Peptide Extract from Red Kidney Beans, Phaseolus vulgaris (Fabaceae), Shows Promising Antimicrobial, Antibiofilm, and Quorum Sensing Inhibitory Effects

The rapid spread of multidrug-resistant bacteria has led to an increased risk of infectious diseases. Pseudomonas aeruginosa, in particular, poses a significant obstacle due to its propensity to rapidly acquire resistance to conventional antibiotics. This has resulted in an urgent need for the development of new classes of antibiotics that do not induce resistance. Antimicrobial peptides (AMPs) have been studied as potential small-molecule antibiotics due to their unique mode of action. In this study, peptides were extracted from the seeds of Phaseolus vulgaris (Fabaceae), and the antimicrobial activities of the extract were evaluated using microbroth dilution against five different microorganisms. The extract showed antimicrobial activity against all tested organisms with minimum inhibitory concentrations (MIC) of 2.5 mg/mL, except for Candida albicans and Pseudomonas aeruginosa, which had MICs of 1.25 mg/mL. The extract was also bacteriostatic for all tested organisms. The crude peptide extract from Phaseolus vulgaris was further studied for its antibiofilm activity against Pseudomonas aeruginosa, a common nosocomial pathogen associated with biofilm formation. The extract showed good antibiofilm activity at 1/2 MIC. The extract also inhibited the expression of pyocyanin and pyoverdine (virulence factors of P. aeruginosa whose expression is mediated by quorum sensing) by 82% and 66%, respectively. These results suggest that the peptide mix from Phaseolus vulgaris may inhibit biofilm formation and virulence factor expression by interfering with cell-to-cell communication in Pseudomonas aeruginosa. The ability of the extract to inhibit the growth and biofilm formation of all tested organisms indicates its potential as an antimicrobial agent that could be further studied for drug discovery.


