Murine Pneumonia Model Research Articles

Activation of inflammasome signaling mediates pathology of acute P. aeruginosa pneumonia

The respiratory tract is exceptionally well defended against infection from inhaled bacteria, with multiple proinflammatory signaling cascades recruiting phagocytes to clear airway pathogens. However, organisms that efficiently activate damaging innate immune responses, such as those mediated by the inflammasome and caspase-1, may cause pulmonary damage and interfere with bacterial clearance. The extracellular, opportunistic pathogen Pseudomonas aeruginosa expresses not only pathogen-associated molecular patterns that activate NF-κB signaling in epithelial and immune cells, but also flagella that activate the NLRC4 inflammasome. We demonstrate that induction of inflammasome signaling, ascribed primarily to the alveolar macrophage, impaired P. aeruginosa clearance and was associated with increased apoptosis/pyroptosis and mortality in a murine model of acute pneumonia. Strategies that limited inflammasome activation, including infection by fliC mutants, depletion of macrophages, deletion of NLRC4, reduction of IL-1β and IL-18 production, inhibition of caspase-1, and inhibition of downstream signaling in IL-1R- or IL-18R-null mice, all resulted in enhanced bacterial clearance and diminished pathology. These results demonstrate that the inflammasome provides a potential target to limit the pathological consequences of acute P. aeruginosa pulmonary infection.

Kinetic Characterization of PB1-F2-Mediated Immunopathology during Highly Pathogenic Avian H5N1 Influenza Virus Infection

The PB1-F2 protein encoded by influenza A viruses can contribute to virulence, a feature that is dependent of its sequence polymorphism. Whereas PB1-F2 from some H1N1 viruses were shown to exacerbate the inflammatory response within the airways, the contribution of PB1-F2 to highly pathogenic avian influenza virus (HPAIV) virulence in mammals remains poorly described. Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia. The mean time of death was 10 days for the two viruses, allowing us to perform global transcriptomic analyses and detailed histological investigations of the infected lungs at multiple time points. At day 2 post-infection (pi), while no histopathological lesion was observed, PB1-F2 expression resulted in a significant inhibition of cellular pathways involved in macrophage activation and in a transcriptomic signature suggesting that it promotes damage to the epithelial barrier. At day 4 pi, the gene profile associated with PB1-F2 expression revealed dysfunctions in NK cells activity. At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils. These transcriptomic data were fully supported by the histopathological analysis of the mice lungs which evidenced more severe inflammatory lesions and enhanced recruitment of neutrophils in the context of PB1-F2 expression, and thus provided a functional corroboration to the insight obtained in this work. In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.

Design, Synthesis, and Biological Evaluations of Novel 7-[7-Amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-8-methoxyquinolines with Potent Antibacterial Activity against Respiratory Pathogens

Novel 7-[7-amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]- 8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic acid 2a and 2b were designed and synthesized to obtain potent antibacterial drugs for the treatment of respiratory tract infections. Among these, compound 2a possessing (S)-configuration for the asymmetrical carbon on the pyrolidine moiety at the C-7 position of the quinolone scaffold exhibited potent in vitro antibacterial activity against respiratory pathogens including gram-positive (Streptococcus pneumoniae and Staphylococcus aureus), gram-negative (Haemophilus influenzae and Moraxcella catarrhalis), and atypical strains (Chalmydia pneumoniae and Mycoplasma pneumoniae), as well as multidrug-resistant Streptococcus pneumoniae and quinolone-resistant and methicillin-resistant Staphylococcus aureus). Furthermore, compound 2a showed excellent in vivo activity against the experimental murine pneumonia model due to multidrug resistant Streptococcus pneumoniae (MDRSP) and favorable profiles in preliminary toxicological and nonclinical pharmacokinetic studies.

Choline catabolism to glycine betaine contributes to Pseudomonas aeruginosa survival during murine lung infection.

