Mouse Model Of Pneumonia Research Articles

Characterization of Pseudomonas aeruginosa bacteriophages and control hemorrhagic pneumonia on a mice model.

Pseudomonas aeruginosa is one of the most common pathogens causing hemorrhagic pneumonia in Chinese forest musk deer. Multidrug-resistant P. aeruginosa is frequently isolated from the lungs of affected musk deer in Shaanxi Province, China. With the increasing bacterial drug resistance, commonly used antibiotics have shown limited efficacy against drug-resistant P. aeruginosa. Therefore, phages have garnered attention as a promising alternative to antibiotics among researchers. In this study, phages vB_PaeP_YL1 and vB_PaeP_YL2 (respectively referred to as YL1 and YL2) were isolated from mixed sewage samples from a farm. YL1 and YL2 exhibit an icosahedral head and a non-contractile short tail, belonging to the Podoviridae family. Identification results demonstrate good tolerance to low temperatures and pH levels, with minimal variation in potency within 30 min of UV irradiation. The MOI for both YL1 and YL2 was 0.1, and their one-step growth curve latent periods were 10 min and 20 min, respectively. Moreover, both single phage and phage cocktail effectively inhibited the growth of the host bacteria in vitro, with the phage cocktail showing superior inhibitory effects compared to the single phage. YL1 and YL2 possess double-stranded DNA genomes, with YL1 having a genome size of 72,187 bp and a total G + C content of 55.02%, while YL2 has a genome size of 72,060 bp and a total G + C content of 54.98%. YL1 and YL2 are predicted to have 93 and 92 open reading frames (ORFs), respectively, and no ORFs related to drug resistance or lysogeny were found in both phages. Genome annotation and phylogenetic analysis revealed that YL1 is closely related to vB_PaeP_FBPa1 (ON857943), while YL2 is closely related to vB_PaeP_FBPa1 (ON857943) and Phage26 (NC041907). In a mouse model of hemorrhagic pneumonia, phage cocktail treatment showed better control of the disease and significantly reduced lung bacterial load compared to single phage treatment. Therefore, YL1 and YL2 have the potential for the prevention and treatment of multidrug-resistant P. aeruginosa infections.

Single-dose of hybrid eukaryotic-prokaryotic nanovesicles to drive a rapid and controllable immune response against Klebsiella pneumoniae induced nosocomial pneumonia

Klebsiella pneumoniae is the main pathogen of hospital-acquired infections causing acute pneumonia, which is characterized by drug resistance, virulence and mortality, and for which there is no clinically approved vaccine formulations. Here, we designed biomimetic hybrid membrane vesicles (HMVs), which consist of outer membrane vesicles of Klebsiella pneumoniae and cell membrane vesicles of alveolar macrophages. The HMVs were administered via intratracheal instillation to mimic the delivery of an inhaled formulation, where immune cells could be rapidly mobilized in the lungs to promote pathogen-specific immunity and to establish local mucosal immunity. In a mouse model of acute pneumonia with experimental strain 43,816 and clinical strain 7,29,088, immunization of HMVs via the lungs provided significant protection, reduced the number of colonies in deep tissues, improved survival, and reduced symptoms of acute inflammation. As expected, HMVs not only retained the immunogenicity of OMVs, but also effectively reduced the excessive immune response induced by OMVs. In conclusion, as a proof-of-concept for the design of antibacterial vaccines, the HMVs is an effective platform for activating bacteria-specific immunity, which can rapidly and effectively protect against nosocomial pneumonia caused by Klebsiella pneumoniae.

Luteolin-7-O-glucoside promotes macrophage release of IFN-β by maintaining mitochondrial function and corrects the disorder of glucose metabolism during RSV infection

