Capsaicin: An Important Alkaloid with Potential Against Klebsiella pneumoniae

1. Introduction

Klebsiella pneumoniae is an opportunistic pathogen of great global relevance. Over the last decade, it has emerged as a major clinical threat, due to the increasing prevalence of healthcare-associated infections caused by multidrug-resistant (MDR) strains and the emergence of severe community-acquired infections caused by "hypervirulent" strains (hvKP); these pathogens can exceptionally acquire exogenous genetic elements that encode resistance and hypervirulence. The rapid dissemination of these clinical strains, associated with therapeutic failure and high mortality, is alarming. [1,2,3,4,5,6,7]. Klebsiella pneumoniae is a global pathogen with remarkable genetic, phenotypic and pathogenic diversity; there are increasing reports of convergence between these pathotypes and their genetic determinants, raising significant public health concerns. [8]. In the infection process, K. pneumoniae adapts to different environmental conditions, employing the two-component regulatory system (TCS), composed of a sensor histidine kinase and a response regulator, which is an important bacterial regulatory system in response to external stimuli [9]. In recent years, with the widespread use of carbapenems, carbapenem-resistant K. pneumoniae (CRKP) infections have been increasing. Data from the China Antimicrobial Surveillance Network (CHINET) show that the resistance rate of K. pneumoniae to imipenem and meropenem increased eightfold, from 3.0% and 2.9% in 2005 to 25.0% and 26.3% in 2018, respectively. Furthermore, their drug resistance rate to imipenem in four children's hospitals in the report ranged from 32.1% to 45.5%, meaning that K. pneumoniae resistance to carbapenems in the pediatric group deserves more attention [10].

Furthermore, the ability to form biofilms is a crucial virulence trait in the pathogenesis of K. pneumoniae disease, as it promotes increased resistance against environmental stressors and provides a reservoir for dissemination and increased exchange of genes associated with antimicrobial resistance; several virulence factors contribute to biofilm formation by K. pneumoniae, either directly, by promoting increased adherence and/or maturation of the biofilm, or indirectly, by inhibiting biofilm formation by bacterial competitors in its colonizing niche [11,12,13]. Capsule, type 1 and 3 fimbriae (mrkA gene), quorum sensing system type 2 (luxS), D-galactan I synthesis (wbbM), LPS transport (wzm) and polybeta-1,6-N-acetyl-D-glucosamine (pgaA) appear to be involved in K. pneumoniae biofilm [12]. The demonstrated ability of K. pneumoniae to survive and regrow in these biofilms, as well as the high mortality it causes, is of great global concern [14].

In this context, the search and development of novel compounds with potential against K. pneumoniae that are safe, tolerable and effective is urgent today. As is well known, since ancient times products of natural origin, particularly from plants, have made a valuable contribution to pharmacotherapy, especially in infectious diseases and cancer; It is estimated that between 65 and 80% of the world's population in developing countries relies essentially on plants for primary health care due to lack of access to medicine. Furthermore, between 25 and 30% of all drugs available as therapy are derived from natural products, which moves around 20 billion dollars annually in the global pharmaceutical market [15,16]. In this scenario, plants play a primary role as a source of specialized metabolites with recognized medicinal properties [17,18]. Due to their wide chemical diversity, these metabolites can be used directly as bioactive compounds, as drug prototypes or used as pharmacological tools for different targets [19], so they are an excellent alternative to be investigated.

