Quorum-quenching potential of recombinant PvdQ-engineered bacteria for biofilm formation

Quorum sensing (QS) is a core mechanism for bacteria to regulate biofilm formation, and therefore, QS inhibition or quorum quenching (QQ) is used as an effective and economically feasible strategy against biofilms. In this study, the PvdQ gene encoding AHL acylase was introduced into Escherichia coli (DE3), and a PvdQ-engineered bacterium with highly efficient QQ activity was obtained and used to inhibit biofilm formation. Gene sequencing and western blot analysis showed that the recombinant pET-PvdQ strain was successfully constructed. The color reaction of Agrobacterium tumefaciens A136 indicated that PvdQ engineering bacteria had shown strong AHL signal molecule quenching activity and significantly inhibited the adhesion (motility) of Pseudomonas aeruginosa and biofilm formation of activated sludge bacteria in Membrane Bio-Reactor (MBR; inhibition rate 51–85%, p < 0.05). In addition, qRT-PCR testing revealed that recombinant PvdQ acylase significantly reduced the transcription level of QS biofilm formation-related genes (cdrA, pqsA, and lasR; p < 0.05). In this study, QQ genetically engineered bacteria enhanced by genetic engineering could effectively inhibit the QS signal transduction mechanism and have the potential to control biofilm formation of pathogenic bacteria in the aquaculture environment, providing an environmentally friendly and alternative antibiotic strategy to suppress biofilm contamination.