Respiratory syncytial virus (RSV) pneumonia is the main cause of acute bronchiolitis in infants. Luteolin-7-O-glucoside (LUT-7G) is a natural flavonoid, which exists in a variety of plants and has the potential to treat viral pneumonia. We established RSV pneumonia mouse models and RSV-infected cell models. Clodronate liposomes were used to deplete macrophages. We used HE staining and immunohistochemistry to determine inflammatory damage and virus replication. We detected the expression levels of inflammatory factors and IFN-β through qPCR and ELISA. JC-1 kit was used for detecting the cell mitochondrial Membrane potential (MMP). ROS, SOD, and MDA kits were used for detecting intracellular oxidative stress damage. Metabolites of TCA in lung tissue and serum of mice were detected by GC-MS. Pharmacodynamic studies have shown that intervention with LUT-7G can alleviate lung tissue damage caused by RSV infection, inhibit RSV replication, and downregulate TNF-α, IL-1β, and IL-6 mRNA expression. LUT-7G upregulated the IFN-β content and the expression of IFN-β, ISG15, and OAS1 mRNA. In vitro, LUT-7G inhibited RSV-induced cell death, reversed the RSV-induced decrease of MMP and decreased intracellular oxidative stress. Target metabonomics showed that RSV infection upregulated the levels of glycolysis and TCA metabolites in lung tissue and serum, while LUT-7G could improve the disorder of glucose metabolism. The results indicate that LUT-7G can promote the release of IFN-β in the lung, alleviate inflammatory damage, and inhibit RSV replication during RSV infection. These effects may be achieved by protecting the mitochondrial function of alveolar macrophages and correcting the disorder of glucose metabolism.

Differential effects of microRNAs miR-21, miR-99 and miR-145 on lung regeneration and inflammation during recovery from influenza pneumonia.

In a mouse model of influenza pneumonia, we previously documented that proliferating alveolar type II (AT2) cells are the major stem cells involved in early lung recovery. Profiling of microRNAs revealed significant dysregulation of specific ones, including miR-21 and miR-99a. Moreover, miR-145 is known to exhibit antagonism to miR-21. This follow-up study investigated the roles of microRNAs miR-21, miR-99a, and miR-145 in the murine pulmonary regenerative process and inflammation during influenza pneumonia. Inhibition of miR-21 resulted in severe morbidity, and in significantly decreased proliferating AT2 cells due to impaired transition from innate to adaptive immune responses. Knockdown of miR-99a culminated in moderate morbidity, with a significant increase in proliferating AT2 cells that may be linked to PTEN downregulation. In contrast, miR-145 antagonism did not impact morbidity nor the proliferating AT2 cell population, and was associated with downregulation of TNF-alpha, IL1-beta, YM1, and LY6G. Hence, a complex interplay exists between expression of specific miRNAs, lung regeneration, and inflammation during recovery from influenza pneumonia. Inhibition of miR-21 and miR-99a (but not miR-145) can lead to deleterious cellular and molecular effects on pulmonary repair and inflammatory processes during influenza pneumonia.

Elizabethkingia anophelis outer membrane vesicles as a novel vaccine candidate against infection: insights into immune response and potential for passive immunity.

Elizabethkingia anophelis, a Gram-negative pathogen, causes infections such as bacteraemia, pneumonia, and neonatal meningitis. The pathogen resists most antimicrobial classes, making novel approaches urgently needed. In natural settings, Gram-negative bacteria secrete outer membrane vesicles (OMVs) that carry important molecules in the bacterial life cycle. These OMVs are enriched with proteins involved in virulence, survival, and carbohydrate metabolism, making them a promising source for vaccine development against the pathogen. This study investigated the efficacy of imipenem-induced OMVs (iOMVs) as a vaccine candidate against E. anophelis infection in a mouse pneumonia model. Mice immunized with iOMVs were completely protected during lethal-dose challenges. Passive immunization with hyperimmune sera and splenocytes conferred protection against lethal pneumonia. Further investigation is needed to understand the mechanisms underlying the protective effects of iOMV-induced passive immunity, such as the action on specific antibody subclasses or T cell subsets.

Inhibition of influenza a virus infection by natural stilbene piceatannol targeting virus hemagglutinin.

Given the magnitude of influenza pandemics as a threat to the global population, it is crucial to have as many prevention and treatment options as possible. Piceatannol (PIC) is a tetrahydroxylated stilbenoid (trans-3,4,3',5'-tetrahydroxystilbene), also known as 3'- hydroxy resveratrol, which has demonstrated many different biological activities such as anti-inflammatory and antiviral activities. In this study, the anti-influenza A virus (IAV) activities and mechanisms of PIC in vitro and in vivo were investigated in order to provide reference for the development of novel plant-derived anti-IAV drugs. The viral plaque assay, RT-PCR and western blot assay were used to evaluate the anti-IAV effects of PIC in vitro. The anti-IAV mechanism of PIC was determined by HA syncytium assay, DARTS assay and Surface Plasmon Resonance assay. The mouse pneumonia model combined with HE staining were used to study the anti-IAV effects of PIC in vivo. PIC shows inhibition on the multiplication of both H1N1 and H3N2 viruses, and blocks the infection of H5N1 pseudovirus with low toxicity. PIC may directly act on the envelope of IAV to induce the rupture and inactivation of IAV particles. PIC can also block membrane fusion via binding to HA2 rather than HA1 and cleavage site of HA0. PIC may interact with the two residues (HA2-T68 and HA2-I75) of HA2 to block the conformational change of HA so as to inhibit membrane fusion. Importantly, oral therapy of PIC also markedly improved survival and reduced viral titers in IAV-infected mice. PIC possesses significant anti-IAV effects both in vitro and in vivo and may block IAV infection mainly through interaction with HA to block membrane fusion. Thus, PIC has the potential to be developed into a new broad-spectrum anti-influenza drug for the prevention and treatment of influenza.

