Murine Model Of Pulmonary Infection Research Articles

A chronic murine model of pulmonary Acinetobacter baumannii infection enabling the investigation of late virulence factors, long-term antibiotic treatments, and polymicrobial infections.

Acinetobacter baumannii can cause prolonged infections that disproportionately affect immunocompromised populations. Our understanding of A. baumannii respiratory pathogenesis relies on an acute murine infection model with limited clinical relevance that employs an unnaturally high number of bacteria and requires the assessment of bacterial load at 24-36 hours post-infection. Here, we demonstrate that low intranasal inoculums in immunocompromised mice with a tlr4 mutation leads to reduced inflammation, allowing for persistent infections lasting at least 3 weeks. Using this "chronic infection model," we determined the adhesin InvL is an imperative virulence factor required during later stages of infection, despite being dispensable in the early phase. We also demonstrate that the chronic model enables the distinction between antibiotics that, although initially reduce bacterial burden, either lead to complete clearance or result in the formation of bacterial persisters. To illustrate how our model can be applied to study polymicrobial infections, we inoculated mice with an active A. baumannii infection with Staphylococcus aureus or Klebsiella pneumoniae. We found that S. aureus exacerbates the infection, while K. pneumoniae enhances A. baumannii clearance. In all, the chronic model overcomes some limitations of the acute pulmonary model, expanding our capabilities to study of A. baumannii pathogenesis and lays the groundwork for the development of similar models for other important opportunistic pathogens.

Biomimetic protein structural transitions regulate activation and inhibition of the broad-spectrum bactericidal activity of cationic nanoparticles

The development of cationic polymers as alternative materials to antibiotics necessitates addressing the challenge of balancing their antimicrobial activity and toxicity. Here we propose a precise switching strategy inspired by biomimetic voltage-gated ion channels, enabling controlled activation and inhibition of cationic antimicrobial functions through protein conformational transitions in diverse physiological environments. Following thermodynamic studies on the specific recognition between mannose end groups on polycations and concanavalin A (ConA), we synthesized a type of ConA-polycation nanoparticle. The nanoparticle was inhibited under neutral conditions, with cationic moieties shielded by ConA's β-sheet. This shielding suppresses their antimicrobial activity, thereby ensuring satisfactory biocompatibility. In mildly acidic environments, however, the transition of a portion of ConA to an α-helix conformation exposed cations at the particle periphery, activating antibacterial functionality. Compared to inhibited nanoparticles, those in the activated state exhibited a 32–256 times reduction in the minimum bactericidal concentration against bacteria and fungi (2–16 µg/mL). In a murine acute pulmonary infection model, intravenous administration of inhibited nanoparticles effectively reduced bacterial counts by 4-log within 12 h. The biomimetic design, regulating cationic antimicrobial functionality through the alteration in protein secondary structure, significantly retards bacterial resistance development, holding great promise for intelligent antimicrobial materials. Statement of significanceCationic antimicrobial polymers exhibit advantages distinct from antibiotics due to their lower propensity for resistance development. However, the presence of cationic moieties also poses a threat to healthy cells and tissues, significantly constraining their potential for clinical applications. To address this challenge, we propose a biomimetic strategy that mimics voltage-gated ion channels to activate the antimicrobial functionality of cations selectively in bacterial environments through the conformational transitions of proteins between β–sheets and α–helices. In healthy tissues, the antimicrobial functionality is inhibited, ensuring satisfactory biocompatibility. Antimicrobial cationic materials capable of intelligent switching between an activated state and an inhibited state in response to environmental changes offer an effective strategy to prevent the development of resistance and mitigate potential side effects.

Murine Models of Cryptococcus Infection.

Cryptococcus is recognized as one of the emerging fungal pathogens that have major impact on diverse populations worldwide. Because of the high mortality rate and limited antifungal therapy options, there is an urgent need to understand the impact of dynamic processes between fungal pathogens and hosts that influence cryptococcal pathogenesis and disease outcomes. With known common limitations in human studies, experimental murine cryptococcosis models that can recapitulate human disease provide a valuable tool for studying fungal virulence and the host interaction, leading to development of better treatment strategies. Infection with Cryptococcus in mice via intranasal inhalation is mostly used because it is noninvasive and considered to be the most common mode of infection, strongly correlating with cryptococcal disease in humans. The protocols described in this article provide the procedures of establishing a murine model of Cryptococcus infection by intranasal inhalation and assessing the host immune response and disease progression during Cryptococcus infection. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Murine model of pulmonary cryptococcal infection via intranasal inhalation Basic Protocol 2: Assessment of the pulmonary immune response during Cryptococcus infection Support Protocol: Evaluation of pulmonary gene expression by real-time PCR Basic Protocol 3: Enumeration of survival rate and organ fungal burden.

