Antibacterial Efficacy of Crude Postbiotics from Enterobacter sp., Bacillus thuringiensis, and Lactobacillus plantarum against Marine Fish Bacterial Pathogens

2.1 Bacterial culture

Three probiotic isolates previously recovered from the gastrointestinal tract of pond-cultured Litopenaeus vannamei in Tuaran, Sabah, Malaysia, and characterized in our earlier work [16], namely Enterobacter sp. P17, Bacillus thuringiensis P18, and Lactobacillus plantarum P20 were used throughout the present study. These three isolates were the sole probiotic candidates examined; no additional isolates were screened or excluded. All subsequent assays (antibacterial screening, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), growth curve, and thermal-stability test) were performed on crude postbiotic extracts derived from these three strains. Bacterial cultivation was conducted with minor modifications of previously described methods [17]. Briefly, bacterial glycerol stocks were inoculated into de Man, Rogosa and Sharpe (MRS) broth (Merck, Darmstadt, Germany) and incubated for 96 h at room temperature (28 ± 2 ℃) using an orbital shaker operating at 130 rpm to obtain the working cultures.

Cell free supernatant (CFS) was obtained by centrifuging the producer cultures at 5,000 rpm for 15 min and filtering the supernatant through a 0.45 µm syringe filter. The sterile CFS was then partitioned against an equal volume of ethyl acetate (1:1, v/v) as described by Burianek and Yousef [18] and Islam et al. [19], with solvent substitution. The mixture was vigorously shaken in a separatory funnel and allowed to stand until three distinct phases formed: (i) an upper organic phase containing predominantly hydrophobic metabolites partitioned into the ethyl-acetate layer, (ii) an interphase layer in which amphiphilic peptide- or lipopeptide-like molecules tend to accumulate, and (iii) a lower aqueous phase retaining water-soluble metabolites such as organic acids, low-molecular-weight peptides, and polar secondary metabolites. The organic and interphase fractions were collected separately and concentrated to dryness under reduced pressure on a rotary evaporator at 40 ℃ until no visible solvent remained; residual ethyl acetate was further removed by overnight evaporation under a gentle nitrogen stream in a fume hood, after which each dried residue was resuspended in sterile Tris buffer (pH 7.0) at a CFS-equivalent volume ratio of 5:1 (CFS: buffer). The aqueous fraction was sterile-filtered (0.22 µm) and stored without further concentration. In this study, all three fractions are collectively referred to as crude postbiotic extracts because no purification step beyond solvent partitioning was performed; each fraction was evaluated independently against the four target pathogens so that the contribution of hydrophobic (organic), amphiphilic (interphase), and water-soluble (aqueous) metabolites to the overall antibacterial activity could be compared. All preparations were stored at −20 ℃ until use.

Crude postbiotic extracts obtained from the organic and interphase layers were resuspended in Tris buffer adjusted to pH 7 at a ratio of 5:1 (CFS: buffer). All crude postbiotic preparations were stored at −20 ℃ until further analysis.

2.2 Antibacterial screening and spectrum assays

The antibacterial effect of postbiotic extracts was determined through an antibacterial screening assay against four aquaculture bacterial pathogens, Vibrio parahaemolyticus SS07 [20], V. alginolyticus ATCC 17749, V. anguillarum ATCC 19264, and Photobacterium damselae SS16 [20]. Both the screening test for antibacterial effect and the antibacterial spectrum determination test were done using the agar spot assay method [21]. Briefly, bacterial culture of the pathogen was prepared with inoculation of bacterial glycerol stock in 2% NaCl supplemented Tryptone Soy Broth (TSB), followed by incubation at room temperature for 24 h in an orbital shaker at 130 rpm. About 100 µL of bacterial pathogen culture was inoculated in molten 0.5% Tryptone Soy Agar (TSA), swirled gently to mix, and poured onto 2% NaCl TSA plate. The molten 0.5% TSA was allowed to evenly coat the surface of the 2% NaCl TSA plate. After the molten TSA solidified, 10 µL of the postbiotic extract was pipetted onto the agar plate. The agar plate was set aside for the postbiotic extract to dry. Once the postbiotic extract had dried out, the agar plate was sealed with parafilm and incubated at room temperature for 16 h. The positive and negative controls were oxytetracycline and tris buffer at pH 7, respectively. Each postbiotic extract from organic, interphase, and aqueous layers was tested for each bacterial pathogen. After incubation, a transparent inhibition zone was observed and recorded as a positive result. Postbiotics with the highest spectrum were selected for further characterization.

