Murine Pneumonia Model Research Articles

The TLR4-PAR1 Axis Regulates Bone Marrow Mesenchymal Stromal Cell Survival and Therapeutic Capacity in Experimental Bacterial Pneumonia.

Bone marrow derived mesenchymal stromal cells have been shown to have significant therapeutic effects in experimental models of pneumonia and lung injury. The current study examined the roles of the toll like receptor 4 (TLR4) and protease activated receptor 1 (PAR1) pathways on mesenchymal stromal cell (MSC) survival and therapeutic activity in a murine model of pneumonia. MSCs from TLR4 -/- and R41Q-PAR1 mutated mice were isolated to test the effect of mutating these specific pathways on MSC survival when exposed to cytotoxic stimuli in vitro. An Escherichia coli pneumonia model was used to assess the effect of these specific pathways on MSC therapeutic activity in vivo. Our results showed that mutation of either the TLR4 or PAR1 pathways in MSCs impaired cell survival under conditions of inflammatory stress in vitro, and eliminated their therapeutic efficacy in vivo. Also, stimulation of the TLR4 pathway on MSCs led to secretion of low levels of prothrombin by MSCs, while disrupting the TLR4 pathway impaired canonical signaling through PAR1 in response to thrombin. Therefore, this study demonstrates that both TLR4 and PAR1 are required for MSC survival under inflammatory conditions in vitro and therapeutic capacity in vivo, and that the TLR4 pathway regulates signaling through PAR1 on MSCs. Stem Cells 2018;36:796-806.

Peptidoglycan Recognition Protein 3 Does Not Alter the Outcome of Pneumococcal Pneumonia in Mice.

Pneumococci frequently cause community-acquired pneumonia, a disease with high mortality rates, particularly in young children and in the elderly. Endogenous antimicrobial peptides and proteins such as PGLYRP3 may contribute to the progression and outcome of this disease. Since increasing antibiotic resistant strains occur all over the world, these endogenous antimicrobial molecules are interesting new targets for future therapies. In this study, the expression pattern of PGLYRP3 was analyzed in alveolar epithelial cells, alveolar macrophages and neutrophils. Additionally, the function of PGLYRP3 during Streptococcus pneumoniae-induced pneumonia was investigated in a murine pneumococcal pneumonia model using PGLYRP3KO mice. PGLYRP3 is expressed in all selected cell types but pneumococcus-dependent induction of PGLYRP3 was observed only in neutrophils and alveolar macrophages. Interestingly, there were no significant differences in the bacterial loads within the lungs, the blood or the spleens, in the cytokine response, the composition of immune cells and the histopathology between wild type and PGLYRP3KO mice. Finally, we could neither observe significant differences in the clinical symptoms nor in the overall survival. Collectively, PGLYRP3 seems to be dispensable for the antibacterial defense during pneumococcal pneumonia.

Digital Image Analysis in Murine Models of Acute Pneumonia With Pathogen-Specific Histopathologies and Lesion Quantification

Digital Image Analysis in Murine Models of Acute Pneumonia With Pathogen-Specific Histopathologies and Lesion Quantification

A novel bivalent fusion vaccine induces broad immunoprotection against Staphylococcus aureus infection in different murine models

With more and more drug-resistant Staphylococcus aureus strains emerging in hospitals, there is an urgent need to develop an effective vaccine to combat S. aureus infection. In this study, we constructed a novel bivalent fusion vaccine, SpA-DKKAA-FnBPA37-507 (SF), based on the D domain of staphylococcal protein A (SpA) and the A domain of fibronectin-binding protein A (FnBPA). Immunisation with SF induced a more ideal protective effect compared with the single components alone in a sepsis model. It also showed broad immunoprotection against seven FnBPA isotypes. Vaccination with SF induced strong antibodies responses and Th1/Th17 polarized cellular responses. Further we demonstrated the protective effect of antibodies by the opsonophagocytic assay (OPA) and passive immunisation. Moreover, vaccination with SF showed protective efficacy in a murine pneumonia model and skin abscess model. These results suggest that SF can be regarded as a promising vaccine candidate for the prevention of S. aureus infections.

