Analysis of the stepwise acquisition of bla(CTX-M-2) and subsequent acquisition of either bla(IMP-1) or bla(IMP-6) in highly conserved IncN-pST5 plasmids

OBJECTIVES: ESBL and carbapenemase genes in Enterobacterales spread via plasmids. Nosocomial outbreaks caused by Enterobacterales producing both CTX-M-2 and either IMP-1 or IMP-6-type carbapenemases have been reported. These organisms carry the incompatibility type N plasmid belonging to plasmid ST 5 (IncN-pST5). We investigated the construction process of the ESBL and carbapenemase genes co-carrying IncN-pST5. METHODS: We retrospectively performed draft WGS analysis for bla(IMP)- or bla(CTX-M)-positive Enterobacterales in our strain collection (n = 281). RESULTS: We selected four types of Escherichia coli plasmids for our study: type A, which carries both bla(CTX-M-2) and bla(IMP-1) (n = 6); type B, which carries both bla(CTX-M-2) and bla(IMP-6) (n = 2); type C, which carries bla(CTX-M-2) (n = 10); and type D, which carries no β-lactamase genes (n = 1). It should be noted that type D plasmid was only detected in E. coli TUM2805, which carries the bla(CTX-M-14) on the IncB/O/B/Z plasmid. Long-read sequencing using MinION revealed that all types of IncN-pST5 were highly conserved and carried a class 1 integron. Integron numbers were type A for In798, type B for In1690, type C for In127 and type D for In207. Because the gene cassettes downstream of bla(IMP) were different between In798 and In1690, the change from bla(IMP-1) to bla(IMP-6) by point mutation was unlikely. Representative plasmids from types A, B and C were conjugatively transferred with quite a high frequency between 1.3 × 10(−1) and 2.5 × 10(−2). CONCLUSIONS: This study suggested that IncN-pST5 acquired bla(CTX-M-2) by ISEcp1 in a stepwise manner, followed by either bla(IMP-1) or bla(IMP-6) into a class 1 integron.