Characterization of a Novel Klebocin from Klebsiella pneumoniae: Broad-Spectrum Antimicrobial and Anti-biofilm Activities with High Stability and Selective Toxicity

The alarming global spread of antimicrobial resistance (AMR) has become one of the greatest threats to public health of the population in the 21^st century. Carbapenem-resistant Klebsiella pneumoniae is listed by the World Health Organization as a priority pathogen, underscoring the need for new effective treatments [1]. Multi-drug resistant (MDR) K. pneumoniae is a significant source of hospital-acquired infections, such as pneumonia, bloodstream, and urinary tract infections, with poor treatment options and high mortality [2, 3]. In the search for alternative strategies to combat MDR K. pneumoniae, attention has increasingly turned to bacterial toxins, particularly bacteriocins, as promising candidates for targeted antibacterial therapies.

Bacterial toxins are one of the most vital virulence factors that help bacteria infect, protect themselves from the immune defenses of the host, and outcompete other microbial organisms in the same ecological niche. These toxins, in general, are biologically active molecules that can cause various cellular and molecular perturbations, such as interference in major metabolic pathways, membrane perturbations, inhibition of macromolecules, or immune modulation of the host organisms [4]. Bacterial toxins are classified into endotoxins and exotoxins, with exotoxins being more diverse and specific. Among them, bacteriocins are ribosomally synthesized protein toxins that inhibit or kill closely related or competing bacteria. Unlike most exotoxins, bacteriocins have a narrow spectrum and do not target eukaryotic cells, making them biologically and ecologically significant [5].

With the deteriorating effectiveness of the traditional antibiotics, there is an urgent need to discover alternative antimicrobial agents. Bacteriocins are ribosomally expressed antimicrobial peptides that have been identified as good candidates because of their high potency, selective targeting, and low propensity to induce resistance [6, 7]. They display broad-spectrum antimicrobial properties due to distinct mechanisms of action, which primarily involve cytoplasmic membrane pore formation, cell wall degradation/ biosynthesis inhibition, protein translational inhibition, as well as direct damage to biological nucleic acids like DNA or RNA. These mechanisms lead to rapid growth inhibition or death in targeted susceptible microbes [8]. Recently, bacteriocins have also received considerable interest as novel alternative or supplementary antimicrobial agents to traditional antibiotics, in particular against the rising incidence of multidrug-resistant (MDR) bacterial isolates across the world [9]. In this scenario, klebocins are one of the most interesting bacteriocin types produced by Klebsiella pneumoniae. This is an important Gram-negative facultative pathogen causing hospital-acquired infections, such as pneumonia, urinary tract infections, and septicemia. Klebocins that K. pneumoniae generate are a comparatively neglected type of bacteriocins among Gram-negative pathogens. Despite genomic studies revealing the presence of bacteriocin gene clusters within K. pneumoniae species complexes [10], there is a scarcity of experimental data characterizing klebocin in clinical isolates, especially in the areas of purification, antibiofilm activity, physicochemical stability, and selective toxicity. Furthermore, the possibility of klebocin as a stable antimicrobial agent that can be used to treat planktonic cells and biofilms has not been effectively studied.

Molecular diagnostic approaches, particularly polymerase chain reaction (PCR)-based detection of virulence and bacteriocin-associated genes, have become indispensable tools for characterizing pathogenic K. pneumoniae isolates. Klebsiella pneumoniae is known to possess an arsenal of gene-encoded virulence factors, including capsular polysaccharides, adhesins, and siderophores, as well as holotoxins, exported from these pathogenic microbes through secretion systems. Klebsiella pneumoniae is known to possess an impressive array of antimicrobial traits to survive and colonize human hosts to cause disease due to this diversity of virulence factors [11]. The secretion of klebocins into bacterial pathogens gives them an impressive survival advantage over competing bacterial strains within this particular biological niche [12]. At the molecular level, klebocins are generally protein or peptide toxins that have special genetic loci, encoded by genetic loci located on plasmids or in specific chromosomal regions. The biosynthesis of klebocins is often accompanied by the simultaneous biosynthesis of immunity proteins that can protect the producing strain from the toxicity of the bacteriocins. Klebocin gene clusters, or bacteriocin genes, such as klebocin, can provide insights into interbacterial competition, as they can strongly affect microbial ecology and infectivity. Therefore, comprehensive characterization of klebocins requires reliable and robust analytical techniques. Chromatographic methods are essential tools for the purification and characterization of bacteriocins such as klebocin [13]. Gel permeation chromatography allows the separation of proteins by size and accordingly performs preliminary protein purification and the initial estimation of the protein's size. Ion exchange chromatography, on the other hand, enables the precise separation of proteins by the presence of certain surface charges. By using a combination of the two, the overall degree of purity and the precision of biochemical analysis can be significantly improved, and the precise biochemical analysis of the bacteriocin, klebocin, in this case, can be undertaken.

