The Role of Yinqiao Powder in Modulating Pseudomonas aeruginosa Biofilm and Virulence Factors

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative, opportunistic pathogen that ranks among the leading causes of nosocomial infections. Human health and food processing industries like dairy, poultry, ready-to-eat food, meat, and aquaculture have been severely affected by biofilm-producing P. aeruginosa.^1–3 This bacterium is particularly prevalent in immunocompromised individuals with cystic fibrosis (CF), implanted medical devices, and chronic wound infection.⁴ The primary challenges in treating P. aeruginosa infections are the secretion of multiple virulence factors, the formation of biofilm, and the development of multi-drug resistance (MDR).⁵ The biofilms of P. aeruginosa confer bacterial drug resistance by impeding antimicrobial penetration, inducing a slow-growth phenotype through microenvironmental alterations, and stimulating adaptive stress responses and cellular differentiation.^6,7

In addition, the bacterium’s motility and secretion of extracellular virulence factors, such as lectin, elastase, alkaline protease, pyocyanin, rhamnolipids are critical to its pathogenesis. Consequently, it is widely believed that elucidating the mechanisms by which P. aeruginosa regulates the expression of virulence genes is essential for developing alternative therapeutic strategies to control and prevent bacterial infections.⁸

Quorum sensing (QS) is a cell-density-dependent intercellular communication system that relies on the synthesis and detection of diffusible signal molecules, including the LasI/LasR (las), and RhlI/RhlR (rhl), Pseudomonas quinolone signal (pqs), and iqs systems.^9–11 An increasing body of evidence suggests that these QS systems are critical regulators of virulence, swarming motility, biofilm formation, and antibiotic resistance in P. aeruginosa.^12–14 Notably, two homologous N-acylhomoserine lactone (AHLs) QS systems, the LasI/LasR and RhlI/RhlR systems, are primarily responsible for activating these associated behaviors.¹⁵

In the P. aeruginosa QS systems LasI/LasR and RhlI/RhlR, the signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), respectively, interact with the transcriptional regulators LasR and RhlR to activate multiple virulence factors, including lectin, elastase, pyocyanin, alkaline protease, rhamnolipids, and exotoxin A.¹⁶ In addition, the pqs system, a group of compounds known as 4-hydroxy-2-alkylquinolines (HAQs), upregulates the expression of virulence-associated genes by interacting with the LasI/LasR and RhlI/RhlR systems. Furthermore, QS systems interact with small regulatory RNAs (sRNAs) to modulate virulence factor expression. Studies have shown that sRNA PhrD stimulates the translation of rhlR, while another sRNA, P27, directly represses the translation of rhlI mRNA by P. aeruginosa.^17,18

In recent years, the widespread application of antibiotics in clinical practice has led to the emergence of multi-drug resistant P. aeruginosa, which has caused great difficulties in clinical treatment. With the onset of the “post-antibiotic era”, there has been a growing inclination towards non-antibiotic therapeutic treatments. Anti-QS drugs are a novel anti-virulence and anti-biofilm approach to target pseudomonal infections.¹⁰ Inhibition of the QS could be achieved by-quorum quenching enzymes (such as AHL-lactonases, AHL-acylases, and AHL-oxidoreductase) and quorum sensing inhibitor which could be synthesized or derived from microorganisms, animals, or bioactive phytochemicals (such as curcumin, geraniol, and ajoene).^10,11,19 Yinqiao Powder, a classical formulation of Traditional Chinese Medicine (TCM) renowned for its heat-clearing and detoxification properties, exhibits low toxicity and a reduced propensity to induce antibiotic resistance.²⁰ Contemporary research has unveiled its anti-inflammatory and anti-allergic attributes.²¹ Previous studies suggest Yinqiao Powder may inhibit P. aeruginosa biofilm formation, potentially influencing the pathogen’s virulence.^22,23 Nevertheless, comprehensive clinical and fundamental investigations are imperative to elucidate the precise mechanism of Yinqiao Powder’s impact on P. aeruginosa virulence and to ascertain its clinical utility.