Introduction
Studies have shown that more than 70% of bacterial infections involve biofilms (Lamin et al. 2022;Zhang et al. 2022). As a structured, assortative, and functional microbial community, biofilms render microorganisms highly resistant to antibiotics and disinfectants (Ciofu et al. 2010;Koo et al. 2017;Gebreyohannes et al. 2019). The abuse of antibiotics not only poses a serious threat to the environment (Klein et al. 2018;Ondon et al. 2020), but also exerts selective pressure on microorganisms. This induces the development of microbial multiple drug resistance (Ben et al. 2018), leading to the enhancement of bacterial drug resistance (Christiaen et al. 2014;Q. Jiang et al. 2019;Khan et al. 2021;Raju et al. 2022). Furthermore, some bacteria in biofilms are a thousand times more resistant to antibiotics than bacteria in the planktonic state (Rasmussen and Givskov 2006a;Liao et al. 2019). Thus, the limitations of antibiotics against membranous bacteria have prompted the search for new methods to solve the challenge of biofilm formation by specific bacteria.
Quorum sensing inhibition or quorum quenching (QQ) is an effective and economically feasible anti-biofilm strategy. Quorum sensing (QS) is a phenomenon whereby microorganisms "communicate" with one another through chemical signaling molecules and can regulate changes in their population density by recognizing themselves and the concentration of signaling molecules released by other bacteria (Pawar and Lahiri 2018;Wu et al. 2020). Studies have shown that the drug resistance exhibited by most pathogenic 1 3 microorganisms is closely related to QS (Sakr et al. 2018;Haque et al. 2018;Taşkan et al. 2022). Pathogens such as Pseudomonas aeruginosa PAO1 and Pantoea ananatis regulate biofilm formation and the expression of virulence factors by secreting acyl-homoserine lactone (AHL) signaling molecules, thereby enhancing their pathogenicity and resistance to conventional drugs and the host's immune system (Hazan et al. 2016;Choi et al. 2021). Thus, researchers mediated bacterial QS systems by regulating the concentration of AHL molecules, thereby utilizing QQ as a potential antibacterial strategy (Murugayah and Gerth. 2019;Okafor et al. 2022).
In nature, QQ bacteria have been widely used for the prevention and treatment of QS pathogenic microorganisms and biofilms, such as Bacillus SDC-U1 (Noori et al. 2022), Lactobacillus crustorum ZHG 2-1 (Cui et al. 2020), and Bacillus safensis M146 (Yu et al. 2020). These QQ bacteria achieve QS inhibition by producing enzymes with AHL degradation activity and reducing AHL signal concentrations (Bzdrenga et al. 2017;Ghanei-Motlagh et al. 2021). Interestingly, the QQ enzyme does not kill the pathogens; however, it reduces the concentration of signal molecules (Lin et al. 2003;Yao et al. 2022). The mRNA expression of the AHL-mediated QS circuit and AHL-mediated virulence factors in P. aeruginosa was investigated in presence of QQs, and qPCR analysis showed that QQs actively reduce the expression of the LasI and RhlI synthase protein, and prevent production of 3-oxo-C12-HSL and C4-HSL, respectively (Kalgudi et al. 2022). Thus, the transcription regulator of QS-related genes is absent, thereby inhibiting pathogen biofilm formation or the expression of virulence factors of the pathogen, and avoiding the generation of bacterial drug resistance (Rasmussen and Givskov 2006b).
The PvdQ acylase has strong AHL degradation activity and can degrade multiple AHL signals (such as C4-HSL, C8-HSL, C12-HSL, 3-oxo-C12-HSL) (Liu et al. 2019). Vogel et al. (2020) immobilized a recombinant PvdQ acylase on PDMS silica gel tablets, resulting in approximately 50% inhibition of P. aeruginosa biofilm formation. Malesevic et al. (2020) cloned and expressed two kinds of AHL lactone enzymes, YtnP and Y2-aiiA, in the Escherichia coli prokaryotic system through genetic engineering. The two enzymes were isolated from Burkholderia cepacia strain BCC4135 and were shown to inhibit biofilm formation and the production of virulence factors by interfering with the las, iqs, rhl, and pqs genes in the QS pathways of P. aeruginosa. Khalid et al. (2022) isolated PvdQ acylase from P. aeruginosa QSP01 strain which inhibited P. aeruginosa biofilm formation by approximately 50%. However, there is no systematic report of the QQ effects of in vitro recombinant PvdQ-engineered bacteria on P. aeruginosa, or on the molecular mechanisms of recombinant PvdQ-engineered bacteria on QQ. Furthermore, the potential of recombinant PvdQ-engineered bacteria resisting biofilm pollution in bioreactors also needs to be evaluated. Although P. aeruginosa is very important in medical research, the formation of biofilms and the water pollution it causes are also very common in nature (Sanz-García et al. 2021). Interestingly, long-chain AHL signal molecules (such as C10-HSL and C12-HSL) were key AHLs during wastewater biofilm development process, which was closely related with the biofilm initial attachment process (Wang et al. 2019). So, researching and addressing PvdQ-engineered bacteria may help to build an efficient and novel QQ genetic engineered bacterium that may be used to control biofilm formation by resistant pathogenic bacteria in the medical departments and may also aid in controlling biofilm formation on the Membrane Bio-Reactor (MBR) (Paluch et al. 2020).
To address the above challenges, this paper evaluated the QQ effect of recombinant PvdQ-engineered bacteria on bacterial biofilm formation. A PvdQ-engineered strain was constructed using the E. coli prokaryotic expression system, and the degradation activity of the AHL signal molecules of the PvdQ-engineered bacteria was verified by the Agrobacterium tumefaciens biosensor bacteria. The effect of PvdQengineered bacteria on biofilm attachment and formation of P. aeruginosa as well as biofilm scaling in MBR was examined by co-culturing at different cell proportions. Finally, the expression levels of QS-related genes cdrA, pqsA, and lasR by qRT-PCR on bacterial biofilm formation of PvdQengineered bacteria were analyzed.

