Early diagnosis of drug susceptibility for tuberculosis (TB) patients could guide the timely initiation of effective treatment. We evaluated a novel multiplex xMAP TIER (Tuberculosis-Isoniazid-Ethambutol-Rifampicin) assay based on the Luminex xMAP system to detect first-line anti-tuberculous drug resistance. Deoxyribonucleic acid samples from 353 Mycobacterium tuberculosis clinical isolates were amplified by multiplex polymerase chain reaction, followed by hybridization and analysis through the xMAP system. Compared with the broth microdilution method, the sensitivity and specificity of the xMAP TIER assay for detecting resistance was 94.9% (95%CI, 90.0–99.8%) and 98.9% (95%CI, 97.7–100.0%) for rifampicin; 89.1% (95%CI, 83.9–94.3%) and 100.0% (95%CI, 100.0–100.0%) for isoniazid; 82.1% (95% CI, 68.0–96.3%) and 99.7% (95% CI, 99.0–100.0%) for ethambutol. With DNA sequencing as the reference standard, the sensitivity and specificity of xMAP TIER for detecting resistance were 95.0% (95% CI, 90.2–99.8%) and 99.6% (95% CI, 98.9–100.0%) for rifampicin; 96.9% (95% CI, 93.8–99.9%) and 100.0% (95% CI, 100.0–100.0%) for isoniazid; 86.1% (95% CI, 74.8–97.4%) and 100.0% (95% CI, 100.0–100.0%) for ethambutol. The results achieved showed that the xMAP TIER assay had good performance for detecting first-line anti-tuberculosis drug resistance, and it has the potential to diagnose drug-resistant tuberculosis more accurately due to the addition of more optimal design primers and probes on open architecture xMAP system.

Mycobacterial membrane proteins play a pivotal role in the bacterial invasion of host cells; however, the precise mechanisms underlying certain membrane proteins remain elusive. Mycolicibacterium smegmatis (Ms) msmeg5257 is a hemolysin III family protein that is homologous to Mycobacterium tuberculosis (Mtb) Rv1085c, but it has an unclear function in growth. To address this issue, we utilized the CRISPR/Cas9 gene editor to construct Δmsmeg5257 strains and combined RNA transcription and LC-MS/MS protein profiling to determine the functional role of msmeg5257 in Ms growth. The correlative analysis showed that the deletion of msmeg5257 inhibits ABC transporters in the cytomembrane and inhibits the biosynthesis of amino acids in the cell wall. Corresponding to these results, we confirmed that MSMEG5257 localizes in the cytomembrane via subcellular fractionation and also plays a role in facilitating the transport of iron ions in environments with low iron levels. Our data provide insights that msmeg5257 plays a role in maintaining Ms metabolic homeostasis, and the deletion of msmeg5257 significantly impacts the growth rate of Ms. Furthermore, msmeg5257, a promising drug target, offers a direction for the development of novel therapeutic strategies against mycobacterial diseases.