Pseudomonas aeruginosa can acquire and metabolize a variety of molecules including choline, an abundant host-derived molecule. In P. aeruginosa, choline is oxidized to glycine betaine which can be used as an osmoprotectant, a sole source of carbon and nitrogen, and as an inducer of the virulence factor, hemolytic phospholipase C (PlcH) via the transcriptional regulator GbdR. The primary objective was to determine the contribution of choline conversion to glycine betaine to P. aeruginosa survival during mouse lung infection. A secondary objective was to gain insight into the relative contributions of the different roles of glycine betaine to P. aeruginosa survival during infection. Using a model of acute murine pneumonia, we determined that deletion of the choline oxidase system (encoded by betBA) decreased P. aeruginosa survival in the mouse lung. Deletion of the glycine betaine demethylase genes (gbcA-B), required for glycine betaine catabolism, did not impact P. aeruginosa survival in the lung. Thus, the defect of the betBA mutant was not due to a requirement for glycine betaine catabolism or dependence on a downstream metabolite. Deletion of betBA decreased the abundance of plcH transcript during infection, which suggested a role for PlcH in the betBA survival defect. To test the contribution of plcH to the betBA mutant phenotype a betBAplcHR double deletion mutant was generated. The betBA and betBAplcHR double mutant had a small but significant survival defect compared to the plcHR single mutant, suggesting that regulation of plcH expression is not the only role for glycine betaine during infection. The conclusion was that choline acquisition and its oxidation to glycine betaine contribute to P. aeruginosa survival in the mouse lung. While defective plcH induction can explain a portion of the betBA mutant phenotype, the exact mechanisms driving the betBA mutant survival defect remain unknown.

Dendritic Cells Are the Major Antigen Presenting Cells in Inflammatory Lesions of Murine Mycoplasma Respiratory Disease

Mycoplasmas cause chronic respiratory diseases in animals and humans, and to date, development of vaccines have been problematic. Using a murine model of mycoplasma pneumonia, lymphocyte responses, specifically T cells, were shown to confer protection as well as promote immunopathology in mycoplasma disease. Because T cells play such a critical role, it is important to define the role of antigen presenting cells (APC) as these cells may influence either exacerbation of mycoplasma disease pathogenesis or enhancement of protective immunity. The roles of APC, such as dendritic cells and/or macrophages, and their ability to modulate adaptive immunity in mycoplasma disease are currently unknown. Therefore, the purpose of this study was to identify individual pulmonary APC populations that may contribute to the activation of T cell responses during mycoplasma disease pathogenesis. The present study indeed demonstrates increasing numbers of CD11c− F4/80+ cells, which contain macrophages, and more mature/activated CD11c+ F4/80− cells, containing DC, in the lungs after infection. CD11c− F4/80+ macrophage-enriched cells and CD11c+ F4/80− dendritic cell-enriched populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC containing CD11c+ F4/80− cell populations from the lungs of infected mice were most capable of stimulating mycoplasma-specific CD4+ Th cell responses in vitro. In vivo, these CD11c+F4/80− cells were co-localized with CD4+ Th cells in inflammatory infiltrates in the lungs of mycoplasma-infected mice. Thus, CD11c+F4/80− dendritic cells appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these dendritic cells likely contribute to responses impacting disease pathogenesis.

Peroxisome proliferator-activated receptor agonist rosiglitazone improves host defense against Pseudomonas aeruginosa in a murine model of pneumonia

Peroxisome proliferator-activated receptor agonist rosiglitazone improves host defense against Pseudomonas aeruginosa in a murine model of pneumonia

Redox-Active Protein Thioredoxin-1 Administration Ameliorates Influenza A Virus (H1N1)-Induced Acute Lung Injury in Mice

Influenza virus infections can cause severe acute lung injury leading to significant morbidity and mortality. Thioredoxin-1 is a redox-active defensive protein induced in response to stress conditions. Animal experiments have revealed that thioredoxin-1 has protective effects against various severe disorders. This study was undertaken to evaluate the protective effects of recombinant human thioredoxin-1 administration on influenza A virus (H1N1)-induced acute lung injury in mice. Prospective animal trial. Research laboratory. Nine-week-old male C57BL/6 mice inoculated with H1N1. The mice were divided into a vehicle-treated group and recombinant human thioredoxin-1-treated group. For survival rate analysis, the vehicle or recombinant human thioredoxin-1 was administered intraperitoneally every second day from day -1 to day 13. For lung lavage and pathological analyses, vehicle or recombinant human thioredoxin-1 was administered intraperitoneally on days -1, 1, and 3. Lung lavage and pathological analyses were performed at 24, 72, and 120 hrs after inoculation. The recombinant human thioredoxin-1 treatment significantly improved the survival rate of H1N1-inoculated mice, although the treatment did not affect virus propagation in the lung. The treatment significantly attenuated the histological changes and neutrophil infiltration in the lung of H1N1-inoculated mice. The treatment significantly attenuated the production of tumor necrosis factor-α and chemokine (C-X-C motif) ligand 1 in the lung and oxidative stress enhancement, which were observed in H1N1-inoculated mice. H1N1 induced expressions of tumor necrosis factor-α and chemokine (C-X-C motif) ligand 1 in murine lung epithelial cells MLE-12, which were inhibited by the addition of recombinant human thioredoxin-1. The recombinant human thioredoxin-1 treatment started 30 mins after H1N1 inoculation also significantly improved the survival of the mice. Exogenous administration of recombinant human thioredoxin-1 significantly improved the survival rate and attenuated lung histological changes in the murine model of influenza pneumonia. The protective mechanism of thioredoxin-1 might be explained by its potent antioxidative and anti-inflammatory actions. Consequently, recombinant human thioredoxin-1 might be a possible pharmacological strategy for severe influenza virus infection in humans.