The pathogenesis of experimental Emergomycosis in mice.

Emergomyces africanus is a recently identified thermally-dimorphic fungal pathogen that causes disseminated infection in people living with advanced HIV disease. Known as emergomycosis, this disseminated disease is associated with very high case fatality rates. Over the last decade, improved diagnostics and fungal identification in South Africa resulted in a dramatic increase in the number of reported cases. Although the true burden of disease is still unknown, emergomycosis is among the most frequently diagnosed dimorphic fungal infections in Southern Africa; and additional species in the genus have been identified on four continents. Little is known about the pathogenesis and the host's immune response to this emerging pathogen. Therefore, we established a murine model of pulmonary infection using a clinical isolate, E. africanus (CBS 136260). Both conidia and yeast forms caused pulmonary and disseminated infection in mice with organisms isolated in culture from lung, spleen, liver, and kidney. Wild-type C57BL/6 mice demonstrated a drop in body weight at two weeks post-infection, corresponding to a peak in fungal burden in the lung, spleen, liver, and kidney. An increase in pro-inflammatory cytokine production was detected in homogenized lung supernatants including IFN-γ, IL-1β, IL-6, IL12-p40 and IL-17 at three- and four-weeks post-infection. No significant differences in TNF, IL-12p70 and IL-10 were observed in wild-type mice between one and four-weeks post-infection. Rag-1-deficient mice, lacking mature T-and B-cells, had an increased fungal burden associated with reduced IFN-γ production. Together our data support a protective T-helper type-1 immune response to E. africanus infection. This may provide a possible explanation for the susceptibility of only a subset of people living with advanced HIV disease despite hypothesized widespread environmental exposure. In summary, we have established a novel murine model of E. africanus disease providing critical insights into the host immune components required for eliminating the infection.

The Aspergillus fumigatus UPR is variably activated across nutrient and host environments and is critical for the establishment of corneal infection.

The Aspergillus fumigatus unfolded protein response (UPR) is a two-component relay consisting of the ER-bound IreA protein, which splices and activates the mRNA of the transcription factor HacA. Spliced hacA accumulates under conditions of acute ER stress in vitro, and UPR null mutants are hypovirulent in a murine model of invasive pulmonary infection. In this report, we demonstrate that a hacA deletion mutant (ΔhacA) is furthermore avirulent in a model of fungal keratitis, a corneal infection, and an important cause of ocular morbidity and unilateral blindness worldwide. Interestingly, we demonstrate that A. fumigatus hacA is spliced in infected lung samples, but not in the cornea, suggesting the amount of ER stress experienced by the fungus varies upon the host niche. To better understand how the UPR contributes to fungal cell biology across a spectrum of ER-stress levels, we employed transcriptomics on the wild-type and ΔhacA strains in glucose minimal media (low stress), glucose minimal media with dithiothreitol (high stress), and gelatin minimal media as a proxy for the nutrient stress encountered in the cornea (mid-level stress). These data altogether reveal a unique HacA-dependent transcriptome under each condition, suggesting that HacA activity is finely-tuned and required for proper fungal adaptation in each environment. Taken together, our results indicate that the fungal UPR could serve as an important antifungal target in the setting of both invasive pulmonary and corneal infections.

The anti-inflammatory effect of tedizolid on carrageenan-induced footpad edema rat model

Tedizolid (TZD) is an oxazolidinone anti-methicillin-resistant Staphylococcus aureus (MRSA) drug. Linezolid (LZD), another oxazolidinone, has been shown to have an anti-inflammatory effect. TZD has been shown to exhibit an anti-inflammatory effect in a murine model of hematogenous pulmonary infection. In this study, we further investigated the anti-inflammatory effect of TZDs using a carrageenan-induced rat footpad edema model. TZD was administered at 0, 10, 20, and 40 mg/kg to the carrageenan-induced rat footpad edema model, and the edema rate was measured over time up to 9 h later. The area under the time curve of the edema rate profile (AUCedema0→9) decreased in a TZD dose-dependent manner. In addition, the correlation between AUCedema0→9 and the area under the time curve of free TZD plasma concentration (fAUCblood) obtained from the pharmacokinetic study of TZD in the carrageenan-induced rat footpad edema model was examined. fAUCblood and AUCedema0→9 showed a good negative correlation. These results indicate that TZD suppresses carrageenan-induced footpad edema and that TZD exerts its anti-inflammatory effects in a plasma concentration-dependent manner.

