BACKGROUND: Antimicrobial resistance (AMR) is a major global health threat, with the highest burden in low-income and middle-income countries where diagnostic and surveillance capacity are often limited. Over the past decade, Timor-Leste has made substantial progress in strengthening microbial diagnostics and AMR surveillance capacity. We aimed to describe antimicrobial susceptibility patterns and temporal trends among priority bacterial pathogens in Timor-Leste between 2020 and 2025.

METHODS: We retrospectively analysed antimicrobial susceptibility patterns for WHO priority pathogens isolated from clinical samples processed at the Laboratory of Health, Instituto Nacional de Saúde Pública de Timor-Leste (INSP-TL), between June 2020 and January 2025. WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS) priority pathogens were identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-ToF) with automated antimicrobial susceptibility testing (AST) and interpreted using European Committee on AST (EUCAST) breakpoints. Duplicates within 12 months were excluded. Susceptibility proportions, temporal trends, and associations of AMR with sex, age, specimen type, and care were analysed.

FINDINGS: Of 4169 isolates, 2170 (52%) were from male and 1943 (47%) from female patients. Among Enterobacterales (1146/4169, 27%) ceftriaxone susceptibility of 65% (410/631) was observed in and 25% (135/541) in . Carbapenem resistance was detected in 235 isolates, including 61 multidrug-resistant complex. Meropenem susceptibility in decreased from 100% (33/33) in 2020 to 87% (155/178) in 2024 (p = 0.0002). Inpatient isolates of and showed lower ceftriaxone susceptibility than outpatient isolates. remained predominantly methicillin-susceptible (83%, 712/858).

INTERPRETATION: AMR burden in Timor-Leste is characterised by third-generation cephalosporin resistance, emerging carbapenem-resistant Enterobacterales, and multidrug-resistant . Sustained investment in infection prevention, antimicrobial stewardship, and laboratory systems is essential to mitigate further resistance emergence and guide treatment.

FUNDING: This work was supported by a Fleming Fund Country Grant (UK Aid).