Background: Mycobacterium abscessus is notorious for being intrinsically resistant to most antibiotics. Antibiotic efflux is one of the mechanisms used by M. abscessus to pump out antibiotics from their cells. Inhibiting efflux pumps (EPs) can be an attractive strategy to enhance the activity of drugs. The objective of this study is to determine the activity of EP inhibitors (EPIs) to enhance the efficacy of the new drug bedaquiline against M. abscessus clinical isolates. Methods: A total of 31 phenotypically and genotypically identified M. abscessus subsp. abscessus, M. abscesss subsp. massiliense, and M. abscessus subsp. bolletii clinical isolates were studied. The contribution of EPs was determined by investigating the minimum inhibitory concentration (MIC) levels of bedaquiline reduction in the absence and presence of EPIs verapamil and reserpine using the resazurin microtiter assay. Results: The observed bedaquiline MIC reduction by verapamil was observed in 100% isolates and by reserpine in 54.8% isolates. Bedaquiline MIC was 4–32-fold using verapamil with M. abscessus subsp. bolletii showing the highest fold change and between 2- and 4-fold using reserpine. Conclusions: The results obtained in this study confirm that bedaquiline MIC decreased in the presence of EPIs verapamil and reserpine in clinical isolates of M. abscessus. Verapamil was the most effective EPI. As shown in previous studies, verapamil may have clinical potential as adjunctive therapy to enhance the effect of bedaquiline.

Background: Nontuberculous mycobacteria (NTM) have been identified in human pulmonary and extrapulmonary infections and are increasing globally, which makes it challenging to identify them. This article reports our experience with the laboratory identification of NTM in clinical practice among pulmonary and extrapulmonary samples received in our routine work. Methods: The study was conducted at the Université Catholique de Louvain at the Cliniques Universitaires Saint-Luc, Brussels, Belgium, from 2015 to 2018. A total of 386 clinical samples were collected from patients suspected of having pulmonary or extrapulmonary mycobacterial infections. Routine laboratory methods phenotypic and molecular tests were performed. Results: The majority of NTM species were isolated from pulmonary samples (68%). The most prevalent species identified were Mycobacterium chimaera_intracellulare group (32%), followed by Mycobacterium avium complex (21%), Mycobacterium abscessus complex (18%), Mycobacterium gordonae (9%), and Mycobacterium chelonae (4%). In extrapulmonary samples, M. avium and M. chimaera_intracellulare were the most frequently isolated. Conclusion: The species diversity of NTM found in our setting suggests the importance of the use of new modern methods for accurate identification of NTM at species level and in some case at subspecies level for the proper treatment and management of patients.

PURPOSE:Pulmonary infections due to non-tuberculous mycobacteria (NTM) are an emerging issue in the cystic fibrosis (CF) population. Due to bacterial and fungal overgrowth, isolation of mycobacteria from the sputum samples of these patients remains challenging. RGM medium, a novel agar-based culture medium was evaluated for the isolation of NTM from sputum samples of CF patients. METHODOLOGY:Sputum samples were inoculated onto RGM medium and conventional Mycobacterial Growth Indicator Tube (MGIT™, Becton Dickinson, USA). Agar plates were incubated at 35 °C and growth was recorded once a week during 42 days. We compared the yield of the two media. RESULTS:217 samples were obtained from 124 CF patients. 20 samples (13 patients) had a positive culture for NTM. 79/217 (36.4%) MGIT had to be discontinued due to contamination compared to 18/217 (8.3%) for RGM. We reported equivalent NTM detection performances for RGM and MGIT (P = 0.579): these media enabled the isolation of 15 and 12 NTM strains respectively. CONCLUSION:RGM medium increases the proportion of interpretable results and the number of NTM cultured. Taking into account the non-inferiority compared to conventional methods and ease of use of RGM medium, we estimate that this test can replace current approaches for the screening of NTM among people with CF. Additionally, RGM provides semi-quantitative results (number of colonies) and information on the morphology of colonies, which may be clinically relevant information.

Introduction: Diagnosis of infections due to Mycobacterium tuberculosis has been recently facilitated with the worldwide implementation of rapid molecular tests. However, the diagnosis of non-TB mycobacteria (NTM) remains a major challenge for laboratories, as this group of bacteria contains over 150 different species with highly variable bacteriological properties. The development and implementation in clinical practice of techniques allowing rapid identification of NTM at the species level is, therefore, important for clinical laboratories. We report the case of an HIV-1 positive patient presenting with severe NTM infection who benefited from a novel rapid diagnostic technique, based on the direct sequencing of the rpoB gene from smear-positive clinical samples. Case Presentation: A 43-year-old HIV-1 positive woman was hospitalized for bicytopenia in the presence of an inflamed supraclavicular lymph node. The diagnosis of disseminated Mycobacterium genavense infection, which involved the bone marrow and several lymph nodes, was missed by conventional mycobacteriological techniques. Identification was obtained by performing direct sequencing of the rpoB gene. Conclusion: We describe a novel technique that allows the rapid diagnosis of NTM infections directly from biological samples. This technique is now implemented in our routine workflow for smear and culture-positive samples, after the exclusion of TB, by rapid molecular assays.