prior to reading. For size exclusion experiments, 10 mL of cell-free supernatant was added to a 3 kDa molecular weight cut off protein concentrator (ThermoFisher, UK) and centrifuged at 4,000 × g for 1 h. Apical and basal volumes were subsequently added to 2.0 mL sterile microcentrifuge Determination of total protease production. Protease production was determined using skimmed milk agar. Cell-free supernatants (40 µL) from overnight cultures were loaded into wells in agar plates and incubated at 37 °C for 24 h. Hydrolysis of the milk protein casein results in a clear zone surrounding the bacterial supernatant and would show evidence of protease production. LBN medium was also loaded as a negative control. Clearance zone diameters were measured with a ruler in millimetres (mm).
Staphylolytic activity. This method was adapted from 92,93 . An overnight culture of S. aureus grown under static normoxia was centrifuged at 4,000 × g for 10 min at 4 °C, prior to the pellet being resuspended in 250 µL of 25 mM diethanolamine buffer (Sigma, UK), pH 9.5. The bacteria were then heated to 100 °C for 10 min, before being diluted to a final optimal density OD 595 of 1.0. 400 µL of the adjusted heat-killed S. aureus was then added to each microtube (Fisher, UK). The cell-free supernatant from each P. aeruginosa isolate was diluted 1:10 with diethanolamine buffer, prior to 100 µL being added to the heat-killed S. aureus. Bacterial lysis was determined by measuring decreases in heat killed S. aureus OD 595 after 60 min on a MultiSkan Go plate reader (ThermoFisher, UK). LBN broth alone was used as a control.
Pyocyanin extraction and quantification. This method was adapted from 94,95 . Overnight cultures of P. aeruginosa grown under normoxia or anoxia were pelleted, resuspended in fresh medium and adjusted to an OD 470 of 1.0. For single cultures, 500 µL of P. aeruginosa was then added to a 250 mL conical flask containing 50 mL of LBN broth (1:100 dilution). The flasks were then incubated at 37 °C for 24 h, under static normoxia or anoxia. To quantify pyocyanin production, bacterial cells were pelleted by 4,000 × g centrifugation for 25 min at 4 °C and the supernatant sterile filtered with a low-binding 0.22 µm polyethersulfone membrane filter (Corning, USA). 7.5 mL of the sterile supernatant was added to 4.5 mL of chloroform and vortexed for ten, 2 s intervals. The sample was centrifuged at 4,000 × g for 1 min at 4 °C, prior to 3 mL of the blue-green phase (chloroform phase) being aspirated into a new tube. 1.5 mL of 0.2 M hydrochloric acid was then added to the tube and vortexed again for ten, 2 s intervals, prior to centrifugation at 4,000 × g for 1 min at 4 °C. 100 µL of the pink coloured phase was then transferred into a 96-well plate in triplicate. 100 µL of hydrochloric acid was added in triplicate as a control. The plate was then read at OD 520 and multiplied by the extinction co-efficient 17.072 to determine the concentration of pyocyanin per mL of supernatant 94 . Pyoverdine fluorescence spectroscopy. A total of 100 µL of each P. aeruginosa cell-free supernatant was added to a black 96-well plate (Griener Bio One, UK) in triplicate and read at excitation and emission wavelengths of 400/460 nm as performed previously 96,97 on a Gemini XS Spectramax (Molecular Devices, USA) fluorescence plate reader. The background level of fluorescence was measured using 100 µL LBN broth only.
Drop collapse assay. Cell-free supernatants from overnight cultures of P. aeruginosa were serially diluted (1:1) in sterile dH 2 O containing 0.005% crystal violet to aid visualisation. A total of 20 µL of each dilution (including neat supernatant) were spotted onto the underside of a lid of a 96-well plate and the plate titled at 90°, as performed previously 82 . The assay works on the principle that if the droplet contains surfactants, the drops spread. However, as the quantity of surfactants decrease by dilution, the droplet eventually beads up due to an increase in surface tension. Surfactant scores are equal to the reciprocal of the greatest dilution at which there was surfactant activity (a collapsed drop).  www.nature.com/scientificreports www.nature.com/scientificreports/ Secretome analysis. Pooled overnight cultures of P. aeruginosa grown under either normoxia or anoxia were pelleted following centrifugation at 4,000 × g for 30 min at 4 °C. The supernatant containing extracellular proteins was sterile filtered through a 0.22 µm polyethersulfone membrane filter (Corning, USA). Supernatants were concentrated using Amicon 3 kDa cut-off filters (Millipore, UK) prior to precipitation with 25% trichloroacetic acid (TCA; w/v) for 15 min on ice. Proteins were pelleted at 14000 × g for 10 min, and pellets washed with acetone. Protein pellets were solubilised in 50 mM pH 7.4 Tris-HCl containing 2 mM CHAPS, 7 M urea and 7 M thiourea using an ultrasonicating probe (30 s sonication per cycle, 65% full power, 2 cycles), and quantified against a BSA calibration curve using the Bradford assay (BioRad, UK). Proteins (30 µg) were reduced in Laemmli buffer (Sigma Aldrich, UK) for 15 min at 65 °C, and separated by SDS-PAGE on a 10% gel. Separated proteins were stained with Coomassie G250 blue (0.5% w/v in 40% aqueous methanol and 10% glacial acetic acid) for 1 h and destained in an aqueous solution of 10% ethanol and 7.5% glacial acetic acid.
Protein in-gel digestion was performed following the method of Schevchenko et al. 98 , with minor modifications. Each sample lane was divided into 5 bands, gel bands were excised and diced in a clean polypropylene tube using a sterile scalpel. Gel pieces were subsequently destained with 50% acetonitrile in 50 mM ammonium bicarbonate, dehydrated with acetonitrile and vacuum dried. Proteins were in-gel digested using trypsin in 3 mM ammonium bicarbonate (25:1 protein to trypsin ratio; Trypsin Gold, sequencing, Promega, UK) coupled with shaking at 550 rpm at 37 °C overnight. Peptides were extracted for 15 min in an ultrasonic bath initially using a volume of acetonitrile equivalent to 50% of sample volume, two times with 150 µL of 50% acetonitrile in 50 mM ammonium bicarbonate. Finally 400 µL of pure acetonitrile was used to fully dehydrated the gel pieces and maximise peptide extraction. Each time the complete peptide extract was collected in a polypropylene tube. Finally, peptide extracts were vacuum dried and stored at −20 °C prior to analysis.
Bacterial motility. Swimming motility of P. aeruginosa was investigated using 0.3% (w/v) nutrient agar plates supplemented with nutrient broth and 1% (w/v) potassium nitrate. Swarming of P. aeruginosa was determined using 0.5% (w/v) nutrient agar plates supplemented with nutrient broth, dextrose and 1% (w/v) potassium nitrate. Overnight cultures of P. aeruginosa grown under normoxia or anoxia were adjusted to an OD 470 of 1.0 with a total of 5 µL of culture added to the centre of each plate. To measure the effects of S. aureus on P. aeruginosa motility, a 1:100 diluted S. aureus cell-free culture supernatant was added to the agar plate. Plates were incubated under normoxia or anoxia for 24 h at 37 °C. The diameter of the zone travelled by P. aeruginosa was then measured using a ruler in mm.

Statistical analysis.
All results unless otherwise stated are expressed as mean ± S.E.M. Data for each experiment were collected from three independent experiments (N = 3), each performed in triplicate. All statistical analyses were performed using GraphPad Prism 6 software (Graphpad, La Jolla, CA, USA) with significance being set to P < 0.05. The specific tests used for each experiment are described in the figure legends.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.