Lung Tissue Culture Research Articles

P-763. Assessment of the in vitro potency of anti-mycobacterial agents against clinical Mycobacterium avium complex (MAC) pulmonary isolates: implications of intravenous and inhaled amikacin breakpoint reporting

Abstract Background MAC pulmonary disease is associated with significant morbidity and requires multi-drug treatment for extended periods. The novel dosage form amikacin liposome inhalation suspension (ALIS) represents a much-needed treatment option and is noted to have success at MICs of ≤64mg/L, higher than the intravenous (IV) amikacin (AMK) breakpoint (≤16 mg/L) due to optimized pulmonary exposure. Herein, we assessed the in vitro potency of AMK utilizing both ALIS and IV breakpoints as well as other standard antimycobacterial agents. Methods MAC isolates obtained from sputum, bronchoalveolar lavage, or lung tissue culture between 1/1/2018 - 3/1/24 were assessed. The first isolate per patient per year was assessed. MICs were collected from the medical record as determined by standard methods at one of two reference laboratories. If a single sample had two MIC values reported, the more conservative was used for analysis. MICs to AMK, clarithromycin (CLR), moxifloxacin (MXF), and linezolid (LNZ) were collected. Data were assessed as MIC50/90 (mg/L) and categorized per CLSI breakpoints. Patients were classified as receiving MAC directed treatment based on documentation in the medical record. If treatment data was unknown, patients were classified as indeterminate treatment. Results 218 samples were included from 178 patients. CLR was the most active agent with 97% of isolates testing as susceptible (MIC50/90, 0.5/2). MXF and LNZ had relatively poor activity with 82% and 66% of isolates susceptible to each agent, respectively. Considering AMK, high susceptibility to the IV breakpoint was observed with 94% of isolates meeting this criterion (MIC50/90, 8/16). Among the 14 isolates that were intermediate or resistant to IV AMK, 13 remained susceptible to ALIS (MIC range 32-64). 54%, 26%, and 20% of patients were treated, untreated, or indeterminate, respectively. The percent of isolates susceptible to each agent between treated and untreated were similar for all agents. Conclusion AMK and CLR remain highly active against MAC pulmonary isolates. Amongst isolates intermediate or resistant to IV AMK, most isolates remained susceptible to ALIS. These data highlight the need for laboratories to report susceptibility to both IV and ALIS to avoid clinicians erroneously disregarding a therapeutic option. Disclosures Christian Mark Gill, PharmD, BCIDP, Cepheid: Grant/Research Support|Entasis: Grant/Research Support|Everest Medicines: Grant/Research Support|Shionogi: Grant/Research Support

Abstract PR017: Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics

Abstract Introduction: Tumors systemically remodel the immune system during metastasis. Developing anti-metastatic immunotherapies that target these tumor-immune interactions to make the metastatic niche hostile colonization would represent a paradigm shift in cancer treatment. However, traditional high-throughput screening (HTS) platforms that use simple read-outs and contrived in vitro systems are ill-suited to identify such therapies. In contrast, HTS platforms that accurately model the metastatic niche and use high-dimensional read-outs such as multiplexed single-cell RNA-sequencing (scRNA-seq) can accurately profile nuanced perturbation responses in individual immune cell types and therefore have the potential to discover anti-metastatic immunotherapies. In this study, we describe the development of an ex vivo lung tissue culture HTS platform that preserves immune gene expression signatures observed in the in vivo metastatic niche and is amenable to single-cell chemical transcriptomic analysis using MULTI- seq (McGinnis et al., Nature Methods, 2019). Inspired by our recent description of myeloid cell TLR-NFκB inflammation during breast cancer lung metastasis (McGinnis et al., Cancer Cell, 2024), we then performed the first anti-metastatic immunotherapy drug screen and identified TLR-NFκB inhibitors that effectively operate on all desired myeloid cell types in the lung metastatic niche. Methods and Results: Building upon existing techniques for culturing precision-cut lung slices (Wu et al., Journal of Experimental Medicine, 2024) and patient- derived tumor fragments (Voabil et al., Nature Medicine, 2021), we established and optimized an ex vivo lung tissue culture system that enables 48-hour culturing of lung slices isolated from 4T1 tumor-bearing mice. scRNA-seq profiling revealed successful capture of all relevant immune cell types and retention of metastasis-associated gene expression programs (e.g., myeloid TLR-NFκB inflammation and neutrophil degranulation) following culture. We then scaled our platform to address how myeloid cells in the lung metastatic niche respond to perturbations targeting every component of the canonical TLR-NFκB signaling cascade. Cell-type-specific perturbation modeling and quantification of TLR-NFκB signaling inhibition identified compounds that optimally perturb all intended myeloid cell types (e.g., tissue-resident macrophages, neutrophils, and monocytes) and represent prioritized candidates for future in vivo validation studies to assess impact on metastatic disease progression. Conclusions: In this study, we describe the development and application of a novel HTS platform that couples ex vivo lung tissue cultures with single-cell chemical transcriptomics to identify anti-metastatic immunotherapy candidates. We demonstrate how our platform can identify TLR-NFκB signaling inhibitors that optimally operate in the lung metastatic niche, paving the way for future interrogation of additional metastasis-associated immune cell signaling pathways in different disease and tissue backgrounds. Citation Format: Chris McGinnis, Winnie Yao, Ansuman Satpathy. Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR017.

