Mouse Model Of Lung Infection Research Articles

Efficacy of epetraborole against Mycobacteroides abscessus in a mouse model of lung infection.

Mycobacteroides abscessus (Mab or Mycobacterium abscessus) is a fast-growing mycobacterium that is ubiquitous in the environment and can cause opportunistic disease in people with lung comorbidity and immunodeficiency. There are no Food and Drug Administration-approved drugs for this disease, and repurposed antibiotics have a poor microbiological response. To address the need for effective new antibiotics, we determined the efficacy of epetraborole (EBO) against three Mab clinical isolates in a mouse model of lung Mab infection. Reduction in lung Mab burden over 4 weeks of treatment was the study end point. EBO was administered orally once daily at doses of 25 and 50 mg/kg, which achieved exposures approximating the once-daily dosing of 250 mg and 500 mg, respectively, in humans. EBO administration led to a gradual reduction in the lung Mab burden. After 4 weeks of treatment, the efficacies of 25 and 50 mg/kg EBO against isolates ATCC 19977 and M9501 were comparable. However, against isolate M9530, 50 mg/kg EBO was more efficacious than 25 mg/kg and comparable with parenteral imipenem, one of the most efficacious antibiotics against Mab. We also undertook a dose-ranging study by evaluating the efficacies of once-daily oral administration of 0.5, 5, 10, 25, and 100 mg/kg EBO against M9501 over 4 weeks. Once-daily oral 100 mg/kg EBO was as effective as twice-daily 100 mg/kg imipenem injection. Our study suggests that EBO could address the unmet need for effective oral treatment options for Mab lung disease, given the high rates of Mab drug resistance and limited tolerable intravenous options.

Dry powder inhalation containing muco-inert ciprofloxacin and colistin co-loaded liposomes for pulmonary P. Aeruginosa biofilm eradication

Pseudomonas aeruginosa (PA), a predominant pathogen in lung infections, poses significant challenges due to its biofilm formation, which is the primary cause of chronic and recalcitrant pulmonary infections. Bacteria within these biofilms exhibit heightened resistance to antibiotics compared to their planktonic counterparts, and their secreted toxins exacerbate lung infections. Diverging from traditional antibacterial therapy for biofilm eradication, this study introduces a novel dry powder inhalation containing muco-inert ciprofloxacin and colistin co-encapsulated liposomes (Cipro-Col-Lips) prepared using ultrasonic spray freeze drying (USFD) technique. This USFD dry powder is designed to efficiently deliver muco-inert Cipro-Col-Lips to the lungs. Once deposited, the liposomes rapidly diffuse into the airway mucus, reaching the biofilm sites. The muco-inert Cipro-Col-Lips neutralize the biofilm-secreted toxins and simultaneously trigger the release of their therapeutic payload, exerting a synergistic antibiofilm effect. Our results demonstrated that the optimal USFD liposomal dry powder formulation exhibited satisfactory in vitro aerosol performance in terms of fine particle fraction (FPF) of 44.44 ± 0.78 %, mass median aerodynamic diameter (MMAD) of 4.27 ± 0.21 μm, and emitted dose (ED) of 99.31 ± 3.31 %. The muco-inert Cipro-Col-Lips effectively penetrate the airway mucus and accumulate at the biofilm site, neutralizing toxins and safeguarding lung cells. The triggered release of ciprofloxacin and colistin works synergistically to reduce the biofilm’s antibiotic resistance, impede the development of antibiotic resistance, and eliminate 99.99 % of biofilm-embedded bacteria, including persister bacteria. Using a PA-beads induced biofilm-associated lung infection mouse model, the in vivo efficacy of this liposomal dry powder aerosol was tested, and the results demonstrated that this liposomal dry powder aerosol achieved a 99.7 % reduction in bacterial colonization, and significantly mitigated inflammation and pulmonary fibrosis. The USFD dry powder inhalation containing muco-inert Cipro-Col-Lips emerges as a promising therapeutic strategy for treating PA biofilm-associated lung infections.

Synergistic D-Amino Acids Based Antimicrobial Cocktails Formulated via High-Throughput Screening and Machine Learning.

