An In-Depth Study on the Inhibition of Quorum Sensing by Bacillus velezensis D-18: Its Significant Impact on Vibrio Biofilm Formation in Aquaculture

Amid growing concerns about antibiotic resistance, innovative strategies are imperative in addressing bacterial infections in aquaculture. Quorum quenching (QQ), the enzymatic inhibition of quorum sensing (QS), has emerged as a promising solution. This study delves into the QQ capabilities of the probiotic strain Bacillus velezensis D-18 and its products, particularly in Vibrio anguillarum 507 communication and biofilm formation. Chromobacterium violaceum MK was used as a biomarker in this study, and the results confirmed that B. velezensis D-18 effectively inhibits QS. Further exploration into the QQ mechanism revealed the presence of lactonase activity by B. velezensis D-18 that degraded both long- and short-chain acyl homoserine lactones (AHLs). PCR analysis demonstrated the presence of a homologous lactonase-producing gene, ytnP, in the genome of B. velezensis D-18. The study evaluated the impact of B. velezensis D-18 on V. anguillarum 507 growth and biofilm formation. The probiotic not only controls the biofilm formation of V. anguillarum but also significantly restrains pathogen growth. Therefore, B. velezensis D-18 demonstrates substantial potential for preventing V. anguillarum diseases in aquaculture through its QQ capacity. The ability to disrupt bacterial communication and control biofilm formation positions B. velezensis D-18 as a promising eco-friendly alternative to conventional antibiotics in managing bacterial diseases in aquaculture.


Introduction
Aquaculture is a vital industry with global significance, providing a substantial source of protein worldwide.However, the imposition of forced practices on aquatic ecosystems can induce stress in fish, detrimentally impacting aquaculture production [1].Due to their sensitivity to stress factors, fish are highly susceptible to bacterial diseases [2], notably Vibrio spp.This vulnerability is a significant concern, considering that Vibrio species are responsible for causing vibriosis, a major epizootic disease that affects a broad spectrum of both wild and cultured fish species on a global scale [3].The clinical presentation of vibriosis in fish encompasses lethargy, anorexia, exophthalmia, hemorrhages, ulcerations, and congestion in internal organs [4].
Vibrio spp.have evolved to employ biofilm production as a survival strategy in response to the diverse challenges posed by the aquatic environment [5].Biofilm is char-acterized by a self-produced matrix of extracellular polymeric substances and serves as a bacterial sessile-building mechanism, providing protection against environmental conditions and various agents [6,7].The formation of biofilm is intricately linked to infection, virulence, and pathogenicity, emphasizing the role of biofilm in bacterial adaptation and resilience [8].Furthermore, biofilm formation enhances bacterial resistance to different agents, including antibiotics, thereby complicating the effectiveness of conventional treatment methods [7].Paradoxically, while antibiotics remain the primary treatment for bacterial infections in aquaculture, their indiscriminate use has led to an alarming increase in multidrug-resistant bacteria, necessitating a shift in research focus towards alternative control methods [9].
Bacteria, as remarkable communicators, produce, release, and sense extracellular signaling molecules, facilitating interaction with their environment [10,11].When the concentration of those signaling molecules reaches a critical threshold, known as a "quorum", bacteria adjust their gene expression to elicit a specific response [12].This process of cell-tocell communication, which can occur both within and between species, is referred to as "quorum sensing" (QS) [10].QS plays a pivotal role in various bacterial activities, including adhesion, biofilm formation, stress adaptation, and the production of virulence factors [13].The extracellular signaling molecules involved in QS are termed "autoinducers" (AIs) [14].There are various types of AIs, with N-acyl homoserine lactones (AHLs) being the primary AI for Gram-negative bacteria [15,16].
Recently, the disruption or inhibition of QS has emerged as a viable strategy to counteract the challenges caused by bacteria [17].This process, known as quorum quenching (QQ), involves the use of chemical or enzymatic means to inhibit QS [16].The various forms of QQ include the degradation of AI molecules, the inhibition of AI synthesis, and the blocking/inhibition/competition of AI binding to receptors [11,18].QQ offers a unique perspective compared to conventional treatments, which primarily focus on bacterial growth inhibition.Through QQ, pathogenic bacteria can be transformed into harmless microorganisms, effectively eliminating their pathogenicity [11,19].As such, QQ serves as an eco-friendly and effective alternative to antibiotics and other chemical control agents for managing bacterial diseases [20] and offers a potential solution to multidrug-resistant pathogens [7].
Remarkably, the disruptive capacity of QQ extends to bacteria classified as probiotics [21].Probiotics, defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" [22], have demonstrated several benefits in aquaculture.These include immunomodulation, increased utilization of digestive enzymes, and improvements in both gut health and water quality [23].Bacillus spp.are among the most widely used probiotic bacteria in aquaculture [24] and exhibit the capacity to degrade AHLs, underscoring their potential as effective agents in managing bacterial diseases [25,26].As the scientific community strives to address the challenges in aquaculture sustainably, understanding the intricate interplay between forced practices, bacterial communication, and innovative control strategies becomes paramount in shaping the future of this vital industry.
We successfully isolated and characterized the Bacillus velezensis D-18 strain in our prior work [27].We subsequently delved into its advantageous effects on the innate immune status of European seabass in our last study [28].Despite these advancements, the intricate interaction dynamics between this probiotic bacterium and potential pathogens remain uncertain.Consequently, drawing from the existing literature, the primary objective of the present study was to assess the QQ potential of B. velezensis D-18.We also aimed to explore its practical application as a biofilm disruptor specifically targeting Vibrio spp.and further enhance our understanding of the multifaceted roles played by B. velezensis D-18, particularly in disrupting Vibrio anguillarum 507 biofilms, thereby contributing valuable insights to the broader field of probiotic research and aquaculture management.

