For the experiments conducted in this study, various antibiotics, media, and reagents were utilized. The antibiotic disks were obtained from MAST (Mast Co., UK), while the powders of oxacillin and penicillin were acquired from Sigma-Aldrich (St. Louis, MO, USA). Media and agar supplies were obtained from Merck (Merck Co., USA). Specifically, mannitol salt agar, blood agar, Mueller-Hinton agar, Mueller-Hinton broth (MHB), sodium chloride (NaCl), and magnesium chloride (MgCl2) were sourced from Merck (Merck Co., USA). Chemical reagents including crystal violet, glucose, and agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). In addition, 96-well sterile polystyrene, non-treated microplates, available in both flat-bottom and round-bottom variants, were obtained from NEST (Biotechnology Co., Ltd., China), and Jet Biofil (Guangzhou, China).

Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ) was synthesized by DGpeptides Company (Wuhan, Hubei, China) using solid-phase synthesis techniques and ensured purity greater than 96% (Mirzaei et al., 2022b). The purity of the synthetic peptides was verified through reversed-phase high-performance liquid chromatography, and the accuracy of the synthesis was confirmed through liquid chromatography-mass spectrometry (LC-MS) equipment. The peptide content was validated using the bicinchoninic acid assay (BCA) (Bevalian et al., 2021).

All experiments in this study utilized eight strains of S. aureus, which included clinical VRSA, MRSA, and MSSA, as well as ATCC strains. Six clinical isolates were obtained from human sources at Motahhari burns hospital in Tehran, Iran (Table 1). Moreover, S. aureus ATCC 25923, and S. aureus ATCC 29213 were procured from the Pasteur Culture Collection of Tehran, Iran. The isolates were collected from a diverse range of individuals in terms of age and gender, ensuring that there were no duplications; only a single sample per patient was taken. To characterize S. aureus isolates, several biochemical tests were employed, including assessments of colony morphology, gram-positive properties, clustering of cocci, catalase activity, mannitol fermentation, DNase production, and coagulase activity (Bevalian et al., 2021). Then PCR amplification was done to confirm the species of the isolates previously identified phenotypically as S. aureus. To extract bacterial DNA from staphylococcal strains, a phenol-chloroform technique was employed in the presence of sodium dodecyl sulfate (SDS), preceded by pretreatment with higher doses of lysozyme (200 mg/ml final concentration) and proteinase K (300 mg/ml final concentration). The extracted DNA was subsequently purified using phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol), chloroform-isoamyl alcohol (24:1, vol/vol), and then precipitated with sodium acetate and ethanol at −20°C. The precipitated DNA was washed with 70% ethanol and resuspended in 100 μl of Milli-Q water. PCR amplification of 16srRNA and nuc genes was done (Abbasi-Montazeri et al., 2013; Zhang et al., 2021; Supplementary Table 1). The confirmed isolates were then stored at −70°C in brain heart infusion (BHI) broth with 30% glycerol.

The primary objective of this research was to delve into the antimicrobial attributes of melittin, concentrating particularly on its efficacy against S. aureus isolates. Moreover, we investigated the synergistic potential of melittin when combined with penicillin and oxacillin. This investigation aimed to shed light on the remarkable properties of melittin and its cooperative effects with conventional antibiotics in combating S. aureus infections.

To determine the Minimum Inhibitory Concentration (MIC) values of melittin, penicillin, and oxacillin in S. aureus isolates, a standard microtiter dilution method was employed using Muller Hinton Broth (MHB) medium, in accordance with Clinical and Laboratory Standards Institute 2021 (CLSI) (Kowalska-Krochmal and Dudek-Wicher, 2021). Fresh colonies of S. aureus were grown overnight at 37°C in 5 mL of MHB medium. The quantity of bacterial cells was calibrated to the 0.5 McFarland standard using spectrophotometry at 625 nm, and to improve the accuracy of cell quantification, the optical density (OD)was set at 0.09 in this study, instead of the typical range of 0.08 to 0.1, which is equivalent to 10⁸ CFU/mL. The bacterial cell quantities were then adjusted to 10⁶ CFU/mL in the same medium. In 96-well microplates, 100 μL of MHB medium was added to each well. Melittin (from 40 to 0.039 μg/mL) and antibiotics (from 512 to 1 μg/mL), produced in a normal saline solution, were serially diluted in the microplates. Next, 100 μL of the provided suspension, containing 2 × 10⁶ bacteria, was added to each well of the microplates and incubated at 37°C for 18–24 h, the lowest value of the investigated drugs that completely inhibited observable bacterial growth, was used to determine the MIC, and also, 100 μL of the provided suspension, containing 2 × 10⁶ bacteria, were cultured on MHB medium and counted, with the MBC values identified as the lowest concentrations of agents required to kill 100% of the cultured isolates. The concentration of the peptide or antibiotic that resulted in complete eradication of bacteria was considered as MBC (Thabaut and Meyran, 1984). All experiments were conducted in accordance with CLSI guidelines. Finally, the MBC/MIC ratio was computed as a means of determining whether isolates exhibited tolerance to antimicrobial agents.

In summary, the synergistic effects of melittin, penicillin, and oxacillin were evaluated using the broth microdilution method. The antibacterial agents were serially diluted and added to microtiter plates, followed by the addition of a bacterial suspension. The microplates were incubated for 24 h at 37°C. The fractional inhibitory concentration index (FICi) was calculated for the combined antibacterial agents, using the formula FIC = (MIC drug A in combination/MIC drug A alone) + (MIC drug B in combination/MIC drug B alone). The FIC indices were used to determine the type of drug interaction, with values of n ≤ 0.5 indicating synergy, values of 0.5 < n < 1 indicating partial synergy, a value of n = 1 indicating an additive effect, values of 1 < n < 4 indicating an indifferent effect, and values of 4 ≤ n indicating an antagonistic effect (Mirzaei et al., 2023). All experiments were conducted using the broth microdilution method in accordance with established protocols.

To investigate the interactions between antibacterial drugs, the MBC values were used in conjunction with the broth microdilution checkerboard method, also known as the fractional bactericidal concentration index (FBCi). The antibacterial agent interaction method was carried out in a manner similar to the FIC method. A 100 μL volume of the diluted bacterial stock containing 10⁵ CFUs/mL was added to each well, and the microplate was incubated at 37°C for 24 h. After incubation, 10 μL from each well was cultured on MHA, and the MBC values of melittin, penicillin, and oxacillin were determined as the lowest concentrations of agents required to kill 100% of the cultured isolates. Finally, the FBCi was calculated using the formula FBCi = (MBC drug A in combination/MBC drug A alone) + (MBC drug B in combination/MBC drug B alone). The FBCi indices were used to determine the type of interaction between the antibacterial agents, as mentioned for FICi (Mirzaei et al., 2023).

In this study, the microplate assay technique was used to study biofilm formation. Fresh colonies of S. aureus ATCC 25923 were cultured overnight at 37°C in 5 mL of TSB with 1% Glucose (Khandan Del et al., 2019). The bacterial suspension’s absorbance was measured at a wavelength of 625 nm to create a 0.5 McFarland standard suspension, with a value of 0.09. A prepared bacterial suspension containing 10⁷ CFUs was created by adding 100 μL of the suspension to 900 μL of TSB under varied conditions in a sterile tube. Next, 200 μL of the bacterial suspension, equivalent to 2 × 10⁶ cells, was added to each well of a 96-well microplate and incubated at 37°C and 70 rpm for 24 h. After incubation, the wells were washed thrice with normal saline and allowed to air dry. Methanol (200 μL) was added to fix the biofilm, and the microplates were allowed to dry at room temperature. Crystal violet (0.05%) was added to each well and incubated for 5 min. The microplates were washed thrice with normal saline and allowed to dry at room temperature. Finally, 200 μL of absolute ethanol was added to each well and shaken at 37°C for 30 min. The contents of each well were transferred to another microplate, and an absorbance measurement was taken at 595 nm using a Synergy HTX Multi-Mode Microplate Reader (BioTek Co., Winooski, VT, USA). All experiments were repeated three times, and the biofilm formation assay based on optimized conditions. The OD cut-off value (ODc) was established using the following formula: ODc = average OD of negative control + (3 × SD of negative control), where the ODc is three standard deviations (SD) above the mean of the OD of the negative control. Finally, the results were sorted into four groups based on their ODs: (1) strong biofilm producer (4 × ODc < OD); (2) medium biofilm producer (2 × ODc < OD ≤ 4 × ODc); (3) weak biofilm producer (ODc < OD ≤ 2 × ODc); and (4) non-biofilm producer (OD ≤ ODc) (Mirzaei et al., 2023).

Besides, to acquire a more precise understanding of biofilm formation in VRSA, MRSA, and MSSA, we conducted a comprehensive analysis of the kinetics involved. It has been established that biofilm formation in S. aureus strains is a strain-dependent process, meaning that the development of mature biofilms varies across different strains and over varying periods of time. For a thorough examination, we evaluated the biofilm development kinetics of all isolates for a duration of 48 h (Khandan Del et al., 2019), Considering this information, we proceeded with the biofilm experiments, focusing on evaluating the OD of each sample at specific time intervals: 3, 6, 9, 12, 18, 24, and 48 h. By measuring the OD, we aimed to gain insights into the progression of biofilm formation for each strain. To ensure the reliability and accuracy of our results, we repeated the experiments three times. By employing this rigorous approach, we aimed that would shed light on the intricate processes involved in biofilm formation across various strains of S. aureus.

Several studies have provided evidence supporting the disruptive and inhibitory properties of melittin in relation to biofilm formation. Melittin has exhibited promising outcomes in reducing the viability and biomass of biofilms when administered alone. In light of these findings, we conducted experiments to assess the antibiofilm potential of this peptide against biofilms formed by S. aureus isolates. Additionally, we evaluated the synergistic effects of melittin in combination with penicillin and oxacillin against S. aureus biofilms.

In summary, the inhibitory effects of melittin, penicillin, and oxacillin on pre-formed biofilms were examined by determining the Minimum Biofilm Inhibitory Concentration (MBIC) (Anbarasi et al., 2020). S. aureus colonies were grown in TSB medium supplemented with 1% Glu, and the bacterial quantity was adjusted to the 0.5 McFarland standard. A suspension of 2 × 10⁶ CFUs was added to each well of 96-well microplates and incubated for 24 h at 37°C with shaking at 70 rpm. After incubation, the contents of the wells were washed with normal saline solution. Next, 100 μL of melittin (ranging from 40 to 0.039 μg/μL), penicillin, and oxacillin (ranging from 1,024 to 2 μg/μL) were added to the wells at a volume of 100 μL and incubated at 37°C for 24 h, and the quantity of biofilm was measured. The MBIC was determined as the least quantity of melittin and antibiotics that resulted in at least 90% inhibition in biofilm biomass compared to the untreated control. The percentage of biofilm inhibition was calculated using a standard formula. All experiments were repeated three times for each isolate.

The Minimum Biofilm Eradication Concentration (MBEC) assay was conducted using 96-well microplates, following the previously described protocol with modifications to determine the effectiveness of melittin, penicillin, and oxacillin in killing and degrading bacteria within biofilms (Mirzaei et al., 2022b). As noted previously (Bardbari et al., 2018), the isolates were given 24 h to produce a pre-formed biofilm. The wells were then gently discarded and washed three times with normal saline solution. Next, 100 μL of melittin (ranging from 40 to 0.039 μg/μL), penicillin, and oxacillin (ranging from 1,024 to 2 μg/μL) were added to the wells at a volume of 100 μL and incubated at 37°C for 24 h. The Minimum Biofilm Eradication Concentration (MBEC) assay was performed as previously described by using 96 Well microplates (Mirzaei et al., 2022b). Modifications were made to determine how well melittin, penicillin, and oxacillin could kill bacteria that were surrounded in biofilms as well as degrade them. In summary, as previously noted, the studied isolates were initially given 24 h to produce a pre-formed biofilm (Bardbari et al., 2018). Following, the wells were gently discarded and washed three times with the normal saline solution. Then, 100 μL melittin (from 40 to 0.039 μg/μL), penicillin, and oxacillin (from 1,024 to 2 μg/μL) were added into the wells at the volume of 100 μL and incubated at 37°C for 24 h.

Staphylococcus aureus isolates were cultured for 24 h at 37°C with shaking at 70 rpm in 5 mL of 1% Glu TSB. As mentioned above, the number of bacteria was adjusted to 0.5 McFarland standard. A suspension of 2 × 10⁶ CFUs as above prepared was added to each well of 96 Well microplate and incubated at overnight incubation, then the contents of the wells were slowly discarded, and the wells were washed three times with normal saline solution. the dilutions of each of melittin (from 40- to 0.039 μg/μL), penicillin (from 1,024 to 2 μg/μL), and oxacillin (from 1,024 to 2 μg/μL) were provided at a volume of 100 μL and added into the wells, and plates were incubated at 37°C for 24 h with shaking at 70 rpm, and as previously stated the quantity of biofilm was then measured. Then, MBIC was considered as discussed above, and FBICi for the two combined anti-bacterial agents was calculated as follows: FBICi = (MBIC drug A in combination/MBIC drug A alone) + (MBIC drug B in combination/MBIC drug B alone). FBIC indices are pointed to the kind of drug interaction if the following data are established: Synergy, values n ≤ 0.5; Partial synergy, values 0.5 < n < 1; Additive effect, for a value n = 1; Indifferent effect, for values 1 < n < 4; Antagonistic effect, for a value 4 ≤ n (Mackay et al., 2000).

Staphylococcus aureus isolates were cultured for 24 h at 37°C with shaking at 70 rpm in 5 mL of 1% Glu TSB. As mentioned above, the number of bacteria was adjusted to 0.5 McFarland standard. A suspension of 2 × 10⁶ CFUs as above prepared was added to each well of 96 Well microplate and incubated at overnight incubation, then the contents of the wells were slowly discarded, and the wells were washed three times with normal saline solution. the dilutions of each of melittin (from 40 to 0.039 μg/μL), penicillin (from 1,024 to 2 μg/μL), and oxacillin (from 1,024 to 2 μg/μL) were provided at a volume of 100 μL and added into the wells, and plates were incubated at 37°C for 24 h with shaking at 70 rpm, and as previously stated, following three times of saline solution washing after discarding the contents of the wells, 100 μL of the solution was poured to the wells. After scratching and mixing, 10 μL of the well contents were cultured on MHA at 37°C for 24 h, and the growing colonies were counted. The MBEC values for melittin and penicillin, and oxacillin were defined as mentioned above. Afterward, FBECi for the two combined antibacterial agents was calculated as follows: FBECi = (MBEC drug A in combination/MBEC drug A alone) + (MBEC drug B in combination/MBEC drug B alone) (Mirzaei et al., 2022b). FBEC indices are pointed to the kind of drug interaction if the following data are established: Synergy, values n ≤ 0.5; Partial synergy, values 0.5 < n < 1; Additive effect, for a value n = 1; Indifferent effect, for values 1 < n < 4; Antagonistic effect, for a value 4 ≤ n (Mirzaei et al., 2022b).

Field emission scanning electron microscopy (FE-SEM) was used to visualize the effect of synergistic effects of melittin with penicillin and oxacillin antibiotics in their FBIC amounts on the biofilm formation of S. aureus. In this assay, we used one VRSA isolate. The sample preparation was performed based on the protocol previously described by Shams Khozani et al. (2019) with some modification. Briefly, the fresh bacteria were cultured in TSB with 1% glucose at 37°C for 24 h and then melittin, melittin-penicillin, and melittin-oxacillin was incubated with a bacterial suspension containing 1.5 × 10⁷ CFU/mL at 37°C for 24 h, in accordance with its predefined FBIC dose. Before incubation, sterile slides were cut and put into the wells. Briefly, the slides were gently washed three times with sterile distilled water and the sample was fixed in glutaraldehyde (2.5% in PBS 1 × ) for 3 h at room temperature. The slides were then rinsed three times in distilled water and post-fixed in 1.5% osmium tetroxide for 1 h. Then the slides were rinsed three times in distilled water, and they were dehydrated in a series of ethanol solutions (20–100%). The specimens were then mounted on conductive copper SEM tape for sample preparation, coated with gold nanoparticles, and then examined in an FE-SEM instrument (MIRA3, TESCAN Co., Czechia).

Statistical analysis was conducted using SPSS Statistics (IBM SPSS Statistics for Windows, V.21.) to assess the data obtained from the experiments. To evaluate the significance of the findings, a paired-sample t-test was utilized, specifically comparing the concentrations of melittin and antibiotics in combination in terms of their anti-biofilm effect. Additionally, an ANOVA test was employed to compare values such as FIC, FBC, FBIC, and FBEC. The results were reported as the mean ± standard deviation (SD), unless specified otherwise. We conducted various tests to ensure the appropriateness of our data for statistical analysis. First, we assessed the normality of the data distribution using both the Shapiro-Wilk and Kolmogorov-Smirnov tests, and the data met the criteria for a Gaussian distribution. Next, for conducting t-tests, we verified several essential assumptions, including the scale of measurement, random sampling, normality of data distribution, adequacy of sample size, and equality of variance in standard deviation. All these prerequisites were confirmed before performing the t-tests. To use the ANOVA test, we ensured that each group’s sample was drawn from a population with a normal distribution, that all populations had a common variance, and that all samples were drawn independently of each other. Furthermore, we conducted Tukey’s post-hoc test to compare the mean of each group with the mean of every other group. The experiments were conducted with a confidence level of 95%, and a p-value less than 0.05 was considered statistically significant. To examine the correlation between examined concentrations and activity percentages, a non-linear regression test was performed. It is important to note that all experiments were repeated three times to ensure accuracy and reliability of the results.

This study analyzed six samples from a total of 50 verified S. aureus isolates previously collected from patients with burn infections in a separate investigation (Bevalian et al., 2021). Additionally, two ATCC strains were included in the analysis. The results of the disk diffusion method used to test the antibacterial susceptibility of S. aureus isolates to gentamicin, clindamycin, rifampin, and DXT, as well as the MIC test for penicillin and oxacillin, are presented in Table 1.

This section presents the antimicrobial findings regarding the efficacy of melittin, both alone and in combination with penicillin, and oxacillin against the planktonic form of S. aureus to evaluate the potential synergistic effects of melittin and antibiotics in combating planktonic bacteria. To reach this aim, various experiments were conducted, including tests for MIC, MBC, FIC, and FBC. The effects of different concentrations of melittin, as well as melittin-antibiotic combinations, were examined.

Six clinical isolates that collected from burn wound infections were tested for their MIC and MBC against penicillin, oxacillin, and melittin. Based on the MIC results, all of isolates resistant to penicillin (≥0.25 μg/mL) and 66% (n = 4) of isolates were resistant to oxacillin In addition, four of isolates were susceptible to vancomycin while unfortunately, we found two vancomycin-resistant MDR-MRSA isolate with MIC equal to 16 μg/ml. The findings indicated that melittin inhibited the growth of all S. aureus isolates, with MIC values ranging from 0.625 to 5 μg/ml. Besides, the results also showed the bactericidal activity for melittin against all S. aureus isolates, with MBC values ranging from 2.5 to 20 μg/ml.