Bilobetin attenuates Staphylococcus aureus virulence by targeting Von Willebrand factor-binding protein and staphylocoagulase.

Staphylococcus aureus (S. aureus) induces a variety of infectious diseases in humans and animals and is responsible for hospital- and community-acquired infections. The aim of this study was to investigate how bilobetin, a natural compound, attenuates S. aureus virulence by inhibiting two key virulence factors, von Willebrand factor-binding protein (vWbp) and staphylocoagulase (Coa). The results showed that bilobetin inhibited Coa- or vWbp-induced coagulation without affecting S. aureus proliferation. The Western blotting and fluorescence quenching assays indicated that bilobetin did not affect the expression of vWbp and Coa but directly bound to the proteins with KA values of 1.66 × 104L/mol and 1.04 × 104L/mol, respectively. To gain further insight into the mechanism of interaction of bilobetin with these virulence factors, we performed molecular docking and point mutation assays, which indicated that the TYR-6 and TYR-18 residues on vWbp and the ALA-190 and ASP-189 residues on Coa were essential for the binding of bilobetin. In addition, the in vivo studies showed that bilobetin ameliorated lung tissue damage and inflammation caused by S. aureus, thereby improving the survival of mice. Furthermore, the use of bilobetin as an adjuvant in combination with vancomycin was more effective in the treatment of a mouse model of pneumonia. Taken together, bilobetin had a dual inhibitory effect on vWbp and Coa by reducing the virulence of S. aureus, suggesting that it is a viable lead compound against S. aureus infections.

Multifunctional Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-Based Nanobomb against Carbapenem-Resistant Acinetobacter baumannii Infection through Cascade Reaction and Amplification Synergistic Effect.

Carbapenems have been considered to be the preferred antibiotics against Acinetobacter baumannii thus far. However, carbapenem-resistant Acinetobacter baumannii (CRAB) has gradually escalated worldwide, and it frequently causes respiratory and bloodstream infections. Its resistance may lead to high mortality. Thus, there is an urgent need to develop antibacterial drugs. In our research, the pH-sensitive sgRNA-I/L@ZS nanosystem delivered imipenem and better released it in infected tissues to synergistically damage bacteria with nanoparticles. Gene editing of the CRISPR-Cas9 nanosystem amplified the synergistic effect by reversing the drug-resistance of imipenem. Nitric oxide, which l-arginine reacted with ROS to produce in cascade reaction and bacterial infection sites, was beneficial to heal the infected tissues and induce bacteria death for further enhancing antibacterial effects. In addition, this nanocomposite influenced host-bacteria interactions and restrained and destroyed biofilms. The sgRNA-I/L@ZS nanosystem, similar to a nanobomb, was a high-efficiency bactericide against CRAB. Eventually, in acute pneumonia and peritonitis mouse models, the sgRNA-I/L@ZS nanosystem could combat bacteria and protect tissues from infection. It had marked suppressive effects on inflammation and promoted healing and proliferation of infected tissues. This multifunctional nanosystem is expected to be an effective antibacterial agent in the clinic based on good biocompatibility and no toxic side effects. Therefore, developing the nanocomposites will take a favorable step toward solving intractable public health issues.

