Discovery and Characterization of a Novel Bacteriocin That Strongly Inhibits Staphylococcus aureus

Abstract

Drug resistance in Staphylococcus aureus is a serious problem, and the development of new antimicrobial drugs to circumvent drug resistance has become a trend. In this study, we isolated a strain of Bacillus subtilis with high tolerance to alcohol, pH, NaCl, bile salt, surfactants, temperature, and simulated intestinal fluids. We optimized culture parameters to obtain the best fermentation conditions for the production of inhibitory compounds in cell-free culture media. The crude extract showed excellent stability when exposed to temperature, pH, and ultraviolet radiation, with almost no loss of bacteriostatic activity after treatment. After isolation and purification, the peptide sequences were identified using ultraperformance liquid chromatography–mass spectrometry (UPLC–MS), and the antibacterial sequences were analyzed using bioinformatics. The results of the identification showed that there was one novel bacteriocin (NSGGSYGSGGGGGGGNSHGY) with a molecular weight of 1513.58 Da. The minimum inhibitory concentration (MIC) of the B5 bacteriocin was 31.25 μg/mL against S. aureus, and it is noteworthy that bacteriocin B5 also showed weak antibacterial activity against Vibrio parahaemolyticus. In conclusion, this study developed a novel bacteriocin that has the potential to be used as an alternative to S. aureus antibiotics.

1. Introduction

Staphylococcus aureus is a common Gram-positive bacterium that is widely found in the human body and the environment, and it can cause a variety of infections, including skin, myocardial, bloodstream, bone, and joint [1]. S. aureus is widely recognized as one of the major causative agents of hospital-acquired infections (HAIs). Its pathogenic mechanisms are diverse, and it produces a variety of exotoxins, including hemagglutinins, hemolysins [2,3], toxic shock syndrome toxin 1 (TSST-1) [4], and leukocytocides [1], etc. These toxins can disrupt the structure and function of the host cells, leading to inflammatory responses and tissue damage [5]. In addition, S. aureus is capable of forming biofilms, which are formed in four steps: (i) initiation, (ii) colonization, (iii) replication, and (iv) dispersion [6], which provide a protective matrix around the encapsulated bacterium and are highly resistant to host immune defenses and antimicrobial drugs [7,8,9]. The prevalence of methicillin-resistant S. aureus (MRSA) has made the management of S. aureus in healthcare settings more difficult, increasing the challenges of infection control and treatment [10]. In summary, the development of novel antimicrobial drugs for S. aureus is necessary.

Antimicrobial peptides (AMPs) are intrinsic proteins produced by microorganisms as secondary metabolites. Members of the genus Bacillus produce several types of AMPs through ribosomal and nonribosomal mechanisms [11]. Bacteriocins are biologically active antimicrobial peptides produced by the ribosomes of Gram-positive (GPB) and Gram-negative bacteria (GNB) that inhibit the growth of other bacterial strains of the same species (narrow spectrum) or across genera (broad spectrum) [12]. These compounds are particularly attractive alternatives to antibiotics because they are natural, can be genetically modified, and are often highly stable and noncytotoxic [13]. In addition, bacteriocins produced by Bacillus sp. have been widely used in the food industry, animal feed additive, and agricultural sectors with a wide range of biological activities and application potential [14,15]. Future studies will focus on their antimicrobial mechanisms, structure–activity relationships, and the expansion of their application areas with the aim of being able to better utilize these natural antimicrobial peptides to combat the growing problem of bacterial drug resistance [16,17].

Bacillus subtilis, a Gram-positive bacterium widely found in soil and water, is notable for its ability to produce peptides, including lipopeptides, volatiles, and bacteriocins, with bacteriostatic activity [18]. The B. subtilis antimicrobial peptides can be categorized into different types, such as α-helical, β-folded, and hybrid structures [19]. These antimicrobial peptides usually consist of 10–50 amino acids and have specific conserved sequences [20]. These structural features allow the antimicrobial peptides to interact with bacterial cell membranes, leading to cell membrane disruption and cell death [21].

