Klebsiella pneumoniae is a Gram-negative bacterium belonging to the Enterobacteriaceae family, recognized as a significant opportunistic pathogen responsible for a range of infections, including pneumonia, septicemia, and urinary tract infections (Choby et al., 2020). This organism primarily resides in the human gastrointestinal tract but poses a serious risk, particularly to immunocompromised individuals (Kar et al., 2023; Kuo et al., 2022). Its notorious ability to rapidly acquire antibiotic resistance has made it a leading cause of nosocomial infections globally (Abbas et al., 2024; Uddin et al., 2021).

The emergence of multidrug-resistant Klebsiella pneumoniae strains presents an escalating public health challenge, contributing to extended hospital stays and heightened mortality rates. A key factor influencing the pathogenicity of this bacterium is its array of virulence factors alongside its antibiotic resistance genes (Assefa, 2022). Notable virulence determinants include the production of polysaccharide capsules, adhesins, and various tissue-damaging enzymes. The capsule plays a crucial role in defending against phagocytosis by the immune system (Riwu et al., 2022; Gao et al., 2024). Additionally, siderophores are vital for iron acquisition, which is critical for bacterial survival during infection (Khasheii et al., 2021). Compounding the challenges in treating Klebsiella pneumoniae infections is the presence of antibiotic resistance genes, such as blaKPC, which confers resistance to carbapenems, and those associated with extended-spectrum beta-lactamases (ESBLs), complicating treatment efforts (Aminov, 2009; Denk-Lobnig and Wood, 2023).

A relatively recent area of research is the effect of antibiotic exposure on the expression of both virulence and resistance genes in Klebsiella pneumoniae. As antibiotics impose selective pressure, understanding how sub-lethal antibiotic concentrations may induce gene expression changes that enhance virulence or promote resistance acquisition has become increasingly important (Andersson and Hughes, 2014; Sanchez-Cid et al., 2023; Li et al., 2019).

As our knowledge of Klebsiella pneumoniae evolves, studying the consequences of antibiotic-induced changes in gene expression is essential for developing effective therapeutic strategies. It is crucial to explore how integrating antibiotics into treatment regimens may inadvertently enhance the pathogenicity of these organisms.

In this study, we examined the alterations in the expression of virulence and antibiotic resistance genes in Klebsiella pneumoniae following exposure to various classes of antibiotics. Using quantitative real-time PCR, we assesed the expression levels of key virulence factors and resistance genes under different antibiotic conditions. Our findings aim to deepen the understanding of how Klebsiella pneumoniae adapts to antibiotic pressure and to inform clinical practices regarding antibiotic use, especially in environments where this pathogen poses a significant threat to patient safety. By exploring the dynamics of virulence and resistance under antibiotic pressure, we hope to contribute valuable insights for prevention and treatment strategies against infections caused by this formidable pathogen.

Klebsiella pneumoniae is a significant pathogen responsible for a wide range of infections, particularly in immunocompromised patients. In recent years, there has been increasing concern regarding its rising antibiotic resistance. In the current susceptibility testing, CRKP was found to be resistant to most cephalosporin antibiotics, while CSKP exhibited resistance rates between 10 and 30%. Additionally, resistance to other antibiotics has also shown varying increases. Consequently, the presence of Klebsiella pneumoniae poses a significant challenge to our healthcare environment (Kot et al., 2023; Wysocka et al., 2021).

To conduct further analytical research, the detection of virulence genes provided additional insights into the pathogenic potential of CSKP and CRKP. In our study, we assessed a total of 100 strains of Klebsiella pneumoniae for seven virulence genes: rmpA, rmpA2, iucA, iutA, Peg - 344, ybts, and iroB. The virulence genes rmpA and rmpA2 enhanced capsular polysaccharide production, helping Klebsiella pneumoniae evade the immune system (Cheng et al., 2010). The iucA and iutA genes facilitated iron acquisition through the synthesis of aerobactin and its receptor, promoting bacterial growth in iron - limited conditions (Burbank et al., 2015). Additionally, Peg - 344 and ybts were involved in enhancing virulence and biofilm formation, while iroB was part of the siderophore gene cluster that aided in iron uptake (Hwang et al., 2024; Hogle et al., 2022). Together, these genes significantly contributed to the pathogenicity of Klebsiella pneumoniae. Our study showed that the positive detection rates among CSKP were evidently high, particularly for rmpA and rmpA2 at 50.0% and 46.0% respectively. In contrast, CRKP exhibited significantly lower rates, with rmpA at only 12.0%, although a remarkably higher positivity rate of iroB at 82.0% was observed. This pattern correlated with findings by Kochan et al. (2023), who emphasized the importance of virulence factors in determining pathogenicity within hypervirulent strains (Debroas and Siguret, 2019; Schroeder et al., 2017).