This study aimed to extract, purify, and characterize klebocin produced by clinical isolates of Klebsiella pneumoniae, with a particular focus on evaluating its antibacterial and antibiofilm activities, stability under varying physicochemical conditions, and selective toxicity to determine its potential as a safe and effective antimicrobial agent.

3.1 Molecular confirmation of klebocin-producing Klebsiella pneumoniae

To identify a source for the production of klebocin, a clinical isolate of Klebsiella pneumoniae was screened for the presence of genes coding for bacteriocins. PCR confirmation of the presence of the kleB and kleC genes enabled the identification of the isolate of choice for the toxin induction, production, and purification. As shown in Table 3 and Figure 1, a single band of the predicted size of 249 bp and 2,300 bp was obtained, respectively, to confirm the specificity of the kleB and kleC genes. An auxiliary marker in the screening process was the micC gene, with a size of 141 bp. This gene is, however, absent in the isolated K. pneumonia strain. Gene screening was performed primarily to identify the klebocin-producing isolate and did not include further genetic analyses; micC screening was performed for confirmation only.

Table 3. Polymerase chain reaction (PCR) confirmation of klebocin-associated genes

Figure 1. Polymerase chain reaction (PCR) detection of the klebB gene in the Klebsiella pneumoniae isolate

3.2 Antimicrobial and antibiofilm activity of crude Klebocin extract

The crude and cell-free extract of Klebsiella pneumoniae demonstrated broad-spectrum antimicrobial activity as assessed by the agar well diffusion assay. Maximum inhibition was observed against E. coli (20 ± 0.8 mm, 1800 AU), followed by S. aureus (18 ± 1.2 mm, 1600 AU), P. aeruginosa (15 ± 1.0 mm, 1400 AU), C. albicans (12 ± 0.5 mm, 1200 AU), and Streptococcus pyogenes (10 ± 0.7 mm, 1100 AU). These results confirm the broad-spectrum antimicrobial potential of the crude klebocin extract, with the highest efficacy observed against Gram-negative bacteria, while measurable inhibition was also observed against Gram-positive strains and C. albicans (Table 4 and Figure 2).

Table 4. Antimicrobial activity of crude klebocin extract

Figure 2. Agar well diffusion assay demonstrating antimicrobial activity of purified klebocin, (a) C. albicans, (b) E. coli, (c) S. aureus, (d) S. pyogenes, (e) P. aeruginosa

3.3 Purified klebocin

Carboxymethyl (CM)-Sepharose ion exchange chromatography separated klebocin based on net charge. Sharp peaks indicating high purity were achieved in the final purification stage (gel filtration), resulting in a 15.69-fold increase in purity with a specific activity of 1806.4 AU/mg, as shown in Table 5 and Figure 3.

Table 5. Klebocin purification steps, protein, activity, yield, and purification fold

The gel filtration on Sephadex G-100 separated proteins based on molecular size. Klebocin activity peaked in mid-column fractions, indicating successful size-based purification, as shown in Figure 4.

Although the total protein concentration decreased significantly during the stages, the specific activity showed a steady increase, peaking in the final gel filtration stage. This inverse relationship confirms the successful removal of non-target proteins and the concentration of the active antimicrobial fraction, resulting in a final purification ratio of 15.69-fold.

3.4 Stability of purified klebocin

Scanning electron microscopy (SEM) images revealed significant morphological changes, including membrane deformation and the formation of bubbles. These changes suggest a mechanism of action likely resulting from membrane rupture, which could lead to a significant change in the formation of the membrane in Table 6 and Figure 5.

Table 6. Comparative properties of klebocin and selected bacteriocins

Figure 5. Scanning electron microscopy (SEM) of E. coli cell (A) untreated control cells showing intact rod-shaped morphology, (B) klebocin-treated cells exhibiting severe membrane damage, surface blebbing, and cell lysis, suggesting a membrane-targeting mechanism

3.5 Stability of purified klebocin at variable pH and temperature

Biofilm percentage inhibition and relative retention activity of klebocin under different pH and temperature regimes show varied responses. Purified klebocin demonstrated broad-spectrum antimicrobial activity and significant biofilm inhibition [23]. These thermal and pH stabilities are characteristic of many bacteriocins and support potential application in diverse environments. Klebocin fractions retained antibiofilm activity under different pH and temperature conditions. Similar stability profiles have been reported for other bacteriocins, which often remain active after exposure to extreme environments, as shown in Table 7 and Figure 6.