The circadian control of tryptophan metabolism regulates the host response to pulmonary fungal infections.

The environmental light/dark cycle has left its mark on the body's physiological functions to condition not only our inner biology, but also the interaction with external cues. In this scenario, the circadian regulation of the immune response has emerged as a critical factor in defining the host-pathogen interaction and the identification of the underlying circuitry represents a prerequisite for the development of circadian-based therapeutic strategies. The possibility to track down the circadian regulation of the immune response to a metabolic pathway would represent a unique opportunity in this direction. Herein, we show that the metabolism of the essential amino acid tryptophan, involved in the regulation of fundamental processes in mammals, is regulated in a circadian manner in both murine and human cells and in mouse tissues. By resorting to a murine model of pulmonary infection with the opportunistic fungus Aspergillus fumigatus, we showed that the circadian oscillation in the lung of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO)1, generating the immunoregulatory kynurenine, resulted in diurnal changes in the immune response and the outcome of fungal infection. In addition, the circadian regulation of IDO1 drives such diurnal changes in a pre-clinical model of cystic fibrosis (CF), an autosomal recessive disease characterized by progressive lung function decline and recurrent infections, thus acquiring considerable clinical relevance. Our results demonstrate that the circadian rhythm at the intersection between metabolism and immune response underlies the diurnal changes in host-fungal interaction, thus paving the way for a circadian-based antimicrobial therapy.

Tartrate resistant acid phosphatase 5 (TRAP5) mediates immune cell recruitment in a murine model of pulmonary bacterial infection.

During airway infection, upregulation of proinflammatory cytokines and subsequent immune cell recruitment is essential to mitigate bacterial infection. Conversely, during prolonged and non-resolving airway inflammation, neutrophils contribute to tissue damage and remodeling. This occurs during diseases including cystic fibrosis (CF) and COPD where bacterial pathogens, not least Pseudomonas aeruginosa, contribute to disease progression through long-lasting infections. Tartrate-resistant acid phosphatase (TRAP) 5 is a metalloenzyme expressed by alveolar macrophages and one of its target substrates is the phosphoglycoprotein osteopontin (OPN). We used a knockout mouse strain (Trap5-/-) and BALB/c-Tg (Rela-luc)31Xen mice paired with siRNA administration or functional protein add-back to elucidate the role of Trap5 during bacterial infection. In a series of experiments, Trap5-/- and wild-type control mice received intratracheal administration of P.aerugniosa (Xen41) or LPS, with mice monitored using intravital imaging (IVIS). In addition, multiplex cytokine immunoassays, flow cytometry, multispectral analyses, histological staining were performed. In this study, we found that Trap5-/- mice had impaired clearance of P. aeruginosa airway infection and reduced recruitment of immune cells (i.e. neutrophils and inflammatory macrophages). Trap5 knockdown using siRNA resulted in a decreased activation of the proinflammatory transcription factor NF-κB in reporter mice and a subsequent decrease of proinflammatory gene expression. Add-back experiments of enzymatically active TRAP5 to Trap5-/- mice restored immune cell recruitment and bacterial killing. In human CF lung tissue, TRAP5 of alveolar macrophages was detected in proximity to OPN to a higher degree than in normal lung tissue, indicating possible interactions. Taken together, the findings of this study suggest a key role for TRAP5 in modulating airway inflammation. This could have bearing in diseases such as CF and COPD where excessive neutrophilic inflammation could be targeted by pharmacological inhibitors of TRAP5.

Combining Gallium Protoporphyrin and Gallium Nitrate Enhances In Vitro and In Vivo Efficacy against Pseudomonas aeruginosa: Role of Inhibition of Bacterial Antioxidant Enzymes and Resultant Increase in Cytotoxic Reactive Oxygen Species.