In this study, a novel strain of B. subtilis was obtained from soil, and a possible novel bacteriocin was identified and purified in its fermentation supernatant. This novel bacteriocin inhibited S. aureus, and this study evaluated its stability and biosafety and explored its possible antimicrobial mechanism, aiming to provide experimental and theoretical bases for expanding the application of bacteriocins in S. aureus therapy.

2. Materials and Methods

2.1. Strains

In this study, an unknown strain of Bacillus sp. B1 was screened from soil. The strain was identified morphologically and molecularly (16S rRNA genes were identified). S. aureus (ATCC 25923), Salmonella typhimurium (ATCC 14028), Escherichia coli (ATCC 25922), and Vibrio parahaemolyticus (ATCC 17802) were kept at −80 °C in our laboratory.

2.2. Stability Analysis

2.2.1. Alcohol

The strains were cultured on LB agar plates an incubated at 37 °C for 24 h. Single colonies were picked into 25 mL of LB sterilized medium and incubated at 37 °C and 180 rpm for 24 h to obtain the seed solution. Anhydrous ethanol was added to each LB fermentation solution to produce mass fractions of 0%, 2%, 4%, 6%, 8%, and 10% (wt/v), and the solutions were sterilized at 121 °C and 0.1 MPa for 15 min. The 4% seed solution was inoculated into the fermentation medium at 37 °C and 180 rpm for 24 h to determine the OD₆₀₀ value. Three parallel tests were conducted.

2.2.2. pH

The pH of the medium was adjusted to 1 to 12 using 1 M HCl and 1 M NaOH and sterilized at 121 °C, 0.1 MPa for 15 min. The seed solution was inoculated at 4% into the medium and incubated at 37 °C and 180 rpm for 24 h to determine the OD₆₀₀ value. Three parallel tests were conducted.

2.2.3. Salt

NaCl was added to the fermentation medium to obtain mass fractions of 0%, 2%, 4%, 6%, 8%, and 10% (wt/v). The fermentation solution was then sterilized at 121 °C, 0.1 MPa for 15 min. The 4% seed solution was inoculated into the fermentation medium and incubated at 37 °C and 180 rpm for 24 h to determine the OD₆₀₀ value. Three parallel tests were conducted.

2.2.4. Pig Bile Salt

Porcine bile salt was added to the fermentation medium to obtain mass fractions of 0%, 0.2%, 0.4%, 0.6%, 0.8% and 1% (wt/v). The fermentation solution was then sterilized at 121 °C, 0.1 MPa for 15 min. The seed solution was inoculated at a rate of 4% (v/v) into the medium and incubated at 37 °C and 180 rpm for 24 h to determine the OD₆₀₀ value. Three parallel tests were conducted.

2.2.5. Surfactants

Several common surfactants, sodium alcohol ether sulphate (AES), Tween 20, sodium lauryl sulfate (SLS), and linear alkylbenzene sulfonates (LAS), were added to the liquid medium to produce final concentrations of 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% (v/v). They were sterilized at 121 °C, 0.1 MPa for 15 min. The seed solution was inoculated at a rate of 4% (v/v) into the medium and incubated at 37 °C and 180 rpm for 24 h to determine the OD₆₀₀ value. Three parallel tests were conducted.

2.2.6. High Temperature

The seed solution was inoculated into the liquid medium at a rate of 4% (v/v) and then heated for 5 min at different temperatures (such as 60, 70, 80, 90, and 100 °C). The solution was then incubated at 37 °C and 180 rpm for 66 h. The OD₆₀₀ value was determined after a 10-fold dilution of the culture solution every 6 h. Three parallel tests were conducted.

