Determination of critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against para-aminosalicylic acid with clinical isolates with thyA, folC and dfrA mutations

Abstract

Background & objectives: Accurate determination of antimicrobial resistance profiles is of great importance to formulate optimal regimens against multidrug-resistant tuberculosis (MDR-TB). Although para-aminosalicylic acid (PAS) has been widely used clinically, the reliable testing methods for PAS susceptibility were not established. Herein, we aimed to establish critical test concentration for PAS on the Mycobacterial Growth Indicator Tube (MGIT) 960 in our laboratory settings.

Methods: A total of 102 clinical isolates were included in this study, including 82 wild-type and 20 resistotype isolates. Minimum inhibitory concentration (MIC) was determined by MGIT 960. Whole-genome sequencing was used to identify the mutation patterns potentially conferring PAS resistance. Sequence alignment and structure modelling were carried out to analyze potential drug-resistant mechanism of folC mutant.

Results: Overall, the Minimum inhibitory concentration (MIC) distribution demonstrated excellent separation between wild-type and resistotype isolates. The wild-type population were all at least 1 dilution below 4 μg/ml, and the resistotype population were no lower than 4 μg/ml, indicating that 4 μg/ml was appropriate critical concentration to separate these two populations. Of 20 mutant isolates, 12 (60.0%) harbored thyA mutations, 2 (10%) had a mutation on upstream of dfrA, and the remaining isolates had folC mutations. Overall, thyA and folC mutations were scattered throughout the whole gene without any one mutation predominating. All mutations within thyA resulted in high-level resistance to PAS (MIC > 32 μg/ml); whereas the MICs of isolates with folC mutations exhibited great diversity, ranged from 4 to > 32 μg/ml, and sequence and structure analysis partially provided the possible reasons for this diversity.

Conclusions: We propose 4 μg/ml as tentative critical concentration for MGIT 960. The major mechanism of PAS resistance is mutations within thyA and folC in MTB isolations. The whole-gene deletion of thyA locus confers high-level resistance to PAS. The diversity of many distinct mutations scattered throughout the full-length folC gene challenges the PCR-based mutation analysis for PAS susceptibility.

Keywords: Critical Concentration; Drug Susceptibility Testing; Mycobacterium tuberculosis; Para-Aminosalicylic Acid.

Fig. 1

Distribution of PAS MICs (μg/ml) in the MGIT for wild-type and resistotype MTB isolates. ECOFF epidemiological cutoff

Fig. 2

Sequence and structure analysis of wild-type FolC and mutants. A Sequence alignment of FolC orthologs in mycobacterial species. Magenta triangles indicated the position of mutants found in our study. FolC_MTB, FolC from MTB (UniProt entry I6Y0R5); FolC_Eco, FolC from E. coli (UniProt entry P08192); FolC_Hin, FolC from H. influenzae (UniProt entry P43775); FolC_Bsu, FolC from B. subtilis (UniProt entry Q05865). B Three-dimensional structure of wild-type MTB FolC in complex with DHP-P and ADP. The LeuRS protein (cyan) was displayed in cartoon mode. The six different residues (magenta) were shown as sticks. Ligands DHP-P (yellow), ADP (red) and magnesium ions (green) were represented in CPK mode. C, D The 2D diagram showing the interactions between wild-type FolC and DHP-P (C) and ADP (D). The ligand molecules, namely DHP-P and ADP, were shown in the middle with a display style of ball and stick. The colored balls indicated the residues involved in the direct interactions between FolC and ligand. The green, purple and yellow dash line connecting ligand and corresponding residue indicated intermolecular hydrogen bond, hydrophobic interaction and attractive charge, respectively. Residues involved in hydrogen bond, van der Waals interactions or polar interactions were represented by green balls. Residues involved hydrophobic interactions and attractive charge were displayed by purple and orange balls, respectively