Effect of Biogenic Silver Nanoparticles on the Quorum-Sensing System of Pseudomonas aeruginosa PAO1 and PA14

The increase in multidrug-resistant microorganisms represents a global threat requiring the development novel strategies to fight bacterial infection. This study aimed to assess the effect of silver nanoparticles (bio-AgNPs) on bacterial growth, biofilm formation, production of virulence factors, and expression of genes related to the quorum-sensing (QS) system of P. aeruginosa PAO1 and PA14. Biofilm formation and virulence assays were performed with bio-AgNPs. RT-qPCR was carried out to determine the effect of bio-AgNPs on the QS regulatory genes lasI, lasR, rhlI, rhlR, pqsA, and mvfR. Bio-AgNPs had an MIC value of 62.50 μM, for both strains. Phenotypic and genotypic assays were carried out using sub-MIC values. Experimental results showed that treatment with sub-MICs of bio-AgNPs reduced (p < 0.05) the motility and rhamnolipids and elastase production in P. aeruginosa PAO1. In PA14, bio-AgNPs stimulated swarming and twitching motilities as well as biofilm formation and elastase and pyocyanin production. Bio-AgNP treatment increased (p < 0.05) the expression of QS genes in PAO1 and PA14. Despite the different phenotypic behaviors in both strains, both showed an increase in the expression of QS genes. Demonstrating that the bio-AgNPs acted in the induction of regulation. The possible mechanism underlying the action of bio-AgNPs involves the induction of the rhl and/or pqs system of PAO1 and of the las and/or pqs system of PA14. These results suggest that exposure to low concentrations of bio-AgNPs may promote the expression of QS regulatory genes in P. aeruginosa, consequently inducing the production of virulence factors such as elastase, pyocyanin, and biofilms.


Introduction
Pseudomonas aeruginosa is a Gram-negative, mobile, aerobic, non-spore-forming bacterium. Due to its versatile metabolism, P. aeruginosa can grow in aquatic environments and in soil, causing infections in plants and animals and a variety of diseases in humans, particularly in immunocompromised individuals [1][2][3].
The World Health Organization recognized the difficulty in treating P. aeruginosa infections and included the bacterium in its priority list of pathogens for which new drugs are urgently required, particularly for the treatment of carbapenem-resistant isolates [4], which are considered a major threat to public health.
Compounds with antivirulence activity, that is, those that can control pathogens without exerting bacteriostatic or bactericidal action, have been proposed as potential therapeutic agents [5,6]. The quorum-sensing (QS) system can be affected by attenuation of QS communication, disruption of receptor proteins, degradation of autoinducing signals, or

Growth Curve Analysis
The effect of bio-AgNPs on the growth kinetics of P. aeruginosa PAO1 and PA14 was assessed by the plating method, as described by Jorgensen [20]. Bacterial inoculum was cultivated in LB broth (Neogen, Lansing, MI, USA) with bio-AgNPs at concentrations of 1/2, 1/4, and 1/8 MIC at 37 • C for 48 h. A control growth curve was constructed by incubating bacterial strains in the absence of bio-AgNPs. Bacterial growth was analyzed at six incubation times (0, 4,8,24,32, and 48 h).

Assessment of Virulence Factors 2.5.1. Biofilm Formation
Biofilm formation capacity was analyzed in 96-well polystyrene plates using the crystal violet method described by Ramos-Vivas et al. [21], with some modifications (Supplementary Table S1). Bacterial isolates were cultured on LB agar (Neogen, Lansing, MI, USA) at 37 • C for 24 h. Then, 180 µL of LB broth (Neogen, Lansing, MI, USA) and 20 µL of P. aeruginosa inoculum (initial concentration of 1.5 × 10 6 CFU/mL) were added to each well and incubated at 37 • C for 24 h in the presence (1/2, 1/4, and 1/8 MIC) and absence (control) of bio-AgNPs. The supernatant of each well was discarded, and the cell layer was washed three times with phosphate buffer solution (PBS, pH 7.2), fixed with 250 µL of absolute methanol PA (Merck, Darmstadt, Germany) for 10 min, and stained with a 1.0% w/v aqueous solution of crystal violet (Merck, Darmstadt, Germany) for 15 min. Subsequently, the dye solution was discarded, and each well was washed three times with ultrapure water and treated with 250 µL of 33% v/v glacial acetic acid (Merck, Darmstadt, Germany). Assays were conducted in the presence (1/2, 1/4, and 1/8 MIC) and absence of bio-AgNPs. Absorbance readings were taken at 620 nm on a spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA). Experiments were repeated four times per isolate, and results are presented as mean and standard deviation.