Organotypic lung tissue culture as a preclinical model to study host- influenza A viral infection: A case for repurposing of nafamostat mesylate

Reliable and effective models for recapitulation of host-pathogen interactions are imperative for the discovery of potential therapeutics. Ex vivo models can fulfill these requirements as the multicellular native environment in the tissue is preserved and be utilized for toxicology, vaccine, infection and drug efficacy studies due to the presence of immune cells. Drug repurposing involves the identification of new applications for already approved drugs that are not related to the prime medical indication and emerged as a strategy to cope with slow pace of drug discovery due to high costs and necessary phases to reach the patients. Within the scope of the study, broad-spectrum serine protease inhibitor nafamostat mesylate was repurposed to inhibit influenza A infection and evaluated by a translational ex vivo organotypic model, in which human organ-level responses can be achieved in preclinical safety studies of potential antiviral agents, along with in in vitro lung airway culture. The safe doses were determined as 10 µM for in vitro, whereas 22 µM for ex vivo to be applied for evaluation of host-pathogen interactions, which reduced virus infectivity, increased cell/tissue viability, and protected total protein content by reducing cell death with the inflammatory response. When the gene expression levels of specific pro-inflammatory, anti-inflammatory and cell surface markers involved in antiviral responses were examined, the significant inflammatory response represented by highly elevated mRNA gene expression levels of cytokines and chemokines combined with CDH5 downregulated by 5.1-fold supported the antiviral efficacy of NM and usability of ex vivo model as a preclinical infection model.

Evaluation of combination therapies with colistin in an experimental mouse pneumonia model induced by carbapenem-resistant Acinetobacter baumannii strain.

The treatment options are limited in Acinetobacter baumannii infections. In this study, the effectiveness of colistin monotherapy and combinations of colistin with different antibiotics were investigated in an experimental pneumonia model induced by carbapenem-resistant A.baumannii strain. Mice in the study were divided into five groups as control (no treatment), colistin monotherapy, colistin + sulbactam, colistin + imipenem, and colistin + tigecycline combinations. The modified experimental surgical pneumonia model of Esposito and Pennington was applied to all groups. The presence of bacteria in blood and lung samples was investigated. Results were compared. In blood cultures, while there was no difference between the control and colistin groups, there was a statistical difference between the control and the combination groups (P = 0.029). When the groups were compared in terms of lung tissue culture positivity, there was a statistical difference between the control group and all treatment groups (colistin, colistin + sulbactam, colistin + imipenem, and colistin + tigecycline) (P = 0.026, P < 0.001, P < 0.001, and P = 0.002, respectively). The number of microorganisms that grew in the lung tissue was found to be statistically significantly lower in all treatment groups in comparison with the control group (P = 0.001). Both monotherapy and combination therapies of colistin were found to be effective in the treatment of carbapenem-resistant A.baumannii pneumonia, but the superiority of combination therapies over colistin monotherapy has not been demonstrated.

