Inter-plasmid transfer of antibiotic resistance genes accelerates antibiotic resistance in bacterial pathogens

Abstract

Antimicrobial resistance is a major threat for public health. Plasmids play a critical role in the spread of antimicrobial resistance via horizontal gene transfer between bacterial species. However, it remains unclear how plasmids originally recruit and assemble various antibiotic resistance genes (ARGs). Here, we track ARG recruitment and assembly in clinically relevant plasmids by combining a systematic analysis of 2420 complete plasmid genomes and experimental validation. Results showed that ARG transfer across plasmids is prevalent, and 87% ARGs were observed to potentially transfer among various plasmids among 8229 plasmid-borne ARGs. Interestingly, recruitment and assembly of ARGs occur mostly among compatible plasmids within the same bacterial cell, with over 88% of ARG transfers occurring between compatible plasmids. Integron and insertion sequences drive the ongoing ARG acquisition by plasmids, especially in which IS26 facilitates 63.1% of ARG transfer events among plasmids. In vitro experiment validated the important role of IS26 involved in transferring gentamicin resistance gene aacC1 between compatible plasmids. Network analysis showed four beta-lactam genes (blaTEM-1, blaNDM-4, blaKPC-2, and blaSHV-1) shuffling among 1029 plasmids and 45 clinical pathogens, suggesting that clinically alarming ARGs transferred accelerate the propagation of antibiotic resistance in clinical pathogens. ARGs in plasmids are also able to transmit across clinical and environmental boundaries, in terms of the high-sequence similarities of plasmid-borne ARGs between clinical and environmental plasmids. This study demonstrated that inter-plasmid ARG transfer is a universal mechanism for plasmid to recruit various ARGs, thus advancing our understanding of the emergence of multidrug-resistant plasmids.

Keywords: ESKAPE pathogens; antibiotic resistance genes; antimicrobial resistance; bioinformatics; horizontal gene transfer; plasmids.

Figure 1

Workflow for identification of recently transferred ARGs in plasmids.

Figure 2

ARGs transfer across clinically relevant plasmids; (A) the number of ARGs in clinical plasmids; (B) the network connecting plasmids with recently transferred ARGs; (C) correlation of transfer frequency and number of ARGs in each plasmid.

Figure 3

Horizontal gene transfer events among different plasmid types; (A) ARG transfers among compatible or incompatible plasmids; node indicate plasmid taxonomic units; the edges indicate ARG transfers between two nodes; (B) ARG occurrences in compatible or incompatible plasmids.

Figure 4

HGT frequency of recent transferred ARGs mediated by MGEs; (A) prevalence of ARGs mediated by mobile genetic elements, the dots indicate a specific ARG subtype was mediated by MGEs; (B) network analysis illustrates ARG transfer mediated by MGEs (IS and integrons); the dots represent ARG or plasmids; the ARG connecting two plasmids indicated that ARG transfer occurred in two plasmids; the edges depict the MGE-mediated transfer; (C) a boxplot is plotted showing frequency of ARG transfer mediated by MGEs; frequency of top five ARG transfer mediated by (D) integron and (E) ISs.

Figure 5

Experimental validation IS26-mediated inter-plasmid ARG transfer; (A) experimental design; (B) confocal laser scanning microscopy of IS26-mediating ARG transfer between plasmids; (C) gel electrophoresis of aacC1 gene in the pTDC28 and RP4; M: DNA ladder; p: positive control (pUC57-lacI^q-IS26-Plac-gfp_mut3b-Gm⁺-IS26); c1: pDTC28; t1: pDTC28-IS26-Plac-gfp_mut3b-Gm⁺-IS26; c2: RP4; t2: RP4- IS26-Plac-gfp_mut3b-Gm⁺-IS26; the full length of aacC1 is 813 bp.

Figure 6

Transfer of ARGs among plasmids that carried by pathogens; (A) network illustrated ARG transfer among plasmid carried by pathogens; the dots represent ARGs, plasmids, and pathogens; the edges connecting ARGs and plasmids depict that ARG transfer among plasmids, and edge connecting plasmid and pathogens showed plasmids carried by pathogens; (D) network showed ARGs transfer among ESKAPE pathogens; representative ARGs of (C) bla_TEM-1 and (D) bla_KPC-2 transfer among plasmid that carried by pathogens.

Figure 7

Transfer of ARGs in plasmids across clinical and environmental strains; (A) plasmid taxonomic units identified in clinical and environmental plasmids; (B) network of ARG in all plasmids; the nodes indicate plasmid taxonomic units and ARGs; the edges present ARG transfer events between two plasmids; (C) network reveals ARG transfer among plasmids isolated from clinic and environment; the nodes indicate clinical plasmids, environmental plasmids, and ARGs; the edges represent ARG transfer events between clinical and environmental plasmids.