Capsaicinoids (Figure 1) are secondary metabolites of plants, with capsaicin (CAP) (trans-8-methyl-N-vanillyl-6-nonenamide C₁₈H₂₇NO₃) being the main responsible for the spiciness of chili peppers; it is biosynthesized through two pathways involved in the metabolism of phenylpropanoids and fatty acids. CAP is used as a food additive and in pharmaceutical applications; it can also act as a cancer preventive agent and shows broad applications against various types of cancer [20]. It has recently attracted attention due to its antimicrobial and antiviral activity. Its antimicrobial potential has been reported against group A hemolytic streptococci [21], Staphylococcus aureus, Listeria monocytogenes [22,23], Vibrio cholerae [23], Acinetobacter baumanii [24], Helicobacter pylori [25], Salmonella typhimurium, Escherichia coli, and Pseudomonas spp., affecting their growth and biofilm formation; likewise, its potential against Candida spp., Toxoplasma gondi and Trypanosoma cruzi has been reported; its action against some viruses has also been documented, whose invasiveness has been affected by the action of this compound [26]. However, the antibacterial potential of this alkaloid against clinical isolates of K. pneumoniae had not been documented, so we hypothesized that CAP could have an effect against K. pneumoniae, and the biofilms formed by these pathogens. The purpose of this research was to evaluate the antibacterial activity of CAP, estimate its capacity to inhibit biofilms and explore its possible effect against the membranes of clinical isolates of K. pneumoniae, contributing to the search for new compounds of natural origin that can serve as adjuvants in the treatment of drug-resistant pathogenic bacteria.

2. Results

2.1. Susceptibility Testing

CAP showed antibacterial activity against all clinical isolates of K. pneumoniae studied; we observed a reduction in the percentage of growth of bacteria treated with CAP, compared to untreated isolates used as a control. Figure 2 shows a similar trend among isolates, with the percentage of growth reduction increasing as the CAP concentration increases. Table 1 shows the MIC values; MIC₉₀ values of CAP were obtained between 28.44 and 1696 µg/mL and it was shown that this effect on K. pneumoniae depended on the CAP concentration, with the percentage of growth reduction increasing as its concentration increased.

We observed that after 24 h the MIC₉₀ values of CAP varied between 28.44 and 1696 µg/mL; as evidenced, the efficacy of CAP was different between strains of the same species.

2.2. Biofilm Reduction

All K. pneumoniae isolates tested produced biofilms on polystyrene microplates as shown in Figure 3a; isolates A10, A42, A38 and A48 were strong biofilm producers while isolates A41 and A49 were moderate biofilm biomass producers. When CAP MIC₉₀ was added to mature biofilms from each isolate, a percentage reduction in biomass in the biofilm ranged from 1.97 to 40.04% after 1 h of exposure to CAP (Figure 3b); while the percentage reduction in biomass formation in the biofilms ranged from 4.26 to 72.48% (Figure 3c). In some cases, these percentages were similar to those obtained with the antibiotics used as control (CIP and GEN) and in others, they were higher, as shown in Table 2 and Table 3.

Table 2 shows the percentages of inhibition of CAP biofilm formation in K. pneumoniae, during 24 hours of exposure.

Table 3 shows the percentages of CAP inhibition against mature K. pneumoniae biofilms after 24 hours of incubation.

2.3. Leakage of Nucleic Acids and Proteins Through the Klebsiella pneumoniae Membrane

The action of CAP on the integrity of K. pneumoniae membranes was evaluated by release assays of intracellular constituents that absorb at 260/280 nm, such as nucleic acids and proteins. These assays were performed at 0, 30, 60 and 120 min after CAP treatment (MIC₉₀ for each isolate). As seen in Figure 4, the OD₂₆₀/OD₂₈₀ values in the CAP-treated groups are significantly higher from the start of CAP exposure, compared to the GEN-treated groups, where minimal release and in some cases no leakage of material was observed with this antibiotic with K. pneumoniae A10 (the other isolates showed similar behavior). These results show damage to the cell membrane permeability of K. pneumoniae caused by CAP.

2.4. Effect of CAP on Membrane Integrity

Damage to K. pneumoniae cell membranes by CAP was also evidenced using Evans blue staining. As indicated in Figure 5, when cells were treated with CAP and observed under a light microscope, they stained blue, suggesting damage to cell membrane permeability after 1 h of CAP treatment, thus suggesting that the membrane is a target of the CAP mode of action against clinical isolates of K. pneumoniae.