Pseudomonas aeruginosa is a highly antibiotic-resistant opportunistic pathogenic bacteria that is responsible for thousands of deaths each year. Infections with P. aeruginosa disproportionately impact individuals with compromised immune systems as well as cystic fibrosis patients, where P. aeruginosa lung infection is a leading cause of morbidity and mortality. In previous work, we showed that a combination of gallium (Ga) nitrate and Ga protoporphyrin worked well in several bacterial infection models but its mechanism of action (MOA) is unknown. In the current work, we have investigated the MOA of Ga combination therapy in P. aeruginosa and its analysis in the in vivo model. In P. aeruginosa treated with Ga combination therapy, we saw a decrease in catalase and superoxide dismutase (SOD) activity, key antioxidant enzymes, which could correlate with a higher potential for oxidative stress. Consistent with this hypothesis, we found that, following combination therapy, P. aeruginosa demonstrated higher levels of reactive oxygen species, as measured using the redox-sensitive fluorescent probe, H2DCFDA. We also saw that the Ga combination therapy killed phagocytosed bacteria inside macrophages in vitro. The therapy with low dose was able to fully prevent mortality in a murine model of P. aeruginosa lung infection and also significantly reduced lung damage. These results support our previous data that Ga combination therapy acts synergistically to kill P. aeruginosa, and we now show that this may occur through increasing the organism's susceptibility to oxidative stress. Ga combination therapy also showed itself to be effective at treating infection in a murine pulmonary-infection model.

Patient-Derived Antibody Data Yields Development of Broadly Cross-Protective Monoclonal Antibody against ST258 Carbapenem-Resistant Klebsiella pneumoniae.

The most pressing challenge for the development of anti-capsular antibodies is maximizing coverage against the heterogenous capsular polysaccharide (CPS) of carbapenem-resistant Klebsiella pneumoniae (CR-Kp). So far, only CR-Kp with wzi154 CPS has been successfully targeted by antibodies. Here, we present murine antibody 24D11, which was developed by vaccinating mice with purified wzi50-type CPS. Cross-reactivity and protective efficacy of MAb 24D11 were confirmed against CR-Kp that express the 3 most prevalent CPS types (wzi29, wzi154, wzi50) using both in vitro and in vivo infection models. 24D11 induced complement-mediated and independent opsonophagocytosis in macrophages as well as killing of all CR-Kp strains in whole blood cells derived from healthy donors. In a murine intratracheal infection model, 24D11 reduced lung burden and dissemination of CR-Kp strains when administered 4 h pre- or postinfection. The protective efficacy of 24D11 remained effective in neutropenic mice. This is the first antibody which exhibits cross-protective efficacy against clade 1 and 2 ST258 CR-Kp strains. It overcomes a major barrier to successfully target wzi29, a major CPS expressed by ST258 CR-Kp. The finding that 24D11 also exhibits potent protective efficacy against wzi154 CR-Kp strains highlights its high potential as a lead agent for the development of broadly active immunotherapy. IMPORTANCE Here, we present in vitro and in vivo data for the wzi50 CPS-specific monoclonal antibody MAb 24D11, demonstrating its cross-protective efficacy against three prominent win types (wzi29, wzi154, and wzi50) of the carbapenem-resistant clonal group CG258. In a murine pulmonary infection model, MAb 24D11 reduced bacterial lung burden and dissemination to other organs even if administered 4 h postinfection. Its protective efficacy was also observed in neutropenic mice, which highlights its potential value in clinical settings where oncology patients with CG258 infections may also be neutropenic.

An ionophore breaks the multi-drug-resistance of Acinetobacter baumannii.

Within intensive care units, multi-drug resistant Acinetobacter baumannii outbreaks are a frequent cause of ventilator-associated pneumonia. During the on-going COVID-19 pandemic, patients who receive ventilator support experience a 2-fold increased risk of mortality when they contract a secondary A. baumannii pulmonary infection. In our recent paper (De Oliveira et al. (2022), Mbio, doi: 10.1128/mbio.03517-21), we demonstrate that the 8-hydroxquinoline ionophore, PBT2 breaks the resistance of A. baumannii to tetracycline class antibiotics. In vitro, the combination of PBT2 and zinc with either tetracycline, doxycycline, or tigecycline was shown to be bactericidal against multi-drug-resistant A. baumannii, and any resistance that did arise imposed a fitness cost. Using a murine model of pulmonary infection, treatment with PBT2 in combination with tetracycline or tigecycline proved efficacious against multidrug-resistant A. baumannii. These findings suggest that PBT2 may find utility as a resistance breaker to rescue the efficacy of tetracycline-class antibiotics commonly employed to treat multi-drug resistant A. baumannii infections.