Inhibition of Macrophage Complement Receptor CRIg by TRIM72 Polarizes Innate Immunity of the Lung.

The complement system plays a critical role in immune responses against pathogens. However, its identity and regulation in the lung are not fully understood. This study aimed to explore the role of tripartite motif protein (TRIM) 72 in regulating complement receptor (CR) of the Ig superfamily (CRIg) in alveolar macrophage (AM) and innate immunity of the lung. Imaging, absorbance quantification, and flow cytometry were used to evaluate in vitro and in vivo AM phagocytosis with normal, or altered, TRIM72 expression. Pulldown, coimmunoprecipitation, and gradient binding assays were applied to examine TRIM72 and CRIg interaction. A pneumonia model was established by intratracheal injection of Pseudomonas aeruginosa. Mortality, lung bacterial burden, and cytokine levels in BAL fluid and lung tissues were examined. Our data show that TRIM72 inhibited CR-mediated phagocytosis, and release of TRIM72 inhibition led to increased AM phagocytosis. Biochemical assays identified CRIg as a binding partner of TRIM72, and TRIM72 inhibited formation of the CRIg-phagosome. Genetic ablation of TRIM72 led to improved pathogen clearance, reduced cytokine storm, and improved survival in murine models of severe pneumonia, specificity of which was confirmed by adoptive transfer of wild-type or TRIM72KO AMs to AM-depleted TRIM72KO mice. TRIM72 overexpression promoted bacteria-induced NF-κB activation in murine alveolar macrophage cells. Our data revealed a quiescent, noninflammatory bacterial clearance mechanism in the lung via AM CRIg, which is suppressed by TRIM72. In vivo data suggest that targeted suppression of TRIM72 in AM may be an effective measure to treat fatal pulmonary bacterial infections.

Virulence Characteristics of Carbapenem-Resistant Klebsiella pneumoniae Strains from Patients with Necrotizing Skin and Soft Tissue Infections

Two types of Klebsiella pneumoniae (KP) strains are currently emerging: hypervirulent (hvKP) strains and carbapenem-resistant (CR-KP) strains. To date, these two strain types rarely overlap. Recent reports, however, suggest that CR-KP strains are increasing in virulence. hvKP strains frequently present as highly invasive infections, such as necrotizing skin and soft tissue infections (NSSTI). To examine whether CR-KP strains with features of hvKP were present in our U.S. hospital, we retrospectively identified four cases of CR-KP NSSTI diagnosed between January 2012 and January 2016. Whole-genome sequencing was used to perform multilocus sequence typing, capsular typing, and identification of virulence and antimicrobial resistance genes. Additionally, the virulence of each isolate was determined in vitro and using murine pneumonia and subcutaneous infection models. We identified one CR-KP isolate that possessed features of hypervirulent KP, including a hypermucoviscous phenotype, K2 capsule, and resistance to phagocytosis. Of the four CR-KP isolates, two had no evidence of enhanced pathogenicity in either mouse model, demonstrating that low-virulence strains can cause NSSTI in immunosuppressed patients. The remaining two isolates exhibited low virulence in the pneumonia model but high virulence in the subcutaneous infection model, suggesting that the virulence attributes of these isolates are adapted to causing NSSTI.

Immunoprotective potential of BamA, the outer membrane protein assembly factor, against MDR Acinetobacter baumannii

