Harnessing Interspecies Antagonism to Enhance Antibiotic Efficacy

Beyond genetically encoded mechanisms of resistance, the factors that contribute to antibiotic treatment failure within the host are poorly understood. Traditional susceptibility assays fail to account for extrinsic determinants of antibiotic susceptibility present during infection and are therefore poor predictors of treatment outcome. To maximize the reach of current therapeutics, we must develop a more sophisticated understanding of antibiotic efficacy in the infection environment. Here we demonstrate that interspecies interactions between two important opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, alters S. aureus response to antibiotics. We show that the P. aeruginosa-produced endopeptidase LasA potentiates lysis of S. aureus by vancomycin, rhamnolipids facilitate proton-motive force-independent aminoglycoside uptake, and that small molecule 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) induces multidrug tolerance in S. aureus through respiratory inhibition and reduction of cellular ATP. We further demonstrate rhamnolipid-mediated potentiation of aminoglycoside uptake and killing of S. aureus restores susceptibility to otherwise tolerant persister, biofilm, small colony variant, anaerobic, and resistant S. aureus populations. Furthermore, bacterial pathogens that replicate within the intracellular niche are protected from antibiotics that cannot penetrate the eukaryotic membrane. Identifying and disrupting the pathways used by these pathogens to modify the intracellular niche in order to survive is an alternative strategy for limiting bacterial proliferation. Here, we use Francisella tularensis as a model intracellular bacterial pathogen to identify and describe the bacterial metabolic pathways and host-derived nutrients necessary for intracellular and in vivo growth. These findings reveal potential new therapeutic targets for disrupting bacterial nutrient acquisition that may be broadly applicable for treating other important intracellular pathogens. Overall, the findings presented here suggest that antibiotic susceptibility is contingent on a multitude of factors including interspecies interaction and the physiological replicative niche. Further elucidation of key antibiotic susceptibility determinants in vivo, as well as of strategies to overcome barriers to antibiotic efficacy may lead to a more holistic and personalized approach to therapy that will aid in the resolution of persistent infection.Doctor of Philosoph

To breathe or not to breathe?

Listeria monocytogenes uses respiration to sustain a risky fermentative lifestyle during infection

Defining the Metabolic Pathways and Host-Derived Carbon Substrates Required for Francisella tularensis Intracellular Growth

The widespread onset of antibiotic resistance prioritizes the need for novel antimicrobial strategies to prevent the spread of disease. With its low infectious dose, broad host range, and high rate of mortality, F. tularensis poses a severe risk to public health and is considered a potential agent for bioterrorism. F. tularensis reaches extreme densities within the host cell cytosol, often replicating 1,000-fold in a single cell within 24 hours. This remarkable rate of growth demonstrates that F. tularensis is adept at harvesting and utilizing host cell nutrients. However, like most intracellular pathogens, the types of nutrients utilized by F. tularensis and how they are acquired is not fully understood. Identifying the essential pathways for F. tularensis replication may reveal new therapeutic strategies for targeting this highly infectious pathogen and may provide insight for improved targeting of intracellular pathogens in general.Francisella tularensis is a Gram-negative, facultative, intracellular bacterial pathogen and one of the most virulent organisms known. A hallmark of F. tularensis pathogenesis is the bacterium’s ability to replicate to high densities within the cytoplasm of infected cells in over 250 known host species, including humans. This demonstrates that F. tularensis is adept at modulating its metabolism to fluctuating concentrations of host-derived nutrients. The precise metabolic pathways and nutrients utilized by F. tularensis during intracellular growth, however, are poorly understood. Here, we use systematic mutational analysis to identify the carbon catabolic pathways and host-derived nutrients required for F. tularensis intracellular replication. We demonstrate that the glycolytic enzyme phosphofructokinase (PfkA), and thus glycolysis, is dispensable for F. tularensis SchuS4 virulence, and we highlight the importance of the gluconeogenic enzyme fructose 1,6-bisphosphatase (GlpX). We found that the specific gluconeogenic enzymes that function upstream of GlpX varied based on infection model, indicating that F. tularensis alters its metabolic flux according to the nutrients available within its replicative niche. Despite this flexibility, we found that glutamate dehydrogenase (GdhA) and glycerol 3-phosphate (G3P) dehydrogenase (GlpA) are essential for F. tularensis intracellular replication in all infection models tested. Finally, we demonstrate that host cell lipolysis is required for F. tularensis intracellular proliferation, suggesting that host triglyceride stores represent a primary source of glycerol during intracellular replication. Altogether, the data presented here reveal common nutritional requirements for a bacterium that exhibits characteristic metabolic flexibility during infection

Stochastic Variation in Expression of the Tricarboxylic Acid Cycle Produces Persister Cells

Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now.Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation

<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> 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>-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> 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

<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