Antimicrobial resistance (AMR) from pathogenic bacterial biofilms has become a global health issue while developing novel antimicrobials is inefficient and costly. Combining existing multiple drugs with enhanced efficacy and/or reduced toxicity may be a promising approach to treat AMR. D-amino acids mixtures coupled with antibiotics can provide new therapies for drug-resistance infection with reduced toxicity by lower drug dosage requirements. However, iterative trial-and-error experiments are not tenable to prioritize credible drug formulations, owing to the extremely large number of possible combinations. Herein, a new avenue is provide to accelerate the exploration of desirable antimicrobial formulations via high-throughput screening and machine learning optimization. Such an intelligent method can navigate the large search space and rapidly identify the D-amino acid mixtures with the highest anti-biofilm efficiency and also the synergisms between D-amino acid mixtures and antibiotics. The optimized drug cocktails exhibit high antimicrobial efficacy while remaining non-toxic, which is demonstrated not only from in vitro assessments but also the first in vivo study using a lung infection mouse model.

Novel Indane-Containing NBTIs with Potent Anti-Gram-Negative Activity and Minimal hERG Inhibition.

Novel bacterial topoisomerase inhibitors (NBTIs) make up a promising new class of antibiotics with the potential to combat the growing threat of antimicrobial resistance. Two key challenges in the development of NBTIs have been to obtain broad spectrum activity against multidrug-resistant Gram-negative bacteria and to diminish inhibition of the hERG cardiac ion channel. Here we report the optimization of a series of NBTIs bearing a novel indane DNA intercalating moiety. The addition of a basic, polar side chain connected to the indane by an ether or an N-linked secondary amide linkage together with a lipophilicity-lowering modification of the enzyme binding moiety led to compounds such as 2a and 2g which showed excellent broad spectrum potency and minimal hERG inhibition. Compound 2a demonstrated robust bactericidal in vivo activity in a mouse lung infection model with the strain P. aeruginosa ATCC 27853 which is resistant to several clinically relevant antibiotics. Rodent pharmacokinetic studies with 2a revealed an unusual profile characterized by rapid tissue distribution and a prolonged, flat terminal phase. This profile precluded further development of these compounds as potential new antibiotics.

Near-infrared light and pH-responsive hyaluronic acid-enveloped ZIF-8 nanoparticles for the treatment of pneumonia caused by methicillin-resistant Staphylococcus aureus

There is a serious threat to public health posed by the rapid spread of methicillin-resistant Staphylococcus aureus (MRSA). Photodynamic sterilization is the most effective method of combating bacterial infection, especially multidrug-resistant bacterial infection. In this study, zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, one of the most attractive metal–organic framework material, were used to encapsulate the indocyanine green (ICG, a potent PS); after that, hyaluronic acid (HA) coating was applied to the nanoparticle to create a host cell-tracking nanoplatform (ICG@ZIF-8/HA). Upon near-infrared light irradiation, ICG@ZIF-8/HA exhibit excellent efficacy in producing reactive oxygen species (ROS) and killing multidrug-resistant bacteria in vitro. After the tail vein injection, ICG@ZIF-8/HA can precisely deliver to lung infection sites, and display an effective PDT-based elimination of multidrug-resistant bacteria in mouse lung infection models. These results suggest that the ICG@ZIF-8/HA nanoplatform may exhibit potent curative effects on infectious diseases.

SPA4 peptide suppresses the lipopolysaccharide (LPS)-stimulated lactate secretion in lung epithelial cells through modulation of Toll-like receptor-4 (TLR4)

Abstract Lung infection and inflammation can cause severe injury leading to acute respiratory distress syndrome. An increased expression and activity of the TLR4 is associated with inflammatory response. Simultaneous elevated level of lactate is used as the marker of disease severity and is associated with poor outcomes in critically ill patients. Therapeutic treatment with TLR4-interacting SPA4 peptide (amino acid sequence: GDFRYSDGTPVNYTNWYRGE) suppresses inflammation in immune cells and in mouse models of lung infection and inflammation. In this study, we evaluated the effect of SPA4 peptide on lactate production by H441 human lung epithelial cells against Pseudomonas aeruginosa-derived LPS. Briefly, the H441 cells were challenged with LPS (1 μg/ml) and treated with SPA4 peptide (100 μM) after 1 h of LPS challenge. Next day, the cell-free supernatants and cell lysates were harvested. We also transfected the cells with TLR4 siRNA to achieve suppression of TLR4. Levels of protein and lactate were quantitated in cell lysates and in filtered supernatants using biochemical assays. The lactate levels were normalized with cell protein. The unchallenged, vehicle-treated cells served as control. Our results demonstrated reduced secretion of lactate by SPA4 peptide-treated cells (187.5% of control) versus vehicle-treated cells (324.7% of control) against LPS stimuli (p<0.05). The immunostaining and confocal imaging showed knockdown of TLR4 in up to 50% cells. The LPS-stimulated lactate secretion was reduced and SPA4 peptide treatment did not further affect the lactate levels in TLR4 siRNA transfected cells. Together, our results suggest that the SPA4 peptide suppresses lactate secretion through modulation of TLR4 in lung epithelial cells. Supported by grant from NIH (R01HL136325) and 2021 American Association of Immunologists Careers in Immunology Fellowship Program.

