Pseudomonas aeruginosa exoproducts determine antibiotic efficacy against Staphylococcus aureus

Chronic coinfections of Staphylococcus aureus and Pseudomonas aeruginosa frequently fail to respond to antibiotic treatment, leading to significant patient morbidity and mortality. Currently, the impact of interspecies interaction on S. aureus antibiotic susceptibility remains poorly understood. In this study, we utilize a panel of P. aeruginosa burn wound and cystic fibrosis (CF) lung isolates to demonstrate that P. aeruginosa alters S. aureus susceptibility to bactericidal antibiotics in a variable, strain-dependent manner and further identify 3 independent interactions responsible for antagonizing or potentiating antibiotic activity against S. aureus. We find that P. aeruginosa LasA endopeptidase potentiates lysis of S. aureus by vancomycin, rhamnolipids facilitate proton-motive force-independent tobramycin uptake, and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) induces multidrug tolerance in S. aureus through respiratory inhibition and reduction of cellular ATP. We find that the production of each of these factors varies between clinical isolates and corresponds to the capacity of each isolate to alter S. aureus antibiotic susceptibility. Furthermore, we demonstrate that vancomycin treatment of a S. aureus mouse burn infection is potentiated by the presence of a LasA-producing P. aeruginosa population. These findings demonstrate that antibiotic susceptibility is complex and dependent not only upon the genotype of the pathogen being targeted, but also on interactions with other microorganisms in the infection environment. Consideration of these interactions will improve the treatment of polymicrobial infections.

Introduction S. aureus is responsible for numerous chronic and relapsing infections such as osteomyelitis, endocarditis, and infections of the cystic fibrosis (CF) lung, as well as many penetrating trauma and burn infections, venous leg ulcers, pressure ulcers, and diabetic foot ulcers. These infections are notoriously difficult to treat, despite isolates frequently exhibiting full sensitivity to administered antibiotics, as measured in vitro using a Minimum Inhibitory Concentration (MIC) assay. This suggests that environmental factors present in vivo may influence the pathogen's susceptibility to antibiotic killing. While these factors can include physical barriers to antibiotic activity, such as tissue necrosis and low vascularization at a site of infection, or bacterial replication within host phagocytes, treatment failure cannot be fully explained by poor drug penetration [1]. Instead, environmental determinants, such as interactions with the host, can induce phenotypic responses or genetic adaptations in bacteria that reduce antibiotic sensitivity [2,3].
Similarly, within complex polymicrobial communities such as those encountered in chronic skin infections, burn wound infections, and chronic colonization of the CF lung, inter-and intraspecies interactions can influence the pathogenicity and antibiotic susceptibility of individual organisms [4][5][6]. The presence of the fungal pathogen Candida albicans, for instance, can induce S. aureus biofilm formation and thus decrease the bacterium's susceptibility to antibiotic killing [4]. Furthermore, antibiotic deactivation by resistant organisms within a population can lead to de facto resistance of all members of the community [7][8][9][10].
These anti-staphylococcal compounds allow P. aeruginosa to quickly eliminate S. aureus during in vitro coculture but do not prevent co-colonization in vivo. Recent findings suggest that within the CF lung, P. aeruginosa strains evolve to be less competitive with S. aureus, resulting in more stable coinfection of the same spatial niche [21]. Additionally, work by Wakeman et al. has shown that the presence of the abundant innate immune protein, calprotectin, induces a phenotypic switch in P. aeruginosa that promotes stable P. aeruginosa and S. aureus interaction through the chelation of zinc and manganese ions at the site of infection. This in turn represses P. aeruginosa metabolic toxin production, resulting in significantly less HQNO and pyocyanin [22]. Similarly, Smith et al. recently demonstrated that S. aureus can tolerate in vitro coculture with P. aeruginosa in the presence of serum albumin through the inhibition of P. aeruginosa lasR quorum sensing and thus LasA expression [23]. Despite these findings, P. aeruginosa LasA, rhamnolipids, HQNO, and pyocyanin are routinely detected at significant concentrations in burn wounds and in CF sputum samples and thus likely influence S. aureus physiology [24][25][26][27][28].
We hypothesized that interaction with P. aeruginosa may antagonize or potentiate S. aureus antibiotic susceptibility and could explain the frequent occurrence of treatment failure in infections involving otherwise drug-susceptible strains. Furthermore, we hypothesized that such interactions could be exploited to improve antibiotic treatment outcome. Here we demonstrate that secreted P. aeruginosa factors dramatically alter S. aureus susceptibility to killing by multiple antibiotic classes, and identify several mediators of S. aureus antibiotic antagonism or potentiation. Importantly, the production of these molecules is highly strain dependent, thus implicating the genotype of coinfecting P. aeruginosa strains as critical determinants of antibiotic treatment outcomes for S. aureus infections. Ultimately, we demonstrate in a mouse model of S. aureus, P. aeruginosa coinfection that the presence of P. aeruginosa can significantly alter the outcome of S. aureus antibiotic treatment. Overall, this work highlights the importance of considering the microbial context of the infection environment during the treatment of polymicrobial infection.