Herein, we demonstrated that Yinqiao Powder effectively suppressed P. aeruginosa biofilm formation and virulence, as evidenced by reduced pyocyanin and rhamnolipid production and impaired swarming motility. Notably, sRNAs PhrD and P27 may contribute to Yinqiao Powder’s suppression of P. aeruginosa virulence.

Materials and Methods

Bacterial Strains and Growth Conditions

Strain utilized in this study is P. aeruginosa wild-type strain PAO1 as described in our previous work.¹² Unless otherwise specified, P. aeruginosa clones isolated from blood plates were cultured overnight at 37°C in Luria-Bertani broth (LB, Oxoid) with 200 rpm agitation, subsequently washed twice with 0.9% NaCl, added 100 μL of cultures to 3 mL LB, and incubated to mid-log phase (0.5 McFarland, 1.5 × 10⁸ CFU/mL) prior to experimental procedures. When required, Yinqiao Powder (Fengchun, Zhongjiang, Sichuan, China) was added at concentrations of 256, 128, 64, or 32 mg/mL.

MIC of Yinqiao Powder

The MIC of Yinqiao Powder was determined using the agar dilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (M100). Yinqiao Powder was incorporated into Mueller–Hinton agar (MHA, Oxoid) at concentrations ranging from 16 to 512 mg/mL. About 2 μL of a P. aeruginosa suspension standardized to a 0.5 McFarland turbidity standard was inoculated onto each agar plate. After incubation at 37°C for 20 hours, the minimum drug concentration that exhibited no visible bacterial growth was defined as the MIC.

Biofilm Formation Assay

The detection of biofilm was performed as described previously by Jieying Pu.²⁴ Briefly, 100 μL of bacteria suspension at 0.5 McFarland were inoculated into LB supplemented with varying concentrations of Yinqiao Powder in 96-well plates and grown at 37°C for 24 hours. Non-adherent cells were removed by washing with 0.9% NaCl, and adherent biofilms were fixed with methanol, stained with crystal violet, and quantified by measuring the absorbance at OD600 of ethanol-extracted crystal violet.

Pyocyanin Production Assay

Pyocyanin was extracted as described previously.²⁵ P. aeruginosa cultures (0.5 McFarland) were added to the LB medium supplemented with Yinqiao Powder (60 mL culture, 6 mL LB) and incubated at 37°C with shaking (200 rpm) for 20 hours. Subsequently, 3 mL of chloroform was added to 5 mL of the culture supernatant containing pyocyanin, followed by vigorous shaking and centrifugation at 4000 rpm for 5 minutes. The chloroform layer was transferred to a test tube containing 0.5 mL of 0.2 N HCL and shaken vigorously until a pink color developed before centrifugation. The absorbance of the culture and the extracted upper layer solution was measured at 600 and 520 nm, respectively. Pyocyanin production, expressed as micrograms per milliliter of culture supernatant, was calculated using the formula: (A520/A600) × 17.072.²⁶

Rhamnolipid Assay

A rhamnolipid assay was conducted following our previous experimental method.¹² Briefly, the indicated bacteria suspension at 0.5 McFarland were inoculated into M9 medium supplemented with varying concentrations of Yinqiao Powder and incubated at 37°C, with 200 rpm shaking for 16 hours. Subsequently, the culture supernatant was acidified to pH 2.5 ± 0.2 using 40 μL of 1 N HCl, followed by extraction of rhamnolipids with 4 mL of chloroform through vigorous shaking for 4 minutes. After the phase separation, 100 µL of 1 g/l methylene blue and 2 mL of distilled water were added to 3 mL of the chloroform extract. The absorbance of the chloroform layer at 638 nm was measured to determine the rhamnolipid concentration.

Motility Assay

Bacterial strains were cultured to mid-log phase in LB before a 5 μL aliquot was inoculated onto swarming medium, composed of LB supplemented with varying concentrations of Yinqiao Powder, 5 g/l glucose, and 0.5% agar.²⁷ After a 10-hour incubation at 37°C, swarming motility was quantified by measuring the maximum diameter of the circular bacterial migration pattern.