Pyocyanin
A liquid medium consisting of 2.0% w/v peptone (Neogen, Lansing, MI, USA), 0.14% w/v magnesium chloride (Synth, Diadema, SP, Brazil), and 1.0% w/v magnesium sulfate (Synth, Diadema, SP, Brazil) was used to assess pyocyanin production, as described by El-Mowafy et al. [28]. Briefly, 1 mL of a 0.5 McFarland-equivalent suspension of P. aeruginosa grown in LB broth (Neogen, Lansing, MI, USA) was inoculated in 30 mL of liquid medium in the presence (1/2, 1/4, or 1/8 MIC) or absence of bio-AgNPs. Cells were incubated at 37 • C for 24 h. Then, cultures were centrifuged, and 7.5 mL of the supernatant was transferred to a tube containing 4.5 mL of chloroform (Merck, Steinheim am Albuch, Germany). This mixture was vigorously homogenized on a vortex mixer for 20 s. The organic phase was collected (3 mL), acidified with 1.5 mL of 0.2 M hydrochloric acid, homogenized for 20 s, and centrifuged at 4600 rpm for 10 min. The absorbance of the resulting pink-colored solution was measured at 520 nm on a SPECORD S600 UV/VIS spectrophotometer (Analytica Jena, Germany). Pyocyanin concentrations were converted to µg/mL, by multiplying the optical density at 520 nm by 17,072 × 1.5. For analysis of the expression of QS-related genes, total RNA was extracted from P. aeruginosa PAO1 and PA14 cells grown in the absence (reference) or presence of bio-AgNPs (1/2 MIC). Extraction was performed using TRIzol reagent (Invitrogen, Waltham, MA, USA) and the RNeasy Mini kit (QIAGEN, Germantown, MA, EUA ). DNA contamination was eliminated by using the RQ1-DNAse kit (Promega, USA). The quality of extracted RNA was verified by agarose gel electrophoresis and calculation of the A260/A280 ratio. Gene sequences were obtained from GenBank, and primers were designed and analyzed using Primer-BLAST and BioEdit Sequence Alignment Editor. Primer sequences are listed in Table 1. RT-qPCR was performed using the QuantiNova SYBR Green RT-PCR kit (QIAGEN, Germantown, MA, EUA) in a final volume of 20 µL, consisting of 10 µL of SYBR Green RT-PCR Master Mix, 0.2 µL of RT mix, 1 µL (20 µM) of each primer, 5 µL of bacterial RNA (50 ng/µL), and 2.8 µL of RNase-free water. Reactions were performed on a Rotor-Gene Q 2plex (QIAGEN, Germantown, MA, EUA) using the following steps: reverse transcription for 10 min at 50 • C, initial denaturation for 2 min at 95 • C, 40 cycles of 95 • C for 5 s, and hybridization and extension at 60 • C for 10 s. The proC gene was selected as an internal control and was used to normalize the expression of target genes. Table 1. List of genes and their respective primers used in the RT-qPCR assay.

