The main aim of this study was to determine the prevalence of plasmid-mediated colistin resistance mcr-1 to mcr-5 genes among colistin and multi-drug resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter sp strains isolated from patients in a tertiary hospital in the city of Toluca, Mexico. 241 strains were included in the study. The presence of mcr genes among these strains was assessed by PCR and sequencing. In the case of mcr-carrying E. coli, further PCR tests were performed to determine the presence of blaCTX-M and whether the strains belonged to the O25b-ST131 clone. Conjugation experiments were carried to assess plasmid-mediated colistin resistance horizontal transmission. 12 strains (5.0%), of which four were E. coli; four, P. aeruginosa; three, K. pneumoniae and one, E. cloacae, were found to be resistant to colistin. Of these strains, two E. coli isolates were found to carry mcr-1. Both mcr-1-carrying E. coli strains were found to co-express blaCTX-M, belong to the O25b-ST131 clone and horizontally transmit their colistin resistance. The results of this study confirm the presence of plasmid-mediated colistin resistance in hospitalized patients in Mexico and demonstrated that the multidrug-resistant O25b-ST131 E. coli clone can acquire mcr genes and transmit such resistance trait to other bacteria.