Tracking inter-institutional spread of NDM and identification of a novel NDM-positive plasmid, pSg1-NDM, using next-generation sequencing approaches

<span class="paragraphSection"><div class="boxTitle">Objectives</div>Owing to gene transposition and plasmid conjugation, New Delhi metallo-β-lactamase (NDM) is typically identified among varied Enterobacteriaceae species and STs. We used WGS to characterize the chromosomal and plasmid molecular epidemiology of NDM transmission involving four institutions in Singapore.<div class="boxTitle">Methods</div>Thirty-three Enterobacteriaceae isolates (collection years 2010–14) were sequenced using short-read sequencing-by-synthesis and analysed. Long-read single molecule, real-time sequencing (SMRTS) was used to characterize genetically a novel plasmid pSg1-NDM carried on <span style="font-style:italic;">Klebsiella pneumoniae</span> ST147.<div class="boxTitle">Results</div>In 20 (61%) isolates, <span style="font-style:italic;">bla</span>_NDM was located on the pNDM-ECS01 plasmid in the background of multiple bacterial STs, including eight <span style="font-style:italic;">K. pneumoniae</span> STs and five <span style="font-style:italic;">Escherichia coli</span> STs. In six (18%) isolates, a novel <span style="font-style:italic;">bla</span>_NDM-positive plasmid, pSg1-NDM, was found only in <span style="font-style:italic;">K. pneumoniae</span> ST147. The pSg1-NDM–<span style="font-style:italic;">K. pneumoniae</span> ST147 clone (Sg1-NDM) was fully sequenced using SMRTS. pSg1-NDM, a 90 103 bp IncR plasmid, carried genes responsible for resistance to six classes of antimicrobials. A large portion of pSg1-NDM had no significant homology to any known plasmids in GenBank. pSg1-NDM had no conjugative transfer region. Combined chromosomal–plasmid phylogenetic analysis revealed five clusters of clonal bacterial NDM-positive plasmid transmission, of which two were inter-institution clusters. The largest inter-institution cluster involved six <span style="font-style:italic;">K. pneumoniae</span> ST147-pSg1-NDM isolates. Fifteen patients were involved in transmission clusters, of which four had ward contact, six had hospital contact and five had an unknown transmission link.<div class="boxTitle">Conclusions</div>A combined sequencing-by-synthesis and SMRTS approach can determine effectively the transmission clusters of <span style="font-style:italic;">bla</span>_NDM and genetically characterize novel plasmids. Plasmid molecular epidemiology is important to understanding NDM spread as <span style="font-style:italic;">bla</span>_NDM-positive plasmids can conjugate extensively across species and STs.</span>

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