Materials
The TIANprep Midi Plasmid Kit was used for plasmid or genomic DNA extractions, and the Efficom BL21 (DE3) Chemically Competent Cell was the host-expressing cell; the Uniclone One Step Seamless Cloning Kit recombinant plasmid construction kit and the total RNA Extraction Kit were purchased from Genesand company. T4 DNA Ligase, BamH I restriction endonuclease, and other reagents were used to construct the expression vectors. Kanamycin (30 mg/mL), ampicillin (10 mg/mL), 5-bromo-4chloro-3-indolyl β-d-galactoside (X-Gal), and isopropyl β-d-thiogalactoside (IPTG) solutions were purchased from Takara and used as the recombinant strain selection medium. The Mag-Beads His-Tag protein purification beads were used for the purification of the His-Tag target protein, and the target protein concentration was determined by BCA Protein Assay Kit. Western blot analysis was performed on the target protein (PvdQ enzyme). Anti-6 His-Tag mouse monoclonal antibody was used as the primary antibody, HRP-conjugated rabbit anti-mouse IgG was the second antibody, and the EasyBlot ECL kit was used to observe the color reaction. The AHL standard reagents were purchased from Sigma (C4-HSL, C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, 3-oxo-C10-HSL, C12-HSL, 3-oxo-C12-HSL, C14-HSL, 3-oxo-C14-HSL) and dissolved with methanol (HPLC grade, 99.9%), sealed, and stored at − 20 °C.

Strain source and culture
Agrobacterium tumefaciens A136 (NTL4) was purchased from Hongsai Biotech (Hangzhou, China) and was used for detecting the activity of AHL signaling molecules. Pseudomonas aeruginosa (QS009B) was purchased from the National Center for Medical Culture Collections (CMCC). Sludge bacteria samples were collected from six Membrane Bio-Reactors (MBR) in the laboratory (Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China). Six groups of samples were collected in different glass bottles, mixed, and 5 mL of sludge samples removed and mixed with 50 mL of stroke-physiological saline solution. This was then sonicated at 20% power for 15 s and subjected to centrifugation at 3000 rpm for 1 min to remove large particulate material. The supernatant was then subjected to centrifugation at 4500 rpm for 5 min, and the precipitate was re-suspended in 15 mL of saline for culturing the biofilm.

DNA extraction and PCR amplification
The PvdQ gene sequence (ID: NC002516.2) was obtained from NCBI GenBank as a reference, and the upstream and downstream homology arms (containing BamH I and Hind III restriction sites with His-tag) were selected in pET-28a plasmid polyclonal region (MCS). The primers were designed (Table 1, online Oligo) and synthesized by Sangon Biotech (Shanghai, China). The restriction sites are underlined in Table 1. Total genomic DNA from P. aeruginosa was extracted and utilized for PCR amplification. The PCR amplification conditions were 95 °C pre-denaturation 3 min; 35 cycles of 95 °C denaturation 30 s, 57 °C annealing 90 s, 72 °C extension 1 min; followed by a final extension of 72 °C for 10 min. An adequate volume of the PCR amplicons was resolved using gel electrophoresis, viewed, and purified. The amplicons were stored at 4 °C.

Construction of pMD-PvdQ cloning vector
The PvdQ gene fragment was ligated with the pMD19-T cloning vector and transformed into E. coli JM109 competent cells using the heat shock method. The transformation steps were incubation of 100 μL of JM109 competent cells on ice for dissolution, addition of 10 μL of connection system to JM109, and mixed gently. This was incubated on the ice for 30 min, then placed in a 42 °C water bath for 90 s, and then immediately placed on ice for another 3 min. Then, sterile SOC medium (800 μL) was added and incubated for 1 h at 37 °C for oscillatory activation. The appropriate coating was applied to solid medium containing X-Gal, IPTG, and ampicillin and incubated at 37 °C for 24 h. Positive monoclones were selected for gene sequencing, and the correctly verified recombinant cloning vector was named pMD-PvdQ.

Construction of pET-PvdQ expression vector
The Uniclone One Step Seamless Cloning Kit was performed according to the manufacturer's instructions. An appropriate amount of linearized pET-28a vector was combined with the target gene PvdQ to generate a 10 μL pET-PvdQ recombinant vector. The upstream and downstream of the experimental target gene contained approximately 15-20 base pair (bp) homology arms from the vector restriction site, which improved the recombination efficiency according to the principle of base complementary pairing. The recombinant vector (10 μL) was transformed into 100 μL BL21 competent cells, using the transformation conditions described above. An appropriate amount of bacterial broth was plated on LB solid medium containing X-Gal, IPTG, and kanamycin, and cultured at 37 °C for 24 h. The white colonies on the plates were selected. The positive clones were verified by colony PCR, and samples requiring enzyme digestion were selected and then sequenced at Sangon Biotech (Shanghai, China). The sequencing was performed to ensure that the expression sequence constructed was not mutated and had no frameshift mutations. The correctly sequenced recombinant expression vector was named pET-PvdQ.

