Comprehensive Mechanisms of Bacterial Antibiotic Tolerance in Mycobacterium Tuberculosis

Periodic Reporting for period 1 - COMBATTB (Comprehensive Mechanisms of Bacterial Antibiotic Tolerance in Mycobacterium Tuberculosis)

Berichtszeitraum: 2019-04-01 bis 2021-03-31

Tuberculosis (TB), a disease that is mostly caused by Mycobacterium tuberculosis, was the leading cause of death from a single infectious agent before Sars-CoV2 swept the globe. In 2020, 9.9 million people fell ill with TB and 1.5 million died from it. Treatment for drug-susceptible TB is largely effective but 6.8% of patients receiving standard treatment develop recurrent TB and treatment options are limited for rifampicin resistant and multidrug-resistant TB. Antimicrobial resistance (AMR) not only confounds treatment for TB but also for a growing number of other infections, making it a threat to human health, security and economic advancement.
AMR refers to the ability of bacteria to avoid or delay being killed by an antibiotic and can manifest into three forms: resistance, tolerance and persistence. In resistance, bacteria that acquired a stable and heritable mutation, or new genetic material, grow in the presence of an antibiotic, resulting in a shift in the minimum inhibitory concentration (MIC) that inhibits bacterial growth. Tolerance and persistence describe phenomena where bacteria survive but don’t grow in the presence of an antibiotic. In tolerance, the entire bacterial population has a slower rate of killing by an antibiotic, and it can be genetic or phenotypic in nature. In persistence, only a subpopulation of bacteria—so-called persisters—have a slower rate of killing upon exposure to lethal concentration of an antibiotic. Persisters form stochastically—at rare frequencies—in unstressed bacterial population. The frequency increases in bacterial population exposed to stresses imposed by antibiotics themselves or by host immunity.
Tolerance and persistence have major clinical consequences, including long treatment times for TB, recurrence of disease after conclusion of antibiotic therapy for many infections, and emergence of resistance. Importantly, standard clinical antibiotic susceptibility assays do not assess tolerance or persistence, and development of tools to detect these phenomena in TB patients and new drugs to target bacteria displaying them is hindered by lack of understanding of the molecular underpinnings of tolerance and persistence in Mtb. To fill this gap, we aimed to exploit the fact that some mutations (high survival mutations) lead to genetic tolerance or high persistence. The latter can affect the frequency at which persisters arise—still stochastically—in bacterial populations. To isolate high survival mutants, we developed a method that allows for their isolation and identification in vitro and in more complex biological settings. This method, called ReMIND (Recombination mediated isolation of non-dividers), allows for discrimination of resisters from non-growing survivors based on the expression of two selection markers. Using it, we are now equipped with a powerful tool to inform the molecular mechanisms by which persistent and tolerant bacteria form during infection.

The overarching aim of this work was to identify pathways and molecular mechanisms fostering antibiotic tolerance and persistence during infection. To do so, we aimed to isolate high survival (HS) mutants that display genetic tolerance and/or high persistence in Mycobacterium tuberculosis in conditions that recapitulate stresses imposed by a combination of host immunity and chemotherapy. To achieve this goal, we developed a method—ReMIND for Recombination-mediated isolation of non-dividers—that allows for the separation of persisters from resisters, based on the ability of the former population to retain certain phenotypic traits (GFP fluorescence and streptomycin resistance).
We first validated ReMIND in non-pathogenic Mycobacterium smegmatis prior to transferring it to the human pathogen M. tuberculosis. In both species, we showed efficient discrimination and isolation of non-growing survivors from growing resisters after exposure to lethal concentration of an antibiotic. The use of a fluorescent marker as a selection marker allowed us to perform cycling experiments, which consist of the repeated exposure of a bacterial population to an antibiotic prior to the sorting of live GFP positive bacteria, to enrich bacterial population for HS mutants. To evaluate the contribution of host immunity in fostering tolerance and persistence, we have started to implement the use of ReMIND in an immortalized cell line that can be differentiated in mouse bone marrow macrophage-like cells for use in infection with M. tuberculosis. Any pathway in this cell line can be easily knock-down so that we can interrogate the role of different host pathways in the emergence of bacterial populations recalcitrant to antibiotic treatment. Overall, the identification and characterization of HS mutations in M. tuberculosis, in vitro and ex vivo, will inform on bacterial and host pathways critical for M. tuberculosis to survive exposure to lethal concentration of antimicrobials and cause relapse of infection. This will help guide the development of new combination therapies that prevent relapse of infection and the development of resistance.
We anticipate that several publications will come out of this work: one that will be submitted upon completion of some aspects of the work that has been conducted over the past years, and others that include the identification of bacterial and host pathways that foster tolerance and persistence in human infections once the screening campaign has been completed.

Persistent and tolerant bacteria are difficult to study because resisters grow in the presence of an antibiotic and quickly outnumber any non-growing survivors. To overcome this challenge, we developed a unique method, ReMIND, that allows for the specific isolation of strains with mutations conferring high persistence or genetic tolerance. This method can be used in vitro as well as in complex biological settings that mimic conditions found in host environments. Application of this method is currently used to identify bacterial and host pathways that foster tolerance and persistence in human infections. Providing fundamental knowledge on the emergence of tolerance and persistence in M. tuberculosis will be critical to inform screening campaigns to search for untapped small molecule classes that kill persisters, but also for the development of new diagnostic tools that could help prevent the emergence of genetic resistance and allow for better husbandry of the current antibiotic arsenal.
This method has the potential to be applied to other microorganisms that are genetically trackable to help broaden the study of tolerance and persistence to other infectious diseases. Strengthening the knowledge on AMR is one of the main objectives of the WHO Global Action Plan on Antimicrobial Resistance adopted in May 2015; further, the United Nations high-level meeting on AMR in 2016 continued to highlight that AMR is slowly penetrating the political agenda. We can only hope for the systematic analysis of bacterial AMR in 2019 published by the Antimicrobial Resistance Collaborators—that suggested AMR as the third cause of death from infection after SarS-CoV2 and tuberculosis—will be another incentive to climb up in the priority list of problems that our leaderships must tackle. We humbly believe that this work can contribute to it.

Antimicrobial resistance and its impact on public health and economy

Periodic Reporting for period 1 - COMBATTB (Comprehensive Mechanisms of Bacterial Antibiotic Tolerance in Mycobacterium Tuberculosis)

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