Silver nanoparticles synthesized from Pseudomonas aeruginosa pyoverdine: Antibiofilm and antivirulence agents

Abstract

The increasing incidence of antimicrobial resistance exhibited by biofilm-forming microbial pathogens has been recognized as one of the major issues in the healthcare sector. In the present study, nanomaterial-based controlling the biofilm and virulence properties has been considered an alternative approach. Pyoverdine (PVD) isolated from the Pseudomonas aeruginosa was utilized as a biological corona to synthesize silver nanoparticles (AgNPs), which will be helpful in a targeted action to microbial pathogens due to the recognition of the corona of the nanoparticles by the pathogenic membrane. Synthesized PVD-AgNPs were spherical to irregular, with an average size value of 251.87 ± 21.8 nm and zeta potential with a value of −36.51 ± 0.69 mV. The MIC value of PVD-AgNPs towards P. aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Candida albicans in the standard and host-mimicking media were observed in decreasing order in a multi-fold, such as standard growth media > sputum > synthetic human urine > saliva. Both the initial stage and the well-established biofilms of these microbial pathogens have been effectively inhibited and eradicated by PVD-AgNPs. PVD-AgNPs increase the susceptibility of tetracycline, PVD, and amphotericin B towards established mature mono- and mixed-species biofilms of S. aureus and C. albicans. Additionally, PVD-AgNPs attenuate several virulence properties, such as inhibition of protease activity, motility, and PVD and pyocyanin production in P. aeruginosa. The inhibition of gene expression of biofilm and virulence-associated genes in P. aeruginosa validates its phenotypic effects.

Highlights

• •Pyoverdine from Pseudomonas aeruginosa was used as a biological corona to synthesize AgNPs.
• •PVD-AgNPs effectively inhibited and eradicated initial stage and mature biofilms.
• •PVD-AgNPs increased the susceptibility of tetracycline and amphotericin B towards mature biofilm.
• •PVD-AgNPs attenuated several virulence properties in P. aeruginosa.
• •The suppression of genes associated with biofilm and virulence validates its phenotypic effects.

1. Introduction

Worldwide, issues of antimicrobial resistance (AMR) by a diverse range of pathogens, such as bacteria, viruses, and fungi, have been raised at an alarming rate [1,2]. Treating patients with AMR stuck situations is financially challenging for the patients but also for the economy worldwide [2,3]. Around > $7.2 billion costs for health care in the USA each year, where there is a significant rate of diseases and deaths reported [4]. The estimated death rate reported due to fungal infection in the USA was >13,000 during 2020–2021, particularly since this death was found to be associated with COVID-19 [5]. Similarly, recent studies investigated that about 4.95 million individuals died due to the AMR occurring by bacterial pathogens [6]. With the increasing risk of microbial resistance and high prevalence of death associated with AMR, the World Health Organization (WHO) has prioritized several pathogenic microorganisms for which the antimicrobial agent must be urgently identified [[7], [8], [9]]. Most of the antimicrobial efficacy of the antimicrobial agents has been assessed by determining the minimum inhibitory concentration (MIC) using Clinical and Laboratory Standards Institute (CLSI) guidelines in vitro in standard growth media towards the planktonic mono-species microbial cell culture [10]. However, these antimicrobial drugs did not demonstrate any activity at the specified MIC in the host-mimicking medium or in vivo when administered under physiological conditions [11,12]. In addition, the failure to administer antimicrobials in vivo was also attributed to the presence of polymicrobial interaction between different species of microorganisms in the tissues and organs of the host [[13], [14], [15], [16]]. Furthermore, the self-protection of microorganisms under the biofilm has been recognized as another adaptive mechanism of the resistance properties towards antimicrobial agents [17]. Furthermore, the horizontal transmission of genes, the existence of persister cells, antibiotic-modifying enzymes, low metabolic activity, and the efflux pump are all factors that contribute to the induction of the AMR mechanism by biofilm [18]. The biofilm is the microbial-produced extracellular polymeric substance (EPS) composed of extracellular DNA, proteins, and polysaccharides [19]. These components of EPS play a crucial role in the protection of biofilm cells against the influence of the host immune system [17]. Additionally, they give resistance to dehydration, exposure to ultraviolet light, salt, metal toxicity, and antimicrobial agents [20]. A recent review summarizes the influence of pathogenic microbial biofilms on the health sector, the food industry, and animal husbandry [21]. The AMR properties were reported to increase by the biofilm cells compared to the planktonic cells [22]. Several reports prove that defensive cooperation among interspecies or cross-kingdom level bacterial and fungal pathogens enhances their tolerance against germicidal agents, host immune responses, and stressors surrounding their niche [23]. Thus, the increasing prevalence of AMR exhibited by biofilm-forming microbial pathogens at the single- and mixed-species levels highlights the need to develop an alternative therapeutic approach for combating these infections.

With the several advantages of nanotechnology, interest in fabricating various nanomaterials by employing green chemistry to manage microbial pathogens has increased [24]. In addition, the application of nanomaterials has been used in a variety of applications, such as an anti-oxidant, nano-sensor, anticancer, tissue regeneration, immunotherapies, gene therapies, drug delivery system, and coating of medical devices [25,26]. The employing of green chemistry as an alternative to physical and chemical methods for the synthesis of nanoparticles (NPs) has a number of benefits, including the fact that it is more economical, less complicated, less harmful to the environment, and reduces the amount of hazardous chemicals that are released into the environment [24]. Furthermore, NPs have a number of additional properties that make them a more viable approach to the management of microbial infections. These qualities include their tiny size, vast surface area, stability, biocompatibility, targeting to particular areas, and increased strength of cellular interactions [27,28].

Recently, green chemistry has enabled the synthesis of biocompatible NPs that are as effective in managing biofilm-related infections and ditching traditional antibiotics [24]. Furthermore, a few studies have proposed applying green-synthesized NPs to overtake mono- and polymicrobial-species biofilms of deadly pathogens [29,30]. A diverse range of biological materials, such as plants, animals, microbes, and algae, have been employed to synthesize the NPs [24]. Several biomolecules from these biological sources act as key players in the synthesis of the NPs [24]. These biomolecules also act as a corona that surrounds the NPs, supporting their endurance and bio-compatibility [31,32]. In addition, the corona layer over the NPs enhances its biological effectiveness by interacting with the cell membrane, followed by internalization [33]. Hence, to control biofilm-forming microbial pathogens, if the corona is derived from the microbial species, that can help in the interaction with the cell surface of the microbial pathogen that might have similar components matching with the corona [32].