Bacterial Strains
The probiotic strain Bacillus velezensis D-18, previously isolated and characterized in our laboratory in prior studies, underwent routine cultivation via two methods: culturing on Luria-Bertani (LB) broth at 26 • C overnight or on brain-heart infusion (BHI) broth with 1.5% NaCl supplementation, following a standardization procedure.Bacterial growth was meticulously monitored at 3, 6, 9, 12, and 24 h using optical density measurements (OD600) and serial dilutions at each time point.These standardization procedures were performed in triplicate to ensure the reliability and reproducibility of the observed growth patterns.
The fish pathogenic strain isolated within our laboratory (Vibrio anguillarum 507) was routinely cultured at 26 • C on BHI broth with 1.5% NaCl supplementation.This process adhered to the following standardization: Similar to the Bacillus velezensis study, the growth of Vibrio anguillarum 507 was monitored at key time points (3,6,9,12, and 24 h) using optical density measurements (OD600) and serial dilutions.This iteration of the experiment was also conducted in triplicate to ensure robustness and consistency in the results.
Chromobacterium violaceum MK, a wild-type strain (CECT494, obtained from the Spanish Type Culture Collection-CECT) producing quorum sensing (QS)-dependent purple pigment violacein, served as a key component in bioassays.This strain was routinely cultured on LB broth at 26 • C overnight.
The biosensor strain Chromobacterium violaceum CV026, a mini-Tn5 mutant of the wild-type ATCC31532 deficient in QS-dependent violacein production (from our laboratory collection), was employed to detect exogenous AHLs.C. violaceum CV026 produces the purple pigment violacein in response to short-chain AHLs and was cultured on LB broth at 26 • C overnight.
Chromobacterium violaceum VIR24 was provided by Instituto de Investigación Marqués de Valdecilla (IDIVAL, Santander, Cantabria) and was used as a biosensor to detect exogenous long-chain AHLs due to its production of violacein.The strain was routinely cultured on LB broth at 26 • C overnight.
Bacillus subtilis subsp.subtilis CECT39 (ytnP-homolog lactonase) and Bacillus cereus CECT148 (aiiA-lactonase) were utilized as control strains for lactonase genes.These strains were sourced from the Spanish Type Culture Collection (CECT) (Paterna, Spain) and were routinely cultured on LB broth at 37 • C overnight.

Quorum Quenching Assay
A quorum quenching assay was carried out in accordance with the protocol outlined in [21], with specific adjustments.Initially, two overnight cultures were initiated: one featuring B. velezensis D-18 at 26 • C and 140 rpm in LB broth and the other with C. violaceum MK under identical conditions.Then, 1.5 mL of the B. velezensis culture was subjected to centrifugation (14,000 rpm, 10 min), and the resulting supernatant was filtered through a 0.22 mm membrane to isolate extracellular products (ECPs).Simultaneously, the B. velezensis pellet was resuspended in 1.5 mL of PBS.Following this, 1 mL of ECPs and 1 mL of the B. velezensis culture were subjected to heat inactivation (99 • C/15 min).A culture medium was prepared by combining 1 mL of C. violaceum MK broth with 49 mL (1:50) of LB soft agar (0.4%), which was thoroughly mixed, agitated, and poured into 6-well plates.Once solidified, 10 µL of the B. velezensis culture, the B. velezensis pellet, ECPs, heat-inactivated B. velezensis, heat-inactivated ECPs, and PBS were added to each respective plate.The 6-well plates were then cultured for 24 h at 26 • C, and the entire experiment was conducted in triplicate to ensure experimental repeatability.

