scholarly journals Pseudomonas aeruginosa Triggers Macrophage Autophagy To Escape Intracellular Killing by Activation of the NLRP3 Inflammasome

2015 ◽  
Vol 84 (1) ◽  
pp. 56-66 ◽  
Author(s):  
Qiuchan Deng ◽  
Yi Wang ◽  
Yuanqing Zhang ◽  
Meiyu Li ◽  
Dandan Li ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Immune Cells ◽  
Nlrp3 Inflammasome ◽  
Critical Event ◽  
Oxygen Species ◽  
Intracellular Killing ◽  
Bacterial Killing ◽  
Content Type ◽  
Human Macrophages

Assembly of the inflammasome has recently been identified to be a critical event in the initiation of inflammation. However, its role in bacterial killing remains unclear. Our study demonstrates thatPseudomonas aeruginosainfection induces the assembly of the NLRP3 inflammasome and the sequential secretion of caspase1 and interleukin-1β (IL-1β) in human macrophages. More importantly, activation of the NLRP3 inflammasome reduces the killing ofP. aeruginosain human macrophages, without affecting the generation of antimicrobial peptides, reactive oxygen species, and nitric oxide. In addition, our results demonstrate thatP. aeruginosainfection increases the amount of the LC3-II protein and triggers the formation of autophagosomes in human macrophages. TheP. aeruginosa-induced autophagy was enhanced by overexpression of NLRP3, ASC, or caspase1 but was reduced by knockdown of these core molecules of the NLRP3 inflammasome. Treatment with IL-1β enhanced autophagy in human macrophages. More importantly, IL-1β decreased the macrophage-mediated killing ofP. aeruginosa, whereas knockdown of ATG7 or Beclin1 restored the IL-1β-mediated suppression of bacterial killing. Collectively, our study explores a novel mechanism employed byP. aeruginosato escape from phagocyte killing and may provide a better understanding of the interaction betweenP. aeruginosaand host immune cells, including macrophages.

scholarly journals Antibacterial Action of Nitric Oxide-Releasing Chitosan Oligosaccharides against Pseudomonas aeruginosa under Aerobic and Anaerobic Conditions

2015 ◽  
Vol 59 (10) ◽  
pp. 6506-6513 ◽  
Author(s):  
Katelyn P. Reighard ◽  
Mark H. Schoenfisch
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Anaerobic Conditions ◽  
No Donor ◽  
Bacterial Killing ◽  
Content Type ◽  
Bacterial Physiology ◽  
Chitosan Oligosaccharides ◽  
Aerobic And Anaerobic ◽  
Aerobic And Anaerobic Conditions

ABSTRACTChitosan oligosaccharides were modified withN-diazeniumdiolates to yield biocompatible nitric oxide (NO) donor scaffolds. The minimum bactericidal concentrations and MICs of the NO donors againstPseudomonas aeruginosawere compared under aerobic and anaerobic conditions. Differential antibacterial activities were primarily the result of NO scavenging by oxygen under aerobic environments and not changes in bacterial physiology. Bacterial killing was also tested against nonmucoid and mucoid biofilms and compared to that of tobramycin. Smaller NO payloads were required to eradicateP. aeruginosabiofilms under anaerobic versus aerobic conditions. Under oxygen-free environments, the NO treatment was 10-fold more effective at killing biofilms than tobramycin. These results demonstrate the potential utility of NO-releasing chitosan oligosaccharides under both aerobic and anaerobic environments.

scholarly journals Activity of Meropenem-Vaborbactam againstPseudomonas aeruginosaandAcinetobacter baumanniiin a Neutropenic Mouse Thigh Infection Model

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Mojgan Sabet ◽  
Ziad Tarazi ◽  
David C. Griffith
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Clinical Isolates ◽  
Infection Model ◽  
Model Data ◽  
Bacterial Killing ◽  
Content Type

ABSTRACTWe have evaluated the activity of meropenem-vaborbactam against clinical isolates ofPseudomonas aeruginosaandAcinetobacter baumanniiin a neutropenic mouse thigh infection model. Data show that meropenem-vaborbactam regimens equivalent to 3-h infusions every 8 h with 2 g meropenem and 2 g vaborbactam produced bacterial killing against strains with MICs of 2 to 16 mg/liter and suggests that this combination may have utility in the treatment of infections caused byP. aeruginosaandA. baumannii.

scholarly journals Meropenem Combined with Ciprofloxacin Combats Hypermutable Pseudomonas aeruginosa from Respiratory Infections of Cystic Fibrosis Patients

