1. P0291
25th ECCMID
25 – 28 April 2015
Copenhagen, Denmark
Complete sequence of plasmid pNDM-32, harbouring blaNDM-1 and armA, from
A. baumannii and sequence variation of plasmids and resistance islands in closely related isolates.
Lim Jones1,2, Karthikeyan Kumarasamy3, Tom Connor4, Mark Toleman1, Kathy Raven5, M. Estée Török5, Robin Howe2, Sharon J. Peacock5 and Tim Walsh1 
1 Department of Medical Microbiology and Infectious Diseases, Cardiff University, Heath Park, Cardiff UK 2 Public Health Wales, Microbiology, Cardiff UK 3 Department of Microbiology, Univeristy of Madras, India
4 Cardiff School of Biosciences, Sir Martin Evans Building, Cardiff UK 5 Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge UK
Introduction. Multi-drug resistant Acinetobacter spp. producing NDM-1-like enzymes are increasingly recognized but relatively little is known about the genetic contexts and mobile genetic elements associated with blaNDM-1-like genes in these organisms.
Results cont. The complete sequence of an ~85 kb plasmid, pNDM-32, was assembled for A. baumannii CHI-32 (see Figure 1). This plasmid contained a replicase, identical to one previously associated with plasmids harbouring blaOXA-58, but no identifiable relaxase or type IV secretion system. pNDM-32 contains eight antimicrobial resistance genes and numerous insertion sequences. The local genetic context of blaNDM-1 is similar to that in Tn125 but ISCR27 is disrupted by IS15-Δ and a rearrangement has occurred so that the 3’ prime fragment of ISCR27 is upstream of blaNDM-1 and is bracketed by two copies of IS15-Δ to form a composite transposon.
The local genetic context of blaNDM-1 is similar to that in Tn125 but ISCR27 is disrupted by IS15-Δ and a rearrangement has occurred so that the 3’ prime fragment of ISCR27 is upstream of blaNDM-1 and is bracketed by two copies of IS15-Δ to form a composite transposon (see Figure 2). In addition the plasmid contains a context found in a number of other resistance plasmids containing a class-1-integron harbouring the aminoglycoside and trimethoprim resistance genes, aadA2 and dfrA12, with armA and the macrolide resistance gene msrE and mphE downstream.
Methods. Whole genome sequences of three clinical A. baumannii isolates from India were assembled de novo using Velvet. Annotation was performed using RAST and edited in Artemis. Plasmid assembly gaps were closed by PCR and amplicon sequencing. Antimicrobial resistance (AMR) genes were identified using Resfinder. Typing was performed using Pasteur MLST and ribosomal MLST schemes. Susceptibility testing was performed using gradient strip methods.
Figure 1 – Gene map of pNDM-32 (LN833432). from A. baumannii CHI-32.
Arrowed boxes indicate ORFs. The blue inner line indicates the %GC which is calculated using a sliding window of 180 bp.
Results. All isolates were ST1 and their rMLST loci were identical to the ST1 A. baumannii AYE strain. They were extensively drug resistant but susceptible to colistin, with A. baumannii CHI-34 also being amikacin susceptible. There were significant differences in the AMR genes present in the three isolates.
Figure 3 – ACT comparison of contigs from A. baumannii CHI-32, CHI-34 and CHI-45-1 with AbaR3 from A. baumannii A85.
Further analysis of WGS contigs suggests that CHI-45-1 and CHI-34 contain plasmids very similar to pNDM-32 but that in CHI-34 a large section is missing including the context containing armA and the class-1-integron. All 3 isolates also contained a resistance island interrupting the comM gene. In CHI-34 and CHI-45-1 this is probably identical to AbaR3 but in CHI-32 the entire section containing the AMR genes is absent (see Figure 3).
Conclusions. These results show that blaNDM-1 is located in another novel genetic element in these isolates. The plasmid containing blaNDM-1 and armA is likely to be non-mobilizable but the resistance genes are associated with multiple elements which could facilitate their introduction into new genetic locations. The differences in the AMR regions in these three very closely related strain backgrounds suggest that these contexts are undergoing rapid evolutionary change.
The 16s RNA methylase gene, armA, was absent in A. baumannii CHI-34. All isolates contained blaOXA-23 and a blaADC-30-like genes downstream of ISAba1 and its promoter.
A. baumannii 161/07
A. baumannii CHI-32
Figure 2 –
Comparison of full Tn125 containing blaNDM-1 from A. baumannii 161/07 with context from pNDM-32