2.2.7. Enteric Liquid and Gastric Fluid

The seed solution was inoculated at 4% into the medium and incubated at 37 °C and 180 rpm for 24 h. An amount of 1.5 mL of culture liquid was centrifuged in Eppendorf tubes (1.5 mL centrifuge tube) at 4 °C and 7000 rpm for 10 min. The supernatant was discarded. Another 1.5 mL of culture solution was added and centrifuged at 4 °C and 7000 rpm for 10 min. The supernatant was discarded and the precipitate was retained. A solution of 100 mL of 0.68% KH₂PO₄ was prepared in a flask, sterilized, and cooled. Then, 1% trypsin was added to simulate the intestinal fluid environment. The 0.68% KH₂PO₄ solution without the 1% trypsin was the control group. To simulate the gastric fluid environment, 100 mL of the diluted HCl solution (pH 2.5, 3.0, and 3.5) with 1% pepsin was prepared; the control group only contained 1% pepsin at pH 7.4. The precipitate was added into the flask, mixed, and incubated for 3 h. The OD₆₀₀ value of the culture solution was measured every 30 min. Three parallel tests were conducted.

2.3. Fermentation Optimization

2.3.1. Screening Method

In order to explore the applicability of the strain, the Oxford cup method was used to detect the bacterial inhibition effect [22]. With the inhibition circle as the only indicator and S. aureus as the indicator bacteria, a one-way experiment was designed to explore the optimal conditions for fermentation. The seed medium consisted of 5 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and 1000 mL of water, with pH 7.0~7.2. This was used as the basic condition for adjustment when optimizing the conditions. The plate of indicator bacteria for the Oxford cup inhibition method was produced using NA solid plate (18 g nutrient broth medium (NB), 15 g agar, 1000 mL water, pH 7.0~7.2). The seed solution of the indicator bacteria was diluted to 1 × 10⁵~6 × 10⁵ CFU/mL in gradient, and 200 μL was extracted for spreading on the plate. After the indicator bacteria dried, the Oxford cup was extracted using sterile tweezers and lightly cauterized in flame 3–4 times. The Oxford cup was cooled down and placed vertically on the medium so that the Oxford cup fit the medium completely without any gap. An amount of 200 μL of fermentation medium supernatant was added to the Oxford cup. The plates were then transferred to a constant temperature incubator at 37 °C for 24 h. The diameter of the inhibition zone (DIZ) was measured using the crosshatch method, and the average value was calculated.

2.3.2. pH

The initial pHs of the seed medium were adjusted to 5, 6, 7, 8, 9, and 10, with 0.1 M NaOH and 0.1 M HCl. The seed solution of Bacillus subtilis (at 4.5–6 × 10⁶ CFU/mL) was inoculated at 1% inoculum and incubated at 37 °C for 24 h. The fermentation supernatant was extracted, and the size of the circle of inhibition was determined using the Oxford cup method, as above.

2.3.3. Inoculum Quantity

The seed solution of B. subtilis was adjusted to 4.5~6 × 10⁶ CFU/mL, and the inoculum amounts for fermentation were set to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10% (v/v). The Oxford cup method was used to determine the size of the circle of inhibition to determine the optimum fermentation inoculum.

2.3.4. Inorganic Salt

The NaCl within the medium was replaced with K₂HPO₄, MgSO₄, and KCl at the same concentration (10 g/L). The circle of inhibition size was determined using the Oxford cup method to determine the optimal inorganic salts for the fermentation medium.

2.3.5. Carbon Source

The yeast extract within the medium was replaced with glucose, soluble starch, sucrose, lactose, maltose, and arabinose at the same concentration (5 g/L). The size of the circle of inhibition was determined using the Oxford cup method.

2.3.6. Nitrogen Source

The nitrogen source (peptone) in the medium was replaced with beef paste, peptone, yeast extract, wheat bran, and NH₄Cl at the same concentration (5 g/L). The size of the circle of inhibition was determined using the Oxford cup method.

2.4. Stability Analysis of Bacteriostatic Active Ingredients

Temperature stability of the bacteriostatic substances was achieved by treating the samples at −20, 0, 20, 40, 60, 80, 100, and 121 °C for 30 min and then cooling to room temperature. The pH stability of the bacteriostatic substances was determined by adjusting the cell-free supernatants to pHs 2–12 using 1 M HCl and 1 M NaOH, incubating the samples at 37 °C for 120 min, and then readjusting all samples to their original pHs. The UV stability of the bacteriostatic substances was determined by exposing the cell-free supernatants to UV light with the wavelength of 257.3 nm (149 μw/cm²) for 10, 20, 30, 40, 50, and 60 min. All experiments were performed using untreated bacteriostatic substances as a positive control, and the bacteriostatic activity of the substances before and after treatment was determined. Each experiment was performed in three replicates.