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Vanessa E. Rees ◽  
Rajbharan Yadav ◽  
Kate E. Rogers ◽  
Jürgen B. Bulitta ◽  
Veronika Wirth ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Time Course ◽  
Respiratory Infections ◽  
Resistant Bacteria ◽  
Infection Model ◽  
Time Curve ◽  
Bacterial Killing ◽  
Content Type ◽  
Concentration Time

ABSTRACT Hypermutable Pseudomonas aeruginosa organisms are prevalent in chronic respiratory infections and have been associated with reduced lung function in cystic fibrosis (CF); these isolates can become resistant to all antibiotics in monotherapy. This study aimed to evaluate the time course of bacterial killing and resistance of meropenem and ciprofloxacin in combination against hypermutable and nonhypermutable P. aeruginosa. Static concentration time-kill experiments over 72 h assessed meropenem and ciprofloxacin in mono- and combination therapies against PAO1 (nonhypermutable), PAOΔmutS (hypermutable), and hypermutable isolates CW8, CW35, and CW44 obtained from CF patients with chronic respiratory infections. Meropenem (1 or 2 g every 8 h [q8h] as 3-h infusions and 3 g/day as a continuous infusion) and ciprofloxacin (400 mg q8h as 1-h infusions) in monotherapies and combinations were further evaluated in an 8-day hollow-fiber infection model study (HFIM) against CW44. Concentration-time profiles in lung epithelial lining fluid reflecting the pharmacokinetics in CF patients were simulated and counts of total and resistant bacteria determined. All data were analyzed by mechanism-based modeling (MBM). In the HFIM, all monotherapies resulted in rapid regrowth with resistance at 48 h. The maximum daily doses of 6 g meropenem (T>MIC of 80% to 88%) and 1.2 g ciprofloxacin (area under the concentration-time curve over 24 h in the steady state divided by the MIC [AUC/MIC], 176), both given intermittently, in monotherapy failed to suppress regrowth and resulted in substantial emergence of resistance (≥7.6 log10 CFU/ml resistant populations). The combination of these regimens achieved synergistic killing and suppressed resistance. MBM with subpopulation and mechanistic synergy yielded unbiased and precise curve fits. Thus, the combination of 6 g/day meropenem plus ciprofloxacin holds promise for future clinical evaluation against infections by susceptible hypermutable P. aeruginosa.

scholarly journals Effect of Nitroxides on Swarming Motility and Biofilm Formation, Multicellular Behaviors in Pseudomonas aeruginosa

2013 ◽  
Vol 57 (10) ◽  
pp. 4877-4881 ◽  
Author(s):  
César de la Fuente-Núñez ◽  
Fany Reffuveille ◽  
Kathryn E. Fairfull-Smith ◽  
Robert E. W. Hancock
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Wild Type ◽  
Planktonic Cells ◽  
Swarming Motility ◽  
Content Type ◽  
Exogenous Addition ◽  
Biofilm Dispersion ◽  
Dispersal Activity

ABSTRACTThe ability of nitric oxide (NO) to induce biofilm dispersion has been well established. Here, we investigated the effect of nitroxides (sterically hindered nitric oxide analogues) on biofilm formation and swarming motility inPseudomonas aeruginosa. A transposon mutant unable to produce nitric oxide endogenously (nirS) was deficient in swarming motility relative to the wild type and the complemented strain. Moreover, expression of thenirSgene was upregulated by 9.65-fold in wild-type swarming cells compared to planktonic cells. Wild-type swarming levels were substantially restored upon the exogenous addition of nitroxide containing compounds, a finding consistent with the hypothesis that NO is necessary for swarming motility. Here, we showed that nitroxides not only mimicked the dispersal activity of NO but also prevented biofilms from forming in flow cell chambers. In addition, anirStransposon mutant was deficient in biofilm formation relative to the wild type and the complemented strain, thus implicating NO in the formation of biofilms. Intriguingly, despite its stand-alone action in inhibiting biofilm formation and promoting dispersal, a nitroxide partially restored the ability of anirSmutant to form biofilms.

scholarly journals Staphylococcal DNA Repair Is Required for Infection

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Kam Pou Ha ◽  
Rebecca S. Clarke ◽  
Gyu-Lee Kim ◽  
Jane L. Brittan ◽  
Jessica E. Rowley ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Staphylococcus Aureus ◽  
Human Blood ◽  
Immune Cells ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Gram Positive ◽  
Content Type ◽  
Strand Breaks

ABSTRACT To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection. IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks in the staphylococcal DNA, which must be repaired by a two-protein complex known as RexAB; otherwise, the bacterium is killed, and it cannot sustain infection. This provides information on the type of damage that neutrophils cause S. aureus and the mechanism by which this damage is repaired, enabling infection.

