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.

Keywords: Salmonella enterica; antibiotic resistance; colistin; mcr genes; mcr-9; mobilized colistin resistance; multidrug resistance; plasmid-mediated resistance.

FIG 1

(A) Comparison of mcr-9 to all previously described mcr homologues, based on amino acid sequence. The maximum likelihood phylogeny was constructed using RAxML version 8.2.12 with the amino acid sequences of novel mobilized colistin resistance gene mcr-9 (in blue) and all previously described mcr genes (mcr-1 to -8 [in black]). The phylogeny is rooted at the midpoint, with branch lengths reported in substitutions per site. Branch labels correspond to bootstrap support percentages out of 1,000 replicates. (B) Colistin killing assay of E. coli NEB5α harboring a pLIV2 empty vector (negative control), mcr-3 (positive control), or mcr-9, expressed under the control of the IPTG-controlled SPAC/lacOid promoter. Cells were grown in MH-II (Mueller-Hinton II) medium with IPTG to the mid-exponential phase. Colistin was added at concentrations of 0, 1, 2, 2.5, or 5 mg/liter, and the bacteria were incubated at 37°C for 1 h. The samples were diluted in phosphate-buffered saline (PBS) and plated on LB agar plates for the determination of CFU. Log CFU reduction was calculated by comparing CFU after each treatment to CFU levels obtained at 0 mg/liter colistin, using three independent biological replicates. Asterisks denote significant differences compared to empty vector treatment (P < 0.05 by Student's t test relative to the concentration's respective negative control after a Bonferroni correction). (C) Similarity matrix (composed of Dali Z-scores) of all previously described Mcr groups (Mcr-1 to -8) and Mcr-9, based on protein structure. The Dali server was used to perform all-against-all comparisons of 3D structural models based on all mcr homologues (Fig. 2A); for this analysis, amino acid sequences of mcr-5.3 and mcr-8.2, which were not available in ResFinder, were additionally included from the National Database of Antibiotic Resistant Organisms (NDARO).

FIG 2

(A) Structural models of all published Mcr proteins (Mcr-1 to -8) and Mcr-9, based on lipooligosaccharide phosphoethanolamine transferase EptA. Models were constructed using the Phyre2 server, and structures were viewed and edited using UCSF Chimera. Structural models show conservation of two EptA domains: transmembrane-anchored and soluble periplasmic domains. (B) Location of Mcr-9 secondary structure elements within the alignment of Mcr amino acid sequences, constructed using the ESPript 3 server. The top track denotes Mcr-9 secondary structure elements (alpha helixes and beta sheets). Green digits below the alignment denote cysteine residues forming a disulfide bridge (e.g., 1 forms a bridge with 1, 2 with 2, etc.). Within the amino acid sequence alignment itself, a strict identity (i.e., identical amino acid residue at a site) is denoted by a red box and a white character. A yellow box around an amino acid residue denotes similarity across groups, where groups were defined using the default “all” specification in ESPript 3 (ESPript 3 total score [TSc] > in-group threshold [ThIn]), while a residue in boldface denotes similarity within a group (ESPript 3 in-group score [ISc] > ThIn). (C) Organization of the mcr-9 locus in S. Typhimurium. An unknown function cupin fold metalloprotein is encoded by the gene downstream of mcr-9 (unlabeled black arrow). The mcr-9 locus is flanked by two different terminal repeat sequences (IRR) from the IS5 (orange box) and IS6 (red box) families. The mcr-9 upstream region contains highly conserved putative −35 and −10 σ⁷⁰-dependent promoter elements (blue boxes and blue text). Moreover, the mcr-9 promoter region contains an inverted repeat motif (green box, green text, and sequence logo) that is conserved in more than 95% of 321 mcr-9 genes, as shown by the sequence logo (constructed using WebLogo) (24).