Abstract
Mycobacterium abscessus is an emerging health risk to immunocompromised individuals and to people with pre-existing pulmonary conditions. As M. abscessus possesses multiple mechanisms of drug resistance, treatments of M. abscessus are of poor efficacy. Therefore, there is an urgent need for new therapeutic strategies targeting M. abscessus. We describe an experimental system for screening of compounds for their antimicrobial activity against intracellular M. abscessus using flow cytometry and imaging flow cytometry. The assay allows simultaneous analysis of multiple parameters, such as proportion of infected host cells, bacterial load per host cell from the infected population, and host cell viability. We verified the suitability of this method using two antibiotics with known activity against M. abscessus: clarithromycin and amikacin. Our analysis revealed a high degree of infection heterogeneity, which correlated with host cell size. A higher proportion of the larger host cells is infected with M. abscessus as compared to smaller host cells, and infected larger cells have higher intracellular bacterial burden than infected smaller cells. Clarithromycin treatment has a more pronounced effect on smaller host cells than on bigger host cells, suggesting that heterogeneity within the host cell population has an effect on antibiotic susceptibility of intracellular bacteria.
Highlights
Mycobacterium abscessus is a non-tuberculous mycobacteria (NTM) species, an opportunistic pathogen that mainly causes a tuberculosis-like lung disease in immunocompromised individuals and in people with pre-existing pulmonary conditions, such as cystic fibrosis [1]
Treatment with 10 or 2.5 μM clarithromycin reduced the percentage of mCherry+ cells by approximately half, and significantly decreased the mCherry mean fluorescent intensity within the mCherry+ population (Figure 1B,C), as determined using both conventional flow cytometry and imaging flow cytometry (IFC). These results indicate that clarithromycin treatment in this infection model reduces both the proportion of infected cells, as well as the bacterial load within the infected macrophages
We describe here an experimental system for rapid and robust screening of compounds for their antimicrobial activity against intracellular M. abscessus
Summary
Mycobacterium abscessus is a non-tuberculous mycobacteria (NTM) species, an opportunistic pathogen that mainly causes a tuberculosis-like lung disease in immunocompromised individuals and in people with pre-existing pulmonary conditions, such as cystic fibrosis [1]. M. abscessus infections is rising worldwide [2], and M. abscessus is the most prevalent NTM species in patients in some parts of Asia [3,4,5,6]. M. abscessus infections have been thought to result from independent acquisitions of environmental bacteria. Recent analyses of M. abscessus sequence diversity provide evidence for genetic clustering of patient isolates, suggesting human-to-human transmission [7,8]. M. abscessus infections can be mis-diagnosed as tuberculosis (TB, caused by M. tuberculosis) [9]; but M. abscessus is resistant to first-line anti-TB drugs [10].
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