High Persister Mutants in <i>Mycobacterium tuberculosis</i>

<div><p><i>Mycobacterium tuberculosis</i> forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in <i>M</i>. <i>tuberculosis</i> is not well understood. In this study, we selected for high persister (<i>hip</i>) mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the <i>hip</i> mutants obtained <i>in vitro</i> with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical <i>hip</i> isolates exhibited greater <i>ex vivo</i> survival than the low persister isolates. Our data suggest that <i>M</i>. <i>tuberculosis</i> persister formation involves multiple pathways, and <i>hip</i> mutants may contribute to the recalcitrance of the infection.</p></div

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Genetic analysis of <i>hip</i> mutants obtained <i>in vitro</i>.

<p>Representative antibiotic survival plots of 18 <i>hip</i> mutant strains, obtained from 12 independent mutageneses, are presented along with lists of genes containing non-synonymous mutations within each strain.</p

Non-synonymous mutations identified in individual <i>in vitro hip</i> mutant strains by whole genome sequencing.

<p>Non-synonymous mutations identified in individual <i>in vitro hip</i> mutant strains by whole genome sequencing.</p

Growth and persister assays of <i>fadD26</i> PDIM mutant strains.

<p>PDIM mutant strain KL2849 (<i>fadD26</i> G74*) and wild type (mc²6020) (A) or <i>fadD26</i>::Tn and wild type (Erdman) (B) were grown (solid lines) and treated with streptomycin (10 μg/ml) and rifampicin (1 μg/ml) at the indicated time points for 14 days (dashed lines). Bacterial survival was determined by plating for CFU. Growth curves were also monitored based on OD₆₀₀ readings. The values are an average of three biological replicates and error bars represent standard deviation.</p

Characterization of clinical isolates.

<p>Longitudinal pairs of isolates from four cases of recalcitrant tuberculosis infection (A) or individual drug sensitive clinical isolates (B) were grown to stationary phase and treated with kanamycin (125 μg/ml) for 14 days and survival monitored by CFU counts. Genes containing non-synonymous SNPs differentiating the early and late isolate from Case 3 (C), unique to the four <i>hip</i> isolates (D), or unique the four low persister isolates (E) are presented. The values are an average of three biological replicates, error bars represent standard deviation, and stars represent significant difference (<i>p</i>-value < 0.001).</p

Characterization of <i>hip</i> mutants obtained <i>in vitro</i>.

<p>Persister assays, performed by antibiotic treatment with streptomycin (10 μg/ml) and rifampicin (1 μg/ml) for 14 days, reveal the number of drug tolerant persister cells based on CFU counts. Exponential (A) and stationary phase (B) treatment of mutagenized strain mc²6020 at each stage of the <i>hip</i> mutant selection process. Time-dependent persister assays in exponential (C) and stationary phase (D) with independent mutants KL2801, KL2825, KL2849, and wild type strain (mc²6020). Late exponential phase cultures were treated with various concentrations of streptomycin (E) or rifampicin (F) or with antibiotics not used in the selection process, kanamycin (50 μg/ml) or ofloxacin (10 μg/ml) (G). Cultures grown in minimal media with glycerol, butyrate, or propionate as the sole carbon source were treated in exponential phase (H). Data represent the average of three biological replicates and the error bars represent standard deviation.</p

Summary of the study of <i>hip</i> mutants in <i>M</i>. <i>tuberculosis</i>.

<p>Schematic depicting the comparative analysis of <i>hip</i> mutants, generated <i>in vitro</i> and identified in clinical isolates, by whole genome sequencing and transcriptome analysis to identify candidate persister genes.</p

Antibiotic tolerance of <i>hip</i> and low persister clinical isolates.

<p>Eight individual clinical isolates were grown exponential phase and treated with D-cycloserine (125 μg/ml) for 21 days and survival monitored by CFU counts (A). Macrophages were infected with either a <i>hip</i> or a low persister clinical isolate for 12 hrs and then treated with D-cycloserine (125 μg/ml) for 5 to 6 days and bacterial survival was determined by lysing the macrophages and plating for CFU (B). The values are an average of three biological replicates for each sample and the error bars represent standard deviation.</p