In terms of antibiotic resistance, the vast presence of resistance genes such as KPC, SHV, and CTX-M3 in CRKP, with detection rates of 86.0%, 94.0%, and 38.0%, respectively, highlights an alarming trend of resistance among these strains. This aligns with observations by Pulingam et al. (2022) who correlated the increasing prevalence of resistance genes with the overuse of antibiotics in healthcare environments. KPC was particularly alarming as it conferred high - level resistance to carbapenems, often regarded as the last - line antibiotics for treating severe infections. The widespread detection of SHV and CTX - M3 reinforced the challenge presented by extended - spectrum beta - lactamases (ESBLs), which could compromise the effectiveness of penicillins and cephalosporins (Yu et al., 2024).

The previous assessments of virulence and resistance genes aimed to provide a comprehensive evaluation of the pathogenicity and resistance profiles of the bacteria, thereby effectively guiding clinical treatment, monitoring disease trends, and controlling the spread of infections. Conversely, the drug induction experiments sought to explore the effects of antibiotics on bacteria, including the mechanisms of induced resistance, the identification of effective antimicrobial agents, and the adaptive changes of bacteria under antibiotic pressure. In this drug induction experiment, we selected eight representative strains from each of 50 CSKP and 50 CRKP isolates to ensure coverage of most virulence and resistance genes that required further investigation.

By using the agar dilution method to determine the minimum inhibitory concentration (MIC) of antimicrobial agents, we found that half of the original CRKP strains were resistant to ciprofloxacin and imipenem, while the other half exhibited sensitivity to these drugs, similar to CSKP strains. After 4 days of drug induction, all strains demonstrated resistance to ciprofloxacin and imipenem, with MIC values exceeding 64 μg/mL. As shown in Figure 1, after 4, 7, and 10 days of induction with ceftazidime-avibactam, the MIC values for both CSKP and CRKP increased from <0.5 to 32 μg, eventually surpassing 64 μg/mL. This indicates that prolonged exposure to antibiotics can lead to the development of multidrug resistance among these pathogens (Ching and Zaman, 2020; Stanton et al., 2020). However, it is noteworthy that after 10 days of induction with polymyxin B, the MIC changes for both CSKP and CRKP were more modest, shifting from <0.5 to around 8 μg. This minimal change may be attributed to the structural characteristics of polymyxin B itself. Because it exerted its lethal effect by interacting with the lipopolysaccharides of the bacterial outer membrane, disrupting its integrity. For bacteria to develop resistance, multiple genetic mutations would have needed to occur simultaneously to alter the outer membrane structure, which was a low-probability event (Yang et al., 2023).

To further compare the virulence and resistance of CSKP and CRKP after antibiotic induction, we conducted RT-qPCR to analyze the original strains as well as their antibiotic-induced counterparts. The analysis revealed that the expression levels of rmpA and rmpA2 in CSKP strains were significantly higher than those in CRKP strains. After 4 days of antibiotic induction, the expression levels of virulence genes in CSKP exhibited minimal changes, whereas CRKP showed a marked decrease in iutA expression alongside a significant increase in resistance gene expression, particularly SHV and KPC. By day seven of induction, rmpA expression in CSKP continued to rise, while CRKP demonstrated a slight recovery in iutA expression compared to day four, although it remained lower than in the original strains. By day ten of induction, rmpA expression in CSKP showed a slight increase, while rmpA2 expression in CRKP increased significantly, with iutA expression stabilizing and KPC and SHV expression rising dramatically compared to the original strains.