Memory CD4+ T Cells Are Required for Optimal NK Cell Effector Functions against the Opportunistic Fungal Pathogen Pneumocystis murina

Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4(+) T cell-depleted mice, there were significantly fewer NK cells in the lung tissue compared with CD4(+) T cell-intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, coincubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4(+) T cells compared with either cell alone, which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4(+) T cells are required for significant NK cell upregulation of the activation marker NK group 2D and production of IFN-γ, granzyme B, and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, as well as to establish a functional relationship between CD4(+) T cells and NK cells in the host response to an opportunistic fungal pathogen.

Assessment of Antimicrobial Combinations for Klebsiella pneumoniae Carbapenemase–Producing K. pneumoniae

The prevalence of bla(KPC) among gram-negative bacteria continues to increase worldwide. Limited treatment options exist for this multidrug-resistant phenotype, often necessitating combination therapy. We investigated the in vitro and in vivo efficacy of multiple antimicrobial combinations. Two clinical strains of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae were studied. The killing activities of six 2-agent combinations of amikacin, doripenem, levofloxacin, and rifampin were quantitatively assessed using a validated mathematical model. Combination time-kill studies were conducted using clinically relevant concentrations; observed bacterial burdens were modeled using 3-dimensional response surfaces. Selected combinations were further validated in a neutropenic murine pneumonia model, using human-like dosing exposures. The most enhanced killing effect in time-kill studies was seen with amikacin plus doripenem. Compared with placebo controls, this combination resulted in significant reduction of the bacterial burden in tissue at 24 hours, along with prolonged animal survival. In contrast, amikacin plus levofloxacin was found to be antagonistic in time-kill studies, showing inferior animal survival, as predicted. Our modeling approach appeared to be robust in assessing the effectiveness of various combinations for KPC-producing isolates. Amikacin plus doripenem was the most effective combination in both in vitro and in vivo infection models. Empirical selection of combinations against KPCs may result in antagonism and should be avoided.

High Mobility Group Box–1 Mediates Hyperoxia-Induced Impairment ofPseudomonas aeruginosaClearance and Inflammatory Lung Injury in Mice

Mechanical ventilation with supraphysiological concentrations of oxygen (hyperoxia) is routinely used to treat patients with respiratory distress. However, a significant number of patients on ventilators exhibit enhanced susceptibility to infections and develop ventilator-associated pneumonia (VAP). Pseudomonas aeruginosa (PA) is one of the most common species of bacteria found in these patients. Previously, we demonstrated that prolonged exposure to hyperoxia can compromise the ability of alveolar macrophages (AMs), an essential part of the innate immunity, to phagocytose PA. This study sought to investigate the potential molecular mechanisms underlying hyperoxia-compromised innate immunity against bacterial infection in a murine model of PA pneumonia. Here, we show that exposure to hyperoxia (≥ 99% O2) led to a significant elevation in concentrations of airway high mobility group box-1 (HMGB1) and increased mortality in C57BL/6 mice infected with PA. Treatment of these mice with a neutralizing anti-HMGB1 monoclonal antibody (mAb) resulted in a reduction in bacterial counts, injury, and numbers of neutrophils in the lungs, and an increase in leukocyte phagocytic activity compared with mice receiving control mAb. This improved phagocytic function was associated with reduced concentrations of airway HMGB1. The correlation between phagocytic activity and concentrations of extracellular HMGB1 was also observed in cultured macrophages. These results indicate a pathogenic role for HMGB1 in hyperoxia-induced impairment with regard to a host's ability to clear bacteria and inflammatory lung injury. Thus, HMGB1 may provide a novel molecular target for improving hyperoxia-compromised innate immunity in patients with VAP.