Table 7. Stability and biofilm inhibition of purified Klebocin

Figure 6. Stability profiles of klebocin activity graph comparing % activity retention of purified klebocin at varying (a) pH and (b) temperature conditions

In this study, the bacteriocin klebocin produced by Klebsiella pneumoniae was isolated, purified, and characterized. Its antibacterial and antifungal activity were evaluated. Also, its effect on biofilm formation and biochemical stability under a wide range of physical and chemical conditions was investigated. The results show that klebocin is a potent and stable biopeptide capable of functioning even under harsh environmental conditions, making it a promising candidate for use in medical and industrial applications. Recent studies indicate that bacteriocins are promising biomolecules as alternatives to conventional antibiotics, given their high efficacy, broad spectrum of resistance to microbial diversity, and ability to target bacteria without significantly affecting animal cells [24]. Klebocin possesses a broad spectrum of antibacterial and antifungal activity, with the largest inhibition zones observed against E. coli (20 ± 0.8 mm), along with significant inhibition against S. aureus, P. aeruginosa, S. pyogenes, and Candida albicans. This reflects a broad spectrum of activity similar to well-known bacteriocins, such as pediocin [25, 26]. Purification results showed that the multiple steps in the protocol (ion-exchange chromatography and molecular filtration) resulted in a concentration of biological activity while minimizing inactive proteins. Statistical analyses (ANOVA, p < 0.01) supported the correlation between purity and the specific activity of the peptide, reflecting the removal of inactive components and the concentration of the active peptide. This finding is corroborated by literature on bacteriocin purification protocols [14].

Regarding biofilm formation, klebocin demonstrated strong biofilm inhibition, reaching a peak of 78% with the purified peptide. Biofilm inhibition is crucial because biofilms constitute a bacterial defense mechanism, preventing antibiotics and the immune system from eradicating microbial colonies [27]. Crude extracts also exhibited good inhibition (65–70%), suggesting a potential synergistic effect of secondary components in the crude extract that enhances biofilm inhibition. This is supported by the positive correlation between the presence of the kleB/kleC genes and the level of biofilm inhibition (r = 0.82, p < 0.01) [28]. Klebocin demonstrated high stability over a wide pH range and temperatures up to 100°C, with optimal efficacy at neutral pH and 37 ℃. This level of physical and chemical stability enhances its potential use in industrial and medical applications, including food preservation and applications exposed to fluctuating environmental conditions. These findings are consistent with other reports on bacteriocins, such as plantarcin and pediocin variants, which are known for their resistance to temperature and pH variations [29, 30].

In terms of selectivity, toxicity tests showed that klebocin has very low toxicity to human cells (< 5% at 1× MIC) and a high STI value (> 16), indicating a wide safety margin for treatment. SEM images revealed bacterial membrane deformation with the formation of bubbles and membrane gaps, demonstrating that the mechanism of action relies on direct action on the cell membrane, a characteristic common among cationic bacteriocins, which preferentially target the negatively charged membranes of bacteria compared to animal cells [31]. When compared to other bacteriocins, such as pediocin, lactococcin, and plantarcin, Klebocin has been shown to possess a broader spectrum, higher stability, and deeper biofilm activity, whereas pediocin and lactococcin are often limited to Gram-positive bacteria only, with only moderate stability. This highlights the potential application advantages of klebocin [31].

Klebocin has a broad-spectrum antimicrobial effect on Gram-positive as well as Gram-negative bacteria, with moderate antifungal activity on Candida albicans. This justifies its capability to act on two different kingdoms. Klebocin also has high antibiofilm ability, as evident from the uninhibited ability to hinder biofilm formation. This could result from its inhibition of extracellular polymeric substance biosynthesis as well as fimbriae-mediated adhesions, resulting in making lesser bacterial adherence. Klebocin also remained stable in high pH as well as temperature values when compared to the stability in conventional bacteriocins like pediocin and lactococcin. However, hemolysis selectivity assays showed a minimum hemolytic effect relative to eukaryotic cells. This justifies its large cytotoxicity index. Klebocin also has the ability to target bacterial membranes, as evident from SEM. This action makes way for its high potency as a bacteriocin since its mechanism of action targets cationic bacterial membranes with a negative charge.

Based on these findings, it can be concluded that klebocin has significant potential for medical and industrial applications, including the treatment of MDR bacteria, coating medical surfaces to prevent biofilm formation, and preserving food against microbial spoilage. Future research may focus on developing advanced expression systems, nano-formulation, and improving shelf life to maximize the peptide's application value [32].

This study has a number of limitations, even though its results are promising. First, the antimicrobial and cytotoxicity tests were only performed using in vitro tests and not in vivo. Second, molecular characterization included only PCR validation of the kleB and kleC genes; no sequencing, gene expression profiling, or structural analysis was carried out. Third, the purification 15.69-fold denotes moderate enrichment, which implies that it can be further optimized to increase yield and purity. The mechanism of action was also deduced based on SEM observations, which was not confirmed at the molecular level. Lastly, the spectrum of activity was also validated on a few clinical strains, which might not be reflective of MDR diversity. The findings presented are promising in the context of developing new antibacterial treatments that could improve patient outcomes.