In Vitro Synergistic Effects of Omadacycline with Other Antimicrobial Agents against Mycobacterium abscessus.

The clinical importance of Mycobacterium abscessus species (MABS) infections has been increasing. However, the standard treatment regimens recommended in the current guidelines often result in unfavorable outcomes. Therefore, we investigated the in vitro activity of omadacycline (OMC), a novel tetracycline, against MABS to explore its potential as a novel therapeutic option. The drug susceptibilities of 40 Mycobacterium abscessus subsp. abscessus (Mab) clinical strains obtained from the sputum of 40 patients from January 2005 to May 2014 were investigated. The MIC results for OMC, amikacin (AMK), clarithromycin (CLR), clofazimine (CLO), imipenem (IPM), rifabutin (RFB), and tedizolid (TZD) alone and their combined effects (with OMC) were examined using the checkerboard method. Additionally, we studied the differences in the effectiveness of the antibiotic combinations based on the colony morphotype of Mab. The MIC50 and MIC90 of OMC alone were 2 and 4 µg/mL, respectively. The combinations of OMC with AMK, CLR, CLO, IPM, RFB, and TZD showed synergy against 17.5%, 75.8%, 25.0%, 21.1%, 76.9%, and 34.4% of the strains, respectively. Additionally, OMC combined with CLO (47.1% versus 9.5%, P = 0.023) or TZD (60.0% versus 12.5%, P = 0.009) showed significantly higher synergy against strains with rough morphotypes than those with smooth morphotypes. In conclusion, the checkerboard analyses revealed that the synergistic effects of OMC were observed most frequently with RFB, followed by CLR, TZD, CLO, IPM, and AMK. Furthermore, OMC tended to be more effective against rough-morphotype Mab strains.

Left-Right Reversal Recurrently Evolved Regardless of Diaphanous-Related Formin Gene Duplication or Loss in Snails.

Bilateria exhibit whole-body handedness in internal structure. This left-right polarity is evolutionarily conserved with virtually no reversed extant lineage, except in molluscan Gastropoda. Phylogenetically independent snail groups contain both clockwise-coiled (dextral) and counterclockwise-coiled (sinistral) taxa that are reversed from each other in bilateral handedness as well as in coiling direction. Within freshwater Hygrophila, Lymnaea with derived dextrality have diaphanous related formin (diaph) gene duplicates, while basal sinistral groups possess one diaph gene. In terrestrial Stylommatophora, dextral Bradybaena also have diaph duplicates. Defective maternal expression of one of those duplicates gives rise to sinistral hatchlings in Lymnaea and handedness-mixed broods in Bradybaena, through polarity change in spiral cleavage of embryos. These findings led to the hypothesis that diaph duplication was crucial for the evolution of dextrality by reversal. The present study discovered that diaph duplication independently occurred four times and its duplicate became lost twice in gastropods. The dextrality of Bradybaena represents the ancestral handedness conserved across gastropods, unlike the derived dextrality of Lymnaea. Sinistral lineages recurrently evolved by reversal regardless of whether diaph had been duplicated. Amongst the seven formin gene subfamilies, diaph has most thoroughly been conserved across eukaryotes of the 14 metazoan phyla and choanoflagellate. Severe embryonic mortalities resulting from insufficient expression of the duplicate in both of Bradybaena and Lymnaea also support that diaph duplicates bare general roles for cytoskeletal dynamics other than controlling spiralian handedness. Our study rules out the possibility that diaph duplication or loss played a primary role for reversal evolution.

Genome-wide association study in patients with pulmonary Mycobacterium avium complex disease.

RATIONALE: Nontuberculous mycobacteria (NTM) are environmental mycobacteria that can cause a chronic progressive lung disease. Although epidemiological data indicate potential genetic predisposition, its nature remains unclear. OBJECTIVES: We aimed to identify host susceptibility loci for Mycobacterium avium complex (MAC), the most common NTM pathogen. METHODS: This genome-wide association study (GWAS) was conducted in Japanese patients with pulmonary MAC and healthy controls, followed by genotyping of candidate single-nucleotide polymorphisms (SNPs) in another Japanese cohort. For verification by Korean and European ancestry, we performed SNP genotyping. RESULTS: The GWAS discovery set included 475 pulmonary MAC cases and 417 controls. Both GWAS and replication analysis of 591 pulmonary MAC cases and 718 controls revealed the strongest association with chromosome 16p21, particularly with rs109592 (p=1.64×10-13, OR 0.54), which is in an intronic region of the calcineurin-like EF-hand protein 2 (CHP2). Expression quantitative trait loci analysis demonstrated an association with lung CHP2 expression. CHP2 was expressed in the lung tissue in pulmonary MAC disease. This SNP was associated with the nodular bronchiectasis subtype. Additionally, this SNP was significantly associated with the disease in patients of Korean (p=2.18×10-12, OR 0.54) and European (p=5.12×10-03, OR 0.63) ancestry. CONCLUSIONS: We identified rs109592 in the CHP2 locus as a susceptibility marker for pulmonary MAC disease.