Editorial Commentary: Saving Lives With Optimal Antimicrobial Chemotherapy

The introduction of antimicrobial chemotherapy was an unprecedented advance in the practice of medicine. Previously fatal infections became treatable and antimicrobials were the agents of that salvage of life. In the more modern era, these agents also became the drugs that were permissive for many of the other modern medical miracles that we currently enjoy. The ability to treat serious infections in neutropenic cancer patients allowed the use of intensive oncologic chemotherapy. In the same vein, immunosuppressive therapy for organ and bone marrow transplants are made possible by antimicrobial therapy, as has the routine use of interventions that cross natural anatomic boundaries. In the early days of antimicrobial therapy, pioneers such as Harry Eagle recognized that certain administration profiles of drug prompted better therapeutic effect. This was demonstrated in a landmark paper published in the New England Journal of Medicine [1]. Certain agents such as penicillin had a better therapeutic effect when administered on very short administration intervals, whereas drugs such as the tetracyclines had antimicrobial effects that were somewhat independent of administration schedule. Unfortunately much of this information became lost in the 1960s and 1970s. In the late 1980s and early 1990s, these principles were rediscovered by the laboratory of William Craig [2–4]. These studies linked the effect of different antimicrobial classes, doses, and schedules to the reduction in colony-forming units (CFUs) in murine thigh or pneumonia models. Shortly thereafter, the burgeoning science of pharmacodynamics and pharmacometrics allowed identification of relationships between drug exposure indexed to the minimum inhibitory concentration (MIC) of the infecting pathogen and clinical and/ or microbiological outcomes [5–7]. The first study was a retrospective evaluation, but the last 2 were prospectively designed with analysis plans filed with the Food and Drug Administration (FDA). Such studies demonstrated conclusively that it was relatively straightforward to derive exposure-response relationships in the midst of clinical trials, employing a number of different mathematical techniques. The next step was to demonstrate the link between the animal model findings and the clinical trial pharmacodynamics relationships. Ambrose and colleagues [8] examined outcomes from clinical trials relative to the effect breakpoints determined from murine pharmacodynamic studies. They demonstrated a strong concordance between the preclinical and clinical pharmacodynamic studies for a number of different antimicrobial classes. Consequently, we can say that another brick was laid in the edifice of antimicrobial dynamics. While these data are convincing, it is also important to demonstrate that attainment of the “correct” antimicrobial targets has an impact on endpoints other than traditional clinical and microbiological outcomes in “real world” clinical practice settings. The clinical benefits of prolonged β-lactam infusion among critically ill patients were highlighted by the study performed at Albany Medical Center Hospital by Lodise and colleagues. Based on the results of a Monte Carlo simulation, prolonged infusion of piperacillin-tazobactam (3.375 g administered over a 4-hour period every 8 hours) was adopted as the standard hospital-wide piperacillin-tazobactam dosing scheme at their institution in February 2002. To evaluate the real-world effectiveness of this automatic dose substitution program, 14-day mortality and hospital length of stay after culture collection were Received 3 September 2012; accepted 11 September 2012; electronically published 16 October 2012. Correspondence: G. L. Drusano, MD, Professor and Director, Institute for Therapeutic Innovation, Department of Medicine, University of Florida, 6550 Sanger Boulevard, Lake Nona, FL, 32827 (gdrusano@ufl.edu). Clinical Infectious Diseases 2013;56(2):245–7 © The Author 2012. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals. permissions@oup.com. DOI: 10.1093/cid/cis863

