Fresh intestinal tissue from grouper and sediment samples from marine grouper aquaculture ponds were collected. The intestinal tissues were homogenized using a tissue grinder, and stored at 4°C for further strain isolation. 1 mL of each sample was serially diluted (10⁶ to 10⁸) using sterile saline. A 100 μL aliquot from each dilution was spread onto MRS agar (HuanKai Microbial Technology Co., Ltd., China) plates and incubated at 37°C for 48 h. The single colonies were selected, purified by repeated streaking, and then inoculated into MRS liquid medium for 48 h. The antimicrobial activity of the isolates was tested using the Oxford cup assay. Briefly, the indicator strain B. contaminans was mixed with nutrient agar (HuanKai Microbial Technology Co., Ltd., China) at a 1:25 ratio at 45°C, poured into plates containing Oxford cups, and after solidification, 200 μL of each bacterial culture was added to the cups. After 48 h of incubation at 37°C, the diameters of the inhibition zones were measured to assess the inhibitory effects of the strains against B. contaminans.

To identify the strain with significant antibacterial activity, bacterial DNA was extracted using the Bacterial genomic DNA extraction kit (Vazyme, China) following manufacturer’s protocols. The extracted DNA was subjected to bacterial 16S rRNA sequencing using universal bacterial primers (27F, 5′-AGAGTTTGATCCTG GCTCAG-3′; 1492R, 5′-GGTTACCTTGTTACGACTT-3′). The PCR reaction mixture (50 μL) contained 25 μL 2 × PCR buffer (Vazyme, China), 1 μL dNTPs (10 μM) (Vazyme, China), 2 μL of each primer (10 μM), 1 μL DNA polymerase (Vazyme, China), and 1 μL template DNA. PCR conditions were as follows: initial denaturation at 98°C for 10 min, followed by 30 cycles of denaturation at 95°C for 20 s, annealing at 50°C for 20 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min. The PCR products were sequenced by Xiamen Borui Biotechnology Co., Ltd. The 16S rRNA gene sequences were analyzed using BLAST to compare homology with known bacterial strains. Phylogenetic analysis was conducted using MEGA11 software to construct a neighbor-joining tree.

L. plantarum Dys01 was activated in MRS liquid medium for optimal viability and then inoculated into fresh MRS medium at a 2% (v/v) ratio. The bacterium was cultured at 37°C for 16–18 h, followed by centrifugation at 8,000 rpm for 10 min at 4°C. The supernatant, containing L. plantarum Dys01 metabolites, was collected, and filtered through a 0.22 μm sterile membrane to remove remaining bacteria. Then the supernatant was either used directly or subjected to lyophilization for later use and determination of metabolite concentrations. B. contaminans, used as the indicator strain, was activated on nutrient agar. A single colony was inoculated into 5 mL of sterile nutrient broth (10.0 g/L peptone, 3.0 g/L beef extract powder, 5.0 g/L sodium chloride, final pH 7.3 ± 0.2) and incubated at 37°C for 18–24 h. The culture was then diluted with sterile nutrient broth to a final concentration of 1–2 × 10⁷ CFU/mL for subsequent assays.

The minimum inhibitory concentration (MIC) of L. plantarum Dys01 metabolites against B. contaminans was determined using a two-fold serial dilution method modified from Zhou et al. (2019). Briefly, in a 96-well microtiter plate, each well was loaded with 100 μL of B. contaminans suspension prepared in nutrient broth and 100 μL of L. plantarum Dys01 metabolites prepared in sterile deionized water at final concentrations of 32, 16, 8, 4, 2, 1, 0.5, and 0.25 mg/mL, with the metabolite concentrations determined by the lyophilized L. plantarum Dys01 metabolites. As a control, 100 μL sterile deionized water was added in B. contaminans suspension. Plates were incubated at 37°C for 24 h. Bacterial growth was assessed visually by observing turbidity. The minimum concentration of L. plantarum Dys01 metabolites that completely cleared the B. contaminans suspension was defined as the minimum inhibitory concentration (MIC) of the L. plantarum Dys01 metabolites against the B. contaminans.

