A Simple In Vitro Method to Determine Bactericidal Activity Against Mycobacterium abscessus Under Hypoxic Conditions

<b>Background/Objectives</b>: Non-replicating persisters (NRPs) of <i>Mycobacterium abscessus</i> are a bacterial subpopulation that can survive in the host under unfavorable conditions, such as hypoxia or nutrient starvation. The eradication of these bacteria is difficult, which is one reason for the long treatment duration and treatment failure. The drug discovery process should therefore contain methods to screen activity against NRPs. <b>Methods</b>: A hypoxic environment is used to generate NRPs of <i>M. abscessus</i> that are termed low-oxygen persisters (LOPs). For this, an oxidation process is used to transition a replicating culture of <i>M. abscessus</i> distributed in microtiter plates within a sealable box into LOPs. Colony counting, automated object counting, bactericidal activity determination of known agents, and confocal laser scanning microscopy are used to study the obtained culture. <b>Results</b>: The obtained culture shows typical attributes of non-replicating cells, such as significantly reduced replication, the reversibility of the LOP state under aerobic conditions, delayed regrowth on solid medium, altered morphological patterns on a single-cell level, and phenotypical resistance against a variety of clinically relevant antimycobacterial compounds. The study reveals metronidazole and niclosamide as bactericidal against <i>M. abscessus</i> LOPs. These compounds can be used as LOP verification compounds within the described model. <b>Conclusions</b>: Our model is easily implemented and quickly identifies compounds that are inactive under hypoxic conditions. It can therefore accelerate the identification of clinically effective antimycobacterial drug substances, and can be a helpful tool during the drug development process.