Editor’s Note: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a significant challenge in nosocomial infections, with its resistance escalating, drawing global attention. At the 34th European Congress of Clinical Microbiology and Infectious Diseases (ESCMID Global 2024), a study by Professor Yunsong Yu‘s team from the Zhejiang Provincial People’s Hospital presented the latest findings on the genomic epidemiology and resistance mechanisms of CRKP (Abstract No: P1673). The study indicates that ST20 CRKP carrying multiple resistance genes poses a threat to the effectiveness of novel enzyme inhibitor combination therapy, highlighting its significance in clinical anti-infective treatment and antimicrobial management strategies.
Chromosomal Integration and Plasmid Fusion Induce Differential Propagation, Resistance, and Adaptation in Carbapenem-Resistant Klebsiella pneumoniae (CRKP) Strains Co-harboring blaNDM-1 and blaIMP-4 (Abstract No: P1673)
Objective
To elucidate the prevalence of newly identified CRKP clone (ST20) in China and analyze strains co-harboring IMP-4 and NDM-1 plasmids to determine the impact of different plasmids on transmission, resistance, and adaptation.
Methods
Seven CRKP strains were collected nationwide for susceptibility testing to commonly used antimicrobial agents. Whole-genome sequencing was performed on these strains to analyze resistance and virulence genes and determine their phylogenetic relationships. S1-PFGE and Southern blot were conducted to locate carbapenem resistance genes on plasmids. Conjugation experiments were performed on strains co-harboring two carbapenem resistance genes to assess the horizontal transferability of resistance plasmids. Integration patterns of resistance plasmids were determined using third-generation sequencing, plasmid sequence alignment, and reverse PCR. Lastly, CRISPR-Cas9 gene editing was employed to individually eliminate resistance plasmids, and the impact of different plasmids on resistance and adaptation was assessed through growth curves and susceptibility testing.
Results
Among the seven ST20 CRKP strains, four carried both IMP-4 and NDM-1, with gene localization experiments revealing three different combinations: IMP-4 and NDM-1 located on separate plasmids, IMP-4 and NDM-1 on the same plasmid, and IMP-4 on the chromosome while NDM-1 on a plasmid. Plasmids carrying the NDM-1 resistance gene were capable of horizontal transfer, while those carrying the IMP-4 resistance gene could not transfer horizontally on their own.
Plasmid alignment revealed that the IMP-4 plasmid integrates into the NDM-1 plasmid via IS26, forming a fused plasmid. Meanwhile, the IMP-4 plasmid recombines with the bacterial chromosome via ltrA, resulting in chromosomal “fixation” of resistance. Both integration forms of these resistance plasmids are associated with transposon units (TU).
Through plasmid elimination, strains carrying only a single resistant plasmid and strains with IMP-4 carried on the chromosome were obtained. However, in the elimination of fused plasmids, despite multiple attempts, only partial fragments of the NDM-1 carrying plasmid were eliminated, and the sizes of the eliminated plasmids varied. PCR validation revealed that this phenomenon was due to recombination events between IS26 elements.
After complete elimination of resistant plasmids, the bacteria showed full sensitivity. Strains carrying only the IMP-4 resistant plasmid exhibited lower resistance compared to strains carrying only the NDM-1 resistant plasmid, with a difference of at least 8-fold. The elimination of resistant plasmids did not affect the growth rate of bacteria on regular culture media. However, strains carrying only the IMP-4 resistant plasmid showed significantly delayed growth in media containing 2 mg/L meropenem, indicating a decrease in adaptability.
Conclusion
This study found that IMP-4 provides baseline resistance to bacteria, while NDM-1 confers major resistance, adaptability, and transmissibility. However, IMP-4 plasmids exist in various integration forms and may serve as vehicles for the transfer of resistance genes. ST20 CRKP represents a novel multidrug-resistant high-risk clone, carrying multiple resistance genes, especially carbapenemases, which could lead to the failure of novel enzyme inhibitor combination therapies (such as ceftazidime-avibactam) and should be given attention.
References :
1] Shi Q, Hu H, Yu Q, et al. Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaIMP-4 induce resistance transmission and fitness variation. Emerg Microbes Infect. 2024;13(1):2339942. doi:10.1080/22221751.2024.2339942
[2] Qiucheng Shi, WeiyiHuang, YinpingWang, et al. Chromosomal integration and plasmid fusion induce the transmission, resistance, fitness differences in carbapenem-resistant Klebsiella pneumoniae isolates co-harboring blaNDM-1 and blaIMP-4. ESCMID Global 2024; Poster P1673.
Professor Yunsong Yu
Infectious Diseases Professor, Chief Physician
Vice President of Zhejiang Provincial People’s Hospital
Zhejiang University Distinguished Physician
Leading Talent of Health Commission of Zhejiang Provincial
Director of the Infectious Diseases Specialty Committee of the China Medical Education Association;
Deputy Director of the Bacterial Infection and Antimicrobial Resistance Prevention Committee of the Chinese Medical Association;
Chairman of the Bacterial Infection and Antimicrobial Resistance Prevention Committee of the Zhejiang Medical Association;
Deputy Director of the Antimicrobial Rational Use and Resistance Evaluation Professional Committee of the National Health Commission;
Director of the Key Laboratory of Microbial Technology and Bioinformatics Research in Zhejiang Province;
Standing Committee Member of the Hospital Infection Control Committee of the Chinese Preventive Medicine Association;
Standing Committee Member of the Infectious Diseases Branch of the Chinese Medical Association;
Chairman of the Hospital Infection Control Professional Committee of the Zhejiang Provincial Preventive Medicine Association;
Deputy Director of the Infectious Disease Specialty Committee of the Zhejiang Medical Association;
Published 145 SCI-indexed papers as first author or corresponding author;
Has led seven projects funded by the National Natural Science Foundation of China (including 2 key projects).
Doctor of Medicine
Postdoctoral Fellow at Zhejiang University School of Medicine
Engaged in research related to the resistance and virulence mechanisms of Klebsiella pneumoniae, including pneumonia, with 16 SCI papers published as the first author or co-author. Additionally, led one National Natural Science Foundation of China Youth Project.
