Exploring the Antimicrobial and Anticancer Potential of Pyocyanin Produced by Pseudomonas aeruginosa Strain ONO14782

doi:10.21203/rs.3.rs-3996369/v1

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Exploring the Antimicrobial and Anticancer Potential of Pyocyanin Produced by Pseudomonas aeruginosa Strain ONO14782

https://doi.org/10.21203/rs.3.rs-3996369/v1

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Background

Pseudomonas aeruginosa is a clinically and epidemiologically important bacterium that causes both acute and chronic infections. The pathogenesis of P. aeruginosa depends on the virulence factors, The important virulence factors of P. aeruginosa included biofilm formation, pigment (pyocyanin), cytotoxins, phospholipases, elastases, and proteases. Pyocyanin is a chloroform soluble blue-green pigment produced by P. aeruginosa, has an antibacterial activity against a wide range of drug-resistant bacteria and pathogenic bacteria, also it was exhibited antifungal activity against different species of mycotoxigenic fungi. and we can use it as an anticancer agent is advised .

Methods

In this study, 46 out of 66 P. aeruginosa isolates were selected based on their pigmentation on cetrimide agar. Pseudomonas isolates were collected from urinary tract infection (urine), burned skin infection and diabetic foot wound (pus) and respiratory infections (sputum). Pseudomonas Cetrimide Agar was used as selective media. TLC technique was used for purification, while UV-Vis, FTIR and GC-MS techniques analysis were used for characterization properties for P73 (ONO14782) strain. Anticancerous effect has been determined by MTT assay established against HepG2, MCF-7 and HCT-116 cell lines.

Results

46 pigmented isolates were selected from 66 isolates. TLC plates showed a blue color in visible light with R_f = 0.81 for pyocyanin. A P73 (ONO14782) strain was used as an experimental strain to study the role of antimicrobial activity of pure pyocyanin, revealing resistance of Klebsiella pneumoniae and Staphylococcus aureus, with Escherichia coli showing intermediate sensitivity. Additionally, pyocyanin demonstrated antifungal efficacy against various yeast and fungi. Furthermore, pyocyanin showed promising anticancer activity against tested cancer cell lines, with strain P73 (ONO14782) displaying activity against HepG2, MCF-7, HCT-116, and A-549 cell lines.

Conclusions

The objective of this study is extraction and purification of pyocyanin from local clinical isolates and choose high productivity strain of pyocyanin studying characterization properties produced from P73 ONO14782 strain and studying its antimicrobial and anticancerous effect. Pyocyanin showed a very high cytotoxic effect on cancerous cell lines leads to reduction in viability of these cells.

Pyocyanin

Extraction

Purification

Antimicrobial activity

Anticancer

Pseudomonas aeruginosa is an aerobic, rod-shaped, gram-negative, opportunistic pathogenic bacteria that grows in both normal and hypoxic air conditions. Its dimensions are 0.5–0.8 µm in width and 1.5–3.0 µm in length [1–2]. According to Green and Crone, its habitat is quite diverse and includes soil, water, people, animals, plants, sewage, and hospital environments [3–4]. P. aeruginosa can infect immunocompromised and immune-deficient hosts' respiratory and urinary tracts [5–6]. Nevertheless, P. aeruginosa is one of the most useful microbes for biotechnology and commerce, even if it is a harmful bacterium [7].

Multi-drug resistant (MDR) P. aeruginosa can cause acute or persistent infection in immunocompromised patients suffering from cancer, cystic fibrosis, traumatic brain injuries, burns, sepsis, and ventilator-associated pneumonia (VAP), including COVID-19-related cases [8–9].

A vast range of colors are produced by P. aeruginosa as secondary metabolites. Pyocyanin, fluorescein, pyorubrin, and pyomelanin are the four primary pigments of redox-active phenazine chemicals that are generated by P. aeruginosa [10]. P. aeruginosa produces the blue-green pigment known as pyocyanin [11–12], which is soluble in chloroform. A pigment that dissolves in water and has strong siderophore action is fluorescein.Pyorubrin is employed as an antibacterial agent; with low oxygen concentration, it permanently reduces to a colorless form [13]. Aeruginosin A and B, water-soluble red pigments with multiple biological functions generated by certain P. aeruginosa strains, also exhibited a notable phenazine group [14]. One efficient method for identifying P. aeruginosa infections in patients is pyocyanin detection [15].