AHL Degradation by Bacillus velezensis D-18
The degradation of short-and long-chain AHLs (C6 and C12 AHLs, respectively) by B. velezensis D-18 was assessed with a methodology inspired by Santos et al. [29], with certain refinements.A single colony from a freshly cultivated and uncontaminated B. velezensis was cultured overnight in 25 mL of LB at 26 • C with continuous agitation at 140 rpm.From this 25 mL culture, 10 mL was subjected to centrifugation (12,000 rpm, 15 min, 4 • C), and the supernatant was filtered through a 0.2 µm membrane to obtain ECPs, some of which were also separated and tested to prevent any interference with violacein production by the biomarkers.The resulting pellet was resuspended in PBS, constituting the B. velezensis pellet.Additionally, 15 mL of the original B. velezensis culture was preserved for subsequent use.
Then, 1.5 mL each of the B. velezensis pellet, ECPs, and PBS (as a control) were deposited in three separate 50 mL centrifuge tubes (Falcon ® ).Measurements of 0.5 µL of C6 AHLs (10 µg/µL) or 0.2 µL of C12 AHLs (10 µg/µL) were added to each Falcon tube.In parallel, 5 µL of C6 AHLs (10 µg/µL) or 2 µL of C6 AHLs (10 µg/µL) was introduced into the preserved 15 mL B. velezensis culture.All Falcon tubes were cultured overnight at 26 • C with continuous agitation at 140 rpm.Following the presumed degradation, the resulting cultures were transferred to 1.5 mL tubes and were centrifuged (14,000 rpm, 15 min) to eliminate bacteria.In addition, 10 mL of the presumed degraded B. velezensis cultures were employed for pH reconstitution, as described below.
Subsequently, 100 µL of the B. velezensis culture, the B. velezensis pellet, ECPs, PBS, and AHLs were individually added to separate wells of a 6-well plate containing soft agar (0.4%) with the biomarkers C. violaceum CV026 for short-chain AHLs and C. violaceum VIR24 for long-chain AHLs.Six-well plates were cultured overnight at 26 • C for 48 h.The entire experiment was conducted in triplicate to ensure the reliability of the results.

AHL Reconstitution via pH Adjustment
The pH reconstitution process was adapted from the methodology outlined by Santos et al. [29] and Singh et al. [30].The overnight cultures resulting from the interaction between B. velezensis D-18 and the respective AHLs were subjected to centrifugation as part of the previously described AHL degradation assay to avoid probiotic bacteria.Aliquots measuring 100 mL of both presumed degraded cultures were utilized in the previously described degradation assay, while the remainder was allocated for the pH reconstitution assay.
Supernatants were adjusted to pH 2 using hydrochloric acid (HCl).Then, 100 µL aliquots of both pH 2 supernatants were carefully added to the wells of the previously prepared 6-well plates containing soft agar (0.4%) with the respective biomarkers, C. violaceum CV026 and VIR24.The plates were incubated for 48 h at 26 • C.
Post-PCR cleanup was accomplished using the ExoSAP-IT enzymatic system to eliminate unincorporated primers and dNTPs.Electrophoresis involved the use of diluted 1/10 PCR products, a 2% agarose gel, and GelRed ® Nucleic Acid Gel Stain (Biotium, San Francisco, CA, USA) and was conducted under conditions of 80 V for 60 min.The marker employed for reference was the DL2000 Plus DNA Marker.

Vibrio anguillarum 507 Quorum Sensing Signaling Molecules
To elucidate the QS mechanisms of V. anguillarum 507, an assay was conducted employing both short-chain and long-chain AHL biomarkers, C. violaceum CV026, and VIR24.These biomarkers were separately incorporated into liquid 0.4% LB agar and evenly spread onto Petri dishes.Following solidification, three wells were established in each plate.Subsequently, 10 µL of C12AHL (1 µg/µL), serving as the positive control; 10 µL of PBS, serving as the negative control; 10 µL of an overnight culture of V. anguillarum 507 were added to each respective well.The plates were then incubated overnight at 26 • C. The experiment was replicated three times to ensure assay reproducibility.

Bacillus velezensis D-18 Quorum Quenching Effects on Vibrio anguillarum 507
An effective assay was conducted to assess the potential QQ effects of B. velezensis D-18 on the marine pathogenic strain V. anguillarum 507.Briefly, the probiotic strain, the pathogenic strain, and the long-chain AHL biomarker C. violaceum VIR24 were cultured at 26 • C and 140 rpm overnight.Subsequently, 1 mL of C. violaceum VIR24 and another mL of V. anguillarum were added to 48 mL of 0.4% soft LB agar.This agar was spread onto a Petri dish.Once solidified, 10 µL of the probiotic strain was added to the center of the plate.The plate was then incubated at 26 • C for 24 h.The experiment was performed in triplicate to ensure experimental repeatability.