scholarly journals Optimization of Synergistic Combination Regimens against Carbapenem- and Aminoglycoside-Resistant Clinical Pseudomonas aeruginosa Isolates via Mechanism-Based Pharmacokinetic/Pharmacodynamic Modeling

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Rajbharan Yadav ◽  
Jürgen B. Bulitta ◽  
Roger L. Nation ◽  
Cornelia B. Landersdorfer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
Viable Count ◽  
Resistant Isolate ◽  
Pharmacodynamic Modeling ◽  
Combination Regimen ◽  
Bacterial Killing ◽  
Content Type ◽  
Dosage Regimens ◽  
Combination Regimens

ABSTRACT Optimizing antibiotic combinations is promising to combat multidrug-resistant Pseudomonas aeruginosa. This study aimed to systematically evaluate synergistic bacterial killing and prevention of resistance by carbapenem and aminoglycoside combinations and to rationally optimize combination dosage regimens via a mechanism-based mathematical model (MBM). We studied monotherapies and combinations of imipenem with tobramycin or amikacin against three difficult-to-treat double-resistant clinical P. aeruginosa isolates. Viable-count profiles of total and resistant populations were quantified in 48-h static-concentration time-kill studies (inoculum, 107.5 CFU/ml). We rationally optimized combination dosage regimens via MBM and Monte Carlo simulations against isolate FADDI-PA088 (MIC of imipenem [MICimipenem] of 16 mg/liter and MICtobramycin of 32 mg/liter, i.e., both 98th percentiles according to the EUCAST database). Against this isolate, imipenem (1.5× MIC) combined with 1 to 2 mg/liter tobramycin (MIC, 32 mg/liter) or amikacin (MIC, 4 mg/liter) yielded ≥2-log10 more killing than the most active monotherapy at 48 h and prevented resistance. For all three strains, synergistic killing without resistance was achieved by ≥0.88× MICimipenem in combination with a median of 0.75× MICtobramycin (range, 0.032× to 2.0× MICtobramycin) or 0.50× MICamikacin (range, 0.25× to 0.50× MICamikacin). The MBM indicated that aminoglycosides significantly enhanced the imipenem target site concentration up to 3-fold; achieving 50% of this synergistic effect required aminoglycoside concentrations of 1.34 mg/liter (if the aminoglycoside MIC was 4 mg/liter) and 4.88 mg/liter (for MICs of 8 to 32 mg/liter). An optimized combination regimen (continuous infusion of imipenem at 5 g/day plus a 0.5-h infusion with 7 mg/kg of body weight tobramycin) was predicted to achieve >2.0-log10 killing and prevent regrowth at 48 h in 90.3% of patients (median bacterial killing, >4.0 log10 CFU/ml) against double-resistant isolate FADDI-PA088 and therefore was highly promising.

scholarly journals Cooperation between Reactive Oxygen and Nitrogen Intermediates in Killing of Rhodococcus equi by Activated Macrophages

2000 ◽  
Vol 68 (6) ◽  
pp. 3587-3593 ◽  
Author(s):  
Patricia A. Darrah ◽  
Mary K. Hondalus ◽  
Quiping Chen ◽  
Harry Ischiropoulos ◽  
David M. Mosser
Keyword(s):  
Nitric Oxide ◽  
Prolonged Exposure ◽  
Rhodococcus Equi ◽  
Activated Macrophages ◽  
Intracellular Killing ◽  
Oxygen Intermediates ◽  
Bacterial Killing ◽  
Ifn Γ

ABSTRACT Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-γ), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO−), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS−/−) or the oxidative burst (gp91 phox−/− ) are more susceptible to lethalR. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O2 −) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO− efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO− in situ. Inhibition of nitric oxide production by activated macrophages by usingN G-monomethyl-l-arginine blocks their production of ONOO− and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equiby IFN-γ-activated macrophages.

scholarly journals Interaction of Interferon Gamma-Induced Reactive Oxygen Species with Ceftazidime Leads to Synergistic Killing of Intracellular Burkholderia pseudomallei

2014 ◽  
Vol 58 (10) ◽  
pp. 5954-5963 ◽  
Author(s):  
Kara Mosovsky ◽  
Ediane Silva ◽  
Ryan Troyer ◽  
Katie Propst-Graham ◽  
Steven Dow
Keyword(s):  
Reactive Oxygen Species ◽  
Interferon Gamma ◽  
Burkholderia Pseudomallei ◽  
Actin Polymerization ◽  
Reactive Oxygen ◽  
Buthionine Sulfoximine ◽  
Oxygen Species ◽  
Intracellular Killing ◽  
Content Type ◽  
Ifn Γ