In order to apply this hypothesis, the present proposal aimed to utilize the synthesis of microbial-inspired NPs that can inhibit the biofilm and attenuate the virulence properties of the microbial pathogens [34]. In particular, bacteria have the ability to synthesize NPs due to their diversity across a wide range of strains and their adaptability to extreme environments [35]. Bacteria-mediated NPs are mainly synthesized by capturing metal ions in microbial cells or microbial metabolites and reducing the metal ions to NPs [36]. Production of NPs synthesized using bacteria emerges as a good candidate for green synthesis due to the fast propagation rate and easy cultivation [37]. Several potential bioactive molecules from the bacterial species exhibit diverse biological roles [[38], [39], [40]]. These molecules can be utilized as a reducing agent to synthesize NPs, which can be easily targeted to microbial pathogens due to the recognition of the corona of the NPs by the pathogenic membrane [32]. Among them are the applications of siderophore from Pseudomonas aeruginosa as a corona in order to synthesize NPs as potential antibiofilm and antivirulence agents. Because the cell receptor and transport system can readily detect the siderophore, producing various types of NPs using the siderophore may also be identified by these receptor proteins and transport system, allowing the NPs to enter the cells and cause cell death [41]. Under iron-limiting conditions, P. aeruginosa regulates its nutrition mainly by two siderophores named pyoverdine (PVD) and pyochelin, which scavenge the iron from the environment to deliver inside the cells via membrane receptor-mediated. The present study will elaborate on seven main points: (1) Isolation of PVD in the iron limiting media from P. aeruginosa, (2) synthesis of silver nanoparticles (AgNPs) using PVD and its detail characterization, (3) antibacterial and antifungal activity of PVD-AgNPs, (4) antibiofilm activity of PVD-AgNPs towards bacterial and fungal species, (5) dispersal of mature biofilm, (6) attenuation of the virulence properties of P. aeruginosa and Staphylococcus aureus, and (7) suppression of the biofilm and virulence related genes in P. aeruginosa.

2. Materials and methods

2.1. Microbial strain, reagents, and culture media

2.1.1. Microbial strain

The bacterial and fungal pathogens used in the present study and their MIC values against PVD-AgNPs are as listed in Table 1.

Table 1.

MIC value of PVD-AgNPs towards different microbial pathogens in the standard and host-mimicking media.

Microbial pathogens	MIC of PVD-AgNPs in different media (μg/mL)
	TSB/PDB	Sputum	Saliva	Synthetic human urine
Klebsiella pneumoniae ATCC 4352	16	4	1	0.0125
Listeria monocytogenes KCTC 3569	32	4	1	0.5
Staphylococcus aureus KCTC 1916	64	8	2	2
Streptococcus mutans KCCM 40105	16	2	0.5	0.5
Escherichia coli KCTC 1682	16	2	0.5	0.0125
Pseudomonas aeruginosa KCTC 1637	16	8	2	1
Candida albicans KCCM 11282	>32	>64	1	0.5

2.1.2. Reagents

Silver nitrate (CAS# 7761-88-8; ≥99.0% pure) and AgNPs (<100 nm particle size with the Cat. No. 576832-5G) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

2.1.3. Standard growth media

Except for Streptococcus mutans (grown in BHI), tryptic soya broth (TSB) supported the growth of all other bacterial cells, whereas Candida albicans were cultured in potato dextrose broth (PDB). Also, the mixed cell culture was prepared by mixing bacterial cells and C. albicans, each with the OD₆₀₀ = 0.05 in equal ratios.

2.1.4. Artificial sputum media

Artificial sputum was prepared following the earlier study [42]. For preparing a 1 L volume mixture of sputum, adding sequentially, mucin (5 g), DNA (from salmon sperm) (4 g), diethylene triamine penta-acetic acid (5.9 g), NaCl (5 g), and KCl (2.2 g), and tris base (1.81 g) in distilled water followed by continuous stirring.

2.1.5. Artificial human saliva

The human saliva was ordered from Sigma Aldrich Co. (SAE0149-200 ML), and its chemical makeup was previously reported [43].

2.1.6. Synthetic human urine

The Brooks-Keevil method was used to produce synthetic human urine with a pH of 6.5 [44].

2.2. Isolation of PVD from P. aeruginosa

PVD was isolated from P. aeruginosa on the steps followed [45]. The seed culture of P. aeruginosa (OD₆₀₀ = 0.05) was inoculated in a sterile flask holding 200 mL of the minimal salt medium (MSM) with a 2% concentration of sodium succinate (SS). The composition of the MSM has been previously reported [45]. The flask was shaken (250 rpm) at 37 °C for 24 h. The appearance of a green-colored pigment indicated the effective production of PVD as checked every 2 h of incubation. In addition, monitoring the increasing absorption spectra of the PVD (400 nm) by scanning from 200 to 600 nm was also checked at every 2 h of incubation. After 24 h incubation, the cultured cell was centrifuge (13,000 rpm, 10 min) and collected the supernatant. The supernatant was filter sterilize twice using 0.2 μM filter followed by the UV-light treatment for 15 min. The sterilized supernatant was used for the synthesis of the AgNPs. Furthermore, the supernatant was also kept freezing at −70 °C for 2 h followed by the freeze drying using a freeze dryer. The dried sample were used for the Fourier transform infrared spectrometer (FTIR) and X-Ray diffractometer (XRD) analysis.

2.3. Green synthesis of PVD-AgNPs

The AgNPs were synthesized with a modified version of the previous study of AgNP synthesis [27]. 2 mM of silver nitrate was dissolved in 10 mL of deionized water. The filter-sterilized PVD containing supernatant (200 mL) from the P. aeruginosa was placed in the 500 mL bottle. A bottle wrapped in aluminum foil was taken to keep a 10 mL solution of silver nitrate at 37 °C overnight in the dark. The accumulation of the white precipitate at the bottom of the bottle indicates the formation of PVD-AgNPs. In addition, an increased absorption peak at a specific wavelength marked its formation. It was centrifuged at 13,000 rpm for 30 min at 4 °C to pellet down the sample. That was rinsed with deionized water and freeze-dried into powder for further characterization.

2.4. Characterization of PVD-AgNPs

Several instruments were employed to characterize the PVD-AgNPs [27]. The morphological features of PVD-AgNPs were thoroughly investigated using field emission transmission electron microscopy (FETEM; JEM-F200, JEOL, Japan). The FTIR (JASCO FT-4100, Tokyo, Japan) provided a remarkable degree of resolution to characterize the surface chemistry of PVD-AgNPs with a range of frequency (4000 to 400 cm⁻¹). The particle distribution and the size were carried out by dynamic light scattering using a particle analyzer (Litesizer 500, Anton Paar, German). The zeta potential of the NPs, which provides surface charge dynamics, was also analyzed using the same particle analyzer. The existence of the Ag metal in the synthesized NP matrix was carried out using energy-dispersive X-ray spectroscopy (EDS; VEGA II LSU, TESCAN, Czech). The XRD (Ultima IV, Rigaku, Japan) was used to analyze the crystalline nature and elemental composition of PVD-AgNPs.

