Genetic determinants of hypervirulence with attenuated pathogenicity in a <i>bla</i> _NDM-1 -encoding <i>Klebsiella pneumoniae</i> causing neonatal sepsis

K39 Klebsiella pneumoniae, a previously unrecognized hypervirulent serotype, was isolated from a septicemic neonate. It was assessed for the acquisition of hypervirulence determinants and virulence potential. Transmissibility of plasmids, functional expression of virulence determinants, capsule visualization (TEM), comparative genome analysis and virulence potential (in vitro, in vivo assays) were studied. Carbapenem-resistant ST985-K39 K. pneumoniae (Sil80), harbored rmpA, rmpA2, iucA, iroB, peg-344 in IncFIB(K)/IncHI1B(pNDM-MAR) hypervirulent plasmid; blaNDM-1 in IncFIB(pQIL)/IncFII(K) plasmid and other antimicrobial resistance determinants in IncFII plasmid. Comparative genomic analysis of global K39 isolates revealed, for the first time, the acquisition of a hypervirulent plasmid in the K39 study isolate. IncFIB(pQil)/IncFII(K) carrying blaNDM-1 showed successful conjugation independently. However, hypervirulent plasmid [IncFIB(K)/IncHI1B(pNDM-MAR)] could not successfully transmit alone, but was co-transmitted along with IncFII plasmid, because of nic site similarity. The strain was serum-resistant and produced a strong biofilm. Despite possessing hypervirulence determinants, Sil80 showed a negative string test, a thin capsule, and attenuated virulence in adult mice. Deletion of 10 amino acids in RmpD and mutation in rmpADC promoter having 10T track (P10T) were observed. Expression of rmpA, rmpD, and rmpC in Sil80 was also downregulated. Mutations in the rmpADC promoter and truncated RmpD of Sil80 caused attenuated virulence in adult mice; nonetheless, the isolate was capable of causing sepsis in an immunocompromised neonate. The transfer of the non self-conjugative virulence plasmid was facilitated by the helper plasmid with a similar nic site. This phenomenon could facilitate the spread of such plasmids to hitherto unknown serotypes.