P. aeruginosa alters S. aureus susceptibility to antibiotic killing
To investigate the impact of P. aeruginosa on S. aureus antibiotic susceptibility, we measured the bactericidal activity of 3 antibiotics against S. aureus in the presence of supernatants from 12 P. aeruginosa clinical isolates; 7 from the lungs of CF patients and 5 from burn wounds, as well as 2 laboratory strains; PAO1 and PA14. We were interested in examining how P. aeruginosa-secreted exoproducts can impact the susceptibility of S. aureus to vancomycin, tobramycin, and ciprofloxacin. Vancomycin is the frontline antibiotic for the treatment of methicillinresistant S. aureus (MRSA). Ciprofloxacin and tobramycin are commonly used to treat P. aeruginosa during coinfection.
S. aureus strain HG003 was grown to exponential phase and treated with 500 μL of sterile supernatant from overnight (18 h) cultures of HG003 (control) or one of the 14 P. aeruginosa strains prior to antibiotic challenge. After 24 h, cells were washed and plated to enumerate survivors. We found that the individual bactericidal activities of all 3 antibiotics against S. aureus were affected by P. aeruginosa supernatants. More specifically, we observed 3 P. aeruginosa isolates that significantly protected S. aureus from killing by tobramycin (BC239, BC312, and BC252) and one P. aeruginosa isolate (BC310) induced over a 10-fold increase in tobramycin killing of S. aureus (Fig 1A). We also observed that the majority of P. aeruginosa supernatants were antagonistic towards ciprofloxacin killing ( Fig 1B). Furthermore, supernatants from 8 P. aeruginosa strains (PAO1, PA14, BC238, BC310, BC249, BC250, BC251, and BC252) dramatically potentiated vancomycin killing of S. aureus, resulting in 100-1,000 times more killing than the control culture ( Fig 1C). These data highlight the variable and strain-dependent influence of P. aeruginosa on the susceptibility of S. aureus to different antibiotics; however, the S. aureus strain HG003 was grown to mid-exponential phase and exposed to sterile supernatants from S. aureus HG003 (red), P. aeruginosa laboratory strains PAO1 and PA14 (grey), P. aeruginosa CF clinical isolates (blue) or P. aeruginosa burn isolates (green) for 30 min prior to addition of (A) 50 μg/ml vancomycin, (B) 58 μg/ml tobramycin or (C) 2.34 μg/ml ciprofloxacin concentrations similar to the Cmax in humans. An aliquot was removed after 24 h, washed, and plated to enumerate survivors. The dotted red line represents the number of survivors in the control culture. All experiments were performed in biological triplicate and the number of survivors following antibiotic challenge in the presence of P. aeruginosa supernatant was compared to the HG003 supernatant-treated control. Underlying data can be found in S1 Data. *p<0.05 (one-way ANOVA with Tukey's multiple comparisons post-test analysis of surviving CFU). Error bars represent mean + sd. CF, cystic fibrosis; CFU, colony-forming units. mechanism(s) by which P. aeruginosa alters S. aureus antibiotic susceptibility remained unclear.

P. aeruginosa rhamnolipids increase tobramycin uptake and efficacy against S. aureus
Previous studies have shown that during coculture the presence of P. aeruginosa results in increased S. aureus resistance to tobramycin through the activity of HQNO [5]. In agreement with this, we observed that supernatants from BC239 and BC312 and BC252 protected S. aureus from tobramycin killing ( Fig 1A). Paradoxically, however, we observed that the majority of our clinical isolates had no significant impact on tobramycin bactericidal activity. Even more striking, isolate BC310 appeared to potentiate tobramycin bactericidal activity against S. aureus (Fig 1A). We hypothesized that the impact of P. aeruginosa on S. aureus tobramycin susceptibility was multifactorial, with an unidentified factor increasing tobramycin bactericidal activity.
Tobramycin uptake is dependent on proton-motive force (PMF) [29]. P. aeruginosa HQNO collapses S. aureus PMF by inhibiting electron transport, thus abolishing tobramycin uptake into the cell [5]. To explore the possibility that an additional factor within P. aeruginosa supernatant may influence the bactericidal activity of tobramycin against S. aureus, we examined S. aureus susceptibility to tobramycin in the presence of supernatant from a PA14 ΔpqsLphzShcnC strain. This strain cannot produce the respiratory toxins HQNO, pyocyanin, or HCN, all of which inhibit S. aureus respiration and deplete PMF. Strikingly, we found that PA14 ΔpqsLphzShcnC mutant supernatant led to the rapid eradication of a S. aureus population following tobramycin treatment (Fig 2A) (S1A Fig). Heat-inactivation of P. aeruginosa PA14 supernatant had no impact on its ability to alter tobramycin activity, ruling out heatlabile proteins as potentiators of tobramycin killing (S1B Fig). P. aeruginosa produces surfactant molecules called rhamnolipids that inhibit growth of competing gram-positive bacteria. These amphiphilic molecules increase cell permeability by interacting with the plasma membrane [30]. We hypothesized that rhamnolipid interaction with the membrane may facilitate tobramycin entry into otherwise tolerant, PMF-depleted persister subpopulations. To investigate this possibility, we deleted the rhlA gene in PA14, which is essential for rhamnolipid biosynthesis. Supernatant from a PA14 ΔrhlA mutant conferred full protection to S. aureus against tobramycin killing (Fig 2A) (S1A Fig). Furthermore, during tobramycin treatment, the exogenous addition of a 50/50 mix of purified P. aeruginosa mono-and di-rhamnolipids at 30 μg/ml facilitated the rapid eradication of the S. aureus population and decreased the MIC of tobramycin for S. aureus 8-fold (Fig 2A) (S1 Table). This concentration is within the physiological range of rhamnolipids likely encountered by S. aureus during coinfection with P. aeruginosa, as previous work by Bjarnsholt et al. found that clinical isolates produce a range of 2.4 μg/ml to 72.8 μg/ml rhamnolipids when grown in vitro, and Read et al. reported rhamnolipid concentrations as high as 64 μg/ml in a CF lung explant [24,31]. At the concentrations used in this study, rhamnolipids did not display antibacterial activity in the absence of antibiotic (S1C Fig). Further, incubation with a similar concentration of L-rhamnose, the glycosyl head constituent of rhamnolipids, had no effect on tobramycin killing, ruling out metabolite-stimulated PMF generation as the mechanism of tobramycin potentiation (S1D Fig). Finally, we found that 30 μg/ml purified P. aeruginosa rhamnolipids led to increased uptake of Texas Red-conjugated tobramycin as determined by flow cytometry (Fig 2B).
We next measured the relative amount of HQNO and rhamnolipids produced by each P. aeruginosa isolate using mass spectrometry [32] and a drop-collapse assay, respectively [33]. aureus HG003 was grown to mid-exponential phase and exposed to (A) sterile supernatants from P. aeruginosa or S. aureus or exogenous addition of rhamnolipids (30 μg/ml) before addition of tobramycin 58 μg/ml. At indicated times, an aliquot was washed and plated to enumerate survivors. (B) Texas Redconjugated tobramycin was added to S. aureus cultures with or without 30 μg/ml rhamnolipids. Following 1 h, Texas Red-tobramycin uptake was measured by flow cytometry. (C) Rhamnolipid production present in the supernatant of P. aeruginosa PAO1, PA14, PA14 ΔrhlA, CF isolates (blue) or burn isolates (green) were quantified by a drop-collapse assay. Experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. CF, cystic fibrosis.