In vitro and in vivo antimicrobial potential of lithium complex against multi-drug resistant Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen, accountable for lethal nosocomial infections, owing to high pathogenicity and multi-drug resistance (MDR). Hence, the World Health Organization has prioritized this bacterium against which new pharmacophores are required urgently. Therefore, this study evaluates the antimicrobial potential of lithium complex based on salicylic acid, and 1,10-phenanthroline {[Li(phen)2sal]} against MDR A. baumannii (MDRAB) through growth inhibition, growth curve assay, biofilm inhibition, and eradication alongside microscopy. The mechanism of action was proposed by flow cytometry and proteomic analysis. Moreover, in vivo efficacy was assessed using an A. baumannii-induced pneumonia model in mice. Results showed that lithium complex significantly inhibited the bacterial growth at 16-32 µg/mL. Growth kinetics revealed that it retarded the bacterial growth after 2-3 h of treatment. Lithium complex significantly inhibited and reduced >70% of biofilms at the MIC level which is a key problem of indwelling and prosthetic devices. Light and atomic force microscopy further indicated disarticulated biofilm and ruptured cellular membrane post-lithium complex treatment. Proteomic analysis revealed that the compound exerted oxidative stress on A. baumannii which may contribute to cell rupturing. Data are available via ProteomeXchange with identifier PXD044293. Flow cytometry confirmed its role in cytoplasmic membrane disintegration. The lithium complex ameliorated the pneumonia symptoms at non-toxic doses of 20 and 40 mg/kg in the pre-clinical A. baumannii-induced pneumonia model as evident by the significant reduction in bacterial burden and parenchymal inflammation. Taken together, these findings suggest that the lithium complex can be a potential drug candidate against MDRAB.IMPORTANCEMulti-drug resistance (MDR) by virtue of evolving resistance and virulence mechanisms among A. baumannii is a global concern which is responsible for lethal hospital-acquired infections. Therefore, it is crucial to develop new therapeutics against it. Metal complexes are compact structures with diverse mechanisms that the pathogens cannot evade easily which make them a strong drug candidate. In this study, we assessed the in vitro and in vivo efficacy of lithium complex {[Li(phen)2 sal]} against biofilm-forming MDR A. baumannii. The lithium complex displayed strong antimicrobial activity and reduced the pre-formed mature biofilm which is key barrier for antimicrobial action. Moreover, it employs oxidative stress as one of its mode of actions and causes cellular rupturing. Lithium complex was non-toxic and was significantly effective to overcome pneumonia in mice model. These results highlight the untapped potential of metal complexes that can be explored and utilized for combating notorious A. baumannii infections.

Impact of the new membrane-targeting lipoglycopeptide antibiotic MCC5145 on the treatment of bacteremic pneumococcal pneumonia in mice.

Bacteremic Streptococcus pneumoniae pneumonia is one of the most severe forms of invasive pneumococcal disease (IPD) and with particularly high case-fatality rates among the elderly and individuals with comorbidities, exacerbated by rising antibiotic resistance and time to initiation of therapy. Here, we examined the efficacy of the preclinical "vancapticin" glycopeptide MCC5145 against fulminant infection by S. pneumoniae serotype 2 strain D39 in a bioluminescent, neutropenic mouse model of bacteremic pneumonia. MCC5145 is a semisynthetic vancomycin derivative chemically modified at the C-terminus with a membrane-targeting motif designed to preferentially bind the anionic bacterial surface. We show that similar to vancomycin, subcutaneous administration of MCC5145 to mice 1 day after intranasal infection with a bioluminescent derivative of S. pneumoniae D39 elicited time and concentration-dependent reduction in total flux in the lungs and blood. Together, our finding supports the further development of MCC5145 as a potential new treatment option for pneumonia and/or bacteremic pneumonia in clinical settings, particularly for immunocompromised individuals. IMPORTANCE S. pneumoniae (the pneumococcus) causes severe community acquired lung and blood infection, especially among the elderly and people with underlying medical conditions and/or weakened immune systems. The rising incidence of antibiotic resistance and delays between diagnosis of infection and commencement of effective therapy make treatment difficult and result in high mortality rates. In this work, we show that a new derivative (MCC5145) of an existing antibiotic (vancomycin) rapidly eradicated lethal pneumococcal challenge from the lungs and blood of mice with a suppressed immune system. Our findings support that MCC5145 is a promising option for the treatment of lung and blood infections caused by the pneumococcus at point-of-care settings, particularly for the elderly and individuals with a weakened immune system.

Inhibitory effect of bilobalide on Staphylococcus aureus von Willebrand factor-binding protein and its therapeutic effect in mice with pneumonia.

