Extensive antimicrobial resistance mobilization via multicopy plasmid encapsidation mediated by temperate phages

Abstract

Objectives: To investigate the relevance of multicopy plasmids in antimicrobial resistance and assess their mobilization mediated by phage particles.

Methods: Several databases with complete sequences of plasmids and annotated genes were analysed. The 16S methyltransferase gene armA conferring high-level aminoglycoside resistance was used as a marker in eight different plasmids, from different incompatibility groups, and with differing sizes and plasmid copy numbers. All plasmids were transformed into Escherichia coli bearing one of four different lysogenic phages. Upon induction, encapsidation of armA in phage particles was evaluated using qRT-PCR and Southern blotting.

Results: Multicopy plasmids carry a vast set of emerging clinically important antimicrobial resistance genes. However, 60% of these plasmids do not bear mobility (MOB) genes. When carried on these multicopy plasmids, mobilization of a marker gene armA into phage capsids was up to 10000 times more frequent than when it was encoded by a large plasmid with a low copy number.

Conclusions: Multicopy plasmids and phages, two major mobile genetic elements (MGE) in bacteria, represent a novel high-efficiency transmission route of antimicrobial resistance genes that deserves further investigation.

Figure 1.

Histogram of 16702 fully sequenced and circular plasmids. Plasmids follow a bimodal distribution: small plasmids (<20 kb) and large plasmids (>20 kb). Plasmids were predicted to be conjugative (with T4SS), MOB (without T4SS, with a MOB gene) and non-MOB (without T4SS or a MOB gene). Note that most plasmids are not transmissible. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

Representative experiment showing the detection of armA in the phage DNA fraction of E. coli strains WG5 and DH5α lysogenized with Stx phages 933W, 557 or 312 or Cdt phage and DH5α lysogenized with Stx phages 933W or 312 transformed with the different plasmids. Shaded bars correspond to MCPs and white bars to large, low-copy-number plasmids.

Figure 3.

High efficiency spread of AMR through multicopy plasmid transduction. AMR genes borne on small MCPs (right) are encapsidated up to 10000 times more efficiently than when borne on large low-copy plasmids (left). As a consequence, the phages can disseminate over distance to transduce susceptible bacteria and transfer resistance genes. We propose a model where multicopy plasmid transduction is a major powerful route for AMR gene dissemination in nature, in which AMR spreads with high efficiency and over distance between bacteria from humans, animals and the environment. As the cargo of plasmids can also be other genes apart from AMR genes, this phenomenon represents a striking coordination between MGEs driving the evolution of bacterial populations. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.