Pyocyanin has shown antioxidant and anticancer properties against cancer cell lines, pyocyanin has demonstrated antibacterial and antifungal action against numerous pathogenic bacteria and mycotoxigenic fungi Consequently, these activities find application in the food sectors, including medicinal applications and food preservation [10–16]. Pyocyanin has demonstrated encouraging potential as an anticancer drug due to its capacity to impose lethal effects on tumor cells. Pyocyanin pigment has antitumor bioactivity because it can produce reactive oxygen species. Additionally, it has been found to possess antioxidant and radical scavenging capabilities at low concentrations [17–18]. Consequently, Pyocyanin is a pigment with a wide range of potential uses in the fields of pharmaceuticals, food, medicine, biocontrol, nanotechnology, textiles, and phytochemistry. Pyocyanin is a very promising material in many different domains because of its adaptability and multifunctionality [19].

Isolation and identification of P. aeruginosa from clinical samples

Pseudomonas Cetrimide Agar was utilized as a selective medium to isolate P. aeruginosa from a variety of clinical samples (urine, sputum, and pus). Initially, 46 isolates were chosen from 66 initial isolates based on visually striking coloration for additional investigation. Samples were cultivated on cetrimide agar using the streak plate method. These isolates were recognized as P. aeruginosa by comparing their microscopic features, physiological, and biochemical properties with the standard description provided in the eightieth edition of Bergey's Manual of Determinative Bacteriology [20–21] .

Extraction and purification of the pigment produced by Pseudomonas isolates

To extract pigment, the chosen Pseudomonas isolates were cultivated in King's medium broth and shaking (200 rpm) for two to three days at 37 ºC. The creation of pigment is indicated by the color pigment shifting to a blue-greenish colour [22]. After cooling centrifuging the green cultures for 15 minutes at 10,000 rpm, 3 mL of chloroform was added to the cell-free filtrate, and the mixture was centrifuged again at 10,000 rpm. The blue chloroformic layer was moved to a new tube that held one milliliter of 0.2 N HCl. mixed until the aqueous layer started to turn pink, and then centrifuged one more. To keep the pink solution's blue-green hue, 0.1M NaOH was added before chloroform was added once again. Pyocyanin powder was prepared by evaporating the chloroform [22].

Using methanol and chloroform in a 1:1 ratio as the mobile phase, TLC was used to purify the extracted pigment. The Rf value of the partially purified pigment was also measured. In addition, 0.1 N NaOH was applied to the percentage of the eluted material. A UV-visible spectrophotometer was used to examine extracted pigment solutions from the 46 P. aeruginosa isolates [23–24]. Following the extraction process, the concentration (µgmL^− 1) of the pigment in the extracted solution was calculated by measuring the optical density (absorbance) at 520 nm wavelength using a UV-visible spectrophotometer (Spectronic Molton Roy Co., 20 D) and multiplying the optical density value (OD520) with 17.072 [25–26]. Maximum absorbance was recorded using a Spectronic Molton Roy Co., 20 D recording spectrophotometer, and maximum absorbance was recorded by this device.

Molecular identification of the most active isolates for pyocyanin-producing ability by 16S rDNA sequencing

By analyzing 16S rRNA gene sequences with extracted DNA in the presence of universal primers 5'-GGGGATCTTCGGACCTCA-3' and 5'-TCCTTAGAGTGCCCACCCG − 3', which are intended to amplify a 956 base pair fragment of the 16S rDNA region reverse that has previously been used [27], the identification of the three most active isolates for pyocyanin production bacterial was confirmed. Microseq PCR & Microseq Cycle Sequencing (Applied Biosystem); Prep Man Ultra (Applied Biosystem) were used for the PCR-mediated amplification of the 16S rDNA and purification.

Initially, the DNA was denaturated at 95°C for five minutes. This was followed by 34 cycles of 95°C for thirty seconds, 55°C for thirty seconds, and 72°C for forty-five seconds. The mixture was then stored for ten minutes at 72°C to allow for full extension, and then it was kept at 4°C until it was purified using the QIAquick Gel Extraction Kit (QIAGEN, USA) [28]. The identification process involved utilizing the BLAST tool (National Centre for Biotechnology Information) to compare the contiguous 16S rDNA sequence with the information found in GenBank databases [29], the strain sequencing was entered into GenBank and assigned an accession number.

Characterization of Pyocyanin produced by P73 (ONO14782) strain

UV-Vis spectrophotometer

Spectroscopic examination was performed on the pure pyocyanin. Over a wavelength range of 200–800 nm, the purified pyocyanin dissolved in chloroform and 0.1N HCl was subjected to ultraviolet and visible absorption spectra analysis. UV spectrophotometric analysis was used to identify absorbance maxima using a UV spectrophotometer (Shimadzu 1601, Japan) [30].

