A large self-transmissible resistance plasmid from Nigeria contains genes that ameliorate a carrying cost

Abstract

Antimicrobial resistance is rapidly expanding, in a large part due to mobile genetic elements. We screened 94 fecal fluoroquinolone-resistant Escherichia coli isolates from Nigeria for six plasmid-mediated quinolone resistance (PMQR) genes. Sixteen isolates harbored at least one of the PMQR genes and four were positive for aac-6-Ib-cr. In one strain, aac-6-Ib-cr was mapped to a 125 Kb self-transmissible IncFII plasmid, pMB2, which also bears bla_CTX-M-15, seven other functional resistance genes and multiple resistance pseudogenes. Laboratory strains carrying pMB2 grew faster than isogenic strains lacking the plasmid in both rich and minimal media. We excised a 32 Kb fragment containing transporter genes and several open-reading frames of unknown function. The resulting 93 Kb mini-plasmid conferred slower growth rates and lower fitness than wildtype pMB2. Trans-complementing the deletion with the cloned sitABCD genes confirmed that they accounted for the growth advantage conferred by pMB2 in iron-depleted media. pMB2 is a large plasmid with a flexible resistance region that contains loci that can account for evolutionary success in the absence of antimicrobials. Ancillary functions conferred by resistance plasmids can mediate their retention and transmissibility, worsening the trajectory for antimicrobial resistance and potentially circumventing efforts to contain resistance through restricted use.

Figure 1

Circular plot illustrating the general features of pMB2. The figure shows the organization of pMB2 ORFs in black and a BLASTn comparison between pMB2 and the five best hits E. coli plasmids retrieved from NCBI (from the innermost to the outermost circles, plasmids p6409, pECAZ146_1, p109, pRCS52 and pRCS57 with the GenBank accession numbers CP010372.1, CP018990.1, CP023372.1, LO017736.1 and LO017738.1, respectively). A brown star marks the likely location of the origin of replication.

Figure 2

Genetic context of the pMB2 antimicrobial resistance region. Arrows indicate the direction of transcription for genes. Gene cassettes are shown by pale blue boxes, the conserved sequences (5′ and 3′-CS) of integrons as orange boxes and insertion sequences as white block arrows labelled with the IS number/name, with the pointed end indicating the inverted right repeat (IRR). Unit transposons are shown as boxes of different colors and their IRs are shown as flags, with the flat side at the outer boundary of the transposon. Direct repeats are shown as ‘lollipops’ of the same color. The zig-zag line indicates which end of the feature is missing. Gaps > 50 bp are indicated by dashed red lines and the length in bp given.

Figure 3

18 h growth time-course of DH5α (orange), DH5α carrying 125 Kb plasmid pMB2 (grey) and 90 Kb plasmid pLMJ50 (yellow) in LB broth. The blue circles represent a no bacteria control.

Figure 4

18 h growth time-course of DH5α carrying the pRMKO miniplasmid derived from pMB2 (green) compared to growth of DH5α (blue), DH5α carrying pMB2 (yellow) and 90 Kb control plasmid pLMJ50 (orange) in LB broth. Brown circles represent a no bacterial control.

Figure 5

18 h growth time-course in Davis minimal media pre-treated with deferrated EDDA to deplete iron. Wildtype M63c strain (grey) compared to DH5α (orange), DH5α carrying pMB2 (green), pRMKO miniplasmid (yellow), pRMKO miniplasmid plus the cloned sit genes in pINK2301 and 90 Kb control plasmid pLMJ50 (blue).

Figure 6

Relative fitness of the initially listed strain compared to the subsequently listed strain in rich media (LB) and minimal media (DMEM). Each data point is the mean of duplicate experiments and the error bars are the computed standard deviations.

Figure 7

Plasmid stability in the absence of selection (A) stability of pMB2 in M63c, its natural host (dark line) and control plasmid pBR322 in DH5α (grey line) (B) stability of pMB2 (grey line) and pRMKO (dark line) in DH5α.