AbstractObjectivesZoos are environments where species of highly valued animals are kept largely separated from others and the wider world. We report the molecular ecology of critically important antibiotic resistant (ABR)Escherichia colicarried by 28 mammalian species housed in a zoo located in an urban residential district.MethodsOver 3 months we collected 167 faecal samples from captive mammals and processed forE. coliresistant to third-generation cephalosporins (3GC-R) and fluoroquinolones (FQ-R). Isolates were sequenced using Illumina.ResultsWe identified high rates of faecal sample-level positivity, with 50%, 57% and 36% of mammalian species excreting 3GC-R, FQ-R or dual 3GC-R/FQ-RE. coli, respectively. Isolates represented multiple ST and ABR mechanisms; CTX-M-15 and CMY-2 dominated for 3GC-R, and target-site mutation caused 75% of FQ-R. We identified multiple examples of ABRE. colitransmission between mammalian species in separate enclosures, and a variant of the epidemic plasmid pCT within the zoo. There was no evidence for ABRE. colileaving the zoo, based on comparative analysis withE. colifrom humans, cattle and dogs isolated from the 50 × 50 km region in which the zoo is located. Amoxicillin/clavulanate was the most widely used antibiotic in the zoo, and we identified four widely disseminated amoxicillin/clavulanate resistance mechanisms, including a previously unreported inhibitor-resistant TEM, and the carbapenemase OXA-181.ConclusionsWe conclude that the zoo studied here is a ‘melting pot’ for the selection and circulation of 3GC-R and FQ-RE. coli, but these circulatingE. coliappear captive within the zoo.