Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells

Giovanni Monteiro Ribeiro,Cristiane De Souza Carvalho-Wodarz,Daniela Teixeira,Renato Arruda Mortara,Fernando Real,Cristianne Kayoko Matsumoto,Sylvia Cardoso Leão,Rafael Silva Duarte

doi:10.1186/s12866-017-1102-7

Abstract

BackgroundOutbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection.ResultsCRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h.ConclusionM. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by avoiding phagosome maturation. Once recovered from infected macrophages, CRM0019 remains infective and displays greater intracellular growth in A549 cells compared to the ATCC 19977 strain. This evasion strategy in alveolar epithelial cells may contribute to the long survival of the CRM0019 strain in the host and thus to the inefficacy of in vivo treatment.

Highlights

Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures
Despite the similar infection rate in A549 cells, CRM0019 showed higher survival and proliferative capability compared to the ATCC 19977 strain, resulting in sustained infection after 24 h (Fig. 2a)
Reinfection of M. abscessus strains obtained from macrophages Because of the evidence that mycobacteria become more infective after the first intracellular infection [61, 62], we evaluated whether this holds for M. abscessus obtained from macrophages

Summary

Introduction

Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus is a nontuberculous mycobacterium (NTM) widely distributed in the environment This bacterium is responsible for lung diseases [1, 2] and healthcare-associated extrapulmonary infections [3,4,5]. As for other NTM, M. abscessus is present in environmental reservoirs (e.g. water and soil) and has been recently isolated from drinking water [9,10,11] The acquisition of this bacterium is most likely to occur from the environment, rather than via person-toperson transmission [12]. Despite sharing genes typically found in environmental organisms [13], M. abscessus harbors genes characteristic of pathogenic bacteria [14, 15] Likewise, it is an intracellular pathogen of macrophages and free-living amoebas [16, 17]

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Discussion

Conclusion