2.5. Separation and Purification of Bacteriostatic Substances

Ammonium sulfate was slowly added to 200 mL of cell-free fermentation supernatant to a final saturation of 50% (wt/v). After stirring slowly with a magnetic stirrer in an ice bath at 4 °C for about 4 h, the sample was placed in a refrigerator at 4 °C for 12 h. The precipitate was centrifuged at 4 °C for 30 min at 7800 rpm and then suspended in a 1/10 volume of the original bacterial broth in 10 mM phosphate buffer solution (pH 7.4). The resulting suspension was dialyzed at 4 °C for 48 h using a dialysis bag with a pore size of 500 Da. The protein crude was obtained by concentrating the suspension to the original volume using an ultrafiltration tube.

Afterwards, the preparative liquid chromatography (Agilent 1290) using an XBridge Prep C18 column (19 × 250 mm, 5 μm OBD^TM) was performed to seperate the crude extract. The mobile-phase water–acetonitrile gradient system was used, ranging linearly (0–5 min, 30% acetonitrile; 5–20 min, 30–60% acetonitrile; 20–26 min, 60–0% acetonitrile; 26–28 min, 0% acetonitrile). The main component was collected and dried into a powder using a vacuum freeze dryer (Scientz, China). The powder was the redissolved in sterile water. The inhibition rate was tested using the Oxford cup inhibition method.

The purification of the main component was evaluated using liquid chromatography (LC). The samples were filtered through a 0.2 μm filter before the LC process. The Agilent 1260 HPLC system equipped with a MAbPac SEC-1 column 300 Å (particle size 5 μm, pore size 300 Å, 4.6 mm × 30 mm, Thermo Fisher Scientific, Sunnyvale, CA, USA) was used. The mobile phase consisted of 50 mM of phosphate buffer, pH 6.8. The temperature of the column was 25 °C, and the flow rate was 0.8 mL/min. The detection was performed at 280 nm using a diode array detector.

The obtained protein was sent to Bio-Tech Pack Technology company Ltd., China, for sequencing analysis. The processed samples were analyzed using liquid–liquid mass spectrometry (LC–MS/MS) using a PEAKS Studio 10.6 De novo to obtain the peptide sequence results.

2.6. Antibacterial Spectrum and Minimum Inhibitory Concentration of the Bacteriocin

2.6.1. Antibacterial Spectrum

Using Vibrio parahaemolyticus, Escherichia coli, and Salmonella typhimurium as indicator bacteria, the Oxford cup inhibition method was used to determine the diameter of the inhibition circle using the crisscross method, and the average value was calculated.

2.6.2. Minimum Inhibitory Concentration

The minimum inhibitory concentration (MIC) was measured in a 96-well plate using 1 × 10⁵ CFU/mL S. aureus in LB medium as the indicator. The different concentrations of purified bacteriocin were 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 μg/mL. Each concentration measurement of the bacteriocin was repeated in triplicate. Pure water was used as the control. After incubating the plate at 37 °C for 16–18 h, the lowest concentration at which no bacterial growth was observed using the naked eye was selected as the MIC.

2.7. Growth Rate and Sterilization Kinetic Curve

A single colony of B. subtilis B was inoculated into 25 mL of fresh LB liquid medium and incubated at 37 °C and 180 rpm for 24 h. Then, a 3% ratio was inoculated into 25 mL of fresh LB liquid medium and incubated under the same conditions for 24 h. Samples were extracted at 3 h intervals during this period, and the absorbance of the bacterial solution at 600 nm (OD₆₀₀) was measured. The growth curve of the bacterium was plotted.

A bacterial solution of S. aureus cultured to mid-exponential stage was extracted and configured as a 1 × 10⁶ CFU/mL solution. Purified proteins were added to produce final concentrations of 0.5×MIC, 1×MIC, and 8×MIC. The solution was then incubated for 18 h. Samples were extracted at intervals of 2 h, and the OD₆₀₀ was measured. The number of viable bacteria was determined simultaneously using the smear method.