Cytotoxicity Assay

A549 cells (ATCC: CCL-185) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2 for 14 hours. P. aeruginosa cultures (0.5 McFarland) were added to LB medium supplemented with Yinqiao Powder (0, 32, 64 or 128 mg/mL) and incubated at 37°C with shaking (200 rpm) for 2 hours to reach the exponential growth phase, subsequently washed and resuspended with PBS. The resuspended PAO1 strains were inoculated into A549 cells (2 × 10⁵ per well) in 24-well plates at a multiplicity of infection (MOI) of 100. The cultures were incubated in DMEM medium supplemented with 1% FBS for 10 hours at 37°C with 5% CO2. Supernatants were collected from 24-well plates, and cytotoxicity was determined with the LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China).

RT-qPCR Assay

Primer sequences are provided in Table S1. To assess gene expression, we added 30 μL P. aeruginosa cultures at 0.5 McFarland to 3 mL of LB supplemented with Yinqiao Powder (0, 32, 64 or 128 mg/mL) and re-incubated at 37°C, with 200 rpm shaking for 4, 12, or 20 hours. Total RNA was extracted using RNAiso Plus reagent (Takara, Dalian, Liaoning, China), quantified using a NanoDrop 2000 BioSpectrometer (Eppendorf), and converted to cDNA using PrimeScript RT Reagent Kit (Takara, Dalian, Liaoning, China). RT-qPCR was performed with SYBR Green qPCR Master Mix (Takara, Dalian, Liaoning, China) on a ViiA^TM 7 Dx system (Applied Biosystems, Foster, CA, USA), using rpoD as a reference gene for normalization of target gene expression via the 2^−ΔΔCt method.²⁸

Statistical Analysis

Data were analyzed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA) and are presented as mean ± standard deviation (SD). Statistical significance was determined by Student’s t-test. ns, not significant; *0.01 < P < 0.05; **P < 0.01; ***P < 0.001.

Results

The Minimum Inhibitory Concentration (MIC) of Yinqiao Powder

To determine the MIC of Yinqiao Powder against P. aeruginosa strain PAO1, a microdilution assay was performed. Serial dilutions of Yinqiao Powder were prepared and inoculated with a standardized suspension of PAO1. Following 20 hours of incubation, bacterial growth was assessed visually. Colony formation was observed at concentrations ranging from 0 to 256 mg/mL, with a corresponding decrease in colony diameter as drug concentration increased. No visible growth was detected at 512 mg/mL (Figure 1). Based on these results, the MIC of Yinqiao Powder against PAO1 was established as 512 mg/mL.

Figure 1 The MIC of Yinqiao Powder. PAO1 was inoculated quantitatively onto plates containing various drug concentrations and grown to the mid-log phase. Growth of P. aeruginosa was assessed after 20 hours of incubation at 37°C.

Yinqiao Powder Inhibits the Biofilm Formation and the Virulence of P. aeruginosa

Yinqiao Powder Inhibits the Biofilm Formation and the Virulence Factors of P. aeruginosa

To delve into the effects of sub-MIC levels of Yinqiao Powder on P. aeruginosa virulence, P. aeruginosa strain PAO1 was exposed to concentrations of 128 mg/mL, 64 mg/mL, and 32 mg/mL, corresponding to 1/4th, 1/8th, and 1/16th of the minimum inhibitory concentration, respectively. A dose-dependent decrease in biofilm formation was observed (Figure 2A). This finding suggests that Yinqiao Powder can inhibit biofilm formation by P. aeruginosa. To further clarify the impact of Yinqiao Powder on virulence, the synthesis of rhamnolipid and pyocyanin, as well as swarming motility, were assessed in PAO1. When exposed to various sub-MIC concentrations of Yinqiao Powder, PAO1 exhibited a dose-dependent decrease in pyocyanin and rhamnolipid synthesis compared to the control group (0 mg/mL) (Figure 2B and C). Moreover, the swarming motility assay revealed a concentration-dependent reduction in colony diameter compared to the control group (Figure 3). Collectively, these findings indicate that Yinqiao Powder exerts a negative regulatory influence on the synthesis of rhamnolipid and pyocyanin, as well as swarming motility in P. aeruginosa.

