Persistence of plasmid and tet(X4) in an Escherichia coli isolate coharboring bla NDM-5 and mcr-1 after acquiring an IncFII tet(X4)-positive plasmid

Abstract

The prevalence of plasmid-mediated tigecycline resistance gene tet(X4) is presenting an increasing trend. Once tet(X4)-bearing plasmids are captured by multidrug-resistant bacteria, such as bla _NDM and mcr-coharboring bacteria, it will promote bacteria to develop an ultra-broad resistance spectrum, limiting clinical treatment options. However, little is known about the destiny of such bacteria or how they will evolve in the future. Herein, we constructed a multidrug-resistant bacteria coharboring tet(X4), bla _NDM-5, and mcr-1 by introducing a tet(X4)-bearing plasmid into a bla _NDM-5 and mcr-1 positive E. coli strain. Subsequently, the stability of tet(X4) and the plasmid was measured after being evolved under tigecycline or antibiotic-free circumstance. Interestingly, we observed both tet(X4)-bearing plasmids in tigecycline treated strains and non-tigecycline treated strains were stable, which might be jointly affected by the increased conjugation frequency and the structural alterations of the tet(X4)-positive plasmid. However, the stability of tet(X4) gene showed different scenarios in the two types of evolved strains. The tet(X4) gene in non-tigecycline treated strains was stable whereas the tet(X4) gene was discarded rapidly in tigecycline treated strains. Accordingly, we found the expression levels of tet(X4) gene in tigecycline-treated strains were several times higher than in non-tigecycline treated strains and ancestral strains, which might in turn impose a stronger burden on the host bacteria. SNPs analysis revealed that a myriad of mutations occurred in genes involving in conjugation transfer, and the missense mutation of marR gene in chromosome of tigecycline treated strains might account for the completely different stability of tet(X4)-bearing plasmid and tet(X4) gene. Collectively, these findings shed a light on the possibility of the emergence of multidrug resistant bacteria due to the transmission of tet(X4)-bearing plasmid, and highlighted that the antibiotic residues may be critical to the development of such bacteria.

Keywords: blaNDM-5; mcr-1; plasmid stability; tet(X4) stability; tet(X4)-bearing plasmid.

Figure 1

tet(X4)-positive plasmid imposes fitness cost on E. coli N31. (A) Relative fitness of tet(X4)-positive plasmid-carrying strains versus isogenic plasmid-free strains in vitro. (B) Comparison of growth curve and generation time (lower right corner) between tet(X4)-positive plasmid-carrying and plasmid-free strains.

Figure 2

Stability of the focal plasmid and tet(X4) gene. (A) Stability of the focal plasmid and tet(X4) gene in five selected strains evolved in antibiotic-free broth and the ancestral strain at the 30^th day. (B) Stability of the focal plasmid and tet(X4) gene in five selected strains evolved in tigecycline containing broth and the ancestral strain at the 30^th day.

Figure 3

Evolved strains with different resistance levels to tigecycline under selective and non-selective pressure. (A) Comparison of MIC of tigecycline between ancestral and evolved strains. (B–D) Time-kill curve of tigecycline for the ancestral strain (B), evolved strains without tigecycline (C), and evolved strains with tigecycline (D). (E) Comparison of the expression levels of tet(X4) gene between ancestral and evolved strains.

Figure 4

Improved conjugation frequencies observed in the evolved strains. (A) Comparison of tet(X4)-positive plasmid conjugation frequency between ancestral and evolved strains. (B) Comparison of the expression levels of conjugative transfer regulatory-related genes between ancestral and evolved strains. (C) Comparison of the expression levels of conjugative transfer genes between ancestral and evolved strains. (D) Comparison of the expression levels of conjugative transfer pilus assembly genes between ancestral and evolved strains.

Figure 5

Linear comparison among pF65-tet(X4) and two evolved plasmids. Two segments were discarded during evolution, and the AMR genes and insertion sequences in deleted regions were marked in red and blue, respectively.

Figure 6

Genomic mutations associated with conjugative transfer-related genes and antibiotic resistance genes on plasmid and chromosome. (A) Mutations observed on plasmid of evolved clones. (B) Mutations observed on chromosome of evolved clones. Blue rings represent the antibiotic evolved clones and the green rings represent the antibiotic-free evolved clones. Dots represent the mutations.