The roles of HicBA and a novel toxin-antitoxin-like system, TsxAB, in the stability of IncX4 resistance plasmids in Escherichia coli.

Objectives: To identify toxin-antitoxin (TA)-like plasmid stability loci on IncX4 plasmids.

Methods: TA-like loci were identified bioinformatically and their contribution to stability of the IncX4 plasmid pJIE143 was tested in optimal growth conditions in vitro. The conservation of the TA-like loci identified was analysed within an updated IncX plasmid database.

Results: A novel TA-like locus, tsxAB, was identified on the IncX4 plasmid pJIE143, carrying the important plasmid-borne antibiotic resistance gene blaCTX-M-15. pJIE143 (the WT plasmid) and its tsxA mutant are stable for 80 bacterial generations in the absence of selective pressure but a tsxB deletion mutant of pJIE143 is relatively quickly lost without positive selection (91.1% ± 1.5% loss after 50 generations). Nine IncX subclasses were identified among 272 fully sequenced IncX plasmids, dominated by those identified as IncX3, IncX1 and IncX4 subclasses in PlasmidFinder. The novel TA-like locus, tsxAB, appears to be a feature of IncX4 plasmids, being present in 64 of 67 so identified, but only present in a single IncX1 plasmid (of 79 identified) and present in no other IncX plasmids.

Conclusions: tsxAB, a novel TA-like stability locus, is highly conserved in IncX4 plasmids associated with transmission of important antibiotic resistance genes. Previous in silico analysis indicated that IncX4 encodes only HicBA among the known TA systems. Here we show that HicBA does not contribute to plasmid stability in optimal growth conditions for Escherichia coli and instead demonstrate this role for a completely novel TA-like system, TsxAB, that appears both necessary and sufficient for E. coli addiction to IncX4 resistance plasmids.