2.5. Determination of MIC value of PVD-AgNPs

The CLSI guidelines were adopted to evaluate the MIC of PVD-AgNPs towards several microbial pathogens in the standard and host-mimicking growth media [46]. The bacterial or fungal cells were diluted 100 folds in the standard growth media, aiming for an OD₆₀₀ = 0.05. Similarly, the seed culture of these microbes was also diluted (1:100) in the host-mimicking media (e.g., artificial saliva, sputum, and synthetic human urine). The 96-well microtiter plate was filled with 300 μL of diluted cell cultures in triplicates with concentrations ranging from 0.5 to 64 μg/mL of PVD-AgNPs. Incubation at 37 °C for 24 h supported their growth. The MIC of PVD-AgNPs assayed in their respective growth media were checked based on the visible growth and by measuring the OD₆₀₀ value. However, the MIC value in the host-mimicking media was determined by colony-counting approaches. The cell culture (100 μL) from the host-mimicking media was serially diluted in the standard growth media up to the dilution of 10⁻⁸. The spreading (100 μL) of the cell culture on the agar plates was carried out and incubated for 24 h at 37 °C. In the case of the standard growth media, the MIC value was determined when there was no visible growth at the lowest concentration of PVD-AgNPs. However, in the case of the host-mimicking media, the MIC value was determined when cell growth was reduced above 90%.

2.6. Biofilm assays

The inhibition of biofilm was carried out by crystal violet [47]. For the biofilm assays, the respective growth media were marked for OD₆₀₀ = 0.05 after dilution of seed culture to its 100 folds. The 96-well microtiter plate was loaded with 300 μL of cell cultures with PVD-AgNPs at a 0.25–64 μg/mL concentration. Further, an optimal temperature of 37 °C for 24 h of incubator accelerated their growth. The crystal-violet staining method was opted to study biofilm inhibition. This required the removal of planktonic cells and washing them three times with distilled water. After 20 min of staining, they were rinsed thrice with distilled water and filled with ethyl alcohol. The quantification was carried out by measuring the OD₅₇₀.

2.7. Examination of microbial biofilm-architecture exposed by PVD-AgNPs

Each NP-treated microbial cell's biofilm architecture was observed using scanning electron microscopy (SEM) [48]. A nylon membrane (0.5 × 0.5 cm) was put in a 24-well microplate. Cell culture was deposited on the membrane in a 24-well microplate according to the biofilm assay. Cells were additionally treated with PVD-AgNPs, while some were left untreated for the control group. The plate was incubated for 24 h at 37 °C. Biofilm cells were fixed with formaldehyde (2%) and glutaraldehyde (2.5%) in the well overnight at 4 °C. Each well was washed three times with phosphate-buffered saline (pH: 7.4) to remove unattached cells. Increasing ethanol concentrations were used to eliminate moisture from these attached cells. The membranes were applied to the SEM stub after freeze drying (FD8518, ilShinBiobase Co. Ltd., Korea). Both treated and untreated biofilm cells were seen using VEGA II LSU (TESCAN, Czech) microscopy at 10 kV voltage and 8.00 kx (10 μm) magnification.

2.8. Eradicating of mature biofilms by PVD-AgNPs

The eradication efficacy of the PVD-AgNPs towards the mature biofilm of the bacterial and fungal cells was carried out as described earlier [46]. The diluted seed culture (300 μL with OD₆₀₀ = 0.05) of each microbial cell was placed in the 96-well microtiter plate and incubated at 37 °C for 24 h. The planktonic cells were removed, and surface attached biofilm cells were washed three times using sterile TSB. The varied concentration of PVD-AgNPs (1–64 μg/mL) was prepared in the sterile TSB and treated these cells. Further microplate incubation was carried out at 37 °C for 24 h. After removing the planktonic cells, the biofilm cells were quantified in the same way as carried out in the biofilm inhibition experiment.

2.9. Combinatorial application of PVD-AgNPs with antibiotics, PVD, and amphotericin B (AmpB) towards mature biofilm

The susceptibility of antibiotics and antifungals in the presence of PVD-AgNPs towards bacterial (S. aureus) and fungal (C. albicans) pathogens was carried out [49]. The efficacy was tested against the mono- and mixed-species mature biofilm of S. aureus and C. albicans in the standard growth media. The antibiotic tetracycline (Tet) and antifungal AmpB were used to check the efficacy. The sub-MIC value of Tet and AmpB (reported previously) was used to apply in combination with the sub-MIC value of PVD-AgNPs [49]. In addition, the efficacy of PVD-AgNPs, Tet, and AmpB towards mono- and mixed-species mature biofilm was also checked in the presence of the filter-sterilized PVD.

The mature biofilm of the mono-species was prepared the same way as performed in the biofilm inhibition experiment section. However, the mixed-species biofilm assays were carried out by mixing equal volumes of the bacterial and C. albicans cell (OD₆₀₀ = 0.05) cultures prepared in the standard growth media. The cell culture (300 μL) of mono- and mixed-species was placed in the 96-well microplate and incubated for 24 h at 37 °C. The planktonic cells were removed from the wells, and biofilm cells were washed three times using sterile standard growth media. The two-fold dilution of Tet (0.25 and 0.5 μg/mL), AmpB (0.25 and 0.5 μg/mL), PVD-AgNPs (16 and 32 μg/mL), and filter-sterilized PVD was carried out in the sterile standard growth media. Similarly, different combinations of Tet + PVD-AgNPs, AmpB + PVD-AgNPs, PVD + PVD-AgNPs, and Tet + AmpB + PVD-AgNPs at their sub-MIC value were also 2-fold diluted. These 2-fold diluted samples (300 μL) were placed in the 96-well microtiter plate containing washed mature established biofilm cells. Furthermore, these plates were incubated at 37 °C for another 24 h. The residual planktonic cells were removed and washed three times with the sterile growth media and scraped off using a pipette tip. The cell suspension was serially diluted (10⁻⁸) and spread-plated on agar plates. In the case of the mono-species biofilm, the spreading of the S. aureus and C. albicans cells was carried out on the TSA and PDA plates. However, for the mixed-species biofilm, the spreading of the cell culture (100 μL) was carried out on TSA plate (containing fluconazole 10 μg/mL for the selective growth of S. aureus cells) and PDA plates (containing Tet 10 μg/mL for the selective growth of C. albicans cells). The colonies that appeared on the plates were counted, the CFU values were calculated, and the CFU values of the control group were compared. The experiment was carried out in triplicates and repeated two times.

2.10. Antivirulence properties of PVD-AgNP

Pyocyanin and PVD production from P. aeruginosa in the presence of PVD-AgNPs was evaluated in the same manner as described earlier [27]. The pyocyanin production was carried out by first growing the cell culture (OD₆₀₀ = 0.05) for 24 h at 37 °C in the absence and presence of PVD-AgNPs (1–8 μg/mL). The supernatant was extracted using chloroform and separated in a fresh tube. The acidification using 0.2 N HCl was carried out, resulting in the appearance of pink. The pink color pyocyanin was quantified at OD₅₂₀. PVD production was carried out by incubating the P. aeruginosa cell culture (OD₆₀₀ = 0.05) in MSM along with 2% SS. These cells were also treated with varying doses of PVD-AgNPs (0.5–8 μg/mL). The treated and untreated cell cultures were incubated under shaking (250 rpm) at 37 °C for 24 h. The measurement of the PVD in the supernatant collected from the cell culture was carried out at OD₄₀₅.