To identify the key antimicrobial components in the cell-free supernatant (CFS) of L. plantarum Dys01 responsible for inhibiting B. contaminans, three different treatments were applied respectively: (1) catalase (10 U/mL) was added to degrade hydrogen peroxide; (2) acidic protease (200 μg/mL) was used to break down proteinaceous antimicrobial substances; and (3) the CFS was adjusted to pH 7.0 using 1 M NaOH to neutralize the effect of organic acids. After treatment, each sample was co-incubated with B. contaminans in nutrient broth at 37°C for 12 h. Antibacterial activity was evaluated by measuring the OD₆₀₀ of the culture.

The influence of L. plantarum Dys01 metabolites on B. contaminans biofilm formation was assessed using a 96-well microtiter plate assay. Each well was filled with 100 μL of B. contaminans suspension prepared in nutrient broth and 100 μL of L. plantarum Dys01 motabolite solution prepared in sterile deionized water at final metabolite concentrations of 0.5 × MIC, 1.0 × MIC, and 2.0 × MIC. The control group was supplemented with 100 μL of B. contaminans and 100 μL of sterile deionized water. After 24 h incubation at 37°C to allow biofilm development, the wells were gently washed three times with phosphate-buffered saline (PBS) to remove planktonic bacteria. Biofilms were fixed with 200 μL of methanol for 15 min, followed by methanol removal. Then, 200 μL of 0.1% crystal violet was added for 20 min of staining. Excess dye was removed by PBS washing, and plates were air-dried. Finally, 200 μL of 95% ethanol was added to dissolve the retained dye. The absorbance at 590 nm for each well was measured using a microplate reader, and the biofilm inhibition effect was evaluated by calculating the absorbance values. The biofilm inhibition rate was calculated as follows:

1 mL of B. contaminans suspension in nutrient broth was mixed with 1 mL of L. plantarum Dys01 metabolites prepared in water, achieving final metabolite concentrations of 0.5 ×, 1 ×, and 2 × MIC. Sterile deionized water was used as a control. After incubation at 37°C for 4 h, the mixtures were centrifuged at 8,000 rpm for 15 min at 4°C, and then the resulting supernatants were collected for the detection of alkaline phosphatase (AKP) activity by AKP assay kit (Nanjing Saihongrui Biotechnology Co., Ltd., China) to assess the cell wall integrity of B. contaminans.

1 mL of B. contaminans suspension was mixed with L. plantarum Dys01 metabolites to reach final metabolite concentrations of 0.5 ×, 1 ×, and 2 × MIC. Sterile deionized water was used as a control. After incubation at 37°C for 4 h, the samples were centrifuged at 8,000 rpm for 15 min at 4°C, and the bacterial pellets were collected. The pellets were washed three times with PBS and then resuspended in PBS. For cell membrane integrity assessment, the bacteria were stained with propidium iodide (PI, 10 μg/mL) in the dark for 30 min. After staining, cells were washed twice with PBS and resuspended in PBS. A 10 μL aliquot of each sample was observed under a fluorescence microscope.

10 mL of B. contaminans suspension was mixed with 10 mL of L. plantarum Dys01 metabolites at final concentrations of 0.5 ×, 1 ×, and 2 × MIC in sterile 50 mL centrifuge tubes. Sterile deionized water was used as a control. After incubation at 37°C for 12 h, samples were centrifuged at 8,000 rpm for 15 min, and the bacterial pellets were collected. After washing by PBS, the pellets were fixed overnight at 4°C with 2.5% glutaraldehyde. Samples were post-fixed with 1% osmium tetroxide for 30 min, dehydrated through a graded ethanol series, dried using a critical point dryer, and gold-coated using a sputter coater. The morphological changes of B. contaminans were observed using a field emission scanning electron microscopy (FESEM).

All experiments were performed with at least three biological replicates, each including at least three technical replicates. All numerical data were presented as the mean ± standard deviation. Statistical significance analysis of numerical data was performed using Student’s t-test, and the analysis was carried out using Origin 2021 software. The p values (P) < 0.05 are considered to indicate a statistically significant difference.