TSLP and IL25 variants are related to asthma and atopy

Asthma is a chronic inflammatory disease that affects millions of people worldwide. There are several phenotypes and endophenotypes of this condition, and some are associated with increased eosinophil counts in blood and sputum. Studies have shown that severe asthma may be associated with genes related to type 2 innate lymphoid cells (ILC2). This study evaluated the association between ILC2-related gene variants (TSLP and IL25) with asthma endophenotypes and atopy markers. Individuals belonging to the Programa para Controle da Asma na Bahia (ProAR) cohort were diagnosed according to the Global Initiative for Asthma (GINA) criteria, and divided into three subgroups: controls with no asthma, mild to moderate asthma (MMA), and severe asthma (SA). Blood collection, DNA extraction, genotyping, blood count, total IgE dosage, and cytokine dosage were performed. Logistic regression models were conducted for the association analysis. We found significant protective associations between the alternative alleles of variants in the TSLP gene with asthma and its severity. Notably, rs10062929 (A), rs11466741 (T), and rs149540660 (T) were related to eosinophil counts, IgE levels, and cytokine levels of our patients. We found significant associations between IL25 gene variants with asthma endophenotypes and atopy markers. The rs10143597 (G) and rs3811178 (G) showed a protective association against atopy. We also evaluated the in silico expression levels of these variants in lung tissue, skin, and fibroblast culture. Our results indicate that variants in these genes may regulate atopy, asthma, and their different endophenotypes in an admixed Brazilian population.

Abstract 14698: Identifying Sources of Prostacyclin Outside the Vascular Wall and Their Contribution to Cardiovascular Protection

Introduction: Prostacyclin is a key anti-thrombotic hormone synthesised through cyclooxygenase (COX) enzymes. Historically, its production has been almost universally associated with the endothelium however the role of non-vascular sources has not been systematically evaluated due to a lack of suitable tools. This has resulted in an underappreciation of its cardiovascular biology and role in other contexts. Hypothesis: We hypothesised that cells outside the vascular wall contribute to generation of prostacyclin which can enter the circulation to provide anti-thrombotic protection. Methods: Global, endothelial and fibroblast COX1 or COX2 knockout mice were studied alongside freshly isolated healthy human lung tissue and primary cell cultures. Prostacyclin levels in plasma and released from cells/tissue ex vivo were measured by ELISA. Individual cell populations were isolated by FACS. Thrombosis in vivo was measured using a FeCl 3 -carotid artery injury model. Results: Vascular endothelial COX1 deletion strongly reduced prostacyclin release from mouse aorta and heart, but not from isolated lung, colon, kidney or spleen (Fig A). This non-vascular prostacyclin required COX1 as it was abolished by global COX1 and not COX2 deletion. Freshly isolated lung fibroblasts exhibited greater prostacyclin synthesis than endothelial or other cell types (Fig B) and this was confirmed in primary human lung cell cultures. Deletion of COX1 from fibroblasts in mice reduced lung prostacyclin release (Fig C) and plasma prostacyclin levels (Fig D) and this was associated with accelerated thrombotic occlusion after carotid artery injury (Fig E). Conclusions: These findings define a new paradigm in prostacyclin biology in which fibroblast/non-vascular-derived prostacyclin works in parallel with endothelium-derived prostanoids to control thrombosis. This may have particular relevance to understanding how local diseases of the lung result in cardiovascular risk.

Pulmonary Granulomas and Mycobacterial Infection: Concordance between the Results of Special Stains Performed on Lung Tissue Sections and Tissue Cultures.

The most common cause of infectious pulmonary granulomas worldwide is Mycobacterium tuberculosis. The diagnosis is based on clinical presentation, histopathologic findings, detection of acid-fast bacilli (AFB) in tissue or sputum using special stains, and/or isolation of mycobacteria in cultures or via PCR-based methods. Different studies have shown that high levels of discrepancy exist between these diagnostic approaches in lung tissue specimens. To assess the degree of concordance between the results of special stains and cultures on lung tissue specimens in the diagnosis of mycobacterial infections. Eighty-seven patients with a diagnosis of granulomas (necrotizing and non-necrotizing) on lung tissue specimens were identified. Cohen's kappa was used to measure the general concordance between the results of the histopathological examination (special stains) and bacteriological tissue cultures. With Kinyoun acid-fast stains, 8/48 (16.7%) cases were positive for AFB. With FITE stains, 10/57 (17.5%) cases were positive for AFB. There was strong agreement between Kinyoun acid-fast and FITE stains (Kappa = 0.806; p-value &lt; 0.001). Tissue cultures were performed on 38/87 cases (43.7%), and 10/38 (26.3%) of the cultures were positive for mycobacteria. There was no concordance between Kinyoun acid-fast stains or FITE stains and tissue cultures results. Our observations represent an initial step in the process of reviewing the two methods used at our institution to diagnose mycobacterial infections on lung tissue specimens and highlight the need of incorporating more advanced diagnostic methods such as PCR to confirm mycobacterial infections and improve patient management. Importantly, species-level identification of mycobacteria is necessary to guide treatment.