Target Gene
Primer Sequence Amplicon Size (bp)

Statistical Analysis
Statistical analysis was performed using R Studio version 1.2.5001 (R, Boston, MA, USA), and graphs were constructed using GraphPad Prism version 8.4.2 (GraphPad Software Inc., San Diego, CA, USA). Values of p < 0.05 were considered significant. Data were subjected to analysis of variance followed by Tukey's test. For RT-qPCR data, randomization tests were conducted using REST software version 2.0.13 (Qiagen, Hilden, Germany) to identify statistically significant differences between groups.

Biofilm Formation
The crystal violet method revealed no significant change in biofilm formation in PAO1 after treatment with bio-AgNPs (Table 2). However, all three bio-AgNP treatments significantly increased (p < 0.05) biofilm formation in PA14, by 41.22% to 127.86%.

Biofilm Formation
The crystal violet method revealed no significant change in biofilm formation in PAO1 after treatment with bio-AgNPs (Table 2). However, all three bio-AgNP treatments significantly increased (p < 0.05) biofilm formation in PA14, by 41.22% to 127.86%.

Effect on Swarming, Swimming, and Twitching Motilities
In the present study, the effects of sub-MIC levels of bio-AgNPs on the swarming, swimming, and twitching motilities in P. aeruginosa were investigated by determining the diameter (mm) of the motility halo. As depicted in Figure 2A1, bio-AgNPs inhibited the swarming motility in PAO1. After treatment with 31.25 µM bio-AgNPs, the mean diameter of the motility halo was 45.67 mm, representing an inhibition rate of 22.45% compared with the untreated control (58.89 mm diameter). In PA14, bio-AgNPs induced a significant increase (p < 0.05) in the swarming motility ( Figure 2A2). The diameter of the swarming motility halo increased from 13.89 mm (untreated control) to 45.00-45.89 mm, representing an increase of up to 230.40%. diameter (mm) of the motility halo. As depicted in Figure 2A1, bio-AgNPs inhibited the swarming motility in PAO1. After treatment with 31.25 μM bio-AgNPs, the mean diameter of the motility halo was 45.67 mm, representing an inhibition rate of 22.45% compared with the untreated control (58.89 mm diameter). In PA14, bio-AgNPs induced a significant increase (p < 0.05) in the swarming motility ( Figure 2A2). The diameter of the swarming motility halo increased from 13.89 mm (untreated control) to 45.00-45.89 mm, representing an increase of up to 230.40%. Similarly, PAO1 swimming motility was significantly (p < 0.05) inhibited by bio-AgNP treatments ( Figure 2B1). The control exhibited a motility halo of 41.00 mm in diameter, whereas treated bacteria had a swimming motility halo of 29.33 to 32.78 mm, with a reduction of 20.05% to 28.46%. In PA14, treatments did not influence the swimming motility ( Figure 2B2). PAO1 twitching motility was significantly inhibited (p < 0.05) by treatment with 7.81 and 15.62 µM bio-AgNPs, leading to reductions of 22.58% and 12.90%, respectively  Figure 2C1). By contrast, in PA14, the twitching motility increased by 34.15% and 39.84% (p < 0.05) after treatment with 15.62 and 31.25 µM bio-AgNPs, respectively ( Figure 2C2).

Alkaline Protease
Alkaline protease production was not affected by bio-AgNP treatments ( Figure  3B1,B2). The mean halo diameters of PAO1 and PA14 were 19.67 and 19.78 mm, respectively, before treatment, and 18.33-18.89 and 19.67-20.22 mm, respectively, after treatment.

Pyocyanin
Pyocyanin production by P. aeruginosa in the presence of different concentrations of bio-AgNPs was determined spectrophotometrically at 520 nm. In both PAO1 and PA14, there was an increase in pyocyanin production with all treatments (p < 0.05). In the absence of bio-AgNPs, PAO1 and PA14 produced 13.51 and 14.74 µg/mL pyocyanin, respectively. PAO1 treated with 31.25 µM bio-AgNPs showed the highest pyocyanin production (16.16 µg/mL, representing an increase of 19.70%. In PA14, the highest pyocyanin production (16.39 µg/mL) was achieved by treatment with 31.25 µM bio-AgNPs, affording an increase of 11.21% ( Figure 3D).