Antibiotics and interspecies interaction
We observed a large variance in the production of both HQNO (Table 1) and rhamnolipids between isolates ( Fig 2C). Importantly, the potentiator of tobramycin activity, BC310, was the only strain shown to be a high rhamnolipid producer without detectable HQNO production. In contrast, strain BC239, the strongest tobramycin antagonist, was among the highest HQNO producers, and did not produce rhamnolipids. Together, these data show that P. aeruginosa has the capacity to both positively and negatively influence S. aureus tobramycin uptake and bactericidal activity through the action of rhamnolipids and respiratory toxins, respectively. The presence of these 2 opposing factors may be responsible for the apparent disconnect between the P. aeruginosa-mediated increase in tobramycin resistance reported previously [5], and lack of protection from tobramycin killing following treatment with supernatant from the majority of P. aeruginosa strains observed in this study. Indeed, deletion of either P. aeruginosa respiratory toxins or rhamnolipids in a P. aeruginosa laboratory strain resulted in supernatants that facilitate complete sterilization or protection of S. aureus cultures, respectively (Fig 2A). Furthermore, similar trends were observed when S. aureus MRSA strain JE-2 was challenged with tobramycin following treatment with P. aeruginosa supernatant, supporting the relevance of this phenomenon in the clinical treatment of S. aureus infection (S6A Fig).

P. aeruginosa induces multidrug tolerance in S. aureus through respiratory inhibition
In addition to the ability of HQNO to inhibit uptake of aminoglycosides, we made an interesting and somewhat unexpected observation during our investigation. HQNO production in P. aeruginosa isolates correlated perfectly with protection against ciprofloxacin killing ( Fig 1B) ( Table 1). As ciprofloxacin uptake is PMF-independent, we wondered if P. aeruginosa HQNO was conferring ciprofloxacin tolerance in S. aureus via an alternate mechanism.
Antibiotic tolerance generally refers to a population-wide decrease in antibiotic susceptibility, often following exposure to external mediators of bacterial metabolism or physiology. In contrast, persister cells are generally described as antibiotic-tolerant subpopulations that form stochastically in an otherwise susceptible population. We recently demonstrated that both phenomena are specifically associated with cells entering a low ATP state [34,35]. Subpopulations of low energy cells give rise to persisters, while changes in the environment can lead to a low energy antibiotic-tolerant state in the entire population. HQNO inhibits respiration, the most efficient mechanism for ATP generation in S. aureus. We hypothesized that P. aeruginosa inhibition of S. aureus respiration induces a low ATP, multidrug-tolerant state of the entire population. In support of this, no protection from antibiotic killing was observed following pretreatment with PA14 supernatant during anoxic growth (S2A Fig). We then cloned the fermentation-specific promoter for pyruvate acetyltransferase (pflB) from S. aureus upstream of gfp in a low-copy plasmid. Expression of pflB only occurs under anaerobic conditions or when respiration is inhibited [36]. We found that transcription of the pflB promoter was activated in response to supernatant from all of the P. aeruginosa strains with the exception of PA14 ΔpqsLphzShcnC (negative control) and 4 of the clinical isolates, BC236, BC308, BC310, and BC251. Importantly, these were the only clinical isolates that did not induce significant protection from ciprofloxacin killing ( Fig 1B). Activation of pflB during aerobic growth demonstrates that respiration is inhibited in these conditions ( Fig 3A). Direct intracellular ATP quantification of cultures treated with P. aeruginosa or S. aureus supernatant revealed that P. aeruginosa supernatant induces significant depletion of S. aureus intracellular ATP ( Fig 3B). S. aureus strain HG003 harboring plasmid PpflB::gfp was grown to mid-exponential phase and treated with supernatant from P. aeruginosa PAO1, PA14, CF isolates (blue) or burn isolates (green), for 30 min. OD 600 and gfp expression levels were determined after 16 h using a Biotek Synergy H1 microplate reader. (B) Intracellular ATP was measured after 1.5 h incubation with supernatant. ***p < 0.0005 (one-way ANOVA with Tukey's multiple comparison post-test). (C) S. aureus strain HG003 was grown to mid-exponential phase in MHB media and pre-treated with sterile supernatants from P. aeruginosa strains PA14 wild-type or its isogenic mutants or (D) physiologicallyrelevant concentrations of HQNO, PYO, or NaCN for 30 min prior to antibiotic challenge [26,27]. At indicated times, an aliquot was washed and plated to enumerate survivors. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. CF, cystic fibrosis; CFU, colony-forming units; GFP, green fluorescent protein; HQNO, 4-hydroxyquinoline N-oxide; MHB, Mueller-Hinton broth; NaCN, sodium cyanide; OD, optical density; PYO, pyocyanin. https://doi.org/10.1371/journal.pbio.2003981.g003 We found that mutation of pqsL (HQNO negative) drastically reduced the capacity of PA14 supernatant to protect S. aureus from ciprofloxacin killing, suggesting tolerance to ciprofloxacin killing is mediated by HQNO ( Fig 3C). Individually, mutations to the biosynthetic pathways for pyocyanin (phzS) and hydrogen cyanide (hcnC) had no influence on P. aeruginosaconferred protection from ciprofloxacin killing. However, supernatants from a ΔpqsLphzS and a respiratory toxin-null mutant (ΔpqsLphzShcnC) were further reduced in their capacity to protect S. aureus from ciprofloxacin killing (S2B Fig) (Fig 3C). Together, these data demonstrate that P. aeruginosa confers protection from ciprofloxacin killing to S. aureus through respiration inhibition and depletion of ATP. Further, treatment with HQNO, pyocyanin, and HCN at concentrations detected within the sputum of CF patients with active P. aeruginosa infection [26,27,37] induced tolerance of S. aureus to ciprofloxacin ( Fig 3D). Surprisingly, similar levels of tolerance were observed for other classes of antibiotics including tobramycin and vancomycin, with HQNO inducing the most robust tolerance to antibiotic killing (S2C and S2D Fig).