AbstarctAcinetobacter baumannii infections are responsible for major health problems in immunocompromised patients particularly in intensive care units. Due to rapid acquisition of and also inherent drug resistance, a vaccine is an effective treatment option against this pathogen. BamA, an outer membrane β-barrel assembly protein, was identified in A. baumannii as potential vaccine candidate by in silico analysis. The immunoprotective efficacy of this highly conserved protein was investigated against a virulent multidrug resistant clinical isolate using murine pneumonia model. Recombinant BamA elicited a high IgG antibody titer (160000) in mice. Opsonophagocytic killing assay showed non-neutrilizing, opsonizing antibodies with combinatorial bactericidal activity of antibodies and complement components. Active and passive immunization protected 80 and 60% mice respectively against intranasal challenge with lethal dose (109 CFU) of virulent A. baumannii along with efficient clearance of bacteria in mice lungs and reduction in levels of pro-inflammatory cytokines viz. TNF-α, IL-6 and IL-1β in sera and lung tissue homogenate. Increase in levels of IL-10, an anti-inflammatory cytokine and reduction of neutrophils in lungs facilitated the control of infection. This study demonstrates the potential of BamA as effective vaccine candidate and a promising target for antibody-based therapy to protect against MDR A. baumannii infections.

A global Staphylococcus aureus proteome resource applied to the in vivo characterization of host-pathogen interactions

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 105 S. aureus cells, 578 proteins were identified. Increased abundance of proteins required for oxidative stress response, amino acid biosynthesis, and fermentation together with decreased abundance of ribosomal proteins and nucleotide reductase NrdEF was observed in post-infection samples compared to the pre-infection state.

Protective Efficacy of the Trivalent Pseudomonas aeruginosa Vaccine Candidate PcrV-OprI-Hcp1 in Murine Pneumonia and Burn Models

Pseudomonas aeruginosa is a formidable pathogen that is responsible for a diverse spectrum of human infectious diseases, resulting in considerable annual mortality rates. Because of biofilm formation and its ability of rapidly acquires of resistance to many antibiotics, P. aeruginosa related infections are difficult to treat, and therefore, developing an effective vaccine is the most promising method for combating infection. In the present study, we designed a novel trivalent vaccine, PcrV28-294-OprI25-83-Hcp11-162 (POH), and evaluated its protective efficacy in murine pneumonia and burn models. POH existed as a dimer in solution, it induced better protection efficacy in P. aeruginosa lethal pneumonia and murine burn models than single components alone when formulated with Al(OH)3 adjuvant, and it showed broad immune protection against several clinical isolates of P. aeruginosa. Immunization with POH induced strong immune responses and resulted in reduced bacterial loads, decreased pathology, inflammatory cytokine expression and inflammatory cell infiltration. Furthermore, in vitro opsonophagocytic killing assay and passive immunization studies indicated that the protective efficacy mediated by POH vaccination was largely attributed to POH-specific antibodies. Taken together, these data provided evidence that POH is a potentially promising vaccine candidate for combating P. aeruginosa infection in pneumonia and burn infections.

CcpA Affects Infectivity of Staphylococcus aureus in a Hyperglycemic Environment.

Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA). Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM) mice were challenged with the mouse pathogenic S. aureus strain Newman and the isogenic ccpA deletion mutant (MST14), and the effects on infectivity were determined. Diabetic NOD mice challenged with the ccpA deletion mutant enhanced the symptoms of infection in an acute murine pneumonia model relative to the parental strain. Interestingly, when diabetic NOD mice were used in footpad or catheter infection models, infectivity of the ccpA mutant decreased relative to the parental strain. These differences greatly diminished when normoglycemic NOD mice (blood glucose level ≤ 10 mM) were used. These data suggest that CcpA is important for infectivity of S. aureus in hyperglycemic individuals.

E3 ubiquitin ligase NKLAM is important for bacterial killing in a murine model of streptococcal pneumonia

Abstract Natural killer lytic associated molecule (NKLAM) is an important component of the innate immune system. Previous in vitro studies using NKLAM-KO macrophages have demonstrated that NKLAM positively affects the transcriptional activity of STAT1, a transcription factor crucial for proper immune function. Therefore, we hypothesized that NKLAM-KO mice would be unable to mount an effective immune response when exposed to Streptococcus pneumoniae. Using a nasal inhalation infection model we found that the lungs of NKLAM-KO mice had a significantly higher bacterial load than WT mice. This observation was associated with increased lung phosphatase activity and decreased STAT1 phosphorylation. STAT1 regulates a large number of genes, including iNOS and MCP-1, which are important for successful pathogen eradication. In our model, we observed that NKLAM-KO mice expressed less iNOS in their lungs at 24 and 48 hr post-infection in comparison to WT mice. Additionally, infected NKLAM-KO lungs contained less TNF-alpha, IL-12, IFN-gamma and MCP-1 than lungs from WT mice. NKLAM-KO mice had significantly less neutrophil lung infiltration than WT mice, an effect that is likely due to the decreased expression of chemokines including MCP-1. In summary, these novel findings demonstrate the importance of NKLAM in innate immunity and begin to define a role for NKLAM in leukocyte trafficking to the lung upon S. pneumoniae infection.

