Role of Disputed Mutations in the rpoB Gene in Interpretation of Automated Liquid MGIT Culture Results for Rifampin Susceptibility Testing of Mycobacterium tuberculosis

Abstract

Low-level rifampin resistance associated with specific rpoB mutations (referred as "disputed") in Mycobacterium tuberculosis is easily missed by some phenotypic methods. To understand the mechanism by which some mutations are systematically missed by MGIT phenotypic testing, we performed an in silico analysis of their effect on the structural interaction between the RpoB protein and rifampin. We also characterized 24 representative clinical isolates by determining MICs on 7H10 agar and testing them by an extended MGIT protocol. We analyzed 2,097 line probe assays, and 156 (7.4%) cases showed a hybridization pattern referred to here as "no wild type + no mutation." Isolates harboring "disputed" mutations (L430P, D435Y, H445C/L/N/S, and L452P) tested susceptible in MGIT, with prevalence ranging from 15 to 57% (overall, 16 out of 55 isolates [29%]). Our in silico analysis did not highlight any difference between "disputed" and "undisputed" substitutions, indicating that all rpoB missense mutations affect the rifampin binding site. MIC testing showed that "undisputed" mutations are associated with higher MIC values (≥20 mg/liter) compared to "disputed" mutations (4 to >20 mg/liter). Whereas "undisputed" mutations didn't show any delay (Δ) in time to positivity of the test tube compared to the control tube on extended MGIT protocol, "disputed" mutations showed a mean Δ of 7.2 days (95% confidence interval [CI], 4.2 to 10.2 days; P < 0.05), providing evidence that mutations conferring low-level resistance are associated with a delay in growth on MGIT. Considering the proved relevance of L430P, D435Y, H445C/L/N, and L452P mutations in determining clinical resistance, genotypic drug susceptibility testing (DST) should be used to replace phenotypic results (MGIT) when such mutations are found.

Keywords: Mycobacterium tuberculosis; diagnostics; drug susceptibility testing; multidrug resistance.

FIG 1

Structural representation of the residues involved in the RpoB and rifampin interaction. (Left) Molecular surface of the RpoB protein (shown in white) with the bound rifampin depicted as sticks. The mutated amino acids formed are all located within 5 Å of the drug and define a contiguous surface in the binding cavity. Both “undisputed” (colored cyan) and “disputed” (yellow) mutations affect amino acids directly involved in shaping the rifampin binding site. The coordinates of RpoB and rifampin were taken from the PDB deposition 5UHB. (Right) The side chains of the mutated amino acids are shown as sticks. Since the resolution of the X-ray data used in the structure determination was lower than 3.5 Å, hydrogen bonds between RpoB and rifampin could not be unequivocally assigned. (The PDB numbering system = the MTB numbering system + 6).

FIG 2

(A) MIC (MIC₉₉) values by 7H10 agar proportion phenotypic drug susceptibility testing for selected mutations. (B) Delay (Δ) in the time to positivity (TTP) results between the control and the test tube using the TB-eXiST protocol on MGIT for selected mutations. Note that in 1 case of H445C, 1 case of H445N, 2 cases of D435Y, and 2 cases of H445S, isolates for which rifampin susceptibility was found were tested twice. Similarly, 1 case of rifampin resistance (H445S) was tested twice. Squares represent mutations associated with a Δ of >0 using the MGIT TB-eXiST protocol, and circles represent mutations not associated with any delay by the MGIT TB-eXiST protocol. Black indicates phenotypically resistant to rifampin according to standard MGIT drug susceptibility testing, and gray indicates phenotypically susceptible to rifampin according to standard MGIT drug susceptibility testing. CC, critical concentration. Δ = TTP_{test tube} – TTP_{control tube}. In panel B, mean values are also indicated (horizontal bars).

FIG 3

Comparison between the delay (Δ) in the time to positivity (TTP) results using the TB-eXiST protocol on MGIT for “undisputed” and “disputed” mutations and wild-type isolates. The wild-type results include as well the P454P silent mutation. Δ values are reported together with mean and 95% confidence intervals. Squares represent mutations associated with a Δ of >0 on the MGIT TB-eXiST protocol, and circles represent mutations not associated with any delay on MGIT TB-eXiST protocol. Black indicates phenotypically resistant to rifampin according to standard MGIT drug susceptibility testing, and gray indicates phenotypically susceptible to rifampin according to standard MGIT drug susceptibility testing. Δ = TTP_{test tube} – TTP_{control tube}. Mean values are indicated by horizontal bars.