P. aeruginosa LasA endopeptidase potentiates vancomycin bactericidal activity against S. aureus
The presence of purified HQNO protects S. aureus from vancomycin killing (S2D Fig). However, P. aeruginosa supernatant from the majority of isolates tested significantly potentiated vancomycin killing of S. aureus (Fig 1C). We hypothesized that, similar to what was observed with S. aureus susceptibility to tobramycin, an additional factor present in P. aeruginosa supernatant is capable of overcoming the protective effects of HQNO to potentiate vancomycin killing of S. aureus. Heat denaturation of PAO1 supernatant completely abrogated the potentiating effect, suggesting the involvement of heat-labile extracellular protein(s) in the phenotype (Fig 4A). Bacteriolytic assays revealed that the PAO1 supernatant combined with vancomycininduced dramatic lysis of the population that was absent in the presence of either factor alone ( Fig 4B). This led us to examine the potential role of the P. aeruginosa extracellular lytic enzyme, LasA, in mediating vancomycin killing. LasA cleaves pentaglycine cross bridges in S. aureus peptidoglycan and has been shown to attack the cell wall of S. aureus during in vivo competition [18]. We examined the capacity of supernatant from a PAO1 lasA mutant to potentiate vancomycin killing. The lasA mutant supernatant did not potentiate killing by vancomycin compared to a 3-log reduction in S. aureus cfu in the presence of the PAO1 wild-type supernatant ( Fig 4A) (S4A Fig). Similar trends were observed in a S. aureus MRSA strain JE-2 (S6B Fig). As it was previously shown that S. aureus can degrade HQNO [38], the absence of which could result in a more dramatic LasA-dependent potentiation effect in our supernatant experiments, we examined vancomycin killing in a coculture model where P. aeruginosa is present to continually produce HQNO. Again, we found that the presence of wild-type PAO1 resulted in a 3-Log reduction in cfu following vancomycin challenge, and that this potentiation was not observed in the presence of a PAO1 lasA mutant, where we observed 100-fold more survivors at 24 h (S7 Fig). Next, we measured the levels of LasA in the supernatants of each clinical isolate via western blot and an additional assay developed previously to quantify LasA activity [39] (Fig 4C). Seven of the clinical isolates and both laboratory strains were positive for LasA. Of these, only BC253, the lowest LasA producer, and BC312, a high HQNO producer, did not induce at least a 10-fold increase in killing by vancomycin (Fig 1C). Of the 5 LasA negative strains, only one, BC251, significantly potentiated vancomycin killing, although no lysis of the culture was observed (S4B Fig). These data suggest that P. aeruginosa potentiates the vancomycin killing of S. aureus via at least 2 distinct mechanisms, only one of which is LasA-dependent. aureus HG003 was grown to mid-exponential phase and exposed to sterile supernatants for 30 min prior to addition of vancomycin 50 μg/ml. Where indicated, PAO1 supernatant was heat inactivated at 95˚C for 10 min. (A) At indicated times, an aliquot was removed, washed, and plated to enumerate survivors or (B) 100 μl cells were added to a 96-well plate and lysis was measured at OD 600 every hour for 16 h. (C) LasA present in the supernatant of P. aeruginosa PAO1, PA14, CF isolates (blue) or burn isolates (green) was quantified by western blot and the ability of each supernatant to lyse heat-killed S. aureus HG003 cells after 2 h. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. CF, cystic fibrosis.

P. aeruginosa potentiates vancomycin killing in a mouse model of P. aeruginosa/S. aureus coinfection
Our observation that purified HQNO induces multidrug tolerance in S. aureus agrees with recent findings that P. aeruginosa protects S. aureus biofilm from vancomycin killing [40]. However, we have demonstrated that under planktonic growth conditions the protective effects of P. aeruginosa HQNO on S. aureus vancomycin susceptibility (S2D Fig) can be overcome by the lytic activity of LasA to potentiate vancomycin killing. In order to determine whether the protective effects of HQNO or the potentiating effects of LasA predominated in vivo, we adapted a previously described murine model of burn injury for S. aureus and P. aeruginosa coinfection [41]. Briefly, groups of mice were inflicted with a 20% total body surface area burn, then after 24 h were infected subcutaneously at the wound site with approximately 10 5 CFU S. aureus, HG003 alone, or in combination with 10 3 PAO1 or 10 3 PAO1 lasA::tet. Mice were then treated daily with vancomycin and harvested 72h post infection.
LasA has been shown to mediate P. aeruginosa epithelial cell invasion and has been shown to be essential for corneal infections [42,43]. Interestingly, in our burn model, it appeared that the presence of PAO1 resulted in a higher burden of S. aureus, which is also dependent on lasA. However, for this study, we were interested solely on the impact of PAO1 presence on vancomycin sensitivity of S. aureus. While we observed no significant vancomycin efficacy in S. aureus mono-infected mice, relative to an untreated control group, we observed a 2-Log reduction in S. aureus burden following vancomycin treatment in mice coinfected with P. aeruginosa PAO1 (Fig 5A and 5B). Furthermore, no potentiation of vancomycin killing was observed in mice coinfected with the PAO1 lasA transposon mutant (Fig 5A and 5B). Importantly, P. aeruginosa appeared to be unaffected by vancomycin treatment, and burden was similar for both wild type and PAO1 lasA::tet-infected mice (S8 Fig). Finally, we observed that PAO1 transcription of lasA is strongly up-regulated (approximately 200-fold) during in vivo coinfection (Fig 5C). Up-regulation of lasA transcription was also observed during P. aeruginosa monoinfection, suggesting that lasA expression is independent of the presence or absence of S. aureus during burn wound infection.
Together, these data demonstrate that the presence of P. aeruginosa can potentiate vancomycin killing of S. aureus during infection through the production of LasA. To our knowledge, these data represent the first evidence of P. aeruginosa altering S. aureus antibiotic susceptibility in vivo and underlines the importance of deciphering interspecies interactions to improve the antibiotic treatment of polymicrobial infections.