Introduction
Despite advancements, infectious diseases remain a global health challenge.Pathogenic organisms like bacteria, fungi, parasites, and viruses cause infectious diseases.Microbial pathogens are responsible for over 400 million years of life lost annually, surpassing cancer and cardiovascular diseases.Antibiotics have demonstrated efectiveness in the treatment of infections by targeting pathogens while causing minimal harm to host cells.Tey have saved countless lives and are vital in treating various infections and life-threatening conditions.However, bacterial resistance has risen due to antibiotic overuse and limited development of new antibiotics.Multi-drug-resistant (MDR) and extremely drugresistant (XDR) bacteria have emerged, posing a major global threat [1].Projections indicate that antibiotic resistance could lead to 10 million annual deaths globally and economic costs of $100 trillion by 2050.It also hinders universal health coverage and the attainment of some of the sustainable development goals.Addressing antimicrobial resistance and developing new treatments are critical for global health progress [2].Pathogenic microorganisms employ diverse mechanisms to develop resistance against antibacterial agents, including efux pumps, target modifcation, and the formation of bioflms [3].Pseudomonas aeruginosa is a model bacterium known for its profciency in bioflm formation and its role as an opportunistic pathogen in cystic fbrosisrelated respiratory infections, as well as chronic infections in immunocompromised individuals [4].Additionally, P. aeruginosa is associated with tissue degradation and spoilage of meat and protein-rich foods [5].Te formation of bioflms in P. aeruginosa is intricately regulated by quorum sensing (QS).QS serves as a cell-to-cell communication system that governs the expression of genes related to bioflm formation.Studies have underscored that P. aeruginosa strains with defective QS mechanisms are unable to form bioflms.Moreover, QS has been shown to exert a signifcant impact on the expression of various virulence factors in P. aeruginosa, including pyoverdine, pyocyanin, and proteases.Two interconnected QS systems in P aeruginosa, Las and Rhl, are of utmost importance for virulence and bioflm production, as well as tolerance to antibiotics, detergents, and biocides [6].Te Las system, under the guidance of LasR, governs other QS systems and infuences bioflm formation and the activation of virulence genes.LasI, within this system, produces N-(3-oxododecanoyl) homoserine lactone (3-O-C12-HSL), which triggers LasR to activate virulence genes, such as lasB, lasA, apr, and toxA [7].Two additional QS systems, namely, Pseudomonas quinolone signal (PQS) and integrated quorum system (IQS), exist, with the latter's specifcs yet to be fully elucidated [8].Te Las system emerges as a central player in QS, with LasR orchestrating various systems and genes critical for the bacterium's behavior [9].
One promising avenue for fnding novel antibacterial remedies lies in the exploration of natural products that are commonly consumed.Antimicrobial peptides (AMPs) have emerged as a focal point of study, and applications in both agriculture and medicine have been touted [10].AMPs are a widespread class of molecules found in diverse organisms, ranging from microbes to animals, and are key components of natural defense systems against pathogens and pests.While AMPs come in diferent molecular forms, most of them are linear peptides derived from insects, animals, and plants.Plants, in particular, possess an extensive distribution of AMPs, which can be extracted and isolated from various plant organs, such as stems, roots, seeds, fowers, and leaves.Tese AMPs exhibit diverse physiological defense mechanisms against viruses, bacteria, fungi, and parasites, making them potential candidates for therapeutic and preservative applications [11].
Generally, AMPs derived from plants share several common features with those obtained from microorganisms, insects, and animals.Some of these features include the molecular form they exhibit, the overall positive charge of the peptide, and their amphipathic nature.All of these characteristics play a critical role in their function as defensive agents, acting as membrane-active antimicrobials.Two plant AMP families that exhibit these features are thionins and plant defensins.In addition to these characteristics, both thionins and plant defensins are notably rich in cysteine residues.It is worth noting that other plant AMP families typically exert their antimicrobial efects on pathogenic microbes in a manner distinct from AMPs derived from animals [12].As an example, hevein-like peptides are known to function by binding to chitins, whereas knottin-type peptides play a critical role in inhibiting key enzymes, such as proteases.Lipid transfer peptides interact strongly with anionic lipopolysaccharides found in the outer membranes of many Gram-negative bacteria (such as P. aeruginosa), leading to destabilization and permeabilization of the membrane [12].Te expression of AMP in plants is induced and usually tissue-specifc, just as it is observed in animals.Once more, plant-derived AMPs exhibit adaptability through the presence of highly variable sequences housed within a specifc scafold unique to each AMP family.Tis phenomenon bears some resemblance, albeit on a considerably smaller scale, to the molecular diversity observed in the immune system's immunoglobulin-based immunity among vertebrates [12].
Red kidney beans, known as Phaseolus vulgaris (Fabaceae), have garnered increasing interest in recent research.Tese beans are recognized for their positive health benefts.Rich in amino acids, red kidney beans ofer a cost-efective source of dietary protein, typically comprising 20% to 30% protein content based on dry weight.Previous investigations on peptides isolated from related species have demonstrated various biological activities [10,13].Antimicrobial protein hydrolysate was isolated from P. vulgaris, which is active against some Grampositive and Gram-negative bacteria [14], setting a foundation for exploring the antimicrobial potential of crude peptides from red kidney beans.In this study, the antimicrobial, bioflm inhibition, and quorum sensing efects' crude peptides extracted from Phaseolus vulgaris were investigated, contributing to their application on health in addition to their nutritional values.

Sample Collection.
Red kidney beans (Figure 1) were collected from the local market of Ayeduase, Kumasi, in the Ashanti Region of Ghana and helped verifed by a plant botanist at the Department of Herbal Medicine, Kwame Nkrumah University of Science and Technology.Beans with uniform size and shape were selected for this work.Te beans were air-dried and stored in a cool-dried container at room temperature (27 °C) until further analysis.

Peptide Extraction.
Crude peptide was extracted from red kidney beans using method described by Roy and coworkers [13] with slight modifcation.Te beans were frst grounded into four, after which the resulting four was mixed with distilled water in a ratio of 1 : 10 (w/v).Te pH of the mixture was then adjusted to 9.0 using 1N NaOH and stirred at room temperature (27 °C) for 2 hours.Te mixture was then centrifuged at 6000 rpm for 20 minutes, and the precipitates were discarded.Te supernatant was acidifed to 2 Biochemistry Research International a pH of 4.5 using 1N HCl and centrifuged to be obtained the precipitates.Te resulting precipitates were washed twice with distilled water at a ratio of 1 : 5 (w/w).After washing, the obtained precipitates were freeze-dried to obtain the crude peptide extracts.Subsequently, subcultures were obtained and used to prepare colony suspensions of the microorganisms, which were then adjusted to a 0.5 McFarland standard.Tese colony suspensions were further diluted into sterile double-strength CM001 nutrient broth (Oxoid, UK), resulting in a concentration of approximately 2 × 10 5 CFU/mL.

