Emergence of Extensively Drug-Resistant and Hypervirulent KL2-ST65 Klebsiella pneumoniae Harboring bla _KPC-3 in Beijing, China

Multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKp) has been emerging worldwide. However, the clinical, microbiological, and genomic characteristics of newly emerged MDR sequence type 65 (ST65) hvKp are unclear. We conducted active longitudinal genomic surveillance of K. pneumoniae in the hospital starting in 2017. Clinical characteristics, including demographic data, infection type, and outcomes, were collected. Whole-genome sequencing was performed to clarify phylogenetic and plasmid features, and phenotype determined by growth curves, plasmid transferability and stability, hypermucoviscosity, biofilm formation, and serum survival were analyzed to microbiologically characterize ST65 in depth. Ten ST65 (1.4%, 10/720) isolates were detected from 720 K. pneumoniae isolates in total. Nine patients (90%, 9/10) were older than 60 years and had multiple underlying diseases. All ST65 K. pneumoniae isolates harbored iucA, rmpA, rmpA2, iroB, and peg344 and were identified as hvKp. Surprisingly, two MDR-hvKp isolates that grew slowly were observed. Isolate PEKP4222 harbored a pLVPK-like plasmid and a conjunctive MDR plasmid. Isolate P1 harbored blaKPC-3 in a new plasmid, pP1-54, resulting in an extensively drug-resistant (XDR) phenotype; this isolate, which might have evolved from a strain harboring blaKPC-2, resulted in fatal infection. The pP1-54 plasmid could not be transferred to Escherichia coli by conjugation but could be stably inherited vertically. Interestingly, P1 also carried the pLVPK-like plasmid and acquired various antimicrobial resistance genes, and blaCTX-M-3 was detected in the IncB/O/K/Z plasmid. The convergence of XDR and hypervirulence within classical ST65 hvKp is emerging, highlighting the need for enhanced genomic surveillance. IMPORTANCE XDR-hvKp poses a great challenge to public health. ST65, a classical hvKp subtype, mostly presented with hypermucoviscosity, which restricts antimicrobial resistance acquisition. However, few studies have demonstrated the clinical, microbiological, and genomic characteristics of ST65, especially MDR-ST65 hvKp. Here, we first reported that ST65 hvKp acquired blaKPC-3 and then conferred the XDR-hvKp phenotype. Genomic context analysis concluded that the blaKPC-3 gene might have evolved from blaKPC-2. Additionally, the pLVPK-like plasmid seemed to acquire more resistance genes, and blaCTX-M-3 located in the IncB/O/K/Z plasmid was observed. The XDR-hvKp phenotype could be stably inherited vertically, indicating that strains harboring blaKPC-3 and pLVPK-like plasmids could persistently exist in hospital settings. These data suggest that genomic adaptation is rapid and that enhanced surveillance is essential.