<i>Pseudomonas aeruginosa</i> exoproducts determine antibiotic efficacy against <i>Staphylococcus aureus</i>

<div><p>Chronic coinfections of <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i> frequently fail to respond to antibiotic treatment, leading to significant patient morbidity and mortality. Currently, the impact of interspecies interaction on <i>S</i>. <i>aureus</i> antibiotic susceptibility remains poorly understood. In this study, we utilize a panel of <i>P</i>. <i>aeruginosa</i> burn wound and cystic fibrosis (CF) lung isolates to demonstrate that <i>P</i>. <i>aeruginosa</i> alters <i>S</i>. <i>aureus</i> susceptibility to bactericidal antibiotics in a variable, strain-dependent manner and further identify 3 independent interactions responsible for antagonizing or potentiating antibiotic activity against <i>S</i>. <i>aureus</i>. We find that <i>P</i>. <i>aeruginosa</i> LasA endopeptidase potentiates lysis of <i>S</i>. <i>aureus</i> by vancomycin, rhamnolipids facilitate proton-motive force-independent tobramycin uptake, and 2-heptyl-4-hydroxyquinoline <i>N</i>-oxide (HQNO) induces multidrug tolerance in <i>S</i>. <i>aureus</i> 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 <i>S</i>. <i>aureus</i> antibiotic susceptibility. Furthermore, we demonstrate that vancomycin treatment of a <i>S</i>. <i>aureus</i> mouse burn infection is potentiated by the presence of a LasA-producing <i>P</i>. <i>aeruginosa</i> 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.</p></div

<i>P</i>. <i>aeruginosa</i>-mediated alteration of <i>S</i>. <i>aureus</i> antibiotic susceptibility.

<p><i>P</i>. <i>aeruginosa</i> exoproducts PYO, HQNO, and HCN inhibit <i>S</i>. <i>aureus</i> electron transport, leading to collapse of PMF and inhibition of the F₁F₀ ATPase leading to a decrease in <i>S</i>. <i>aureus</i> antibiotic susceptibility. Conversely, <i>P</i>. <i>aeruginosa</i> RLs intercalate into the plasma membrane-forming pores that permit aminoglycoside entry into the cell in a PMF-independent manner, while <i>P</i>. <i>aeruginosa</i> endopeptidase LasA cleaves pentaglycine crosslinks between peptidoglycan molecules of the cell wall, increasing vancomycin-mediated lysis of <i>S</i>. <i>aureus</i>. HCN, hydrogen cyanide; HQNO, 2-heptyl-4-hydroxyquinoline <i>N</i>-oxide; NAG, <i>N</i>-acetylglucosamine; NAM, <i>N</i>-acetylmuramic acid; PMF, proton-motive force; PYO, pyocyanin; RL, rhamnolipids.</p

<i>P</i>. <i>aeruginosa</i> secondary metabolites inhibit <i>S</i>. <i>aureus</i> aerobic respiration resulting in a drop in intracellular ATP and protection from ciprofloxacin killing.

<p>(A) <i>S</i>. <i>aureus</i> strain HG003 harboring plasmid P<i>pflB</i>∷<i>gfp</i> was grown to mid-exponential phase and treated with supernatant from <i>P</i>. <i>aeruginosa</i> PAO1, PA14, CF isolates (blue) or burn isolates (green), for 30 min. OD₆₀₀ and <i>gfp</i> 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. ***<i>p</i> < 0.0005 (one-way ANOVA with Tukey’s multiple comparison post-test). (C) <i>S</i>. <i>aureus</i> strain HG003 was grown to mid-exponential phase in MHB media and pre-treated with sterile supernatants from <i>P</i>. <i>aeruginosa</i> strains PA14 wild-type or its isogenic mutants or (D) physiologically-relevant concentrations of HQNO, PYO, or NaCN for 30 min prior to antibiotic challenge [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.ref026" target="_blank">26</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.ref027" target="_blank">27</a>]. 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.s003" target="_blank">S1 Data</a>. Error bars represent mean ± sd. CF, cystic fibrosis; CFU, colony-forming units; GFP, green fluorescent protein; HQNO, 4-hydroxyquinoline <i>N</i>-oxide; MHB, Mueller-Hinton broth; NaCN, sodium cyanide; OD, optical density; PYO, pyocyanin.</p

<i>P</i>. <i>aeruginosa</i> supernatant alters <i>S</i>. <i>aureus</i> antibiotic susceptibility.

<p><i>S</i>. <i>aureus</i> strain HG003 was grown to mid-exponential phase and exposed to sterile supernatants from <i>S</i>. <i>aureus</i> HG003 (red), <i>P</i>. <i>aeruginosa</i> laboratory strains PAO1 and PA14 (grey), <i>P</i>. <i>aeruginosa</i> CF clinical isolates (blue) or <i>P</i>. <i>aeruginosa</i> 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 <i>P</i>. <i>aeruginosa</i> supernatant was compared to the HG003 supernatant-treated control. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.s003" target="_blank">S1 Data</a>. *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.</p

<i>P</i>. <i>aeruginosa</i> supernatant potentiates killing by vancomycin via the LasA endopeptidase.

<p><i>S</i>. <i>aureus</i> 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 OD₆₀₀ every hour for 16 h. (C) LasA present in the supernatant of <i>P</i>. <i>aeruginosa</i> PAO1, PA14, CF isolates (blue) or burn isolates (green) was quantified by western blot and the ability of each supernatant to lyse heat-killed <i>S</i>. <i>aureus</i> HG003 cells after 2 h. All experiments were performed in biological triplicate. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003981#pbio.2003981.s003" target="_blank">S1 Data</a>. Error bars represent mean ± sd. CF, cystic fibrosis.</p

LC-MS/MS quantification of HQNO production in <i>P</i>. <i>aeruginosa</i> strains.

<p>LC-MS/MS quantification of HQNO production in <i>P</i>. <i>aeruginosa</i> strains.</p

CCP recipient and non-recipient characteristics.

CCP recipient and non-recipient characteristics.</p

Comparisons of recipient factors of time from admission to CCP infusion of EAP participants at UNCMC.

(A) those that were consented in English vs those that required use of a Spanish interpreter, (B) sex, (C) race, (D) ethnicity, (E) age, and (F) blood type. Medians are reported. P values obtained via a non-parametric Mann-Whitney U test. (TIF)</p

Proportions and cumulative titered units collected and transfused during the EAP era at UNCMC.

(A) Monthly proportions of low (gray) versus standard (blue) versus high (black) titered units collected. Percentages at tops of bars are cumulative proportions of standard + high titered units. (B) Monthly proportions of low (gray) versus standard (blue) versus high (black) titered units transfused. Percentages at tops of bars are cumulative proportions of standard + high titered units. (C) Cumulative total units collected and transfused over time, cumulative units with titers ≥1:160 collected and transfused over time, cumulative national supplier units transfused over time.</p

CCP recipient characteristics.

CCP recipient characteristics.</p