Baicalein Resensitizes Multidrug-Resistant Gram-Negative Pathogens to Doxycycline.

As multidrug-resistant pathogens emerge and spread rapidly, novel antibiotics urgently need to be discovered. With a dwindling antibiotic pipeline, antibiotic adjuvants might be used to revitalize existing antibiotics. In recent decades, traditional Chinese medicine has occupied an essential position in adjuvants of antibiotics. This study found that baicalein potentiates doxycycline against multidrug-resistant Gram-negative pathogens. Mechanism studies have shown that baicalein causes membrane disruption by attaching to phospholipids on the Gram-negative bacterial cytoplasmic membrane and lipopolysaccharides on the outer membrane. This process facilitates the entry of doxycycline into bacteria. Through collaborative strategies, baicalein can also increase the production of reactive oxygen species and inhibit the activities of multidrug efflux pumps and biofilm formation to potentiate antibiotic efficacy. Additionally, baicalein attenuates the lipopolysaccharide-induced inflammatory response in vitro. Finally, baicalein can significantly improve doxycycline efficacy in mouse lung infection models. The present study showed that baicalein might be considered a lead compound, and it should be further optimized and developed as an adjuvant that helps combat antibiotic resistance. IMPORTANCE Doxycycline is an important broad-spectrum tetracycline antibiotic used for treating multiple human infections, but its resistance rates are recently rising globally. Thus, new agents capable of boosting the effectiveness of doxycycline need to be discovered. In this study, it was found that baicalein potentiates doxycycline against multidrug-resistant Gram-negative pathogens in vitro and in vivo. Due to its low cytotoxicity and resistance, the combination of baicalein and doxycycline provides a valuable clinical reference for selecting more effective therapeutic strategies for treating infections caused by multidrug-resistant Gram-negative clinical isolates.

Copper Efflux System Required in Murine Lung Infection by Haemophilus influenzae Composed of a Canonical ATPase Gene and Tandem Chaperone Gene Copies.

Copper is an essential micronutrient but is toxic at high concentrations. In Haemophilus influenzae mechanisms of copper resistance and its role in pathogenesis are unknown; however, our previous genetic screen by transposon insertion-site sequencing implicated a putative cation transporting ATPase (copA) in survival in a mouse lung infection model. Here, we demonstrate that H. influenzae copA (HI0290) is responsible for copper homeostasis involving the merR-type regulator, cueR, as well as six tandem copies of the metallochaperone gene, copZ. Deletion of the ATPase and metallochaperone genes resulted in increased sensitivity to copper but not to cobalt, zinc, or manganese. Nontypeable H. influenzae (NTHi) clinical isolate NT127 has the same locus organization but with three copies of copZ. We showed that expression of the NTHi copZA operon is activated by copper under the regulatory control of CueR. NTHi single copA and copZ mutants and, especially, the double deletion copZA mutant exhibited decreased copper tolerance, and the ΔcopZA mutant accumulated 97% more copper than the wild type when grown in the presence of 0.5 mM copper sulfate. Mutants of NT127 deleted of the ATPase (copA) alone and deleted of both the ATPase and chaperones (copZ1-3) were 4-fold and 20-fold underrepresented compared to the parent strain during mixed-infection lung challenge, respectively. Complementation of cop locus deletion mutations restored copper resistance and virulence properties. NTHi likely encounters copper as a host defense mechanism during lung infection, and our results indicate that the cop system encodes an important countermeasure to alleviate copper toxicity.