Study on the pharmacodynamic activity of combinations with the new anti-tuberculosis drug pyrifazimine in vitro and in vivo in mouse

Objective: To evaluate two-drug combination interaction between pyrifazimine(TBI-166) and anti-drug-resistant tuberculosis group A drugs Bedaquiline (BDQ), Moxifloxacin (MFX) and the new anti-tuberculosis drug Delamanid (DLM), SQ109, Q203, and PBTZ169 in vitro and in vivo in mouse, so as to provide basis for TBI-166 combination therapy. Methods: This study was performed from September 2020 to July 2021. The chessboard method was used to evaluate the interaction between TBI-166 and BDQ, MFX, DLM, SQ109, and PBTZ169. The time-killing kinetics method was used to evaluate the anti-tuberculosis activity of the two-drug combination with partial synergy. The BALB/c mouse acute infection model was used to evaluate the anti-tuberculosis activity at 4 and 8 weeks in the two-drug combination group (TBI-166+BDQ, TBI-166+SQ109, TBI-166+PBTZ169, TBI-166+Q203) and monotherapy groups (TBI-166, BDQ, SQ109, PBTZ169, Q203). Data analysis was performed using an independent sample t-test. Results: After TBI-166 combined with anti-tuberculosis drugs, MIC was reduced to 6.25% to 25.00% of TBI-166 monotherapy. After TBI-166 combined with BDQ, SQ109 and PBTZ169, the partial inhibitory concentration index (FICI) values were 0.53, 0.75 and 0.75, respectively; the time sterilization experiment showed that the viable population of Mycobacterium tuberculosis treated with two-drug combination of TBI-166 and BDQ, SQ109, PBTZ169 for 14 days decreased at least 3 log10 CFU/ml. In the mouse experiments, it was found that, the amount of viable bacteria in lung tissue of BDQ, SQ109 and PBTZ169 combined with TBI-166 groups was lower than that of the monotherapy group,respectively. The lung tissue culture of mice in the TBI-166+BDQ group was negative after 4 weeks of treatment, and the number of live bacteria in the lungs of the TBI-166+BDQ group was 1.49 log10CFU lower than that of the BDQ monotherapy group(P<0.01). Conclusion: In vitro and in vivo experiments in mice revealed that TBI-166 had synergistic anti-tuberculosis activity after being combined with BDQ, SQ109 and PBTZ169, respectively.

Congenital Pulmonary Lymphangiectasia impersonating pulmonary interstitial emphysema in an extremely low gestational age neonate

Background: The purpose of this case report is to illustrate the clinical course of an extremely rare case of Congenital Pulmonary Lymphangiectasia (CPL) presenting as an early pulmonary interstitial emphysema (PIE) complicating respiratory distress syndrome, in an extremely low gestational age neonate (ELGAN) along with a short review of the literature. Case presentation: This 24 weeks ELGAN male with radiologic changes consistent with pulmonary interstitial emphysema (PIE) complicating respiratory distress syndrome which later progressed to bilateral cystic lung changes, expired at 23 days of age. Maternal history was complicated by E. coli urinary tract infection and Group-B Strep chorioamnionitis. The infant remained intubated throughout the hospital course and received antibiotics initially and terminally. His tracheal aspirate cultures also grew Mycoplasma hominis, and Ureaplasma urealyticum, treated with azithromycin. An autopsy revealed diffuse bilateral congenital lymphangiectasia. Postmortem blood and lung tissue cultures were positive for methicillin-resistant Staphylococcus aureus (MRSA). Conclusion: Despite its extreme rarity, the authors recommend considering congenital pulmonary lymphangiectasia in the differential diagnosis, in an extremely preterm neonate, presenting with early pulmonary interstitial emphysema (PIE), and respiratory failure refractory to surfactant, antibiotic therapy, and complex ventilator management.