Pyocyanin
Pyocyanin production by P. aeruginosa in the presence of different concentrations of bio-AgNPs was determined spectrophotometrically at 520 nm. In both PAO1 and PA14, there was an increase in pyocyanin production with all treatments (p < 0.05). In the absence of bio-AgNPs, PAO1 and PA14 produced 13.51 and 14.74 μg/mL pyocyanin, respectively. PAO1 treated with 31.25 μM bio-AgNPs showed the highest pyocyanin production (16.16 μg/mL, representing an increase of 19.70%. In PA14, the highest pyocyanin production (16.39 μg/mL) was achieved by treatment with 31.25 μM bio-AgNPs, affording an increase of 11.21% ( Figure 3D).

Discussion
Given the need for advances in the control of multidrug-resistant bacteria and antivirulence therapeutic strategies, we investigated the potential of bio-AgNPs as bacterial control agents. Research efforts have been directed toward the use of nanotechnology and nanoparticles to target the QS system and/or virulence of microorganisms such as P. aeruginosa [16,29,30]. One of the advantages of using bio-AgNPs is related to their low

Discussion
Given the need for advances in the control of multidrug-resistant bacteria and antivirulence therapeutic strategies, we investigated the potential of bio-AgNPs as bacterial control agents. Research efforts have been directed toward the use of nanotechnology and nanoparticles to target the QS system and/or virulence of microorganisms such as P. aeruginosa [16,29,30]. One of the advantages of using bio-AgNPs is related to their low cytotoxicity. Scandorieiro et al. [31] showed that this biogenic silver nanoparticle did not show cytotoxicity at concentrations up to 97.22 µM in human RBC and HEp-2 cells. In the present study, we used concentrations up to 31.25 µM of bio-AgNPs.
In the current study, we identified significant differences in phenotypic characteristics between the evaluated reference strains. In PAO1, the swarming, swimming, and twitching motilities, as well as rhamnolipid and elastase production, were significantly reduced in the presence of bio-AgNPs at sub-MIC levels. However, in PA14, bio-AgNP treatment significantly enhanced swarming motility, twitching motility, biofilm formation, and elastase and pyocyanin production.
The reference strains exhibit different genotypic characteristics. P. aeruginosa PAO1 is a moderately virulent strain belonging to a relatively rare clonal group (ST 549). PA14, on the other hand, is highly virulent and belongs to the most common clonal group (ST 253). Furthermore, PA14 has two pathogenicity islands (PAPI-1 and PAPI-2), which are absent in PAO1 [32][33][34] The strains also differ in genome size (6.3 Mbp in PAO1 and 6.5 Mbp in PA14) and have unique genomic regions (54 regions in PAO1 and 58 regions in PA14) [35]. Such factors could explain the different responses of strains to bio-AgNP treatment.
As evidenced by the biofilm formation assay, untreated PAO1 showed greater capacity for biofilm formation than untreated PA14. The reduced capacity of PA14 for biofilm formation might be related to mutation of the ladS gene, which has a deleterious effect on biofilm production [33]. Kasetty et al. [36] also showed that in microfluidic biofilm culture conditions, PAO1 quickly outcompetes PA14 in density. Furthermore, PA14 exhibits a competitive fitness advantage when invading a preformed biofilm and is better able to tolerate starvation than PAO1 in the biofilm context. Surprisingly, after bio-AgNP treatment, PA14 exhibited a significant increase in biofilm formation. The mechanism of biofilm formation differs between PAO1 and PA14, given that each strain uses a different exopolysaccharide as the predominant structural biofilm component. Whereas PAO1 uses the Wsp system to produce Psl, PA14 uses the Pil-Chp system for Pel production [37,38]. Biofilm formation is dependent on several factors, such as synthesis of exopolysaccharides Psl and Pel, alginates, extracellular DNA, and adhesins (flagella and type IV pili). These factors contribute to maturation, antibiotic resistance, and biofilm persistence [39]. Exopolysaccharides are responsible for bacterial adhesion, biofilm formation, and architecture stability [40]. Extracellular DNA contributes to twitching motility and the supply of nutrients to bacterial cells [41].