Virulence of Mycobacterium intracellulare clinical strains in a mouse model of lung infection – role of neutrophilic inflammation in disease severity

BackgroundMycobacterium intracellulare is a major etiological agent of Mycobacterium avium-intracellulare pulmonary disease (MAC-PD). However, the characteristics of the virulence of M. intracellulare and the in vivo chemotherapeutic efficacy remain unclear. In this study, we examined the virulence of nine M. intracellulare strains with different clinical phenotypes and genotypes in C57BL/6 mice.ResultsWe classified three types of virulence phenotypes (high, intermediate, and low) based on the kinetics of the bacterial load, histological lung inflammation, and neutrophilic infiltration. High virulence strains showed more severe neutrophilic infiltration in the lungs than intermediate and low virulence strains, with 6.27-fold and 11.0-fold differences of the average percentage of neutrophils in bronchoalveolar lavage fluid, respectively. In particular, the high virulence strain M.i.198 showed the highest mortality in mice, which corresponded to the rapid progression of clinical disease. In mice infected with the drug-sensitive high virulence strain M019, clarithromycin-containing chemotherapy showed the highest efficacy. Monotherapy with rifampicin exacerbated lung inflammation with increased lymphocytic and neutrophilic infiltration into the lungs.ConclusionsThe virulence phenotypes of clinical strains of M. intracellulare were diverse, with high virulence strains being associated with neutrophilic infiltration and disease progression in infected mice. These high virulence strains were proposed as a useful subject for in vivo chemotherapeutic experiments.

Codelivery of synergistic antimicrobials with polyelectrolyte nanocomplexes to treat bacterial biofilms and lung infections.

Bacterial biofilm infections, particularly those of Pseudomonas aeruginosa (PA), have high rates of antimicrobial tolerance and are commonly found in chronic wound and cystic fibrosis lung infections. Combination therapeutics that act synergistically can overcome antimicrobial tolerance; however, the delivery of multiple therapeutics at relevant dosages remains a challenge. We therefore developed a nanoscale drug carrier for antimicrobial codelivery by combining approaches from polyelectrolyte nanocomplex (NC) formation and layer-by-layer electrostatic self-assembly. This strategy led to NC drug carriers loaded with tobramycin antibiotics and antimicrobial silver nanoparticles (AgTob-NCs). AgTob-NCs displayed synergistic enhancements in antimicrobial activity against both planktonic and biofilm PA cultures, with positively charged NCs outperforming negatively charged formulations. NCs were evaluated in mouse models of lung infection, leading to reduced bacterial burden and improved survival outcomes. This approach therefore shows promise for nanoscale therapeutic codelivery to treat recalcitrant bacterial infections.

Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1β in Cellular and Animal Infection Models.

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1β. Furthermore, inhibition of LasB-mediated IL-1β activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen.

1704. Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, Demonstrates Potent Efficacy and Improves Efficacy of Standard of Care Regimen Against Mycobacterium avium complex in a Chronic Mouse Lung Infection Model

Abstract Background Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. These studies evaluated oral doses (PO) of EBO against 5 M. avium complex (MAC) strains in a chronic mouse infection model either as monotherapy or in combination with standard of care [SOC; clarithromycin (CLR), rifabutin (RFB), ethambutol (EMB)]. Methods A pilot chronic efficacy study against M. avium 2285R evaluated EBO at 1, 10, 30, 100, 300 and 500 mg/kg PO once daily (QD) compared to 250 mg/kg CLR PO QD. C57BL/6 mice were infected with a pulmonary aerosol of 1x1011 CFU. Treatment was administered for 56 days starting on day 28 post-infection. The bacterial burden (CFU) in lungs was evaluated on days 1, 28 and 84 post-infection by plating serial dilutions of homogenates on Middlebrook 7H11 charcoal agar plates. An additional 4 strains of MAC were evaluated with EBO doses of 100, 200, 300 or 400 mg/kg QD compared with the SOC therapy for MAC (CLR 250 mg/kg, RFB 100 mg/kg, EMB 100 mg/kg) QD and SOC plus EBO 200mg/kg QD. Oral exposures of EBO were determined in a group of uninfected mice (Table 1).Table 1:C57BL/6 Murine Pharmacokinetic Parameters Results In a study with M. avium 2285R, a biofilm-forming strain, EBO at all doses tested was significantly better than CLR dosed at 250 mg/kg (Figure 1), and no CFU were detected on agar plates containing EBO (16 mg/L). In subsequent studies, SOC was compared to EBO in 4 additional MAC strains (Figure 2). Efficacy of EBO monotherapy was better than SOC against M. avium ATCC 700898, while it was as good as SOC with M. intracellulare 1956, M. intracellulareDNA00055, and M. intracellulare DNA00111 with CFU reductions ranging from 2 - 4.8 log10 compared to day 28 controls. In all four strains tested, 200 mg/kg EBO, which approximates the human oral equivalent dose of 500 mg, combined with SOC increased bacterial killing from 1.4 - 3.0 log10 CFU compared to SOC alone resulting in total lung CFU reductions of 4.6 - 5.6 log10. Figure 1Figure 2 Conclusion In this chronic mouse lung infection model, no EBO resistance development was detected with M. avium 2285R at day 84. EBO demonstrated potent in vivo efficacy against 5 MAC strains and significantly improved efficacy when combined with SOC, supporting further clinical development for EBO. Disclosures Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn Shoen, PhD, AN2 Therapeutics: Grant/Research Support Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds.