2.10.1. Protease assay

The effect of PVD-AgNPs on the protease activity of P. aeruginosa was checked by making a skim milk agar plate. The cell culture of P. aeruginosa (OD₆₀₀ = 0.05) was incubated (shaking at 250 rpm overnight and at 37 °C) along with the sub-MIC value of PVD-AgNPs (from 1 to 8 μg/mL). The cell cultures, both treated with PVD-AgNPs and untreated, underwent centrifugation, and the resulting supernatant was subjected to filter sterilization using a 0.2 μM filter. The 30 μL of supernatant was carefully added to the well created in the skim-milk agar plate. The measurement of the protein-digested zone in diameter was carried out.

2.10.2. Hemolysis assay

The study involved the assessment of hemolytic activity by PVD-AgNPs towards S. aureus and P. aeruginosa as described earlier [27]. The cell culture of these bacteria (OD₆₀₀ = 0.05) was incubated with PVD-AgNPs (from 0.5 to 8 μg/mL) at 37 °C overnight. The diluted sheep blood was incubated with the PVD-AgNPs treated and untreated cell culture (100 μL) of S. aureus and P. aeruginosa. These cell cultures were incubated under shaking conditions at 37 °C for 3 h. After incubation, the samples were centrifugated (16,000 rpm for 10 min), and the absorbance of the supernatant was measured at 543 nm.

2.10.3. Motility assays

The attenuation of flagella-mediated motility, such as swarming and swimming in P. aeruginosa by PVD-AgNPs, was carried out as described earlier [27]. The swarming and swimming were checked on the surface of the agar plates containing different ingredients along with the sub-MIC value of PVD-AgNPs (8 μg/mL). The agar media used for swarming was made using Luria-Bertani medium, which consisted of 0.5% casamino acid, glucose, and 0.5% bacto agar. The swimming medium contained 1% sodium chloride, 0.25% tryptone, and 0.3% bacto agar. A volume of 3 μL overnight grown cell culture was carefully deposited in the middle of the swarming and swimming agar plates. After a 24-h incubation at 37 °C, the diameter of the migrated cells was measured.

2.11. Gene expression analysis of biofilm and virulence gene

The expression of genes linked with the biofilm, quorum sensing (QS) signaling, virulence factors, and motilities in P. aeruginosa was carried out using qRT-PCR as described earlier [50]. The primers used in this study include QS signaling genes (rhlR-rhlI and lasR-lasI systems) [51], biofilm matrix forming-exopolysaccharide gene (algA) [52], virulence genes (phzC, phzE, pvdA, pvcC, and lasB) [51,53,54], flagellar gene (flgG), and pili genes (pilB and pilI) [54,55]. The proC was used as a housekeeping gene for the normalization of expression of all genes [53]. For the isolation of mRNA, the cell culture of P. aeruginosa (OD₆₀₀ = 0.05) was incubated along with PVD-AgNPs (4 and 8 μg/mL) under shaking conditions overnight at 37 °C [54]. The cell cultures were harvested by centrifugation (13,000 rpm for 10 min), and the obtained pellet was utilized for the isolation of the mRNA using an AccuZol™ reagent kit (Bioneer, South Korea). The complementary DNA (cDNA) was synthesized using a high-capacity cDNA Synthesis Kit (Appliedbiosystems, Thermaofisher Scientific). As described earlier, the synthesized cDNA was used as a template for performing the RT-PCR [56]. The SYBR™ Green PCR Master Mix (Appliedbiosystems, Thermaofisher Scientific) was used for making the reaction mixture. The obtained gene expression data was normalized with the housekeeping gene (proC), and the relative expression level of each gene was determined as described earlier [57].

2.12. Statistical analysis

The graph was plotted using GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA). Each experimental data was analyzed using one-way ANOVA and Dunnett's multiple comparisons tests. ***p < 0.0001, **p < 0.01 and * p < 0.05 indicated significance.

3. Results

3.1. Isolation of PVD and synthesis of PVD-AgNPs

The production of PVD from P. aeruginosa was carried out by growing cells in MSM supplemented with 2% SS. A control culture of P. aeruginosa was carried out by simply growing in the TSB. The growth of P. aeruginosa in MSM + SS was found to be seven times lower than in rich media (TSB) (Fig. 1A and B). The production of PVD in MSM at different time intervals was confirmed by spectral scanning of UV–visible absorption ranging from 200 to 700 nm (Fig. 1C). The characteristic absorbance spectra of PVD were observed at 400 nm in the sample collected from the different time intervals from the sample containing MSM + SS (Fig. 1C). After the complete production, the sample was harvested when there was no increment of the absorption spectra at 400 nm. The PVD was separated by centrifugation of the sample at 13,000 rpm for 30 min at 4 °C. The collected supernatant was further filtered using a 0.2 μM filter followed by UV sterilization for 30 min. The supernatant was freeze-dried in the form of powder using a freeze-dryer.

Fig. 1.

(A–B) Growth properties of P. aeruginosa in TSB and minimum salt media containing SS and (C) UV–vis absorption spectra of PVD at different time intervals produced by P. aeruginosa.

3.2. Characterization of PVD-AgNPs

Characterizing the instrumental properties of PVD-AgNPs is essential for understanding their physical and chemical attributes. The UV–visible absorption spectra analysis indicates an increasing absorption peak at the wavelength of 460 nm when different concentrations of PVD-AgNPs were scanned (Fig. 2A). These characterizations involve examining their average particle size, shape, morphology, and zeta potential. The FTIR analysis of PVD-AgNPs shows a peak of 2971 cm⁻¹, typically associated with the stretching vibrations of C–H bonds in aliphatic hydrocarbons (Fig. 2B). The average size of PVD-AgNPs, as examined using a particle analyzer, was 251.87 ± 21.8 nm (Fig. 2C). The zeta potential measurement carried out by the same particle analyzer instrument shows a value of −36.51 ± 0.69 mV (Fig. 2D). It also suggests the presence of saturated carbon-hydrogen bonds, often found in organic compounds like alkanes.

Fig. 2.

(A) UV–visible absorption spectra of PVD-AgNPs, (B) FTIR spectra of PVD and PVD-AgNPs, (C) Average particle size of PVD-AgNPs, and (D) Zeta potential of PVD-AgNPs.