3. Discussion

Klebsiella pneumoniae infections represent a major challenge in medical science. This pathogen has a large accessory genome of plasmids and chromosomal gene loci; this accessory genome divides K. pneumoniae strains into opportunistic, hypervirulent and multidrug-resistant groups [28], its ability to evade the immune system, its increasing antimicrobial multi-resistance and the emergence of hypervirulent pathotypes have become a great challenge, increasingly limiting the drugs available for its control and increasing morbidity and mortality rates, not only in community infections but also in healthcare-associated infections [1,4,7,29,30,31]. This has sparked our interest in the search for novel, safe and effective compounds with antibacterial potential, particularly against K. pneumoniae. In this context, plant-based compounds are an excellent alternative to be investigated.

In this investigation, we demonstrated that the CAP alkaloid has antibacterial activity against clinical isolates of K. pneumoniae. This effect being concentration dependent, these results are consistent with the results documented [26], indicating that, depending on the concentration and strain of the bacteria, CAP can exert bacteriostatic or even bactericidal effects against a wide range of bacteria. Likewise, the inhibitory effect of CAP against H. pylori strains has been demonstrated, this effect being doce-dependent, at concentrations greater than 10 µg/mL and showing bactericidal activity after 4 hours of exposure [25]. It was also evidenced [21] the effect of CAP against group A Streptococcus, showing MIC values of 64 to 128 µg/mL, and early detection of dead cells in the live/dead cell assay; CAP also inhibited intracellular invasion and hemolytic activity, so it has been suggested that it could thus prevent both the formation of an intracellular reservoir that is difficult to eradicate and the spread of infection to deep tissues. The effect of CAP against bacteria and Candida albicans was documented, showing that L. monocytogenes and S. aureus were more susceptible to the antimicrobial effects of CAP than Salmonella and E. coli O157:H7, while C. albicans were significantly more susceptible than all bacterial species examined [22]. Other studies [24], they have also demonstrated the effect of CAP against colistin-resistant A. baumanii strains, with MIC values > 512 μg/mL and the synergistic effect of CAP, capable of reducing colistin MICs to values below the susceptibility breakpoint. The effect of CAP on the inhibition of cholera toxin production in V. cholerae strains belonging to various serogroups has also been reported, suggesting that CAP could act as a potent repressor to produce this toxin [23]. Alkaloids are widely known for their antibacterial and antifungal potential, the most active ones are mainly planar in structure, amphiphilic, with a molecular mass between 200 and 400 g/mol, and a polar surface of approximately 50 Ų, and they target DNA and/or topoisomerase, as well as the cytoplasmic membrane [32,33].

Biofilms formed by K. pneumoniae represent an important virulence factor that crucially contributes to its pathogenesis and to the multidrug resistance expressed by these pathogens. Natural plant-derived compounds have been shown to have significant antibiofilm properties. This study shows the effect of CAP against biofilm formation and mature biofilms of K. pneumoniae. The effect against mature biofilms was in some cases superior to the effect shown with the evaluated antibiotics CIP and GEN; likewise, the effect shown against mature biofilms was superior to that shown against the formation of these biofilms, in which the effect was in some cases similar to that shown with CIP and GEN. These coincide with reports [26], which indicate that CAP can reduce the pathogenicity of these bacteria, through a variety of mechanisms, such as mitigating the release of toxins or inhibiting the formation of biofilms. On the other hand, the study of natural compounds such as curcumin, eugenol, linoleic acid, chitosan, reserpine and berberine against K. pneumoniae biofilms has been documented [13,34], as well as the effect of thyme and mint essential oils and their active components have been studied as promising antibiofilm agents alone and/or in combination with CIP to inhibit/eradicate K. pneumoniae biofilms [35]. The results of this research are the first report of the antibacterial and antibiofilm effect of CAP against clinical isolates of K. pneumoniae.

This study also explored the effect of CAP on K. pneumoniae membranes, showing that the antibacterial effect of CAP is likely associated with damage to the integrity of K. pneumoniae membranes caused by this alkaloid, as evidenced by the intracellular material efflux experiments at 260/280 nm, which show a significant and early release of intracellular material in K. pneumoniae treated with CAP, compared to untreated cells. This is in agreement with other results [21], which show early detection of dead group A Streptococcus cells when exposed to CAP. Besides, microscopy with Evans blue staining showed blue stained cells when treated with CAP (MIC₉₀), in contrast to untreated cells used as a control, which remained transparent; these results are consistent with documented studies [31] indicating that one of the targets of the antimicrobial action of these compounds is the cell membrane.