Efficacy of Ceftaroline Fosamil in a Staphylococcal Murine Pneumonia Model

Ceftaroline fosamil is a cephalosporin with activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). The objective of this study was to characterize the dose-response relationship of ceftaroline fosamil against S. aureus in an immunocompromised murine pneumonia model, as well as to evaluate the efficacy of the humanized regimen of 600 mg intravenously (i.v.) every 12 h. Seventeen S. aureus (2 methicillin-susceptible Staphylococcus aureus [MSSA], 15 MRSA) isolates with ceftaroline MICs of 0.5 to 4 μg/ml were utilized. The pharmacokinetics of ceftaroline in serum and epithelial lining fluid (ELF) were evaluated to determine bronchopulmonary exposure profiles in infected and uninfected animals, using single and human-simulated doses. Serum fT>MIC (the percentage of time that free drug concentrations remain above the MIC) of 17% to 43% was required to produce a 1-log(10) kill in the dose-ranging studies. These targets were readily achieved with the humanized exposure profile, where decreases of 0.64 to 1.95 log(10) CFU were observed against 13 MRSA and both MSSA isolates tested. When taken as a composite, the fT>MICs required for stasis and a 1-log(10) kill were 16% and 41%, respectively. ELF concentrations were similar to serum concentrations across the dosing interval in infected and uninfected animals. The serum fT>MIC targets required in this lung infection model were similar to those observed with ceftaroline against S. aureus in a murine thigh infection model. Exposures simulating the human dose of 600 mg i.v. every 12 h achieved pharmacodynamic targets against MRSA and MSSA considered susceptible by current U.S. FDA breakpoints.

Comparative Pharmacodynamics of the New Oxazolidinone Tedizolid Phosphate and Linezolid in a Neutropenic Murine Staphylococcus aureus Pneumonia Model

Tedizolid phosphate (TR-701) is a novel oxazolidinone prodrug (converted to the active form tedizolid [TR-700]) with potent Staphylococcus aureus activity. The current studies characterized and compared the in vivo pharmacokinetic/pharmacodynamic (PD) characteristics of TR-701/TR-700 and linezolid against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in the neutropenic murine pneumonia model. The pharmacokinetic properties of both drugs were linear over a dose range of 0.625 to 40 mg/kg of body weight. Protein binding was 30% for linezolid and 85% for TR-700. Mice were infected with one of 11 isolates of S. aureus, including MSSA and community- and hospital-acquired MRSA strains. Each drug was administered by oral-gastric gavage every 12 h (q12h). The dosing regimens ranged from 1.25 to 80 mg/kg/12 h for linezolid and 0.625 to 160 mg/kg/12 h for TR-701. At the start of therapy, mice had 6.24 ± 0.40 log(10) CFU/lungs, which increased to 7.92 ± 1.02 log(10) CFU/lungs in untreated animals over a 24-h period. A sigmoid maximum-effect (E(max)) model was used to determine the antimicrobial exposure associated with net stasis (static dose [SD]) and 1-log-unit reduction in organism relative to the burden at the start of therapy. The static dose pharmacodynamic targets for linezolid and TR-700 were nearly identical, at a free drug (non-protein-bound) area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) of 19 and 20, respectively. The 1-log-unit kill endpoints were also similar, at 46.1 for linezolid and 34.6 for TR-700. The exposure targets were also comparable for both MSSA and MRSA isolates. These dosing goals support further clinical trial examination of TR-701 in MSSA and MRSA pneumonia.

Efficacy of ceftolozane in a murine model of Pseudomonas aeruginosa acute pneumonia: in vivo antimicrobial activity and impact on host inflammatory response

To assess the activity of ceftolozane, a novel oxyimino-cephalosporin, in comparison with ceftazidime and piperacillin/tazobactam against a multidrug-resistant Pseudomonas aeruginosa strain using a murine model of pneumonia. Quantitative bacteriology, survival, histological examination, myeloperoxidase activity, proinflammatory cytokine levels in lungs and endothelial permeability were evaluated to determine the effects of ceftolozane and comparators on P. aeruginosa-induced pneumonia. After 48 h of treatment, ceftolozane reduced the bacterial load by 3-4 log(10) cfu/g of lung. Systemic dissemination of the pulmonary infection and development of lung damage were inhibited in all β-lactam-treated animals. P. aeruginosa-induced pneumonia led to elevated concentrations of tumour necrosis factor-α, interleukin (IL)-1β and macrophage inflammatory protein (MIP)-2 in the lungs. While the levels of proinflammatory cytokines decreased following ceftazidime and piperacillin/tazobactam therapy, ceftolozane exhibited increased concentrations of IL-1β and MIP-2 after 24 h of infection, resulted in significantly increased levels of recruited neutrophils within the infected lung without increasing lung endothelial permeability. These data strongly support ceftolozane as an effective option for the treatment of severe P. aeruginosa respiratory infections by improving the early pulmonary inflammatory response without impairing 48 h post-infection homeostasis.