2.3 Minimum inhibitory concentration and minimum bactericidal concentration

The MIC of the selected crude postbiotic extract against the tested pathogens was determined by adapting previously described methods by researchers [22-24]. As no standardized method is currently available for quantifying crude postbiotic concentrations, the undiluted extract was defined as 100% concentration. The 100% is the extract volume that equivalent to the original CFS. Pathogenic bacteria were cultured in TSB supplemented with 2% NaCl and incubated at room temperature for 24 h. Serial two-step dilutions of the crude postbiotic extract were prepared in 2%-NaCl TSB to obtain a broader final-concentration series of 50%, 40%, 30%, 25%, 20%, 15%, and 10% (v/v) for MIC determination. The dilution series was narrowed to 5%-interval steps between 10% and 30% after initial trials showed that MIC values fell within this range.

The pathogenic bacterial suspension was adjusted to an optical density of 1.0 at 600 nm (≈ 1 × 10⁸ CFU mL⁻¹) and diluted 1:100 in fresh 2%-NaCl TSB to a final working inoculum of ≈ 1 × 10⁶ CFU mL⁻¹, which was added 1:1 (v/v) to each crude postbiotic dilution in sterile glass tubes. Cultures were incubated at room temperature for 24 h. Bacterial growth was scored both visually (turbidity) and spectrophotometrically by measuring OD₆₀₀ before and after incubation; the MIC was defined as the lowest crude postbiotic concentration at which (i) no turbidity was visible and (ii) the post-incubation OD₆₀₀ increase relative to the t = 0 reading was ≤0.05 absorbance units (≥90% growth inhibition compared with the growth control). Each assay was carried out in three independent biological replicates, each with two technical replicates. Controls included: (i) growth control pathogen in TSB without postbiotic; (ii) sterility control TSB without pathogen; (iii) extract control postbiotic dilution series without pathogen, to verify extract sterility and to allow background-OD subtraction; and (iv) solvent control Tris buffer (pH 7) at the highest equivalent concentration carried over from the ethyl-acetate extraction step. Data are reported as mean ± SD of the three biological replicates.

The MBC of the selected crude postbiotic extract was also evaluated with modifications based on the methods reported by Parvekar et al. [23] and Fasolato et al. [24]. Following MIC determination, an aliquot from MIC tubes showing no visible bacterial growth was streaked onto TSA supplemented with 2% NaCl and incubated at room temperature for 24 h. MBC was defined as the lowest crude postbiotic concentration at which no bacterial colonies were observed on the TSA plates.

2.4 Growth curve assays

The bacterial growth curve assay was performed based on the method described by Huang et al. [25], with minor modifications. Vibrio parahaemolyticus was cultivated in TSB supplemented with 2% NaCl and incubated at room temperature for 24 h. The resulting bacterial suspension was standardized to an optical density of 1.0 at 600 nm. The adjusted culture was then inoculated into fresh TSB containing 2% NaCl and supplemented with crude postbiotics at concentrations equivalent to 1× and 2× the previously determined MIC. The cultures were incubated at room temperature for 12 h, during which optical density readings were recorded at 2 h intervals. All experiments were carried out in triplicate.

2.5 Temperature stability test

The influence of temperature and buffer type on the antimicrobial activity of the selected crude postbiotics was evaluated by subjecting the extracts to different thermal conditions and by suspending them in various buffer systems. To assess thermal stability, the crude postbiotics were heated using a block heater (Cole-Palmer, Stone, UK) at 40 ℃, 60 ℃, 80 ℃, and 100 ℃ for 20 min [26], as well as at 121 ℃ for 15 min [27]. Following thermal treatment, the remaining antibacterial activity was assessed against Vibrio parahaemolyticus SS07 using the soft overlay agar method. Untreated crude postbiotic served as the positive control. All assays were conducted in triplicate.

This study demonstrated that postbiotics derived from probiotic bacteria exhibit antibacterial activity against a range of aquaculture bacterial pathogens. Crude postbiotics produced by isolates P17 (Enterobacter sp.), P18 (Bacillus thuringiensis), and P20 (Lactobacillus plantarum) showed inhibitory effects against all tested pathogens. These findings indicate that the postbiotics evaluated in this study are not species-specific. The antibacterial activities observed from these three isolates were not limited to Vibrio spp. or Photobacterium damselae, suggesting a broader antimicrobial spectrum.

