Diversified mcr-1-Harbouring Plasmid Reservoirs Confer Resistance to Colistin in Human Gut Microbiota

Abstract

Colistin is an ultimate line of refuge against multidrug-resistant Gram-negative pathogens. Very recently, the emergence of plasmid-mediatedmcr-1colistin resistance has become a great challenge to global public health, raising the possibility that dissemination of themcr-1gene is underestimated and diversified. Here, we report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients. Structural and functional analyses determined that the colistin resistance is conferred purely by the singlemcr-1gene. Genetic and sequence mapping revealed thatmcr-1-harbouring plasmid reservoirs are present in diversity. Together, the data represent the first evidence of diversity inmcr-1-harbouring plasmid reservoirs of human gut microbiota.

Importance: The plasmid-mediated mobile colistin resistance gene (mcr-1) challenged greatly the conventional idea mentioned above that colistin is an ultimate line of refuge against lethal infections by multidrug-resistant Gram-negative pathogens. It is a possibility that diversified dissemination of themcr-1gene might be greatly underestimated. We report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients and functionally define the colistin resistance conferred purely by the singlemcr-1gene. Genetic and sequence mapping revealed unexpected diversity among themcr-1-harbouring plasmid reservoirs of human gut microbiota.

FIG 1

Global distribution of the mcr-1 colistin resistance gene. The countries where the mcr-1 gene was discovered are highlighted in blue.

FIG 2

Genetic, structural, and molecular characterization of the mcr-1-harbouring plasmids from gut microbiota of diarrhea patients. (a) Scheme for the four mcr-1-harbouring plasmids. pHNSHP45 is a plasmid from the Chinese pig isolate of E. coli with a known genome sequence (5); the other three plasmids (referred to as pE15004, pE15015,and pE15017) were isolated from three clinical E. coli strains (E15004, E15015, and E15017) collected from diarrhea patients admitted to hospitals in Shenzhen City, China, in 2015. Arrows denote the known (and/or putative) genes. A mobile element, ISApl1-mcr-1 (or an mcr-1-containing mobile element), is highlighted with a yellow background. The gray arrow illustrated with plasmid pE15004 (in panel a) represents a DNA fragment of 1,189 bp that shows 100% identity to the equivalent sequence of Salmonella enterica serovar Heidelberg plasmid pSH146-32, whereas the mcr-1 gene of plasmid pE15004 (and/or plasmid pE15015 plus pE15017) is 100% identical to the counterpart gene of E. coli SHP45 strain plasmid pHNSHP45 (5). The mcr-1-harbouring mobile element candidate from the clinical pE15017 plasmid exhibited 99% identity to that of E. coli plasmid pHNSHP45 (5). The broken arrow (tnpA*) denotes the intergenic sequence that is closely next to the tnpA gene at the 3-terminus determined in our trials, and the dots indicate that point mutations are present in tnpA* and a hypothetical protein-encoding locus (hp gene). PCR-based fine mapping suggested that the pE15017 plasmid carries all four genes (nikB, pilP, virD4, and virB4), similarly to E. coli plasmid pHNSHP45, but that such is not the case for the other two clinical mcr-1-harbouring plasmids (pE15004 andpE15015) that we have reported here. The results validated the idea that the diversified plasmid background is linked to a mcr-1 colistin resistance gene. (b) Modeled structure of the enzymatic domain of MCR-1 protein. Structural modeling was performed using the program of Swiss model and Neisseria lipooligosaccharide phosphoethanolamine transferase A (LptA) as the structural template (PDB: 4KAV), and the resultant output (ribbon photograph) was given using PyMol software. (c) mcr-1-based screening of clinical isolates from gut microbiota of diarrhea patients. The expected amplicon of the full-length (~1.6-kb) mcr-1 gene is indicated with a red arrow, and the clinical mcr-1-positive isolates (E15004, E15015, and E15017) are highlighted in red. (d) Profile for plasmids extracted from the clinical mcr-1-positive isolates (E15004, E15015, and E15017) determined on the basis of 0.7% agarose gel electrophoresis. (e to i) Molecular detection of the acquired three plasmids using five pairs of specific primers that separately target the mcr-1 colistin resistance gene (e), nikB (f), pilP (g), and type IV secretion system-encoding genes virD4 (h) and virB4 (i). (j and k) PCR screening for the two neighboring genes of mcr-1, the transposase-encoding tnpA gene (j) and the hypothetical protein (hp)-encoding gene (k). M denotes a Trans2K Plus II DNA ladder (TransGen Biotech, Beijing, China), and minus refers to the negative control.

FIG 3

16S-based identification and Gram staining analyses of the three mcr-1-harbouring isolates. (a) 16S-based phylogenetic tree of the three mcr-1-containing isolates. (b) Gram staining analyses for the E15004 isolate. (c) Gram staining analyses for the E15015 isolate. (d) Gram staining analyses for the E15017 isolate.

FIG 4

Determination of colistin resistance conferred by the plasmid-borne mcr-1 gene. (a) Determination of colistin MIC of the clinical E. coli isolates from diarrhea patients. (b) Expression of mcr-1 augments resistance of colistin-susceptible strain E. coli MG1655 to the colistin antibiotics. The three clinical mcr-1-positive E. coli strains (E15004, E15015, and E15017) are highlighted in red, whereas the prototypical wild-type strain (also mcr-1-negative strain) of E. coli, MG1655, is referred to the negative control (in blue). To determine the MIC of colistin, the mid-log-phase cultures (optical density at 600 nm [OD₆₀₀] = 0.7) in serial dilution were spotted on LBA plates supplemented with colistin at various levels (0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, and 32.0 mg/liter) and maintained overnight at 37°C. Expression vector pBAD24 is used for functional cloning of the mcr-1 gene in E. coli. Expression of mcr-1 is induced by the addition of 0.2% arabinose to LBA media.