In this regard, CAP being a phytochemical with numerous physiological and therapeutic effects, including its important antimicrobial properties and having shown its effect against clinical isolates of K. pneumoniae, further studies are recommended to elucidate the antibacterial action mechanisms of CAP against K. pneumoniae isolates.

4. Materials and Methods

4.1. Reagents

Mueller–Hinton broth (MHB) (Sigma, Mendota Heights, MN, USA) was used for the determination of the MIC and cultures of bacterial isolates. Tryptic soy agar (TSA) and Tryptic soy broth (TSB) (Becton, Dickinson and Company, San Diego, CA, USA), Mueller– Hinton agar (MHA) (Sigma, Mendota Heights, MN, USA), and Brain heart infusion (BHI) broth (Sigma-Aldrich, St. Louis, MO, USA) were also used for the bacterial cultures. The dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), crystal violet (CV) and the antibiotics, ciprofloxacin (CIP) y gentamicin (GEN) used in this study were obtained from Sigma-Aldrich, St. Louis, MO, USA. Meanwhile, the glacial acetic acid was obtained from Carlo Erba Reagents, Milan, Italy.

4.2. Capsaicin

Capsaicin was purchased from Sigma-Aldrich Inc, M2028, 8-Methyl-N-vanillyl-trans-6-nonenamide (C₁₈H₂₇NO₃) 305.41 g/mol.

4.3. Strains

Eleven clinical isolates of K. pneumoniae (A70, A10, A48, A42, A41, A38, A47, A39, A49, A100, A40) were used in this study. The isolates were cultured from the tracheal aspiration, bronchoalveolar lavage, blood and urine culture samples of patients hospitalized at the Social Health Service S.A.S. in the city of Sincelejo, Colombia. All microorganisms were identified using standard methods: Vitek® 2 Compact. Biomerieux SA (Marcy-l’Étoile,m France). BHI medium was used to maintain the cultures until testing was performed.

4.3. Susceptibility Testing

The MICs of CAP against K. pneumoniae strains were determined by broth microdilution assay using 96-well microtiter plates, following the protocol established in the M07-A9 method of the Clinical and Laboratory Standards Institute (CLSI), [36]. For this purpose, double serial dilutions were made in MHB until reaching final concentrations of the compound (CAP) of 1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90 µg/mL in each reaction well. The assay was performed in a total volume of 200 μL per well, distributed as follows: 100 μL of CAP (at previously described concentrations) and 100 μL of the bacterial inoculum were added at a concentration of 1 × 10⁸ CFU/mL. Each strain was tested in triplicate. The commercial antibiotics CIP and GEN were used as positive controls, and untreated cells as negative controls. Absorbance readings were immediately measured using a ChroMate 4300 ELISA reader at a wavelength of 630 nm, and readings were subsequently taken after 24 hours of incubation at 37 °C. The inhibition percentages were calculated from the following equation:

% Inhibition = (1 - (ODT24 - ODT0/ODGC24 - ODGC0)) × 100

where ODT24 is the optical density of the test well at 24 h post inoculation; ODT0 the optical density of the test well at 0 h post inoculation; ODGC24 the optical density of the growth control well at 24 h post inoculation and ODGC0: optical density of the growth control well at 0 h post inoculation.

