P005

1:Tuberculosis Genomics Unit, Instituto de Biomedicina de Valencia, CSIC, Valencia, 46011 Spain; 2:CIBER de Epidemiología y Salud Pública, CIBERESP, Madrid, Spain; 3:Micorbiología Clínica, Hospital Universitario y Polítecnico La Fe

Isoniazid is a key drug for treating drug-susceptible tuberculosis (TB), with strong early bactericidal activity. However, ~5% of isoniazid-resistant (INH-R) cases have unknown resistance mechanisms. The main gene linked to INH-R is katG, though mutations in inhA have also been associated through mutations, some of them, such as inhA_S94A, with limited evidence. This mutation confers low-level resistance, confirmed in allele exchange experiments, but often produces discordant results in phenotypic drug susceptibility testing (pDST). As a result, it is rarely detected in front-line resistance tests, especially since it responds to combined therapy or high-dose isoniazid. In the Valencia Region, a genomic epidemiology study has sequenced ~80% of culture-positive TB cases since 2014. A large cluster of 32 cases carrying inhA_S94A has circulated from 2016 to 2024. The role of this mutation in clinical outcomes and the cluster’s spread remains unclear. We analyzed this cluster in depth to: (1) confirm the minimum inhibitory concentration linked to inhA_S94A using various phenotypic methods and isolates; (2) use PacBio sequencing to improve transmission resolution; (3) apply Bayesian methods to reconstruct transmission trees and estimate the cluster's origin; and (4) analyze clinical, microbiological, and epidemiological data to assess the mutation’s impact on TB spread. Results confirm that inhA_S94A shows borderline resistance in microdilution tests, while BACTEC has limitations. Although treatment outcomes were not significantly affected, routine diagnostic oversight of this mutation likely contributed to the spread of the mutation, compromising control efforts.