Figure 2 The sub-MIC concentrations of Yinqiao Powder influence the biofilm formation, pyocyanin and rhamnolipid synthesis of P. aeruginosa. (A) The sub-MIC concentrations of Yinqiao Powder inhibited biofilm formation. The indicated PAO1 strains were cultured in LB in the 96-well plates at 37°C for 24 hours, and biofilm in the supernatant was determined at OD600. (B) The sub-MIC concentrations of Yinqiao Powder inhibited pyocyanin production. The indicated PAO1 strains were cultured in LB at 37°C for 20 hours, and pyocyanin in the supernatant was determined at OD520. (C) The sub-MIC concentrations of Yinqiao Powder inhibited rhamnolipid synthesis. The indicated PAO1 strains were cultured in M9 medium at 37 °C for 16 hours, and rhamnolipid in the supernatant was measured. Values are the mean ± SD of three independent experiments. *P< 0.05; ***P< 0.001.

Figure 3 The sub-MIC concentrations of Yinqiao Powder influence swarming motility of P. aeruginosa. 5 μL cultures of the indicated strains were spotted onto the swarming medium and incubated at 37°C for 10 hours, and swarming motility was quantified by measuring the maximum diameter. (A) Image of swarming medium. (B) The swarming diameter statistics. Values are the mean ± SD of three independent experiments. ***P < 0.001. Abbreviation: ns, not significant.

Yinqiao Powder Protects Cell From Inactivation by P. aeruginosa

To evaluate the cytotoxic effects of P. aeruginosa treated with varying concentrations (0, 32, 64, and 128 mg/mL) of Yinqiao Powder on A549 cells, a lactate dehydrogenase (LDH) release assay was performed. The results demonstrated that the cytotoxicity of A549 cells induced by P. aeruginosa exposed to sub-MIC concentrations of Yinqiao Powder exhibited a dose-dependent reduction (Figure 4). Specifically, compared with the control group (P. aeruginosa without Yinqiao Powder), the cytotoxicity of A549 cells induced by P. aeruginosa treated with 64 mg/mL and 128 mg/mL of Yinqiao Powder was significantly decreased to 53.40% and 51.92%, respectively. These findings suggest that Yinqiao Powder can effectively attenuate the virulence of P. aeruginosa in a dose-dependent manner.

Figure 4 The cytotoxic effects of P. aeruginosa treated by Yinqiao Powder on cells. After the P. aeruginosa strains treated by Yinqiao Powder (0, 32, 64 and 128 mg/mL) were co-cultured with the A549 cells for 10 hours, the supernatants from the 24-well plates were collected, and cytotoxicity was detected by LDH Cytotoxicity Assay Kit. Cytotoxicity was quantified by measuring LDH release. Values are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01. Abbreviation: ns, not significant.

Yinqiao Powder Suppresses the Expression of lasI, rhlI, pqsA, and phzA Genes in P. aeruginosa

In P. aeruginosa, the synthesis of virulence factors and the formation of biofilm are primarily regulated by the QS system.^15,29 Based on the outcomes of the virulence tests, we hypothesized that Yinqiao Powder might exert a direct regulatory effect by modulating QS-associated virulence genes. To investigate the underlying molecular mechanism of Yinqiao Powder’s influence on virulence traits, we quantified the gene expression relative levels of lasI, rhlI, pqsA, and phzA in PAO1 exposed to sub-MIC concentrations of Yinqiao Powder at 4, 12, and 20 hours (Figure 5A–C). As illustrated in Figure 5, the expression of virulence-associated genes such as lasI, rhlI, pqsA, and phzA were downregulated following sub-MIC concentrations of Yinqiao Powder treatment. The Yinqiao Powder-treated group had the strongest inhibitory effect on QS-related gene expression at 20 h.