SDS-PAGE gel electrophoresis
Ten microliters of recombinant bacteria containing pET-PvdQ and pET-28a empty plasmids was inoculated into a conical flask containing 100 mL LB medium with 10 mM kanamycin and cultured overnight at 37 °C (OD 600 was approximately 0.4-0.6). The temperature was reduced to 25 °C, and 0.5 mM IPTG and 0.5 g α-lactose were added and cultured for an additional 8 to 16 h. The bacterial cells were collected by centrifugation at 5000 rpm for 15 min and sonicated at 50% power. The sonicator was operated for 4 s and then intermittently for 10 s, for a total of 60 min. After sonication, centrifugation was performed to collect the supernatant, and an appropriate volume of the supernatant was used for SDS-PAGE protein gel electrophoresis.

Protein purification and western blot analysis
The PvdQ protein was purified according to the Mag-Beads His-Tag protein purification beads manufacturer's instructions, and the protein concentration was determined by BCA Protein Assay Kit. Purified PvdQ protein was quantified by SDS-PAGE 15% gel electrophoresis, followed by western blotting. Membrane transfer conditions were 150 V for 150 min. The primary antibody (Anti-6 His-Tag mouse monoclonal antibody) was incubated overnight at 4 °C, and the secondary antibody (HRP-conjugated Rabbit anti-mouse IgG) was incubated at room temperature for 2 h. A color rendering reaction was performed by the EasyBlot ECL kit and observed in an imaging system to detect target protein expression.

AHL signal degradation testing
The QQ activity of the PvdQ-engineered bacteria was detected by the chromogenic reaction of the A. tumefaciens A136, which was performed according to the method of Khalid et al. (2022) with slight modifications. Briefly, the A136 bacterial culture (OD 600 = 0.8 − 1.0) containing X-Gal (10 mg/mL) was added to a 96-well microplate or 1.5-mL centrifuge tube. The C4-HSL, C8-HSL, C12-HSL, 3-xox-C12-HSL signal molecule solution, and recombinant bacterial solution (OD 600 = 0.8) were mixed in specific proportions (each AHL total concentration about 500 ng/L), incubated for 6 h at 28 °C, and then added to the A136 indicator bacterial sample well. Each AHL pure dilution and E. coli transformed with plasmid blank were used as the controls. Each group was performed in triplicate with a final liquid volume of 200 μL per well, incubated at 28 °C, and the depth of color change (blue) in the wells was recorded.

Movability measurement
The motility of swarming assays of P. aeruginosa was performed according to the method provided by Yin et al. (2021). Briefly, overnight cultures of P. aeruginosa were inoculated onto LB agar (1%) centers containing serial dilutions of PvdQ acylase (0, 0.1, 0.3, and 0.5 mg/mL), incubated at 37 °C for 24 h, and the diffusion (coverage) radius of P. aeruginosa on agar plates was recorded.

Biofilm microscopy analysis
The biofilm formation of P. aeruginosa was analyzed using light microscopy according to Meena et al. (2021). Briefly, P. aeruginosa (OD 600 = 0.8) was added to a 6-well plate at different concentrations of PvdQ acylase (0, 0.1, 0.3, and 0.5 mg/mL, 2 mL of the total volume per well), and a coverslip (20 × 20 mm area, 0.17 mm thick) was placed over each well. The mixture was incubated at 37 °C for 24 h. The coverslips were removed, carefully washed with distilled water, and stained with 0.1% (m/v) crystal violet. The biofilm was observed at 600 (40 × 15) times using the high-powered lens of a light microscope.