The peak at 1616 cm⁻¹ is often attributed to C C stretching vibrations, indicating the presence of carbon-carbon double bonds. This suggests the presence of unsaturated organic compounds or aromatic rings. Peaks in this region 1516 cm⁻¹ are often associated with aromatic ring vibrations. It also suggests the presence of aromatic compounds in the sample. This region 1400 cm⁻¹ is often associated with various vibrational modes, including bending vibrations of C–H bonds and vibrational modes of C–O bonds. The specific assignment of this peak would depend on the overall spectrum and the chemical context of the sample. 1623 cm⁻¹ is a close match to the 1616 cm⁻¹ peak mentioned earlier, and it is also indicative of C C stretching vibrations, suggesting the presence of double bonds in the sample. Peaks in this region 968 cm⁻¹ can be associated with a variety of vibrations, including bending vibrations of C–H bonds and out-of-plane vibrations of aromatic rings. The precise assignment of this peak would depend on the overall spectrum and the chemical context. As examined using FETEM, PVD-AgNPs exhibited spherical and irregular shapes (Fig. 3A, B, and C).

Fig. 3.

(A) FETEM micrograph at a resolution of 500 nm, (B) FETEM micrograph at a resolution of 200 nm, (C) FETEM image at 100 nm resolution, and (D) Selected area electron diffraction of PVD-AgNPs.

X-ray diffraction was employed to determine the crystal structure and crystalline phase of PVD and PVD-AgNPs (Fig. 4A). The characteristic diffraction peaks at 2θ of AgNPs were identified at 29.8°, 33.2°, 36.5°, 47.8°, 52.7°, 5.0°, 61.6°, 69.9°, 71.8°, and 87.3°. Whereas the PVD exhibits diverse types of peaks in XRD spectra (Fig. 4B). The crystalline character of PVD-AgNPs is further shown by the existence of comparable Debye–Scherrer rings in SAED, as shown in Fig. 3D. Based on the EDS spectrum investigation, it was determined that PVD-AgNPs contain silver (Fig. 4C). In addition, the elemental mapping image of Ag that corresponds to it may be seen in Fig. 4D. The spherical and irregular forms were also visible in the SEM picture obtained during the EDS examination (Fig. 4E).

Fig. 4.

(A) XRD spectra of PVD-AgNPs, (B) XRD spectra of PVD, (C) EDS spectra of PVD-AgNPs, (D) Elemental mapping of Ag from PVD-AgNPs, and (E) SEM image of PVD-AgNPs.

3.3. MIC values of PVD-AgNPs towards microbial pathogens

The MIC value of PVD-AgNPs against bacterial and fungal pathogens in the standard and host-mimicking growth media, such as artificial saliva, sputum, and synthetic human urine, has been summarized in Table 1. The MIC value of PVD-AgNPs in artificial sputum, saliva, and synthetic human urine media was found to be comparatively low compared to the tested in standard growth media. Against bacterial pathogens such as P. aeruginosa, S. aureus, Listeria monocytogenes, Klebsiella pneumoniae, S. mutans, Escherichia coli, and C. albicans, the MIC value of PVD-AgNPs in the standard growth media was found to be in the range of 16–64 μg/mL. The MIC value of PVD-AgNPs towards bacterial pathogens in the sputum media was found to be in the range of 2–8 μg/mL, however, against the C. albicans it was > 64 μg/mL. The MIC value of PVD-AgNPs in the artificial saliva media against all bacterial and C. albicans pathogens was determined to be in the range of 0.5–2 μg/mL. The MIC value of PVD-AgNPs in the synthetic human urine was very low compared to the standard, sputum, and saliva media, and the MIC range against all pathogens was found to be 0.0125–2 μg/mL. Based on the results, it is concluded that the MIC values were found to be lower than those of the standard growth medium. The MIC value of PVD-AgNPs falls in the decreasing order from the standard to host-mimicking media, such as standard-growth media > sputum > saliva > synthetic human urine. The MIC values of the commercially manufactured silver nanoparticles (C-AgNPs) against these pathogens in the standard and host-mimicking conditions were different from those of the PVD-AgNPs, as shown in the supplemental material (Tables S1 and S2). The MIC value of the C-AgNPs in the standard medium was discovered to be very high, measuring at > 4096 μg/mL, as shown in Table S1. The MIC value of C-AgNPs in the sputum, saliva, and synthetic human urine was found to be variable, and the values were also quite high when compared to the MIC value of PVD-AgNPs (Table S2).

3.4. Inhibitory effect of PVD-AgNPs towards biofilms

3.4.1. Inhibition of biofilm

The sub-MIC of PVD-AgNPs was used to check the initial stage biofilm inhibition of different microbial pathogens. The inhibition of biofilm of P. aeruginosa, K. pneumoniae, C. albicans, E. coli, L. monocytogenes, S. aureus, and S. mutans was found to be significantly inhibited at the sub-MIC levels of PVD-AgNPs (Fig. 5A). The maximum biofilm inhibition was found in such way like 90% (at 8 μg/mL) towards P. aeruginosa, 70% (at 8 μg/mL) towards K. pneumoniae, 70% (at 4 μg/mL) C. albicans, 50% (at 4 μg/mL) E. coli, 70% (at 4 μg/mL) L. monocytogenes, 40% (at 2 μg/mL) S. aureus, and 70% (at 2 μg/mL) S. mutans. An examination of the biofilm architecture using SEM was used to validate the initial stage biofilm inhibition effects of the PVD-AgNPs towards various microbial pathogens (Fig. 6). The SEM image of C. albicans (Fig. 6A and B), S. aureus (Fig. 6C and D), P. aeruginosa (Fig. 6E and F), and K. pneumoniae (Fig. 6G and H) that were treated with PVD-AgNPs and non-created control are shown in Fig. 6. The SEM images showed that the PVD-AgNPs treatment results in very little or negligible attachment to the cell on the surface membrane, whereas the nontreated (control) cells are maximally attached in the aggregates form on the surface.

Fig. 5.

Inhibition of the initial stage biofilm and eradication of the mature biofilms of different microbial pathogens in the standard media. (A) Biofilm inhibition and (B) Eradication of the mature biofilms. ***p < 0.0001 and **p < 0.01 indicated significance.

Fig. 6.

Imaging of the biofilm architecture of microbial cells treated with PVD-AgNPs. (A)C. albicans treated with PVD-AgNPs, (B)C. albicans cell (control), (C)S. aureus treated with PVD-AgNPs, (D)S. aureus (control), (E)P. aeruginosa cell treated with PVD-AgNPs, (F)P. aeruginosa (control), (G)K. pneumoniae treated with PVD-AgNPs, and (H)K. pneumoniae (Control).

3.4.2. Eradication of mature biofilm

The established mature biofilm of bacterial and fungal pathogens was found to be also significantly dispersed by the PVD-AgNPs (Fig. 5B). The eradication of the mature biofilm occurred maximally at the MIC and above the MIC of PVD-AgNPs. The eradication of mature biofilm of P. aeruginosa, K. pneumoniae, E. coli, and S. mutans was found to be 70.83%, 70.91%, 72.28%, and 85.88% at the concentration of PVD-AgNPs (64 μg/mL), which is four-times higher to their MIC value (16 μg/mL). The eradication of L. monocytogenes and C. albicans was found to be maximal 84.36% and 81.79% at the concentration (64 μg/mL), two-fold higher than the MIC value of PVD-AgNPs. Also, eradication in S. aureus was highest i.e., 83.65%, at its MIC value of 64 μg/mL. The results showed that the eradication of mature biofilm occurred maximally at the above MIC value and MIC value of the PVD-AgNPs. The percentage increase values were calculated with reference to the negative control group, which was only bacteria/fungal culture.

