Efflux Pump Gene Expression in Multidrug-Resistant <i>Mycobacterium tuberculosis</i> Clinical Isolates

<div><p>Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical <i>Mycobacterium tuberculosis</i> isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in <i>rpoB</i> (RIF), <i>katG</i> (INH), the <i>inhA</i> promoter (INH), and <i>oxyR-ahpC</i> (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. <i>drrA</i>, <i>drrB</i>, <i>efpA</i>, <i>jefA (Rv2459)</i>, <i>mmr</i>, <i>Rv0849</i>, <i>Rv1634</i>, and <i>Rv1250</i> were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type <i>katG</i>, <i>inhA</i>, and <i>oxyR-ahpC</i> associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (<i>efpA</i>, <i>Rv0849</i>, <i>Rv1250</i>, <i>P55 (Rv1410c)</i>, <i>Rv1634</i>, <i>Rv2994</i>, <i>stp</i>, <i>Rv2459</i>, <i>pstB</i>, <i>drrA</i>, and <i>drrB</i>) without drug inducement were significantly higher (<i>P</i> < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR <i>M</i>. <i>tuberculosis</i>, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.</p></div

The MIC breakpoints (µg/mL) of the 15 antimicrobial agents.

<p>MIC, minimum inhibitory concentration.</p

The susceptibility distributions to 15 antimicrobial agents of 12 standard rapidly growing mycobacteria strains.

<p>The susceptibility distributions to 15 antimicrobial agents of 12 standard rapidly growing mycobacteria strains.</p

The susceptibility distributions to 15 antimicrobial agents of 12 standard slowly growing mycobacteria strains.

<p>The susceptibility distributions to 15 antimicrobial agents of 12 standard slowly growing mycobacteria strains.</p

MIC (µg/mL) of the 15 antimicrobial agents^* to the 24 standard NTM strains.

<p>MIC, minimum inhibitory concentration; NTM, nontuberulous mycobacteria; RGM, rapidly growing mycobacteria; SGM, slowly growing mycobacteria; RIF, rifampicin; INH, isoniazid; STR, streptomycin; AK, amikacin; KM, kanamycin; CP, ciprofloxacin; OF, ofloxacin; LOF, levofloxacin; CAP, capreomycin; CEF, cefoxitin; DOX, doxycycline; MIN, minocycline; ETH, ethionamide; PAS, P-aminosalicylic acid; DIP, dipasic.</p><p>bold typeface indicates that the species was susceptible to the antimicrobial drug, while underlining indicates that the species was moderately susceptible to the antimicrobial drug.</p

Assay performance with <i>M</i>. <i>tuberculosis</i> and non-tuberculosis mycobacteria (NTM).

<p><i>M</i>. <i>tuberculosis</i> was diagnosed with no probe dropouts (RFP-susceptible <i>M</i>. <i>tuberculosis</i>) or no more than two dropouts (RFP-resistant <i>M</i>. <i>tuberculosis</i>). The 12 NTM DNA samples were identified with three or more LNA probe dropouts.</p

Schematic description of the assay and mutation discrimination capability of the six LNA probes.

<p>(a) The assay is performed in a two-tube form with three LNA probes and one IAC probe in each tube to detect <i>M</i>. <i>tuberculosis</i> and RFP-resistant <i>M</i>. <i>tuberculosis</i>. The two tubes share the same real-time PCR components except the included probes. (b) The LNA probes used in the assay could discriminate single-base mutations. The single-base variation in the target sequence prevents the corresponding probe matching and combining with the target; as a result, probe dropout from the sequence occurs (Cq = 0).</p

Assay performance with 154 clinical isolates.

<p>Assay performance with 154 clinical isolates.</p

Sensitivity of the probe assay for the detection of <i>M</i>. <i>tuberculosis</i> H37Rv.

<p>(a) Amplification curves of <i>M</i>. <i>tuberculosis</i> (10 GE/tube to 1.0 × 10⁶ GE/tube) through the multiple-probe real-time PCR. The LOD could be increased to 10 GE/tube through the combination of nested PCR amplification. (b) Quantification curve for <i>M</i>. <i>tuberculosis</i>. The curve displayed good linearity (R² = 0.987) for <i>M</i>. <i>tuberculosis</i> at a range of 1.0 × 10² GE/tube to 1.0 × 10⁶ GE/tube.</p