Previous studies have reported that Enterobacter spp. possess antibacterial activity against several major aquaculture pathogens. For instance, supernatants from Enterobacter amnigenus and Enterobacter sp. PIC15 was shown to inhibit Flavobacterium psychrophilum [28-30]. Similarly, unfractionated CFS and ≤3 kDa fractions derived from Enterobacter sp. C6-6 exhibited inhibitory effects against F. psychrophilum [31]. In addition, a partially purified N-acyl-homoserine lactone lactonase enzyme obtained from the cell-free lysate of Enterobacter sp. was reported to suppress biofilm formation by Aeromonas hydrophila [32]. Antibacterial activity of B. thuringiensis has also been documented, where the supernatant of B. thuringiensis G5-8-3T02 inhibited Vibrio mimicus XQ [33]. Likewise, the supernatant of B. thuringiensis KL1 demonstrated inhibitory activity against other important aquaculture pathogens, including Aeromonas caviae ATCC 15468 [34]. The antimicrobial properties of L. plantarum have been widely investigated, with reports showing inhibition against V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. mimicus, V. harveyi, and V. anguillarum [35-38]. However, to date, there are no published studies reporting antibacterial activity of Enterobacter sp. against Vibrio spp. and P. damselae. Therefore, the present study provides the first evidence of antibacterial activity of crude postbiotics derived from Enterobacter sp. against these aquaculture pathogens.

Among the three isolates examined, the inhibitory potential of L. plantarum against major aquaculture bacterial pathogens has been the most extensively studied. Neutralized and filtered supernatants of L. plantarum were reported to inhibit Pseudomonas fluorescens [39]. Moreover, CFS from L. plantarum exhibited antimicrobial activity against Edwardsiella tarda, Streptococcus iniae, Aeromonas veronii, Pse. fluorescens, and A. hydrophila [40-44]. Further investigation by Tremonte et al. [44] revealed that most L. plantarum strains produced organic acids as the primary inhibitory compounds, with only strain W_TA8 retaining antimicrobial activity against Pse. fluorescens after CFS neutralization to pH 7. Additional studies showed that CFS adjusted to pH 6 [45] or pH 5.5 [46] was still effective in inhibiting Streptococcus agalactiae and Pse. fluorescens, respectively. Enzyme-treated and pH-adjusted CFS of L. plantarum was also reported to inhibit Lactococcus garvieae [47]. Giri et al. [40] further demonstrated that intracellular products of L. plantarum VSG3, obtained through cell lysis and filtration, exhibited inhibitory activity against A. hydrophila. In addition, crude ethyl acetate extracts of L. plantarum CY 1-1 showed biofilm inhibition against Aeromonas sobria [48]. Purified bacteriocins produced by L. plantarum have also been reported to inhibit Pse. fluorescens [49-51], A. sobria, and A. hydrophila [48, 52].

The observed MIC and MBC values demonstrate that the selected crude postbiotics are effective against the tested bacterial pathogens. Specifically, crude postbiotics from isolates P17, P18, and P20 required concentrations of 10–25% to inhibit bacterial growth and 10–30% to achieve complete bactericidal effects. Determination of MIC and MBC is essential for subsequent application of postbiotics as disease control agents in aquaculture systems. Given that industrial-scale extraction and purification of postbiotic metabolites remain technically challenging [53, 54], establishing in vitro MIC and MBC values for crude extracts provides a first reference range for the relative potency of different producer–fraction combinations; these crude-extract percentages should not, however, be used directly as application doses in vivo until the active components have been identified and quantified. This approach is consistent with recommendations by Sahoo et al. [55], who emphasized the importance of dose determination for bacteriocin-based disease management in aquaculture.