Inhibition of Biofilm Formation and Growth of Vibrio anguillarum 507 by Bacillus velezensis D-18
After monitoring the growth dynamics of the probiotic bacteria and the pathogen, we assessed the capacity to inhibit biofilm formation and growth through the following procedure.B. velezensis was subjected to a 12-h incubation at 37 • C and 140 rpm in 20 mL of BHI supplemented with 1.5% NaCl, yielding a concentration of 10 8 CFU/mL.Simultaneously, the pathogen V. anguillarum was cultivated at 26 • C and 140 rpm for 3 h in 20 mL of BHI supplemented with 1.5% NaCl, resulting in a concentration of 10 7 CFU/mL.Serial dilutions were conducted to validate these concentrations.
A 1 mL sample was collected from each culture and subjected to centrifugation.The supernatant was removed and then resuspended in 100 µL of sterile PBS.A 12-well plate was employed in the experiment, and an enriched and filtered medium inspired by O'Toole [33] (using BHI supplemented with 1.5% NaCl instead of LB) was prepared to facilitate biofilm formation for both species.
Different well compositions were devised as follows in order to establish the desired concentration of each bacterium for assessing the biofilm formation and growth of both strains.The first comprised 2895 µL of enriched medium, 100 µL of B. velezensis (final concentration: 10 8 CFU/mL), and 5 µL of V. anguillarum (final concentration: 10 5 CFU/mL).The second consisted of 2900 µL of enriched medium and 100 µL of B. velezensis (final concentration: 10 8 CFU/mL).The third included 2995 µL of medium and 5 µL of V. anguillarum (final concentration: 10 5 CFU/mL).The last only included 3000 µL of medium and served as a control.The remaining wells of the 12-well plate were used as controls of the different treatments (B.velezensis and V. anguillarum, B. velezensis, V. anguillarum, and control) to confirm biofilm formation using crystal violet (0.1%) after incubation.
The plate was cultured at 26 • C and 100 rpm for 48 h.After this incubation period, the supernatant was removed, and 1 mL from each well was saved for quantification through serial dilutions.Each well underwent three washes with sterile PBS.The well surfaces were scraped, and the material was resuspended in 1 mL of PBS for further serial dilutions to quantify the biofilm amount in UFC/mL.
Serial dilutions of both biofilm formation and culture growth were plated on oxytetracycline (180 µg/mL) plates to quantify the selective growth of B. velezensis and on lincomycin (80 µg/mL) plates for V. anguillarum.The plates were incubated at 26 • C overnight.This entire experiment was conducted in triplicate to ensure the robustness and reliability of the results.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism software version 8.4.2 for macOS (GraphPad Software, San Diego, CA, USA).The unpaired t-test was used to test the differences between the groups.p < 0.0001 was defined as statistical significance for all tests that necessitated statistical analyses.

Quorum Quenching Assay
The synthesis of violacein by C. violaceum MK is a consequence of QS.The QS inhibition by B. velezensis (culture and pellet) exhibits an opaque coloration (Figure 1).Importantly, no discernible inhibition was observed in wells containing heat-inactivated B. velezensis, PBS, ECPs, heat-inactivated ECPs, and PBS (Figure 1).
dilutions to quantify the biofilm amount in UFC/mL.
Serial dilutions of both biofilm formation and culture growth were plated on oxytetracycline (180 µg/mL) plates to quantify the selective growth of B. velezensis and on lincomycin (80 µg/mL) plates for V. anguillarum.The plates were incubated at 26 °C overnight.This entire experiment was conducted in triplicate to ensure the robustness and reliability of the results.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism software version 8.4.2 for macOS (GraphPad Software, San Diego, CA, USA).The unpaired t-test was used to test the differences between the groups.p < 0.0001 was defined as statistical significance for all tests that necessitated statistical analyses.

Quorum Quenching Assay
The synthesis of violacein by C. violaceum MK is a consequence of QS.The QS inhibition by B. velezensis (culture and pellet) exhibits an opaque coloration (Figure 1).Importantly, no discernible inhibition was observed in wells containing heat-inactivated B. velezensis, PBS, ECPs, heat-inactivated ECPs, and PBS (Figure 1).

AHL Degradation by Bacillus velezensis D-18 and AHL Reconstitution via pH Adjustment
Following 48 h of growth at 26 °C, the results for the degradation of short-chain AHLs distinctly revealed that wells containing the B. velezensis pellet, ECPs, and PBS did not inhibit purple pigment production (Figure 2C,E,F).This implies the absence of inhibitors for short-chain AHL (C6 AHL) components in these conditions.However, the notable inhibition of violacein was observed in the well containing the B. velezensis culture (Figure 2A), indicating C6AHL degradation.