Minimum Inhibitory Concentration (MIC).
Te minimum inhibitory concentration (MIC) of the peptide extract was determined by the broth microdilution method [16].Eight serial twofold dilutions of peptide extract were prepared, resulting in a concentration range of 2.5 to 1.953 × 10 −3 mg/mL.Gentamicin and fuconazole were used as the positive controls with concentrations ranging from 50 to 1.96 × 10 −1 μg/mL for bacterial and fungal strains, respectively.Each well of the microtiter plate was flled with 40 μL of double-strength broth containing an inoculum size of 2 × 10 5 CFU/mL.Te fnal volume in each well was 200 μL.Te plates were then covered and incubated at 37 °C for 24 hours.

Minimum Bactericidal Concentration (MBC).
MBC/MFC values were determined by pipetting 40 μL of microbial suspension from subculture demonstrating no visible growth from the MIC experiment and inoculating nutrient agar plates.Te MBC or MFC was determined with the wells with concentrations greater than the MIC.Plates were incubated at 37 °C for a total period of 24 hours.Each experiment was done in triplicate.Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were recorded as the lowest extract concentration killing 99.9% of the bacterial or fungal inocula after 24-h incubation at 37 °C [3].

Evaluation of Microbicidal and Microbistatic Capacity of
Peptide Extract.Te antimicrobial activity of the compounds was assessed using the MBC (or MFC)/MIC ratio.An MBC (or MFC)/MIC ratio ≤2 indicated a microbicidal efect, while a microbistatic efect was recorded when the MBC (or MFC)/MIC ratio was ≥4 [17].

Bioflm Inhibition Assay.
Te bioflm inhibition capacity of the peptide extract was determined using the crystal violet assay [18].Sterile microtiter plates were flled with both MIC and sub-MIC concentrations of the peptide extract and a standard drug (gentamicin).Inoculum suspensions adjusted to 0.5 McFarland standard were added to each well, resulting in a fnal volume of 200 μL.Te plates were incubated at 37 °C for 24 hours.After incubation, the contents of the wells were discarded and thoroughly washed with deionized water.Tis was to remove any unbound or loosely attached bacterial cells.Te wells were stained with 0.1% crystal violet, and the contents were eluted with 30% acetic acid onto a new sterile plate.Te absorbance of the eluates was measured at 595 nm, allowing for the evaluation of bioflm inhibition using the following equation: Biochemistry Research International 3 Multimode Microplate Reader, Germany) at an excitation wavelength of 405 nm and an emission wavelength of 465 nm [19].Te inhibition percentages were determined by comparing the treated cultures to the untreated culture (control) using the following equation: 2.6.2.Pyocyanin Quantifcation.P. aeruginosa was incubated as described in the pyoverdine inhibition assay.Cell-free supernatants were obtained by centrifuging the culture at 4000 rpm for 45 minutes.To 8 mL of the supernatant, 4 mL of chloroform was added, and the mixture was vortexed 10 times for 2 seconds each to separate the greenblue chloroform layer, which settled at the bottom of the tube.Te samples were then centrifuged for 2 minutes at 4000 rpm, and the supernatant above the green-blue chloroform layer was carefully removed.Next, 3 mL of 0.2 M HCl was added to each tube, and the mixture was vortexed again 10 times for 2 seconds.After centrifugation for 2 minutes at 4000 rpm, the supernatant (pink layer) was transferred into a cuvette, and the absorbance was measured at 520 nm.Te concentration of pyocyanin (μg/mL) was determined by multiplying the absorbance value at 520 nm by 17.072 and the molar extinction coefcient of pyocyanin at 520 nm [3].
Percentage inhibition was calculated relative to the untreated culture (control) using the following expression:  2).In the UV characterization of the peptides, the peptide solution demonstrated a maximum absorbance of 1.219 A at 203 nm, accompanied by a minor shoulder at 194 nm (0.075 A), and a weaker band at 280 nm (Figure 3).Te peptide extract showed positive results for the biuret test.Te formation of purple color after the addition of copper sulfate indicated the presence of peptide bonds in the extract.