3.5. PVD-AgNPs enhanced the efficacy of the antibiotics and antifungal toward mature biofilm

The combination effect of PVD-AgNPs with the antibioticsor antifungals towards the mature biofilm was checked using their MIC as well as at sub-MIC values. Multiple combinations of PVD-AgNPs with antibiotics (tetracycline; Tet) and antifungal (amphotericin B; AmpB) were tested to prove the efficiency of combinational therapy against the eradication of single and mixed-species S. aureus and C. albicans biofilms, respectively (Fig. 7). When PVD-AgNPs were administered either alone or in combination with PVD, PVD + Tet, and Tet, it was shown that the eradication of S. aureus in single species biofilm varied (Fig. 7A). The application of 32 μg/mL of PVD-AgNPs, either by alone or in combination with PVD, PVD + Tet, and Tet, resulted in a considerable elimination of S. aureus biofilm in every single instance (Fig. 7A). It was discovered that the maximal eradication of S. aureus biofilm in the presence of PVD-AgNPs (32 μg/mL) alone and combination with the Tet (0.5 μg/mL) was 30.10% and 31.57%, respectively, which is practically same (Fig. 7A).

Fig. 7.

Combined effect of PVD-AgNPs with AmpB, Tet, and PVD towards established mature mono- and mixed-species biofilms of S. aureus and C. albicans. (A) CFU of S. aureus treated by PVD + Tet, PVD + PVD-AgNPs, PVD + PVD-AgNPs + Tet, PVD-AgNPs, PVD-AgNPs + Tet, PVD, and control, (B) CFU of C. albicans treated by AmpB, PVD + AmpB, PVD + PVD-AgNPs + AmpB, PVD + PVD-AgNPs, PVD-AgNPs, PVD-AgNPs + AmpB, PVD, and control, (C) CFU of S. aureus from mixed-biofilm treated by PVD + Tet, PVD + PVD-AgNPs, PVD + PVD-AgNPs + Tet, PVD-AgNPs, PVD-AgNPs + Tet, PVD, and control, and (D) CFU of C. albicans from mixed-biofilm treated by PVD + AmpB, PVD + PVD-AgNPs, PVD + PVD-AgNPs + AmpB, PVD-AgNPs, PVD-AgNPs + AmpB, PVD, and control. ***p < 0.0001, **p < 0.01 and * p < 0.05 indicated significance.

An intriguing finding was discovered when PVD-AgNPs were applied to the mature biofilm of C. albicans either alone or in combination with PVD, PVD + AmpB, and AmpB (Fig. 7B). When PVD-AgNPs were applied in conjunction with PVD + AmpB, there was the complete eradication of the mature biofilm that was produced by C. albicans (Fig. 7B). At a concentration of 32 μg/mL, PVD-AgNPs effectively eliminated C. albicans biofilm by 78.17% compared to the control. Surprisingly, when PVD was applied alone or in combination with AmpB and PVD-AgNPs, there was maximal eradication of C. albicans biofilm, suggesting that PVD increased the effectiveness of PVD-AgNPs and AmpB against C. albicans cells.

There was also a considerable eradication of S. aureus from the mixed-species biofilm with C. albicans in the presence of PVD-AgNPs, either on their own, in combination with PVD, or with PVD + Tet (Fig. 7C). In mixed culture of S. aureus and C. albicans, PVD-AgNPs at a dose of 32 μg/mL eradicated S. aureus by 16.12%. Using 32 μg/mL of PVD-AgNPs in combination with 0.5 μg/mL of Tet or PVD resulted in 27.84% and 25.52% eradication of S. aureus biofilms, respectively (Fig. 7C), which is nearly the same. C. albicans was effectively eliminated from the mixed-species culture of C. albicans and S. aureus when treated with PVD-AgNPs alone, a combination of PVD-AgNPs with PVD or AmpB, or PVD + AmpB (Fig. 7D). PVD-AgNPs completely eradicated C. albicans biofilm when used with PVD + AmpB or AmpB alone (Fig. 7D). PVD combined with PVD-AgNPs (32 μg/mL) and AmpB (0.5 μg/mL) resulted in the highest eradication of C. albicans (77.0% and 78.3%, respectively) (Fig. 7D), indicating that PVD increased the effectiveness of PVD-AgNPs and AmpB against C. albicans.

3.6. PVD-AgNPs attenuated several virulence characteristics of P. aeruginosa

The virulence-disarming properties of PVD-AgNPs towards P. aeruginosa and S. aureus have been investigated. The results showed that there was significant inhibition (77.37%) of the pyocyanin production from P. aeruginosa by PVD-AgNPs at the sub-MIC value of 8 μg/mL (Fig. 8A). However, the inhibition of PVD production from P. aeruginosa was greatly inhibited by PVD-AgNPs at 2 μg/mL concentration i.e., 97.40% of the control group (Fig. 8B). The protease activity of P. aeruginosa was inhibited entirely at the concentration of 8 μg/mL of PVD-AgNPs (Fig. 8C). Similarly, the hemolytic activity of P. aeruginosa and S. aureus was also significantly inhibited at the sub-MIC value of PVD-AgNPs (Fig. 8D). The antihemolytic activity towards P. aeruginosa was found to be 89.26% at the concentration of 4 μg/mL, whereas, in case of the S. aureus the inhibition was 48.73% at the concentration of 8 μg/mL (Fig. 8D). Different types of motility in P. aeruginosa were significantly inhibited by the sub-MIC of PVD-AgNPs. The percentage of the swarming motility inhibition was found to be 72.61% at the concentration of 8 μg/mL (Fig. 8E). The representative image shows the inhibition of swarming motility on the surface of the agar plates in the presence of PVD-AgNPs and non-inhibition in the absence of the PVD-AgNPs (Fig. 8F and G). Similarly, the inhibition of swimming motility at 8 μg/mL was 82.89% (Fig. 8H). The plate image shows the inhibition of the swimming motility by PVD-AgNPs compared to the control cells, which were not treated with PVD-AgNPs (Fig. 8I and J).

Fig. 8.

Antivirulence properties of PVD-AgNPs towards P. aeruginosa. (A) Inhibition of pyocyanin, (B) Inhibition of PVD, (C) Inhibition of protease activity, (D) Antihemolytic activity of PVD-AgNPs towards P. aeruginosa and S. aureus, (E) Bar diagram showing inhibition of swarming, (F–G) Agar plate showing inhibition of swarming in the absence and presence of PVD-AgNPs, (H) Bar diagram showing inhibition of swimming, and (I–J) Agar plate showing inhibition of swimming in the absence and presence of PVD-AgNPs. ***p < 0.0001, **p < 0.01 and * p < 0.05 indicated significance.