AHL Degradation by Bacillus velezensis D-18 and AHL Reconstitution via pH Adjustment
Following 48 h of growth at 26 • C, the results for the degradation of short-chain AHLs distinctly revealed that wells containing the B. velezensis pellet, ECPs, and PBS did not inhibit purple pigment production (Figure 2C,E,F).This implies the absence of inhibitors for short-chain AHL (C6 AHL) components in these conditions.However, the notable inhibition of violacein was observed in the well containing the B. velezensis culture (Figure 2A), indicating C6AHL degradation.
Regarding the degradation of long-chain AHLs, no pigment production was observed using C. violaceum VIR24 after 48 h in the B. velezensis culture well (Figure 2G).The partial degradation of C12AHL was observed in the well containing the B. velezensis pellet (Figure 2H).Nevertheless, violacein production was noted in the well containing ECPs, confirming the absence of degradation (Figure 2I).
degrading enzymes.This method distinguishes between enzymatic degradation by lactonases, which can be reversed under acidic pH conditions, and by acylases which cannot.Next, 10 mL of both short-and long-chain AHLs degraded by the B. velezensis culture were presented to the respective biomarkers, C. violaceum CV026 and VIR24.After 48 h, violacein production occurred, confirming the successful reconstitution of short-and longchain AHLs (Figure 2D,J).ECPs previously isolated for both AHL degradation assays to test any possible effects on C. violaceum CV026 and VIR24 did not interfere in the production of violacein by both biomarkers (Supplementary Figure S2).
AHL reconstitution via the pH technique serves as a valuable tool for identifying degrading enzymes.This method distinguishes between enzymatic degradation by lactonases, which can be reversed under acidic pH conditions, and by acylases which cannot.Next, 10 mL of both short-and long-chain AHLs degraded by the B. velezensis culture were presented to the respective biomarkers, C. violaceum CV026 and VIR24.After 48 h, violacein production occurred, confirming the successful reconstitution of short-and long-chain AHLs (Figure 2D,J).

Genetic Analysis
Conventional PCR and electrophoresis were conducted to confirm the existence of lactonase genes and compare the in vitro findings.The examination of the results on a 2% agarose gel confirmed the absence of the aiiA gene (756 bp) in the probiotic B. velezensis D-18.However, B. velezensis D-18 exhibited the presence of the homologous lactonase gene ytnP (559 bp) (Figure 3).The identification of lactonase-producing genes justifies the outcomes observed in the AHL reconstitution via acidic pH.

Genetic Analysis
Conventional PCR and electrophoresis were conducted to c lactonase genes and compare the in vitro findings.The examinatio agarose gel confirmed the absence of the aiiA gene (756 bp) in th D-18.However, B. velezensis D-18 exhibited the presence of the hom ytnP (559 bp) (Figure 3).The identification of lactonase-producing comes observed in the AHL reconstitution via acidic pH.

Vibrio anguillarum 507 Quorum Sensing Signaling Molecules
In this experimental study, conspicuous evidence of violacein of QS signals, was exclusively observed in the 10 µL V. anguillaru on the C. violaceum VIR24 plate (Figure 4A).Conversely, no disce were detected in the plate containing C. violaceum CV026 (Sup These observations strongly imply the targeted liberation of lon guillarum 507, thereby reaffirming its pivotal involvement in Q strain.
M BS -BV BC M BC BS BV -ytnP gen test aiiA gen test

Vibrio anguillarum 507 Quorum Sensing Signaling Molecules
In this experimental study, conspicuous evidence of violacein production, indicative of QS signals, was exclusively observed in the 10 µL V. anguillarum 507 overnight culture on the C. violaceum VIR24 plate (Figure 4A).Conversely, no discernible violacein signals were detected in the plate containing C. violaceum CV026 (Supplementary Figure S3).These observations strongly imply the targeted liberation of long-chain AHLs by V. anguillarum 507, thereby reaffirming its pivotal involvement in QS within this bacterial strain.

Bacillus velezensis D-18 Quorum Quenching Effects on Vibrio anguillarum 507
Following the incubation of the plate in which V. anguillarum was imbibed with the biomarker C. violaceum VIR24 with 10 µL of the probiotic added to its surface, an inhibition zone was observed, indicating QQ activity.C. violaceum VIR24 did not produce violacein due to the absence of AHL molecules, attributed to the presence of the probiotic (Figure 5).

