Dynamics of a large multidrug-resistant plasmid encoding New Delhi metallo-β-lactamase-1 and oxacillinase-58 carbapenemases in Acinetobacter baumannii clinical isolates from a tertiary hospital in Malaysia

Acinetobacter baumannii carrying the bla _NDM-1 gene, which encodes New Delhi metallo-β-lactamase-1 (NDM-1), exhibits resistance to nearly all β-lactams and is not affected by β-lactamase inhibitors, limiting treatment options. The bla _NDM-1  gene is often associated with other antimicrobial resistance (AMR) genes, resulting in multidrug-resistant (MDR) phenotypes. We previously reported a large, circa (ca.) 170 kb plasmid co-harbouring bla _NDM-1 - and bla _OXA-58-encoded carbapenemases in clinical MDR Acinetobacter nosocomialis and A. baumannii isolates from a tertiary hospital in Terengganu, Malaysia, in 2015 and 2016, respectively. In this study, we identified four additional MDR A. baumannii isolates from the same hospital (2018-2020) carrying bla _NDM-1 on a similar plasmid. Complete genome sequences were obtained using a hybrid assembly of short-read DNA Nanoball Sequencing(DNBSeq) and long-read (Oxford Nanopore Technologies) data. All isolates belonged to distinct, unrelated clonal lineages, indicating the ongoing horizontal transmission of this plasmid. Comparative analysis revealed substantial structural variability among the plasmids, largely driven by insertion sequence elements and mobile xrs (also known as pdif) modules. In two A. baumannii isolates (i.e. AC1932 and AC2014), bla _OXA-58 and several adjacent xrs modules were absent. Despite this, carbapenem minimum inhibitory concentrations remained comparable across all isolates, strongly indicating that bla _NDM-1 is the primary carbapenem resistance determinant. Notably, in A. baumannii AC1839, we identified a 15,434 bp presumptive transposon carrying a type III cyclic oligonucleotide-based anti-phage signalling system, inserted within the xrs-rich region of the plasmid. AC1839 also carried a separate 11.1 kp plasmid carrying the tetracycline resistance genes tetA(39)-tetR within an xrs module. Identical xrs modules were identified in other unrelated Acinetobacter plasmids. These results underscore the mobility and potential roles of xrs modules in AMR gene dissemination and, more generally, in shaping Acinetobacter plasmid evolution.