Comparison of MIC and MBC values across the three solvent fractions (Tables 2 and 3) provides preliminary indications about the chemical nature of the active metabolites in each crude postbiotic preparation. For all three producer isolates, the aqueous-layer extracts consistently produced the lowest MIC (10% v/v) and, in most cases, the lowest MBC against the four target pathogens, whereas the organic- and interphase-layer extracts required higher crude-extract concentrations (15–25% v/v) to achieve equivalent inhibition. The disproportionately high activity of the aqueous fraction is consistent with the well-documented role of water-soluble metabolites, particularly short-chain organic acids (lactic, acetic, and formic acid) and small polar peptides as the principal antibacterial effectors of L. plantarum CFS [39, 44-46], and with the observation that pH-neutralized CFS often loses the bulk of its inhibitory activity [44]. For Bacillus thuringiensis P18, the additional activity recovered in the organic and interphase fractions is compatible with the presence of hydrophobic or amphiphilic secondary metabolites such as cyclic lipopeptides (e.g., iturin- and surfactin-family compounds [10]) and proteinaceous bacteriocin-like substances [33, 34], which partition preferentially into ethyl acetate or accumulate at the water/solvent interphase. For Enterobacter sp. P17, the observation that all three fractions retain measurable activity is consistent with the broader metabolite spectrum reported for this genus, including siderophore-like polar molecules and quorum-sensing-disrupting AHL-lactonases recovered from cell-free lysates [30, 32]. We emphasize that these assignments remain inferential; targeted chemical fractionation (HPLC-MS, NMR), heat- and protease-sensitivity profiling, and pH-neutralization assays of each layer are needed to confirm the identity of the active components in each fraction, and are listed in the Conclusion as priority follow-up work.

The growth curve assay is conducted to understand how the crude postbiotics affect the pathogens’ growth throughout the culture period. Although the growth of the bacterial pathogens treated with 1× and 2× MIC of postbiotics is almost the same, the presence of viable and non-viable cells might be different. Since 2× MIC has a higher concentration than the MBC of crude postbiotics, it is bactericidal to bacterial pathogens, and it is expected that no viable cells will be available. However, when it comes to an aquaculture environment, it is almost impossible to have an environment free of disease-causing agents. Assefa and Abunna [56] agreed that due to the aquaculture environment, the disease is difficult to detect and diagnose, and it allows the disease to transmit quickly. But the disease-causative agents can be brought to a controlled level to prevent any occurrence of disease outbreaks. Therefore, Assefa and Abunna [56] suggested that disease prevention strategies should be focused on instead. Based on the results of this study, postbiotics are a potential disease control strategy in aquaculture and have multiple benefits in comparison with other disease control strategies [7, 9-11]. However, the combination of different disease control strategies with postbiotics may give maximum disease protection to aquaculture animals [56].

Comparable findings have been reported for bacteriocins produced by lactic acid bacteria. Lim [26] reported that bacteriocins from Pediococcus acidilactici MCL11, Leuconostoc mesenteroides MCL12, and Enterococcus faecium MCL13 remained stable up to 80 ℃ after 10 min of heating, whereas bacteriocins from Lactobacillus sakei MCL14 and L. acidophilus MCL15 were stable up to 100 ℃ and 120 ℃, respectively. However, partial inactivation of bacteriocins from P. acidilactici MCL11, Leu. mesenteroides MCL12, and Ent. faecium MCL13 was observed at 100 ℃ [26].

Similarly, bacteriocin-like substances (BLS) produced by Ent. faecium Arla-18, ASR-6, JFR-1, OKA-14, and Yog-3S, Streptococcus thermophilus ASR-1 and TSB-8, as well as Lactobacillus casei JFR-5, exhibited thermal stability at 100 ℃ for up to 90 min [28]. Nevertheless, the activity of BLS from S. thermophilus ASR-1 declined following prolonged exposure at 100 ℃, and heating at 121 ℃ for 15 min resulted in complete inactivation of BLS from both S. thermophilus ASR-1 and L. casei JFR-5 [28]. Thermal stability up to 100 ℃ for 120 min has also been reported for bacteriocins produced by Lactococcus lactis subsp. lactis [57]. In contrast, bacteriocin-like inhibitory substances (BLIS) from Ent. faecium B3-8 showed a marked reduction in activity after 30 min at 90 ℃ and 15 min at 121 ℃, while BLIS from Ent. thailandicus B3-22 rapidly decreased after 15 min exposure at 121 ℃ [58]. Bacteriocins produced by Lactobacillus murinus AU06 were stable between 30 ℃ and 80 ℃, although a gradual decline in activity was observed with increasing temperature and exposure time starting from 40 ℃ [59]. In addition, bacteriocin from Weissella confusa A3 remained heat stable at 100 ℃ for 20 min [29].

Despite the extensive literature on the thermal stability of bacteriocins, studies addressing the temperature stability of postbiotic components, particularly organic acids, remain limited. Therefore, to the best of our knowledge, this study provides novel evidence regarding the thermal stability of bacteria-derived organic acids across a wide range of temperatures, supporting their potential application as robust antimicrobial agents in marine fish aquaculture systems.