Antimicrobial Activity.
Peptide extracts derived from P. vulgaris displayed good antimicrobial activity, as evidenced by their low minimum inhibitory concentrations (MICs) against Gram-positive and Gram-negative microorganisms.C. albicans, a fungus, and P. aeruginosa, a Gramnegative bacterium, displayed particularly notable susceptibility, with MICs recorded at 1.25 mg/mL.Additionally, for the Gram-negative bacterium E. coli and the Gram-positive bacteria S. aureus and E. faecalis, the MICs were observed to be 2.5 mg/mL (Table 1).Te minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC) of the peptide extract against all tested microbes exceeded 2.5 mg/mL, indicating that higher concentrations were required to achieve microbicidal activity (Table 1).Furthermore, the MBC/MIC and MFC/MIC ratio for all organisms was greater than 1, suggesting a microbistatic efect of the peptide extract.Tis implies that the peptide extract inhibited the growth of the organisms without completely eradicating them.

Efect of Crude Peptide on Bioflm Formation.
Te antibioflm activity of the peptide extract was tested against the wild type, widely used bioflm-forming clinical isolate P. aeruginosa PAO1.Gentamicin was used as a positive control.Optical density (600 nm) measurements in treated cultures showed that bioflm formation was reduced in a largely dose-dependent manner (Figure 4).At 1/2 MIC and 1/32 MIC, gentamicin inhibited bioflm formation by 64% and 13%, respectively (Figure 4), whereas at the MIC of the peptide extract, bioflm formation was inhibited to about 4 Biochemistry Research International 87% (Figure 4).Tis reduced to 42% at 1/4 MIC and dropped to 18% at 1/32 MIC.A high inhibition rate was shown at MIC/2 (62%).

Efect of Crude Peptide on Secretion and Inhibition of
Virulence Factors.Te efect of the peptide extracts in inhibiting quorum sensing (QS) in P. aeruginosa was evaluated in the PAO1 strain.As QS regulates the expression of genes involved in the production and secretion of virulence factors in P. aeruginosa, the ability of the peptide extract in interfering with the production of pyoverdine and pyocyanin by P. aeruginosa was investigated.Again, gentamicin was used as a positive control.Te crude peptide extract demonstrated signifcant anti-QS activity, efectively inhibiting the production of pyoverdine and pyocyanin in a dose-dependent manner at both MIC and sub-MIC concentrations.Te levels of pyocyanin   produced varied inversely with the concentration of the peptide extract or standard gentamicin used (Figure 5).In the absence of any treatment, the average pyocyanin production in the growth control was 12 μg/mL.At the lowest concentration of the extract or drug, pyocyanin production decreased to approximately 7 μg/mL, further reducing to around 1 μg/mL and 2 μg/mL, respectively, for gentamicin and the peptide extract at their respective MIC concentrations.Percentage inhibition analysis revealed that the peptide extracts inhibited pyocyanin production by 81%, 76%, 64%, 53%, and 41% at sub-MIC concentrations of 1/2, 1/4, 1/8, 1/16, and 1/32, respectively.Notably, at 1/16 and 1/ 32 MICs, both the extract and standard gentamicin exhibited similar levels of pyocyanin inhibition.Te corresponding percentage inhibitions are graphically presented in Figure 6.
Regarding pyoverdine, fuorescence emissions demonstrated a dose-dependent relationship, with signifcantly lower fuorescence in the treated groups compared to the growth control (Figure 7).Both gentamicin and the peptide extract exhibited similar inhibitory efects on pyoverdine production at various concentrations.At the MIC, pyoverdine production was maximally inhibited by 65%.Te inhibition of pyoverdine between MIC/2 and MIC/32 remained relatively consistent, ranging between 44% and 25% for both the gentamicin and peptide extract treatments (Figure 8).
Assessing the growth rate of Pseudomonas aeruginosa in the presence of MIC and sub-MIC concentrations of the peptide extract, a marginal decrease was observed compared to the growth control.While the MIC treatment resulted in no bacterial growth, as evidenced by consistent OD measurements over 24 hours, the sub-MIC treatments followed a growth pattern similar to that of the control, albeit at a slower rate during the lag phase.Te impact of the peptide extract on the growth rate of P. aeruginosa was concentration-dependent.