Discussion
Polymicrobial infections are associated with exacerbated morbidity, accelerated disease progression and poor treatment outcome [44][45][46][47]. Antibiotic therapies are often selected to specifically target individual pathogens within a polymicrobial community without consideration of how interspecies interactions may alter a target organism's antibiotic susceptibility. S. aureus and P. aeruginosa are 2 major human pathogens that frequently coexist within chronically colonized patients, and these infections are often impossible to resolve through conventional antibiotic therapy. We find that P. aeruginosa dramatically alters the susceptibility of S. aureus to the killing activities of commonly used and clinically relevant antibiotics through 3 distinct pathways governed by rhamnolipids, HQNO, and LasA, and that these molecules are produced at different levels by P. aeruginosa clinical isolates resulting in vastly different impacts on antibiotic efficacy against S. aureus (Fig 6; summarized in S2 Table). We found that P. aeruginosa staphylolytic activity correlates with vancomycin potentiation, and that P. aeruginosa HQNO production correlates with ciprofloxacin antagonism (S9A and S9B Fig). Correlation analysis with rhamnolipid production is not appropriate as the measurement of biosurfactant activity is qualitative. Overall, our results imply that antibiotic efficacy is strongly influenced by interactions between bacterial species, which may have major implications for future susceptibility determination and antibiotic treatment of polymicrobial infection. Aminoglycosides are used routinely for the treatment of P. aeruginosa infection. Though aminoglycosides are effective against susceptible populations of S. aureus, bactericidal activity is limited against anaerobic, small colony variant (SCV), biofilm-associated, or persister subpopulations due in part to decreased respiration and thus PMF-dependent drug uptake [48,49]. Stimulating tobramycin uptake has been proposed as a way to eradicate these recalcitrant populations [49]. We have observed that P. aeruginosa-produced rhamnolipids sensitize the entire S. aureus population to tobramycin killing, leading to total eradication of otherwise tolerant populations. P. aeruginosa rhamnolipids may represent a promising new avenue for potentiating aminoglycoside killing of recalcitrant S. aureus and possibly other bacterial populations. Interestingly, a recent study has revealed that S. aureus increases tobramycin resistance in P. aeruginosa in an in vitro biofilm model, further emphasizing the importance of interaction between these organisms in dictating aminoglycoside susceptibility [50].
Vancomycin is a frontline antibiotic in the treatment of MRSA. To exert bactericidal activity against S. aureus, vancomycin must specifically bind the D-Ala-D-Ala residues of lipid II during cell wall biosynthesis [51]. However, vancomycin will also bind D-Ala-D-Ala residues of mature peptidoglycan. Thus, vancomycin exhibits limited bactericidal activity against dense populations of S. aureus cells due to the increased number of "decoy" targets available in late exponential or stationary phase populations of cells. Our data demonstrate that through LasA, P. aeruginosa can restore vancomycin efficacy against otherwise tolerant S. aureus populations. In support of this, we found that strains capable of increasing vancomycin lysis of S. aureus were LasA producers while the inert strains, generally, were not. This variance in LasA production may be due to mutations in lasR, an activator of lasA expression, which acquires mutations at high frequency during chronic P. aeruginosa infection [52]. We hypothesize that the combined action of cell wall degradation by LasA and inhibition of de novo peptidoglycan biosynthesis by vancomycin leads to cell wall destruction and a potent bactericidal effect.
P. aeruginosa HQNO induces tolerance of S. aureus to multiple antibiotic classes through respiration inhibition and depletion of intracellular ATP. Recent work by Orazi et al. found that in a bronchial epithelial tissue culture system, P. aeruginosa inhibited the killing activity of vancomycin through HQNO [40]. In agreement with these findings, we observed that the addition of exogenous HQNO protects S. aureus from vancomycin killing (S2D Fig) However, during planktonic growth the protective effect of HQNO was overshadowed by LasA-mediated potentiation of vancomycin killing. We were interested in examining whether P. aeruginosa antagonized or potentiated vancomycin in vivo, and found that P. aeruginosa expresses LasA at high levels during infection, and significantly potentiates the activity of vancomycin against S. aureus. This effect was abrogated in a P. aeruginosa lasA mutant suggesting that LasA plays a role in potentiating vancomycin killing of S. aureus during polymicrobial infection.
Similarly, we observed in vitro that P. aeruginosa-produced rhamnolipids can negate the protective effect of HQNO to restore or even increase S. aureus susceptibility to tobramycin killing. The opposing influences of HQNO and rhamnolipids on aminoglycoside activity against S. aureus may have major implications for aminoglycoside treatment of S. aureus during coinfection where production of one factor may dominate, leading to inhibition or potentiation of tobramycin activity against S. aureus. Indeed, we observed that clinical isolates produce a range of HQNO, LasA, and rhamnolipids, and production of each factor determines an isolate's ability to potentiate or antagonize antibiotic killing.
P. aeruginosa strain variation and the impact of this variation on S. aureus antibiotic susceptibility suggests that a personalized approach to antibiotic therapy may be necessary to identify the ideal therapy to eradicate infection in an individual patient based on the genotype of S. aureus and the genotype of the bacteria it's interacting with. Recent work has demonstrated that within the infectious environment, the production of HQNO, LasA, and rhamnolipids is highly variable. P. aeruginosa isolates from chronic CF infections frequently harbor mutations associated with decreased quorum-sensing activities and increased alginate production [52]. These mutations are attributed to the conversion to a mucoidal phenotype of P. aeruginosa that is significantly less competitive with S. aureus [53]. P. aeruginosa mucoidy is rarely associated with acute infection, thus the impact of P. aeruginosa on antibiotic susceptibility of S. aureus may differ during acute versus chronic coinfection. Future studies are necessary to identify genetic hallmarks of P. aeruginosa strains that potentiate or antagonize the activities of different antibiotic classes against S. aureus towards the goal of improving antibiotic efficacy against currently unresolvable coinfections.
It has long been observed that S. aureus is the dominant pathogen in the early life of CF patients with P. aeruginosa eventually dominating later in life [54]. It is interesting to consider a possible role of altered antibiotic susceptibility of S. aureus contributing to these dynamics, where vancomycin or tobramycin treatment in the presence of LasA-or rhamnolipid-producing P. aeruginosa strains may be particularly efficacious, resulting in a decrease in relative S. aureus abundance.
In summary, we characterized 3 distinct P. aeruginosa-mediated pathways altering S. aureus antibiotic susceptibility. HQNO induces a low-energy multidrug-tolerant state while LasA and rhamnolipids overcome this tolerance in cooperation with vancomycin and tobramycin, respectively. Exploitation of these newly discovered pathways may lead to better prediction of antibiotic efficacy in vivo and improved treatments for chronic S. aureus infection.