Bacillus velezensis D-18 Quorum Quenching Effects on Vibrio anguillarum 507
Following the incubation of the plate in which V. anguillarum was imbibed with the biomarker C. violaceum VIR24 with 10 µL of the probiotic added to its surface, an inhibition zone was observed, indicating QQ activity.C. violaceum VIR24 did not produce violacein due to the absence of AHL molecules, attributed to the presence of the probiotic (Figure 5).

Bacillus velezensis D-18 Quorum Quenching Effects on Vibrio anguillarum 507
Following the incubation of the plate in which V. anguillarum was imbibed with the biomarker C. violaceum VIR24 with 10 µL of the probiotic added to its surface, an inhibition zone was observed, indicating QQ activity.C. violaceum VIR24 did not produce violacein due to the absence of AHL molecules, attributed to the presence of the probiotic (Figure 5).

Inhibition of Biofilm Formation and Growth of Vibrio anguillarum 507 by Bacillus velezensis D-18
Following a 48-h co-culture of probiotic bacteria and the pathogen, the quantification of biofilm formation and culture growth was conducted in terms of CFU/mL.The evaluation of biofilm formation in the co-culture of B. velezensis and V. anguillarum compared to the control revealed that the presence of the pathogen did not influence B. velezensis.The biofilm formation via B. velezensis remained comparable to the control, evidencing the probiotic's robust culture.Conversely, the introduction of the probiotic significantly impacted the biofilm formation of V. anguillarum (10 3 CFU/mL) in contrast to the control, demonstrating the pathogen's solitary culture (10 CFU/mL) (Figure 6A).The assessment of Bacillus velezensis D-18 and Vibrio anguillarum 507 biofilm formation in control wells stained with 0.1% crystal violet is depicted in Supplementary Figure S4.

Inhibition of Biofilm Formation and Growth of Vibrio anguillarum 507 by Bacillus velezensis D-18
Following a 48-h co-culture of probiotic bacteria and the pathogen, the quantification of biofilm formation and culture growth was conducted in terms of CFU/mL.The evaluation of biofilm formation in the co-culture of B. velezensis and V. anguillarum compared to the control revealed that the presence of the pathogen did not influence B. velezensis.The biofilm formation via B. velezensis remained comparable to the control, evidencing the probiotic's robust culture.Conversely, the introduction of the probiotic significantly impacted the biofilm formation of V. anguillarum (10 3 CFU/mL) in contrast to the control, demonstrating the pathogen's solitary culture (10 6 CFU/mL) (Figure 6A).The assessment of Bacillus velezensis D-18 and Vibrio anguillarum 507 biofilm formation in control wells stained with 0.1% crystal violet is depicted in Supplementary Figure S4.
The culture growth exhibited analogous results.The co-culture data clearly indicate that the growth of B. velezensis was unaffected by the presence of V. anguillarum.However, the growth of V. anguillarum in the presence of the probiotic was significantly inhibited in comparison to the pathogen control (Figure 6B).

Discussion
Traditional methods, such as the use of antibiotics, can have detrimental effects on health by fostering the growth of multidrug-resistant bacteria.Therefore, there is an escalating focus on investigating alternative strategies [34].As described earlier, bacteria employ a signaling process to communicate with each other or with the environment: QS.QS in bacteria is responsible for regulating various processes, including virulence, biofilm formation, sporulation, and the production of secondary metabolites [30,35].Consequently, QQ, which is the enzymatic disruption of this communication, is emerging as a novel mechanism for bacterial control [19,36].
The application of probiotics with QQ capabilities has recently been on the rise.In this study, we assessed the QQ capacity of the probiotic B. velezensis D-18 using a co- The culture growth exhibited analogous results.The co-culture data clearly indicate that the growth of B. velezensis was unaffected by the presence of V. anguillarum.However, the growth of V. anguillarum in the presence of the probiotic was significantly inhibited in comparison to the pathogen control (Figure 6B).