Discussion
Phaseolus vulgaris (Figure 1), an essential legume with its roots in Central and South America, holds great signifcance in human nutrition.It has achieved global dietary prominence due to its nutrient-rich composition and versatility in cooking.Beyond being a protein source, it provides vital vitamins, minerals, and dietary fber, contributing to sustainable agriculture and addressing global nutrition challenges [10,14].Te extraction of antimicrobial peptides (AMPs) can be performed from various parts of the plant.In this study, peptides were extracted from the seeds, as they represent the proteinaceous component of the plant.
To enhance peptide stability and minimize degradation during the extraction process, the pH of the seed slurry was adjusted to 9.0.Tis higher pH value is known to promote peptide stability and reduce the risk of degradation or hydrolysis [21].Precipitates were obtained by further adjusting the pH to 5.0 as the peptides may exhibit reduced solubility due to alterations in intermolecular forces, such as hydrophobic interactions or hydrogen bonding, which impact their solubilization in the surrounding solution [22].Tus, adjusting the pH to 5.0 may induce changes in these intermolecular forces, resulting in decreased peptide solubility and subsequent precipitation.
Following peptide precipitation, the biuret test was used to confrm the presence of peptides in the precipitate.Te biuret test is a chemical assay used to detect the presence of peptide bonds in a substance.It relies on the biuret reaction,   Biochemistry Research International where a substance containing at least two links turns violet when treated with alkaline copper sulfate.In an alkaline solution, a blue-colored Cu 2+ forms a complex with peptide bonds due to unshared electron pairs on nitrogen and oxygen.Tis complexation occurs between Cu 2+ ions and the carbonyl oxygen (>C�O) and amide nitrogen (�NH) of the peptide bond, changing the solution from blue to purple.Te depth of purple indicates the quantity of peptide-copper complexes, and the reaction applies to compounds with at least two H 2 N-C, H 2 N-CH 2 -, H 2 N-CS-, or similar groups directly linked or via a carbon or nitrogen atom.Each copper ion likely forms coordinate bonds with six nearby peptide linkages.Te color intensity correlates with the number of peptide bonds in the reacting protein molecule and the quantity of protein molecules in the system [15].Tus, the precipitate obtained from the seeds of P. vulgaris was found to be peptide-rich.Mim employed the biuret test in determining the presence of amino acids (peptide bonds) in wool and limed hair [23].Valdoz also determined the presence of peptides in coconut and macapuno fruits using the biuret test [24].Te presence of peptides in both samples in each analysis was confrmed by the violet color observed after the analysis.Te extract's FTIR spectrum (Figure 2) exhibited consistent vibrational spectra with reported peptide spectra.Peptides display various vibrational frequencies, with the amide I and amide II bands being prominent in the peptide IR spectrum.Te amide I region, spanning from 1,700 to 1,600 cm −1 , is particularly responsive in detecting secondary structure compositions.It arises from the stretching Biochemistry Research International vibration of the C�O bond in the amide group, coupled with the in-phase bending of the N-H bond and stretching of the C-N bond.Each frequency within this range corresponds to a particular protein structure.Te amide II band, which is more complex than amide I, primarily originates from inplane N-H bending and C-N stretching vibrations.While it is conformationally sensitive, it has been less commonly utilized for protein structure analysis.Other amide bands have limited utility in protein structure analysis due to their complexity and their dependence on factors, such as the force feld, side chain nature, and hydrogen bonding [25].
Te FTIR spectrum of the peptide obtained was consistent with other spectrums reported in literature [3,20,26,27].Te similarities in vibrational stretches observed in both the reported works, and the spectrum obtained from this study confrms that the extract obtained confrms that the extract is peptidic.Peptides possess inherent chromophores that exhibit various responses to UV light [28].Te electronic absorption spectra of peptides are commonly investigated within the ultraviolet range of the electromagnetic spectrum, spanning from 185 nm to 320 nm.An attenuated absorption at 194 nm was detected (Figure 2), indicating the presence of peptide bonds within the extract.Te absorption 194 nm is a characteristic π⟶π * of the peptide backbone, aiding in peptide bond identifcation.Additionally, a prominent absorption peak at 203 nm, corresponding to peptide bonds, was also observed.Te peak at 203 nm corresponds to 8 Biochemistry Research International a n⟶π * transition of the peptide bond, providing a distinct spectral marker for peptide bond identifcation [11,29,30].Similar absorptions at 194 nm and 240 nm of peptide extract were reported by Gasu and coworkers [26], indicating the presence of peptides in the extract.Again, Wang and his research team observed UV absorptions at 191 nm in a solution of oyster protein hydrolysate and deduced that these absorptions originated from the linkages present within the hydrolysate [31].Antimicrobial peptides (AMPs) are often efective against a wide range of microorganisms and are considered a potential solution to combat antimicrobial resistance.Since AMPs primarily target cell membranes, any resistance from microorganisms would likely require signifcant and costly modifcations to their entire lipid membrane