Ethics statement
P. aeruginosa CF isolates were provided by an IRB-approved biospecimen bank (IRB#02-0948). P. aeruginosa burn wound isolates were from a previous study and use was deemed exempt by IRB study number #17-0836. All mice used in the study were maintained under specific pathogen-free conditions in the Animal Association of Laboratory Animal Careaccredited University of North Carolina Department of Laboratory Animal Medicine Facilities. All protocols were approved by the Institutional Animal Care and Use Committee at the University of North Carolina, protocol number 17-141, and all experiments were performed in accordance with the National Institutes of Health. Animals were anesthetized by inhalation of vaporized isoflurane. A subcutaneous injection of morphine was given prior to burn injury for pain control, and an intraperitoneal injection of lactated Ringer's solution was given immediately after burn injury for fluid resuscitation. Animals were provided morphinated water ab libitum and monitored twice a day.

Bacterial strains and growth conditions
S. aureus strains HG003 and JE-2 were cultured aerobically in Mueller-Hinton broth (MHB) at 37˚C with shaking at 225 rpm. HG003 is a well-characterized model strain of S. aureus, while JE-2 is a well-characterized USA300 S. aureus associated with community-acquired MRSA infection. For anaerobic growth, overnight cultures were washed twice with PBS and diluted into 5 ml of pre-warmed (37˚C) TSB + 100 mM MOPS (pH7) to an OD 600 of 0.05. Cultures were prepared in triplicate in 16 x 150 mm glass tubes containing 1 mm stir bars. Following dilution, cultures were immediately transferred into a Coy anaerobic chamber and grown at 37˚C with stirring. P. aeruginosa strains were grown aerobically in MHB at 37˚C with shaking at 225 rpm. Burn wound isolates represent the first positive Pseudomonal wound cultures obtained from 5 unique patients admitted to the NC Jaycee Burn Center between Nov 2015 and April 2016 with a total body surface area burn ! 20% and/or inhalational injury after obtaining informed consent. CF isolates were collected from 5 patients at the UNC medical center. Isolates were cultured from sputum or bronchoalveolar lavage (BAL) from patients with CF after obtaining informed consent. All burn and CF isolates were grown on Pseudomonas isolation agar (BD Difco) and verified with 16S ribosomal sequencing using the primer pair 5'-AGTATTGAACTGAAGAGTTTGATCATGG-3' and 5'-CTGAGATCTTCGATTAAG GAGGTGA-3' for PCR amplification.

Strain construction
The PAO1 lasA mutant (PW4282 lasA-H03::ISlacZ/hah) was from the PAO1 knockout library [55]. PA14 deletion mutants were constructed by singly or sequentially deleting the coding sequences of rhlA, pqsL, phzS, and hcnC. Briefly, flanking primers were designed to anneal 800-1,200 bp upstream and downstream of the coding region. The resulting PCR product was inserted into plasmid pEX18Gm in accordance with the NEBuilder HiFi DNA Assembly protocol (New England Biolabs). Mutant alleles were integrated onto the chromosome of PA14 as described previously [56]. Briefly, pEX18Gm containing the inframe deletion and gene-specific flanking regions was mated into P. aeruginosa via Escherichia coli S17-λpir. Primary integrants were selected for with gentamycin and irgasan, then grown for 4 h in LB without selection to allow for recombination. Dilutions of P. aeruginosa were plated on LB containing 8% sucrose for counterselection (loss of plasmid). Deletion strains were confirmed through PCR and sequencing (Genewiz). Plasmid PpflB::gfp was constructed as follows, 298 bp upstream of the pflB coding region was amplified from HG003 genomic DNA using primers flanked with EcoRI and XbaI sites and cloned upstream of gfpuvr in plasmid pALC1434 [57].

Antibiotic survival assays
To prepare sterile supernatants, S. aureus and P. aeruginosa strains were grown in MHB at 37˚C with shaking at 225 rpm for approximately 20 h. The cultures were pelleted and supernatants were passed through a 0.2 μm filter. HG003 or JE-2 was grown to approximately 5 x 10 7 (for cell wall acting antibiotics) or approximately 2 x 10 8 cfu/ml (for all other antibiotics) in 3 ml MHB under aerobic or in 5 ml TSB + 100 mM MOPS under anaerobic conditions. Cells were pre-treated with 0.5 ml sterile supernatant (or 0.83 ml for anaerobic cultures) and returned to the incubator for a further 30 min. A 30-min pre-exposure was routinely used as we attempted to emulate the situation in vivo, where a population that has encountered and reacted to the relevant metabolites is subsequently exposed to antibiotic treatment. Where appropriate, cells were treated with 0.5 ml of P. aeruginosa culture taken directly from stationary phase (18 h) cultures in place of sterile supernatant. Coculture experiments were performed in the presence of 5% bovine serum albumin (BSA) to facilitate S. aureus/P. aeruginosa coexistence. An aliquot was plated to enumerate cfu before the addition of antibiotics. Antibiotics were added at concentrations similar to the Cmax in humans at recommended dosing; ciprofloxacin 2.34 μg/ml [58], tobramycin 58 μg/ml [59], vancomycin 50 μg/ml [60]. The Cmax of vancomycin is physiologically relevant for bacteremia and infections with good blood supply. The Cmax of vancomycin in serum is likely higher than that reached in the lung during IV infusion, however, it is certainly within the range experienced during inhaled therapy where clinical trials observed a Cmax of 270 μg/ml in sputum of CF patients. The Cmax of tobramycin is 58 μg/ml. Regarding lung concentrations, work by Ruddy et al. has found that inhaled tobramycin therapy results in sputum concentrations of between 17.2 and 327.3 μg/ ml. The concentration we use is well within this range. Also, this therapy fails to eradicate P. aeruginosa in vivo and thus, we believe, is physiologically relevant for this study [61]. The ciprofloxacin blood Cmax used in this study is 2.34 μg/ml. This is also well within the physiologically relevant concentration for lung infection where ciprofloxacin concentration is actually higher than corresponding blood serum levels [62].
Ciprofloxacin concentration was increased to 4.68 μg/ml when cells were grown in TSB + 100 mM MOPS to account for any decrease in pH where ciprofloxacin killing activity is reduced. At indicated times, an aliquot was removed and washed with 1% NaCl. Cells were serially diluted and plated to enumerate survivors. We routinely used 2 time points to enumerate survivors, 16 h and 24 h after antibiotic challenge as we previously found that, in S. aureus, susceptible cells are killed and a stable subpopulation of survivors emerges between 16 and 24 h of exposure to antibiotics [35,63]. Where indicated sterile supernatant was heat-inactivated at 95˚C for 10 min before addition to culture. Where indicated, pyocyanin 100 μM, HQNO 11.5 μM, sodium cyanide 150 μM, or rhamnolipids 10-50 μg/ml (50/50 mix of mono-and dirhamnolipids, Sigma) or L-rhamnose 10-50 μg/ml were added in place of supernatant. Concentrations of respiratory toxins represent levels detected in the sputum of CF patients [26,27].