Genomic characterization of a new phage BUCT541 against Klebsiella pneumoniae K1-ST23 and efficacy assessment in mouse and Galleria mellonella larvae.

Over the past decades, the spread of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) is becoming a new threat and new effective therapies against this pathogen are needed. Bacteriophage (phage) therapy is considered to be a promising alternative treatment for MDR-KP infections compared with antibacterial drug usage. Here, we reported a new phage BUCT541 which can lyse MDR-KP ST23. The genome of BUCT541 is a double-stranded linear 46,100-bp long DNA molecule with 48% GC content through the Next generation sequencing (NGS) data. A total of 81 open reading frames and no virulence or antimicrobial resistance genes are annotated in the BUCT541 genome. BUCT541 was able to lyse 7 of the 30 tested MDR-KP according to the host range analysis. And the seven sensitive strains belonged to the K. pneumoniae K1-ST23. BUCT541 exhibited high thermal stability (4–70°C) and broad pH tolerance (pH 3-11) in the stability test. The in vivo results showed that BUCT541 (4 × 105 plaque-forming units (PFU)/each) significantly increased the survival rate of K. pneumoniae infected Galleria mellonella from 5.3% to 83.3% within 48 h. Moreover, in the mouse lung infection model, high doses of BUCT541 (2 × 107 PFU/each) cured 100% of BALB/c mice that were infected with K. pneumoniae. After 30 h of treatment with phage BUCT541 of the multiplicity of infection (MOI) = 10, the K. pneumoniae in the lungs of mice was lower than 104 CFU/mL, compared to the control group 109 CFU/mL. Together, these findings indicate that phage BUCT541 holds great promise as an alternative therapy with excellent stability and a wide lysis range for the treatment of MDR-KP ST23 infection.

The oncogene ABL1 regulates the inflammatory response of innate immunity via mediating TRAF6 ubiquitination

The oncogene ABL1 plays an important role in various cancers, while its roles remain unclear in pneumonia. This study aims to investigate the roles of ABL1 in pneumonia and the underlying mechanisms. RNA sequencing was used to determine the expressions of multiple kinases in the PBMCs. A series of overexpression and knockout cell lines were constructed. Besides, an intranasal lung infection mouse model was pre-treated with asciminb. ELISAs and qPCR were used to determine the levels of target genes. In addition, STRING Interaction Network and Immunoblotting assays were used to determine the interaction between target proteins. An elevation in ABL1 was observed in the infant with Ecoli pneumonia. ABL1 was positively correlated to the levels of inflammatory cytokines and the activation of the NF-kB pathways. In vivo data demonstrated that the inhibition of ABL1 suppressed the inflammatory cytokines, reduced the lung bacterial burden, and ameliorated the lung injury score. ABL1 inhibited the phosphorylation of IκBα and p38 and regulated the ubiquitination of TRAF6. ABL1 regulates the inflammatory response in pneumonia in part by the regulation of MAPK and NF-κB pathways and TRAF6 ubiquitination.

Pneumolysin suppresses the initial macrophage pro-inflammatory response to Streptococcuspneumoniae.