structure.Consequently, AMPs ofer a promising therapeutic choice as such modifcations would be challenging for microorganisms to achieve [32].Te susceptibility of the microorganisms was evaluated using the broth microdilution method, which is considered more sensitive compared to other assays for screening antimicrobial natural products [16,33].A total of fve microbial strains, consisting of two Gram-positive bacteria, two Gram-negative bacteria, and a fungus, were examined.Te MICs at which the tested organisms are inhibited and the distributions of MICs for these organisms are summarized in Table 1.Peptide extract obtained from P. vulgaris displayed antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as a fungus.Te extract showed low MIC values for all tested organisms, particularly against C. albicans and P. aeruginosa, indicating its broad-spectrum antimicrobial activity [33].In general, the MIC values recorded are much lower than those recorded for peptide extracts from Olivancillaria hiatula [20], Patella rustica, and Galatea paradoxa [34], as well as peptide extracts from an Australian plant mixture [35].Tese results are consistent with the research conducted by Sarnthima and Khanmmuang [28], who also reported signifcant MIC values when testing S. stramoniifolium seed extracts against P. aeruginosa.Furthermore, the MIC values obtained in this study fall within the same range as those recorded for pexiganan, an antimicrobial peptide that has reached advanced stages of clinical trials for treating diabetic foot ulcers [36,37].
Te antimicrobial activity of the peptide extracts from P. vulgaris can be attributed to their capacity to disrupt bacterial membranes by interacting with lipid molecules on the cell surface of the tested microorganisms, as is observed for many AMPs [12,26].Tis interaction is facilitated by the linear α-helical structure present in the peptide extract.Antimicrobial activity has been shown in several cases to correlate closely with an increase in α-helical secondary structure [38].Tis relationship was speculated from the IR data.Antimicrobial peptides (AMPs) employ diverse mechanisms to disrupt cell membranes, making them effective against a wide range of pathogens.Teir amphipathic structure allows them to interact with microbial membranes selectively.AMPs can insert themselves into the lipid bilayer, creating pores and increasing membrane permeability.Tis disrupts the electrochemical balance, leading to ion leakage and cell death [39].Some AMPs target specifc membrane regions, such as lipid rafts or lipopolysaccharides in Gramnegative bacteria, weakening structural integrity.Additionally, certain AMPs induce the generation of reactive oxygen species within microbial cells, causing damage to lipids, proteins, and DNA.Importantly, AMPs often exhibit selectivity for microbial membranes due to diferences in composition compared to mammalian cell membranes, minimizing harm to host cells while efectively combating pathogens [40,41].
Te inhibitory efects of the peptide extract on various bacteria were evaluated by determining the minimum bactericidal concentration (MBC), as shown in Table 1.MBC represents the lowest concentration of the extract that kills 99.9% of the bacterial inoculants after 24-hour incubation at 37 °C.Determining the MBC or MFC and calculating the MBC or MFC to MIC ratio provide an avenue for gaining a comprehensive understanding of the antimicrobial's effectiveness against a specifc pathogen.Tus, to characterize the antimicrobial activity, the MBC to MIC ratio was calculated.A ratio of MBC/MIC ≤2 indicates a bactericidal efect, while a ratio ≥4 suggests a bacteriostatic efect.In our study, the peptide extract exhibited a bacteriostatic efect at the MIC for all microorganisms tested.However, above the MIC, a bactericidal efect was observed.Te concentration of antimicrobial peptides plays a crucial role in their activity.At lower peptide : lipid ratios, cationic antimicrobial peptides remain associated with the membrane, aligned parallel to the lipid bilayer interface.As the peptide : lipid ratio increases, peptides can aggregate or reorient within the membrane, leading to membrane disruption and microbial death.Processes, such as ion channel formation, transmembrane pore formation, and membrane rupture, are more prominent at higher peptide concentrations [42].
Te World Health Organization designates antibioticresistant P. aeruginosa as a top-priority pathogen due to its versatility and adaptability, enabling it to cause various infectious diseases.Pseudomonas aeruginosa stands out among other bacterial strains due to its distinct characteristics such as its ability to form bioflms, utilize quorum sensing, and employ efux pumps among others, all of which enhances its pathogenic potential [43].Bioflm-forming bacteria generally display broad-spectrum resistance to various antimicrobial agents and thus make it more challenging to eliminate bioflm-associated infections [32].It is well documented that quorum sensing and bioflm formation are integral processes that modulate the social behavior of bacterial [44].Bioflm formation in the Gram-negative microbe P. aeruginosa is under the control of the regulatory genes of quorum sensing (QS), and compounds that possess antiquorum sensing properties are routinely examined as prospective antibioflm agents.Tese compounds are thought to interfere in the QS process and therefore inhibit the production of various virulence factors.Some commercially available anti-QS compounds have recently been shown to enhance the susceptibility of bacterial bioflm to various antimicrobial agents, both in in vitro and in vivo experiments [45].Several AMPs have demonstrated signifcant potential as prospective treatments in the Biochemistry Research International management of infectious diseases, with a number of them currently undergoing advanced stages of clinical trials [19,46].Te strain of P. aeruginosa utilized in this investigation exhibited a remarkable ability to form bioflms. Te production of pyoverdine and pyocyanin (both virulence factors) plays a crucial role in QS, a process that triggers bioflm formation.For QS research, the reference strain P. aeruginosa PA01 is renowned for its capacity to produce and pyocyanin when exposed to external AHL and is extensively employed.