Promoter induction measurement
S. aureus strain HG003 harboring gfp promoter plasmid PpflB::gfpwas grown to approximately 2 x 10 8 cfu/ml in 3 ml MHB containing chloramphenicol 10 μg/ml. Cultures were treated with 0.5 ml supernatant from HG003, PAO1, PA14, or P. aeruginosa clinical isolates as indicated. 200 μl culture was added to the wells of a clear bottom, black side 96-well plate. The plate was placed in a Biotek Synergy H1 microplate reader at 37˚C with shaking. Absorbance (OD 600 ) and GFP fluorescence (emission 528 nm and excitation 485 nm) were measured every 1 h for 16 h. GFP values were divided by OD 600 .

ATP assays
HG003 was grown to approximately 2 x 10 8 cfu/ml in 3 ml MHB and pre-treated with 0.5 ml sterile supernatant from S. aureus HG003 or P. aeruginosa PAO1 or PA14. ATP levels of the cultures were measured after 1.5 h, as described previously using a Promega BacTiter Glo kit according to the manufacturer's instructions [35]. P-values are indicated.

Vancomycin lysis assay
HG003 was grown to approximately 2 x 10 8 cfu/ml in 3 ml MHB and pre-treated with 0.5 ml sterile supernatant from S. aureus HG003, P. aeruginosa PAO1, PA14, or P. aeruginosa clinical isolates as indicated. Cells were incubated for a further 30 min before addition of vancomycin 50 μg/ml. 200 μl aliquots were added to the wells of a clear 96-well plate and placed in a Biotek Synergy H1 microplate reader. Absorbance (OD 600 ) was measured every 1 h for 16 h.
Western blot analysis of LasA P. aeruginosa strains were grown in MHB media for~20 h. Cultures were normalized to OD 600 2.0 and pelleted, and supernatants were passed through a 0.2 μm filter. Supernatants were boiled in SDS-sample buffer and run on a 4%-12% bis-tris acrylamide gel (Invitrogen). Protein was transferred onto a PVDF membrane, and LasA was detected using rabbit polyclonal anti-LasA antibodies (LifeSpan BioSciences, Inc.).

Staphylolytic assay
Staphylolytic assay was modified from Grande et al. [39]. Briefly, stationary phase S. aureus strain HG003 was heat killed at 95˚C for 20 min. Cells were pelleted and resuspended in 20 mM Tris-HCl (pH 8.0) at an OD 595 0.8-1. P. aeruginosa strains were cultured in MHB media for approximately 20 h. Cultures were normalized to OD 600 2.0, pelleted and supernatants were passed through a 0.2 μm filter. Seventeen microliters of sterile supernatant were added to a 100 μl heat-killed cells. OD 595 was measured at time 0 and after 2 h, and percent cell lysis was determined. The values shown represent the average of biological triplicates.

Tobramycin-Texas Red uptake
Tobramycin-Texas Red was made as described previously [64,65]. S. aureus strain HG003 was grown to mid-exponential phase and then incubated with or without 30 μg/ml rhamnolipids for 30 min. Cells were plated to enumerate cfu prior to addition of Texas-Red tobramycin at a final concentration of 58 μg/ml. After 1 h, an aliquot of cells was removed, washed twice in 1% NaCl, and plated to enumerate survivors. The remaining aliquot was analyzed for Texas Red uptake on a BD Fortessa flow cytometer. Thirty thousand events were recorded. Figures were generated using FSC Express 6 Flow.

Rhamnolipid quantification
P. aeruginosa rhamnolipid production was quantified utilizing a drop collapse assay, as previously described [33]. Briefly, clarified supernatants from overnight cultures of P. aeruginosa strains were serially diluted (1:1) with deionized water plus 0.005% crystal violet for visualization. Twenty-five microliters of aliquots of each dilution were spotted on to the underside of a petri dish plate and tilted to a 90˚angle. Surfactant scores represent the reciprocal of the highest dilution at which a collapsed drop migrated down the surface of the plate. Burn wound model of P. aeruginosa/S. aureus coinfection Animals were purchased from Taconic Farms and housed in specific pathogen-free conditions in the Animal Association of Laboratory Animal Care at the University of North Carolina's Department of Laboratory Animal Medicine Facilities. All protocols were approved by the Institutional Animal Care and Use Committee at the University of North Carolina, and all experiments were performed in accordance with the National Institutes of Health. Wild type C57BL/6 mice were burned and infected as previously described [41]. Briefly, a 65 g copper rod was heated to 100˚C and used to create a full-contact burn of approximately 20% of total body surface area through 4 applications of the rod to the anesthetized animal's dorsal region. In preparing the inoculum, overnight cultures of bacterial strains were subcultured into fresh MHB and grown for 2.5 h to mid-exponential phase. An aliquot of each culture was centrifuged and washed with 1 ml of PBS + 1% protease peptone. Approximate bacterial density was calculated by absorbance (OD 600 ), and cultures were diluted to obtain the desired concentration. Inoculum was verified through CFU enumeration. Mice were infected subcutaneously in the mid-dorsal region in unburned skin surrounded by the wound 2 4h after burn. Mice were administered vancomycin intraperitoneally at 110 mg/kg once daily for 2 d, then sacrificed 48 h post infection. At the time of sacrifice, 5 mm tissue biopsy of the bacterial infection site were aseptically removed, homogenized with 3.2 mm stainless steel beads and a Bullet Blender (Next Advance; Averill Park, NY), then bacterial burden was enumerated by plating serial dilutions of the homogenates on selective media. Mice that mostly cleared P. aeruginosa (<10 3 CFU/g tissue) were discounted from analysis.