Published data for the Streptococcus pneumoniae virulence factor Pneumolysin (Ply) show contradictory effects on the host inflammatory response to infection. Ply has been shown to activate the inflammasome, but also can bind to MRC-1 resulting in suppression of dendritic cell inflammatory responses. We have used an in vitro infection model of human monocyte-derived macrophages (MDM), and a mouse model of pneumonia to clarify whether pro- or anti-inflammatory effects dominate the effects of Ply on the initial macrophage inflammatory response to S. pneumoniae, and the consequences during early lung infection. We found that infection with S. pneumoniae expressing Ply suppressed tumour necrosis factor (TNF) and interleukin-6 production by MDMs compared to cells infected with ply-deficient S. pneumoniae. This effect was independent of bacterial effects on cell death. Transcriptional analysis demonstrated S. pneumoniae expressing Ply caused a qualitatively similar but quantitatively lower MDM transcriptional response to S. pneumoniae compared to ply-deficient S. pneumoniae, with reduced expression of TNF and type I IFN inducible genes. Reduction of the MDM inflammatory response was prevented by inhibition of SOCS1. In the early lung infection mouse model, the TNF response to ply-deficient S. pneumoniae was enhanced and bacterial clearance increased compared to infection with wild-type S. pneumoniae. Overall, these data show Ply inhibits the initial macrophage inflammatory response to S. pneumoniae, probably mediated through SOCS1, and this was associated with improved immune evasion during early lung infection.

Translational in vitro and in vivo PKPD modelling for apramycin against Gram-negative lung pathogens to facilitate prediction of human efficacious dose in pneumonia

ObjectivesNew drugs and methods to efficiently fight carbapenem-resistant gram-negative pathogens are sorely needed. In this study, we characterized the preclinical pharmacokinetics (PK) and pharmacodynamics of the clinical stage drug candidate apramycin in time kill and mouse lung infection models. Based on in vitro and in vivo data, we developed a mathematical model to predict human efficacy. MethodsThree pneumonia-inducing gram-negative species Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were studied. Bactericidal kinetics were evaluated with time-kill curves; in vivo PK were studied in healthy and infected mice, with sampling in plasma and epithelial lining fluid after subcutaneous administration; in vivo efficacy was measured in a neutropenic mouse pneumonia model. A pharmacokinetic-pharmacodynamic model, integrating all the data, was developed and simulations were performed. ResultsGood lung penetration of apramycin in epithelial lining fluid (ELF) was shown (area under the curve (AUC)ELF/AUCplasma = 88%). Plasma clearance was 48% lower in lung infected mice compared to healthy mice. For two out of five strains studied, a delay in growth (∼5 h) was observed in vivo but not in vitro. The mathematical model enabled integration of lung PK to drive mouse PK and pharmacodynamics. Simulations predicted that 30 mg/kg of apramycin once daily would result in bacteriostasis in patients. DiscussionApramycin is a candidate for treatment of carbapenem-resistant gram-negative pneumonia as demonstrated in an integrated modeling framework for three bacterial species. We show that mathematical modelling is a useful tool for simultaneous inclusion of multiple data sources, notably plasma and lung in vivo PK and simulation of expected scenarios in a clinical setting, notably lung infections.

AZI2 positively regulates the induction of type I interferon in influenza-trigger pediatric pneumonia

5-azacytidine-induced protein 2 (AZI2) is known to have a crucial role in antiviral innate immunity. This study aims to explore the roles of AZI2 in influenza-trigger pediatric pneumonia and its molecular mechanism. qPCR and immunoblotting assays were used to determine the levels of target genes and proteins. The lung infection mouse model was established by using PR8 H1N1 virus in AZI2 germline knockout (AZI2-/-) and wild-type (WT) mice. In addition, HEK293T cell-based luciferase reporter assays were used to investigate the regulatory effects of AZI2 on type I interferon. Immune precipitation and immunofluorescence staining were used to evaluate the interactions between AZI2 and TANK binding kinase 1 (TBK1). We observed an elevation in the expressions of IFN-I and AZI2 in peripheral blood mononuclear cells from the pneumonia patients with mild symptoms. Interestingly, AZI2 deficiency deteriorated the influenza-induced pathological symptoms in the lung as well as reduced the survival rate. It was further showed that AZI2 positively regulated the expressions of type I interferon, inflammatory cytokines, and IFN production-related genes. The molecular mechanism data revealed that AZI2 regulated the interactions between TBK1 and TANK. In summary, AZI2 positively regulates type I interferon production in influenza-induced pediatric pneumonia by promoting the interactions between TBK1 and TANK.

Comparison of L- and D-Amino Acids for Bacterial Imaging in Lung Infection Mouse Model.