Biofilm assay
The effect of PvdQ-engineered bacteria on the extent of P. aeruginosa biofilm formation (biofilm formation index, BFI) was determined using the crystal violet (CV) method . The concentration of QQ-engineered bacteria (PvdQ) and the QS bacterium P. aeruginosa in LB broth was adjusted to an OD 600 = 0.5. The P. aeruginosa and QQ bacterial cells were then co-cultured at varying ratios of 1:0.3, 1:1, and 1:3 or inoculated with the diluted PvdQ enzyme (0.1, 0.3, and 0.5 mg/mL). In addition, the MBR sludge bacteria concentration was adjusted to OD 600 = 0.5, and the sludge bacteria and the PvdQ-engineered bacteria were co-cultured at a cell ratio of 1:1, 1:3, and 1:5. After 24-h incubation at 37 °C in the 96-well plates, CV was added and the cells were stained. The level of biofilm formed on the wells at 570 nm was quantified by a microplate reader (Noori et al. 2022). Cells containing E. coli with the transformed pET-28a empty plasmid, sludge bacteria, and P. aeruginosa were used as controls. All tests were performed six times.

Expression assay of QS-related genes
Genes related to QS-mediated biofilm formation, such as the AHL receptor protein genes lasR, pqsA, and cdrA (Table 2) associated with biofilm formation, were retrieved from GenBank. The rpsl gene was selected as the internal control for normalizing gene expression. The specificity of the qRT-PCR was determined by melting curve analysis, calculating the threshold period (Ct) value; for relative quantification, the Ct values were normalized to the rpsl gene Ct, and (Δct sample -Δct control) method was performed (Yin et al. 2021). The PvdQ enzyme (0.1 mg/mL) was grown overnight, at 28 °C and 1 3 150 rpm, in LB medium containing P. aeruginosa, with three parallel settings for each group. Total RNA was extracted from bacterial somatic cells using the Total RNA Extraction Kit. The mass concentration of RNA was then analyzed by an ultramicro spectrophotometer (TIANGEN, Beijing, China). The cDNA was generated from the total RNA using the PrimeScript™-RT-reagent-Kit (Takara, Dalian, China) and used as a template for the qRT-PCR (LightCycler® 96 SW 1.1). The mRNA expression levels of the QS-related genes were measured using the LightCycler® real-time PCR system and the TB Green® Fast qPCR Mix (2 Conc.) kit (Takara, Dalian, China).

Statistical analysis
All the experiments performed were performed in at least three independent replicates. The data obtained in this experiment, such as Biofilm assay and qRT-PCR, were analyzed by SPSS and Duncan software and expressed as percentage. The p < 0.05 (*) was considered as having a significant level for all statistical tests.

PvdQ-engineered bacterial constructs
The primers for the P. aeruginosa strain PAO1 PvdQ gene (ID: NC002516.2) were designed as a template. Following PCR amplification, appropriate volumes of the amplicons were resolved on a 1% agarose gel. As expected, a single band between 2000 and 3000 bp representing the PvdQ (2280 bp) gene was observed (Fig. 1A).
The PCR amplicons were cloned into the pMD-19 T cloning vector and transformed into E. coli competent receptor cells (JM109). Single clones were selected and verified by colony PCR. The plasmid was extracted and sequenced. Correct sequences indicated the success of PvdQ gene amplification which were submitted to GenBank (ID: 2,642,871). Subsequently, the pET-28a expression vector was constructed and transformed into the E. coli receptor cells (DE3), resulting in a single white colony (Fig. 1B). Single colonies were selected and verified by colony PCR. The plasmids were extracted and analyzed by sequencing and were aligned to the correct recombinant bacteria. The vector was named pET-PvdQ, and the recombinant PvdQ-engineered bacteria were successfully constructed.

