Molecular characterisation of the NDM-1-encoding plasmid p2189-NDM in an Escherichia coli ST410 clinical isolate from Ghana

Abstract

Global dissemination of New Delhi metallo-β-lactamase (NDM)-producing bacteria has become a major health threat. However, there are few reports regarding the identification and characterisation of NDM-producing bacteria from West Africa, including Ghana. An Escherichia coli strain with resistance to meropenem was isolated from the Tamale Teaching Hospital in Ghana. Its identification and determination of antibiotic susceptibility profile were carried out using commercial systems. The antibiotic resistance mechanism was analysed by phenotypic detection kits, PCR, and DNA sequencing. Conjugation experiments, S1 nuclease pulsed field gel electrophoresis, and Southern blotting were performed. Finally, the NDM-1-harbouring plasmid was characterised using next-generation sequencing and phylogenetic analysis. The meropenem-resistant Escherichia coli strain EC2189 harboured blaNDM-1 and belonged to sequence type 410. blaNDM-1 was located on the IncHI type transferrable plasmid p2189-NDM (248,807 bp long), which co-carried multiple resistance genes, such as blaCTX-M-15, aadA1, aac(6')-Ib, sul3, dfrA12, and cmlA1. p2189-NDM phylogenetically differed from previously identified blaNDM-1-positive IncHI type plasmids. A truncated Tn125 containing blaNDM-1 was bracketed by an ISSm-1-like insertion sequence upstream and by a site-specific integrase downstream. To the best of our knowledge, we have, for the first time identified and molecularly characterised an NDM-1-producing Enterobacteriaceae strain in Ghana with blaNDM-1 that had a novel genetic structure. Our findings indicate a possibility of NDM-1 dissemination in Ghana and underscore the need for constant monitoring of carbapenemase-producing bacteria.

Fig 1. S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) of the donor strain EC2189, trans-conjugant TcEC2189 and recipient C600.

(A) PFGE of genomic DNA digested with S1-nuclease. (B) Southern blot hybridisation of the PFGE gel with a bla_NDM-1 specific probe. Lane M: Lambda ladder; Lane 1: EC2189; Lane 2: TcEC2189; Lane 3: C600.

Fig 2. Genetic characteristics of plasmid p2189-NDM.

(A) Comparisons of the plasmid backbone structure of p2189-NDM, with those of bla_NDM-1-positive and negative plasmids with highest homology. The truncated Tn125 region is highlighted in blue. Other coding sequences are coloured in orange. (B) Genetic structure comparison of the bla_NDM-1-surrounding region in p2189-NDM and IncHI type plasmid pPKPN1. Antimicrobial resistance genes are coloured in red. Mobile genetic elements are coloured in yellow, with the exception of IS125. The insertion sequence IS125 or truncated IS125 are coloured in green. The site-specific integrase is coloured in blue. Other coding sequences are coloured in orange. (C) Whole genome maximum phylogenetic analysis for some bla_NDM-1 positive and IncHI type bla_NDM-1 negative plasmids. Phylogenetic tree was constructed using maximum likelihood method with 1,000 bootstrap replicates. Bootstrap values are shown next to branches. Plasmid accession numbers, replicon types included the plasmids and the bla_NDM-1 containing information are listed next to the phylogenetic tree.