| Summary: | Few studies have addressed drug resistance of Enterobacterales in rural communities in developing countries. This study aimed to determine the coexistence of extended-spectrum β-lactamase (ESBL) and carbapenemase genes in <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> strains carrying the <i>mcr-1</i> gene in rural communities in Ecuador from healthy humans and their backyard animals. Sixty-two strains, thirty <i>E. coli</i> and thirty-two <i>K. pneumoniae</i> strains carrying the <i>mcr-1</i> gene were selected from a previous study. PCR were performed for the presence of ESBLs and carbapenemase genes. The strains were further characterized, and the genetic relationship was studied with multi-locus sequencing typing (MLST) of seven housekeeping genes. Fifty-nine of the sixty-two <i>mcr-1</i> isolates (95%) harbored at least on β-lactam resistance gene. The most prevalent ESBL genes were the <i>bla</i><sub>TEM</sub> genes (present in in 80% of the <i>E. coli</i> strains) and the <i>bla</i><sub>SHV</sub> gene (present in 84% of the <i>K. pneumoniae</i> strains). MSLT analysis revealed 28 different sequence types (ST); 15 for <i>E. coli</i> and 12 for <i>K. pneumoniae</i>, with most ST never described in humans and animals. The coexistence of <i>mcr-1</i> and β-lactams resistant genes in <i>E. coli</i> and <i>K. pneumoniae</i> strains is alarming and threatens the efficacy of last-resort antibiotics. Our findings highlight backyard animals as a reservoir of <i>mcr-1/</i>β-lactams resistant genes.
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