To identify bacteria with antimicrobial activity against B. contaminans, bacterial strains were isolated from the intestines of marine grouper and sediment from a grouper aquaculture pond using MRS medium. A total of 17 strains were obtained, with 11 strains isolated from grouper intestines, labeled as Dys01–Dys11, and 6 strains from the sediment of marine aquaculture pond, labeled as N0–N6 (Table 1). These strains were screened for antibacterial activity against B. contaminans using the Oxford cup assay. Among them, strain Dys01 showed the most significant inhibitory effect against B. contaminans, with an inhibition zone diameter of 17 ± 0.23 mm (Table 1; Figure 1A). Therefore, strain Dys01 was chosen for further study.

To identify strain Dys01, this strain was cultured on MRS medium, followed by observation of its morphological characteristics. The results showed that the colonies of strain Dys01 were round and small in shape, white or off-white in color, with a smooth and moist surface, and exhibited some stickiness (Figure 1B). Transmission electron microscopy analysis revealed that strain Dys01 exhibited a rod-shaped morphology with a mean length of 3 μm and a mean width of 1 μm (Figure 1C). The above characteristics were similar to those of the genus Lactiplantibacillus (Corsetti and Gobbetti, 2003). To further establish the taxonomic classification of strain Dys01, the 16S rRNA gene of strain Dys01 was sequenced and subjected to phylogenetic analysis. The results indicated that strain Dys01 belonged to the genus Lactiplantibacillus and clustered with Lactiplantibacillus plantarum MT611725.1^T (Figure 1D). A comparison in the GenBank database showed a 99.93% similarity in the 16S rRNA sequence between strain Dys01 and L. plantarum MT611725.1^T. Taken together, based on the morphological and phylogenetic analyses, strain Dys01 was identified as L. plantarum Dys01, with antimicrobial activity against B. contaminans.

Previous studies have reported that the antimicrobial activity of lactic acid bacteria is largely attributed to their metabolic products, such as organic acids, hydrogen peroxide, and bacteriocins (Wang et al., 2022). To evaluate the antibacterial effect of L. plantarum Dys01 metabolites on B. contaminans, the metabolites of L. plantarum Dys01 were extracted and added to plates containing B. contaminans for antibacterial activity assay, the results showed the metabolites of L. plantarum Dys01 exhibited a significant inhibitory effect on B. contaminans, with an inhibition zone diameter of 16 ± 0.24 mm, which was similar to the inhibition effect produced by L. plantarum Dys01 itself (Figure 2), indicating that the antibacterial effect of L. plantarum Dys01 was mainly attributable to its metabolites. The minimum inhibitory concentration (MIC) assays of L. plantarum Dys01 metabolites against B. contaminans showed that at a metabolites concentration of 4 mg/mL, B. contaminans still grew significantly in the liquid medium, resulting in turbidity in medium (Table 2). However, when the concentration of L. plantarum Dys01 metabolites was increased to 8 mg/mL, the medium stayed clear during cultivation, indicating that the growth of B. contaminans was significantly inhibited (Table 2). Therefore, the MIC of L. plantarum Dys01 metabolites against B. contaminans was 8 mg/mL.

As reported, the antimicrobial activity of the bacterial metabolites may result from the various antimicrobial components they contain, such as organic acids, hydrogen peroxide, proteinaceous substances, or other metabolic byproducts (Fuochi et al., 2019). To identify the active components in the metabolites of L. plantarum Dys01 responsible for the inhibition of B. contaminans, the effects of organic acids, proteinaceous antimicrobial substances, and hydrogen peroxide from L. plantarum Dys01 metabolites were investigated. The results showed that after 12 h of culture with the metabolites of L. plantarum Dys01, the growth of B. contaminans was significantly inhibited, with the OD₆₀₀ value decreasing to 0.13 (Figure 3). When organic acids were neutralized by adjusting the metabolites of L. plantarum Dys01 to pH 7.0 using NaOH, the antibacterial effect was significantly reduced, with the OD₆₀₀ increasing to 0.337 and the inhibition rate decreasing by 37.63%, suggesting that organic acids in L. plantarum Dys01 metabolites played a key role in the inhibition of B.contaminans (Figure 3). To estimate the effect of proteinaceous substances on the growth of B. contaminans, the L. plantarum Dys01 metabolites were treated with acidic protease to digest the proteins, and then co-cultured with B. contaminans. The results indicated the digestion of proteinaceous substances in L. plantarum Dys01 metabolites significantly attenuated its antimicrobial effect on B. contaminans, with the inhibition rate decreasing from 76.36 to 55.09%, suggesting that proteinaceous substances are among the key antimicrobials (Figure 3). For the effect of hydrogen peroxide, given that the optimal activity of catalase occurred at pH 7.0 (Trawczyńska, 2020), the metabolites were first adjusted to pH 7.0, and then treated with catalase to remove hydrogen peroxide. Compared to the metabolites at pH 7.0 without catalase treatment, catalase-treated metabolites showed a slight reduction in inhibitory activity on B. contaminans, with an OD₆₀₀ of 0.39 and a 9.6% decrease in inhibition rate (Figure 3).

