In-Host Co-colonization and Bloodstream Infection by Distinct Classical and Hyper-virulent CRKP Clones Harboring a Homologous blaKPC-2-Harboring Plasmid

Haixing Fang¹Yueliang Chen²Ying Chen²Yan Qi³Rushuang Yan⁴Feng Guo^2*

• ¹Zhejiang University School of Medicine Sir Run Run Shaw Hospital Department of General Surgery, Hangzhou, China
• ²Department of Critical Care Medicine, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, 310016, P. R, China
• ³Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, China
• ⁴Zhejiang Key Laboratory of Precision Diagnosis and Therapy for Major Gynecological Diseases, Women's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, China

Carbapenem-resistant Klebsiella pneumoniae (CRKP) readily colonizes clinical environments as well as the respiratory and intestinal tracts of patients, and can easily cause secondary infections, posing a serious threat to infection treatment and hospital infection control. In this study, we continuously tracked CRKP strains isolated from a single patient over a total of 16 weeks—from admission to the ICU until discharge from the general ward. A total of 21 CRKP strains were obtained, including 1 bloodstream infection (BSI) isolate, 8 respiratory tract colonization isolates, and 12 intestinal colonization isolates. All isolates were evaluated for their phenotypic characteristics related to antimicrobial resistance, pathogenicity, and plasmid transfer through antimicrobial susceptibility testing, mouse infection models, and conjugation experiments. In parallel, whole-genome sequencing was performed to determine their MLST types, capsular serotypes, resistance and virulence genes, and plasmid profiles. The results showed that these isolates belonged to two distinct clones. One BSI isolate, along with one respiratory and one intestinal colonization isolate, belonged to the ST268 with capsular serotype KL20. This clone carried not only the typical hyper-virulence (hv) genes aerobactin, colibactin, and rmpA2, but also a plasmid encoding blaKPC-2, representing a classic CR-hvKP strain. Mouse infection models confirmed its high virulence. The remaining isolates belonged to the ST4496 clone, a member of the CC11 clonal complex commonly found in ICU outbreaks, with serotype KL47, exhibiting lower pathogenicity but carrying the same blaKPC-2-harboring plasmid as ST268, indicating horizontal plasmid transfer. In-host co-colonization by the distinct CRKP clones ST4496 and ST268 may have facilitated horizontal transfer of the blaKPC-2-harboring plasmid, enabling ST268 to evolve from hvKP into CR-hvKP and subsequently cause secondary BSI. This process, in which pathogen clones with different traits co-colonize and mutually promote evolutionary changes, may interfere with clinical treatment decisions and underscores the need for more intensive hospital infection surveillance.