Figure 5 The influence of Yinqiao Powder on the expression of lasI, rhlI, pqsA, and phzA. (A–C) P. aeruginosa strain PAO1 in the exponential growth phase was treated with various concentrations (0, 32, 64, 128 mg/mL) of Yinqiao Powder for 4 hours (A), 12 hours (B) and 20 hours (C). Gene expression levels of lasI, rhlI, pqsA, and phzA were assessed using RT-qPCR. The rpoD gene was used as an internal control. Values are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviation: ns, not significant.

Yinqiao Powder Regulates sRNA P27 and PhrD Expression

Quantitative real-time PCR (RT-qPCR) and transcriptome sequencing analyses suggested a regulatory interplay between multiple small regulatory RNAs (sRNAs) and the QS system.^12,24 Based on these findings, we hypothesized that Yinqiao Powder exerts its pharmacological effects by modulating the sRNA-QS-virulence factor signaling pathway. To validate this hypothesis, we measured the expression of sRNAs P27 and PhrD under sub-MIC concentrations of Yinqiao Powder (Figure 6A–C). Our results demonstrated that sub-MIC Yinqiao Powder upregulated P27 expression while downregulating PhrD expression, suggesting a regulatory influence of Yinqiao Powder on sRNA expression.

Figure 6 The influence of Yinqiao Powder on the expression of sRNA P27 and PhrD. (A–C) P. aeruginosa strain PAO1 in the exponential growth phase was treated with various concentrations (0, 32, 64, 128 mg/mL) of Yinqiao Powder for 4 hours (A), 12 hours (B) and 20 hours (C). Gene expression levels of P27 and phrD were assessed using RT-qPCR. The rpoD gene was used as an internal control. Values are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviation: ns, not significant.

Discussion

In this study, phenotypic experiments demonstrated that Yinqiao Powder inhibited P. aeruginosa biofilm formation and the synthesis of multiple virulence factors, including pyocyanin and rhamnolipid, as well as swarming motility. These processes are known to be linked to QS and sRNAs. Subsequent RT-qPCR analysis confirmed the regulatory effects of Yinqiao Powder on QS-associated genes, their downstream virulence genes, and the sRNAs P27 and PhrD. Our findings elucidate the regulatory impact of Yinqiao Powder on P. aeruginosa virulence and suggest that its pharmacological effects may be mediated through the sRNA-QS-virulence factors signaling pathway.

At the top of the QS systems network hierarchy in P. aeruginosa is the las system, which positively regulates the expression of the other three systems. The rhl system operates hierarchically under the control of the las system.⁹ The pqs system can be positively regulated by las system and negatively by rhl system.³⁰ Similarly, the iqs system has a stimulatory effect on pqs and rhl systems.¹⁴ Together, these QS systems form a complex regulatory network that allows P. aeruginosa to adapt to various environmental conditions. QS systems play a crucial role in regulating biofilm formation and the synthesis of virulence factors, including rhamnolipid, alkaline protease, elastase, pyocyanin, and swarming motility in P. aeruginosa.³¹ QS represents a promising therapeutic target for suppressing biofilm and virulence. Currently, numerous studies have demonstrated that phytochemicals possess significant quorum quenching properties, which in turn inhibit biofilm formation and the production of virulence factors in P. aeruginosa. Carotenoid zeaxanthin has been shown to inhibit the formation of biofilm by targeting the LasI/LasR and RhlI/RhlR systems of P. aeruginosa. Similarly, plant flavonoid quercetin demonstrates medicinal properties by suppressing pyocyanin production and biofilm formation in P. aeruginosa.^32,33 Additionally, study has found that geraniol can suppress the biofilm and virulence factors of P. aeruginosa by inhibiting the three QS systems of las, rhl, and pqs.¹⁹ These findings collectively indicate that the promising role of phytochemicals in the development of novel anti-virulence therapies.