Disabling bacterial virulence with small molecules has been proposed as a potential strategy to prevent bacterial pathogenicity. The von Willebrand factor-binding protein of Staphylococcus aureus was identified previously as a key virulence determinant. Our objective was to discover a von Willebrand-factor binding protein (vWbp) inhibitor distinct from the antibiotics used to prevent infections resulting from S. aureus. Using coagulation assays, we found that the sesquiterpene trilactone bilobalide blocks coagulation mediated by vWbp, but has no impact on the growth of S. aureus at a concentration of 128μg ml-1. Moreover, a mouse model of pneumonia caused by S. aureus indicated that bilobalide could attenuate S. aureus virulence in vivo. This effect is achieved not by interfering with the expression of vWbp but by binding to vWbp, as demonstrated by western blotting, thermal shift assays, and fluorescence quenching assays. Using molecular dynamic simulations and point mutagenesis analysis, we identified that the Q17A and R453A residues are key residues for the binding of bilobalide to vWbp. Overall, we tested the ability of bilobalide to inhibit S. aureus infections by targeting vWbp and explored the potential mechanism of this activity.

QingXiaoWuWei decoction alleviates methicillin-resistant Staphylococcus aureus-induced pneumonia in mice by regulating metabolic remodeling and macrophage gene expression network via the microbiota-short-chain fatty acids axis.

QingXiaoWuWei decoction (QXWWD) exerts a prominent therapeutic effect on the methicillin-resistant Staphylococcus aureus (MRSA)-induced pneumonia model in mice; however, its pharmacological mechanisms remain unclear. This study aimed to investigate the underlying pharmacological mechanisms of QXWWD in MRSA-induced pneumonia. In the present study, 62 compounds were identified using high-resolution mass spectrometry. Network analysis, leveraging mass spectrometry, pinpointed the infection-linked, immunity-associated, and inflammation-related pathways as predominant targets. QXWWD significantly alleviated MRSA-induced pneumonia in mice and decreased the levels of pro-inflammatory cytokines and chemokines. 16S ribosomal RNA (16S rRNA) sequencing revealed that QXWWD regulated gut microbiota composition in mice with MRSA-induced pneumonia, which correlated with the enrichment of certain short-chain fatty acids (SCFAs)-producing strains. Further analysis with targeted metabolomics confirmed that the acetic, propionic, and butyric acid levels in the mice's serum were elevated significantly after QXWWD treatment. The fecal microbiota transplantation experiment suggested that gut microbiota from QXWWD-treated mice and SCFAs treatment may alleviate MRSA-induced pneumonia. Additionally, the untargeted metabolomic analysis further demonstrated that metabolic remodeling is significantly regulated by the QXWWD, particularly by the enhancement of the citrate cycle. In the case of QXWWD treatment, global transcriptome profiling revealed that genes, such as NLRP12 and CYP1A1, associated with macrophage antibacterial and immune activity, were downregulated. The results revealed that QXWWD regulated metabolic remodeling and macrophage gene expression network via the microbiota-SCFAs axis and thus alleviated MRSA-induced pneumonia in mice.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) colonizes the upper respiratory airways and is resistant to antibiotics. MRSA is a frequently acquired infection in hospital and community settings, including cases of MRSA-induced pneumonia. Multidrug-resistant Staphylococcus aureus and the limited efficacy of antibiotics necessitate alternative strategies for preventing or treating the infection. QingXiaoWuWei decoction (QXWWD) protects against both gut microbiota dysbiosis and MRSA-induced pneumonia. Furthermore, the QXWWD-regulated metabolic remodeling and macrophage gene expression network contribute to its protective effects through the microbiota-short-chain fatty acid axis. The results of this study suggest that QXWWD and its pharmacodynamic compounds might have the potential to prevent and treat pulmonary infections, especially those caused by multidrug-resistant organisms. Our study provides a theoretical basis for the future treatment of pulmonary infectious diseases by manipulating gut microbiota and their metabolites via traditional Chinese medicine.

Investigating Pseudomonas aeruginosa Gene Function During Pathogenesis Using Mobile-CRISPRi.

CRISPR interference (CRISPRi) is a robust gene silencing technique that is ideal for targeting essential and conditionally essential (CE) genes. CRISPRi is especially valuable for investigating gene function in pathogens such as P. aeruginosa where essential and CE genes underlie clinically important phenotypes such as antibiotic susceptibility and virulence. To facilitate the use of CRISPRi in diverse bacteria-including P. aeruginosa-we developed a suite of modular, mobilizable, and integrating vectors we call, "Mobile-CRISPRi." We further optimized Mobile-CRISPRi for use in P. aeruginosa mouse models of acute lung infection by expressing the CRISPRi machinery at low levels constitutively, enabling partial knockdown of essential and CE genes without the need for an exogenous inducer. Here, we describe protocols for creating Mobile-CRISPRi knockdown strains and testing their phenotypes in a mouse pneumonia model of P. aeruginosa infection. In addition, we provide comprehensive guide RNA designs to target genes in common laboratory strains of P. aeruginosa and other Pseudomonas species.

