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

Abstract

Mobilized colistin resistance (mcr) genes are plasmid-borne genes that confer resistance to colistin, an antibiotic used to treat severe bacterial infections. To date, eight known mcr homologues have been described (mcr-1 to -8). Here, we describe mcr-9, a novel mcr homologue detected during routine in silico screening of sequenced Salmonella genomes for antimicrobial resistance genes. The amino acid sequence of mcr-9, detected in a multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium (S. Typhimurium) strain isolated from a human patient in Washington State in 2010, most closely resembled mcr-3, aligning with 64.5% amino acid identity and 99.5% coverage using Translated Nucleotide BLAST (tblastn). The S. 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. mcr-9 was cloned in colistin-susceptible Escherichia coli 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 mcr-9 conferred resistance to colistin in E. coli NEB5α at 1, 2, and 2.5 mg/liter colistin, albeit at a lower level than mcr-3. Pairwise comparisons of the predicted protein structures associated with all nine mcr 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 mcr-9 is capable of conferring phenotypic resistance to colistin in Enterobacteriaceae and should be immediately considered when monitoring plasmid-mediated colistin resistance. IMPORTANCE 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, Critically Important Antimicrobials for Human Medicine, 5th revision, 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 mcr 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 mcr genes that can be used to screen for plasmid-mediated colistin resistance is essential for developing effective control strategies.

Massive analysis of 64’628 bacterial genomes to decipher a water reservoir and origin of mobile colistin resistance (mcr) gene variants: is there another role for this family of enzymes?

“…Interestingly, among these MCR-3-like sequences, one of them identified from Salmonella and Buttiauxella spp. has recently been published as the new MCR-9 variant 29–32 . We identified Shewanella spp.…”

“…Interestingly, most of these mcr genes have been discovered into bacteria from animal and/or environmental origin 15–19 . Indeed, mcr -1 and mcr -2 have been suggested being originated from Moraxella species 20 , mcr -3 from Aeromonas veronii 21 , mcr-4 from Shewanella 22 , mcr -8 recently described in Raoultella ornithinolytica 15 and mcr-9 from Salmonella enterica serovar Typhimurium 23 . However, despite the large number of reported publications on these mobilized colistin resistance genes, the sources, origins and roles of these enzymes remain uncertain and not yet described.…”

Recent progress on elucidating the molecular mechanism of plasmid-mediated colistin resistance and drug design

“…As polymyxins are regarded to be the last line of defense against bacterial infections, they have attracted increased worldwide attention after MCR-1 was first reported in late 2015 (Arcilla et al 2016;Hu et al 2016b;Mulvey et al 2016;Rawson et al 2016;Rolain and Olaitan 2016;Ruppe et al 2016;Tse and Yuen 2016;Zhang et al 2016;Zhi et al 2016). At present, researchers have reported eight additional different MCR family members (MCR-2 through MCR-9) (AbuOun et al 2018;Borowiak et al 2017;Carattoli et al 2017;Carroll et al 2019;Wang et al 2018;Xavier et al 2016;Yang et al 2018;Yin et al 2017). Protein sequence analysis suggests that the nine members of the MCR family of proteins have a highly identical secondary structure, with a high degree of conservation observed for many amino acid residues, especially those in the active site and PE-interacting cavities ( Supplementary Fig.…”

Fast and robust detection of colistin resistance in Escherichia coli using the MALDI Biotyper Sirius mass spectrometry system

“…To date a further eight MCR proteins have been described. These enzymes cluster into four main groups: MCR-1-like (MCR-1, −2, −6), MCR-3-like (MCR-3, −7, −8, −9), MCR-4-like (MCR-4) and MCR-5-like (MCR-5) (8–11).…”