In Vivo Pharmacodynamic Evaluation of Omadacycline (PTK 0796) against Streptococcus pneumoniae in the Murine Pneumonia Model.

Omadacycline is a novel aminomethylcycline antibiotic in clinical development for community-acquired bacterial pneumonia (CABP). We used a neutropenic murine pneumonia infection model to characterize the in vivo pharmacodynamic activity of omadacycline against Streptococcus pneumoniae Four strains with various phenotypic resistances to other antimicrobials, including tetracyclines, were utilized. Drug concentration measurements were performed in the plasma and epithelial lining fluid (ELF) after administration of 0.5, 2, 8, and 32 mg/kg. Pharmacokinetic parameters were calculated using a noncompartmental model and were linear over the dose range. Penetration into ELF ranged from 72 to 102%. Omadacycline demonstrated net cidal activity in relation to the initial burden against all four strains. The pharmacokinetic/pharmacodynamic index AUC/MIC correlated well with efficacy (R2 = 0.74). The plasma 24-h static dose AUC/MIC values were 16 to 20 (24-h ELF AUC/MIC of 14 to 18). A 1-log10 kill was achieved at 24-h plasma AUC/MIC values of 6.1 to 180 (24-h ELF AUC/MIC values 6.0 to 200). A 2-log10 kill was achieved at 24-h plasma AUC/MIC values of 19 to 56 (24-h ELF AUC/MIC of 17 to 47). The targets identified in this study in combination with in vitro potency and favorable human pharmacokinetics make omadacycline an attractive candidate for further development and study in patients with CABP.

Pharmacokinetics and Pharmacodynamics of Minocycline against Acinetobacter baumannii in a Neutropenic Murine Pneumonia Model.

Multidrug-resistant (MDR) Acinetobacter baumannii is increasingly more prevalent in nosocomial infections. Although in vitro susceptibility of A. baumannii to minocycline is promising, the in vivo efficacy of minocycline has not been well established. In this study, the in vivo activity of minocycline was evaluated in a neutropenic murine pneumonia model. Specifically, we investigated the relationship between minocycline exposure and bactericidal activity using five A. baumannii isolates with a broad range of susceptibility (MIC ranged from 0.25 mg/liter to 16 mg/liter). The pharmacokinetics of minocycline (single dose of 25 mg/kg of body weight, 50 mg/kg, 100 mg/kg, and a humanized regimen, given intraperitoneally) in serum and epithelial lining fluid (ELF) were characterized. Dose linearity was observed for doses up to 50 mg/kg and pulmonary penetration ratios (area under the concentration-time curve in ELF from 0 to 24 h [AUCELF,0-24]/area under the concentration time curve in serum from 0 to 24 h [AUCserum,0-24]) ranged from 2.5 to 2.8. Pharmacokinetic-pharmacodynamics (PK-PD) index values in ELF for various dose regimens against different A. baumannii isolates were calculated. The maximum efficacy at 24 h was approximately 1.5-log-unit reduction of pulmonary bacterial burdens from baseline. The AUC/MIC ratio was the PK-PD index most closely correlating to the bacterial burden (r2 = 0.81). The required AUCELF,0-24/MIC for maintaining stasis and achieving 1-log-unit reduction were 140 and 410, respectively. These findings could guide the treatment of infections caused by A. baumannii using minocycline in the future. Additional studies to examine resistance development during therapy are warranted.

