Use of WGS data for investigation of a long-term NDM-1-producing Citrobacter freundii outbreak and secondary in vivo spread of bla NDM-1 to Escherichia coli , Klebsiella pneumoniae and Klebsiella oxytoca

<span class="paragraphSection"><div class="boxTitle">Objectives</div>An outbreak of NDM-1-producing <span style="font-style:italic;">Citrobacter freundii</span> and possible secondary <span style="font-style:italic;">in vivo</span> spread of <span style="font-style:italic;">bla</span>_NDM-1 to other Enterobacteriaceae were investigated.<div class="boxTitle">Methods</div>From October 2012 to March 2015, meropenem-resistant Enterobacteriaceae were detected in 45 samples from seven patients at Aalborg University Hospital, Aalborg, Denmark. <span style="font-style:italic;">In silico</span> resistance genes, Inc plasmid types and STs (MLST) were obtained from WGS data from 24 meropenem-resistant isolates (13 <span style="font-style:italic;">C. freundii</span>, 6 <span style="font-style:italic;">Klebsiella pneumoniae</span>, 4 <span style="font-style:italic;">Escherichia coli</span> and 1 <span style="font-style:italic;">Klebsiella oxytoca</span>) and 1 meropenem-susceptible <span style="font-style:italic;">K. oxytoca</span>. The sequences of the meropenem-resistant <span style="font-style:italic;">C. freundii</span> isolates were compared by phylogenetic analyses. <span style="font-style:italic;">In vitro</span> susceptibility to 21 antimicrobial agents was tested. Furthermore, <span style="font-style:italic;">in vitro</span> conjugation and plasmid characterization was performed.<div class="boxTitle">Results</div>From the seven patients, 13 highly clonal ST18 NDM-1-producing <span style="font-style:italic;">C. freundii</span> were isolated. The ST18 NDM-1-producing <span style="font-style:italic;">C. freundii</span> isolates were only susceptible to tetracycline, tigecycline, colistin and fosfomycin (except for the <span style="font-style:italic;">C. freundii</span> isolates from Patient 2 and Patient 7, which were additionally resistant to tetracycline). The <span style="font-style:italic;">E. coli</span> and <span style="font-style:italic;">K. pneumoniae</span> from different patients belonged to different STs, indicating <span style="font-style:italic;">in vivo</span> transfer of <span style="font-style:italic;">bla</span>_NDM-1 in the individual patients. This was further supported by <span style="font-style:italic;">in vitro</span> conjugation and detection of a 154 kb IncA/C2 plasmid with <span style="font-style:italic;">bla</span>_NDM-1. Patient screenings failed to reveal any additional cases. None of the patients had a history of recent travel abroad and the source of the <span style="font-style:italic;">bla</span>_NDM-1 plasmid was unknown.<div class="boxTitle">Conclusions</div>To our knowledge, this is the first report of an NDM-1-producing <span style="font-style:italic;">C. freundii</span> outbreak and secondary <span style="font-style:italic;">in vivo</span> spread of an IncA/C2 plasmid with <span style="font-style:italic;">bla</span>_NDM-1 to other Enterobacteriaceae.</span>

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