Longitudinal phenotypic and genomic evidence revealing increased risk of drug resistance accumulation in tuberculosis patients in the counties of central China.

Drug-resistant tuberculosis (DR-TB) disproportionately affects rural China, yet the molecular and epidemiological drivers of this disparity remain inadequately explored. This study investigates resistance evolution and transmission dynamics in Xianning, China, using longitudinal data from 3,865 culture-positive pulmonary tuberculosis patients (2016-2023). Phenotypic drug susceptibility testing for 14 commonly used anti-tuberculosis drugs showed a stable multidrug-resistant tuberculosis (MDR-TB) rate of 6.6%, while mono-resistance increased from 8.5% to 12.9% over the study period. Notably, 19.3% (53/275) of patients with ≥2 months of culture positivity developed new phenotypic resistance during treatment. Whole-genome sequencing of strains from the last two years identified resistance accumulation through two additional mechanisms: (i) acquisition of resistance via unfixed mutations in individuals and (ii) transmitted strains harboring novel resistance-conferring mutations absent in parental clones within genomic clusters. For the combined cases of resistance accumulation, multivariable logistic regression revealed that baseline drug resistance increased the risk more than threefold (aOR = 3.65-5.28, varying by resistance type), while rural residence independently doubled the risk (aOR = 2.60, 95% confidence interval:1.11-6.49). Furthermore, three of five genomic clusters with resistance accumulation exhibited urban-rural transmission, highlighting risks linked to cross-district care-seeking. These findings underscore how systemic healthcare barriers in rural China drive DR-TB through both treatment failures and strain transmission. Urgent action is needed to decentralize rapid resistance screening and implement tiered care models in primary clinics to curb transmission and mitigate the expanding DR-TB threat.IMPORTANCEThe ongoing epidemic of drug-resistant tuberculosis (DR-TB) in resource-poor settings poses a major public health challenge. This study sheds light on the evolution of DR-TB and its community transmission dynamics in central rural China, suggesting that unequal healthcare may exacerbate resistance accumulation risks by driving acquired resistance through inadequate treatment as well as facilitating strain transmission with escalating drug resistance. These findings emphasize the critical need for decentralized, rapid drug-resistance screening, and enhanced diagnosis and treatment strategies in primary care settings, prioritizing vulnerable populations to curb this growing threat.