Fourier Transform Infra-Red Spectroscopy (FTIR)

The functional groups and chemical bonds in the current molecule were investigated using FTIR. The National Research Center's Jasco FTIR Spectrophotometer (FTIR-6100 Jasco, Japan) was used for the analysis. The sample was made by evenly dispersing one milligram of pyocyanin extract into potassium bromide pellets (Merck, USA). An integrated plotter was used to acquire IR absorption spectra. IR spectra were obtained at a resolution of 4 cm^− 1, spanning the 400–4000 cm^− 1 range. To understand the chemical makeup of the bioactive substance, the spectrum was examined [31].

Gas chromatoghrophy – Mass spectrometery (GC/MS) Analysis

A Thermo Scientific Trace GC Ultra / ISQ Single Quadrupole MS, TG-5MS fused silica capillary column (30 m, 0.251 mm, 0.1 mm film thickness) was utilized to conduct the GC/MS analysis. Helium gas served as the carrier gas for the GC/MS detection system, which employed an electron ionization system with an ionization energy of 70 eV and a steady flow rate of 1 mL min^− 1. The temperature of the MS transfer line and injector was fixed to 280 ºC. The oven is heating up by 7ºC every minute.

The temperature will then rise to 270 ºC at a rate of 5ºC min^− 1 (hold for 2 min), and finally, it will rise to 310 ºC as the final temperature at a rate of 3.5ºC min^− 1 (hold for 10 min). A percent relative peak area was used to evaluate the quantification of all the components that were discovered. By comparing the compounds' respective retention times and mass spectra with those of the NIST, WILLY library data of the GC/MS instrument, a tentative identification of the compounds was carried out [32–33].

Primary screening for the isolates having antimicrobial activity

Researchers tested the pyocyanin extract's inhibitory potential on pathogenic bacterial isolates. Five Gram-negative bacteria (Salmonella typhi, Shigella sp, Listeria sp, Escherichia coli, and Klebsiella pneumoniae) and two Gram-positive bacteria (Bacillus cereus, Staphylococcus aureus) [34], the stock cultures were cultivated on nutrient-agar slants at 37ºC for 24 hours before being refrigerated until needed. For the antifungal assay, Aspergillus flavus and Aspergillus niger were the two fungal isolates that were utilized. After growing for five days at 25ºC on potato dextrose agar slant, the stock cultures were refrigerated until needed. On yeast extract peptone dextrose (YEPD) agar slants, Candida tropicalis and Candida albican were grown for 48 days at 25ºC. The test organisms were then stored in the refrigerator until needed, as they would be a good indicator of the antimicrobial activity of pseumonads [35].

Determination of antimicrobial activity of pyocyanin on bacterial and fungal isolates

Using the agar well diffusion method, the antibacterial activity of the high yielding pyocyanin generated by the chosen strain p73 (ONO14782) against various bacterial and fungal isolates was examined [36]. To assess the antibacterial activity, several concentrations of the pure pigment in water were prepared: 25, 50, 75, and 100 µgmL^− 1.

Prior to pouring the prepared media into the Petri plate, solidifying it, and creating wells in the agar medium, the test organism (OD corresponding to 0.5 McFarland scale) was planted into it. Next, 100 µL of each produced pigment solution were added to various agar plate wells and incubated at 37ºC for a whole day. To ascertain the test agent's antibacterial activity, the diameter of the zone of growth inhibition was determined after incubation.

Every experiment was conducted three times. Positive reference standards for the susceptibility experiments were antibacterial and antifungal antibiotic discs (HiMedia Chemicals Pvt., Ltd., Mumbai, India) that belonged to various classes based on their structural composition. The antifungals comprised amphotericin B, fluconazole, itraconazole, ketoconazole, and nystatin; the antibiotics included cefotaxime, cefpodoxime, Augmantin, Gentamycin, and Amoxycillin. As previously mentioned, the discs were aseptically placed on to inoculated agar plates and the incubation conditions were carried out.

Statistics: The data were expressed as mean ± SD. Using the mini tab program, a one-way analysis of variance (ANOVA) was applied to all the data. If P was less than 0.05, the differences were deemed significant.

Anti-tumor activity of pyocyanin

After being treated with pyocyanin at various concentrations to demonstrate its cytotoxic effect, the MTT Assay was used to evaluate the cell viability of several cell lines, including: Hepatocellular carcinoma (HEPG-2), adenocarcinomic human lung (A-549), colon cancer (HCT-116), and human breast cancer (MCF-7). The reduction of yellow 3-(4,5-dimethythiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase is measured in this colorimetric experiment.