Factors Contributing to Epidemic MRSA Clones Replacement in a Hospital Setting

The mechanisms governing the epidemiology dynamics and success determinants of a specific healthcare-associated methicillin-resistant S. aureus (HA-MRSA) clone in hospital settings are still unclear. Important epidemiological changes have occurred in Europe since 2000 that have been related to the appearance of the ST22-IV clone. Between 2006 and 2010, we observed the establishment of the ST22-IV clone displacing the predominant Italian clone, ST228-I, in a large Italian university hospital. To investigate the factors associated with a successful spread of epidemic MRSA clones we studied the biofilm production, the competitive behavior in co-culture, the capacity of invasion of the A549 cells, and the susceptibility to infection in a murine model of acute pneumonia of the two major HA-MRSA clones, ST22-IV and ST228-I. We showed that persistence of ST22-IV is associated with its increased biofilm production and capacity to inhibit the growth of ST228-I in co-culture. Compared to ST228-I, ST22-IV had a significantly higher capacity to invade the A549 cells and a higher virulence in a murine model of acute lung infection causing severe inflammation and determining death in all the mice within 60 hours. On the contrary, ST228-I was associated with mice survival and clearance of the infection. ST22-IV, compared with ST228-I, caused a higher number of persistent, long lasting bacteremia. These data suggest that ST22-IV could have exploited its capacity to i) increase its biofilm production over time, ii) maintain its growth kinetics in the presence of a competitor and iii) be particularly invasive and virulent both in vitro and in vivo, to replace other well-established MRSA clones, becoming the predominant European clone.

ExoU Activates NF-κB and Increases IL-8/KC Secretion during Pseudomonas aeruginosa Infection

ExoU, a Pseudomonas aeruginosa cytotoxin injected into host cytosol by type III secretion system, exhibits a potent proinflammatory activity that leads to a marked recruitment of neutrophils to infected tissues. To evaluate the mechanisms that account for neutrophil infiltration, we investigated the effect of ExoU on IL-8 secretion and NF-κB activation. We demonstrate that ExoU increases IL-8 mRNA and protein levels in P. aeruginosa-infected epithelial and endothelial cell lines. Also, ExoU induces the nuclear translocation of p65/p50 NF-κB transactivator heterodimer as well as NF-κB-dependent transcriptional activity. ChIP assays clearly revealed that ExoU promotes p65 binding to NF-κB site in IL-8 promoter and the treatment of cultures with the NF-κB inhibitor Bay 11-7082 led to a significant reduction in IL-8 mRNA levels and protein secretion induced by ExoU. These results were corroborated in a murine model of pneumonia that revealed a significant reduction in KC secretion and neutrophil infiltration in bronchoalveolar lavage when mice were treated with Bay 11-7082 before infection with an ExoU-producing strain. In conclusion, our data demonstrate that ExoU activates NF-κB, stimulating IL-8 expression and secretion during P. aeruginosa infection, and unveils a new mechanism triggered by this important virulence factor to interfere in host signaling pathways.

Activity of Tedizolid Phosphate (TR-701) in Murine Models of Infection with Penicillin-Resistant and Penicillin-Sensitive Streptococcus pneumoniae

The in vitro activity of tedizolid (previously known as torezolid, TR-700) against penicillin-resistant Streptococcus pneumoniae (PRSP) clinical isolates and the in vivo efficacy of tedizolid phosphate (torezolid phosphate, TR-701) in murine models of PRSP systemic infection and penicillin-susceptible S. pneumoniae (PSSP) pneumonia were examined using linezolid as a comparator. The MIC(90) against 28 PRSP isolates was 0.25 μg/ml for tedizolid, whereas it was 1 μg/ml for linezolid. In mice infected systemically with a lethal inoculum of PRSP 1 h prior to a single administration of either antimicrobial, oral tedizolid phosphate was equipotent to linezolid (1 isolate) to 2-fold more potent than linezolid (3 isolates) for survival at day 7, with tedizolid phosphate 50% effective dose (ED(50)) values ranging from 3.19 to 11.53 mg/kg of body weight/day. In the PSSP pneumonia model, the ED(50) for survival at day 15 was 2.80 mg/kg/day for oral tedizolid phosphate, whereas it was 8.09 mg/kg/day for oral linezolid following 48 h of treatment with either agent. At equivalent doses (10 mg/kg once daily tedizolid phosphate or 5 mg/kg twice daily linezolid), pneumococcal titers in the lungs at 52 h postinfection were approximately 3 orders of magnitude lower with tedizolid phosphate treatment than with linezolid treatment or no treatment. Lung histopathology showed less inflammatory cell invasion into alveolar spaces in mice treated with tedizolid phosphate than in untreated or linezolid-treated mice. These results demonstrate that tedizolid phosphate is effective in murine models of PRSP systemic infection and PSSP pneumonia.

