Identification of Novel Mobilized Colistin Resistance Gene <i>mcr-9</i> in a Multidrug-Resistant, Colistin-Susceptible Salmonella enterica Serotype Typhimurium Isolate

Abstract

Mobilized colistin resistance (<i>mcr</i>) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known <i>mcr</i> homologues have been described (<i>mcr-1</i> to <i>-8</i>). Here, we describe <i>mcr-9</i>, a novel <i>mcr</i> homologue detected during routine <i>in silico</i> screening of sequenced <i>Salmonella</i> genomes for antimicrobial resistance genes. The amino acid sequence of <i>mcr-9</i>, detected in a multidrug-resistant (MDR) <i>Salmonella enterica</i> serotype Typhimurium (<i>S</i> Typhimurium) strain isolated from a human patient in Washington State in 2010, most closely resembled <i>mcr-3</i>, aligning with 64.5% amino acid identity and 99.5% coverage using Translated Nucleotide BLAST (tblastn). The <i>S.</i> Typhimurium strain was tested for phenotypic resistance to colistin and was found to be sensitive at the 2-mg/liter European Committee on Antimicrobial Susceptibility Testing breakpoint under the tested conditions. <i>mcr-9</i> was cloned in colistin-susceptible <i>Escherichia coli</i> NEB5α under an IPTG (isopropyl-β-d-thiogalactopyranoside)-induced promoter to determine whether it was capable of conferring resistance to colistin when expressed in a heterologous host. Expression of <i>mcr-9</i> conferred resistance to colistin in <i>E. coli</i> NEB5α at 1, 2, and 2.5 mg/liter colistin, albeit at a lower level than <i>mcr-3</i> Pairwise comparisons of the predicted protein structures associated with all nine <i>mcr</i> homologues (Mcr-1 to -9) revealed that Mcr-9, Mcr-3, Mcr-4, and Mcr-7 share a high degree of similarity at the structural level. Our results indicate that <i>mcr-9</i> is capable of conferring phenotypic resistance to colistin in <i>Enterobacteriaceae</i> and should be immediately considered when monitoring plasmid-mediated colistin resistance.<b>IMPORTANCE</b> Colistin is a last-resort antibiotic that is used to treat severe infections caused by MDR and extensively drug-resistant (XDR) bacteria. The World Health Organization (WHO) has designated colistin as a "highest priority critically important antimicrobial for human medicine" (WHO, <i>Critically Important Antimicrobials for Human Medicine</i>, <i>5th revision</i>, 2017, https://www.who.int/foodsafety/publications/antimicrobials-fifth/en/), as it is often one of the only therapies available for treating serious bacterial infections in critically ill patients. Plasmid-borne <i>mcr</i> genes that confer resistance to colistin pose a threat to public health at an international scale, as they can be transmitted via horizontal gene transfer and have the potential to spread globally. Therefore, the establishment of a complete reference of <i>mcr</i> genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.