Once the MTT enters the mitochondria, succinate dehydrogenase reduces it to formazan, an insoluble substance with a dark purple color. A flat bottom 96-well microplate was used to plate cells (0.5X105 cells/well) in serum-free medium. The cells were then treated with 20 µL of various dilutions of the tested sample for 48 hours at 37º C in a humidified 5% CO₂ atmosphere. Following incubation, the media were taken out and 40 µl of MTT solution per well were added. Four more hours were then spent incubating.

Following the solubilization of MTT crystals with 180 µL of acidified isopropanol well and plate shacking at room temperature, the absorbance at 570 nm was measured photometrically using a microplate ELISA reader. For every dilution, three duplicate repeats were made, and the average was determined. The proportion of relative viability between the vehicle control and untreated cells was used to express the data, with cytotoxicity being indicated by less than 100% relative viability. (A treated sample/A untreated sample) x 100 was the formula used to determine the relative cell viability [37].

Isolation and identification of P. aeruginosa from clinical samples

Upon culturing clinical specimens on selective medium Pseudomonas Cetrimide Agar, two distinct forms of colony morphology were observed: pigmented colonies (69.7%) and non-pigmented colonies (30.3%). Among these, 46 isolates exhibiting similar pigmentation characteristics were initially identified as P. aeruginosa. The distribution percentage of these isolates originating from pus burnt wound infections and diabetic foot ulcers was found to be 73.9%. Additionally, sputum respiratory tract infections accounted for 15.22% of isolates, while urine urinary tract infections comprised 10.87%. (Table 1).

Table 1

The distribution percentage of pigmented *P. aeruginosa* from urine, pus and sputum samples
Source of isolation	Pigmented P.aeruginosa isolates
Source of isolation	Number	%
Urine [ urinary tract infection - urethral catheters ]	5	10.87
Pus [ pus burned infection- diabetic foot wound ]	34	73.91
Sputum [ respiratory tract infection ]	7	15.22
Total pigmented	46	100

Extraction and purification of the pigment produced by Pseudomonas isolates

The extract's TLC examination matches the results of the column chromatography. A blue spot with Rf value ranging from 0.70 to 0.81 was observed for the pigment extracted from all the isolates which was characterized as phenazine compound such as Pyocyanin as shown in Fig. (1). The high productivity of pyocyanin recorded by P. aeruginosa ONO14782 strain that isolated from (pus burned wound) recorded 13.5 µg mL^− 1, P. aeruginosa ONO14778 isolated from (diabetic foot ulcer) was identified as 12.2 µg mL^− 1 and P. aeruginosa ONO14780 recorded as 10.2 µgmL^− 1 isolated from urinary tract infection respectively.

Molecular Characterization of Selected Bacterial Isolates:

Through 16S rRNA gene sequencing, the identity of the three specific P. aeruginosa isolates (P73, U51, and p51), exhibiting high pyocyanin production, was confirmed as P. aeruginosa. Subsequently, the sequences were submitted to GenBank with accession codes ONO14782, ONO4780, and ONO14778, respectively, via the NCBI website (www.ncbi.nlm.nih.gov). Polymerase chain reaction (PCR) was utilized with universal primers designed to amplify a 956-base pair segment of the 16S rDNA region, as depicted in Fig. (2).

Characterization of pyocyanin produced by P73 (ONO14782) strain.

UV-visible spectrophotometric analysis

The absorbance spectra of the pyocyanin isolated from P 73 (ONO14782) with the highest productivity was measured using a UV-Vis spectrophotometer in the 200–800 nm range. For pure pyocyanin, two peaks were observed at wavelengths of 368 nm and 687 nm respectively. The 368 nm peak indicated the pyocyanin compound's existence as in Fig. (3).

FTIR analysis

The molecular fingerprint of the material is created by the molecule absorption and transmission that FTIR provides [31]. The side chains of the phenazine-characterized Pyocyanin molecule were shown in Fig. (4). The spectra indicates the presence of an O-H bond at 3429 cm^− 1. The C = N bond was represented by the peak at 1631 cm^− 1, the C-O bond by the peak at 1250 cm^− 1, and the C-H aromatic bond by the other peak, which was measured at 2367 cm^− 1.

GC-MS analysis

The analysis of the tested Pyocyanin revealed that, as shown in Fig. (5) it has a molecular weight of 206 Dalton and a retention period of 52 minutes.

