ABSTRACT
Tuberculous sputum contains multipleMycobacterium tuberculosispopulations with different requirements for isolationin vitro These include cells that form colonies on solid media (plateableM. tuberculosis), cells requiring standard liquid medium for growth (nonplateableM. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependentM. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of theseM. tuberculosispopulations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10reductions), and SN-dependentM. tuberculosiswas more tolerant to streptomycin and isoniazid than the plateable and nonplateableM. tuberculosisstrains. Susceptibility of plateableM. tuberculosisto bactericidal drugs was significantly increased after passagein vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simpleex vivosystem for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection.
Subject(s)
Adaptation, Physiological , Antitubercular Agents/pharmacology , Biological Assay , Isoniazid/pharmacology , Mycobacterium tuberculosis/drug effects , Streptomycin/pharmacology , Cryopreservation , Drug Resistance, Bacterial/drug effects , Humans , Microbial Sensitivity Tests , Multilocus Sequence Typing , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/physiology , Phenotype , Sputum/microbiology , Tuberculosis, Pulmonary/microbiologyABSTRACT
We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria.
Subject(s)
Acyl Coenzyme A/metabolism , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Mycobacterium/metabolism , Acyl Coenzyme A/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites/genetics , DNA, Bacterial/genetics , Fluorescence Polarization , Gene Expression Regulation, Bacterial/drug effects , Microbial Viability/drug effects , Microbial Viability/genetics , Molecular Sequence Data , Molecular Structure , Mutation , Mycobacterium/genetics , Mycobacterium bovis/genetics , Mycobacterium bovis/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Oleic Acid/pharmacology , Palmitoyl Coenzyme A/chemistry , Palmitoyl Coenzyme A/metabolism , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Transcriptome/drug effects , Transcriptome/geneticsABSTRACT
Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis.