Antibiofilm Analysis, Synergistic Potential and Biocompatibility Evaluation of a Bacteriocin from Bacillus subtilis (MK733983)

Abstract

This study emphasizes the potency of a bacteriocin screened from Bacillus subtilis (MK733983) of ethnomedicinal origin. Antibiofilm analysis with 0.5–3x minimal bacteriocin concentrations with critical and highly prioritized standard microbes such as Staphylococcus aureus, Mycobacterium smegmatis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Chromobacterium violecium showed potential biofilm inhibition and eradication of ≥ 5–99%, ≥ 1–86% respectively that correlated with biofilm viable cell-count. The bacteriocin exhibited remarkable synergistic potential with antibiotics like Amikacin, Ampicillin, Bacitracin, Chloramphenicol, Kanamycin, Norfloxacin, Vancomycin, Tetracycline, and Streptomycin. The sum of the fractional inhibitory concentrations was less than 0.5, which corresponded to the preliminary evaluation that included disc diffusion assays and checkerboard assays. In addition to synergism, the time-kill assays revealed a 2 or 3 log10 (1000-fold) reduction, indicating bactericidal potential. Bacteriocin’s effect on the growth dynamics of microorganisms has revealed its ability to intervene early and reduce microbial multiplication within 15 h of administration. Observations with a scanning electron microscope validated the antibiofilm capability. Methyl thiazol tetrazolium assay on 3T3 (normal fibroblast cell lines) up to 100 μg/ml of bacteriocin for 96 h (24 h-interval) revealed that the bacteriocin is not cytotoxic. It was also confirmed by trypan blue staining of the 3T3 cells at 96 h. Many biofilm-forming bacteria are known for causing harmful infections and resistance, and there is a growing need for new treatments. Bacteriocins are potential antibiotic alternatives, and the findings of this study are capable of being examined for larger application prospects.

Introduction

Microorganisms develop several strategies to adapt to their evolving environment, especially to overcome hostile variations and one such challenging strategy is biofilm formation [1]. Biofilms refer to microbial communities that are connected to either living or non-living surfaces, either as aggregates or embedded within an extracellular polymeric matrix. These communities are characterized by their sessile nature and can consist of one or numerous species of microorganisms. Biofilms harbor bacteria that exhibit resistance to unfavorable conditions, including exposure to antibiotics, starvation, and desiccation. This resilience contributes to the persistence of infections in individuals with weakened immune systems, as well as environmental challenges such as fomite contamination and biofouling of lakes, among various other consequences [2]. Antibiotics exhibit their highest efficacy against the planktonic microbial species in their free-floating, single-cell form, hence exerting substantial inhibitory and eradication effects on early infections. Nevertheless, the excessive and prolonged utilization of antibiotics might result in the development of antimicrobial resistance (AMR), a fast-progressing issue observed globally. The limited availability of new antimicrobial agents further exacerbates this matter, giving rise to significant apprehension [3]. Conversely, antibiotics are unable to completely eradicate biofilm-associated chronic infections, and many bacteria present beneath the biofilm layers show resistance to antibiotics and the host immune system. This is because biofilms are typically encapsulated by an extracellular matrix that provides protection to the cell community within [4]. Combinatorial therapy is preferred over antibiotic monotherapy, according to the findings of a number of studies. This treatment approach also has the added benefit of delaying the development of microbe resistance [5].

In recent years, there has been a growing interest in bacteriocins, which are mostly synthesized by bacteria, as promising agents for combating biofilms. These bacteriocins are being considered as alternatives to antibiotics due to their distinctive characteristics, such as their ability to inhibit a wide range of microorganisms including bacteria, fungi, viruses, and other parasites. Furthermore, they comprise low molecular weight antimicrobial peptides (AMPs) [6]. Other notable characteristics encompass the combination of enhanced efficacy and decreased toxicity, adaptability for bioengineering purposes to broaden their range of antimicrobial activity, and the capacity for synergistic interactions with other antibiotics [7]. An increasing amount of empirical data suggests that antimicrobial peptides (AMPs) possess the ability to eradicate bacteria residing within biofilms. This is achieved through their distinctive mechanisms, wherein the positively charged AMPs, possessing both hydrophilic and hydrophobic components, facilitate their penetration into the bilayers of lipids. Consequently, the AMPs induce the formation of multiple pores on the cell membrane, which eventually leads to the disruption of cells integrity [8].

This study showcases the antimicrobial effects of a bacteriocin obtained from Bacillus subtilis (MK733983), a strain derived from a plant with traditional medicinal uses. The focus of this research is on the bacteriocin’s ability to inhibit biofilm formation. The objective of the study was to evaluate the possible synergistic effects of a particular compound when combined with nine commonly used antibiotics against six established bacterial strains that are recognized for their ability to form biofilms. The cytotoxicity of the bacteriocin was evaluated by using the technique of the MTT test, while its mechanism of action against both gram-positive and gram-negative bacteria was explored via inspection employing Scanning Electron Microscopy.

Materials and Methods

Chemicals and Materials

All chemicals were purchased from SD Fine-Chem Ltd (India), HIMEDIA and Thermo Fisher Scientific. Antibiotics and susceptibility test discs of Amikacin (AMK), Ampicillin (AMP), Bacitracin (BAC), Chloramphenicol (CHL), Kanamycin (KAN), Norfloxacin (NOR), Streptomycin (STR), Tetracycline (TET) and Vancomycin (VAN) were procured from HIMEDIA.

Indicator Microorganisms (IMO’s)

Mycobacterium smegmatis (MC2-155 wild type) (ATCC 607)—(MS), Staphylococcus aureus (MTCC 737)—(SA), Pseudomonas aeruginosa (MTCC 3541)—(PA), Klebsiella pneumoniae (ATCC 700721)—(KP), Escherichia coli (ATCC 8739)—(EC) and Chromobacterium violaceum (MTCC 2656)—(CV).

Bacteriocin Activity Assay

Bacteriocin was prepared from the crude fraction of the supernatant of Bacillus subtilis (MK733983) described by Santhi and Aranganathan [9]. C₁₈ SEP-PACK purified eluents of bacteriocin are taken as partially purified bacteriocin (PPB) and HPLC purified eluents as purified bacteriocin (PB) for this study. Their potential was evaluated based on Spot-on-lawn assay and well diffusion assays that were carried out twice in triplicates with indicator organisms [10, 11]. A volume of 30 µl (1 mg/mL) of the bacteriocin was solubilized in dimethyl sulfoxide (DMSO) and thereafter dispensed into individual wells measuring 6 mm in width, which had been created on agar plates that had been previously inoculated with 1 ml of indicator inoculum. The plates were then placed in an incubator set at a temperature of 37 °C for a duration of 24 h. 30 µl of DMSO alone is taken as a blank or negative control for this study except for C. violaceum, where 5% DMSO was used. The anti-bacterial activity was determined based on its potential inhibitory zone (mm) against indicator organisms [12].

In-Vitro Tests for the Characterization of Bacteriocin

Minimal Inhibitory Concentrations (MIC) Assay

The lowest concentrations (MICs) of the bacteriocin (PPB & PB) required to prevent the visible growth of 18–24 h culture of indicator microorganisms (1 × 10⁶ CFU/mL of MS, SA, PA, KP, EC & CV) were determined by broth dilution and micro-dilution methods as recommended by National Committee for Clinical Laboratory Standards [13, 14]. All assays were performed in triplicates. The MICs of Antibiotics (AMK, AMP, BAC, CHL, KAN, NOR, STR, TET, and VAN) with the IMOs (MS, SA, PA, KP, EC & CV) were also determined in a similar manner.

Minimum Bactericidal Concentration (MBC₅₀ & MBC₉₀) Assay

MBCs of the bacteriocin that was required for bactericidal killing with 50 & 90% reduction in the initial inoculum of IMOs (1 × 10⁶ CFU/mL of MS, SA, PA, KP, EC & CV), after their subculture onto an antibiotic-free media was determined according to the standardized methods by Andrews [15].

Bacteriocin Killing Kinetics

The effect of the bacteriocin (PPB) on the growth dynamics of the IMOs (MS, SA, PA, KP, EC, and CV) was studied as indicated by Zhong et al. [16] with minor modifications. IMOs in their exponential phase were diluted in Mueller Hinton Agar (MHA) medium in 1 × 10⁶ CFU/ml. 100 µL of the bacteriocin in the final concentration with their respective MICs in 0.5x, 1x, and 2x were taken in 96-well microtiter plates. Each well received the same volume of bacterial solution, which was subsequently incubated at 37 °C for and at intervals of 6 h up to 24 h, and the growth was measured using turbidometry with an ELISA plate reader (Lisa Plus Plate Reader, Rapid Diagnostics, India) at 600 nm. The untreated bacterial suspension served as the control, and all sets were tested in triplicate in two separate tests. Their mean values were used to construct growth curves to investigate bacteriocin-killing kinetics.

Antibiofilm Analysis of the Bacteriocin

In Vitro Biofilm Formation Assays

Qualitative Biofilm Detection: Congo Red Agar (CRA) Assay and Tube method (TM)

Preliminary assessment of biofilm or slime production by the selected IMOs (1 × 10⁶ CFU/mL of MS, SA, PA, KP, EC & CV) was done by a qualitative method, Congo Red Agar (CRA) medium described by Freeman et al. [17] with minor modifications. To the sterile, autoclaved brain heart infusion (BHI) broth (35 g/L) with added sucrose 60 g/L and agar 9 g/L, a concentrated autoclaved solution of Congo Red indicator 5 g/L was added, and the CRA plates were inoculated with the IMOs. After incubation at 37 °C for 24 h aerobically, the black colonies indicated biofilm production. This experiment was repeated twice with triplicates.

The tube method, an alternative qualitative for biofilm detection also experimented as depicted by Christensen et al. [18] with minimal variations. 8 mL of trypticase soy broth (TSB) with 1.25% glucose in test tubes were inoculated with a loopful of IMOs colonies and the tubes were incubated at 37 °C for 24 h. Later, the tubes were decanted followed by a quick wash with phosphate buffer saline (PBS—pH 7.2) and dried. The dried tubes were then stained with crystal violet (0.125%), the excess stain was washed away, and the tubes were dried. Biofilm formation was considered positive when a visible film lined the wall and the bottom of the tube. The amount of biofilm formed was scored as 1-weak/none, 2-moderate, and 3-high/strong compared to the negative control tube with TSB alone. This experiment was performed in duplicates and repeated thrice.

Quantitative Biofilm Detection: Tissue Culture Plate Method (TCP)

Microtiter plate (MTP) assay, also known as TCP, is a standard method for biofilm detection. For this study, the IMO (1 × 10⁶ CFU/mL of MS, SA, PA, KP, EC & CV) cultures were prepared just as described above in TM, the cultures were then diluted in 1:100 with fresh TSB and the assay was carried out using 96-well flat bottom polystyrene titer plates (Thermo Fisher Scientific, India). Individual wells were filled with 200 μL of the diluted cultures. The sterile broth was taken as negative control and the plates were incubated at 37 °C for 24 h. After incubation, the wells were gently emptied, and washed thrice with 0.25 mL of PBS (pH 7.2) to ensure the removal of planktonic bacteria, the adherent biofilms were fixed by 2% sodium acetate for 1–2 min and were stained by crystal violet (CV) dye (0.125%). Further, the excess stain was removed by using sterile water very carefully to avoid any disruption of the biofilm. The plates were then left to dry for 60 min at room temp (22–25 °C), followed by the addition of 250 μL of 96–99% ethanol. Its optical density (OD) was recorded by an ELISA plate reader at 570 nm. A negative value for optical density (OD) was presented as zero, and the experiment was performed thrice with triplicates. The interpretation of biofilm production was done according to the criteria of Stepanovic et al. [19], and the biofilm formation was reported based on the OD values such as OD > 0.24 is positive biofilm former, OD > 0.12– < 0.24 is a weak biofilm, and OD < 0.12 is a negative biofilm [20].

Evaluation of Biofilm Viable Cells by Colony Count

The IMOs (MS, SA, PA, KP, EC & CV) adherent biofilms were developed as described in the TCA assay, then the plates were washed once with 0.25 mL of PBS (pH 7.2). The wells were then pipetted vigorously with 100 μL of PBS solution-disrupting biofilm. The suspended biofilm was diluted six-fold and later transferred to a fresh 96-well flat-bottom microplate in tenfold dilutions prepared in PBS. Each aliquot of 10 μL was inoculated onto petri plates with Mueller–Hinton agar (MHA), followed by incubation at a temperature of 37 °C for a duration of 24 h. The colony-forming units (CFUs) were then counted.

Determination of Minimum Biofilm Inhibition Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC)

To determine the biofilm inhibition concentration of the bacteriocin, each of the IMOs (1 × 10⁶ CFU/mL of MS, SA, PA, KP, EC & CV) was incubated with bacteriocin concentrations ranging from 0.5 to 3 MIC of both PPB and PB for the respective IMO on 96-well microtiter plate for 48 h at 37 °C. The biofilms were stained with crystal violet (CV) dye and the OD values were recorded as described in the TCA method. Samples without bacteriocin were taken as the controls.

For the biofilm eradication assay, a biofilm of the IMOs (1 × 10⁶ CFU/mL of MS, SA, PA, KP, EC & CV) was established on a 96-well microtiter plate for 48 h at 37 °C. The Petri plates containing biofilms were next subjected to three washes with phosphate-buffered saline (PBS) in order to eliminate the planktonic cells. Following this, the plates were incubated with varying doses of bacteriocin (PPB and PB), specifically within the range of 0.5 to 3 times the minimum inhibitory concentration (MIC) of the respective inoculum, on a 96-well microtiter plate. This incubation period lasted for 48 h at a temperature of 37 °C. The biofilm inhibition assay involved the application of CV staining to biofilms, followed by the measurement of optical density (OD) values at a wavelength of 570 nm using an ELISA plate reader. Control samples were collected without the presence of bacteriocin, and the live cells in the biofilm were quantified using the aforementioned procedure. Using duplicates, the experiment was conducted twice, and the percentage (%) inhibition and disruption concentrations were calculated using the following formula.

$$\%\mathrm{Inhibition}\mathrm{or}\mathrm{Disruption}=\frac{\mathrm{Control}\mathrm{OD}-\mathrm{Sample}\mathrm{OD}}{\mathrm{Sample}\mathrm{OD}}\times 100$$

Morphological Observation by Scanning Electron Microscopy (SEM)

The bacterial cells from cultures of S. aureus (SA) and P. aeruginosa (PA) treated with the bacteriocin (PPB) from MBIC assay at 3 MIC concentration and the bacteriocin untreated samples (control) were taken to observe under SEM for any morphological changes. The bacterial cells were thoroughly washed with phosphate buffer (pH 7.4), resuspended in 2.5% glutaraldehyde, and dehydrated via a gradual ethanol gradient (10–100%). The dehydrated samples were then freeze-dried, gold coated, and observed using a Field-emission scanning electron microscopy (FESEM) with dual beam focused ion beam (FIB), EDS, and monochromator (MonoCL) (Indian Institute of Science, Bengaluru).

Assessment of Synergistic Potential (SP) of the Bacteriocin

Preliminary SP Evaluation by Disc Diffusion Assay

Preliminary evaluation of the synergistic antibacterial activity of the bacteriocin with antibiotics was performed by disc diffusion method with IMOs (1 × 10⁶ CFU/mL of MS, SA, PA, KP, EC & CV) based on the novel procedures performed by Azucena et al. [21], Mickymaray et al. [22] and AZDAST method by Darounkalaei et al. [23] with some modifications. The culture plates were prepared with 25 ml of LB agar medium for gram-positive and MHA medium for gram-negative IMOs that were pre-swabbed with 100 µL of their 24 h culture (1 × 10⁶ CFU/mL or 0.5 McFarland turbidity). Each susceptibility test disc of AMK, AMP, BAC, CHL, KAN, NOR, STR, TET, and VAN in the concentration of 10 µg, was gently dribbled with 30 µL and 50 µL of bacteriocin (PPB in 1 mg/mL) and were left for drying in room temperature for an hour. The antibiotic discs with 30 µL bacteriocin were used for synergy assessment with S. aureus, M. smegmatis, and those with 50 µL bacteriocin for P. aeruginosa, K. pneumoniae, E. coli, and C. violaceum. The antibiotic discs with and without bacteriocin were gently placed over the culture plates. The plates were incubated for 24 h at 37 °C and after incubation, the diameter (mm) of the zone of inhibition (ZOI) was measured. This experiment was performed twice with triplicates and the results were the mean of the observations with the standard deviations.

Checker-Board Assay

The zero Interaction Potency (ZIP) model of checkerboard assays of the PPB with antibiotic combination against planktonic cells was done as previously done by Torres et al. [24] with very minor modifications. Serial dilutions of each antibiotic (AMK, AMP, BAC, CHL, KAN, NOR, STR, TET, and VAN), taken in the range of 0.01–1 μg/mL in sterile milli-Q H2O combined with the serial dilutions of the PPB in the range of 25–150 μg/mL in sterile milli-Q H2O and IMOs were added at a density of 1 × 10⁶ CFU/mL, making a total volume of 250 μL per well and 250 μL of untreated cell controls were taken also taken. The concentration range of antibiotics and the PB were selectively taken based on their MIC values relative to each of the IMO. The test plates were incubated for 24 h at 37 °C and after incubation, the cell survival was recorded based on their turbidity by ELISA plate reader at 570 nm and their fractional inhibitory concentrations (FIC) were interpreted according to Hall et al. [25];

$$\mathrm{\epsilon }\text{FIC}=\text{FIC; of ;agent; A}\left(\text{antibiotic}\right)+\text{FIC; of ;agent; B}\left(\text{bacteriocin}\right)$$

$$\mathrm{FIC}\mathrm{of}\mathrm{agent}\mathrm{A}\left(\text{antibiotic}\right)=\frac{\mathrm{MIC}\mathrm{of}\mathrm{antibiotic}\mathrm{in}\mathrm{combination}}{\mathrm{MIC}\mathrm{of}\mathrm{antibiotic}\mathrm{alone}}$$

$$\mathrm{FIC}\mathrm{of}\mathrm{agent}\mathrm{B}\left(\text{bacteriocin}\right)=\frac{\mathrm{MIC}\mathrm{of}\mathrm{bacteriocin}\mathrm{in}\mathrm{combination}}{\mathrm{MIC}\mathrm{of}\mathrm{bacteriocin}\mathrm{alone}}$$

Interpretation of results was based on the ƐFIC values, < 0.5 is a Synergistic combination with an increase in inhibitory activity, by a decrease in MIC of at least one agent. FIC value of 0.5–4 is additive or indifferent with no or slight increase in inhibitory activity by the compounds combined and antagonism as the combination of compounds increases the MIC or with a lowered activity of the compounds and an FIC value > 4. This assay was done twice with duplicates.

Time-Kill Assay

Time-kill studies were carried out on each of the antibiotics (AMK, AMP, BAC, CHL, KAN, NOR, STR, TET, and VAN) with the bacteriocin (PPB & PB) using concentrations of 0.5 and 1 × MIC in single and combination with IMOs (M S, SA, PA, CV, EC & KP). The IMOs used for this study were sub-cultured in Mueller–Hinton broth till the exponential phase with an OD at 600 nm to be approximately 0.25 and were then standardized to an inoculum size of 5 × 10⁵ CFU/mL. The bacterial cultures were grown in a combination of the bacteriocin, antibiotics, and alone and then the growth was quantified by their total viable count (TVC) by plating on Mueller Hinton agar. The agar plates were incubated at 37 °C and the TVC was estimated after 24 h incubation [26, 27]. The synergistic effect of the Bacteriocin was shown when a decrease of ≥ 2-log 10 was achieved. The study determined that a reduction of less than 3-log10 in colony-forming units per milliliter (CFU/ml) was classified as bacteriostatic, whereas a reduction of 3-log10 or above was classified as bactericidal, based on the initial inoculum, after 24 h [28]. This test was carried out twice using duplicate samples.

Cytotoxicity Studies by MTT Assay and Trypan Blue Staining

Cell Cytotoxicity was assessed by an in-vitro methyl thiazol tetrazolium (MTT) based toxicology assay kit (Sigma). 3T3 (normal fibroblast cell lines) cells were used for the assay and were seeded in a 96-well microtiter plate at a density of 8 × 10⁴ cells/100 μL and incubated overnight. Following incubation for initial cell attachment, the Petri plates were subjected to air drying. Subsequently, the attached cells were treated with the bacteriocin (PB) at concentrations of 10, 25, 50, and 100 μg/mL, which were prepared using milli-Q water. In triplicate, 100 μL of each concentration was added to the wells, while 100 μL of milli-Q water alone was used as a control. After time intervals 24, 48, 72, and 96 h of treatment, the MTT test was performed according to the manufacturer’s protocol, and absorption was measured at 570 nm using a microplate reader to assay the effect of the bacteriocin on 3T3 cells. Results were expressed as mean values ± SD of three determinations. Further, cells treated with the control and bacteriocin samples of 10, 25, 50, and 100 μg/ml at 96 h were trypan blue stained to assess the cell viability, and its biocompatibility was evaluated.

Results

Antibacterial Bacteriocin Concentrations

The lowest inhibitory/ bactericidal concentrations (MICs / MBC_{50 & ≥90}) of the bacteriocin (PPB & PB) and MICs of antibiotics required to prevent the visible growth of 18–24 h culture of IMOs are presented in Table 1 and 2.

Table 1.

MIC and MBC values of the Bacteriocin (PPB & PB) on IMOs in mg/mL

IMO	MIC—PPB	MIC—PB	MBC50—PPB	MBC50—PB	MBC ≥90—PPB	MBC ≥90—PB
SA	0.325 ± 0.02	0.22 ± 0.02	0.45 ± 0.04	0.32 ± 0.02	0.61 ± 0.02	0.38 ± 0.1
MS	0.35 ± 0.004	0.27 ± 0.02	0.5 ± 0.02	0.39 ± 0.03	0.73 ± 0.02	0.58 ± 0.1
PA	0.6 ± 0.02	0.37 ± 0.02	1.25 ± 0.11	0.83 ± 0.11	1.58 ± 0.11	1.08 ± 0.11
KP	0.75 ± 0.02	0.55 ± 0.05	1.5 ± 0.14	1 ± 0.2	3.08 ± 0.2	2.4 ± 0.11
EC	0.7 ± 0.02	0.52 ± 0.07	1.75 ± 0.11	1.1 ± 0.23	3.75 ± 0.20	2.75 ± 0.2
CV	0.75 ± 0.04	0.45 ± 0.05	1.25 ± 0.20	1.08 ± 0.1	3.25 ± 0.20	2.1 ± 0.23

MIC minimum inhibitory concentration, MBC minimum bactericidal concentration, PB partially purified bacteriocin, PB purified bacteriocin

Table 2.

MIC values of antibiotics on IMOs in µg/mL

IMO	AMK	AMP	BAC	CHL	KAN	NOR	STR	TET	VAN
MS	0.74 ± 0.01	0.52 ± 0.04	0.25 ± 0.01	0.74 ± 0.01	0.75 ± 0.01	0.75 ± 0.01	0.25 ± 0.01	0.52 ± 0.04	0.25 ± 0.01
SA	0.25 ± 0.01	0.75 ± 0.01	1 ± 0.01	1 ± 0.01	1 ± 0.01	1 ± 0.01	0.5 ± 0.01	0.75 ± 0.04	0.75 ± 0.02
PA	0.75 ± 0.01	0.76 ± 0.02	0.50 ± 0.01	0.75 ± 0.01	0.76 ± 0.02	1.01 ± 0.02	0.74 ± 0.03	1.25 ± 0.01	0.75 ± 0.01
CV	0.75 ± 0.01	0.25 ± 0.01	0.74 ± 0.01	0.75 ± 0.01	0.50 ± 0.01	0.76 ± 0.02	0.75 ± 0.01	0.76 ± 0.02	0.24 ± 0.01
EC	0.75 ± 0.01	0.24 ± 0.01	0.51 ± 0.01	0.75 ± 0.01	0.74 ± 0.01	0.75 ± 0.02	0.53 ± 0.03	0.50 ± 0.01	0.53 ± 0.03
KP	0.75 ± 0.01	0.5 ± 0.01	0.5 ± 0.02	0.75 ± 0.01	0.5 ± 0.02	0.75 ± 0.01	0.75 ± 0.02	0.75 ± 0.01	0.25 ± 0.01

IMO indicator microorganism, MS M. smegmatis, SA S. aureus, PA P. aeruginosa, CV C. violaceum, EC E. coli, KP K. pneumoniae. AMK amikacin, AMP ampicillin, BAC bacitracin, CHL chloramphenicol, KAN kanamycin, NOR norfloxacin, STR streptomycin, TET tetracycline, VAN vancomycin

Effect of Bacteriocin on IMOs Growth Dynamics

Figures 1, 2, 3, 4, 5 and 6 illustrate the impact of bacteriocin (PPB) on the development of IMOs. The bacteriocin concentration of 0.5 MIC did not have a significant effect on all the IMOs. However, doses of 1 and 2 MIC demonstrate a decline in bacterial cell growth within a fifteen-hour incubation period.

Fig. 1.

Effect of the bacteriocin on growth dynamics of the IMOs. Effect of the bacteriocin (PPB) on indicator microorganism—MS—M. smegmatis

Fig. 2.

Effect of the bacteriocin on growth dynamics of the IMOs. Effect of the bacteriocin (PPB) on Indicator microorganism—SA—S. aureus

Fig. 3.

Effect of the bacteriocin on growth dynamics of the IMOs. Effect of the bacteriocin (PPB) on Indicator microorganism—PA—P. aeruginosa

Fig. 4.

Effect of the bacteriocin on growth dynamics of the IMOs. Effect of the bacteriocin (PPB) on Indicator microorganism—KP—K. pneumoniae

Fig. 5.

Effect of the bacteriocin on growth dynamics of the IMOs. Effect of the bacteriocin (PPB) on Indicator microorganism—EC—E. coli

Fig. 6.

Effect of the bacteriocin on growth dynamics of the IMOs. Effect of the bacteriocin (PPB) on Indicator microorganism—CV—C. violaceum

Antibiofilm Analysis of the Bacteriocin

Qualitative Biofilm Assessment

Following incubation at a temperature of 37 °C for a period of 24 h under aerobic conditions, the CRA plates that were inoculated with all the IMOs exhibited the presence of shiny, thick black colonies. This observation suggests the occurrence of biofilm development. Figure 7 illustrates the biofilm development seen in four different bacterial strains, namely MS, SA, PA, and CV. Biofilm production was seen to be positive, as evidenced by the presence of a distinct film that coated the rim, wall, and bottom of the test tubes used in the experiment. The growth of each microorganism (MS, SA, PA, CV, EC, and KP) was assessed using the tube technique. The biofilm formation exhibited high/strong scores across all the IMOs (MS, SA, PA, CV, EC & KP) in comparison to the negative control tube containing only TSB.

Fig. 7.

Qualitative biofilm detection assays. Congo Red assay (CRA) test plates of MS—M. smegmatis, SA—S. aureus, PA—P. aeruginosa & CV—C. violaceum

Quantitative Biofilm Assessment

Biofilm assessment on TCP method at 570 nm on IMOs were, SA—0.74 ± 0.004, CV—0.70 ± 0.003, EC—0.65 ± 0.004, MS—0.64 ± 0.002, PA—0.61 ± 0.004 and KP—0.61 ± 0.002 with their controls (sterile broth) OD values ranging from 0.0001 to 0.0002 with a standard deviation of 4.93288E−05 to 0.00002 suggesting that all the IMOs taken for this study are strong biofilm producers.

MBIC & MBEC

The antibiofilm efficacy of the purified bacteriocin was shown to be much superior when compared to the partially purified bacteriocin. Specifically, the Purified bacteriocin demonstrated a biofilm-inhibition percentage of over 98% in gram-positive IMOs and over 90% in gram-negative IMOs. Similarly, PB has shown > 85% and > 80% biofilm-eradication in gram-positive and negative (PA, KP & EC) IMOs, respectively. TVC of the IMOs in biofilms was enumerated for the IMOs (SA, MS, PA, KP, EC & CV) were in the range of 2.7–3 × 10⁸ CFU/mL on incubation at 37 °C for 24 h. The PB showed a 100-fold decrease in the TVC in all the IMOs, at 3 × MBIC but under similar concentrations for PPB, only SA, MS, and PA showed a 100-fold TVC reduction. Only gram-positive IMOs showed a 100-fold reduction in TVC at 3 × MBEC of the PPB, whereas PB showed a 100-fold reduction in the range of 5.75–9.5106 CFU/mL under comparable circumstances. Table 3 provides all of the data, including the average values and standard deviations of the duplicates (Table 4).

Table 3.

MBIC and MBEC

IMO	IC≥90	SA	MS	PA	KP	EC	CV
PPB	MBIC	0.5 ± 0.02	0.6 ± 0.02	1.3 ± 0.11	2.2 ± 0.20	2.3 ± 0.23	2.5 ± 0.20
PB	MBIC	0.3 ± 0.04	0.46 ± 0.01	0.8 ± 0.11	1.5 ± 0.2	1.8 ± 0.4	1.75 ± 0.2
PPB	MBEC	1.5 ± 0.02	1.6 ± 0.01	3.5 ± 0.2	4.1 ± 0.1	4 ± 0.2	6.75 ± 0.2
PB	MBEC	0.75 ± 0.2	1.1 ± 0.11	2.25 ± 0.2	3.08 ± 0.11	3.1 ± 0.2	5.1 ± 0.2

IC_≥90 inhibitory concentrations, MBIC & MBEC minimal biofilm inhibitory concentration & eradication concentration

Table 4.

Antibiofilm Analysis- MBIC, MBEC, and TVC of PPB & PB

B-IC		Total viable count (CFU/mL)						Percent inhibition (%)
B	MBIC	SA	MS	PA	KP	EC	CV	SA	MS	PA	KP	EC	CV
PPB	0.5	9.3 ± 0.03 × 105	9.5 ± 0.05 × 105	9.6 ± 0.09 × 105	9.8 ± 0.08 × 105	9.8 ± 0.09 × 105	9.8 ± 0.05 × 105	4.8	6.7	7.2	2.6	14	9.5
PB	0.5	8.9 ± 0.01 × 105	8.1 ± 0.04 × 105	8.9 ± 0.01 × 105	9 ± 0.01 × 105	9.1 ± 0.01 × 105	9.2 ± 0.05 × 105	8.7	10.4	14	2.6	28	56.9
PPB	1	7.4 ± 0.01 × 105	8.6 ± 0.01 × 105	9.1 ± 0.02 × 105	9.1 ± 0.07 × 105	8.9 ± 0.08 × 105	9.2 ± 0.02 × 105	16.5	19.3	43.7	37.3	60.5	63.5
PB	1	6.5 ± 0.03 × 104	6.0 ± 0.01 × 105	3.8 ± 0.04 × 105	4.4 ± 0.05 × 105	6.3 ± 0.01 × 105	7.5 ± 0.02 × 105	28.8	41.5	65.8	51.2	79.4	76.6
PPB	2	9.7 ± 0.02 × 104	1.0 ± 0.09 × 105	1.3 ± 0.01 × 105	1.8 ± 0.05 × 105	1.7 ± 0.04 × 105	1.9 ± 0.01 × 105	90.5	85.8	79.2	74.7	78.9	73.5
PB	2	2.2 ± 0.03 × 104	9.3 ± 0.01 × 104	9.9 ± 0.03 × 104	9.9 ± 0.01 × 104	1.1 ± 0.01 × 105	1.3 ± 0.01 × 105	86.6	81.4	89.3	90.8	87.1	84.9
PPB	3	6.3 ± 0.02 × 104	8.5 ± 0.07 × 104	9.9 ± 0.03 × 104	1.0 ± 0.07 × 105	1.2 ± 0.01 × 105	1.1 ± 0.01 × 105	94.7	91.2	89.7	86.2	85.2	82.3
PB	3	1.1 ± 0.01 × 104	5.1 ± 0.03 × 104	6.6 ± 0.02 × 104	9.9 ± 0.02 × 104	9.9 ± 0.01 × 104	9.8 ± 0.05 × 104	99.2	98.9	94.6	94	91.1	93.5
C	MBIC	1 × 106	1 × 106	1 × 106	1 × 106	1 × 106	1 × 106	0	0	0	0	0	0
C	MBEC	2.8 ± 0.3 × 108	2.9 ± 0.6 × 108	2.8 ± 0.4 × 108	2.72 ± 0.2 × 108	2.8 ± 0.3 × 1 × 108	2.7 ± 0.4 × 108	0	0	0	0	0	0
PPB	0.5	2.8 ± 0.03 × 108	2.9 ± 0.03 × 108	2.8 ± 0.04 × 108	2.7 ± 0.07 × 108	2.8 ± 0.01 × 108	2.6 ± 0.03 × 108	1.2	2.7	5.1	13.1	9.5	13.7
PB	0.5	2.6 ± 0.03 × 108	2.7 ± 0.06 × 108	2.8 ± 0.07 × 108	2.6 ± 0.03 × 108	2.7 ± 0.07 × 108	2.5 ± 0.01 × 108	3.4	11.9	27.1	22.9	15.4	29
PPB	1	1.4 ± 0.03 × 108	1.3 ± 0.09 × 108	1.2 ± 0.06 × 108	1.1 ± 0.04 × 108	1.3 ± 0.03 × 108	1.3 ± 0.09 × 108	12.2	18.7	47.7	36.8	33.5	39.7
PB	1	1.2 ± 0.03 × 108	1.2 ± 0.07 × 108	1.2 ± 0.06 × 108	1.1 ± 0.03 × 108	1.1 ± 0.05 × 108	1.2 ± 0.03 × 108	21.2	57.2	77	52.2	51.2	62
PPB	2	9.4 ± 0.06 × 10 6	1.0 ± 0.04 × 107	1.2 ± 0.03 × 108	1.2 ± 0.04 × 107	1.3 ± 0.07 × 107	1.3 ± 0.03 × 107	71	51.3	53.8	52.5	42.5	52.5
PB	2	8.2 ± 0.03 × 106	8.7 ± 0.07 × 106	1.0 ± 0.03 × 107	1.0 ± 0.02 × 107	1.0 ± 0.08 × 107	1.0 ± 0.02 × 107	76.3	71.2	66	72.6	70.5	61.5
PPB	3	8.2 ± 0.03 × 106	9.7 ± 0.03 × 106	1.0 ± 0.04 × 107	1.1 ± 0.08 × 107	1.2 ± 0.07 × 107	1.3 ± 0.08 × 107	76.4	85.5	62.1	56	51.1	48.7
PB	3	5.7 ± 0.08 × 106	6.1 ± 0.07 × 106	8.5 ± 0.03 × 106	9.3 ± 0.02 × 106	9.4 ± 0.02 × 106	9.1 ± 0.08 × 106	86.5	85.9	82	81.1	80.2	68.5

B bacteriocin, PPB partially purified bacteriocin, PB-PB purified bacteriocin, IMO SA, MS, PA, KP, EC & CV. MBIC minimum biofilm inhibition concentration, MBEC minimum biofilm eradication concentration, IP inhibition percentage. C control, MBIC & MBEC initial inoculum size. IC inhibitory concentration

SEM Analysis

The bacteriocin (PPB) untreated cells from cultures of S. aureus (SA) and P. aeruginosa (PA) showed clear, dense, and smooth symmetry and spatial configuration of the cell walls appearing in a uniform layer with close physical contact like a biofilm, as seen in Fig 8 (SA 1,3 & PA 1,3). The bacterial cells of SA and PA treated with 3x MIC PPB showed clear visible damage to the cell membranes that were severely disrupted and shriveled. A few of the SA and PA cells showed pore formation and thus the disruption of the biofilm (SA & PA) was clear with the SEM morphological analysis (Figure 8: SA 2, 4 & PA 2, 4).

Fig. 8.

Antibiofilm Analysis by Field Emission Scanning Electron Microscopy. SA and PA morphological changes before (SA1,3; PA 1,3) and after (SA2,4; PA2,4) bacteriocin treatment incubated for 48 h

SP Evaluation of the Bacteriocin

Disc Diffusion

The bacteriocin has shown synergistic antimicrobial activity with all the antibiotics used in this study tested with all the IMOs. PPB showed the highest SP with AMP for MS and VAN for SA, on the other hand, PPB showed greater SP in gram-negative IMOs, with ≥ 100% increase with AMP, BAC, and VAN for PA, with AMP for KP, with AMP and BAC for EC and with AMP & VAN for CV (Table 5). Some representative images for SP evaluation by disc diffusion for the IMOs are shown in Fig. 9.

Table 5.

Synergistic potential evaluation by disc diffusion assay

	Antibiotic	A—ZOI (mm)	SP (mm)	%↑		Antibiotic	A—ZOI (mm)	SP (mm)	%↑		Antibiotic	ZOI (mm)	SP (mm)	%↑
MS	AMK	27.08 ± 0.1	34.3 ± 0.28	26.76	SA	AMK	20.25 ± 0.25	30.50 ± 0.5	50.6	EC	AMK	25.16 ± 0.28	33.83 ± 0.76	34.43
	AMP	25.08 ± 0.1	38.08 ± 0.14	51.82		AMP	15.08 ± 0.14	22.58 ± 0.52	49.73		AMP	6.66 ± 0.57	18.41 ± 0.38	176.25
	BAC	25.16 ± 0.2	33.16 ± 0.28	31.78		BAC	25.25 ± 0.25	33.66 ± 0.57	33.3		BAC	15.25 ± 8.80	30.5 ± 0.5	100
	CHL	35.08 ± 0.1	42.08 ± 0.14	19.95		CHL	22 ± 0.25	30.16 ± 0.28	37.09		CHL	35.16 ± 0.28	39.08 ± 0.14	11.13
	KAN	20.08 ± 0.1	28.08 ± 0.14	39.83		KAN	20.25 ± 0.25	25.66 ± 0.57	26.71		KAN	24.25 ± 0.25	33.83 ± 0.28	35.39
	NOR	34.08 ± 0.1	42.08 ± 0.14	23.47		NOR	32.16 ± 0.28	40.33 ± 0.57	25.4		NOR	35.16 ± 0.28	42.16 ± 0.28	19.9
	STR	20.08 ± 0.1	29.08 ± 0.14	44.81		STR	18.33 ± 0.57	27.33 ± 0.28	49.09		STR	23.75 ± 0.66	34.16 ± 0.28	50.18
	TET	30.08 ± 0.1	39.08 ± 0.14	29.91		TET	30.08 ± 0.38	39.5 ± 0.5	30.58		TET	25.33 ± 0.38	34.91 ± 0.14	38.15
	VAN	20.08 ± 0.1	28.9 ± 0.14	43.98		VAN	20.25 ± 0.25	32.66 ± 0.57	61.28		VAN	6.33 ± 0.57	12.16 ± 0.28	92.1
PA	AMK	26.58 ± 0.52	37.5 ± 0.5	41.06	KP	AMK	14.25 ± 0.25	25.5 ± 0.5	78.94	CV	AMK	20.16 ± 0.28	29.41 ± 0.52	45.86
	AMP	6.16 ± 0.28	20.83 ± 1.04	237.83		AMP	6	12.14 ± 0.38	106.94		AMP	6.16 ± 0.28	20.41 ± 0.38	231.08
	BAC	16.08 ± 0.14	34.16 ± 0.28	112.43		BAC	7.08 ± 0.14	12.25 ± 0.43	75.29		BAC	7.08 ± 0.14	11.7 ± 0.66	65.88
	CHL	18.08 ± 0.14	27.58 ± 0.38	52.53		CHL	20.08 ± 0.14	29.16 ± 0.28	45.22		CHL	20.25 ± 0.25	30.08 ± 0.14	48.55
	KAN	23.08 ± 0.14	37.41 ± 0.14	62.09		KAN	15.5 ± 0.43	23.25 ± 0.25	50		KAN	15.33 ± 0.28	25.25 ± 0.25	64.67
	NOR	24.08 ± 0.14	32.08 ± 0.14	33.21		NOR	12.3 ± 0.28	22.33 ± 0.28	81.08		NOR	33.08 ± 0.14	45.41 ± 0.38	37.27
	STR	24.08 ± 0.14	36.25 ± 0.25	50.51		STR	15.0 ± 0.14	22.5 ± 0.43	49.17		STR	15.16 ± 0.14	23.58 ± 0.52	55.49
	TET	22.16 ± 0.28	30.33 ± 0.28	36.84		TET	23.16 ± 0.28	35.08 ± 0.14	51.43		TET	23.08 ± 0.14	30.5 ± 0.5	32.12
	VAN	10.25 ± 0.25	22.16 ± 0.28	116.26		VAN	15.4 ± 0.38	30.33 ± 0.14	96.75		VAN	6.16 ± 0.14	16.16 ± 0.28	162.16

ZOI zone of inhibition (mm), SP synergistic potential. IMO indicator microorganism, MS M. smegmatis, SA S. aureus, PA P. aeruginosa, CV C. violaceum, EC E. coli, KP K. pneumoniae. AMK amikacin, AMP ampicillin, BAC bacitracin, CHL chloramphenicol, KAN kanamycin, NOR norfloxacin, STR streptomycin, TET tetracycline, VAN vancomycin

Fig. 9.

SP evaluation by disc diffusion. The bacteriocin (PPB)—synergistic potential (SP) evaluation is shown with VAN on MS, NOR on SA, CHL on PA, KAN on EC, STR on KP & AMK on CV. A—Antibiotic alone; B—Antibiotic + Bacteriocin

Checkerboard Assay

Checkerboard assays of the PPB revealed a Synergistic combination (ƐFIC values, < 0.5) with an increase in inhibitory activity, by a decrease in MIC of the antibiotic (FIC A) or the bacteriocin (FIC B) or both, with all the antibiotics against all the IMOs used in this study, as depicted in Table 6.

Table 6.

Checkerboard assay results

IMO	Antibiotic	FIC B (µg)	FIC A (µg)	ƐFIC	IMO	Antibiotic	FIC A	FIC B	ƐFIC
MS	AMK	95 ± 7.07/350	0.1 ± 0.03/0.74 ± 0.01	0.43	SA	AMK	45 ± 7.07/325	0.094 ± 0.006/0.25 ± 0.01	0.4
	AMP	95 ± 9.07/350	0.09 ± 0.01/0.52 ± 0.04	0.44		AMP	50/325	0.225 ± 0.03/0.75 ± 0.01	0.45
	BAC	55 ± 7/350	0.08 ± 0.007/0.25 ± 0.01	0.49		BAC	50 ± 14/325	0.275 ± 0.03/1 ± 0.01	0.42
	CHL	90 ± 14/350	0.1 ± 0.04/0.74 ± 0.01	0.42		CHL	37.5 ± 3.5/325	0.237 ± 0.01/1 ± 0.01	0.35
	KAN	90 ± 14/350	0.1 ± 0.05/0.75 ± 0.01	0.41		KAN	45 ± 7.07/325	0.275 ± 0.03/1 ± 0.01	0.41
	NOR	70 ± 14/350	0.1 ± 0.03/0.75 ± 0.01	0.36		NOR	47.5 ± 3.5/325	0.3/1 ± 0.01	0.44
	STR	77.5 ± 10/350	0.06 ± 0.01/0.25 ± 0.01	0.46		STR	40 ± 14/325	0.125 ± 0.03/0.5 ± 0.01	0.37
	TET	85 ± 7.07/350	0.07 ± 0.003/0.52 ± 0.04	0.43		TET	50/325	0.095 ± 0.007/0.75 ± 0.04	0.27
	VAN	32.5 ± 3.5/350	0.08 ± 0.007/0.25 ± 0.01	0.43		VAN	42.5 ± 10/325	0.175 ± 0.03/0.75 ± 0.02	0.36
PA	AMK	110 ± 14/600	0.195 ± 0.01/0.75 ± 0.01	0.44	KP	AMK	130 ± 14/750	0.175 ± 0.03/0.75 ± 0.01	0.40
	AMP	97.5 ± 3/600	0.195 ± 0.01/0.76 ± 0.02	0.41		AMP	135 ± 21/750	0.125 ± 0.03/0.5 ± 0.01	0.43
	BAC	95 ± 7/600	0.125 ± 0.03/0.50 ± 0.01	0.40		BAC	130 ± 14/750	0.11 ± 0.01/0.5 ± 0.02	0.39
	CHL	55 ± 7/600	0.275 ± 0.03/0.75 ± 0.01	0.45		CHL	185 ± 21/750	0.075 ± 0.03/0.75 ± 0.01	0.34
	KAN	62.5 ± 17/600	0.175 ± 0.03/0.76 ± 0.02	0.33		KAN	95 ± 7.07/750	0.15 ± 0.03/0.5 ± 0.02	0.42
	NOR	85 ± 7.07/600	0.175 ± 0.02/1.01 ± 0.02	0.31		NOR	97.5 ± 3.5/750	0.175 ± 0.03/0.75 ± 0.01	0.36
	STR	95 ± 7.07/600	0.15 ± 0.07/0.74 ± 0.03	0.36		STR	82.5 ± 3.5/750	0.25 ± 0.07/0.75 ± 0.02	0.44
	TET	90 ± 14/600	0.25 ± 0.07/1.25 ± 0.01	0.35		TET	140 ± 14/750	0.15 ± 0.07/0.75 ± 0.01	0.38
	VAN	75 ± 7.07/600	0.125 ± 0.03/0.75 ± 0.01	0.29		VAN	115 ± 21/750	0.06 ± 0.01/0.25 ± 0.01	0.39
EC	AMK	97.5 ± 3/700	0.175 ± 0.03/0.75 ± 0.01	0.35	CV	AMK	102 ± 10/750	0.2 ± 0.07/0.75 ± 0.01	0.4
	AMP	92.5 ± 10/700	0.085 ± 0.01/0.24 ± 0.01	0.48		AMP	97.5 ± 3.5/750	0.05 ± 0.01/0.25 ± 0.01	0.35
	BAC	95 ± 7.07/700	0.15 ± 0.07/0.51 ± 0.01	0.43		BAC	95 ± 7.07/750	0.225 ± 0.03/0.74 ± 0.01	0.42
	CHL	92.5 ± 10/700	0.275 ± 0.03/0.75 ± 0.01	0.49		CHL	92.5 ± 3.5/750	0.275 ± 0.03/0.75 ± 0.01	0.48
	KAN	85 ± 7.07/700	0.225 ± 0.03/0.74 ± 0.01	0.42		KAN	132.5 ± 3/700	0.15 ± 0.07/0.50 ± 0.01	0.47
	NOR	110 ± 14/700	0.175 ± 0.03/0.75 ± 0.02	0.38		NOR	125 ± 7/750	0.125 ± 0.03/0.76 ± 0.02	0.33
	STR	122.5 ± 3/700	0.125 ± 0.03/0.53 ± 0.03	0.41		STR	85 ± 7/750	0.275 ± 0.03/0.75 ± 0.01	0.48
	TET	115 ± 21/700	0.075 ± 0.03/0.5 ± 0.01	0.31		TET	95 ± 7.07/750	0.2 ± 0.07/0.76 ± 0.02	0.38
	VAN	92.5 ± 10/700	0.0875 ± 0.01/0.5 ± 0.03	0.29		VAN	92.5 ± 10/ 750	0.065 ± 0.02/0.24 ± 0.01	0.39

IMO indicator microorganism, MS M. smegmatis, SA S. aureus, PA P. aeruginosa, CV C. violaceum, EC E. coli, KP K. pneumoniae. AMK amikacin, AMP ampicillin, BAC bacitracin, CHL chloramphenicol, KAN kanamycin, NOR norfloxacin, STR streptomycin, TET tetracycline, VAN vancomycin, FIC A fractional inhibitory concentration of antibiotic (A), FIC B fractional inhibitory concentration of bacteriocin (B), ƐFIC FIC A + FIC B

Time-Kill Assay

The PPB with the antibiotic combinations with 1MIC showed a 100-fold reduction for MS with AMK, CHL, NOR, and TET, for SA with TET, for CV with NOR, for EC with NOR and STR, and KP with TET signifying synergism. On the other hand, the PB showed a 1000-fold reduction or 3-log10 reductions with the antibiotics used against the IMOs in this study, suggesting their bactericidal potency besides synergism. All the results are depicted in Table 7 with the mean of observations and the standard deviations.

Table 7.

Time-kill assay

B + A	MIC	M S		SA		PA		CV		EC		KP
		PPB	PB	PPB	PB	PPB	PB	PPB	PB	PPB	PB	PPB	PB
B + AMK	0.5	− 3.9 ± 0.01	− 3.8 ± 0.29	− 4.2 ± 0.01	− 3.2 ± 0.01	− 4.0 ± 0.01	− 3.0 ± 0.02	− 4.0 ± 0.01	− 3.0 ± 0.01	− 4.0 ± 0.01	− 3.0 ± 0.03	− 4.1 ± 0.05	− 3.1 ± 0.04
	1	− 3.0 ± 0.04	− 2.0 ± 0.03	− 3.2 ± 0.06	− 2.2 ± 0.07	− 3.1 ± 0.01	− 2.1 ± 0.03	− 3.1 ± 0.03	− 2.1 ± 0.05	− 3.1 ± 0.02	− 1.9 ± 0.07	− 3.2 ± 0.07	− 1.8 ± 0.08
B + AMP	0.5	− 4.0 ± 0.01	− 3.0 ± 0.03	− 4.0 ± 0.01	− 3.1 ± 0.05	− 4.2 ± 0.01	− 3.1 ± 0.04	− 4.2 ± 0.06	− 3.2 ± 0.03	− 4.3 ± 0.02	− 3.1 ± 0.02	− 4.4 ± 0.02	− 3.0 ± 0.02
	1	− 3.1 ± 0.01	− 2.1 ± 0.02	− 3.1 ± 0.02	− 2.1 ± 0.03	− 3.2 ± 0.06	− 2.2 ± 0.07	− 3.3 ± 0.07	− 2.2 ± 0.06	− 3.3 ± 0.06	− 1.9 ± 0.08	− 3.5 ± 0.06	− 1.6 ± 0.08
B + BAC	0.5	− 4.1 ± 0.01	− 3.0 ± 0.01	− 4.0 ± 0.01	− 3.1 ± 0.01	− 4.0 ± 0.01	− 3.0 ± 0.01	− 4.3 ± 0.02	− 3.1 ± 0.06	− 4.1 ± 0.02	− 3.1 ± 0.02	− 4.5 ± 0.02	− 3.2 ± 0.04
	1	− 3.1 ± 0.02	− 2.0 ± 0.01	− 3.1 ± 0.02	− 2.1 ± 0.03	− 3.1 ± 0.04	− 2.1 ± 0.03	− 3.3 ± 0.06	− 2.3 ± 0.07	− 3.1 ± 0.01	− 1.9 ± 0.07	− 3.5 ± 0.03	− 1.8 ± 0.06
B + CHL	0.5	− 3.9 ± 0.01	− 3.3 ± 0.07	− 4.0 ± 0.01	− 3.1 ± 0.02	− 4.1 ± 0.02	− 3.1 ± 0.05	− 4.1 ± 0.05	− 3.0 ± 0.02	− 4.0 ± 0.01	− 3.0 ± 0.03	− 4.2 ± 0.01	− 3.1 ± 0.07
	1	− 2.9 ± 0.03	− 1.9 ± 0.03	− 3.1 ± 0.02	− 2.1 ± 0.03	− 3.2 ± 0.02	− 2.0 ± 0.04	− 3.1 ± 0.03	− 1.9 ± 0.04	− 3.0 ± 0.01	− 1.8 ± 0.04	− 3.1 ± 0.04	− 2.0 ± 0.08
B + KAN	0.5	− 4.1 ± 0.01	− 3.2 ± 0.03	− 4.2 ± 0.05	− 3.1 ± 0.01	− 4.0 ± 0.01	− 3.1 ± 0.05	− 4.1 ± 0.02	− 3.0 ± 0.01	− 4.0 ± 0.01	− 3.1 ± 0.03	− 4.2 ± 0.02	− 3.1 ± 0.03
	1	− 3.2 ± 0.03	− 2.1 ± 0.01	− 3.2 ± 0.02	− 2.0 ± 0.08	− 3.1 ± 0.04	− 2.1 ± 0.02	− 3.2 ± 0.06	− 2.0 ± 0.03	− 3.2 ± 0.02	− 1.9 ± 0.05	− 3.3 ± 0.02	− 1.9 ± 0.03
B + NOR	0.5	− 3.9 ± 0.01	− 3.4 ± 0.03	− 4 ± 0.003	− 3.0 ± 0.03	− 4.1 ± 0.01	− 3.0 ± 0.01	− 3.9 ± 0.02	− 3.2 ± 0.06	− 3.9 ± 0.02	− 3.1 ± 0.04	− 4.2 ± 0.01	− 3.0 ± 0.01
	1	− 2.9 ± 0.03	− 1.9 ± 0.02	− 3.1 ± 0.01	− 2.1 ± 0.03	− 3.1 ± 0.04	− 1.8 ± 0.07	− 2.9 ± 0.04	− 2.0 ± 0.01	− 3.0 ± 0.05	− 1.9 ± 0.06	− 3.2 ± 0.09	− 2.1 ± 0.04
B + STR	0.5	− 3.7 ± 0.01	− 3.5 ± 0.03	− 4.1 ± 0.01	− 3.1 ± 0.01	− 4.0 ± 0.04	− 3.1 ± 0.02	− 4.2 ± 0.03	− 3.1 ± 0.01	− 4.0 ± 0.02	− 3.0 ± 0.03	− 5.2 ± 0.01	− 3.1 ± 0.04
	1	− 3.1 ± 0.02	− 2.1 ± 0.01	− 3.2 ± 0.05	− 2.1 ± 0.02	− 3.1 ± 0.01	− 2.0 ± 0.04	− 3.2 ± 0.05	− 2.1 ± 0.03	− 3.0 ± 0.08	− 1.9 ± 0.08	− 4.2 ± 0.02	− 2.0 ± 0.06
B + TET	0.5	− 3.7 ± 0.01	− 3.2 ± 0.02	− 4.9 ± 0.02	− 3.4 ± 0.06	− 4.1 ± 0.01	− 3.0 ± 0.01	− 4.0 ± 0.02	− 3.0 ± 0.02	− 4.0 ± 0.01	− 3.0 ± 0.03	− 4.0 ± 0.02	− 3.0 ± 0.06
	1	− 2.8 ± 0.02	− 1.9 ± 0.13	− 3.0 ± 0.05	− 2.0 ± 0.05	− 3.2 ± 0.04	− 1.9 ± 0.06	− 3.1 ± 0.07	− 2.0 ± 0.05	− 3.0 ± 0.07	− 2.0 ± 0.03	− 3.0 ± 0.07	− 2.0 ± 0.09
B + VAN	0.5	− 4.1 ± 0.02	− 3.1 ± 0.01	− 4.0 ± 0.01	− 3.0 ± 0.01	− 4.2 ± 0.01	− 3.1 ± 0.09	− 5.4 ± 0.01	− 3.3 ± 0.04	− 4.4 ± 0.06	− 3.2 ± 0.06	− 4.1 ± 0.01	− 3.0 ± 0.08
	1	− 3.1 ± 0.05	− 2.1 ± 0.04	− 3.1 ± 0.06	− 2.1 ± 0.04	− 3.3 ± 0.04	− 2.3 ± 0.05	− 3.4 ± 0.03	− 2.1 ± 0.02	− 3.5 ± 0.06	− 2.2 ± 0.06	− 3.1 ± 0.03	− 2.0 ± 0.04

B bacteriocin, A antibiotic, PPB partially purified bacteriocin, PB purified bacteriocin, MIC minimal inhibitory concentration. MS M. smegmatis, SA S. aureus, PA P. aeruginosa, CV C. violaceum, EC E. coli, KP K. pneumoniae. AMK amikacin, AMP ampicillin, BAC bacitracin, CHL chloramphenicol, KAN kanamycin, NOR norfloxacin, STR streptomycin, TET tetracycline, VAN vancomycin, FIC A fractional inhibitory concentration of antibiotic (A), FIC B fractional inhibitory concentration of bacteriocin (B), ƐFIC FIC A + FIC B

Cytotoxicity Studies

MTT assay revealed that the bacteriocin is not cytotoxic to the 3T3 (normal fibroblast cell lines) cells and its cell viability was further confirmed by trypan blue staining of cells treated with the control and bacteriocin samples of 10, 25, 50, and 100 μg/ml at 96 h as depicted in Fig. 10.

Fig. 10.

MTT assay and Trypan Blue staining determined at 24, 48, 72, and 96 h of incubation

Discussion

The Bacillus genus has the remarkable ability to generate numerous antimicrobial peptides that possess highly encouraging characteristics [29]. Concerning this matter, only a limited number of antimicrobial peptides generated by the bacillus genus possess the capability to effectively combat the formation of biofilms and demonstrate synergistic effects. These antimicrobial peptides have immense potential in the realm of medicine and can potentially transform the methods we employ to combat bacterial infections. The primary objective of this research was to study and explore the antibiofilm characteristics of a bacteriocin derived from Bacillus subtilis (MK733983), which was identified as a temporary endophyte. In addition, the current study aimed to explore the potential synergistic effects of the mentioned bacteriocin when used in combination with commercially available antibiotics.

The findings from multiple qualitative and quantitative experiments conducted in this research study strongly indicate that the bacteriocin displayed a broad spectrum of inhibitory activities and possessed several beneficial characteristics. These include its ability to effectively inhibit at suitable doses, efficient killing kinetics, prevent biofilm formation by creating pores, enhance the effectiveness of antibiotics through synergistic actions, and the absence of any adverse effects. Each of these results will now be discussed in detail.

Typically, bacteriocins that come from closely related groups of bacteria are more effective against species that are also closely related. However, in this particular study, the bacteriocin was produced by gram-positive bacteria, yet it surprisingly showed greater effectiveness in killing gram-negative IMOs, as indicated by higher minimum bactericidal concentrations (MBCs) of MBC50 and ≥ 90. The occurrence of such a coincidence is quite remarkable and unexpected, but it is not unprecedented in the field of bacteriocins. Previous studies on bacteriocins like Subtilein [30], Thuricin 17 [31], Cerein 8A [32], and Gas 101 [33] have also reported similar phenomena. These findings highlight the need for further investigation and evaluation to better understand and interpret these unexpected observations.

