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

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.


Introduction
The carbapenemase-producing Gram-negative bacteria have become a worldwide healthcare concern due to their resistance to carbapenems, which are one of the last resort antibiotics for the treatment of infectious diseases caused by multidrug-resistant bacteria [1].
bla NDM encodes Ambler class B New Delhi metallo-β-lactamase (NDM) that hydrolyses almost all β-lactams, including carbapenems.Since its initial discovery in the last decade, bla NDM-1 and its homologues have rapidly spread worldwide and become a major public health threat [2,3].In the African continent, NDM-producing bacteria had been detected mostly in northern and southern Africa [3], but to date, there have been no reports of NDM-producing bacteria, including Enterobacteriaceae, in Ghana.
Among Enterobacteriaceae, most of bla NDM-1 -containing plasmids can be passed on between different species by horizontal gene transfer.Moreover, bla NDM-1 is found in plasmids with diverse replicon types, including IncHI, and is usually located on mobile genetic elements, such as transposons or insertion sequences [4][5][6].Therefore, it is important to characterise bla NDM -carrying plasmids to understand the mechanism of gene acquisition and to trace their spread.In this study, for the first time, we describe p2189-NDM, a bla NDM-1 -harbouring plasmid, in a clinical isolate of Escherichia coli from Ghana, and demonstrate molecular characteristics of p2189-NDM using whole-genome sequencing.

Ethics approval
This study was approved by both the ethics committee of the Faculty of Medicine, Tokyo Medical and Dental University (M2017-208) and the ethics committee of Noguchi Memorial Institute for Medical Research, University of Ghana (FWA 00001824).Written informed consent was obtained from all participants of the study.

Bacterial isolate
Carbapenem-resistant E. coli strain EC2189 was isolated from a urine sample of a patient who had been hospitalised in Tamale Teaching Hospital in Ghana in 2016.Bacterial identification was performed using MALDI Biotyper (Bruker Daltonics, Karlsruhe, Germany) and VITEK MS (bioMe ´rieux Japan, Tokyo, Japan).

Antimicrobial susceptibility testing
Minimal inhibitory concentrations (MICs) of antibiotics were determined using MicroScan WalkAway system (Beckman Coulter, Tokyo, Japan) and interpreted according to the Clinical and Laboratory Standards Institute guideline [7].Quality control for the MICs was performed using the reference strains E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603.

Conjugation experiment
Conjugation experiments were performed by the agar mating method as described previously with some alterations [10].Briefly, EC2189 was conjugated with the rifampicin-resistant recipient E. coli strain C600 at the donor-to-recipient ratio of 1:10.Transconjugants were subsequently selected on BTB agar plates supplemented with 50 μg/ml rifampicin and 1 μg/ml meropenem.

S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blot hybridisation
Genomic DNA preparations for EC2189, C600 and transconjugant strains were made in agarose plugs and digested with S1 nuclease (Takara Bio, Shiga, Japan).The linearised plasmids and partially digested DNA were separated by using the CHEF-mapper XA system (Bio-Rad, Tokyo, Japan).After staining the PFGE gel, DNA fragments were transferred to Hybond N + membrane (GE Healthcare, Tokyo, Japan), hybridised using DIG-labelled bla NDM-1 probe, and then the signal was detected using DIG high prime DNA labelling and a detection starter kit (Roche Diagnostics, Tokyo, Japan) according to the manufacturer's instructions.

Plasmid analysis
Plasmids were extracted using a NucleoBond Xtra Midi kit (Takara Bio, Shiga, Japan) by following the manufacturer's instructions and sequenced by PacBio RSII (Pacific Biosciences of California, Menlo Park, CA).SMRT analysis v2.3 was used for the de novo assembly of the sequenced data.The plasmid sequences were annotated using online annotation system RAST [11].

Nucleotide sequence accession numbers
The complete plasmid nucleotide sequence of p2189-NDM was deposited under GenBank accession number CP029631.The draft genome sequence of strain EC2189 was deposited in the GenBank whole-genome shotgun database under accession number CP029630.

