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Flies have the capacity to transfer pathogens between different environments, acting as one of the most important vectors of human diseases worldwide. In this study, we trapped flies on a university campus and tested them for mobile resistance genes against colistin, a last-resort antibiotic in human medicine for treating clinical infections caused by multidrug-resistant Gram-negative bacteria. Quantitative PCR assays we developed showed that 34.1% of Musca domestica (86/ 252) and 51.1% of Protophormia terraenovae (23/45) isolates were positive for the mcr-1 gene, 1.2% of M. domestica (3/252) and 2.2% of P. terraenovae (2.2%, 1/45) isolates were positive for mcr-2, and 5.2% of M. domestica (13/252) and 44.4% of P. terraenovae (20/45) isolates were positive for mcr-3. Overall, 4.8% (9/189) of bacteria isolated from the flies were positive for the mcr-1 gene (Escherichia coli: 8.3%, 4/48; Enterobacter cloacae: 12.5%, 1/8; Providencia alcalifaciens: 11.8%, 2/17; Providencia stuartii: 4.9%, 2/41), while none were positive for mcr-2 and mcr-3. Four mcr-1- positive isolates (two P. stuartii and two P. alcalifaciens) from blow flies trapped near a dumpster had a MIC for colistin above 4 mg/ml. This study reports mcr-1 carriage in Providencia spp. and detection of mcr-2 and mcr-3 after their initial identification in Belgium and China, respectively. This study suggests that flies might contribute significantly to the dissemination of bacteria, carrying these genes into a large variety of ecological niches. Further studies are warranted to explore the roles that flies might play in the spread of colistin resistance genes.IMPORTANCE Antimicrobial resistance is recognized as one of the most seriousglobal threats to human health. An option for treatment of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) bacteria with multiple drug resistance was the reintroduction of the older antibiotic colistin. However, a mobile colistin resistance gene (mcr-1) has recently been found to occur widely; very recently, two other colistin resistance genes (mcr-2 and mcr-3) have been identified in Belgium and China, respectively. In this study, we report the presence of colistin resistance genes in flies. This study also reports the carriage of colistin resistance genes in the genus Providencia and detection of mcr-2 and mcr-3 after their initial identification. This study will stimulate more in-depth studies to fully elucidate the transmission mechanisms of the colistin resistance genes and their interaction. [ABSTRACT FROM AUTHOR]