ABSTRACTBurkholderia pseudomallei, a facultative intracellular pathogen, causes severe infections and is inherently refractory to many antibiotics. Previous studies from our group have shown that interferon gamma (IFN-γ) interacts synergistically with the antibiotic ceftazidime to kill bacteria in infected macrophages. The present study aimed to identify the underlying mechanism of that interaction. We first showed that blocking reactive oxygen species (ROS) pathways reversed IFN-γ- and ceftazidime-mediated killing, which led to our hypothesis that IFN-γ-induced ROS interacted with ceftazidime to synergistically killBurkholderiabacteria. Consistent with this hypothesis, we also observed that buthionine sulfoximine (BSO), another inducer of ROS, could substitute for IFN-γ to similarly potentiate the effect of ceftazidime on intracellular killing. Next, we observed that IFN-γ induced ROS-mediated killing of intracellular but not extracellular bacteria. On the other hand, ceftazidime effectively reduced extracellular bacteria but was not capable of intracellular killing when applied at 10 μg/ml. We investigated the exact role of IFN-γ-induced ROS responses on intracellular bacteria and notably observed a lack of actin polymerization associated withBurkholderiabacteria in IFN-γ-treated macrophages, which led to our finding that IFN-γ-induced ROS blocks vacuolar escape. Based on these results, we propose a model in which synergistically reduced bacterial burden is achieved primarily through separate and compartmentalized killing: intracellular killing by IFN-γ-induced ROS responses and extracellular killing by ceftazidime. Our findings suggest a means of enhancing antibiotic activity againstBurkholderiabacteria through combination with drugs that induce ROS pathways or otherwise target intracellular spread and/or replication of bacteria.

scholarly journals Clinically Relevant Plasma Concentrations of Colistin in Combination with Imipenem Enhance Pharmacodynamic Activity against Multidrug-Resistant Pseudomonas aeruginosa at Multiple Inocula

2011 ◽  
Vol 55 (11) ◽  
pp. 5134-5142 ◽  
Author(s):  
Phillip J. Bergen ◽  
Alan Forrest ◽  
Jürgen B. Bulitta ◽  
Brian T. Tsuji ◽  
Hanna E. Sidjabat ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Therapy ◽  
Systematic Study ◽  
Plasma Concentrations ◽  
Multidrug Resistant ◽  
Bacterial Killing ◽  
Content Type ◽  
Resistant Strains ◽  
Time Kill

ABSTRACTThe use of combination antibiotic therapy may be beneficial against rapidly emerging resistance inPseudomonas aeruginosa. The aim of this study was to systematically investigatein vitrobacterial killing and resistance emergence with colistin alone and in combination with imipenem against multidrug-resistant (MDR)P. aeruginosa. Time-kill studies were conducted over 48 h using 5 clinical isolates and ATCC 27853 at two inocula (∼106and ∼108CFU/ml); MDR, non-MDR, and colistin-heteroresistant and -resistant strains were included. Nine colistin-imipenem combinations were investigated. Microbiological response was examined by log changes at 6, 24, and 48 h. Colistin combined with imipenem at clinically relevant concentrations increased the levels of killing of MDR and colistin-heteroresistant isolates at both inocula. Substantial improvements in activity with combinations were observed across 48 h with all colistin concentrations at the low inoculum and with colistin at 4× and 16× MIC (or 4 and 32 mg/liter) at the high inoculum. Combinations were additive or synergistic against imipenem-resistant isolates (MICs, 16 and 32 mg/liter) at the 106-CFU inoculum in 9, 11, and 12 of 18 cases (i.e., 9 combinations across 2 isolates) at 6, 24, and 48 h, respectively, and against the same isolates at the 108-CFU inoculum in 11, 7, and 8 cases, respectively. Against a colistin-resistant strain (MIC, 128 mg/liter), combinations were additive or synergistic in 9 and 8 of 9 cases at 24 h at the 106- and 108-CFU inocula, respectively, and in 5 and 7 cases at 48 h. This systematic study provides important information for optimization of colistin-imipenem combinations targeting both colistin-susceptible and colistin-resistant subpopulations.

scholarly journals A Small Molecule Deubiquitinase Inhibitor Increases Localization of Inducible Nitric Oxide Synthase to the Macrophage Phagosome and Enhances Bacterial Killing

2011 ◽  
Vol 79 (12) ◽  
pp. 4850-4857 ◽  
Author(s):  
Kristin M. Burkholder ◽  
Jeffrey W. Perry ◽  
Christiane E. Wobus ◽  
Nicholas J. Donato ◽  
Hollis D. Showalter ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Small Molecule ◽  
Inducible Nitric Oxide Synthase ◽  
Ubiquitin Ligases ◽  
Bacterial Killing ◽  
Content Type ◽  
Macrophage Response ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACTMacrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogenListeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with theListeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.

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