Pulmonary actinomycosis: cytomorphological features.

Pulmonary actinomycosis is an uncommon infectious disease. Although the gold standard for diagnosis is histological examination with bacterial culture of lung tissue, cytology samples offer a fast and low-cost alternate diagnostic procedure. The cytology literature on this topic is limited to mostly case reports. Therefore, the aim of this study was to review cytological material in a series of patients with a diagnosis of pulmonary actinomycosis to characterize the main cytomorphological findings. Different cytological respiratory samples including sputum smears, bronchoalveolar lavages (BALs), transthoracic or endobronchial fine needle aspiration cytology (FNAC) and cell block preparations were used for retrospective examination. For all cases patient age, gender, symptoms, and radiological chest findings were recorded. A total of 26 cytological respiratory samples (14 sputum smears, 9 FNAC, two BALs) including direct smears and 6 cell blocks from 9 patients were examined. In sputum smears the most remarkable findings were the presence of dark cotton ball masses with projections like spider legs and/or mouse tails (75% of the samples). Sulfur granules were observed in 4 (40%) of the sputum smears and within FNAC cases. Various respiratory cytology samples including sputum smears, FNAC and BALs can reveal cytomorphological findings diagnostic of pulmonary actinomycosis. Characteristic cytological findings compatible with a diagnosis of this infection include cotton ball masses and less frequently sulfur granules.

Metformin Attenuates Silica-Induced Pulmonary Fibrosis by Activating Autophagy via the AMPK-mTOR Signaling Pathway.

Long-term exposure to crystalline silica particles leads to silicosis characterized by persistent inflammation and progressive fibrosis in the lung. So far, there is no specific treatment to cure the disease other than supportive care. In this study, we examined the effects of metformin, a prescribed drug for type || diabetes on silicosis and explored the possible mechanisms in an established rat silicosis model in vivo, and an in vitro co-cultured model containing human macrophages cells (THP-1) and human bronchial epithelial cells (HBEC). Our results showed that metformin significantly alleviated the inflammation and fibrosis of lung tissues of rats exposed to silica particles. Metformin significantly reduced silica particle-induced inflammatory cytokines including transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in rat lung tissue and HBEC culture supernatant. The protein levels of Vimentin and α-smooth muscle actin (α-SMA) were significantly decreased by metfomin while expression level of E-cadherin (E-Cad) increased. Besides, metformin increased the expression levels of phosphorylated adenosine 5′-monophosphate (AMP)-activated protein kinase (p-AMPK), microtubule-associated protein (MAP) light chain 3B (LC3B) and Beclin1 proteins, and reduced levels of phosphorylated mammalian target of rapamycin (p-mTOR) and p62 proteins in vivo and in vitro. These results suggest that metformin could inhibit silica-induced pulmonary fibrosis by activating autophagy through the AMPK-mTOR pathway.

Acute electronic vapour product whole aerosol exposure of 3D human bronchial tissue results in minimal cellular and transcriptomic responses when compared to cigarette smoke

The use of electronic vapour products (EVPs) continues to increase worldwide and with advances in cell culture systems, molecular biology and the computational sciences there is also accumulating evidence of their potential reduced toxicity and reduced potential harm when compared to cigarette smoke. To further understand the potential risks and health effects associated with exposure to EVP aerosols we have assessed the cellular and transcriptomic response from a commercially available lung tissue culture system (MucilAirTM) following a single sub-cytotoxic exposure to cigarette smoke and the equivalent nicotine delivered dose of EVP aerosol. The transcriptomic, cellular (cilia beat frequency (CBF) and percent active area (%AA), trans epithelial electrical resistance (TEER), histology) and cytokine release were assessed at 4- and 48- hours following recovery from air, EVP aerosol (8.4% V/V: mybluTM blueberry flavour, 2.4% nicotine) and 3R4F smoke (3.5% V/V: exposure). No pathological changes were observed at either recovery time point from any exposure. Air and EVP aerosol exposure had no effect on CBF, %AA nor TEER at 48 hours. Exposure to cigarette smoke resulted in a decrease in TEER, an increase in CBF and the release of proinflammatory cytokines at both recovery time points. Although the number of significantly expressed genes was minimal following exposure to EVP aerosol, exposure to 3R4F smoke resulted in a significant upregulation of several disease relevant pathways. These data provide evidence that following an acute exposure to EVP aerosol there is significantly less damage to lung cells in culture than the equivalent, nicotine based, dose of cigarette smoke.