Antimicrobial activity of Pyocyanin

The agar well diffusion experiment was used to screen eleven bacteria strains for sensitivity to the antibiotic activity of pyocyanin (Table 2). Different bacterial strains responded differently to the antibacterial action of pyocyanin. The most pyocyanin-sensitive bacterial strain was Staphylococcus aureus, which was followed by Shigella sp, B. cereus, Salmonella typhi, and E. coli. On the other hand, K. pneumonia responded negatively to pyocyanin. A similar mean diameter of inhibitory zone (16–25 mm at 100 µgmL^− 1) was seen with A. niger, A. flavus, Candida albicans, and Candida tropicalis when the pyocyanin activity was tested on fungi. On the other hand, A. niger had the highest pyocyanin sensitivity, registering a 25 mm inhibitory zone. Additional data revealed that the production of pyocyanin activity occurred at high concentrations (75 and 100 µgmL^− 1) and that there were significant variations (P < 0.05) in the growth inhibition zone widths between the first and second concentrations.

Table 2

Antimicrobial activity of pyocyanin against various pathogenic microorganisms
Microorganism	Inhibition zone (100 µL)	Inhibition zone (75 µL)	Inhibition zone (50 µL)	Inhibition zone (25 µL)
Staphylococcus aureus	25^*ab±0.5	21^{* ab±0.57}	18^{* b±0.28}	0.00
Bacillus cerus	23^*c±0.4	19^{* cd±0.3}	16.5^*bc±0.25	0.00
Escherichia.coli	20^*d±0.5	18^{* d±0.2}	16 ^*c±0.19	0.00
Shigella sp.	24^*ab±0.5	21^{* ab±0.4}	18 ^*a±0.29	0.00
Salmonella typhi	22^*±0.6	19 ^{* a±0.6}	16^{* c±0.3}	0.00
Kleblisella Pneumoniae	0.00	0.00	0.00	0.00
Listeria sp.	16^*e±0.3	13^{* e±0.5}	0.00	0.00
Candida albican	20^*e±0.6	13^{* e ±0.2}	0.00	0.00
Candida tropicalis	16^*e±0.3	14^{* e ±0.31}	0.00	0.00
Aspergillus niger	25 ^*ab±0.3	22^{* a±0.6}	18 ^*±0.19	14 ^*a±0.55
Aspergillus flavus	23 ^*bc±0.2	20^*bc±0.5	17^{* b±0.28}	0.00

Calculated mean is for triplicate measurements from two independent experiments ± SD, ^a−e means different superscripts in the same column are considered statistically different (LSD test, P ≤ 0.05).

Impact of Various Antibiotics on Pathogenic Bacteria and Fungi

To evaluate the resistance of the bacterial strains, several antibacterial agents including cefpodoxime (CPO, 10 mcg), cefotaxime (CTX, 10 mcg), amoxicillin (A, 25 mcg), and gentamycin (Gm, 30 mcg) were utilized as positive reference standards in the study. Table (3) illustrates the diameter of inhibition zones observed against these commonly used antibiotics. Among the antibiotics tested, gentamycin exhibited the broadest spectrum of activity, closely followed by CTX. CPO displayed a moderate range of effectiveness, while amoxicillin showed acceptable antibacterial activity. On the other hand, AUG demonstrated the least efficacy against the tested microorganisms.

In assessing the sensitivity of the fungal strains, various antifungals including itraconazole (IT, 10 mcg), nystatin (N, 100 U), ketoconazole (KT, 10 mcg), amphotericin-B (AMP, 100 U), and fluconazole (FU, 10 mcg) were employed as positive reference standards. Table (4) presents the diameter of inhibition zones observed against these antifungal agents. Among them, amphotericin-B exhibited the broadest spectrum of activity against the fungal strains. However, fluconazole showed inactivity against certain fungal strains. AMP, KT, and NS demonstrated moderate levels of activity. The increasing prevalence of antibiotic resistance has led to a growing interest in natural antibacterial compounds.

Table 3

Antibiotic resistance of tested bacteria species
Tested Bacteria	Inhibition zone diameter (mm)
Tested Bacteria	Gm (10 mcg)	AUG (5 mcg)	A (25 mcg)	CPO (30 mcg)	CTX (30 mcg)
Staphylococcus aureus	18^{* b ±0.5}	21^{* b ±0.6}	17^{* c ±0.3}	0.00	0.00
Bacillus cereus	15^{* c±0.5}	0.00	0.00	13^{* b±0.5}	18^{* a±0.28}
Kleblisella Pneumoniae	0.00	0.00	0.00	0.00	0.00
Escherichia.coli	18^{* b±0.7}	0.0	21^{* a±0.28}	18^{* a±0.27}	0.00
Salmonella typhi	0.00	20^{* b±0.6}	18^{* b±0.25}	0..00	15^{* b±0.57}
Shigella spp.	22^{* b±0.4}	0.00	0.00	13^{* b±0.28}	18^{* a±0.28}
Listeria spp	15^{* c±0.5}	0.00	0.00	13^{* a±0.2}	15^{* b±0.54}

Calculated mean is for triplicate measurements from two independent experiments ± SD, ^{a− c} means with different superscripts in the same column are considered statistically different (LSD test, P ≤ 0.05).