3.7. PVD-AgNPs significantly downregulated biofilm and virulence-related gene expression

The RT-PCR results showed that PVD-AgNPs at their sub-MIC value significantly suppressed the expression of genes that are involved in the formation of the biofilm and production of virulence factors (Fig. 9). The expression of genes involved in the production of exopolysaccharides such as algA and algU were found to significantly inhibited compared to the control (Fig. 9). The expression of flagellar (flgG) and pili gens (pilB and pilI) which play a role in motility were found to be significantly reduced in the presence of PVD-AgNPs (4 and 8 μg/mL) as compared to the control. Similarly, the gene that is associated with virulence factors such as pyocyanin (phzE and phzC), PVD (pvdA and pvcC), and elastase (lasB) were also found to be significantly suppressed in the presence of sub-MIC value of PVD-AgNPs (4 and 8 μg/mL). The expression of the QS-related genes, such as rhlI, lasI, and lasR, were also significantly reduced by the sub-MIC value of PVD-AgNPs. The QS gene, such as rhlR, was significantly affected at both PVD-AgNPs concentrations. The gene expression results showed the difference between the treated and untreated groups to be between 56.00% and 99.94%.

Fig. 9.

The relative expression of genes related to the biofilm and virulence properties of P. aeruginosa in the presence and absence of PVD-AgNPs.

4. Discussion

The current research used several microbial pathogens, including bacteria like K. pneumoniae, L. monocytogenes, S. aureus, S. mutans, E. coli, and P. aeruginosa, as well as fungal pathogens like C. albicans (Table 1). The WHO identified some of the above-listed microbial pathogens, such as P. aeruginosa, S. aureus, E. coli, K. pneumoniae, and C. albicans, as critical and high-priority pathogens because they exhibited multi-drug resistance characteristics [[7], [8], [9]]. These microbial pathogens have been reported to form biofilm and produce several virulence factors. The biofilms produced by these microorganisms are made up of biopolymers known as EPS [58]. EPS allows bacteria to exist and communicate in close proximity, acting as a stress safeguard [59]. Bacteria and fungus cells inside biofilms resist drugs and environmental stressors and may even evade the host's immune system [60]. This poses significant problems in industry, agriculture, and clinical settings [61]. Biofilm-forming microbial pathogens have also been shown to produce chronic infections by producing different virulence factors, motility, and antibiotic-tolerant persister cells [62]. As a result, in addition to reducing the biofilm qualities, disarming the virulence properties has been identified as another potential approach for managing microbial infection [[63], [64], [65], [66]]. In the current work, we used PVD as a reducing agent to synthesize NPs that can be readily targeted to different species of microbial pathogens because the pathogenic membrane recognizes the PVD corona of the NPs. The production of PVD in the MSM from P. aeruginosa has been previously reported to be due to the presence of the characteristic absorption peak at a wavelength of 400 nm, consistent with the previous report [67,68]. The synthesized PVD-AgNPs have both spherical and non-spherical shapes. Previous studies reported that AgNPs generated via PVD from P. aeruginosa have a spherical [69] and face-centered cubic morphology [33]. The produced PVD-AgNPs were very stable, with a high zeta potential (−36.51 ± 0.69 mV), as shown by prior stability studies of AgNPs [70].

The results of the study demonstrated that PVD-AgNPs exhibit lowered MIC values against a variety of bacterial species and C. albicans, with concentrations ranging from 16 to 64 μg/mL in the standard growth conditions (Table 1). According to previous findings, it was discovered that silver nanoparticles synthesized using P. putida KT2440 supernatant (P-AgNPs) and silver nanoparticles synthesized using E. coli supernatant (E-AgNPs), which were synthesized by employing the supernatant of P. putida and E. coli, exhibited a reduced MIC with a value of 1.0 and 8 μg/mL against P. aeruginosa PAO1 and E. coli UTI 89 [32]. They have asserted that the synthesis of these NPs from closely related bacteria leads to a more efficient interaction between the microbial corona present in the P-AgNPs and E-AgNPs and the cell membrane of the pathogenic bacteria. Similarly, Kang et al. [27] produced C1-AgNPs using a lactic acid bacteria strain C1 supernatant. It was found that the C1-AgNPs had lower MIC values in the standard growth medium of 32 μg/mL for S. mutans and 32 μg/mL for P. aeruginosa. However, the MIC value of C-AgNPs against bacterial and C. albicans pathogens was shown to be 6 to 8 times higher than that of PVD-AgNPs in standard growing media (Table S1). Thus, based on the current and prior research, it is inferred that the PVD employed in the production of PVD-AgNPs may interact more efficiently with the microbial cell membrane, resulting in more substantial cell death.

Most NPs or other bioactive compounds for antimicrobial activity have been tested in standard growth medium under in vitro settings; however, when these NPs are applied in an in vivo system or administered to the host, they may fail to exhibit comparable action [71,72]. As a result, in addition to the standard media, host-mimicking media such as artificial saliva, synthetic human urine, and artificial sputum were utilized in the current investigation to assess the antimicrobial activity of PVD-AgNP. The MIC values of PVD-AgNPs in artificial sputum, saliva, and synthetic human urine medium were lower than those evaluated in standard growth media. Similarly, C-AgNPs in the host-mimicking medium have a lower MIC value than standard growth media. However, compared to PVD-AgNPs, C-AgNPs had a much higher MIC value in the host-mimicking medium (Table S2). The lower MIC value in host-mimicking media indicates that the media component or molecules may interact additively or synergistically with PVD-AgNPs [11,46]. It has been discovered that artificial saliva contains a histatin peptide, which is considered to possess antibacterial and antifungal characteristics [73,74]. Furthermore, recent research has shown that phloroglucinol-gold NPs had a lower MIC value against S. aureus and C. albicans in artificial saliva than in a standard medium [46]. However, in order to prove the validity of this concept, it is necessary for future research to investigate the combinatorial application of each component that is present in the host-mimicking medium combined with the PVD-AgNPs. Sub-MIC of PVD-AgNPs was shown to have an inhibitory impact on first-stage biofilm, and this effect was found to be concentration-dependent. This discovery is in line with previous findings [75]. On the other hand, it was discovered that eliminating the established mature biofilm was more significant at the MIC level and above the MIC value of PVD-AgNPs. Several reports have shown that the biofilm matrix is a significant barrier to the entry of antimicrobial agents [76]. In addition, several studies that were conducted in the past have indicated that the high rate of disruption of the mature biofilm occurred at the MIC and above the MIC value of the NPs [75,77]. As a result, it has been shown that PVD-AgNPs have an antibiofilm impact on the early stage of biofilm and can eradicate mature biofilm on various bacterial pathogens and C. albicans. P. aeruginosa infections might be reduced if its virulence traits were disarmed [78]. These characteristics include pyocyanin, oxidative stress, hemolysis, protease activity, synthesis of rhamnolipid, and motility properties (such as swarming and swimming). PVD-AgNPs effectively suppressed flagellar motility in P. aeruginosa at the sub-MIC level, including swarming and swimming, which is similar to reported antivirulence activities of AgNPs produced from cell-free culture filtrate from Fusarium oxysporum [79].

