Antibiotics | Genomic and Phenotypic Characteristics of ST258-blaKPC-2 and ST11-blaNDM-1 Clones in Greek Tertiary Hospitals

This study reveals the coexistence of two high-risk carbapenem-resistant Klebsiella pneumoniae clones, ST258 and ST11, in Greek tertiary hospitals through whole-genome sequencing and phenotypic analysis. It highlights the potential risk of nosocomial dissemination of resistance genes and provides a detailed analysis of the resistance genes, virulence factors, and plasmid types of these two clones, offering critical insights for antibiotic stewardship and infection control strategies.

Literature Overview
This article, titled 'Genomic and Phenotypic Characterization of Two High-Risk Klebsiella pneumoniae Clones (ST258-blaKPC-2 and ST11-blaNDM-1) in a Greek Tertiary Hospital', published in the journal Antibiotics, reviews and summarizes the molecular and phenotypic features of two carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates, INT18S (ST258-blaKPC-2) and INT20U (ST11-blaNDM-1), collected from a tertiary hospital in Heraklion, Greece. The study reveals the coexistence of these two high-risk clones in different clinical samples and compares their similarities and differences in terms of antibiotic resistance, virulence genes, and plasmid types.

Background Knowledge
Klebsiella pneumoniae is one of the primary pathogens responsible for hospital-acquired infections, particularly in high-antibiotic-pressure environments such as ICUs, where its resistance to antibiotics has become a global public health challenge. The emergence of carbapenemases such as KPC and NDM has rendered these strains resistant to multiple antibiotics, limiting treatment options and increasing mortality. This study focuses on the coexistence of two major high-risk clones, ST258 and ST11, in Greek hospitals, exploring their resistance genes, virulence factors, and plasmid types to provide a scientific basis for hospital infection control and antibiotic management strategies. In recent years, advances in genomic sequencing technology have enhanced our understanding of CRKP transmission mechanisms and the dynamic transfer of resistance genes. However, the transmission routes and gene flow within hospitals still require further investigation. This study fills the gap in CRKP epidemiological data in Greece and provides genomic and phenotypic data that will aid future studies on the balance between resistance and virulence.

Research Methods and Experiments
In January 2025, two distinct carbapenem-resistant Klebsiella pneumoniae strains, INT18S (ICU patient respiratory s

Key Conclusions and Perspectives

• INT18S isolate belongs to the ST258 clone and carries the blaKPC-2 carbapenemase gene, consistent with resistant strains prevalent globally, especially in Europe, suggesting a high potential for transmission within the hospital ICU.
• INT20U isolate belongs to the ST11 clone and carries the blaNDM-1 carbapenemase gene, commonly associated with epidemic strains in Asia, showing enhanced resistance in the emergency department.
• Both isolates carry at least one extended-spectrum β-lactamase gene (TEM, SHV, OXA-1, and CTX-M groups), indicating that their resistance to multiple antibiotics could be enhanced through co-transfer of resistance genes.
• Whole-genome sequencing (WGS) analysis revealed that both INT18S and INT20U contain multiple resistance genes, such as aac(6')-Ib, fosA6, and OqxA/B, further confirming their multidrug-resistant phenotypes.
• Both strains exhibit resistance to carbapenem antibiotics (ertapenem, imipenem, meropenem) and third-generation cephalosporins but remain susceptible to tigecycline and aztreonam/avibactam, suggesting that these drugs may serve as viable treatment options.
• Plasmid analysis showed that INT18S carries ColRNAI, IncFIB(K), IncFIB(pQil), and IncX3 plasmid types, while INT20U harbors IncFIA(HI1), IncFIB(K), IncFII(K), and IncR plasmids, indicating that the two strains may utilize different plasmid systems for horizontal transfer of resistance genes.
• Neither strain carries the rmpA/D genes associated with the hypermucoviscosity phenotype, suggesting that their virulence may be linked to adaptive evolution and tissue tropism rather than traditional hypervirulence mechanisms.
• INT18S exhibits higher allelic diversity in the yersiniabactin gene cluster, potentially enhancing its iron acquisition capabilities and survival within the host.

Conclusion
Through genomic and phenotypic analysis of two carbapenem-resistant Klebsiella pneumoniae strains (INT18S and INT20U), this study reveals the coexistence of ST258-blaKPC-2 and ST11-blaNDM-1 clones in a Greek tertiary hospital. Both strains exhibit multidrug resistance, carrying different carbapenemase genes and plasmid types, suggesting that their resistance dissemination may involve distinct genetic mechanisms. The study further identifies differences in virulence factors such as the yersiniabactin gene cluster between the two strains, which may influence their host adaptability and pathogenicity. These findings underscore the necessity of stringent infection control measures in hospital settings and provide genomic resources for future studies on resistance gene transmission and host adaptation.

Humanization

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Monoclonal antibody therapeutics typically originate from non-human sources (usually mice), which may trigger immune responses in humans. Antibody humanization aims to modify the variable region sequences of antibodies to obtain antibodies that do not elicit immune responses. We utilized nearly one billion antibody sequences from the OAS database to establish an antibody humanness evaluation AI model capable of distinguishing between human and non-human antibody variable region sequences. The scores output by the model are negatively correlated with the experimental immunogenicity (ADA) of existing FDA-approved antibody therapies. Following the approach of Marks and Hummer, we combined this model with a Beam Search algorithm to develop an antibody sequence humanization tool. This tool aims to maximize the level of humaness of antibodies while minimizing number of mutations and maintaining key characteristics such as affinity, thereby reducing their immunogenicity.