Characterization of OXA232-Producing Carbapenem-Resistant Klebsiella pneumoniae: Genomic Analysis and Virulence Assessment

The rise of antibiotic-resistant pathogens emerged as a critical global health challenge, complicating the treatment of common infectious diseases. Among these, carbapenem-resistant Klebsiella pneumoniae (CRKP) emerged as a particularly concerning threat, given its significant role in hospital-acquired infections and its resistance to last-resort antibiotics (Lan et al. 2021). This issue was starkly highlighted by the widespread dissemination of strains harboring OXA-48-like carbapenemases, which were identified as a key driver behind the alarming rise in resistance rates observed worldwide (Aslan et al. 2022).

The identification of OXA-48-like enzymes marked a landmark in the understanding of bacterial resistance mechanisms. These enzymes were first discovered in Turkey in the early 2000s, isolated from the urethra of a 54-year-old patient suffering from a urinary tract infection and skin burns at Istanbul College Hospital (Poirel et al. 2004). Since then, OXA-48-like enzymes have been reported in various Klebsiella strains across multiple continents, highlighting their widespread dissemination (Chen et al. 2021; Cerón et al. 2023; Singh et al. 2023). OXA-48-like enzymes belong to a class of broadspectrum β-lactamases capable of hydrolyzing penicillins, cephalosporins, and carbapenems, thus conferring resistance to these key antibiotics (Boyd et al. 2022). Characterized by potent hydrolytic activity against carbapenems and diminished activity against cephalosporins like ceftriaxone, these enzymes pose unique challenges in both diagnosis and treatments (Boyd et al. 2022).

OXA-232, the third most common OXA-48-like variant globally, has garnered attention due to its enhanced activity against a broad spectrum of betalactams, thereby complicating therapeutic protocols. To date, eight sequence types (STs) of OXA-232-producing K. pneumoniae isolates reported worldwide are ST14, ST15, ST16, ST17, ST147, ST231, ST307 and ST395 (Chen et al. 2023). In China, OXA-232-producing K. pneumoniae identified mainly belongs to ST15, which carries a 6.1 kp unconjugated ColKP3 plasmid containing the bla_OXA-232 gene (Wu et al. 2023).

Rising antimicrobial resistance (AMR) in bacterial pathogens is mainly driven by the horizontal transfer of AMR genes and the accumulation of spontaneous mutations, with different mobile genetic elements (MGEs) playing a key role in their dissemination (Mehrotra et al. 2023). In this study, we hypothesized that OXA-232-producing K. pneumoniae strains isolated from a hospital in Kunming may display genomic diversity attributable to regional variations, particularly in their plasmid content and resistance gene profiles. Furthermore, the dissemination of the OXA-232 resistance gene may be further amplified by horizontal gene transfer (HGT).

This study examined the genomic characteristics, resistance mechanisms, and virulence factors of OXA-232-producing carbapenem-resistant K. pneumoniae, focusing on the ST231 clone. In all ST231 isolates, the bla_OXA-232 gene was consistently located on a nonconjugative ColKP3 plasmid, aligning with previous studies identifying this plasmid as a frequent carrier of the OXA-232 gene (Potron et al. 2013; Abdul Momin et al. 2017; Mancini et al. 2018). The stable presence and dissemination potential of the bla_OXA-232 gene indicated that these strains exhibit high resistance levels with the potential for widespread distribution across various clinical settings. Notably, the detection of IncFIB(pQil) plasmids in all ST231 K. pneumoniae isolates supported prior findings that both ColKP3 and IncFIB(pQil) plasmids can carry bla_OXA-232 and bla_TEM-1, highlighting the essential role of these plasmids in spreading multiple resistance genes (Lutgring et al. 2018).

Our study also revealed structural changes in the IncFIB(pQil) plasmid, including deleting the antibiotic resistance gene CTX-M, which mirrors the plasmid structure of an ST231 strain previously identified in China. We found that a 8.6kb region containing bla_TEM-181 and qnrS1 was lost from an MDR plasmid, likely due to surrounding mobile genetic elements (Chen et al. 2023). This dynamic evolution in plasmid structure reflected ongoing bacterial adaptation and survival strategies under antibiotic selective pressure. Detailed analysis of these plasmids enhances our understanding of their role in resistance gene spread, supporting the development of effective intervention strategies.

MLST typing identified ST11 and ST231 as multidrug-resistant clones primarily originating in South and Southeast Asia (Wyres et al. 2020). Phylogenetic analysis confirmed an increasing prevalence of these isolates in Europe, showing significant genetic similarities to Indian isolates (Shankar et al. 2019). This clustering suggests potential transnational dissemination, underscoring the need for strong global surveillance systems. The close genetic relationship to Indian strains implied regional transmission dynamics, likely facilitated by geographical and clinical interactions, even though the patients had no recent travel history. This finding is essential for creating region-specific public health strategies.

We found that two key iron-carrier virulence genes: yersiniabactin and aerobactin. The yersiniabactin genes, implicated in respiratory infections, promoted pneumonia development in mouse models (Bachman et al. 2012; Holt et al. 2015). Yersiniabactin was a critical siderophore system that enables pathogens to acquire iron in iron-limited environments, such as the host, enhancing bacterial fitness during infection and contributing to increased pathogenicity. Aerobactin plays a crucial role in nasopharyngeal colonization, enhancing the bacterium’s ability to establish and maintain infection in this region (Bachman et al. 2009). The combined presence of these virulence factors significantly improved the ability of these isolates to acquire iron, a vital nutrient, allowing them to adapt to various hosts. The presence of yersiniabactin (ybtA, fyuA, irp2) and aerobactin (iucABCD, iutA) was consistent with previous research findings (Shen et al. 2023). A notable observation was the detection of an ST11 isolate carrying high-virulence genes (rmpA, rmpA2, peg-344) along with carbapenemase resistance genes (OXA-232, KPC-2). This combination represented a significant clinical challenge, highlighting K. pneumoniae’s capacity to accumulate multiple resistance and virulence determinants, increasing its public health threat.

