Repeated Occurrence of Mobile Colistin Resistance Gene-Carrying Plasmids in Pathogenic Escherichia coli from German Pig Farms

Abstract

The global spread of plasmid-mediated mobile colistin resistance (mcr) genes threatens the vital role of colistin as a drug of last resort. We investigated whether the recurrent occurrence of specific E. coli pathotypes and plasmids in individual pig farms resulted from the continued presence or repeated reintroduction of distinct E. coli strains. E. coli isolates (n = 154) obtained from three pig farms with at least four consecutive years of mcr detection positive for virulence-associated genes (VAGs) predicting an intestinal pathogenic pathotype via polymerase chain reaction were analyzed. Detailed investigation of VAGs, antimicrobial resistance genes and plasmid Inc types was conducted using whole genome sequencing for 87 selected isolates. Sixty-one E. coli isolates harbored mcr-1, and one isolate carried mcr-4. On Farm 1, mcr-positive isolates were either edema disease E. coli (EDEC; 77.3%) or enterotoxigenic E. coli (ETEC; 22.7%). On Farm 2, all mcr-positive strains were ETEC, while mcr-positive isolates from Farm 3 showed a wider range of pathotypes. The mcr-1.1 gene was located on IncHI2 (Farm 1), IncX4 (Farm 2) or IncX4 and IncI2 plasmids (Farm 3). These findings suggest that various pathogenic E. coli strains play an important role in maintaining plasmid-encoded colistin resistance genes in the pig environment over time.

Keywords: EDEC; ETEC; Escherichia coli; IncHI2; IncI2; IncX4; mcr-1; mobile colistin resistance; plasmid; swine.

Figure 1

Schematic circular representation of E. coli virulence plasmids detected in all three farms over the years (details given in Table 2). For comparison, one representative isolate per year and farm was selected. The reference plasmids used were p15ODTXV (A); NCBI Reference Sequence: MG904998.1) and pUMNK88_K88 (B); NCBI Reference Sequence: CP002730.1), shown in the inner rings. The plasmids detected in isolates from the three farms are arranged according to farm and year of isolation. Isolates from the same farm are color-coded (A): Farm 1 = red, Farm 2 = green, Farm 3 = blue; (B): Farm 1 = orange, Farm 2 = turquoise/blue, Farm 3 = violet). The color of the earliest isolation is displayed as the lightest, while the color of the latest isolation is displayed as the darkest.

Figure 2

Neighbor-joining tree based on the comparison of 2670 core genome genes of 87 E. coli isolates from Farm 1, Farm 2 and Farm 3, with the respective multilocus sequence types (MLST) and isolation years color-coded. The occurrence of mcr genes and their location on different plasmids are shown. Farm specific clustering of isolates from different years can be observed for Farm 1, Farm 2 and Farm 3.

Figure 3

Overview of the phylogenetic grouping of mcr-positive and mcr-negative E. coli isolates sorted by Farms 1 to 3 and in total.

Figure 4

Schematic circular representation of detected MCR-1 plasmids from three farms in our study compared to most similar reference plasmids predicted using the BacWGST database. One representative mcr-positive E. coli isolate per year and farm was selected for comparison (details given in Table 2). If two different mcr-positive pathotypes were detected per year and farm, we selected both pathotypes. The plasmids from our study are arranged in order of isolation year, with the earliest (light color) on the third innermost ring and the latest (dark color) on the outermost ring in (A–D). The reference plasmids are shown in the two innermost rings. Reference plasmids (NCBI Reference Sequence in brackets) included are as follows: pDJB-3 (MK574666.1), pGD27-70 (MN232195.1) for Farm 1 (A); pMCR-1-IHIT35346 (KX894453.1), pGMI17-004_2 (NZ_CP028167.1) for Farm 2 (B); pMCR-1-IHIT35346 (KX894453.1), pCFSAN061769_01 (CP042970.1) for Farm 3 (C); pC2 (LC473131.1) and p778 (MN746292.1) for Farm 3 (D).