Objectives: To investigate epidemiological/drug-resistance characteristics and identify potential factors related to drug-resistant and clustered tuberculosis in Sichuan. Methods: A total of 295 Mycobacterium tuberculosis (MTB) isolates were collected from surveillance sites in Sichuan from 2019 to 2021. The minimum inhibitory concentrations (MICs) of 12 anti-TB drugs were acquired using the broth microdilution method, followed by whole-genome sequencing (WGS) analysis. Results: Of 268 MTB isolates with both WGS and drug-susceptibility testing (DST) results, 159 (59.3%, 159/268) strains belonged to the Beijing lineage (L2). Isoniazid had the highest resistance rate (15.3%, 41/268), followed by rifampicin (9.3%, 25/268). The sensitivity of WGS to predict drug resistance varied from 75% to 97.6%, and the specificity was above 96.0% for all. rpoB Ser450Leu (41.7%, 10/24) and katG Ser315Thr (70%, 28/40) were the most frequent mutations in rifampicin and isoniazid resistance isolates, respectively. The clustering rate in Sichuan was 9.3% (25/268), and patients ≤ 24 years old (aOR = 11.697; 95% CI: 0.817–167.463) had a greater risk of clustering. Conclusions: Our findings prove that WGS is a promising tool for predicting drug resistance to isoniazid, rifampicin, ethambutol, moxifloxacin and levofloxacin in Sichuan. The higher resistance rate to isoniazid emphasizes the urgent need for susceptibility testing surveillance and application management. Improving the diagnosis, treatment and management of patients ≤ 24 years old may reduce the transmission of MTB in Sichuan.

Latent tuberculosis is prevalent in HIV-infected people and has an impact on the progression of AIDS. The aim of this study is to match a more accurate IGRA method for the better detection of latent tuberculosis infection in HIV patients. All 2394 patients enrolled were tested using three IGRA methods. The positive rate consistency of pairwise comparison and risk factors were analyzed. Receiver operator characteristic (ROC) curve analysis was applied to evaluate the diagnostic value of T-SPOTTB. The positive rates of the three methods were statistically different (p < 0.001). The CD4⁺ T cell number statistically impacted the QuantiFERON and Wan Tai tests after the analysis with univariate logistic regression, while no statistical difference was observed in T-SPOT.TB. Additionally, there was a better sensitivity and specificity of T-SPOT.TB if the positive cut-off value of ESAT-6 and CFP-10 was 4.5 and 5.5, respectively. This study provides an insight into the IGRA methods and demonstrated that the positive response detected via QuantiFERON declined with decreased CD4⁺ T cells in the HIV-infected population; T-SPOT.TB functions independently of the CD4⁺ T cell level and Wan Tai was affected in some cases. This will be useful in the diagnosis of LTBI in the HIV-infected population, which will be a key step toward TB elimination in China.

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.