Human iPSC-derived macrophages for efficient Staphylococcus aureus clearance in a murine pulmonary infection model

AbstractPrimary or secondary immunodeficiencies are characterized by disruption of cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients, with Staphylococcus aureus being a common offending organism. We propose here an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S aureus. Our studies, using human-induced pluripotent stem cell-derived macrophages (iMφs), demonstrate efficient antimicrobial potential against methicillin-sensitive and methicillin-resistant clinical isolates of S aureus. Using an S aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφs is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφs compared with monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours postinfection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S aureus infections and offer an alternative treatment strategy for immunodeficient or immunocompromised patients.

Fine Particulate Matter (PM2.5) Promotes CD146 Expression in Alveolar Epithelial Cells and Cryptococcus neoformans Pulmonary Infection.

Air pollution is a leading cause of increasing infectious lung diseases. Pulmonary cryptococcosis is a fatal fungal pneumonia in acquired immunodeficiency syndrome patients. In some cases, the pathogen Cryptococcus neoformans also develops dormant nodules in immunocompetent individuals. In the present study, we demonstrated that fine particulate matter (PM2.5) increased CD146 expression in alveolar epithelial cells and promoted C. neoformans pulmonary infection. Aryl hydrocarbon receptor (AhR) signaling was required for increased expression of CD146 in epithelial cells treated with PM2.5. In a murine model of pulmonary infection, PM2.5 promoted fungal infection, and CD146 deficiency decreased the fugal burden of C. neoformans. Our study may highlight the importance of air pollution to lung mycosis and CD146 as a target for preventing infectious lung diseases.

A Clofazimine-Containing Regimen Confers Improved Treatment Outcomes in Macrophages and in a Murine Model of Chronic Progressive Pulmonary Infection Caused by the Mycobacterium avium Complex.

Treatment outcomes using the standard regimen (a macrolide, ethambutol, and rifampicin) for Mycobacterium avium complex-pulmonary disease (MAC-PD) remain unsatisfactory. Thus, improved treatment regimens for MAC-PD are required. Clofazimine has recently been revisited as an effective drug against mycobacterial infection. We performed a comparison between the standard regimen and an alternative regimen (replacing the rifampicin of the standard regimen with clofazimine) based on the intracellular anti-MAC activities of the individual drugs in a murine model of chronic progressive MAC-pulmonary infection (MAC-PI). The intracellular anti-MAC activities of the individual drugs and their combinations in murine bone marrow-derived macrophages (BMDMs) were determined. The treatment efficacies of the standard and clofazimine-containing regimens were evaluated in mice chronically infected with M. avium by initiating 2- and 4-week treatment at 8 weeks post-infection. Bacterial loads in the lung, spleen, and liver were assessed along with lung inflammation. Insufficient intracellular anti-MAC activity of rifampicin in BMDMs was recorded despite its low in vitro minimum inhibitory concentrations (MICs), whereas optimal intracellular killing activity against all tested MAC strains was achieved with clofazimine. Compared to the standard regimen, the clofazimine-containing regimen significantly reduced CFUs in all organs and achieved marked reductions in lung inflammation. The replacement of rifampicin with clofazimine in the treatment regimen resulted in more favorable outcomes in an animal model of chronic progressive MAC-PI. Intriguingly, 2 weeks of treatment with the clofazimine-containing regimen reduced bacterial loads more effectively than 4 weeks of treatment with the standard regimen in M. avium-infected mice. Thus, the clofazimine-containing regimen also had a treatment-shortening effect.