Decellularized Human Lung Scaffolds as Complex Three-Dimensional Tissue Culture Models to Study Functional Behavior of Fibroblasts.

In vitro culturing of cells in two-dimensional (2D) environments is a widespread used methodology in biomedical research. Most commonly, cells are cultured on artificial plastic dish surfaces, which lead to abnormal functional behaviors, as plastic does not reflect the native microenvironment found in vivo or in situ. Therefore, a multitude of three-dimensional (3D) cell culture systems were developed in the past years, which aim to bridge the gap between 2D cell culture dishes and the in vivo situation. One of the more recent development in the field, the generation of viable precision-cut tissue slices from various organs emerged as an exciting approach to study complex interactions and biological processes ex vivo in 3D. Decellularization of such tissue slices leads to the removal of all functional cells, and leaves behind a scaffold of extracellular matrix (ECM), which closely recapitulates the molecular composition, mechanical properties, topology, and microarchitecture of native ECM. Subsequently, decellularized precision-cut lung slices (PCLS), also called 3D lung tissue culture (3D-LTCs), can be successfully reseeded with a variety of cell types, including fibroblasts, which attach to and engraft into the matrix. Here, we describe the generation of PCLS from resected human lung tissue and their decellularization and recellularization with primary human fibroblasts. This novel 3D tissue culture model allows for various functional studies of fibroblast behavior on native ECM composition and topology.

Elucidating the mechanisms of colistin resistance and the efficacy of combination therapy in MDR/XDR/PDR Acinetobacter baumannii

Background: The rapid dissemination of carbapenem resistance in Gram-negative bacteria, particularly in Acinetobacter baumannii, has gained focus on the “old” drug colistin. Neverthless, the emergence of colistin resistance, last line of defense, causes a serious global threat. Therefore, antimicrobial treatment for these infections remains an important challenge. In this study, we aimed (1) to understand the involvement of the Two-Component system pmrAB and lipid biosynthesis lpx genes in colistin resistance and their corresponding relative expression levels and (2) implement suitable combination therapy options. Methods and materials: Eighty clinical isolates of A. baumannii were identified phenotypically and confirmed by PCR of GyrA gene. Isolates were subjected to antimicrobial susceptibility testing by BMD. The role of pmrAB and lpx genes on colistin resistance was determined by qRT-PCR. Assessment of the effect of combination therapy was done by the checkerboard analysis, Time kill experiments and the neutropenic pneumonia-induced mouse model. The effect of the electrostatic interaction of colistin action was seen by the Scanning Electron Microscopy. Results: Data have shown that 13 of the 80 (16%) isolates were resistant to colistin. The efficacy of colistin in combination with tigecycline, fosfomycin, zerbaxa, tazocin, teicoplanin, amikacin, levofloxacin, rifampicin and meropenem was assessed against colistin resistant A. baumannii isolates. Checkerboard and Time-kill assays demonstrated a synergestic effect between colistin and rifampicin and between colistin and teicoplanin, with bactericidal activity occuring prior to 4 h of incubation. A preliminary neutropenic pneumonia-induced mouse model, demonstrated improved survival and pathogen clearance from the blood and lung tissue cultures in the rifampicin combination. Up-regulation of pmrAB genes was recorded with a down-regulation of the lpx genes in vitro, nonethless this was not the case in in vivo studies. In addition, qRT-PCR did not show any significant differences between resistant and susceptible isolates upon examining the levels of expression of lpx and pmrAB genes. Conclusion: In conclusion, further understanding of the various molecular mechanisms of colistin resistance in A. baumannii is a must. Our current efforts focused on correlating the results of combination therapy in relation to the different resistance mechanisms to colistin, paving the path for potential treatment options against XDR and PDR A. baumannii infections.