Clarithromycin expands CD11b+Gr-1+ cells via the STAT3/Bv8 axis to ameliorate lethal endotoxic shock and post-influenza bacterial pneumonia.

Macrolides are used to treat various inflammatory diseases owing to their immunomodulatory properties; however, little is known about their precise mechanism of action. In this study, we investigated the functional significance of the expansion of myeloid-derived suppressor cell (MDSC)-like CD11b+Gr-1+ cells in response to the macrolide antibiotic clarithromycin (CAM) in mouse models of shock and post-influenza pneumococcal pneumonia as well as in humans. Intraperitoneal administration of CAM markedly expanded splenic and lung CD11b+Gr-1+ cell populations in naïve mice. Notably, CAM pretreatment enhanced survival in a mouse model of lipopolysaccharide (LPS)-induced shock. In addition, adoptive transfer of CAM-treated CD11b+Gr-1+ cells protected mice against LPS-induced lethality via increased IL-10 expression. CAM also improved survival in post-influenza, CAM-resistant pneumococcal pneumonia, with improved lung pathology as well as decreased interferon (IFN)-γ and increased IL-10 levels. Adoptive transfer of CAM-treated CD11b+Gr-1+ cells protected mice from post-influenza pneumococcal pneumonia. Further analysis revealed that the CAM-induced CD11b+Gr-1+ cell expansion was dependent on STAT3-mediated Bv8 production and may be facilitated by the presence of gut commensal microbiota. Lastly, an analysis of peripheral blood obtained from healthy volunteers following oral CAM administration showed a trend toward the expansion of human MDSC-like cells (Lineage-HLA-DR-CD11b+CD33+) with increased arginase 1 mRNA expression. Thus, CAM promoted the expansion of a unique population of immunosuppressive CD11b+Gr-1+ cells essential for the immunomodulatory properties of macrolides.

Obesity worsens the outcome of influenza virus infection associated with impaired type I interferon induction in mice.

Increasing evidence indicates that obesity is a risk factor for increased severity of influenza virus infection. However, its precise immunological mechanism is not fully understood. To investigate this, diet-induced obese (DIO) mice were established by feeding C57BL/6 male mice a high-fat diet for 16 weeks. DIO and lean control mice were infected intranasally with 3000 pfu of influenza A virus (IAV) (PR8/H1N1). Interestingly, we found adipose tissue located along the bronchus in naïve DIO mice. In addition, the Nos2 level was significantly higher and Arg1 level was significantly lower in lung macrophages of naïve DIO mice, consistent with an M1-skewed phenotype. The survival rate and body weight of DIO mice infected with IAV were significantly lower than those of lean control mice and associated with higher viral load in the lungs of DIO mice. Histopathological analysis demonstrated higher numbers of inflammatory cells in the lungs of DIO mice after IAV infection. Levels of cytokines, including TNF-α, IL-6, IL-10, and type I IFN (IFN-α and IFN-ß), in bronchoalveolar lavage fluid (BALF) were altered after IAV infection; in particular, IFN-α and IFN-ß levels were significantly suppressed in the BALF of DIO mice. In vitro, bone marrow-derived macrophages were stimulated with ligands of toll-like receptor (TLR) 7/8, a pattern recognition receptor for single-stranded RNA, and levels of TNF-α, IL-6, and IL-10 were similarly altered. In addition, levels of IFN-α and IFN-ß were significantly lower in culture supernatants of alveolar macrophages sorted from naïve DIO mice and infected with IAV, compared to those in macrophages sorted from lean control mice. Collectively, these results suggest that macrophages may be the main contributors to poor outcomes of influenza virus infection in obesity.

Anti-inflammatory roles of mesenchymal stromal cells during acute Streptococcus pneumoniae pulmonary infection in mice.