Te peptide extract derived from P. vulgaris was assessed for its ability to inhibit the growth of P. aeruginosa, considering its antimicrobial properties.Te MIC (1.25 mg/mL) of the peptide extract was determined to completely hinder bacterial growth, consequently preventing the formation of bioflms and the expression of virulent factors.Considering bioflm formation and production of virulence factors are dependent on bacteria quorum size, it was crucial to demonstrate that sub-MIC (0.625, 0.313, 0.156, 0.078, and 0.039) mg/mL concentrations of peptides did not hinder bacterial growth.Tus, the growth kinetics of bacteria were monitored by measuring the OD 600 .Te absorption levels at OD 600 showed no signifcant diference between the untreated group (control) and the cells treated with sub-MIC concentrations of peptides.While the rate of growth varied, the OD600 after 24 hours demonstrated that bacterial growth was unafected (Figure 9) and quorum sizes could be reached.Tis suggests that any efect on any of the processes tested is due to the peptide extract interference in important cellular processes, rather than bacterial growth inhibition.
Te impact of sub-MIC doses of the peptide mixture on the modulation of bioflm formation in P. aeruginosa was assessed, resulting in a bioflm inhibition rate ranging from 18% to 62%.Analysis of the data indicated that peptide concentrations within the range of the MIC, 1/2 MIC, and 1/ 4 MIC are necessary to achieve approximately 50% bioflm inhibition.It has been proposed that antibioflm peptides operate by preventing microbial adhesion to surfaces, eliminating early surface colonizers, targeting preexisting bioflm-associated cells, and interfering with the microbe's quorum sensing mechanisms [3].It is therefore likely that the peptide mixture derived from P. vulgaris might disrupt QS in P. aeruginosa, leading to its antibioflm efects.
As quorum sensing (QS) in P. aeruginosa plays a role in regulating the expression of virulence factors like pyocyanin and pyoverdine, the peptide extract is expected to interfere with the production and expression of these harmful factors.Consequently, we examined the levels of pyocyanin and pyoverdine in the presence and absence of sub-MIC concentrations of the peptide extract.Te presence of the peptide extract led to a reduction in pyoverdine levels, with inhibitory efects showing a clear dose-dependent pattern.At 1/2 MIC, pyoverdine production was inhibited by more than 44%.Similarly, pyocyanin production was inhibited by 81% at 1/2 MIC concentration.Tese fndings demonstrate that the peptide mixture derived from P. vulgaris does indeed impede the production of virulence factors, using the same mechanism employed for bioflm inhibition.Te probable mechanism of inhibition by the crude extract is interfering with the membrane of the bacteria by perturbing it.Such peptides have a strong afnity for the lipopolysaccharide groups that are the major components of bacterial cell membrane, and this leads to pore formation within the membrane, ultimately exposing intracellular organelles into the extracellular environment.On the other hand, bioflm inhibition is caused by the crude extract binding to the receptors responsible for bioflm formation, such as PqsR and RhlR [9,26].Pyoverdine, a fundamental siderophore produced by P. aeruginosa, serves the dual function of sequestering iron from host depots and acting as a QS signaling molecule.When pyoverdine is bound to iron, it interacts with the P. aeruginosa cell receptor FpvA, forming a complex that subsequently interacts with the antisigma factor FpvR. Tis interaction leads to the upregulation of exotoxin A, an endoprotease, as well as the upregulation of pyoverdine itself.Pyocyanin, on the other hand, induces oxidative stress in the host and stimulates the secretion of airway mucus [3,19,47].All these virulent factors, including pyocyanin and pyoverdine, play crucial roles in the development and maintenance of bioflms, signifcantly contributing to the destructive nature of P. aeruginosa infections.
Te peptide extract derived from P. vulgaris exhibits the ability to hinder the formation of bioflms in P. aeruginosa and also suppresses the expression of virulence factors.Tis indicates that the extract's mode of action involves targeting a shared factor involved in these processes.Since quorum sensing (QS) governs all these activities in P. aeruginosa, it is likely that the peptide extract interferes with cell-to-cell communication mediated by QS.Tere are very few drugs available that can efectively inhibit both quorum sensing and bioflm formation.Te discovery of compounds and extracts that can perform these dual functions reduces the likelihood of antimicrobial resistance and ofers 10 Biochemistry Research International a convenient approach for controlling pathogenic microorganisms.Te promising results demonstrated by the peptide mixture from P. vulgaris in this regard present an opportunity for the development of innovative therapeutics that specifcally target pathogenic bacteria.