Table 4

Effect of different antifungal on pathogenic sensitive fungi
Organism	Diameter of inhibition zone (mm)
Organism	IT (10 mcg)	NS (100 mcg)	KT (10mcg)	AMP (100mcg)	FU (10 mcg)
A. niger	13.33^{*b ± 0.145}	10.8^{*c ± 0.115}	10.9^{*c ± 0.153}	11.5^{*bc ± 0.203}	0.00
A. flauvs	12.1^{*bc ± 0.361}	10.3^{*c ± 0.145}	21.3^{*a ± 0.208}	0.00	0.00
C .trobicalis	12.5^{*c ± 0.145}	0.00	0.00	12.7*^{b ± 0.173}	0.00
C. albicans	14.3^{*a ± 0.173}	15.3^{*a ± 0.173}	12.3^{*b ± 0.173}	11.3^{*a ± 0.173}	0.00

Anti-tumor activity of Pyocyanin

The effect of pyocyanin on the growth of four different cell lines, namely HepG2 (human liver cancer cell line), MCF-7 (human breast cancer cell line), A-549 (adenocarcinomic human lung cell line), and HCT-116 (human colon cancer), was evaluated using the MTT colorimetric assay. After a 48-hour incubation period, the cell lines were treated with varying concentrations of pyocyanin ranging from 0 to 100 µgmL^− 1. The inhibitory effect of pyocyanin on the growth of each cell line is depicted in Fig. (6). HepG2 cells exhibited the highest sensitivity to pyocyanin treatment, displaying a significant reduction in proliferation with an IC50 of 12.5 µgmL^− 1. Similarly, the MCF-7 cell line showed responsiveness to pyocyanin, with an estimated IC50 of 14.33 µgmL^− 1. In contrast, the IC50 values for HCT-116 and A-549 cell lines were higher, at 31.2 µgmL^− 1 and 35 µgmL^− 1 of pyocyanin, respectively. Thus, the sensitivity of cancer cell lines to pyocyanin can be ranked as follows: HepG2 > MCF-7 > HCT-116 > A-549.

The anticancer potential of pyocyanin against these cell lines—HepG2, MCF-7, A-549, and HCT-116 was assessed using the MTT colorimetric assay.

The findings of this study shed light on the potential of pyocyanin, a secondary metabolite produced by P. aeruginosa, as an antimicrobial and anticancer agent. The emergence of a blue-greenish pigment in 46 isolates was observed, consistent with previous studies [38]. Microscopic examination and biochemical analyses confirmed their identity. Our findings align with research by Jameel et al, revealing that the majority of isolates (30%) originated from ear infections, followed by wounds (22%), burns (17%), urine (13%), diabetic foot ulcers, and stools (9%) [39]. This pattern is consistent with the findings Shouman et al who reported that out of 125 clinical isolates of P. aeruginosa, 57 (45.6%) produced pyocyanin, while 68 (54.4%) did not[16]. Among our isolates, a lower level of pyocyanin pigment was observed in forty-six isolates (36.7%). Interestingly, only two isolates produced a substantial amount of pyocyanin, while nine isolates (7.2%) produced intermediate quantities. During the designated incubation period, the pseudomonas broth inoculated with bacteria exhibited vivid coloring, ranging from blue-green to yellowish-green. P. aeruginosa is known to produce various pigments, including pyocyanin (blue-green), pyomelanin (light brown), pyoverdin (yellow, green, and fluorescent), and pyorubrin (reddish-brown) [40]. Interestingly, it was observed that shaking conditions during incubation increased pyocyanin production by 31–63.5% compared to static conditions [41]. The results of TLC analysis of the extracted pigment were consistent with the findings of column chromatography. Each isolated pigment exhibited a blue spot with an Rf value ranging from 0.70 to 0.81, indicative of a phenazine compound, similar to pyocyanin. These findings are in line with previous studies [37] reported an Rf value of 0.83 for purified pyocyanin, and Shouman et al who identified a single area with an Rf of 0.8 in their study of purified pyocyanin[16]. The present work focuses on synthesizing pyocyanin from various clinically isolated strains of P. aeruginosa [13].

