Adaptation of Mycobacterium tuberculosis to Biofilm Growth Is Genetically Linked to Drug Tolerance

Abstract

Mycobacterium tuberculosis spontaneously grows at the air-medium interface, forming pellicle biofilms, which harbor more drug-tolerant persisters than planktonic cultures. The underlying basis for increased persisters in M. tuberculosis biofilms is unknown. Using a transposon sequencing (Tn-seq) approach, we show here that multiple genes that are necessary for fitness of M. tuberculosis cells within biofilms, but not in planktonic cultures, are also implicated in tolerance of bacilli to a diverse set of stressors and antibiotics. Thus, development of M. tuberculosis biofilms appears to be associated with an enrichment of population, in which challenging growth conditions within biofilm architecture select for cells that maintain intrinsic tolerance to exogenous stresses, including antibiotic exposure. We further observed that the intrinsic drug tolerance of constituent cells of a biofilm determines the frequency of persisters. These findings together allow us to propose that the selection of elite cells during biofilm development promotes the frequency of persisters. Furthermore, probing the possibility that the population enrichment is an outcome of unique environment within biofilms, we demonstrate biofilm-specific induction in the synthesis of isonitrile lipopeptide (INLP). Mutation analysis indicates that INLP is necessary for the architecture development of M. tuberculosis biofilms. In summary, this study offers an insight into persistence of M. tuberculosis biofilms under antibiotic exposure, while identifying INLP as a potential biomarker for further investigation of this phenomenon.

The DNA Damage Inducible SOS Response Is a Key Player in the Generation of Bacterial Persister Cells and Population Wide Tolerance

“…Mtb spontaneously grows at the air-liquid interface, forming pellicle biofilms with elevated drug-tolerant persisters compared to planktonic cultures. The intrinsic drug tolerance of biofilm cells was found to determine the frequency of persisters and isonitrile lipopeptide necessary for the architectural development of Mtb biofilms (Richards et al, 2019).…”

Transcriptional portrait of M. bovis BCG during biofilm production shows genes differentially expressed during intercellular aggregation and substrate attachment

“…The formation of biofilms contributes to stress survival, since these dense multicellular aggregates, in which cells are embedded in an extracellular matrix of self-produced polymers, represent a self-constructed protective 'niche' 21 that yet remains metabolically active even after reaching maturity 22 . In M. tuberculosis complex bacteria, biofilm production in vitro has been shown to harbor drug-tolerant bacteria 7 and to be genetically linked to this phenotype 8 . Drug-tolerant mycobacteria may comprise a fraction of the population, as mature M. tuberculosis biofilms showed sensitivity to antibiotic treatment when assessed with microcalorimetry (IMC) and tunable diode laser absorption spectroscopy 22 .…”

“…In M. tuberculosis complex bacteria, biofilm production in vitro has been shown to harbor drug-tolerant bacteria 7 and to be genetically linked to this phenotype 8 . Drug-tolerant mycobacteria may comprise a fraction of the population, as mature M. tuberculosis biofilms showed sensitivity to antibiotic treatment when assessed with microcalorimetry (IMC) and tunable diode laser absorption spectroscopy 22 .…”

“…In support of a stepwise process regulating biofilm production in mycobacteria, it was recently shown that in M. smegmatis , lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation 1 . Additionally, it was found that multiple genes that are necessary for fitness of M. tuberculosis cells within biofilms, were also implicated in tolerance to a diverse set of stressors and antibiotics 8 , something not observed for planktonic cells, further supporting a role for M. tuberculosis biofilms in drug tolerance 7 .…”

Tolerance and Persistence to Drugs: A Main Challenge in the Fight Against Mycobacterium tuberculosis

“…A Tn-seq screening for M. tuberculosis mutants unable to form biofilms revealed that several genes required for biofilm formation were also involved in stress and drug tolerance. It is possible that the harsh environment found in the biofilm, characterized by nutrients and oxygen depletion and mechanical stresses, selects a bacterial population intrinsically tolerant to exogenous stresses, including drug exposure, therefore increasing the frequency of persisters ( Richards et al, 2019 ).…”