Discussion
Traditional methods, such as the use of antibiotics, can have detrimental effects on health by fostering the growth of multidrug-resistant bacteria.Therefore, there is an escalating focus on investigating alternative strategies [34].As described earlier, bacteria employ a signaling process to communicate with each other or with the environment: QS.QS in bacteria is responsible for regulating various processes, including virulence, biofilm formation, sporulation, and the production of secondary metabolites [30,35].Consequently, QQ, which is the enzymatic disruption of this communication, is emerging as a novel mechanism for bacterial control [19,36].
The application of probiotics with QQ capabilities has recently been on the rise.In this study, we assessed the QQ capacity of the probiotic B. velezensis D-18 using a co-cultivation technique with a specific biomarker, C. violaceum MK, which produces violacein in response to QS [20].The disappearance of the purple pigment in C. violaceum MK indicated that B. velezensis inhibits QS, confirming its QQ ability [30].Live B. velezensis D-18 showed QQ activity, while heat-inactivated B. velezensis, ECPs, and heat-inactivated ECPs did not produce an inhibition halo (Figure 2).This suggests that the QQ capacity is associated with live B. velezensis, emphasizing the importance of understanding the specific QQ mechanisms involved.
To explore the B. velezensis D-18 QQ mechanism, we investigated the degradation of AHLs, the main QS molecules of Gram-negative bacteria, using both short (C6) and long (C12) chains [18].AHL-producing and AHL-degrading bacteria coexist and employ contrasting strategies to gain a competitive edge over each other [37].Enzymes catalyzing AHLs can primarily be divided into two groups: (i) those that lead to the degradation of the homoserine lactone ring, known as lactonases, and (ii) those that cause cleavage in the bond between the acyl chain and the homoserine lactone, known as acylases [19].In the case of acylases, enzymatic degradation is conditioned by the length of the carbon rings, making it highly specific.However, lactonases interact directly with AHLs [19,38].The Bacillus genus is known to degrade AHLs due to the presence of genes that produce degrading enzymes [19,38].Therefore, the B. velezensis culture, the B. velezensis pellet, and ECPs were utilized to assess the degradation of long-(C12) and short-chain (C6) AHLs using C. violaceum VIR24 and CV026 as biomarkers, respectively [39].The study revealed that the B. velezensis D-18 culture was responsible for the degradation of both forms of AHLs.The B. velezensis D-18 pellet exhibited partial degradation of C12AHL.However, it was unable to degrade C6AHL.ECPs showed no capability to degrade any AHL molecules, which contrasts with previous research highlighting the extracellular QQ potential of Bacillus spp.mediated by ECPs [29].Nevertheless, other studies on probiotic candidates have demonstrated intracellular QQ activity [21].In this study, the majority or sole degradation occurred with the B. velezensis culture.There is a strong indication of the presence of an inducible lactonase producer gene in B. velezensis D-18 and its subsequent release in the presence of AHL molecules during the biological development of the bacteria.This hypothesis is supported by the well-established communication among bacteria through the emission and uptake of autoinducers (AIs).As previously described, Gram-positive bacteria QS is mediated by autoinducer peptides (AIPs), which are typically released extracellularly and are, therefore, present in the surrounding medium.Gram-positive bacteria can detect and respond to AIPs to regulate their metabolic activities [15].This feedback loop involving AIPs may enhance QS activity, leading to gene regulation that potentially triggers higher QQ activity, resulting in the detection of AHL molecules and the increase in degradation by B. velezensis D-18.
Consequently, the degradation of both long-and short-chain AHLs strongly suggested the presence of lactonase activity.To confirm this, these degraded AHLs were subjected to pH reduction and then exposed to the respective biomarkers.This resulted in the production of violacein, indicating the reconstitution of AHLs.Therefore, the enzyme was confirmed to be a lactonase.Researchers have argued that acylase enzymes have more advantages in practical applications since the AHLs degraded by lactonase could be reconstituted by lowering the pH [17,40].However, lactonases have a wide range of effects on long-and short-chain AHLs, unlike acylases, which are generally most effective against AHLs with side chains longer than 10 carbon atoms [16,40].
We verified the presence of the lactonase-producing gene in Bacillus velezensis D-18 despite confirming that the enzymatic reaction is performed by a lactonase.Several authors support that the aiiA lactonase producer gene is inherent to numerous Bacillus spp.[19,25,32].Recently, researchers have demonstrated the presence of homologous lactonase-producing genes in several Bacillus spp., such as the ytnP gene [41].In this study, PCR was conducted using primers designed and used by previous researchers [29,31,32] to determine the AHL-producing genes of the probiotic.The PCR results confirmed the absence of the aiiA gene in B. velezensis D-18, which aligns with findings in other Bacillus spp.[29,32].However, PCR results confirmed the presence of the homologous lactonase gene ytnP (584 bp) in B. velezensis D-18, suggesting an alternative mechanism for QQ activity in accordance with previous studies that identified homologous lactonase-producing genes in Bacillus species [25,29,32].
As previously mentioned, pathogenic bacteria form biofilms to adapt to environmental conditions and evade antibacterial agents.This bacterial protection mechanism has led to a decrease in the effectiveness of antibiotic treatments [42].In the field of aquaculture, biofilms serve as significant reservoirs for pathogenic microorganisms.In particular, the presence of Vibrio spp. in aquaculture systems is the cause of numerous economic losses, making its control essential [36,43].Therefore, the reduction and control of Vibrio biofilms contribute to an improvement in animal welfare.Several researchers have offered differing perspectives on the implication of QS in biofilm formation [12,18,42].QS is one of the mechanisms responsible for many biofilm stages such as bacterial adhesion, biofilm production, and bacterial dispersion [44].The presence of AIs is also crucial for biofilm formation.In Gram-negative bacteria, AHLs play a crucial role in biofilm development and dispersion [45].Throughout this study, we have verified that Vibrio anguillarum 507 releases long-chain AHLs as QS signal molecules.Numerous investigations have previously delineated the regulatory mechanisms of AHLs in Vibrio spp.and their roles in biofilm formation [12,46,47].Drawing upon the documented QQ effects of Bacillus spp. on Vibrio biofilms [48,49], we explored the potential QQ impacts of B. velezensis D-18 on V. anguillarum 507.
This study unveiled that B. velezensis D-18 exerts QQ effects on V. anguillarum by degrading AHLs.This was evidenced by the absence of violacein production when the long-chain AHL biomarker C. violaceum VIR24 was present.In subsequent assays, the formation of B. velezensis D-18 biofilms remained unaffected by the presence of the pathogen V. anguillarum.Conversely, the presence of B. velezensis significantly impacted the biofilm formation of V. anguillarum, suggesting its potential as a biofilm control agent.These findings were mirrored in culture growth, with B. velezensis thriving in the co-culture, while the growth of V. anguillarum was notably reduced.The evident inhibition of QS in V. anguillarum 507, characterized by a lack of observable growth in the medium without a decrease in colonies, underscores the impactful role of B. velezensis QQ.The production of lactonase by the probiotic serves as a pivotal factor, actively degrading the signaling molecules, AHLs, crucial for the communication network of the pathogen.This disruption deprives V. anguillarum of the vital communication needed to initiate gene expression related to virulence factors, biofilm formation, and growth [19].Our findings shed light on the multifaceted QQ mechanisms employed by B. velezensis D-18.The presence of ytnP further contributes to our understanding of the diverse QQ strategies employed by Bacillus species.These results reinforce the position of Bacillus spp. as promising candidates for preventing Vibrio diseases in aquaculture due to their comprehensive QQ capabilities [24,50].

