Abstract

Persistence of infection despite extensive chemotherapy with antibiotics displaying low MICs is a hallmark of lung disease caused by Mycobacterium abscessus (Mab). Thus, the classical MIC assay is a poor predictor of clinical outcome. Discovery of more efficacious antibiotics requires more predictive in vitro potency assays. As a mycobacterium, Mab is an obligate aerobe and a chemo-organo-heterotroph – it requires oxygen and organic carbon sources for growth. However, bacteria growing in patients can encounter micro-environmental conditions that are different from aerated nutrient-rich broth used to grow planktonic cultures for MIC assays. These in vivo conditions may include oxygen and nutrient limitation which should arrest growth. Furthermore, Mab was shown to grow as biofilms in vivo. Here, we show Mab Bamboo, a clinical isolate we use for Mab drug discovery, can survive oxygen deprivation and nutrient starvation for extended periods of time in non-replicating states and developed an in vitro model where the bacterium grows as biofilm. Using these culture models, we show that non-replicating or biofilm-growing bacteria display tolerance to clinically used anti-Mab antibiotics, consistent with the observed persistence of infection in patients. To demonstrate the utility of the developed “persister” assays for drug discovery, we determined the effect of novel agents targeting membrane functions against these physiological forms of the bacterium and find that these compounds show “anti-persister” activity. In conclusion, we developed in vitro “persister” assays to fill an assay gap in Mab drug discovery compound progression and to enable identification of novel lead compounds showing “anti-persister” activity.

Highlights

  • Non-tuberculous mycobacteria (NTM), mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae, are ubiquitous environmental bacteria that can cause difficultto-cure lung disease (Falkinham, 2013)

  • To determine whether Mycobacterium abscessus (Mab) Bamboo is capable of surviving nutrient starvation, aerated pre-cultures growing exponentially in nutrient rich broth were transferred to PBS devoid of any nutrients and CFU over time were measured

  • Growing aerated nutrient-rich planktonic cultures may not reflect the predominant physiology of the bacteria growing in patients

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Summary

Introduction

Non-tuberculous mycobacteria (NTM), mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae, are ubiquitous environmental bacteria that can cause difficultto-cure lung disease (Falkinham, 2013). Treatment often goes on for years and negative sputum culture for 1 year – while on therapy – is recommended before a patient is declared cured. Despite this extensive chemotherapy, treatment success rates are low (Griffith et al, 2007). Mab lung disease can be considered a chronic infection that is rarely cured by our currently used drugs

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