Recombination of PvdQ expression and western blot assays
The PvdQ-engineered bacteria were cultured in LB medium containing IPTG (0.5 mM). The bacteria were collected by centrifugation, and the harvested protein supernatant was sonicated and used for gel electrophoresis analysis. As shown in Fig. 2A, E. coli-pET-PvdQ (lane 2) was compared with E. coli-pET-28a (lane 1). The bands were compared with the molecular marker which indicated that the size of the target protein band was as expected at 84 kDa (indicated by the red arrow). The recombinant protein yield was improved by adding 0.5 mM IPTG and by incubating at 25 °C for 8 h. The PvdQ crude protein was purified according to the Mag-Beads His-Tag protein purification beads manufacturer's instructions. The purified protein concentration was measured by the BCA method. The formula of the standard curve of the protein concentration is y = 0.5717x − 0.0052, R 2 = 0.9998. The mean concentration of PvdQ after protein purification was approximately 2.870 mg/mL. Western blotting demonstrated that there was a band that the expected size of 84 kDa (Fig. 2B), indicating that the recombinant protein achieved efficient expression in E. coli.

PvdQ-engineered bacteria activity testing
The A. tumefaciens cultures containing X-Gal were used as an indicator bacterium for detecting AHL. AHL is a signal molecule that induces the expression of β-galactosidase enzyme gene in strain A136. The enzyme acts on its substrate, X-Gal, causing the indicator bacteria to produce green, with the degradation effect of AHL being determined by the depth of the blue. The results are shown in Fig. 3 whereby group 1 is pure A. tumefaciens cultures which do not have color, and group 2 represents the well containing C12-HSL mixed with the engineered bacteria. The 28 °C reaction was added to the well containing A. tumefaciens cultures after 6 h which appears dark blue. Group 3 is a control group containing E. coli-pET-28a cultures and appears light blue which is due to the presence of the LacZ gene in E. coli. Figure 3 (1) is a control group containing E. coli-pET-28a with C12-HSL mixed cultures and appears dark blue. Group 4 is the modified engineered PvdQ bacterial culture and does not appear blue. Group 5 is the reaction of PvdQ-engineered bacteria with C12-HSL (500 ng/L) and was faintly blue. From Fig. 3 and the supplementary figure,  Fig. 3 (2) experimental results confirmed that the modified PvdQ-engineered bacteria had efficient QQ activity and degraded long-chain AHL signal molecules with efficient degradation activity against such as C4-HSL, C8-HSL, C12-HSL, and 3-oxo-C12-HSL.  Figure 4A shows that the PvdQ enzyme inhibited the flagellar-mediated motility of P. aeruginosa in a dose-dependent manner. Inhibition of bacterial motility was observed in the 0.1 mg/mL PvdQ enzyme treatment group, indicating that P. aeruginosa motility was significantly decreased by inhibiting initial cell attachment to the surface, or by inhibiting QS in P. aeruginosa. In addition, the level of P. aeruginosa on the coverslips decreased in the presence of PvdQ enzyme (Fig. 4B). Light microscopy showed that the biofilm in the control group completely covered the surface of the coverslip. However, the surface covered by P. aeruginosa noticeably decreased on the coverslip, and cell populations were in the 0.3 mg/mL and 0.5 mg/mL PvdQ enzyme test groups. Flagellar-driven motility (swarming and aggregation movements) plays a crucial role in the initial attachment of bacteria, prompting the spreading of the biofilm which ultimately covers the matrix surface (Mostafa et al. 2020); thus, a decline in motility would be great for the capacity of P. aeruginosa to form biofilms.

Biofilm assay
The effect of the recombinant PvdQ strain on the level of P. aeruginosa biofilm formation was determined by CV in 96-well microplates. Compared with the pure culture of P. aeruginosa and the control (E. coli transformed pET-28a empty plasmid), the addition of the recombinant PvdQ strain group showed substantial inhibition of P. aeruginosa biofilm formation (Fig. 5), with the inhibition effect being cell density-dependent. Furthermore, the PvdQ protein supernatants obtained after cell fragmentation also showed considerable inhibition of biofilm formation in a concentration-dependent manner. As shown in Fig. 6, the addition of recombinant PvdQ engineered strains showed noticeable MBR sludge bacteria biofilm inhibition compared with the transformed pET-28a. Similarly, P. aeruginosa bacteria demonstrated the best inhibition at a cell ratio of 1:3. Overall, approximately 51 to 85% inhibition of bacterial biofilm formation was observed for the PvdQ-engineered strains (p < 0.05).

