Analysis of the virulence of a lethal, carbapenem-resistant hypervirulent KPC-33-producing Klebsiella pneumoniae: Emergence of ST11-KL64 hv-CRKP in ICU.

Objective: Hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses a serious threat to public health. Here, we analyse a case of systemic infection caused by a hv-CRKP, which ultimately led to the patient's death from sepsis. And a total of 30 CRKPs were analyzed to elucidate the molecular epidemiological features of CRKPs in the hospital, and to provide a basis for clinical anti-infective therapy.

Methods: In this case, a total of 7 K. pneumoniae strains were isolated from the blood, sputum, urine, and feces of the patient. The Vitek-2 compact system was used to identify the strains and perform antimicrobial susceptibility testing. Biofilm formation, siderophore production assays and Galleria mellonella infection model were used to verify the virulence phenotypes of the strains in the case. Whole-genome sequencing was conducted on the four hv-CRKP isolated from different samples in the case and 26 other CRKP collected in our hospital from September to November in 2022, using the Illumina Hiseq 6000 high-throughput sequencing platform to analyse the resistance and virulence genes.

Results: In the case, after 7 days of treatment with ceftazidime-avibactam (CZA), the resistance profile of the strains changed. The strain that was initially sensitive to CZA developed to resistant, resistant to imipenem (IPM) developed to sensitive, and resistant to meropenem (MEM) developed to intermediate. Whole-genome sequencing revealed that the four strains in the case were all ST11-KL64 K. pneumoniae, and the change in resistance phenotype was due to the mutation from bla to bla. KPN7 had a total of six plasmids, with siderophore-related genes iucABCD and iutA, and mucoid phenotype-related gene rmpA2 located on plasmid p4-KPN7; resistance genes bla, bla, and bla located on plasmid p5-KPN7; and virulence genes fim, irp, iutA, and ybt located on the chromosome. Biofilm formation and siderophore production assays confirmed that the seven K. pneumoniae strains isolated in this case had strong biofilm formation and siderophore production capabilities. Galleria mellonella Infection Model showed that KPN4 and KPN7 was phenotypically highly virulent and KPN7 performed lower virulence compared to KPN4. Apart from the 4 hv-CRKP strains, other 26 CRKP strains all carried bla, and 69.2% (18/26) were ST-11 and 30.8%(8/26) were ST-15. And 83.3% (15/18) were ST11-KL64 strains, followed by ST11-KL25 strains 11.1%(2/18) and ST11-KL47 strain 5.6%(1/18). All the eight ST-15 strains were KL-19.

Conclusion: The ST11-KL64 hv-CRKP clone spread widely in ICU carried numerous resistance and virulence genes, and under antibiotic pressure, they easily underwent mutations resulting in changes in resistance phenotypes, especially in mutations of bla gene in acquiring resistance to CZA. Therefore, clinical attention should be paid to such strains, and the use of antibiotics should be adjusted promptly based on the susceptibility of the strains to antimicrobial agents.