BackgroundMultidrug-resistant organisms (MDROs) disproportionately affect hospitalized patients due to the combination of comorbidities, frequent antimicrobial use, and in-hospital MDRO transmission. Identification of MDRO transmission by hospital microbiology laboratories is difficult due to limitations of existing typing methods.MethodsWe conducted a prospective multicenter genomics implementation study (8 hospitals, 2800 beds) from 24thApril to 18thJune 2017 in Melbourne, Australia. Clinical and screening isolates from hospital inpatients were collected for six MDROs (vanAVRE, MRSA, ESBLE. coli[ESBL-Ec] andKlebsiella pneumoniae[ESBL-Kp], and carbapenem-resistantPseudomonas aeruginosa[CRPa] andAcinetobacter baumannii[CRAb]), sequenced (Illumina NextSeq) and analyzed using open-source tools. MDRO transmission was assessed by genomics (core SNP phylogeny, grouped by species and ST) and compared to epidemiologic data.Results408 isolates were collected from 358 patients; 47.5% were screening isolates. ESBL-Ec was most common (52.5%), then MRSA (21.6%),vanAVRE (15.7%) and ESBL-Kp (7.6%).We define the transmission rate for each MDRO by genomics and epidemiology; 31.6% of all study patients had potential genomic links to other study isolates; 86% of these were confirmed by epidemiologic links (probable or possible transmission). The highest transmission rates occurred withvanA VRE (88.4% of patients).ConclusionsCombining genomics with high-quality epidemiologic data gives substantial insights into the burden and distribution of critical MDROs in hospitals, including in-hospital transmission. By defining transmission rates by genomics, we hope to enable comparisons over time and between sites, and introduce this as a new outcome measure to assess the efficacy of infection control interventions.