Antibacterial efficacy of an ultra-short palmitoylated random peptide mixture in mouse models of infection by carbapenem-resistant Klebsiella pneumoniae.

Indiscriminate use of antibiotics has imposed a selective pressure for the rapid rise in bacterial resistance, creating an urgent need for novel therapeutics for managing bacterial infectious diseases while counteracting bacterial resistance. Carbapenem-resistant Klebsiella pneumoniae strains have become a major challenge in modern medicine due to their ability to cause an array of severe infections. Recently, we have shown that the 20-mer random peptide mixtures are effective therapeutics against three ESKAPEE pathogens. Here, we evaluated the toxicity, biodistribution, bioavailability, and efficacy of the ultra-short palmitoylated 5-mer phenylalanine:lysine (FK5P) random peptide mixtures against multiple clinical isolates of carbapenem-resistant K. pneumoniae and K. oxytoca. We demonstrate the FK5P rapidly and effectively killed various strains of K. pneumoniae, inhibited the formation of biofilms, and disrupted mature biofilms. FK5P displayed strong toxicity profiles both in vitro and in mice, with prolonged favorable biodistribution and a long half-life. Significantly, FK5P reduced the bacterial burden in mouse models of acute pneumonia and bacteremia and increased the survival rate in a mouse model of bacteremia. Our results demonstrate that FK5P is a safe and promising therapy against Klebsiella species as well as other ESKAPEE pathogens.

Inhibitory Effect of Salicin on Staphylococcus aureus Coagulase.

The massive use of antibiotics has resulted in an alarming increase in antibiotic resistance in Staphylococcus aureus (S. aureus). This study aimed to identify the inhibitory effect of salicin on S. aureus. Coagulase (Coa) activity was assessed using in vitro Coa assays and Western blot, thermal shift assay (TSA), fluorescence quenching and molecular docking experiments were conducted to verify the interaction between salicin and Coa. An in vivo mouse pneumonia model demonstrated that salicin can reduce the virulence of S. aureus. In vitro Coa assays elucidated that salicin directly inhibited Coa activity. The Western blot and TSA results suggested that salicin did not block the expression of Coa but affected the thermal stability of the protein by binding to Coa. The fluorescence quenching, molecular docking and molecular dynamics assays have found that the most promising binding site between salicin and Coa was GLN-97. The pneumonia model of mice infected with S. aureus revealed that salicin could not only reduce the content of lung bacteria in mice but also prolong their survival. Salicin was identified as a novel anti-infective candidate compound with the potential to target Coa and inhibit its activity by binding to it, which would facilitate the development of roadmaps for future research.

Murepavadin induces envelope stress response and enhances the killing efficacies of β-lactam antibiotics by impairing the outer membrane integrity of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can cause a variety of acute and chronic infections. The bacterium is highly resistant to numerous antibiotics. Murepavadin is a peptidomimetic antibiotic that blocks the function of P. aeruginosa lipopolysaccharide (LPS) transport protein D (LptD), thus inhibiting the insertion of LPS into the outer membrane. In this study, we demonstrated that sublethal concentrations of murepavadin enhance the bacterial outer membrane permeability. Proteomic analyses revealed the alteration of protein composition in bacterial inner and outer membranes following murepavadin treatment. The antisigma factor MucA was upregulated by murepavadin. In addition, the expression of the sigma E factor gene algU and the alginate synthesis gene algD was induced by murepavadin. Deletion of the algU gene reduces bacterial survival following murepavadin treatment, indicating a role of the envelope stress response in bacterial tolerance. We further demonstrated that murepavadin enhances the bactericidal activities of β-lactam antibiotics by promoting drug influx across the outer membrane. In a mouse model of acute pneumonia, the murepavadin-ceftazidime/avibactam combination showed synergistic therapeutic effect against P. aeruginosa infection. In addition, the combination of murepavadin with ceftazidime/avibactam slowed down the resistance development. In conclusion, our results reveal the response mechanism of P. aeruginosa to murepavadin and provide a promising antibiotic combination for the treatment of P. aeruginosa infections.IMPORTANCEThe ever increasing resistance of bacteria to antibiotics poses a serious threat to global public health. Novel antibiotics and treatment strategies are urgently needed. Murepavadin is a novel antibiotic that blocks the assembly of lipopolysaccharide (LPS) into the Pseudomonas aeruginosa outer membrane by inhibiting LPS transport protein D (LptD). Here, we demonstrated that murepavadin impairs bacterial outer membrane integrity, which induces the envelope stress response. We further found that the impaired outer membrane integrity increases the influx of β-lactam antibiotics, resulting in enhanced bactericidal effects. In addition, the combination of murepavadin and a β-lactam/β-lactamase inhibitor mixture (ceftazidime/avibactam) slowed down the resistance development of P. aeruginosa. Overall, this study demonstrates the bacterial response to murepavadin and provides a new combination strategy for effective treatment.