According to the research by Hu et al. (2024) on the pathogenic mechanisms of Klebsiella pneumoniae, rmpA and rmpA2 are likely involved in various key physiological processes in CSKP and have significant importance in maintaining bacterial structural integrity and virulence. Their higher expression may be closely related to the composition and functional regulation of the outer membrane, enhancing the bacteria’s ability to withstand external environmental pressures, including a certain level of antibiotic resistance (Sun et al., 2022). This might represent an adaptive strategy that CSKP has developed over time. Pountain et al. (2024) highlighted in their studies on bacterial gene regulation that different strains may possess unique regulatory networks. There could be specific transcriptional regulatory mechanisms in CSKP that keep rmpA and rmpA2 in a relatively active transcriptional state. For instance, there may be positive regulatory factors binding specifically to the promoter regions of rmpA and rmpA2, promoting efficient transcription (He et al., 2023).

The continued increase in rmpA expression throughout the induction period may represent an adaptive response by CSKP. In the presence of antibiotics, CSKP may sense cellular stress and upregulate rmpA expression to enhance its defensive capabilities. RmpA could be involved in the repair mechanisms against antibiotic damage or may improve antibiotic tolerance by modulating the structure and function of the outer membrane (Memar et al., 2020). By day ten, the slight increase in rmpA expression may indicate that CSKP has reached a new equilibrium, where the expression level of rmpA is sufficient to meet its survival and functional needs in an antibiotic environment.

The significant decline in iutA expression in CRKP may be a strategy for adapting to the antibiotic environment. IutA may be associated with certain antibiotic-sensitive metabolic pathways or cellular functions, and reducing its expression could lessen CRKP’s dependency on these potentially vulnerable aspects, thereby enhancing its chances of survival. For example, since iutA is involved in iron uptake, which can sometimes increase bacterial sensitivity to antibiotics, CRKP may lower its iron acquisition by reducing iutA expression, thus decreasing its susceptibility (Sheldon et al., 2016). The antibiotic induction likely triggered a reprogramming of the gene regulatory network within CRKP. Transcriptional regulators may sense the threat posed by antibiotics and initiate a suite of regulatory programs to suppress the expression of genes, like iutA, that may be detrimental to survival while activating the transcription of resistance genes. This remodeling of gene expression is a critical mechanism by which CRKP adapts to an antibiotic environment, reflecting the flexibility of bacterial gene regulatory systems and their rapid response capabilities to environmental changes (Hu et al., 2024).

Additionally, throughout the 10 days of antibiotic induction, the expression of resistance genes such as KPC and SHV significantly increased. A lateral comparison of the induction capabilities of four antibiotics, along with the agar dilution determination of MIC, showed that the expression of resistance genes under polymyxin B induction was not as strong as that under the other three antibiotics, consistent with previous discussions (Yang et al., 2023). The substantial increase in resistance genes like KPC and SHV further enhances the resistance capabilities of CRKP. As antibiotic pressure persists, CRKP must continuously strengthen its resistance to ensure survival. The high expression of resistance genes enables CRKP to more effectively combat antibiotics, thereby improving its competitive survival in antibiotic environments and ensuring the bacteria’s survival and reproduction in adverse conditions (Budia-Silva et al., 2024).

This study acknowledges several limitations, including the relatively small sample sizes of 50 CSKP and 50 CRKP strains, which may not fully encapsulate the genetic diversity of Klebsiella pneumoniae. Additionally, the research being conducted at a single institution may limit the generalizability of the findings. The absence of longitudinal data restricts our ability to observe the evolution of resistance and virulence traits over time. Moreover, while focusing on specific virulence and resistance genes provides valuable insights, the lack of functional studies assessing their direct impacts on pathogenicity may overlook other vital contributing factors. Future research should strive to overcome these limitations to yield more comprehensive insights into the challenges posed by Klebsiella pneumoniae.

This study sheds light on the complex relationship between virulence factors and antibiotic resistance in Klebsiella pneumoniae, highlighting that while CSKP strains exhibit higher expression of virulence genes, CRKP strains are becoming increasingly resilient through enhanced expression of resistance genes at the cost of certain virulence traits. The dynamics of gene expression reveal that Klebsiella pneumoniae responds to antibiotic inhibition with a marked plasticity, navigating between virulence and resistance in a rapidly changing environment. Continuous monitoring and investigation into these aspects are vital for the development of effective treatment strategies and the management of infections caused by Klebsiella pneumoniae.