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Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.
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Antibacterianos , Levofloxacino , Pruebas de Sensibilidad Microbiana , Mycobacterium abscessus , Reserpina , Levofloxacino/farmacología , Antibacterianos/farmacología , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/genética , Reserpina/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Girasa de ADN/genética , Girasa de ADN/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Farmacorresistencia Bacteriana/genética , Humanos , Verapamilo/farmacologíaRESUMEN
BACKGROUND: The integrity of cell wall structure is highly significant for the in vivo survival of mycobacteria. We hypothesized that changes in morphology may indicate changes in cell wall metabolism and identified an aceE gene mutant (aceE-mut) which presented a deficient colony morphology on 7H10 agar by screening transposon mutagenesis in Mycolicibacterium smegmatis, basonym Mycobacterium smegmatis (M. smegmatis). This study aimed to identify the functional role of aceE gene in cell wall biosynthesis in M. smegmatis. RESULTS: We observed that the colony morphology of aceE-mut was quite different, smaller and smoother on the solid culture medium than the wild-type (WT) strain during the transposon library screening of M. smegmatis. Notably, in contrast with the WT, which aggregates and forms biofilm, the aceE-mut lost its ability of growing aggregately and biofilm formation, which are two very important features of mycobacteria. The morphological changes in the aceE-mut strain were further confirmed by electron microscopy which indicated smoother and thinner cell envelope images in contrast with the rough morphology of WT strains. Additionally, the aceE-mut was more fragile to acidic stress and exhibited a pronounced defects in entering the macrophages as compared to the WT. The analysis of mycolic acid (MA) using LC-MS indicated deficiency of alpha-MA and epoxy-MA in aceE-mut strain whereas complementation of the aceE-mut with a wild-type aceE gene restored the composition of MA. CONCLUSIONS: Over all, this study indicates that aceE gene plays a significant role in the mycolic acid synthesis and affects the colony morphology, biofilm formation of M. smegmatis and bacteria invasion of macrophage.
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Biopelículas/crecimiento & desarrollo , Proteínas de la Membrana/metabolismo , Mutación , Mycobacterium smegmatis/fisiología , Ácidos Micólicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Prueba de Complementación Genética , Espectrometría de Masas , Proteínas de la Membrana/genética , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica , Mutagénesis Sitio-Dirigida , Mycobacterium smegmatis/metabolismoRESUMEN
INTRODUCTION: To evaluate the performance of Xpert MTB/RIF Ultra (Xpert-Ultra) in testing pleural tissue and fluid collected by medical thoracoscopy among patients with unexplained exudative pleural effusion. METHODS: Patients with an undiagnosed exudative pleural effusion were prospectively and consecutively recruited. Pleural tissue and fluid were collected by medical thoracoscopy and subjected to culture, Xpert MTB/RIF (Xpert) and Xpert-Ultra assays. Histopathological examination was also performed with the tissue and used as the major reference. RESULTS: Sixty-one patients were enrolled, including: 27 tuberculosis (TB) pleurisy, 15 malignancy and 19 other chronic infection cases. The sensitivity, specificity, positive predictive value, and negative predictive value of Xpert-Ultra for TB diagnosis were 85.19% (23/27), 97.06% (33/34), 95.83% (23/24), and 89.19% (33/37), respectively. Xpert-Ultra testing with the biopsy tissue alone had an equivalent diagnostic capacity to that of pathological examination for the diagnosis of confirmed TB cases. By combining the pathological examination with Xpert-Ultra for biopsy, the percentage of confirmed TB cases greatly increased (i.e. 92.59% (25/27)). The "trace" positive outcome of Xpert-Ultra was highly supportive of TB diagnosis for both biopsy tissue and pleural fluid examinations. CONCLUSION: With the specimens collected by medical thoracoscopy, the Xpert-Ultra assay presented high value in identifying TB among pleurisy patients who had difficulties in etiological diagnosis.
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Derrame Pleural/complicaciones , Rifamicinas/farmacología , Toracoscopía , Tuberculosis Pleural/diagnóstico , Tuberculosis Pleural/patología , Adulto , Biopsia , Estudios de Cohortes , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/fisiología , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sensibilidad y Especificidad , Tuberculosis Pleural/complicaciones , Adulto JovenRESUMEN
OBJECTIVE: Treatment choices for Mycobacterium abscessus (M. abscessus) infections are very limited, and the prognosis is generally poor. Effective new antibiotics or repurposing existing antibiotics against M. abscessus infection are urgently needed. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a member of the lipophilic weak acid class, is known as an efflux pump inhibitor for Mycobacterium tuberculosis. The aim of this study was to determine the inhibitory activity of CCCP as a potential novel antibiotic against M. abscessus. METHODS: A total of 47 reference strains of different mycobacterial species and 60 clinical isolates of M. abscessus were enrolled. In vitro inhibitory activity of CCCP was accessed using microplates alamar blue method with the reference and clinical isolates. The activity of CCCP against intracellular M. abscessus residing within macrophage was also evaluated by intracellular colony numerating assay. RESULTS: CCCP exhibited good activity against M. abscessus clinical isolates in vitro, the minimum inhibitory concentration (MIC) ranged from 0.47 µg/mL to 3.75 µg/mL, with a MIC50 of 1.875 µg/mL and MIC90 of 3.75 µg/mL. At concentrations safe for the cells, CCCP exhibited highly intracellular bactericidal activities against M. abscessus and M. massiliense reference strains, with inhibitory rates of 84.8%±8.8% and 72.5%±13.7%, respectively. CCCP demonstrated bactericidal activity against intracellular M. abscessus that was comparable to clarithromycin, and concentration-dependent antimicrobial activity against M. abscessus in macrophages was observed. In addition, CCCP also exhibited good activities against most reference strains of rapidly growing mycobacterial species. CONCLUSION: CCCP could be a potential candidate of novel antimicrobiological agent to treat M. abscessus infection.
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BACKGROUND: Previous studies have shown that the dysregulation of lncRNAs participates in non-small cell lung cancer (NSCLC) development. The purpose of this study was to research the biological function of lncRNA SNHG14 and its molecular mechanism in NSCLC progression. METHODS: RT-PCR was applied for investigating the expression of SNHG14, miR-206 and G6PD. The progression of NSCLC was detected by CCK-8, Transwell and western blot assays. The targets of SNHG14 and miR-206 were measured by dual-luciferase reporter assay. RESULTS: We found a higher expression of SNHG14 in NSCLC and upregulation of SNHG14 contributed to NSCLC cell proliferation, invasion and migration. However, knockdown of SNHG14 showed the opposite effect on the progression of NSCLC. Specifically, SNHG14 negatively regulated miR-206 expression by binding with it directly. Furthermore, G6PD served as the target of miR-206. Rescue experiments showed that SNHG14 promoted G6PD expression by inhibiting miR-206. CONCLUSIONS: LncRNA SNHG14 contributed to NSCLC progression through miR-206/G6PD axis, providing novel clues for understanding the mechanism of NSCLC.