Genomic Characterization of Two Escherichia fergusonii Isolates Harboring mcr-1 Gene From Farm Environment

Abstract

The prevalence and transmission of mobile colistin resistance (mcr) genes have led to a severe threat to humans and animals. Escherichia fergusonii is an emerging pathogen which is closely related to a variety of diseases. However, the report of mcr genes harboring E. fergusonii is still rare. One study in Brazil reported the E. fergusonii isolates with IncHI2-type plasmids harboring mcr-1. A Chinese study reported two strains carrying mcr-1 gene with the same plasmid type IncI2. Here, we identified two strains of E. fergusonii carrying mcr-1 gene from farm environments with IncX4-type and IncI2-type plasmids, respectively. To our best knowledge, this is the first report about mcr-1 gene located on IncX4-type plasmid in E. fergusonii. We investigate the resistance mechanism of colistin-resistant Escherichia fergusonii strains 6S41-1 and 5ZF15-2-1 and elucidate the genetic context of plasmids carrying mcr-1 genes. In addition, we also investigated chromosomal mutations mediated colistin resistance in these two strains. Species identification was performed using MALDI-TOF MS and 16S rRNA gene sequencing. The detection of mcr-1 gene was determined by PCR and Sanger sequencing. S1-pulsed-field gel electrophoresis (PFGE), Southern blotting, antimicrobial susceptibility testing, conjugation experiments, complete genome sequencing, and core genome analysis were conducted to investigate the characteristics of isolates harboring mcr-1. The mcr-1 genes on two strains were both plasmids encoded and the typical IS26-parA-mcr-1-pap2 cassette was identified in p6S41-1 while a nikA-nikB-mcr-1 locus sites on the conjugative plasmid p5ZF15-2-1. In addition, Core genome analysis reveals that E. fergusonii 6S41-1 and 5ZF15-2-1 have close genetic relationships. The mcr-1 gene is located on conjugative IncI2-type plasmid p5ZF15-2-1, which provides support for its further transmission. In addition, there's the possibility of mcr-1 spreading to humans through farm environments and thereby threatening public health. Therefore, continuous monitoring and investigations of mcr-1 among Enterobacteriaceae in farm environments are necessary to control the spread.

Keywords: IncI2; IncX4; core genome analysis; farm environments; whole-genome sequencing.

Figure 1

Plasmid profiles of E. fergusonii 6S41-1 and 5ZF15-2-1. (A) Plasmid size determination by S1-PFGE, with Salmonella enterica serotype Braenderup H9812 as the size marker. (B) Southern blotting hybridization with an mcr-1-specific probe.

Figure 2

The genetic context of mcr-1 gene on p6S41-1 and p5ZF15-2-1. (A) Comparison of genes surrounding mcr-1 on p6S41-1, pMFDS1318.1 (accession number: MK875282), pWI2-mcr (accession number: LT838201) and pICBEC7Pmcr (accession number: CP017246). (B) Comparison of genes surrounding mcr-1 on p5ZF15-2-1, pAH01-2 (accession number: CP055253) and pGD65-3 (accession number: KY075661). Open reading frames (ORFs) are shown as arrows and indicated according to their putative functions. Purple indicates genes related to mobile elements, red indicates genes related to drug resistance, light yellow indicates genes related to plasmid stability, blue indicates genes involved in conjugation and orange represents other functional genes. Hypothetical protein encoded genes are colored by grey. Regions with a high degree of homology are indicated by pink shading.

Figure 3

The genetic features of plasmid p6S41-1 and p5ZF15-2-1. (A) Circular comparison between mcr-1 bearing IncX4 plasmids p6S41-1 in this study and four similar IncX4 plasmids in NCBI nr/nt database. p6S41-1 was used as the reference in the outermost ring. (B) Circular comparison between mcr-1 bearing IncI2 plasmids p5ZF15-2-1 in this study and four similar IncI2 plasmids in NCBI nr/nt database. p5ZF15-2-1 was used as the reference in the outermost ring.

Figure 4

The maximum likelihood phylogenetic tree based on the core genome sequences of 115 E. fergusonii strains. E. fergusonii 6S41-1 (BioSample: SAMN20239770) and 5ZF15-2-1 (BioSample: SAMN20243900) are indicated in red. The other mcr-1-bearing strains were marked by dark red. The three circles around the phylogenetic tree indicate the location (inner circle), collection date and source (outer circle) of these strains.