The effectiveness of L- and D-amino acids for detecting the early stage of infection in bacterial imaging was compared. We evaluated the accumulation of 3H-L-methionine (Met), 3H-D-Met, 3H-L-alanine (Ala), and 3H-D-Ala in E. coli EC-14 and HaCaT cells. Biological distribution was assessed in control and lung-infection-model mice with EC-14 using 3H-L- and D-Met, and 18F-FDG. A maximum accumulation of 3H-L- and D-Met, and 3H-L- and D-Ala occurred in the growth phase of EC-14 in vitro. The accumulation of 3H-L-Met and L-Ala was greater than that of 3H-D-Met and D-Ala in both EC-14 and HaCaT cells. For all radiotracers, the accumulation was greater in EC-14 than in HaCaT cells at early time points. The accumulation was identified at 5 min after injection in EC-14, whereas the accumulation gradually increased in HaCaT cells over time. There was little difference in biodistribution between 3H-L-and D-Met except in the brain. 3H-L- and D-Met were sensitive for detecting areas of infection after the spread of bacteria throughout the body, whereas 18F-FDG mainly detected primary infection areas. Therefore, 11C-L- and D-Met, radioisotopes that differ only in terms of 3H labeling, could be superior to 18F-FDG for detecting bacterial infection in lung-infection-model mice.

The effects of different doses of inhaled bacteriophage therapy for Pseudomonas aeruginosa pulmonary infections in mice

ObjectivesInhaled phage therapy has been revisited as a potential treatment option for respiratory infections caused by multidrug-resistant Pseudomonas aeruginosa; however, there is a distinct gap in understanding the dose–response effect. The aim of this study was to investigate the dose–response effect of Pseudomonas-targeting phage PEV31 delivered by the pulmonary route in a mouse lung infection model. MethodsNeutropenic BALB/c mice were infected with multidrug-resistant P. aeruginosa (2 × 104 colony-forming units) through the intratracheal route and then treated with PEV31 at three different doses of 7.5 × 104 (Group A), 5 × 106 (Group B), and 5 × 108 (Group C) plaque-forming units, or phosphate-buffered saline at 2 hours postinoculation. Mice (n = 5–7) were euthanized at 2 hours and 24 hours postinfection, and lungs, kidneys, spleen, liver, bronchoalveolar lavage fluid, and blood were collected for bacteria and phage enumeration. ResultsAt 24 hours postinfection, all phage-treated groups exhibited a significant reduction in pulmonary bacterial load by 1.3–1.9 log10, independent of the delivered phage dose. The extent of phage replication was negatively correlated with the dose administered, with log10 titre increases of 6.2, 2.7, and 9 for Groups A, B, and C, respectively. Phage-resistant bacterial subpopulations in the lung homogenate samples harvested at 24 hours postinfection increased with the treatment dose (i.e. 30%, 74%, and 91% in respective Groups A–C). However, the mutants showed increased susceptibility to ciprofloxacin, impaired twitching motility, and reduced blue-green pigment production. The expression of the inflammatory cytokines (IL-1ß and IL-6, and TNF-α) was suppressed with increasing PEV31 treatment dose. DiscussionThis study provides the dose–response effect of inhaled phage therapy that may guide dose selection for treating P. aeruginosa respiratory infections in humans.

Discovery of a Tricyclic β-Lactam as a Potent Antimicrobial Agent against Carbapenem-Resistant Enterobacterales, Including Strains with Reduced Membrane Permeability and Four-Amino Acid Insertion into Penicillin-Binding Protein 3: Structure-Activity-Relationships and In Vitro and In Vivo Activities.

The current worldwide emergence of carbapenem-resistant enterobacterales (CREs) constitutes an important growing clinical and public health threat. Acquired carbapenemases are the most important determinants of resistance to carbapenems. In the development of the previously reported tricyclic β-lactam skeleton which exhibits potent antibacterial activities against several problematic β-lactamase-producing CREs without a β-lactamase inhibitor, we found that these activities were reduced against clinical isolates with resistance mechanisms other than β-lactamase production. These mechanisms were the reduction of outer membrane permeability with the production of β-lactamases and the insertion of four amino acids into penicillin-binding protein 3. Here, we report the discovery of a potent compound that overcomes these resistance mechanisms by the conversion of the alkoxyimino moiety of the aminothiazole side chain in which a hydrophilic functional group is introduced and the carboxylic acid of the alkoxyimino moiety is converted to reduce the negative charge of the whole molecule from 2 to 1. This potent tricyclic β-lactam is a promising drug candidate for infectious diseases caused by CREs due to its potent therapeutic efficacy in the neutropenic mouse lung infection model and low frequency of producing spontaneously resistant mutants.