Strain EC2189 characteristics
E. coli strain EC2189 was resistant to 13 antibiotics, including meropenem, but was intermediate to imipenem, and remained fully susceptible to fosfomycin and cefmetazole (Table 1).EC2189 produced carbapenemases and harboured bla NDM-1 .MLST analysis revealed that EC2189 was related to E. coli ST410 strains with bla NDM-1 that were isolated in several countries, including Norway, UK, Switzerland, France, USA, and Poland [16].In most of these cases, isolation of E. coli ST410 strains with bla NDM-1 was associated with the history of patients' travel to Southeast Asia, Eastern Europe, or North Africa.Moreover, ST410 strains with bla NDM-4 and bla NDM-5 also were identified in China and Egypt, respectively [17,18].
Meropenem-resistant transconjugant strain TcEC2189 was successfully obtained by conjugation and showed almost similar MICs to EC2189 except for fluoroquinolones (Table 1).In S1-PFGE and Southern blot analyses, bla NDM-1 -positive signals were detected in both EC2189 and TcEC2189, indicating that bla NDM-1 was present on the transferrable plasmid of ~250 kb in size (Fig 1).
Furthermore, EC2189 genome was also extracted together with the plasmids and simultaneously sequenced by PacBio RSII.EC2189 possessed the S80I mutation in the quinolone resistance-determining region (QRDR) of parC, as well as S83L and D87N mutations in the QRDR of gyrA.These mutations are well-known causes of the resistance to fluoroquinolone of gram-negative bacilli, including E. coli [19,20].Therefore, the resistance to fluoroquinolone in EC2189 resulted from chromosome mutations in DNA gyrase and topoisomerase IV.
BLAST results indicated that the whole sequence of p2189-NDM had the highest homology (89% query cover and 99% identity) to the Citrobacter sp.CRE-46 strain AR_0157 plasmid unnamed1 (accession no.CP029729), which did not contain bla NDM-1 .Among the bla NDM-1containing plasmids, p2189-NDM showed the highest similarity to plasmid pNDM-CIT, with 60% query cover and 88% identity (accession no.JX182975) (Fig 2A).However, the gene clusters containing bla NDM-1 were inverted in p2189-NDM relative to those in pNDM-CIT.Furthermore, to investigate the genetic relationship between p2189-NDM and previously identified IncHI type plasmids with or without bla NDM-1 as well as with different types of bla NDM-1 -containing plasmids, we conducted phylogenetic analysis.It revealed that p2189-NDM was genetically distinct from all other identified bla NDM-1 -encoding plasmids and, as expected, was closely related to the bla NDM-1 -negative Citrobacter sp.CRE-46 strain AR_0157 plasmid unnamed1 (Fig 2B).Collectively, these findings suggested that p2189-NDM possessed a novel backbone structure, because it was clearly separate from the previously identified bla NDM-1 -encoding plasmids.
Genetic environment surrounding p2189-NDM bla NDM-1 (~25.5 kb) was compared to that of the bla NDM-1 -containing IncHI type plasmid pPKPN1 of K. pneumoniae strain PittNDM01 ST14 (accession no.CP006799), with which p2189-NDM shared the highest homology among IncHI type plasmids with bla NDM-1 (Fig 2C).The genetic context of p2189-NDM was partially conserved in pPKPN1.However, the bla NDM-1 -containing region in p2189-NDM was flanked by the first copy of ΔISAba125 (downstream) and the second copy of ΔISAba125 (upstream) and spanned ~8.3 kb, which shows truncated Tn125.The downstream ΔISAba125 was disrupted by ISSm1-like, whereas the upstream ΔISAba125 was truncated by the site-specific integrase with 100% identity to that of the Citrobacter sp.CRE-46 strain AR_0157 plasmid unnamed1.ISSm-1-like showed 94% identity with the IS110 family insertion sequence ISSm1 from Serratia marcescens.To the best of our knowledge, this is the first identification of ISSm1-like sequence associated with the mobilisation of truncated Tn125.Different elements including flanking insertion sequences, singleton insertion sequences, class 1 integrons, and ISCR elements have been involved in the genetic acquisition of Tn125.An intact Tn125 bounded by two copies of ISAba125 has often been detected in Acinetobacter spp.However, among Enterobacteriaceae, bla NDM-1 is found in the truncated ΔTn125 region with intact or partial ISAba125 with the bla NDM-1 promotor sequence [4,5,16,21,22].Therefore, our results indicated that p2189-NDM also showed a similar organization of a truncated Tn125 surrounded by an insertion sequence.Moreover, IS26, which is one of the most common insertion sequences associated with the mobilisation of antibiotic resistance genes including bla NDM-1 , has also been detected in p2189-NDM [4,23].The first copy of IS26 was revealed between bla CTX-M-15 and bla NDM-1 , and the second copy of IS26 was found ~50 kb downstream of bla NDM-1 .The ISEcp1-bla CTX-M-15 module was also found upstream of bla NDM-1 .Taken together, our findings suggest that p2189-NDM may have acquired bla NDM-1 from pPKPN1 or a closely related IncHI plasmid among Enterobacteriaceae via ΔTn125-mediated gene transfer, whereas the region flanked by IS26 copies had clear traces of IS-mediated homologous recombination event.

Conclusion
In conclusion, for the first time in Ghana, we identified the NDM-1-producing E. coli strain EC2189 that harboured the transferrable plasmid p2189-NDM with multiple genes that caused resistance to antibiotics, including the bla NDM-1 gene.p2189-NDM differed phylogenetically from previously identified bla NDM-1 -positive IncHI type plasmids and contained a ΔTn125 structure known in Enterobacteriaceae as well as a novel IS110 family insertion sequence, ISSm-1-like.As the dissemination of Enterobacteriaceae possessing carbapenemases poses a significant threat to the management of infections worldwide, continuous monitoring should be strengthened to prevent the spread of carbapenemase-producing Enterobacteriaceae in Ghana.

Fig 2 .
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.https://doi.org/10.1371/journal.pone.0209623.g002