Furthermore, the interaction between the drug and the host organism can also affect its overall efficacy. Each individual’s unique physiology and immune response can influence how well the drug is absorbed, distributed, metabolized, and eliminated within the body. These pharmacokinetic and pharmacodynamic factors can greatly impact the drug’s ability to reach its target site and effectively combat the infection. Another factor that influences the effectiveness of antimicrobial drugs is the quantity of inoculum or the initial number of bacteria present in the infected area. Higher levels of bacteria can pose a greater challenge for the drugs to eradicate, as they may require higher dosages or longer treatment durations to effectively eliminate the infection. In other words, the effectiveness of antimicrobial drugs in combating bacterial infections is heavily influenced by various factors. The condition of the bacteria, the quantity of inoculum, and the drug’s interaction with the host organism all play crucial roles in determining the antimicrobial drug’s efficacy. Understanding and considering these factors is essential in developing and administering appropriate treatment regimens for bacterial infections [34]. Microorganisms that have a higher tolerance need to be exposed to antimicrobial substances for a longer period to inhibit and eliminate susceptible microorganisms. Therefore, it is crucial to determine the minimum duration of killing (MDK) through time-kill curves or growth dynamic studies. This is significant because microorganisms that are not tolerant may also have the same minimum inhibitory concentration (MIC) value [35].

The bacteriocin showed a significant inhibitory effect that varied depending on the dosage, that was observed within a 15-h time frame and ranged from 0.5 to 1 minimum inhibitory concentration (MIC). This effect was observed in a growth dynamic study involving standard strains of different microorganisms, including S. aureus, M. smegmatis, P. aeruginosa, C. violecium, E. coli, and K. pneumoniae. These strains were determined to be strong biofilm producers through qualitative and quantitative assessments of biofilm formation. However, when the bacteriocin concentration was increased to 2 MIC, there was a noticeable reduction in SA and KP, but other microorganisms did not show a dose-dependent decrease in growth and had a similar minimum dose for killing (MDK) of the bacteriocin. This finding suggests the need for further investigation to gain a more comprehensive understanding.

The initial step in biofilm formation occurs when cells attach themselves to a surface. Subsequently, microcolonies undergo irreversible growth and advancement, during which they release a substance known as extracellular polysaccharide substance (EPS). This EPS, primarily produced by bacteria, is influenced by the host organism. Over time, the microcolonies merge to form a bio-molecular layer, which becomes embedded within an extracellular polymeric matrix. This matrix possesses distinct methods for quorum sensing [36]. During the process of biofilm formation, specific bacterial species, such as E. coli, Salmonella, B. subtilis, S. aureus, and S. mutans, have been observed to produce amyloid fibers known as ‘curli’ [37]. Some strains of B. subtilis have been observed to create a distinct biofilm center known as the ‘coffee ring’ [38].

Even more interestingly, it has been discovered that many microorganisms can form biofilms through the acquisition of specific quorum-sensing systems. This adaptation allows them to survive in challenging environments. Some microorganisms have been identified as potential causes of chronic clinical infections related to biofilms. They are capable of impeding the host’s phagocytes and the complement system, as well as increasing their resistance to common antibiotics. Given the increasing problem of antimicrobial resistance and limited treatment options, there is an urgent requirement for the development of new antimicrobials that employ diverse strategies to effectively address emerging diseases due to biofilm-forming microbes [39, 40]. The minimal Biofilm Inhibitory Concentration (MBIC) refers to the lowest level of a substance that effectively stops the initial growth of biofilm. On the contrary, the Minimum Biofilm Eradication Concentration (MBEC) indicates the minimum level necessary to eliminate biofilm that has already formed.

In the current research, the MBIC of the bacteriocin (PPB & PB) is lower than its MBEC. This distinction may arise from the fact that a smaller quantity of the antimicrobial agent is necessary to hinder the formation of biofilm initially, in comparison to the amount needed to eradicate or eliminate a pre-existing biofilm.

Bacteriocins have different ways of working and can be broadly classified as either inhibitors of the target cell membrane or inhibitors of cell metabolisms such as DNA, RNA, and protein metabolism [41]. The process of dissimulating biofilms is a complex task that involves destroying the extracellular matrix and modifying cellular metabolism. Through scanning electron microscopy (SEM), it was observed that pore creation occurred in the cell membranes of both S. aureus and P. aeruginosa, suggesting permanent damage to the cell membrane. Moreover, this damage led to the complete misshaping of the cells and the rupture of the biofilms.

Several research studies indicate that bacteriocins are proposed to be a component of cytolytic pore-forming proteins, which function through a mechanism called the barrel-stave mechanism. Furthermore, these studies demonstrate that the way bacteriocins work is connected to the phospholipid makeup of the membrane. When liposomes within cell membranes are composed of phosphatidylcholine (PC), bacteriocin can act as a transporter that selectively carries anions. The effect of bacteriocin on membranes containing anionic phospholipids involves locally disrupting the structure of the bilayer and inserting bacteriocin into the membrane. Additionally, the electrostatic interactions between the bacteriocin molecules and the phospholipids could cause the lipid head groups to align with the pore.

The bacteriocin has undeniably demonstrated its ability to disturb cell membranes, which supports the theory mentioned above. Nonetheless, conducting a more thorough analysis by employing advanced techniques like transmission electron microscopy could potentially unveil the effects of this substance on various cellular organelles.

Furthermore, the bacteriocin displays bactericidal properties against microorganisms that form biofilms, regardless of whether they are gram-positive or gram-negative bacteria. Moreover, the bacteriocin acts on biofilms by causing the formation of pores, leading to irreversible destruction. Thus, the bacteriocin can significantly contribute to the fight against antibiotic-resistant bacteria due to their ability to selectively target pathogenic microbes, their low toxicity levels, and their impressive stability and specificity.

To assess the possible combined benefits of bacteriocin (PPB), disc diffusion experiments were carried out. Various strains of important microorganisms such as SA, MS, PA, KP, EC, and CV were subjected to exposure with nine commonly utilized antibiotics including amikacin (AMK), ampicillin (AMP), bacitracin (BAC), chloramphenicol (CHL), kanamycin (KAN), norfloxacin (NOR), streptomycin (STR), tetracycline (TET), and vancomycin (VAN). The outcomes of these experiments indicated a comparable result in experiments conducted on the checker-board assays, which implies the potential for synergy between the substances being tested. This was evident as the Fractional Inhibitory Concentrations (FIC) were found to be below 0.5. These FIC values were subsequently utilized to assess the combined effects of bacteriocins and antibiotics, as well as to gain insight into the dynamics of their interaction.

Furthermore, the results of time-kill analyses not only confirmed the bactericidal activity of the bacteriocin (PB), but also unveiled its remarkable ability to enhance the effectiveness of antibiotics when used in combination. Moreover, the research findings shed light on the bacteriocin’s remarkable capability to inhibit the proliferation of Chromobacterium, a well-known bacterium that has been extensively investigated for its intricate quorum-sensing mechanisms. This significant discovery also implies that the bacteriocin possesses the potential to impede the formation of biofilms by other microorganisms, thereby offering promising prospects in combating their detrimental effects.

The MTT test yielded empirical evidence indicating that the bacteriocin does not exhibit any indications of toxicity, even when exposed to extended durations such as 96 h. This discovery offers substantiating proof that supports the categorization of the bacteriocin as generally regarded as safe (GRAS) for proteins. The bacteriocin displays immense promise for utilization due to its capability to impede the formation of biofilms and its synergistic impact when combined with antibiotics.

Conclusion

The increasing prevalence of antimicrobial resistance, coupled with a scarcity of effective antimicrobial agents, poses a significant socioeconomic burden. The potential emergence of substantial biofilm infections poses a tremendous threat not only in the medical field but also in agriculture, dairy production, fisheries, and poultry farming. There is a collective need for antimicrobial drugs with distinct mechanisms of action. The bacteriocin analyzed in this study exhibited remarkable efficacy in reducing biofilm formation, indicating its ability to killing broad spectrum bacteria and have beneficial interactions with antibiotics. Furthermore, extensive experimental research has verified that it does not harm healthy cells, thus confirming its potential for future application.

Author Contributions

Dr. SSS & Dr. VA have contributed in designing this study and drafting the manuscript. Dr. SSS performed research, analyzed data, and wrote the article. Special regards to Dr. Salamun, who has contributed in obtaining the multiple purified (HPLC) samples of the bacteriocin.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data Availability

All data, text and results presented in this publication are authors own. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Consent to Participate

The authors give their consent to participate.

Consent for Publication

The authors give their consent for publication.