Each of the 96 clinical strains of P. aeruginosa examined in this study exhibited varying capacities to produce pyocyanin pigment. Consistent with previous research by [14], strains isolated from urine showed the highest pyocyanin production (20.15 µgmL^− 1), while those recovered from sputum exhibited the lowest quantity (3.80 µgmL^− 1). Notably, the P. aeruginosa U3 strain isolated from a urine specimen demonstrated the highest pyocyanin production in another study [42]. Interestingly, only two isolates (1.6%) in our study produced a high amount of pyocyanin (> 10 µgmL^− 1), while forty-six isolates (36.8%) exhibited low-level pigment (< 5 µgmL^− 1), and nine isolates (7.2%) showed moderate levels (5–10 µgmL^− 1) [16].

The identification of the three selected P. aeruginosa isolates (P73, U51, and p51), known for their high pyocyanin production, was confirmed through 16S rRNA gene sequencing [43–44]. The obtained 16S rRNA sequences were submitted to the NCBI online server (https://www.ncbi.nlm.nih.gov/genbank), where they underwent analysis using the BLASTP and BLASTX algorithms to identify similar sequences in the NCBI Genbank database. Furthermore, comparison of the recorded sequences of the P. aeruginosa strains with 16S rDNA gene sequences of organisms cataloged in the GenBank databases revealed a strikingly high degree of similarity (99%) between the two species [45].

The absorbance spectra of pyocyanin isolated from P73 (ONO14782), exhibiting the highest productivity, were measured using a UV-Vis spectrophotometer across the 200–800 nm range. For pure pyocyanin, two distinct peaks were observed at wavelengths of 368 nm and 687 nm, respectively. The presence of the 368 nm peak confirmed the existence of the pyocyanin compound, consistent with earlier research where pyocyanin was identified and quantified based on distinctive absorptions at 370 nm and 690 nm [46–47]. Using chloroform solvent, pure pyocyanin was detected at wavelengths of 699 nm, 529 nm, 310 nm, and 254.5 nm [48]. When utilizing 0.1 N HCl solvent, five absorption maxima were observed at wavelengths of 553 nm, 390 nm, 284 nm, and 246 nm. These peaks closely matched the typical absorption maxima of pyocyanin in 0.1 N HCl (555 nm, 388 nm, 284 nm, 247 nm, and 225 nm) and chloroform (691 nm, 529 nm, 306 nm, and 255.5 nm). Additionally, pyocyanin that had been partially purified was examined and measured at a wavelength of 278 nm [49].

The molecular fingerprint of the material was established through Fourier-transform infrared spectroscopy (FTIR), which revealed the side chains of the phenazine-characterized pyocyanin molecule [31]. The spectra indicated the presence of an O-H bond at 3429 cm^− 1, a C-H aromatic bond at 2367 cm^− 1, a C = N bond at 1631 cm^− 1 and a C-O bond at 1250 cm^− 1. These findings were consistent with previous research [50–51], which also identified similar peaks associated with pyocyanin.

Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis revealed pyocyanin's molecular weight to be 206 Daltons, aligning with previous findings [52–53]. The mass spectrum showed ions at m/z 199, 122, and 138–193, consistent with prior studies [54 − 47]. Additionally, the retention time of pyocyanin was found to be 52 minutes in GC-MS analysis, confirming its molecular weight. These results were corroborated by previous research, which also reported a retention time of 53.08 minutes and an ion at m/z 211 for pyocyanin [55–57].

Our study demonstrates the diverse antibacterial activity of pyocyanin concentrations derived from P73 (ONO14782) against various pathogenic microorganisms.. As the concentration of pyocyanin increased, its effectiveness in inhibiting the tested microorganisms also increased. Notably, pyocyanin did not exhibit any inhibitory effect against K. pneumoniae, while it demonstrated strong antibacterial activity against Staph. aureus, Listeria sp., B. cereus, S. typhi, E. coli, and Shigella sp. This observation aligns with previous studies by Alzahrani and Alqahtani, which reported an increase in antagonistic activity against tested bacteria with increasing pyocyanin concentration [58]. Moreover, El-Shouny et al. found that pyocyanin completely suppressed the growth of all tested Candida spp. and Gram-positive bacteria, while some Gram-negative bacteria, like Salmonella. typhi and Proteus mirabilis, exhibited mild susceptibility, and K. pneumoniae showed resistance [59]. Several other studies, demonstrated the antagonistic activity of pyocyanin against various pathogenic bacteria, such as Salmonella paratyphi, E. coli, and K. pneumonia [53, 60].