Time-course transcriptome analysis of lungs from mice infected with inhaled aerosolized Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia (SMA) has emerged as an important pathogen capable of causing an opportunistic and nosocomial infection. We performed RNA sequencing (RNA-seq) of lung tissues from mice with pulmonary SMA infection over time via aerosolized intratracheal inhalation to investigate transcription profile changes in SMA-infected lungs. A mouse model of acute lethal SMA pneumonia was established in this study using aerosolized intratracheal inhalation, laying the groundwork for future SMA research. RNA-seq was then used to create a transcriptional profile of the lungs of the model mice at 0, 4, 12, 24, 48, and 72 hours post-infection (hpi). Mfuzz time clustering, weighted gene coexpression network analysis (WGCNA), and Immune Cell Abundance Identifier for mouse (ImmuCellAI-mouse) were used to analyze RNA-seq data. A gradual change in the lung transcriptional profile was observed, which was consistent with the expected disease progression. At 4 hpi, the expression of genes related to the acute phase inflammatory response increased, as predicted abundance of innate immune cells. At this stage, an increased demand for energy was also observed, including an increase in the expression of genes involved in circulation, muscle function and mitochondrial respiratory chain function. The expression of genes associated with endoplasmic reticulum stress (ERS) and autophagy increased at 24 hpi. Unlike the number of natural killer (NK) cells following most bacterial lung infections, the abundance of NK cells decreased following infection with SMA. The expression levels of Cxcl10, Cd14, Gbp5, Cxcr2, Tnip1, Zc3h12a, Egr1, Sell and Gbp2 were high and previously unreported in SMA pneumonia, and they may be important targets for future studies. To our knowledge, this is the first study to investigate the pulmonary transcriptional response to SMA infection. The findings shed light on the molecular mechanisms underlying the pathogenesis of SMA pneumonia, which may aid in the development of therapies to reduce the occurrence of SMA pulmonary infection.

A novel phage carrying capsule depolymerase effectively relieves pneumonia caused by multidrug-resistant Klebsiella aerogenes

BackgroundKlebsiella aerogenes can cause ventilator-associated pneumonia by forming biofilms, and it is frequently associated with multidrug resistance. Phages are good antibiotic alternatives with unique advantages. There has been a lack of phage therapeutic explorations, kinetic studies, and interaction mechanism research targeting K. aerogenes.MethodsPlaque assay, transmission electron microscopy and whole-genome sequencing were used to determine the biology, morphology, and genomic characteristics of the phage. A mouse pneumonia model was constructed by intratracheal/endobronchial delivery of K. aerogenes to assess the therapeutic effect of phage in vivo. Bioinformatics analysis and a prokaryotic protein expression system were used to predict and identify a novel capsule depolymerase. Confocal laser scanning microscopy, Galleria mellonella larvae infection models and other experiments were performed to clarify the function of the capsule depolymerase.ResultsA novel lytic phage (pK4-26) was isolated from hospital sewage. It was typical of the Podoviridae family and exhibited serotype specificity, high lytic activity, and high environmental adaptability. The whole genome is 40,234 bp in length and contains 49 coding domain sequences. Genomic data show that the phage does not carry antibiotic resistance, virulence, or lysogenic genes. The phage effectively lysed K. aerogenes in vivo, reducing mortality and alleviating pneumonia without promoting obvious side effects. A novel phage-derived depolymerase was predicted and proven to be able to digest the capsule, remove biofilms, reduce bacterial virulence, and sensitize the bacteria to serum killing.ConclusionsThe phage pK4-26 is a good antibiotic alternative and can effectively relieve pneumonia caused by multidrug-resistant K. aerogenes. It carries a depolymerase that removes biofilms, reduces virulence, and improves intrinsic immune sensitivity.

