Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.

Background: Antimicrobial resistance is a major public health threat, affecting not only humans but also animals and the environment. Although the "One Health" dimension of resistance is well recognized, data are lacking on the circulation of resistance, particularly in low-income countries. The World Health Organization has proposed a protocol called Tricycle, focusing on extended-spectrum beta-lactamase (ESBL)-Escherichia coli surveillance in the three sectors. We implemented Tricycle in Madagascar to assess ESBL-E. coli prevalence and describe intra- and inter-sector circulation of ESBL-E. coli and plasmids. Methods: 289 pregnant women, 246 farm chickens and 28 surface waters were sampled in Antananarivo (the capital city) area and tested for ESBL-E. coli. Isolates were sequenced by short- (Illumina) and long- (Nanopore) read methods. Findings: ESBL-E. coli prevalence was 29.8%, 56.9% and 100% in pregnant women, chickens, and the environment, respectively. The wet season was associated with higher rates of carriage in humans (OR=3.1, 95%CI 1.8-5.3) and animals (OR=2.8 95%CI 1.7-4.8). Sequencing of 277 non-duplicated isolates (82, 118 and 77 from each sector, respectively) showed high genetic diversity (90 STs identified) with differences between sectors. Single nucleotide polymorphism (SNP) analysis revealed 169/277 (61%) isolates grouped into 44 clusters (≥2 isolates) of closely related isolates (<40 SNPs), of which 24 contained isolates from two sectors and five contained isolates from all three sectors. ESBL genes were all blaCTX-M (77.6% blaCTX-M-15), chromosomally integrated in 57.4% (159/277) of isolates, and plasmidic in 40.8% (113/277). The 114 ESBL-carrying plasmids were mainly IncF (55.2%, n=63) and IncY (36.8%, n=42). The F31/36:A4:B1 (n=13) and F-:A-:B53 (n=8) subtypes, and all IncY plasmids, highly conserved, were observed in isolates of differing genetic backgrounds from all sectors. Interpretation: Despite varying strain population structures in the three sectors, both ESBL-E. coli strains and plasmids are circulating among humans, chickens and the environment in the capital of Madagascar. Funding: Fondation Merieux, INSERM, Universite Paris Cite. Keywords: Extended spectrum beta-lactamase (ESBL), Escherichia coli, Tricycle, One Health, circulation