MIC assays
MICs were determined using the microdilution method. Briefly, approximately 5 x 10 5 cfu were incubated with varying concentrations of ciprofloxacin, tobramycin, or vancomycin in a total volume of 200 μl MHB in a 96-well plate. Where indicated, 34 μl MHB was replaced with sterile P. aeruginosa or S. aureus supernatant or purified HQNO or rhamnolipids at final concentrations of 11.5 μM and 30 μg/ml, respectively. MICs were determined following incubation at 37˚C for 24 h.

Statistical analysis
Statistical data analysis was performed using Prism GraphPad software (San Diego, CA) version 5.0 b. Differences in S. aureus intracellular ATP concentration or surviving S. aureus CFU following P. aeruginosa supernatant treatment and antibiotic challenge were compared using a one-way ANOVA with Tukey's multiple comparisons post test or the Student t test where appropriate. Differences in tissue burden following S. aureus and P. aeruginosa coinfection were compared with a Mann-Whitney test. Finally, the statistical significance of each correlation analysis was determined with a two-tailed Pearson's chi-squared test. Differences with a p-value 0.05 were considered significant.
Supporting information S1  S. aureus strain HG003 was grown to mid-exponential phase in MHB media and pre-treated with (A,B) sterile supernatants from P. aeruginosa PA14 wild type or isogenic mutants, S. aureus HG003 or (D) L-rhamnose 10-50 μg/ml before addition of tobramycin at 58 μg/ml. Where indicated, PA14 supernatant was heat inactivated (PA14 HI) at 95˚C for 10 min. (C,D) Cultures were treated with exogenous rhamnolipids or L-rhamnose (10-50 μg/ml) in the absence of antibiotic. At indicated times, an aliquot was washed and plated to enumerate survivors. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. MHB, Mueller-Hinton broth. (TIF) S2 Fig. Pseudomonas-produced toxins inhibit respiration in S. aureus and induce antibiotic tolerance. (A) S. aureus HG003 was grown to mid-exponential phase in TSB + 100 mM MOPS in an anaerobic chamber and pre-treated with sterile supernatants from HG003 or PA14 for 30 min before addition of ciprofloxacin. HG003 was grown aerobically to mid-exponential phase in MHB media and pre-treated with (B) sterile supernatants from P. aeruginosa strains PA14 wild-type or its isogenic mutants or (C-D) physiologically relevant concentrations of HQNO, PYO, or NaCN for 30 min prior to antibiotic challenge. At indicated times, an aliquot was washed and plated to enumerate survivors. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. HQNO, 4-hydroxyquinoline N-oxide; MHB, Mueller-Hinton broth; MOPS, 3-(N-morpholino)propanesulfonic acid; NaCN, sodium cyanide; PYO, pyocyanin; TSB, tryptic soy broth. (TIF) S3 Fig. P. aeruginosa supernatant inhibits S. aureus aerobic respiration. (A-C) S. aureus strain HG003 harboring plasmid PpflB::gfpwas grown to mid-exponential phase and treated with supernatant from P. aeruginosa clinical isolates or laboratory strains. OD 600 and gfp expression levels were measured every 30 min for 16 h using a Biotek Synergy H1 microplate reader. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. (TIF) S4 Fig. P. aeruginosa endopeptidase LasA induces lysis in S. aureus. S. aureus HG003 was grown to mid-exponential phase and exposed to sterile supernatants indicated for 30 min prior to addition of vancomycin 50 μg/ml. (A) At indicated times, an aliquot was removed, washed, and plated to enumerate survivors. (B) At 24 h post antibiotic treatment, the turbidity of cultures treated with supernatant from HG003 (red), P. aeruginosa CF isolates (blue), or burn isolates (green) was measured by absorbance at OD 600 . Ã p < 0.05 by one-way ANOVA and Tukey's multiple comparisons post test. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. CF, cystic fibrosis. (TIF) S5 Fig. P. aeruginosa LasA lyses heat-killed S. aureus. (A,B) Heat killed S. aureus cells were incubated with supernatant from P. aeruginosa isolates in a 96-well plate. Lysis of S. aureus was monitored by measuring OD 595 every 5 min for 2 h. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. (TIF) S6 Fig. Exposure to P. aeruginosa supernatant alters MRSA antibiotic susceptibility. S. aureus strain JE-2 was grown to mid-exponential phase and exposed to sterile supernatants from P. aeruginosa for 30 mins prior to the addition of (A) tobramycin 58 μg/ml or (B) vancomycin 50 μg/ml. At indicated times, an aliquot was removed, washed and plated to enumerate survivors. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. MRSA, methicillin-resistant S. aureus. (TIF) S7 Fig. P. aeruginosa LasA potentiates vancomycin killing of S. aureus during P. aeruginosa/S. aureus coculture. S. aureus strain HG003 was grown to mid-exponential phase, exposed to 0.5 ml of stationary phase culture from P. aeruginosa strains PAO1 or PAO1 lasA::tet and 5% BSA for 30 mins prior to addition of vancomycin (50 μg/ml). At indicated times, an aliquot was removed, washed and plated on selective media to enumerate (A) S. aureus and (B) P. aeruginosa cells. All experiments were performed in biological triplicate. Underlying data can be found in S1 Data. Error bars represent mean ± sd. (TIF)