It is important to highlight that there is an indirect link between biofilm formation and the QS system, related to the control of the swarming and twitching motilities as well as rhamnolipid [42] and pyocyanin [43] production. P. aeruginosa displays three types of motilities (swarming, swimming, and twitching), which allow surface colonization and exploration of new environments [44]. The first step in biofilm formation is bacterial fixation to surfaces via polar flagella and production of adhesion proteins, such as type IV pili, thereby allowing cells to spread to surrounding areas [45].
In the present study, the significant increase in PA14 swarming motility, twitching motility, and pyocyanin production with bio-AgNP treatment contributed to biofilm formation. According to Persat et al. [46], virulence factors can be activated by structural elements (e.g., type IV pili). P. aeruginosa uses these structures for surface recognition and activation of the Chp chemosensory system, which regulates cAMP (second messenger) and induces virulence gene transcription. Therefore, the expressive increase in the swarming and twitching motilities in PA14, mediated by type IV pili, might have favored the increase in other virulence factors.
Ouyang et al. [30] treated Pseudomonas putida KT2440 with low concentrations of zinc nanoparticles (0.5-30 mg/L) and observed an increase in biofilm formation. Protein and sugar contents of biofilm also increased with treatment. Garuglieri et al. [47] reported that subinhibitory concentrations of AgNPs (0.01 µg/mL) might increase the swimming motility in Escherichia coli. In the study of Saeki et al. [48], P. aeruginosa isolates from clinical and environmental sources were found to exhibit increased swarming, swimming, and twitching motilities following treatment with bio-AgNPs. In such cases, bio-AgNPs possibly stimulated a response to stress induced by environmental conditions, as described by Villa et al. [49].
PAO1 showed a significant reduction in LasB production with bio-AgNP treatment, and the opposite was observed in PA14 (p < 0.05). LasB, also known as pseudolysin, is associated with vascular inflammation in P. aeruginosa infections [50]. In agreement with the present study, in which PA14 showed increased biofilm formation with bio-AgNP treatment, Yu et al. [51] demonstrated that LasB is crucial for biofilm formation. The authors found that ∆lasB mutant P. aeruginosa has reduced capacity for biofilm formation owing to a decrease in rhamnolipid synthesis.
The alkaline protease assay revealed no differences in protease production with bio-AgNP treatment in either PAO1 or PA14. Alkaline protease causes severe damage to host tissues by disruption of cytoskeleton structures and degradation of fibronectin and laminin, important components of the endothelium [50]. Both alkaline protease and LasB are able to inhibit neutrophil function, interfering with chemotaxis. As a result, bacteria gain an advantage by escaping phagocytes of the host defense system [52].
To further understand bio-AgNP at the molecular level and to lend support to the outcome of our in vitro results, we followed up with qRT-PCR analysis. lasI, rhlI, and pqsA expression is necessary for the synthesis of QS signaling molecules in P. aeruginosa. Thus, the transcriptional regulators lasR, rhlR, and mvfR were analyzed in this study. Although most PAO1 virulence factors decreased after bio-AgNP treatment, the respective genes were upregulated in both strains (PAO1 and PA14). Bio-AgNP-treated PAO1 bacteria exhibited a higher expression (upregulation) of the evaluated genes compared with the control (p < 0.05). In PA14, only rhlI was downregulated; the other genes were more highly expressed (p < 0.05) than in the control.
Typically, the common mechanisms underlying quorum-sensing interference include inhibition of signal biosynthesis, signal degradation, and interruption of the reception signal molecules [53]. Based on our results, the possible mechanism underlying the action of bio-AgNPs involves the induction of the rhl and/or pqs system of PAO1 and induction of the las and/or pqs system of PA14.