Azithromycin potentiates avian IgY effect against Pseudomonas aeruginosa in a murine pulmonary infection model

Cystic fibrosis (CF) patients are at risk of acquiring chronic Pseudomonas aeruginosa lung infections. The biofilm mode of growth of P. aeruginosa induces tolerance to antibiotics and the host response; accordingly, treatment failure occurs. Supplemental azithromycin has proven beneficial in CF owing to potential immunomodulatory mechanisms. Clinical studies have demonstrated a reduction in exacerbations in CF patients by avian IgY anti-Pseudomonas immunotherapy. We hypothesise that azithromycin pre-treatment could potentiate the observed anti-Pseudomonas effect of IgY opsonisation in vivo. Evaluation of phagocytic cell capacity was performed using in vitro exposure of azithromycin pre-treated human polymorphonuclear neutrophils to IgY opsonised P. aeruginosa PAO3. A murine lung infection model using nasal planktonic P. aeruginosa inoculation and successive evaluation 24 h post-infection was used to determine lung bacteriology and subsequent pulmonary inflammation. Combined azithromycin treatment and IgY opsonisation significantly increased bacterial killing compared with the two single-treated groups and controls. In vivo, significantly increased bacterial pulmonary elimination was revealed by combining azithromycin and IgY. A reduction in the inflammatory markers mobiliser granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein 2 (MIP-2) and interleukin 1 beta (IL-1β) paralleled this effect. Combination of azithromycin and anti-Pseudomonas IgY potentiated the killing and pulmonary elimination of P. aeruginosa in vitro and in vivo. The augmented effect of combinatory treatment with azithromycin and IgY constitutes a potential clinical application for improving anti-Pseudomonas strategies.

Gallium Porphyrin and Gallium Nitrate Synergistically Inhibit Mycobacterial Species by Targeting Different Aspects of Iron/Heme Metabolism.

There is an urgent need for new effective and safe antibiotics active against pathogenic mycobacterial species. Gallium (Ga) nitrate (Ga(NO3)3) and Ga porphyrin (GaPP) have each been shown to inhibit the growth of a variety of mycobacterial species. The Ga(III) ion derived from Ga(NO3)3 has the potential to disrupt the mycobacterial Fe(III) uptake mechanisms and utilization, including replacing iron (Fe) in the active site of enzymes, resulting in the disruption of function. Similarly, noniron metalloporphyrins such as heme mimetics, which can be transported across the bacterial membrane via heme-uptake pathways, would potentially block the acquisition of iron-containing heme and bind to heme-utilizing proteins, making them nonfunctional. Given that they likely act on different aspects of mycobacterial Fe metabolism, the efficacy of combining Ga(NO3)3 and GaPP was studied in vitro against Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium tuberculosis (M. tb). The combination was then assessed in vivo in a murine pulmonary infection model of M. abscessus. We observed that Ga(NO3)3 in combination with GaPP exhibited synergistic inhibitory activity against the growth of M. avium, M. tb, and M. abscessus, being most active against M. abscessus. Activity assays indicated that Ga(NO3)3 and GaPP inhibited both catalase and aconitase at high concentrations. However, the combination showed a synergistic effect on the aconitase activity of M. abscessus. The Ga(NO3)3/GaPP combination via intranasal administration showed significant antimicrobial activity in mice infected with M. abscessus. M. abscessus CFU from the lungs of the Ga(NO3)3/GaPP-treated mice was significantly less compared to that of nontreated or single Ga(III)-treated mice. These findings suggest that combinations of different Ga(III) compounds can synergistically target multiple iron/heme-utilizing mycobacterial enzymes. The results support the potential of combination Ga therapy for development against mycobacterial pathogens.

Production of neutrophil extracellular traps (NETs) in response to Scedosporium apiospermum in a murine model of pulmonary infection

Scedosporium apiospermum is an opportunistic emerging pathogen that can develop in both immunosuppressive and immunocompetent patients with pulmonary infections. Neutrophils are recognized as critical cells in the early response to a fungal infection through different mechanisms that eliminate or control the infection such as neutrophil extracellular traps (NETs). In this work, we investigate the presence of NETs in the lung tissue of immunocompetent mice infected with Scedosporium apiospermum. In the histopathological study the presence of filamentous basophilic material with hematoxylin-eosin and periodic acid Schiff stains suggestive of extracellular DNA was observed. We demonstrated the presence of NETs by immunofluorescence staining of extracellular DNA, myeloperoxidase, and elastase in lung tissue. Our results showed that on days 1 and 3 post-infection extracellular DNA, myeloperoxidase, and elastase correlate with areas of high concentration of cell infiltrates and fungal structures. The observation of fungal structures in the tissue decreased as did the presence of NETs by day 5 post-infection. We suggest that NETs release may play an important role in the early containment of Scedosporium apiospermum lung infection.