Taken together, these findings demonstrated that the organic acids, proteinaceous substances, and hydrogen peroxide from L. plantarum Dys01 metabolites had important roles in inhibiting B. contaminans, with organic acids and proteinaceous substances being the primary contributors.

Bacterial biofilms are complex structures embedded in an extracellular matrix and play important roles in the resistance to bacteriophages, chemical disinfectants and antibiotics (Cheng et al., 2022). To assess whether the metabolites of L. plantarum Dys01 could affect biofilm formation of B. contaminans, the biofilm inhibition assays were performed. The results showed that compared to the control group without any treatment, the addition of L. plantarum Dys01 metabolites at a concentration of 0.5 × MIC significantly inhibited B. contaminans biofilm formation with the inhibition rate of 37.3% (Figure 4). As the concentration increased, the inhibitory effect on B. contaminans biofilm formation was enhanced, with the biofilm inhibition rate reaching 52.2% at 1.0 × MIC and 65.2% at 2.0 × MIC. These findings indicated that the metabolites of L. plantarum Dys01 could inhibit B. contaminans biofilm formation in a dose-dependent manner.

To investigate the effect of different concentrations of L. plantarum Dys01 metabolites on the cell wall integrity of B. contaminans, the activity of alkaline phosphatase (AKP), a periplasmic enzyme served as an indicator of cell wall damage, was measured in the supernatant of B. contaminans culture medium. The results showed that when treated with 1.0 × MIC or 2.0 × MIC of L. plantarum Dys01 metabolites, the AKP activity in the supernatant of B. contaminans culture medium was significantly enhanced compared to the control group, while the metabolites at low concentrations (0.5 × MIC) slightly upregulated the AKP activity (Figure 5). These findings suggested that L. plantarum Dys01 metabolites could impair the cell wall integrity of B. contaminans in a dose-dependent manner, resulting in the increased AKP activity in the supernatant of B. contaminans culture medium.

To explore the influence of L. plantarum Dys01 metabolites on the cell membrane integrity of B. contaminans, propidium iodide (PI), a fluorescent dye widely used to stain bacteria with damaged cell membranes red, was employed to assess the cell membrane integrity of B. contaminans. In the untreated control, almost no fluorescence signal was detected, while the treatment of 0.5 × MIC L. plantarum Dys01 metabolites led to faint red fluorescence in B. contaminans (Figure 6). Moreover, the fluorescence intensity increased with higher metabolite concentrations (Figure 6). Consequently, the metabolites of L. plantarum Dys01 could impair the cell membrane integrity of B. contaminans.

To reveal the effect of L. plantarum Dys01 metabolites on the morphology of B. contaminans, field emission scanning electron microscopy was used to observe B. contaminans after treatment with different metabolite concentrations (Figure 7). In the untreated control, B. contaminans exhibited intact morphology with smooth surfaces and tight arrangement. After treatment with 0.5 × MIC of the metabolites, the bacterial surface became mild roughness and fine wrinkles began to appear. The gaps between bacteria slightly increased, and the bacteria started to exhibit a tendency toward dispersion. At 1.0 × MIC, the bacterial surfaces were severely damaged, exhibiting pore formation, significant shrinkage, deeper grooves, irregular morphology, and an increasingly loose intercellular structure. At 2.0 × MIC, significant cellular disruption and loss of morphological features were observed, with the overall morphology severely deformed and fragmented, displaying obvious cracks and signs of lysis on the bacterial surface (Figure 7). Collectively, these data indicated that the metabolites of L. plantarum Dys01 could exert antimicrobial effects by disrupting and lysing B. contaminans.