Hypermucoviscous/hypervirulent and extensively drug-resistant QnrB2-, QnrS1-, and CTX-M-3-coproducing Klebsiella pneumoniae ST2121 isolated from an infected elephant (Loxodonta africana)

The rapid dissemination of extended-spectrum β-lactamases (ESBLs)-producing Enterobacterales from different spheres worldwide over recent years has become a serious problem in both human and veterinary medicine. CTX-M-3-type ESBL has only been reported on few occasions, and in Brazil the blaCTX-M-3 gene has been identified only once in clinical strains. In this study, we aimed to molecularly characterize a hypermucoviscous (hm), hypervirulent (hv), and extensively drug-resistant (XDR) Klebsiella pneumoniae strain isolated from a lung tissue culture of an infected elephant. The A246 strain belonged to ST2121 and presented hm phenotype, hypervirulence-associated genes, and carried blaCTX-M-3 and plasmid-mediated quinolone resistance genes (qnrB2 and qnrS1) on an IncFII-IncQ1-IncM1 multireplicon plasmid (pA246-CTX-M-3, ∼ 162 kb). A novel genetic context of blaCTX-M-3, in which a 482-bp ISEcp1 was truncated by an IS26, was also harbored by pA246-CTX-M-3. Furthermore, in vivo experiments revealed that the hm/hv A246 strain killed 100 % of the Galleria mellonella larvae at 72 h post-infection. Our findings evidence the intercontinental dissemination of a rare K. pneumoniae ST2121 and the multidrug resistance IncFII-IncQ1-IncM1 plasmid. Therefore, to the best of our knowledge, this is the first report of an XDR K. pneumoniae coproducing CTX-M-3, QnrB2, and QnrS1 isolated from captive wild animals.

Clinical Features, Replication Competence, and Innate Immune Responses of Human Adenovirus Type 7 Infection.

Epidemiologic reports suggest that the most severe or fatal adenoviral disease in children might be associated with human adenovirus (HAdV) type 7. However, the pathogenesis of HAdV-7-induced severe disease remains poorly understood. HAdV-3 and HAdV-7 replication kinetics and the host response to infection were compared using ex vivo human lung tissue cultures. Furthermore, cytokine and chemokine levels and the presence of adenovirus DNA in the serum of hospitalized children infected with HAdV-7 (n = 65) or HAdV-3 (n = 48) were measured (using a multiplex immunoassay and Taqman real-time polymerase chain reaction, respectively). Among 471 HAdV-positive specimens, HAdV-3 or HAdV-7 was the most prevalent genotype during 2014-2016 or 2018, respectively. The incidence of severe pneumonia was higher in HAdV-7-infected than in HAdV-3-infected individuals (30.1% vs 4.5%, respectively). HAdV-7 replicated more efficiently than HAdV-3 ex vivo. Interferon-induced protein 10, interleukin 6, and monocyte chemoattractant protein 1 levels were significantly higher in HAdV-7-infected than in HAdV-3-infected children. Adenovirus DNA was detected in serum samples from 40% and 4.2% of HAdV-7- and HAdV-3-infected children, respectively. Furthermore, viremia was strongly associated with severe clinical presentations. The pathogenesis of HAdV-7-induced severe disease was probably associated with high replication competence and hyperinflammatory responses. The detection of adenovirus DNA in blood may be useful in assessing risk for severe disease.

The accuracy of ultrasound-guided lung biopsy pathology and microbial cultures for peripheral lung lesions.