Conclusions
In conclusion, Bacillus velezensis D-18 presents a promising avenue for the prevention of Vibrio anguillarum 507 diseases in aquaculture due to its quorum quenching capacity as an enzymatic disruptor of AHLs.The ability to disrupt bacterial communication and control biofilm formation positions B. velezensis D-18 as a potential eco-friendly alternative to conventional antibiotics in managing bacterial diseases in aquaculture.

Figure 6 .
Figure 6.Inhibition of biofilm formation and growth of Vibrio anguillarum 507 by Bacillus velezensis D-18.(A) Biofilm formation following a 48-h co-cultivation of B. velezensis (10 8 CFU/mL) and V. anguillarum (10 5 CFU/mL), denoted as "B + V".Solitary cultures of B. velezensis and V. anguillarum were employed as controls.Bacillus (B + V) indicates the biofilm formation of B. velezensis in the co-culture.Vibrio (B + V) indicates the biofilm formation of V. anguillarum in the co-culture.(B) Bacterial culture after 48 h of co-cultivation of B. velezensis (10 8 CFU/mL) and V. anguillarum (10 5 CFU/mL), denoted as "B + V".Controls included individual cultures of B. velezensis and V. anguillarum.Bacillus (B + V) indicates the culture growth of B. velezensis in the co-culture.Vibrio (B + V) indicates the culture growth of V. anguillarum in the co-culture.Student's t-test was used to examine differences in all the parameters tested.Asterisks indicate a significant statistical difference, **** p < 0.0001.

Figure 6 .
Figure 6.Inhibition of biofilm formation and growth of Vibrio anguillarum 507 by Bacillus velezensis D-18.(A) Biofilm formation following a 48-h co-cultivation of B. velezensis (10 8 CFU/mL) and V. anguillarum (10 5 CFU/mL), denoted as "B + V".Solitary cultures of B. velezensis and V. anguillarum were employed as controls.Bacillus (B + V) indicates the biofilm formation of B. velezensis in the coculture.Vibrio (B + V) indicates the biofilm formation of V. anguillarum in the co-culture.(B) Bacterial culture after 48 h of co-cultivation of B. velezensis (10 8 CFU/mL) and V. anguillarum (10 5 CFU/mL), denoted as "B + V".Controls included individual cultures of B. velezensis and V. anguillarum.Bacillus (B + V) indicates the culture growth of B. velezensis in the co-culture.Vibrio (B + V) indicates the culture growth of V. anguillarum in the co-culture.Student's t-test was used to examine differences in all the parameters tested.Asterisks indicate a significant statistical difference, **** p < 0.0001.