Pharmacokinetics and Pulmonary Disposition of Tedizolid and Linezolid in a Murine Pneumonia Model under Variable Conditions

In vivo pharmacokinetics are often evaluated in only one variation of an infection model, and the resulting exposures are assumed to be similar in each model. We evaluated and compared the effect of lung infection and immune status on the murine pharmacokinetics and pulmonary disposition of tedizolid and linezolid. Both factors resulted in differing blood and pulmonary exposure profiles, with similar trends for tedizolid and linezolid. These data highlight the importance of pharmacokinetic confirmation in each model.

In vivo efficacy and pharmacokinetics of biapenem in a murine model of ventilator-associated pneumonia with Pseudomonas aeruginosa

Biapenem (BIPM) has high bactericidal activity against Pseudomonas aeruginosa and similar activity in vitro as meropenem (MEPM). We used a murine model to examine the efficacy of biapenem against ventilator-associated pneumonia (VAP) caused by P. aeruginosa. Mice were treated by intraperitoneal injection with 100 mg/kg BIPM or MEPM every 12 h beginning 12 h after inoculation with P. aeruginosa. Survival was evaluated for 7 days, and 24 h after infection, lung histopathology was analyzed and the number of viable bacteria in the lungs and blood was counted. In addition, the pharmacokinetics of BIPM and MEPM were analyzed after the initial treatment. BIPM and MEPM significantly prolonged survival compared to control (P < 0.05). The lungs of mice treated with BIPM or MEPM had significantly fewer viable bacteria (3.54 ± 0.28 vs. 3.77 ± 0.14 log(10) CFU/ml) than in the lungs of control mice (6.65 ± 0.57 log(10) CFU/ml) (P < 0.05). Furthermore, viable bacteria were not detected in the blood of mice treated with BIPM or MEPM (control 2.85 ± 0.85 log(10) CFU/ml) (P < 0.05). Histopathological examination of lung specimens indicated that BIPM and MEPM prevent the progression of lung inflammation, including alveolar neutrophil infiltration and hemorrhage. The % time above MIC for BIPM and MEPM was 15.4% and 18.3% in plasma and 19.8% and 19.8% in lungs, respectively. These results show that BIPM and MEPM significantly prolongs survival and reduces the number of viable bacteria in a murine model of VAP caused by P. aeruginosa. Therefore, BIPM might be a potent and effective treatment for VAP caused by this bacterium.

Comparative evaluation of intratracheal colistimethate sodium, imipenem, and meropenem in BALB/c mice with carbapenem-resistant Acinetobacter baumannii pneumonia

The identification of the optimal agent for administration via the respiratory tract when treating pneumonia caused by carbapenem-resistant Acinetobacter baumannii (CRAB). A murine model of acute CRAB pneumonia was established by intratracheal (i.t.) inoculation with 2.5 × 10⁷ colony-forming units (CFU) of A. baumannii strain Ab396 plus 10% porcine mucin. After 4h the infected BALB/c mice were treated intratracheally with 25μl of either 0.85% saline (control group), colistimethate sodium (CMS) (166 666 U/kg, CMS group), imipenem/cilastatin (30/30 mg/kg, imipenem group), or meropenem (20mg/kg, meropenem group), every 8h. The therapeutic efficacy of these agents was examined. A. baumannii strain Ab396 was susceptible to CMS only. However, meropenem treatment did give a significantly superior survival rate (100%) compared to treatment with imipenem (50%), CMS (33%), or saline (0%) (p<0.001 vs. the control and CMS groups, p=0.006 vs. the imipenem group, by log-rank test). Furthermore, compared to the other groups, the meropenem group demonstrated significantly more favorable results in terms of tissue penetration of the antibiotic, bacterial clearance, normalization of the wet lung-to-body weight ratio, and down-regulation of pro-inflammatory cytokine levels in the lungs. Administration of meropenem via the respiratory tract proved to have the best therapeutic efficacy among the antibiotics tested when treating advanced murine CRAB pneumonia.