Fig 1.
Addictive antibiotic resistance plasmids.
The replicon (rep, solid circle), antitoxin (AT, arrowhead) and toxin (T, arrow) genes of a PSK/addiction system, an antibiotic resistance gene (AbR) and corresponding antibiotic (Ab, solid blocks) are shown. (A) An addictive plasmid is stable in the absence of antibiotic selection. (B) An addictive plasmid can be displaced by an incompatible plasmid. (C) A compatible plasmid providing specific antitoxin (non-addictive compatible) leads to loss of addictive resistance plasmids from some cells. (D) An incompatible non-addictive interference plasmid providing specific antitoxin (non-addictive incompatible) ensures that all bacterial cells are ultimately free of both plasmid types.
Table 1.
Plasmids used in this study.
Table 2.
Bacterial strains used in this study.
Fig 2.
Construction of conjugative interference plasmids.
pJIMK46 (A, B) was constructed from pJIBE401 by replacing 28.5 kb including the MRR with tetA and then part of the pemK toxin gene with fosA3. pJIMK56 (C, D) was constructed from pJIE512b by replacing the blaCMY-2 gene and flanking IS with fosA3 and then part of the pndA toxin gene with tetA. Numbers indicate the positions of the amplified regions in GenBank accession nos. JX101693.1 (pEl1573) or HG970648.1 (pJIE512b). Blue and red arrows indicate overlapping primers, black arrows indicate other primers.
Table 3.
Protocol for mouse experiments.
Table 4.
Associated replicon types and addiction systems in Enterobacteriaceae plasmids.
Table 5.
Effect of entry exclusion on conjugative transfer efficiency.
Fig 3.
Acquisition and loss of pEl1573 from K. pneumoniae 13883.
Pulsed-field gel electrophoresis of S1-endonuclease treated extracts of Kp13883 before (1) and after (2) acquisition of pEl1573 (horizontal arrow) and after cure (3), showing other ‘bystander’ plasmids. M1, Mid-range and M2, Lambda PFG ladders (New England Biolabs, USA).
Fig 4.
In vivo cure of antibiotic resistance plasmids.
CTXSTETS E. coli (green) were detected in all four groups of mice at the start of the experiments (A-D). All groups of mice were then fed bacteria carrying CTXR target plasmid (pEl1573 or pJIE512b) with CTX, days 4–6 (red lines, A-D) and CTXR E. coli (red) appeared. Two groups of mice (A, B) then received the corresponding TETR interference plasmid (pJIMK46 or pJIMK56) with TET, days 8–10 (blue lines) resulting in a decline in number of CTXR E. coli. TETR E. coli (blue) also appeared and then declined. CTXSTETS E. coli appeared again after curing of target and interference plasmids but were killed by CTX administered on days 23–24 (black lines, A, B). CTXR E. coli persisted to end of protocol in control groups that did not receive interference plasmid (C, D).
Table 6.
In vivo cure of pEl1573-colonized mice.
Table 7.
In vivo cure of pJIE512b-colonized mice.
Fig 5.
Exclusion and incompatibility.
Replicon (solid circle), antitoxin and toxin genes (arrowhead, arrow) and antibiotic resistance genes (CTXR, orange and TETR, black solid blocks). Interference plasmid not excluded by entry exclusion system (EES) is incompatible (INC) with resident CTXR plasmid and is selected by TET.