A comparative analysis was conducted on 264 publicly available ST231 K. pneumoniae genomes to understand the global dissemination of these strains. A phylogenetic tree based on core SNPs grouped these genomes into four distinct clades. The largest clade predominantly included genomes from South Asia, East Asia, and Europe, with most ST231 isolates carrying bla_OXA-232 and other key virulence factors. Although the phylogenetic analysis revealed that these 10 isolates belonged to distinct strains with substantial divergence in their core genomes they exhibited a remarkable convergence in the number and types of plasmids and resistance genes. This striking convergence was likely driven by extensive horizontal gene transfer (HGT) of resistance determinants, facilitated by the selective pressures exerted by frequent antibiotic use in hospital environments. Such conditions created a permissive ecosystem for genetic exchange, enabling even phylogenetically unrelated strains to acquire similar resistance profiles. While the phylogenetic analysis suggested no immediate epidemiological threat, the convergence of resistance genes highlighted the latent risk of their accelerated dissemination through HGT, emphasizing the critical role of hospital settings as reservoirs and amplifiers of antibiotic resistance. These findings underscore the urgency of implementing stringent infection control measures and robust antibiotic stewardship programs to mitigate the spread of resistance genes and their far-reaching impact on clinical outcomes and public health.

CCL2/MCP-1, IL-6, and TNF-α were mediators of intercellular communication during the immune response. IL-6 was produced rapidly and transiently in response to infections and tissue injuries, promoting host defense by stimulating acute phase responses, hematopoiesis, and immune reactions (Tanaka et al. 2014). TNF-α was an inflammatory cytokine produced by macrophages/monocytes during acute inflammation, being responsible for various intracellular signaling events leading to necrosis or apoptosis (Idriss et al. 2000). CCL2/MCP-1 played a critical role in the inflammatory process by attracting and enhancing the expression of other inflammatory factors/cells. It facilitated the migration and infiltration of inflammatory cells, such as monocytes/macrophages, and other cytokines, to the inflammation site, contributing to the progression of various diseases (Singh et al. 2021). Therefore, these cytokines can serve as indicators of inflammatory responses. This enabled us to compare the intensity of inflammatory reactions elicited by different strains.

Consequently, we detected the concentrations of inflammatory factors CCL2/MCP-1, IL-6, and TNF-α in the alveolar lavage fluid and blood of mouse models. The results indicated that all strains presented statistically significant differences from the negative control, and the intensity of inflammatory reactions varied among different strains. For instance, the concentration of inflammatory factors in the alveolar lavage fluid of the Kp7 strain was higher than that of other strains, but the concentration of inflammatory factors in the blood of the Kp9 and Kp10 strains was lower than that of the Kp7 strain (Fig. 6). The lung tissue, as the primary site of respiratory infection, provided crucial information on inflammation and damage following intranasal inoculation. Previous reports on multi-drug-resistant K. pneumoniae isolated from patients with liver and renal abscesses have highlighted the significance of evaluating organ damage (Lee et al. 2008; Qian et al. 2023). These analyses will enhance our understanding of the systemic infection mechanisms of these highly resistant strains. Therefore, we collected the model mice’s lung, liver, and kidney tissues for pathological analysis. It was found that although the strains invaded the blood of the model mice, there was no damage to the kidney tissues, while the lung and liver tissues showed apparent damage, and the degree of damage to organ tissues varied among each strain.

Despite consistent resistance profiles, the virulence of ST231 isolates showed significant variability. The presence of key iron-scavenging systems, such as the iuc cluster – a promising marker for high-virulence phenotypes (Shankar et al. 2021) – indicated their ability to adapt to iron-limited environments, increasing their pathogenic potential. However, mouse model experiments showed that while these isolates can invade the bloodstream and cause substantial inflammatory responses, their overall virulence remains moderate. This aligns with previous studies (Chen et al. 2023), suggesting that despite high resistance, the virulence of these strains may be less severe than expected. This discrepancy could be attributed to variations in virulence gene expression, differences in regulatory pathways, or interactions with other unidentified virulence factors. Additionally, strain-specific traits, such as hypermucoviscosity (e.g., Kp13), may elicit varied host immune responses, leading to differing degrees of tissue damage. These findings highlight the need for further investigation into the interplay between virulence factors and host immunity to better understand the pathogenic mechanisms of K. pneumoniae.

The study’s limitations include a small sample size and limited geographical scope, making it difficult to draw broad conclusions. Additionally, while the respiratory infection model offered insights into virulence, it does not fully replicate the complexity of human infection. Future studies should involve more considerable, geographically diverse cohorts and use advanced genomic and proteomic analyses to clarify these isolates’ transmission pathways and genetic evolution. In conclusion, this study underscores the need for active surveillance and large-scale genomic investigations to understand the distribution and genetic characteristics of OXA-232-producing K. pneumoniae, particularly ST231 strains. Enhanced monitoring will be essential to developing effective strategies to limit the spread of these high-risk clones and mitigate their clinical impact. In our research, the tested pathogenic factors are not the sole determinants of virulence and pathogenicity; other regulatory systems also play crucial roles in modulating these traits. Therefore, conducting further research and monitoring it is of great significance.