2.8. Propidium Iodide Staining

The suspension of S. aureus was treated with the purified bacteriocin at the final concentrations of 1× MIC and 2× MIC and incubated at 37 °C for 24 h, with PBS (pH 7.4) used as the control. Then the S. aureus was washed twice with PBS (pH 7.4) and then resuspended in PBS containing 10 μg/mL of propidium iodide (PI). Then, the suspension was placed in the dark for 1 h and observed using a fluorescence microscope.

3. Results and Discussion

3.1. Strain Identification

One isolate that was purified from soil samples was designated as isolate B. Isolate B was presented on LB plates as flattened colonies, with rough surfaces, folds, and irregular edges; an opaque, grayish-white color; and a moist, nonglossy surface (Figure 1a). Gram staining resulted in a blue–purple color and short, rod-shaped cells (Figure 1b), indicating that the strain was Gram-positive.

According to BLAST analysis, the rDNA of the 16S subunit showed 100% homology between this strain and B. subtilis JCM1465, which is known in GenBank (NR113265). As shown in Figure 1c, strain B was in the same branch as B. subtilis with high confidence, indicating a credible result. Combined with the morphological characteristics of the strains, Gram staining, and the results of the phylogenetic analyses of the 16S rRNA gene sequences, it was finally determined that isolate B belonged to B. subtilis.

3.2. Stability Analysis of B. subtilis

Although the growth of the strain was partially inhibited with an increasing alcohol concentration, the survival rate greater than 70% indicated that the strain was highly tolerant to alcohol (Figure 2a). The optimal growth environment of the strain was weakly alkaline, and it remained tolerant under strong alkaline conditions (pH 10–12). However, the growth of the strain was significantly inhibited under strong acidic conditions (pH 1–4) and was weakly inhibited under weakly acidic and neutral conditions (pH 5–7) (Figure 2b). The strain showed strong tolerance under different concentrations of salt ions, and the inhibition was obvious when the NaCl concentration was 10% (Figure 2c).

Probiotics need to be tolerant to a certain concentration of bile salt in order to survive in the animal’s digestive tract and exert a probiotic effect [23]. The intestinal bile salt content of animals is 0.3%, and the strain possesses a certain tolerance ability under this condition. When the bile salt content was increased to 0.4%, the growth of the strain was inhibited, and its average OD₆₀₀ value was 0.181. When the bile salt content was increased to 1%, the growth of the strain almost stopped (Figure 2d).

Different surfactants had different effects on the strain. Tween 20 (at 0.2%) promoted the growth of the strain and began to inhibit it growth when the concentration was increased to 0.4%; LAS (at 0.2–0.4%) promoted the growth of the strain and began to inhibit it when the concentration was increased by 0.6%. The strains were more tolerant to surfactants such as Tween 20 and LAS (Figure 2e).

After heat treatment, the growth of the strain was inhibited but still grew slowly, demonstrating that the strain was tolerant to high temperatures to some extent (Figure 2f).

The fluid of the small intestine is weakly alkaline, with a pH of about 7.6. With increasing incubation times, the strain was able to adapt to the simulated intestinal fluid environment, displaying tolerance to intestinal fluid (Figure 2g). Under simulated-gastric-fluid conditions (strongly acidic conditions), the OD₆₀₀ of the strain was low and decreased with the increase in incubation time, indicating that the strain had poor tolerance to simulated gastric fluid (Figure 2h).

3.3. Fermentation Optimization and Stability Analysis of Bacteriostatic Substances

The optimal medium consisted of soluble starch 5 g/L, beef paste 5 g/L, and K₂HPO₄ 10 g/L. The maximum yield of the bacteriostatic active substance was obtained through inoculation at 3% and incubation at 37 °C and 180 rpm for 24 h when the initial pH of fermentation was 7 and the concentration of the seed liquid was 4.5~6 × 10⁶ CFU/mL (Figure 3).