Yinqiao Powder, a traditional Chinese medicine, possesses heat-clearing, detoxifying, anti-inflammatory, and detumescent properties. Although its bacteriostatic efficacy may be less potent than antibiotics, it offers several advantages, including holistic conditioning, abundant natural sources, a safe usage profile, and a reduced risk of inducing bacterial drug resistance. This study demonstrated that sub-MIC concentrations of Yinqiao Powder (64mg/mL, 128mg/mL, 256mg/mL) significantly suppressed virulence phenotypes, including biofilm formation, swarming motility, pyocyanin and rhamnolipid production, in a dose-dependent manner. The cytotoxicity assay also demonstrated that the Yinqiao Powder treatment reduced the cytotoxic effects of P. aeruginosa on cells. Forsythia suspense, one of the components of Yinqiao powder, has been found to inhibit the production of QS-regulated virulence factors (such as pyocyanin, elastase and swimming motility) and biofilm formation in P. aeruginosa.³⁴ Their results were similar to ours, but we concomitantly verified the expression of virulence-associated genes, such as lasI, rhlI, pqsA, and phzA, were downregulated upon the Yinqiao Powder treatment. Those results are corroborating a previous study.³⁵ Nevertheless, the variance resides in that Liang’s results demonstrated that the MIC of Yinqiao Powder was 500 mg/mL, while ours was 512 mg/mL. The reason for this difference was that the concentration gradients set by the two were not consistent. Our study was conducted in accordance with the requirements of CLSI, so our MIC should be more accurate. In addition, the downregulation of rhlI and pqsA did not demonstrate a dose-dependent pattern; instead, the inhibitory effect decreased with higher concentrations of Yinqiao Powder. One possible reason is that Yinqiao Powder modulates the expression of rhlI and pqsA by influencing multiple QS systems, or it may regulate the expression of rhlI and pqsA at the post-transcriptional level. In P. aeruginosa, the medicinal herb extracts completely inhibited the pqs system, moderately inhibited the rhl system, and slightly inhibited the las system, suggesting that medicinal herb extracts may selectively target the QS system to inhibit bacterial virulence and ensuring that gene expression remains stable despite external interventions.³⁶ Other studies have demonstrated that the used different doses of carvacrol affected the transcription levels of lasR gene, while, lasI gene was not affected. This result indicated a specific post-translational effect of carvacrol against LasI.³⁷ Our findings substantiate that Yinqiao Powder can effectively inhibit the formation of biofilm and the synthesis of multiple virulence factors by modulating QS system, thereby attenuating bacterial pathogenicity.

In P. aeruginosa, sRNAs are key regulators of virulence phenotype, interacting with the QS system to facilitate adaptation to diverse environments.^38,39 Previous studies have demonstrated that PrrH, regulated by RhlR, controls pyocyanin synthesis through its interaction with phzC/D,¹² while sRNAs P27 and PhrD, in complex with Hfq, respectively, downregulate and upregulate the QS system.^17,18 Our findings indicate that Yinqiao powder alters the expression of P27 and PhrD, suggesting a potential mechanism of action involving the sRNA-QS-virulence factor axis. To elucidate this further, future studies will delve deeper into the molecular underpinnings of Yinqiao powder’s effects on P. aeruginosa virulence and biofilm formation.

Conclusion

In summary, the experimental findings substantiate the hypothesis that Yinqiao Powder possesses antibacterial properties mediated by inhibiting QS and sRNA in P. aeruginosa. This inhibitory effect is manifested in the suppression of biofilm formation, pyocyanin and rhamnolipid synthesis, and swarming motility. Consequently, these results validate the potential of Yinqiao Powder as a quorum-sensing inhibitor and elucidate a mechanistic pathway involving sRNA-QS-virulence factor signaling. This study provides a foundational framework for exploring the therapeutic efficacy of Yinqiao Powder in the management of chronic P. aeruginosa infections.