For S. aureus isolates, the geometric means of the MIC values for penicillin, oxacillin, and melittin were 19.02, 16, and 1.62 μg/ml, respectively, and the geometric means of the MBC values for penicillin, oxacillin, and melittin were 107.63, 49.35, and 5.45 μg/ml, respectively. Also, the geometric means MBC/MIC ratios for penicillin, oxacillin, and melittin were 5.65, 3.08, 3.36 μg/ml respectively. Further details are displayed in Table 2.

In the current investigation, we used the serial dilution method to determine the FICi and FBCi values, which stand for the interaction coefficients, to evaluate the antimicrobial agent interactions (Tables 3–6). These values indicate whether the combined inhibitory and bactericidal effects of drugs are synergistic, additive, indifferent, and/or antagonistic against selected isolates. In general, melittin-penicillin and melittin-oxacillin combination indexes based on FICi values against all isolates were 0.37 and 0.03, respectively. For MSSA 1, MRSA 2, MRSA 3, MRSA 4, MRSA 5, MRSA 6, ATCC 25923, and ATCC 29213, the geometric means of FICi for various melittin-penicillin synergistic concentrations were calculated as 1.25, 0.625, 1.25, 0.312, 0.312, 0.078, 0.625, and 0.078, respectively (Table 3). For MSSA 1, MRSA 2, MRSA 3, MRSA 4, MRSA 5, MRSA 6, ATCC 25923, and ATCC 29213, the geometric means of FICi for various melittin-oxacillin synergistic concentrations were calculated as 0.625, 0.038, 0.312, 0.002, 0.008, 0.004, 0.038, and 0.018, respectively (Table 4). A paired sample t-test revealed a significant difference between MIC values of melittin alone, melittin-penicillin, and melittin-oxacillin combinations against isolates (p = 0.006 and p = 0.005, respectively). In specifically, in MSSA 1, MRSA 2, MRSA 3, MRSA 4, MRSA 5, MRSA 6, ATCC 25923, and ATCC 29213 their MIC values of penicillin and melittin was decreased 8, 8, 8, 4, 8, 16, 8, and 16-fold, respectively, and for oxacillin and melittin was decreased 16, 128, 32, 256, 256, 256, 128, and 64-fold, respectively.

Most significantly, the interactions between melittin-penicillin and melittin-oxacillin combinations were assessed against all of isolates using MBC values. Melittin-penicillin and melittin-oxacillin generally had combination indexes based on FBC values against all isolates of 1.145 and 0.711, respectively. In this regard, for MSSA 1, MRSA 2, MSSA 3, MRSA 4, MRSA 5, MRSA 6, ATCC 25923, and ATCC 29213 strains the geometric means of FBCi for various melittin– penicillin synergistic concentrations were calculated as 2.5, 1.25, 2.5, 0.625, 0.625, 0.625, 2.5, and 0.625, respectively (Table 5), And, for MSSA 1, MRSA 2, MSSA 3, MRSA 4, MRSA 5, MRSA 6, ATCC 25923, and ATCC 29213 strains the geometric means of FBCi for various melittin– oxacillin synergistic concentrations were calculated as 1.25, 1.25, 0.625, 0.44, 0.625, 0.625, 1.25, and 0.312, respectively (Table 6). A paired sample t-test revealed a significant difference between MBC values of melittin alone, melittin-penicillin, and melittin-oxacillin combinations against isolates (p = 0.01 and p = 0.009, respectively). In specifically, in MSSA 1, MRSA 2, MRSA 3, MRSA 4, MRSA 5, MRSA 6, ATCC 25923, and ATCC 29213 their MBC values of penicillin and melittin was decreased 8, 8, 8, 8, 16, 8, 16, and 8-fold, respectively, and for oxacillin and melittin was decreased 16, 8, 32, 16, 16, 8, 32, and 16-fold, respectively.