The las system is the master regulator of QS, as it induces the expression of both rhl and PQS pathways in P. aeruginosa [54]. The synthase proteins LasI and RhlI are responsible for the production of the autoinducers 3-oxo-C12-AHL and C4-AHL, respectively. When the concentration of such molecules reaches a given threshold, 3-oxo-C12-AHL binds to the receptor lasR, thereby inducing the expression of virulence factors, such as LasB, exotoxin, and proteases, and activating the rhlI/R system. The autoinducer C4-AHL binds to rhlR and controls motility and expression of genes that encode LasB, pyocyanin, and rhamnolipid. The third autoinducer, PQS, binds to the receptor mvfR, regulating pyocyanin expression and activating the rhlI system [14]. Therefore, the 2.0-fold increase in pqsA and mvfR gene expression in PAO1 and PA14 was probably associated with increased pyocyanin production resulting from bio-AgNP treatment; the 0.6-fold reduction in rhlI expression in PA14, however, did not lead to a decrease in virulence in phenotypic assays.
Liao et al. [55] demonstrated that the main mechanism of action of AgNPs against multidrug-resistant isolates of P. aeruginosa involves the imbalance of oxidation processes and impairment of the elimination of oxygen reactive species. Cugini et al. [56] proposed that compounds such as farnesol, which promote reactive oxygen species production, may enhance the expression of the PQS system by activating rhlR in P. aeruginosa.
The decrease in motility and rhamnolipid and LasB production in PAO1 might be explained by the effect of bio-AgNPs on lasR expression: expression increased 1.1-fold. Given the interconnection of QS systems and the fact that lasR controls the activation of connected cascades [57], it is suggested that the effect of bio-AgNP treatment on lasR expression was not sufficient to induce a phenotypic increase in virulence factors. By contrast, in PA14, the 1.4-fold increase in lasR expression resulted in increased motility and LasB production.
Our findings were similar to those of previous studies investigating the influence of AgNPs on the QS system of P. aeruginosa. Yang and Alvarez [58] observed that subinhibitory concentrations of AgNPs (21.6 and 108 µg/L) could induce the QS system (lasI, lasR, rhll, and rhlR expression) in PAO1 and increase biofilm formation. Li et al. [29] studied several nanomaterials (e.g., silver, iron, zinc oxide, graphene) and found that AgNPs (100 µg/L) enhanced 3-oxo-C12-AHL synthesis, protease production, biofilm formation, and lasR expression in PAO1. Ouyang et al. [30], in assessing the effect of low concentrations of zinc nanoparticles on P. putida, observed an increase in the expression of QS regulatory genes with treatment.
Given that QS inhibition has shown high potential as an antivirulence strategy [6,9], understanding the mechanisms of pathogenicity in P. aeruginosa is of extreme importance, to ensure that antivirulence compounds are applied efficiently. As highlighted by Mohanty et al. [61], the effects of metal nanoparticles on the QS system are highly dependent on bacterial species. Therefore, given the variability of bacterial responses to different metal compounds, this approach needs to be well-studied and scientifically proven with complementary molecular studies on reference strains and isolates obtained from different sources (environmental and clinical).

Conclusions
Our results suggest that bacterial exposure to low concentrations of bio-AgNPs may enhance the expression of QS regulatory genes in P. aeruginosa. This model indicates that the target compound (bio-AgNP) affected functional genes involved in the biofilms' formation and virulence production, mainly in PA14. This implies that the presence or accidental release of low concentrations of AgNPs may cause ecological imbalances, possibly leading to an increase in P. aeruginosa virulence. Thus, the new concept of antivirulence therapy needs to be carefully studied. Understanding bacterial virulence and pathogenicity mechanisms, which depend on a variety of extracellular and cell-associated factors, is essential for the development of potential targets for antivirulence therapy.