4.4. Quantitative Evaluation of Biofilm Inhibition

The effect of CAP on mature K. pneumoniae biofilms was evaluated following the protocol described by [37], with minor modifications. For biofilm formation, bacterial colonies incubated for 24 h in TSA were used, standardizing the bacterial inoculum to 1 × 10⁸ cells/mL. Then, 200 µL of the bacterial inoculum was poured into 96-well polystyrene microplates in each well and incubated at 37 °C for 24 h. The broth was then removed from the microplates and 200 µL of CAP (MIC₉₀ of each isolate) was added in TSB broth and incubated at 37 °C for 1 h. Subsequently, floating cells were removed, and biofilms were washed from the bottom of the wells with deionized water. Excess moisture was then removed by tapping the microplates on sterile napkins and the plates were dried for 5 minutes. Six replicates of each experiment were performed. Cultures without CAP were used as negative control and cultures with CIP and GEN were used as positive control. Biofilm reductions were quantified by staining the wells with 200 µL of 0.1% CV for 20 minutes. Samples were washed with deionized water until excess dye was removed, excess water was carefully dried, and then the CV was solubilized in 250 µL of 30% glacial acetic acid. Absorbance values were measured at 590 nm (OD₅₉₀) using a SYNERGY LX microplate reader (Biotek). Biofilm production was grouped into the following categories: OD₅₉₀ < 0.1, non-producers (NP); OD₅₉₀ 0.1–1.0, weak producers (WP); OD₅₉₀ 1.1–3.0, moderate producers (MP); OD₅₉₀ > 3.0, strong producers (SP). For biofilm formation inhibition assays the standardized bacterial inoculum was incubated simultaneously with the CAP MIC. Biofilm reduction was calculated using the following equation:

% Biofilm Reduction =(AbsINO-AbsCAP) /AbsCAP × 100

where AbsINO: absorbance of the untreated inoculum and AbsCAP: absorbance of the sample treated with CAP

4.5. Leakage of Nucleic Acids and Proteins Through the Cell Membrane

The release of intracellular material was measured according to the methodology proposed by [36], with some modifications. Klebsiella pneumoniae A10 cells grown in nutrient broth were centrifuged at 3000 g for 20 min, washed three times and resuspended in 20 mL of PBS (pH 7.0). The cell suspension was then treated with CAP (MIC₉₀ for each isolate, separate experiments) and incubated at 37 °C for 0, 30, 60 and 120 min. Subsequently, 2 mL of the samples were collected and centrifuged at 3000 g for 20 min. Then, to determine the concentration of the released constituents, 2 mL of the supernatant was used to measure the absorbance at 260/280 nm with a Spectroquant® Prove 300 UV/Vis spectrophotometer (Merck KGaA, Darmstadt, Germany). Samples without CAP and samples with GEN were used as controls. All assays were performed in triplicate.

4.6. Effect of Extracts on Membrane Integrity

To evaluate the effect of CAP on the membrane integrity of K. pneumoniae, staining was also carried out using Evans blue following a protocol described by [36]. Prior to the assay, a 1% solution of Evans blue in phosphate buffered saline (PBS) was prepared. Klebsiella pneumoniae samples were incubated in BHI broth for 24 hours at 37 °C on slides, in triplicate. They were then treated with CAP (CMI₉₀) for 1 h, followed by the addition of Evans blue for 5 min. Untreated cells were used as a control. Samples were then observed under an Olympus CX31 microscope to assess changes in cell membrane integrity.

4.7. Statistical Analysis

The results were analyzed using GraphPad Prism software version 8.0 and Microsoft Excel version 2024. Initially, the Shapiro-Wilk test was used to determine the distribution of the data. Subsequently, Spearman correlation coefficients were used to measure the degree of linearity, the correlation between CAP concentration and the percentage reduction in bacterial growth. To compare the effects of CAP and antibiotics (CIP and GEN) on biofilm reduction, Holm-Sidak's and Games-Howell's tests were used; the Holm-Sidak's test was used to compare the effects of CAP and antibiotics on the leakage of intracellular material through the membrane (260/280 nm).

5. Conclusions

In this study, we investigated the antibacterial potential of the alkaloid CAP against clinical isolates of K. pneumoniae, as well as its role in biofilm inhibition. Furthermore, we explored the action against the cell membrane of these pathogens. We demonstrated the antibacterial action of CAP against K. pneumoniae, this effect being associated with damage to the integrity of the cell membrane, in addition to its action against bacterial biofilms. These studies need to continue, with the aim of elucidating the antibacterial action mechanisms of CAP, and its possible synergistic action with drugs, since it shows promise as an alternative tool for the treatment and control of multi-resistant nosocomial pathogens such as K. pneumoniae.