BACKGROUND: Pneumonia is the fourth leading cause of death worldwide, and Streptococcus pneumoniae is the most commonly associated pathogen. Increasing evidence suggests that mesenchymal stromal cells (MSCs) have anti-inflammatory roles during innate immune responses such as sepsis. However, little is known about the effect of MSCs on pneumococcal pneumonia. METHODS: Bone marrow-derived macrophages (BMDMs) were stimulated with various ligands in the presence or absence of MSC-conditioned medium. For in vivo studies, mice intranasally-inoculated with S. pneumoniae were intravenously treated with MSCs or vehicle, and various parameters were assessed. RESULTS: After stimulation with toll-like receptor (TLR) 2, TLR9 or TLR4 ligands, or live S. pneumoniae, TNF-α and interleukin (IL)-6 levels were significantly decreased, whereas IL-10 was significantly increased in BMDMs cultured in MSC-conditioned medium. In mice, MSC treatment decreased the number of neutrophils in bronchoalveolar lavage fluid (BALF) after pneumococcal infection, and this was associated with a decrease in myeloperoxidase activity in the lungs. Levels of proinflammatory cytokines, including TNF-α, IL-6, GM-CSF and IFN-γ, were significantly lower in MSC-treated mice, and the bacterial load in the lung after pneumococcal infection was significantly reduced. In addition, histopathologic analysis confirmed a decrease in the number of cells recruited to the lungs; however, lung edema, protein leakage into the BALF and levels of the antibacterial protein lipocalin 2 in the BALF were comparable between the groups. CONCLUSIONS: These results indicate that MSCs could represent a potential therapeutic application for the treatment of pneumonia caused by S. pneumoniae.

Association between six-minute walk test parameters and the health-related quality of life in patients with pulmonary Mycobacterium avium complex disease.

BACKGROUND: Pulmonary Mycobacterium avium complex (pMAC) disease is a chronic, slowly progressive disease. The aim of the present study was to determine the association of six-minute walk test (6MWT) parameters with pulmonary function and the health-related quality of life (HRQL) in patients with pMAC disease. METHODS: This cross-sectional study included adult patients with pMAC and was conducted at Keio University Hospital. We investigated the relationship of 6MWT parameters with clinical parameters, including pulmonary function, and HRQL, which was assessed using the 36-Item Short Form Health Survey (SF-36) and St. George's Respiratory Questionnaire (SGRQ). RESULTS: In total, 103 consecutive patients with pMAC participated in 6MWT (median age, 64 years; 80 women) and completed SF-36 and SGRQ. The six-minute walk distance (6MWD) showed significant negative and positive correlations with all SGRQ domain scores [ρ = (- 0.54)-(- 0.32)] and the physical component summary (PCS) score (ρ = 0.39) in SF-36, respectively; the opposite was observed for the final Borg scale (FBS) score (all SGRQ scores, ρ = 0.34-0.58; PCS score, ρ = - 0.50). The distance-saturation product showed significant negative and positive correlations with all SGRQ scores [ρ = (- 0.29)-(- 0.55)] and the PCS score (ρ = 0.40), respectively. Multivariate analysis revealed that 6MWD and the FBS score were significant predictors of HRQL. CONCLUSIONS: Our findings suggest that 6MWD and the FBS score are useful parameters for evaluating HRQL in patients with pMAC. Further studies should investigate the impact of 6WMT parameters on disease progression, treatment responses, and prognosis.

The efficacy, safety, and feasibility of inhaled amikacin for the treatment of difficult-to-treat non-tuberculous mycobacterial lung diseases.