UV treatment for 10–60 min had no effect on the activity of the substance, indicating that the bacteriostatic active substance displayed UV stability (Figure 4a). The inhibitory activity remained unchanged after 30 min of treatment at −20~100 °C. It remained at 80% after 30 min of treatment at 121 °C, suggesting temperature stability in the inhibitor (Figure 4b), which is also better than the stability of most of the bacteriocins found [24,25]. The bacteriostatic activity remained relatively stable at pHs 2.0–9.0, and slightly decreased at pHs 11.0–12.0, which showed that the bacteriostatic substance maintained stability in various pH conditions (Figure 4c).

3.4. Identification of Bacteriocin

The purification of bacteriocin was conducted using the pre-HPLC (Figure S1). The purity of the main component 5 (named bacteriocin B5) was analyzed using the HPLC method and a MAbPac SEC-1 column (Figure S2). The sole peak indicates that bacteriocin B5 is the sole compound. In addition, the protease K treatment displayed a partial inactivating effect on bacteriocin B5. The results (Figure S3) suggest that protease K could partially inactivate the peptides (bacteriocin B5) after treating for 2 h, but bacteriocin B5 still displayed inhibition activity.

The molecular mass of the peptide was shown to be 1513.58 Da using PEAKS Studio 10.6 De novo analysis [26]. The amino acid sequence was determined using LC–MS/MS and identified as NSGGSYGSGGGGGGNSHGY. A comparison with the APD database (https://aps.unmc.edu/) (7 june, 2024) revealed that the peptide shared 39.29% homology with the antimicrobial Shepherin I, an antimicrobial peptide from Capsella bursa-pastoris.

3.5. Antimicrobial Spectrum and Minimum Inhibitory Concentration of Bacteriocins

The bacteriocin had an inhibitory effect on the Gram-positive bacterium S. aureus, with a MIC of 31.25 µg/mL, which was lower than the MIC previously reported for bacteriocin produced by Lactobacillus spp [27,28]. Bacteriocin showed weak inhibition of the Gram-negative bacterium V. parahaemolyticus, with a MIC of greater than 500 µg/mL, whereas it had no inhibitory effect on E. coli and S.typhimurium.

3.6. Growth and Killing Kinetics Curves

The addition of 1× MIC and 8× MIC of bacteriocin resulted in almost no growth of S. aureus, indicating that the bacteriocin can effectively inhibit the growth of S. aureus (Figure 5a). The number of S. aureus cells at different time points was further determined for bacteriocins at concentrations of 1× MIC and 8× MIC. The results showed that after 18 h, 99.9% of S. aureus bacteria were killed at a concentration of 8× MIC. In addition, the killing effect on S. aureus was concentration-dependent, and the number of viable bacteria gradually decreased with an increase in bacteriocin concentration (Figure 5b).

3.7. Propidium Iodide (PI) Chromatography

In order to study the bacteriostatic mechanism of bacteriocin, PI staining was performed on S. aureus after treatment with different concentrations of bacteriocin. Fluorescence microscopy observation showed that the red fluorescence in the field of view was enhanced after bacteriocin treatment, and a large area of intracellular red fluorescence appeared after 2× MIC bacteriocin treatment. This indicated that the cell membrane integrity of S. aureus was disrupted at this concentration (Figure 6) [16].

4. Conclusions

In the present study, a novel strain of B. subtilis was isolated from soil and was identified as Bacillus spp. morphologically (Gram staining) and molecular biologically. The strain showed tolerance to alcohol, pH, salt, porcine bile salt, surfactant, high temperature, simulated intestinal fluid, and simulated gastric fluid. Further isolation and purification of the cell-free supernatant of the fermentation broth yielded a novel bacteriocin, named bacteriocin B5, with a molecular weight of 1513 Da and an amino acid composition of NSGGSYGSGGGGGGGNSHGY. This bacteriocin showed strong antibacterial activity against S. aureus, with a MIC value of 31.25 ug/mL. This bacteriocin displays temperature, pH, and UV stability. These findings suggest that this bacteriocin has potential as an alternative to antibiotics for the treatment of S. aureus.