Fis Regulates Type III Secretion System by Influencing the Transcription of exsA in Pseudomonas aeruginosa Strain PA14.

Fis is a versatile DNA binding protein in bacteria. It has been demonstrated in multiple bacteria that Fis plays crucial roles in regulating bacterial virulence factors and optimizing bacterial adaptation to various environments. However, the role of Fis in Pseudomonas aeruginosa virulence as well as gene regulation remains largely unknown. Here, we found that Fis was required for the virulence of P. aeruginosa in a murine acute pneumonia model. Transcriptome analysis revealed that expression of T3SS genes, including master regulator ExsA, was defective in a fis::Tn mutant. We further demonstrate that the continuous transcription of exsC, exsE, exsB, and exsA driven by the exsC promoter was required for the activation of T3SS. Fis was found to specifically bind to the exsB-exsA intergenic region and plays an essential role in the transcription elongation from exsB to exsA. Therefore, we found a novel role of Fis in the regulation of exsA expression.

Hypoxia Reduces the Pathogenicity of Pseudomonas aeruginosa by Decreasing the Expression of Multiple Virulence Factors.

Our understanding of how the course of opportunistic bacterial infection is influenced by the microenvironment is limited. We demonstrate that the pathogenicity of Pseudomonas aeruginosa strains derived from acute clinical infections is higher than that of strains derived from chronic infections, where tissues are hypoxic. Exposure to hypoxia attenuated the pathogenicity of strains from acute (but not chronic) infections, implicating a role for hypoxia in regulating bacterial virulence. Mass spectrometric analysis of the secretome of P. aeruginosa derived from an acute infection revealed hypoxia-induced repression of multiple virulence factors independent of altered bacterial growth. Pseudomonas aeruginosa lacking the Pseudomonas prolyl-hydroxylase domain-containing protein, which has been implicated in bacterial oxygen sensing, displays reduced virulence factor expression. Furthermore, pharmacological hydroxylase inhibition reduces virulence factor expression and pathogenicity in a murine model of pneumonia. We hypothesize that hypoxia reduces P. aeruginosa virulence at least in part through the regulation of bacterial hydroxylases.

DnaJ (hsp40) of Streptococcus pneumoniae is involved in bacterial virulence and elicits a strong natural immune reaction via PI3K/JNK.

As a heat shock protein, DnaJ plays an important role in the pathogenesis of pneumococcal infection. However, how the virulence factor-DnaJ elicits host natural immunity still remains unclear. In this study, we investigated the effects of dnaJ deficiency in Streptococcus pneumoniae (S. pneumoniae) on bacterial virulence, and further explored the related molecular mechanisms in vivo and in vitro. By generating dnaJ deficient mutant (ΔdnaJ), the virulence and colonization were detected in murine pneumonia and sepsis models in vivo. Compared with wild-type parent strain, the abilities of rapid colonization and induction of inflammatory responses of ΔdnaJ in mouse lungs were significantly impaired. Simultaneously, recombinant DnaJ purified from E. coli expression system (rDnaJ) induced macrophage strain RAW264.7 to secrete IL-6 by activation of PI3K and JNK signal pathways, which were confirmed by the specific signaling inhibitors. In conclusion, DnaJ, a novel virulence protein, was essential for the virulence and colonization of S. pneumoniae and induced pro-inflammatory cytokine production in macrophages through PI3K/JNK.

An Oxygen-Sensing Two-Component System in the Burkholderia cepacia Complex Regulates Biofilm, Intracellular Invasion, and Pathogenicity

Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence.