Abstract P2192: Growth Hormone-releasing Hormone Receptor Antagonist Attenuates Cardiopulmonary Injury Induced By A Bsl2 Rvsv-sars-cov-2-s Virus Mimic Of Sars-cov-2 Infectivity

Background: COVID-19 causes severe pulmonary injury that can lead to acute respiratory distress syndrome. Growth hormone-releasing hormone receptor (GHRH-R) and its splice variant are expressed in murine and human lung and heart. GHRH-R antagonist, MIA-602, has been shown to modulate cellular immune responses to bleomycin lung injury and decrease inflammation in models of sarcoid granuloma. Using the BSL2-friendly rVSV-SARS-CoV-2-S of K18 hACE2tg mice to mimic native SARS-CoV-2 infection, we tested our hypothesis that MIA-602 attenuates cardiopulmonary injury in this COVID-19 model. Methods: Male and female K18 hACE2tg mice were inoculated with SARS-CoV-2 Washington (WA-1) native strain, recombinant VSV-SARS-CoV-2-Spike virus (rVSV-SARS-CoV-2-S), or PBS and lung viral load, weight loss and histopathology compared between groups (N=5-8). K18 h ACE2 tg mice infected with rVSV-SARS-CoV-2-S were subject to daily subcutaneous injections of 10 μg MIA-602 or vehicle starting at 24h post-infection. Pulmonary function was measured via whole-body plethysmography on day 0, day 3, and day 5 (n=7). Five days after viral infection mice were sacrificed; and blood and tissues collected for histopathological analyses, H&E staining and ICAM-1 immunohistochemistry. T-test or One-way ANOVA-test was used for statistical analysis. Results: Native SARS-CoV-2 and rVSV-SARS-CoV-2-S presented with similar patterns of weight loss, infectivity (~60%) and histopathologic changes. Daily treatment with MIA-602 ameliorated weight loss, reduced lung perivascular inflammation and pneumonia, and decreased lung/heart ICAM-1 expression compared to vehicle. MIA-602 rescued respiratory rate, increased expiratory parameters (Te, PEF, EEP) and mitigated dysregulated measures of airway obstruction (Penh and Rpef) compared to vehicle. Conclusions: rVSV-SARS-CoV-2-S is an accurate and safe alternative to native SARS-CoV-2 for preclinical studies. Daily treatment with the synthetic peptide GHRH-R antagonist MIA-602 attenuates pulmonary dysfunction and heart inflammation in this new preclinical mouse model of COVID-19 pneumonia. We hypothesize that the molecular mechanism involves anti-inflammatory actions of MIA-602.

Bakuchiol reduces the severity of Mycoplasma pneumoniae-induced pneumonia in mice by attenuating inflammatory responses

BackgroundPneumonia has become a leading cause of death due to its high morbidity rates worldwide. Pneumonia is caused by the bacterium Mycoplasma pneumoniae (MP), which is a prominent airway pathogen. MP infection is usually self-limiting, but an alarming increase in cases has recently developed into refractory, severe, and even fatal pneumonia. ObjectiveThe objective of the current work was to study the anti-inflammatory and antioxidant properties of bakuchiol in ameliorating MP-induced pneumonia in mice. MethodologyBALB/c mice were challenged with MP (100 µl) via nasal drops for 2 days to induce pneumonia, and bakuchiol (25 mg/kg) was given concurrently to the mice for 3 days. Thereafter, measurements of lung weight, MPO, and NO concentrations were done. The corresponding kits were used to analyze the GSH, SOD, and MDA concentrations. The DNA contents of MP were assessed by PCR, and the total cells in BALF were counted. The corresponding kits were used to measure NF-κB levels and examine the inflammatory cytokine levels in the BALF samples. The lung histopathology was assessed to identify the histological changes. ResultsIn pneumonia-induced mice, the lung weight was significantly reduced after treatment with 25 mg/kg of bakuchiol. The bakuchiol treatment exhibited an effective antioxidant and anti-inflammatory properties on the pneumonia mice. Bakuchiol also lowered the NF-κB level and attenuated histological alterations in the lungs of pneumonia-induced mice. Conclusion: In summary, our findings exhibited that bakuchiol significantly reduced inflammation induced by MP in a mouse model of pneumonia. As a result, it has potential as a future salutary agent for the pneumonia treatment.