BackgroundUltrasound-guided lung biopsy (USLB) is a minimally invasive, real-time, safe and effective way to diagnose peripheral lung lesions. USLB is now widely used in clinical practice. However, the accuracy of USLB lacks large sample studies, and specimens from USLB are seldom used for microbial cultures. In this study, the efficiency of diagnosing tumours and infectious diseases with USLB is investigated, and the safety of USLB is evaluated.MethodsFrom September 2015 to August 2018, a total of 507 cases of USLB were retrospectively reviewed, including the pathological results, microbial culture results and complications. The final diagnosis was confirmed by clinical diagnosis and follow-up. The technical success rate and accuracy of USLB were calculated.ResultsSix patients were excluded because they were lost to follow-up. A total of 501 patients were finally included (335 males and 166 females). The diagnostic accuracy of USLB was 82.0%. The accuracy of USLB in diagnosing lung tumours was 88.5% and that for diagnosing special infections was 55.2%. The positive rate of USLB lung tissue cultures was 18.3%. USLB can diagnose some microbial infections with high accuracy, such as mycobacterial infections, and Cryptococcus neoformans, Aspergillus fumigatus and Burkholderia cepacia infections.ConclusionsUSLB is a safe and effective method for diagnosing peripheral lung lesions with high accuracy and a low complication rate. Lung tissue pathology and cultures have good diagnostic value for tuberculosis and fungi infections. Lung tissue cultures can also diagnose bacterial infections and can contribute to the selection of antibiotics.

Increased expression of heme oxygenase-1 suppresses airway branching morphogenesis in fetal mouse lungs exposed to inflammation.

Intrauterine inflammation affects fetal lung development. BTB and CNC homology 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1) and interleukin-6 (IL-6) genes. We investigated the role of Bach1 in the development of fetal mouse lungs exposed to lipopolysaccharide (LPS) using a whole fetal lung tissue culture system. We isolated and cultured embryonic day 12.5 fetal mouse lungs from pregnant Bach1 knockout (-/-) and wild-type (WT) mice. Airway branching morphogenesis was assessed by microscopically counting peripheral lung buds after incubation with/without LPS. Expression levels of genes related to inflammation and oxidative stress were evaluated using quantitative PCR. Zinc protoporphyrin, HO-1-specific inhibitor, was used. Branching morphogenesis was observed in Bach1-/- and WT fetal mice lungs without LPS exposure; after exposure to LPS, the number of peripheral lung buds was suppressed in Bach1-/- group only. Basal messenger RNA (mRNA) and protein expression of HO-1 was significantly higher in Bach1-/- group than in WT group; IL-6 and monocyte chemoattractant protein-1 mRNA expression was significantly increased after LPS exposure in both groups. Zinc protoporphyrin mitigated the LPS-induced suppression of branching morphogenesis in Bach1-/- mice. The ablation of Bach1 suppresses airway branching morphogenesis after LPS exposure by increased basal expression levels of HO-1.

In vivo antibacterial activity of Zataria multiflora Boiss extract and its components, carvacrol, and thymol, against colistin-resistant Acinetobacter baumannii in a pneumonic BALB/c mouse model.

Acinetobacter baumannii has emerged as a major cause of nosocomial infections. Various resistance mechanisms of A. baumannii against antibiotics have transformed it into a successful nosocomial pathogen. Because of the limited number of available antibiotics, we used a medicinal plant with an antibacterial effect. Zataria multiflora Boiss (ZMB) extract and its components were used for the treatment of pneumonic mice infected with A. baumannii. The biological effects of this extract and the regulation of the outer membrane protein A (ompA) gene were used in a mouse model. A pneumonic mouse model was prepared using clinical and standard strains (1.5 × 108 colony-forming units/mL) of A. baumannii. BALB/c mice groups were treated with a ZMB extract, carvacrol, thymol, and sensitive antibiotics. The lung tissues of the treated mice were cultured for 5 days and each day, bacterial clearance and the ompA gene expression were assessed by quantitative real-time polymerase chain reaction. In the lung tissue culture of pneumonic mice infected with standard or clinical isolate, no colony was detected when treated with the ZMB extract after 2 and 3 days (P < 0.01), respectively. In the carvacrol-treated group, bacterial clearance was seen at day 4 and day 5 (P < 0.05). Bacterial clearance was seen 5 days after treatment with thymol and imipenem and 6 days after ampicillin/sulbactam treatment. The regulation of ompA gene was significantly decreased in this order: ZMB extract, carvacrol, thymol, imipenem, and ampicillin/sulbactam. The ZMB extract had a potent bactericidal effect against A. baumannii that could downregulate the ompA gene. ZBM extract and carvacrol could be novel therapeutic agents for antibiotic-resistant A. baumannii.

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue's stiffness, which is crucial for subsequent slicing. 500 µm thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.