Delayed Therapy with Plasma Gelsolin Improves Survival in Murine Pneumococcal Pneumonia

BackgroundInnate immune responses contribute to successful resolution of bacterial pneumonia. Bolstering host defense with immunomodulators might be increasingly needed to improve outcomes in antibiotic-resistant infections. One candidate molecule is recombinant human plasma gelsolin (rhu-pGSN), an abundant normal blood protein whose levels fall proportionally with disease severity. Pretreatment with rhu-pGSN has beneficial effects in many pre-clinical models of inflammation and injury, including pneumonia. We evaluated the effects of delaying therapy with rhu-pGSN up to 48 hours after lethal intra-nasal pneumococcal challenge in a mouse model to more closely mimic realistic clinical circumstances.MethodsAdult Bl/6 mice were inoculated intra-nasally with S. pneumoniae serotype 3 on day 0, followed by subcutaneous rhu-pGSN 24 hours later for evaluation of bacterial clearance in lavage fluids. To assess effects on survival, rhu-pGSN was administered on days 2 and 3 after infection and effects monitored for 10 days. No antibiotics or other interventions were given.ResultsTreatment with rhu-pGSN at 24 hours after infection improved bacterial clearance, seen as reduction of bacterial CFU in bronchoalveolar lavage fluid at 48 hours (% of initial inoculum, vehicle vs.. rhu-pGSN (dose range 0.5–2 mg): 30 ± 13 vs. 11 ± 7, n = 6 trials using inocula ranging 0.3–1.8 x 106 CFU, 3 mice/group/trial, P = .001). In 3 separate trials, pGSN (0.5 mg s.c.) reduced weight loss and mortality (% survival, vehicle vs. pGSN: 40 vs. 80, 0 vs. 25, 17 vs. 43; n ≥ 16/group, P =.02). Increasing the dose to 1 mg further improved survival from 17 to 71%.ConclusionRhu-pGSN can substantially improve survival in a murine model of fatal pneumococcal pneumonia, even when administered as single doses on days 2 and 3 after infection without antibiotics. The data support further evaluation of pGSN as adjunctive therapy for serious infections with diverse pathogens and in models of antibiotic-resistant pneumonia.Disclosures Z. Yang, BioAegis: Shared NIH grant to study plasma gelsolin, we receive plasma gelsolin for our lab studies; S. Levinson, BioAegis: BIoAegis shares a grant to investigate plasma gelsolin with HSPH, Employee and Shareholder, Salary; T. Stossel, BioAegis: Consultant and Shareholder, portion of royalties from Hospital IP licensed to BioAegis; M. DiNubile, BioAegis: Employee and Shareholder, Consulting fee; L. Kobzik, BioAegis: Collaborator and We share a NIH grant on pGSN with BioAegis, we receive plasma gelsolin for our lab studies

Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo.

Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

Efficacy of High-Dose Meropenem (Six Grams per Day) in Treatment of Experimental Murine Pneumonia Induced by Meropenem-Resistant Pseudomonas aeruginosa.

High-dose meropenem (MEPM; 6 g/day) has been approved as a treatment for purulent meningitis; however, little is known regarding its in vivo efficacy in refractory lower respiratory tract infections. The purpose of this study was to evaluate the efficacy of MEPM at 6 g/day in a murine model of severe pneumonia caused by MEPM-resistant Pseudomonas aeruginosa Experimental pneumonia induced by MEPM-resistant P. aeruginosa was treated with normal-dose MEPM (150 mg/kg of body weight, simulating a 3-g/day regimen in humans) or high-dose MEPM (500 mg/kg, simulating a 6-g/day regimen in humans). Mice treated with high-dose MEPM showed significantly restored survival relative to that of untreated mice and tended to show a survival rate higher than that of mice treated with normal-dose MEPM. The viable bacterial counts (of two clinical isolates) in the lungs decreased significantly in mice treated with high-dose MEPM from those for untreated mice (P < 0.001) or mice treated with normal-dose MEPM (P, <0.01 and <0.05). The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) was also significantly lower in mice treated with high-dose MEPM than in untreated mice. The free MEPM concentration in the epithelial lining fluid (ELF) exceeded 16 μg/ml for 85 min in mice treated with high-dose MEPM, but not for mice treated with normal-dose MEPM. Our results demonstrate that high-dose MEPM (6 g/day) might provide better protection against pneumonia caused by MEPM-resistant strains of P. aeruginosa than the dose normally administered (less than 3 g/day).