Combination therapy is one of the alternate ways to revitalize multiple antibiotics or antifungals to treat microbiological infections [[80], [81], [82]]. This strategy increases activity, prevents resistance from developing, offers new modes of action, minimizes side effects, and successfully increases the sub-MIC of antimicrobial agents [83]. It has been observed that the antimicrobial effect of the compound is ineffective when it is administered to the host system because of the existence of polymicrobial species [16,84]. It has been revealed that the synergistic interaction of the polymicrobial species demonstrates an increased AMR mechanism in comparison to the biofilm composed of a single species [16,84,85]. It has been determined that S. aureus and C. albicans are the two pathogens co-isolated from the host's tissues and organs. There have been many instances in which these two pathogens employed an example of polymicrobial-species biofilm to test drug antibiofilm or antimicrobial activity [63]. To test the combinatorial activity of PVD-AgNPs with Tet or AmpB against S. aureus and C. albicans, we employed both single-species and polymicrobial-species forms. The findings demonstrated that applying PVD-AgNPs made Tet more effective against S. aureus, regardless of whether it was administered in a single-species or mixed-species culture. In a similar manner, PVD-AgNPs improve the effectiveness of the AmpB against C. albicans in both single-species culture and mixed-species cell culture. In addition, a previous study has shown that the combination of AgNPs and vancomycin had more antibiofilm action than the treatment of AgNPs alone against biofilms of S. aureus, P. aeruginosa, L. monocytogenes, and K. pneumoniae [86]. Furthermore, an intriguing result was seen when PVD-AgNPs were applied to mature C. albicans biofilms produced alone or in combination with S. aureus. PVD-AgNPs completely eradicated C. albicans biofilm cells from single or mixed cultures with S. aureus when used in combination with PVD + AmpB or AmpB, respectively. It was discovered that the presence of PVD increases the effectiveness of PVD-AgNPs or AmpB against the mature biofilm of C. albicans that was produced alone or in combination with S. aureus. Even applied PVD demonstrated substantial eradication of C. albicans biofilm in the case of single- or mixed-species biofilms. Future investigations should be carried out to verify the likely mechanism of PVD enhancing the effectiveness of the PVD-AgNPs and AmpB against C. albicans. There is a significant association between the LasI/LasR and RhlI/RhlR QS systems, which are connected with PQS (Pseudomonas quinolone sensing signal), and the development of biofilm and the synthesis of virulence factors in P. aeruginosa [78]. The LasI/LasR system employs a lactone autoinducer that is 3-oxo-C12-hemoserine, while the RhlI/RhlR system employs a butanoyl homoserine lactone autoinducer [87]. Pyocyanin, siderophores, rhamnolipid, protease enzymes, and swarming motility are all produced and expressed by these systems responsible for their synthesis and expression [87,88]. Therefore, to verify the phenotypic impact of the PVD-AgNPs, the current work has also included investigating gene expression connected to the biofilm and virulence characteristics of P. aeruginosa features. The findings demonstrated a considerable reduction of the LasI/LasR and RhlI/RhlR QS systems, as well as the suppression of specific genes that are related to the motilities (flagellar and pili-mediated), biofilm, pyocyanin, PVD, and protease activity. The results showed significant suppression of the LasI/LasR and RhlI/RhlR QS systems along with the suppression of the specific genes that are associated with the motilities (flagellar and pili-mediated), biofilm, pyocyanin, PVD, and protease activity. A recent study found that utilizing Koelreuteria paniculata extract as a reducing agent in the synthesis of AgNPs lowered the expression of QS- and biofilm-related genes such as lasA, lasB, lasI, rhlA, rhlB, rhlR, and rhlI by up to 53% at a dosage of 15 μg/mL [89].

5. Conclusion

To achieve targeted killing of cells, biofilm inhibition, and virulence attenuation, we used bacterial product as a corona to produce AgNPs. PVD isolated from P. aeruginosa in MSM was used as a source to manufacture AgNPs and test the hypothesis of PVD-AgNPs targeting cells via interactions with the cell membrane, protein receptor, and transport system. The produced PVD-AgNPs were extensively characterized using many pieces of equipment, with spherical-nonspherical shape and a 251.87 ± 21.8 nm size. The high zeta potential value ensured great stability. The MIC value of PVD-AgNPs in the host-mimicking medium was lower than in the standard growth media, indicating that the media composition may have an additive or synergistic impact with the PVD-AgNPs. PVD-AgNPs strongly suppressed the early-stage biofilm at the sub-MIC level. However, the established mature biofilm was impacted at the MIC and above MIC levels of PVD-AgNPs. The sub-MIC value of PVD-AgNPs strongly reduced several virulence qualities of P. aeruginosa (e.g., pyocyanin, PVD, protease activity, hemolytic activity, and motility capabilities) and hemolytic activity of S. aureus. Based on the current findings, it is concluded that PVD-AgNPs are an effective antimicrobial agent for killing bacteria in both normal and host-mimicking conditions. Interstingly, PVD-AgNPs render Tet and AmpB more susceptible to pre-formed mature mono- and mixed-species biofilms of S. aureus and C. albicans. Its inhibitory impact at the phenotypic level is confirmed by the process of suppressing the expression of genes linked with biofilm and virulence. Future study is required for in vivo experiments using some animal model organisms to validate the phenotypic and gene expression level biofilm and virulence inhibition properties.

Ethical approval

This paper does not contain any studies with human participants or animals.

Funding

This research was supported by the Pukyong National University Research Fund in 2023 (202315350001). This research was also supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea grant funded by the Ministry of Education [2022R1A2B5B01001998 and (RS-2023-00241461 to F. Khan)].

CRediT authorship contribution statement

Nazia Tabassum: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Fazlurrahman Khan: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Geum-Jae Jeong: Writing – review & editing, Methodology, Data curation. Du-Min Jo: Methodology, Data curation. Young-Mog Kim: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Formal analysis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviation

AgNPs

silver nanoparticles

AmpB

amphotericin B

AMR

antimicrobial resistance

cDNA

complementary DNA

C-AgNPs

commercially manufactured silver nanoparticles

CLSI

Clinical and Laboratory Standards Institute

E-AgNPs

silver nanoparticles synthesized using Escherichia coli supernatant

EDS

energy-dispersive X-ray spectroscopy

EPS

extracellular polymeric substances

FETEM

field emission transmission electron microscopy

FTIR

Fourier transform infrared spectrometer

MIC

minimum inhibitory concentration

MSM

minimal salt medium

NPs

nanoparticles

P-AgNPs

silver nanoparticles synthesized using Pseudomonas putida KT2440 supernatant

PDA/B

potato dextrose agar/broth

PVD

pyoverdine

QS

quorum sensing

SEM

scanning electron microscopy

SS

sodium succinate

Tet

tetracycline

TSA/B

tryptic soy agar/broth

XRD

X-Ray diffractometer

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.