Additionally, Rahman et al. showed that pyocyanin from P. aeruginosa DSO-129 exerted antimicrobial effects against Staph. aureus, Staph. epidermis, Bacillus subtilis, Micrococcus luteus, and Saccharomyces cerevisiae [61]. Hamad et al. further corroborated these findings by demonstrating the susceptibility of Bacillus cereus, Staph. aureus, Staphylococcus sciuri, E. coli, S. typhi, Salmonella enterica, and K. pneumonia to pyocyanin [47] .

Furthermore, the antifungal activity of pyocyanin against various yeast species, including Candida albicans and Candida trobiocalis, was demonstrated in this study. An increase in pyocyanin pigment concentration enhanced its antagonistic activity against the tested fungi. This aligns with the findings of [62–63], who highlighted the antifungal activity of pyocyanin against Candida spp. and other mycotoxigenic fungi. The emergence of antibiotic resistance has spurred interest in natural antibacterial compounds like pyocyanin. Pyocyanin's potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as yeasts, underscores its potential as an alternative therapeutic agent. Additionally, pyocyanin has shown promising anticancer properties, exhibiting cytotoxic effects against various cancer cell lines, including HepG2, MCF-7, HCT-116, and A-549. These findings suggest that pyocyanin holds significant potential for combating infectious diseases and cancer, warranting further investigation into its mechanisms of action and clinical applications.

In this study, pyocyanin extracted from P. aeruginosa isolates obtained from pus, urine, and sputum exhibited significant antibacterial and antifungal activity against various clinical pathogens. It effectively inhibited both Gram-positive and Gram-negative bacteria, as well as fungi, with strains such as A. niger and Staph. aureus showing high sensitivity to pyocyanin. Comparatively, pyocyanin demonstrated antibacterial efficacy comparable to several commercial antibiotics, surpassing others in effectiveness. These findings underscore the potential of pyocyanin as a valuable antimicrobial agent for therapeutic use against infections caused by pathogenic bacteria and fungi. Furthermore, our study suggests that pyocyanin holds promise as an anticancer agent, exhibiting selective cytotoxicity against cancer cells. This highlights its potential as a novel candidate for cancer therapy. Overall, the multifaceted antimicrobial and anticancer properties of pyocyanin emphasize its importance and potentiality as a therapeutic agent for combating infectious diseases and cancer. Further research is warranted to explore its mechanisms of action and clinical applications in greater detail

Acknowledgements

Not applicable.

Authors’ Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Mohga Tohamy Afifi Mostafa, Prof. Dr. Mohamed Khaled Ibrahim Prof. Dr. Ahmed Abdel Rahman Esmaiel and Prof. Dr. Ahmed Abdel Rahman Askora. The first draft of the manuscript was written by Mohga Tohamy Afifi Mostafa and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research did not receive any fund.

Availability of data and materials

The datasets utilized and/or examined in the present study can be obtained by contacting the corresponding author. Additionally, the genetic sequence of the strain analyzed has been submitted to the GenBank nucleotide sequence database at the National Library of Medicine, National Center for Biotechnology Information (NCBI). The assigned accession number for the sequence is ONO14782, which can be accessed at https://www.ncbi.nlm.nih.gov/nuccore/ONO14782

Ethics approval and consent to participate

The study was approved by the Ethics Committee of Ain Shams University. It was reviewed by the Institutional Review Board under the number ZU-IRB #11105–11/9–2023.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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Exploring the Antimicrobial and Anticancer Potential of Pyocyanin Produced by Pseudomonas aeruginosa Strain ONO14782

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Materials and Methods

Molecular identification of the most active isolates for pyocyanin-producing ability by 16S rDNA sequencing

Characterization of Pyocyanin produced by P73 (ONO14782) strain

UV-Vis spectrophotometer

Fourier Transform Infra-Red Spectroscopy (FTIR)

Gas chromatoghrophy – Mass spectrometery (GC/MS) Analysis

Primary screening for the isolates having antimicrobial activity

Determination of antimicrobial activity of pyocyanin on bacterial and fungal isolates

Anti-tumor activity of pyocyanin

Results

Molecular Characterization of Selected Bacterial Isolates:

UV-visible spectrophotometric analysis

FTIR analysis

GC-MS analysis

Antimicrobial activity of Pyocyanin

Impact of Various Antibiotics on Pathogenic Bacteria and Fungi

Anti-tumor activity of Pyocyanin

Discussion

CONCLUSION

Declarations

References

Additional Declarations

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