QS-associated gene expression testing in qRT-PCR
To further investigate the potential molecular mechanism of biofilm inhibition in P. aeruginosa by PvdQ-engineered bacteria, the transcription levels of QS-related genes were determined using qRT-PCR. For example, the lasR receptor protein gene is responsible for sensing AHL signaling molecules, the cdrA adhesin gene is associated with biofilm formation, and the pqsA gene also plays key regulatory roles in the biosynthesis of multiple virulence factors (Reichhardt et al. 2018;Grossman et al. 2020). As shown in Fig. 7, the transcription levels of the cdrA, pqsA, and lasR genes in the 0.1 mg/mL acylase treatment group were 0.59, 0.50, and 0.84, respectively, which were significantly reduced compared to the control group (1.0) (p < 0.05). Therefore, qRT-PCR analysis of P. aeruginosa confirmed that the genes involved in the synthesis of AHL receptors, virulence factor production, and biofilm formation were significantly reduced (p < 0.05). This could explain the significant QS inhibition effect of PvdQ-engineered bacteria on P. aeruginosa in motility and biofilm formation (Figs. 4 and 5). The biofilm formation and QS-related gene (lasR, cdrA, and pqsA) transcription levels of P. aeruginosa. ▨ represents the control groups without any treatment; ▄ represents P. aeruginosa treated with 0.1 mg/mL PvdQ enzyme concentration (*p < 0.05)