Conclusion
In summary, the study fndings indicate that the peptide extract obtained from P. vulgaris exhibits potent antimicrobial activity with a broad spectrum.At the minimum inhibitory concentrations (MICs), the extract demonstrates bacteriostatic efects.Furthermore, the study demonstrates the ability of the crude peptide extract to inhibit bioflm formation suppress the expression of virulence factors, such as pyocyanin and pyoverdine, in P. aeruginosa.Tese results suggest that the crude peptide extract from P. vulgaris has the potential to serve as a valuable source for the development of new antimicrobial agents.With this, novel peptides and peptidomimetics can be isolated and design using isolated peptides from P. vulgaris crude.Other applications include the design of nanoparticles with efective antimicrobial activity.

Figure 2 :
Figure2: Fourier transform infrared (FTIR) spectrum of peptide extract of Phaseolus vulgaris.Amides A and B bands span 3100-3500 cm −1 , Amide I band is from 1600 to 1700 cm −1 , Amide II band is from 1480 to 1600 cm −1 , and the region from 500 to 1300 cm −1 represents Amides III-VI bands.

Figure 4 :
Figure 4: Antibioflm efect of crude peptide and gentamicin on P. aeruginosa.Mean values of three independent experiments and their standard deviations are shown.When compared with the untreated wells (GC), no signifcant diference was observed.

Figure 5 :Figure 6 :
Figure 5: Pyocyanin inhibition.Relative fuorescence of pyocyanin secreted by Pseudomonas aeruginosa with and without sub-MIC doses of the peptide extract and standard drug, gentamicin.Each bar represents the mean ± SD of fuorescence intensities of three independent.

Figure 7 :Figure 8 :
Figure 7: Percentage inhibition of pyoverdine secretion in Pseudomonas aeruginosa in the presence of sub-MIC doses of the crude peptides and gentamicin.Percentage inhibitions were computed with respect to the fuorescence of the control group.Each bar represents mean ± SD of triplicate experiments.

Figure 9 :
Figure 9: Growth curve of Pseudomonas aeruginosa in the absence (GC) and presence of varying concentrations of crude peptide (MIC, 1/32 MIC).
An overnight culture of P. aeruginosa (50 μL) was inoculated into 2 mL of nutrient broth in the absence (growth control) and presence of sub-MIC (1/2 MIC, 1/4 MIC, 1/8 MIC, 1/16 MIC, and 1/32 MIC) doses of gentamicin and peptide extract (2 mL) and incubated for 24 hours at 37 °C.After incubation, the culture media was then centrifuged at 5000 rpm for 45 minutes.A 96-well microtiter plate was prepared by flling each well with 100 μL of the cell-free supernatant for pyoverdine measurement.Te relative concentration of pyoverdine in all treated supernatants with respect to control (growth control) was measured by fuorescence (BioTek ® Synergy H1 ) 2.6.Inhibition of Quorum Sensing in P. aeruginosa 2.6.1.Pyoverdine Quantifcation.