Development of an immunocompetent murine model of pulmonary infection due to Scedosporium apiospermum

A pulmonary infection model due to Scedosporium apiospermum in immunocompetent mice was developed. BALB/c mice were infected by endotracheal intubation with 5 × 106 conidia/mouse and disease progression was evaluated on days 1, 3, 5, 7, 11, 16, 21, 30, 50 and 60 post-infection through quantitative culture and histopathological analysis of lungs, livers, spleens, brains, and kidneys. There was no extrapulmonary dissemination during the study nor shown to be a lethal infection. The fungal burden in lungs was maintained from day 1–5 and gradually decreased by day 30 post-challenge. On day 60, 30% of mice showed complete elimination of the fungus. Severe alterations in the lung tissue were observed, as well as the presence of conidia and hyphae surrounded by a cellular infiltrate composed mainly of neutrophils in the first days of the infection. The elimination of fungal cells and normal tissue morphology were recovered throughout the study.

The small quinolone derived compound HT61 enhances the effect of tobramycin against Pseudomonas aeruginosa in vitro and in vivo

HT61 is a small quinolone-derived compound previously demonstrated to exhibit bactericidal activity against gram-positive bacteria including methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). When combined with the classical antibiotics and antiseptics neomycin, gentamicin, mupirocin and chlorhexidine, HT61 demonstrated synergistic bactericidal activity against both MSSA and MRSA infections in vitro. In this study, we investigated the individual antimicrobial activity of HT61 alongside its capability to potentiate the efficacy of tobramycin against both a tobramycin sensitive laboratory reference strain (PAO1) and tobramycin resistant clinical isolates (RP73, NN2) of the gram-negative bacteria Pseudomonas aeruginosa (P. aeruginosa). Using broth microdilution methods, the MICs of HT61 were assessed against all strains, as well as the effect of HT61 in combination with tobramycin using both the chequerboard method and bacterial time-kill assays. A murine model of pulmonary infection was also used to evaluate the combination therapy of tobramycin and HT61 in vivo. In these studies, we demonstrated significant synergism between HT61 and tobramycin against the tobramycin resistant P. aeruginosa strains RP73 and NN2, whilst an additive/intermediate effect was observed for P. aeruginosa strain PA01 which was further confirmed using bacterial time kill analysis. In addition, the enhancement of tobramycin by HT61 was also evident in in vitro assays of biofilm eradication. Finally, in vivo studies revealed analogous effects to those observed in vitro with HT61 significantly reducing bacterial load when administered in combination with tobramycin against each of the three P. aeruginosa strains at the highest tested dose (10 mg/kg).

Quercetin, a pneumolysin inhibitor, protects mice against Streptococcus pneumoniae infection

Pneumolysin (PLY), a pore-forming cytotoxin and a major virulence determinant, is a member of the cholesterol-dependent cytolysin (CDC) family and essential for promoting Streptococcus pneumoniae (S.pneumoniae) infection. Due to the action characteristics of hemolysin itself, the pneumolysin released after killing bacteria with conventional antibiotics still has the ability to damage host cells; therefore, drug treatments directly inhibiting hemolysin activity are the most effective. Hemolysis assays were used to confirm that quercetin can inhibit the activity of PLY, protecting cells in vitro, and an oligomerization assay was used to determine the mechanism of quercetin to suppress PLY activity. Live/Dead testing, lactate dehydrogenase (LDH) release analysis and a murine model of endonasal pulmonary infection were used to explore the capability of quercetin to protect cells and mice from S. pneumoniae-mediated damage in vivo and in vitro. The results indicated that quercetin significantly reduced PLY-induced hemolytic activity and cytotoxicity via repressing the formation of oligomers. In addition, treatment with quercetin can reduce PLY-mediated cell injury, improve the survival rate of mice infected with a lethal dose of S. pneumoniae, alleviate the pathological damage of lung tissue and inhibit the release of cytokines (IL-1β and TNF-α) in bronchoalveolar lavage fluid. Considering the importance of these events in antimicrobial resistant S. pneumoniae pathogenesis, our results indicated that quercetin may be a novel potential drug candidate for the treatment of clinical pneumococcal infections.