Discussion
Numerous studies have confirmed that QS systems have the ability to highly regulate microbial virulence (Zhang and Li 2016). QS has been used as an important regulatory mechanism for biofilm formation and drug resistance in pathogenic bacteria (Anwar et al. 1990;Alayande et al. 2018). Pseudomonas aeruginosa is one of the most dangerous malignant strains worldwide, causing serious health problems for both humans and animals (Milivojevic et al. 2018). Furthermore, P. aeruginosa is able to evade antimicrobial agents, antibiotics, and host immune systems due to their ability to form relevant biofilm barriers (Pang et al. 2019), which are mainly controlled by the QS mechanism (Pawar and Lahiri. 2018). In this study, P. aeruginosa was the focus of QS interference. We were able to construct QQ functional strains through genetic engineering, construct QQ functional strains from endogenous block bacterial QS-AHL signal communications (such as C4-HSL, C8-HSL, C12-HSL, 3-oxo-C12-HSL), control biofilm formation, and reduce drug resistance. This study has positive implications for medical treatment, membrane pollution, and other fields, and is crucial for future strategies to prevent and control biofilm pollution.
The recombinant PvdQ acylase enzyme-engineered strain was constructed using E. coli (DE3) and verified for its efficient QQ activity, which was further demonstrated in reducing AHL signaling and inhibiting bacterial biofilm formation. Firstly, the QQ-AHL capacity of PvdQ-engineered bacteria was investigated using the bioindicator strain A. tumefaciens. It was shown that when C12-HSL was added to the PvdQ-engineered bacteria, A. tumefaciens A136 produced cyanine which was qualitatively analyzed. This indicated that PvdQengineered bacteria reduced AHL signaling. Meanwhile, the biofilm formation inhibition experiments of P. aeruginosa also showed that the PvdQ-engineered bacteria and their secreted extracellular proteases can degrade AHL signaling molecules and block the QS communication of P. aeruginosa, thus inhibiting the initial attachment of the biofilms . Biofilm formation in P. aeruginosa occurs in sequence, starting with the initial attachment, followed by reproduction or the mushroom-shaped microcolony stage, and finally by the biofilm maturation stage (Klausen et al. 2003). With advancements in genetic engineering, understanding biological properties has improved, and recombinant Ahl-1 lactonase from Sakr et al. (2018) achieved significant results in inhibiting biofilms in P. aeruginosa (44.3-80%, p < 0.05). Similarly, in this study, biofilm formation measurements showed that when co-cultured with PvdQ-engineered cells (1:3), biofilm formation in P. aeruginosa decreased by approximately 75% (p < 0.05), and biofilm formation in MBR-activated sludge bacteria decreased by approximately 85% (p < 0.05). Notably, PvdQ-engineered bacteria had no effect on the total bacterial biomass, implying that PvdQ acylase induced the blocking of bacterial QS-AHL signal communications and inhibited or delayed biofilm formation, and therefore, QQ probiotic strategies were unlikely to exert selective pressure for resistance to bacteria to reduce the generation of bacterial resistance.
Swarming-related motility mediated by the flagellum is the initiating step of activating cell-to-surface attachment causing biofilm formation, while motility also plays an important role in the level of virulence of pathogenic bacteria (Mostafa et al. 2020); thus, inhibition of P. aeruginosa motility is a key step to prevent biofilm formation. This study showed that PvdQ acylase not only inhibited the diffusion of P. aeruginosa, but also suppressed adhesion (motility). This phenomenon was more pronounced in the optical microscopy images. The P. aeruginosa formed sparse and loose biofilms in the presence of PvdQ acylase (0.3 mg/mL).
Furthermore, the significant QQ effect of the PvdQengineered bacteria was also confirmed at the gene transcription level. The qRT-PCR in this study showed that the PvdQ-engineered bacteria significantly inhibited the transcription level of the QS-related genes (lasR, cdrA, and pqsA) (p < 0.05). The lasR genes are involved in the AHL signaling receptor protein, the cdrA adhesion genes are associated with biofilm formation (Reichhardt et al. 2018), and the pqsA genes play a key regulatory role in the biosynthesis of various virulence factors (Grossman et al. 2020). These genes are essential in regulating P. aeruginosa for host tissue invasion, in the initial stages of biofilm formation, and in promoting virulence expression. Therefore, through the degradation of AHL signals, PvdQ-engineered bacteria inhibit the normal functioning of QS-related gene pathways and from the endogenous inhibition of virulence expression and biofilm formation of P. aeruginosa.
In conclusion, the interference of QS systems by QQengineered bacteria can be a promising strategy to combat biofilm contamination. This study demonstrated considerable inhibition of biofilm attachment to P. aeruginosa cells by recombinant PvdQ acylase. Furthermore, PvdQ-engineered strains had QQ viability in inhibiting MBR reactor membrane contamination, opening the way for PvdQ-engineered bacteria in the application of antibiotic replacement biofilm contamination. Future research prospects are to improve bacterial fixation techniques, conduct better survival in different environmental conditions, and functional stability, and lay the foundation for promoting and applying engineered bacteria.

Conclusion
In this study, a QQ genetically engineered bacterium was successfully constructed by integrating the PvdQ gene into E. coli and recombinantly expressing the PvdQ acylase with a highly efficient QS-AHL quenching capability using the E. coli prokaryotic system. Agar plates and microscopic analysis confirmed that the engineered bacteria had a strong inhibitory effect on the movement and adhesion of P. aeruginosa. Co-culturing of engineered bacteria with MBR sludge bacteria (cell ratio 1:3) significantly reduced the amount of biofilm formation of sludge bacteria in the MBR reactor (about 51-85%, p < 0.05), demonstrating the potential of QQ against adverse bacterial biofilm pollution. By qRT-PCR, we analyzed the molecular mechanisms of the QS-related pathway (cdrA, pqsA, and lasR) inhibition in P. aeruginosa. This paper provides an environmentally friendly and economically feasible probiotic strategy to address the increasing challenges of bacterial resistance and its biofilm contamination in areas such as aquaculture.
Author contribution All authors contributed to the study conception and design. Material preparation and data collection and analysis were performed by Y. X., J.T., Z.L., and K.Z. The first draft of the manuscript was written by J.L., and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
All authors have read and agreed to the published version of the manuscript.

Conflict of interest
The authors declare no competing interests.