BACKGROUND: In multidrug regimens, including an intravenous aminoglycoside (e.g. amikacin [AMK]) is recommended for difficult-to-treat non-tuberculous mycobacterial (NTM) lung diseases. We aimed to evaluate the efficacy, safety, and feasibility of inhaled AMK therapy in patients with difficult-to-treat NTM lung diseases in a retrospective chart review. METHODS: The study population consisted of patients with NTM lung diseases who received combination therapy, including inhaled AMK therapy, at Keio University Hospital (Tokyo, Japan), from January 2014 through May 2016. A total of 26 cases, consisting of 23 Mycobacterium avium complex (MAC) and three Mycobacterium abscessus complex (MABC) infections cases, were included in this study. The efficacy, safety, and feasibility of inhaled AMK therapy were retrospectively investigated. The Research Ethics Committee of Keio University Hospital approved this study, and informed consent was obtained from all patients. RESULTS: All 26 patients were culture-positive at enrolment. Twenty-three of the 26 patients (88.5%), including 21/23 MAC patients (91.3%) and 2/3 MABC patients (66.7%), were administered inhaled AMK therapy for >3 months. The proportion of patients who had clinical symptoms, including, cough and sputum, declined after inhalation AMK therapy. Ten of the 23 patients (43.5%) who received AMK inhalation, including 8/21 MAC (38.1%) and 2/2 MABC patients (100%), showed sputum conversion, defined as at least three consecutive negative sputum cultures. Seven of the 23 patients, including, 5/21 MAC and 2/2 MABC patients, showed improvements in high-resolution computed tomography imaging of the chest. In addition, the serum AMK trough levels before the second inhalation were <1.2 µg/mL in all 26 patients, with no occurrence of severe adverse events, such as renal toxicity. One patient (3.8%) experienced auditory toxicity, in the form of tinnitus. However, this symptom was reversible, after temporary interruption of AMK, the patient was able to safely resume the therapy. CONCLUSIONS: Inhaled AMK therapy is an effective and feasible therapy for difficult-to-treat NTM lung disease.

Pneumococcal Infection Aggravates Elastase-Induced Emphysema via Matrix Metalloproteinase 12 Overexpression.

BACKGROUND: Acute exacerbation of chronic obstructive pulmonary disease (COPD)--typically caused by bacterial or viral infection--is associated with poor prognosis and emphysema progression through unknown mechanisms. We aimed to elucidate the mechanisms responsible for the poor prognosis and emphysema progression associated with COPD exacerbation. METHODS: We established a mouse model mimicking acute human COPD exacerbation, wherein mice with elastase-induced emphysema were intranasally infected with Streptococcus pneumoniae. RESULTS: In mice with elastase-induced emphysema, infection with S. pneumoniae resulted in increased mortality, an increased number of inflammatory cells in bronchoalveolar lavage fluid (BALF), and increased matrix metalloproteinase 12 (MMP-12) production in the lungs, as well as enhanced emphysema progression. The increased MMP-12 production was mostly due to alveolar type II cells, alveolar macrophages, and lymphocytes that aggregated around vessels and bronchioles. Dexamethasone treatment suppressed the mortality rate and number of inflammatory cells in BALF but not emphysema progression, possibly owing to the failure of MMP-12 suppression in the lungs, whereas treatment with the MMP inhibitor ONO-4817 dramatically suppressed both mortality rate and emphysema progression. CONCLUSIONS: These results suggest that MMP-12 production during COPD exacerbation results in increased mortality and emphysema progression. Our study identifies MMP-12 as a target to prevent further aggravation of COPD.

Histone Deacetylase Inhibition Protects Mice Against Lethal Postinfluenza Pneumococcal Infection.

OBJECTIVES: Secondary bacterial pneumonia following influenza virus infection is associated with high mortality, but the mechanism is largely unknown. Epigenetic gene regulation appears to play key roles in innate and adaptive immunity. We hypothesized that histone acetylation, a major epigenetic mechanism associated with transcriptionally active chromatin, might contribute to the poor outcome of postinfluenza pneumonia. DESIGN: Prospective experimental study. SETTING: University research laboratory. SUBJECTS: C57BL/6 male mice. INTERVENTIONS: Mice were infected intranasally with 1.0 × 10 colony-forming units of Streptococcus pneumoniae, 7 days after intranasal inoculation with five plaque-forming units of influenza virus A/H1N1/PR8/34. The mice were intraperitoneally injected with the histone deacetylase inhibitor trichostatin A (1 mg/kg) or vehicle once a day from 1 hour after pneumococcal infection throughout the course of the experiment. The primary outcome was survival rate. MEASUREMENTS AND MAIN RESULTS: Trichostatin A significantly suppressed histone deacetylase activity and significantly improved the survival rate of mice (56.3%) after postinfluenza pneumococcal infection when compared with vehicle-treated mice (20.0%), which was associated with a significant decrease in the total cell count of the bronchoalveolar lavage fluid. The interleukin-1ß level in the serum and the number of natural killer cells in the lungs were significantly lower in the trichostatin A-treated group. CONCLUSIONS: The histone deacetylase inhibitor trichostatin A protects mice against postinfluenza pneumonia possibly through multiple factors, including decreasing local cell recruitment into the lungs and suppressing systemic inflammation.