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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 NEB5? under an IPTG (isopropyl-?-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 NEB5? 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.

SUBMITTER: Carroll LM

PROVIDER: S-EPMC6509194 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Carroll Laura M LM Gaballa Ahmed A Guldimann Claudia C Sullivan Genevieve G Henderson Lory O LO Wiedmann Martin M

mBio 20190507 3

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 ...[more]

PMID: 31064835

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Project description:With the rapid emergence of plasmid-mediated colistin resistance gene mcr-1, the increased resistance of Salmonella has attracted extensive attention. This study reports on 11 multidrug-resistant Salmonella enterica serovar Typhimurium strains harboring mcr-1 in China. They all presented resistance to colistin, and additionally, one that was isolated from a child's stool sample was also resistant to ceftriaxone and azithromycin. We screened 1454 strains of Salmonella for mcr-1 gene through PCR, and these strains are all preserved in our laboratory. Antimicrobial sensitivity analysis was carried out for the screened mcr-1 positive strains. Genetic polymorphism analysis of S. Typhimurium was performed by using the Pulsed-Field Gel Electrophoresis (PFGE). The plasmids harboring mcr-1 were identified by S1-PFGE and southern blotting. Plasmid conjugation assays were used to analyze the transferability of colistin resistance. The plasmids harboring mcr-1 were characterized by sequencing and bioinformatic analysis. Eleven S. Typhimurium strains harboring mcr-1 with colistin resistance (MICs 4μg/ml) were detected, which were isolated from children and pig offal in China. All of them were multidrug-resistant strains. PFGE results revealed that the strains isolated from different samples or locations have identical genotypes. S1-PFGE and southern blotting experiments showed that three plasmids of different sizes (33, 60, and 250 kb) all carried the mcr-1 gene. The plasmid conjugation assays revealed that Salmonella acquired mcr-1 harboring plasmids by horizontal transfer. Sequencing and plasmid type analysis revealed that these plasmids were types IncX4, IncI2, and IncHI2. Among them, IncX4 and IncI2 plasmids had extremely similar backbones and contained one resistant gene mcr-1. IncHI2 plasmid contained multiple resistant genes including bla CTX-M, oqxB, sul, aph, aadA, and bla TEM. We identified 11 mcr-1 harboring S. Typhimurium strains in China and described their characteristics. Our findings indicate that the mcr-1 gene can effectively spread among intestinal bacteria by horizontal transfer of three types of plasmids. Moreover, the IncHI2 plasmid can also mediate the transfer of other drug resistance genes. These results reveal that constant surveillance of mcr-1 harboring S Typhimurium is imperative to prevent the spread of colistin resistance.

Project description:Regulation of flagellum biosynthesis is a hierarchical process that is tightly controlled to allow for efficient tuning of flagellar expression. Flagellum-mediated motility directs Salmonella enterica serovar Typhimurium toward the epithelial surface to enhance gut colonization, but flagella are potent activators of innate immune signaling, so fine-tuning flagellar expression is necessary for immune avoidance. In this work, we evaluate the role of the LysR transcriptional regulator YeiE in regulating flagellum-mediated motility. We show that yeiE is necessary and sufficient for swimming motility. A ΔyeiE mutant is defective for gut colonization in both the calf ligated ileal loop model and the murine colitis model due to its lack of motility. Expression of flagellar class 2 and 3 but not class 1 genes is reduced in the ΔyeiE mutant. We linked the motility dysregulation of the ΔyeiE mutant to repression of the anti-FlhD4C2 factor STM1697. Together, our results indicate that YeiE promotes virulence by enhancing cell motility, thereby providing a new regulatory control point for flagellar expression in Salmonella Typhimurium. IMPORTANCE The ability to finely tune virulence factor gene expression is required for bacterial pathogens to successfully colonize a host. Flagellum-mediated motility is critical for many gut pathogens to establish productive infections. However, flagella activate the immune system, leading to bacterial clearance; therefore, tight control of flagellar gene expression enhances bacterial fitness in the host. Here, we demonstrate that the transcriptional regulator YeiE acts as a control point for flagellar gene expression and is required for Salmonella Typhimurium to establish a productive infection in mammals. The expression of an inhibitor of flagellar biogenesis is repressed in the absence of yeiE. Our work adds a new layer to the tightly controlled cascade regulating control of flagellar gene expression to facilitate the fitness of an enteric pathogen.

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Dataset Information

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

Publications

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

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