All the isolates showed strong biofilm formation, also the ATCC strain. The results of the kinetics of biofilm formation showed that a maximum OD value was reached after 24 h of incubation, which ranged between 1.703 and 3.757, and after 48 h, we saw a decrease in biofilm production, which ranged between 0.919 and 2.245. The geometric means of the OD value of biofilm formation for 3, 6, 9, 12, 18, 24, and 48 h were 0.533, 0.788, 1.398, 1.787, 1.934, 2.190, and 1.293, respectively. The results of the Kinetics of Biofilm Formation are shown in Table 7 and Figure 1.

This section of our work presents the findings regarding the anti-biofilm effects of melittin, both individually and in combination with antibiotics, against S. aureus biofilms. The objective was to evaluate the potential synergistic effects of melittin and antibiotics in targeting biofilms, which are known for their heightened resistance to antimicrobial treatments. To accomplish this goal, a series of experiments were conducted, focusing on assessing the inhibition or eradication of biofilms. This involved evaluating parameters such as the MBIC and MBEC for melittin, penicillin, and oxacillin alone, as well as the combination of melittin with antibiotics, represented by the FBIC and FBEC. These experiments aimed to provide insights into the potential synergistic interactions between melittin and antibiotics, which could enhance their antibiofilm activities.

The results showed that melittin inhibited the biofilm formation of all S. aureus isolates, with MBIC values ranging from 10 to 1.25 μg/mL. Besides, the MBIC results for penicillin and oxacillin were 512–516 μg/mL, and 512–532 μg/mL, respectively. Melittin, penicillin, and oxacillin had the geometric means of 3.85, 181.019, and 165.995 g/mL, respectively. Further details are depicted in Table 8.

The results showed that melittin eradicated all S. aureus isolates with MBEC values ranging from 40 to 10 μg/mL. Besides, the MBEC results for penicillin, and oxacillin were > 1,024–256 μg/mL, and > 1,024–128 μg/mL, respectively. The geometric means of the MBEC values for melittin, penicillin, and oxacillin, were 18.34, 689.1, and 469.506 μg/mL, respectively. The geometric means MBEC/MBIC ratios for melittin, penicillin, and oxacillin were 4.756, 3.622, 2.828 μg/ml. Further details are depicted in Table 8.

In the current study, we used the serial dilution method to determine whether the combined antibiofilm effects of antibacterial agents are synergistic, additive, indifferent, or antagonistic against all of isolates. To do this, we calculated the FBICi and FBECi, which are the interaction coefficients. In general, melittin-penicillin and melittin-oxacillin combination indexes based on FBIC values against all isolates were 0.23 and 0.177, respectively. Melittin-penicillin and melittin-oxacillin generally had combination indexes based on FBEC values against all isolates of 5 and 2.97, respectively.

The geometric means of the FBICi, and FBECi at various melittin– penicillin synergistic concentrations for strong biofilm-forming MRSA 1, 2, 3, 4, 5, 6, ATCC 25923, and ATCC 29213 were calculated as “0.625, 0.39, 0.386, 0.156, 0.883, 0.441, 0.441, and 0.312” for FBICi, and “3.535, 1.767, 14.142, 3.535, 7.071, 3.535, 7.071, and 7.071” for FBECi, respectively. The geometric means of the FBICi, and FBECi at various melittin– oxacillin synergistic concentrations for strong biofilm-forming MRSA 1, 2, 3, 4, 5, 6, ATCC 25923, and ATCC 29213 were calculated as “0.883, 1.767, 0.22, 0.11, 0.027, 0.027, 0.22, and 0.156” for FBICi, and “2.5, 2.5, 5, 2.5, 5, 2.5,2.5, and 2.5” for FBECi, respectively (Tables 9–12). A paired sample t-test revealed a significant difference between MBIC values of melittin alone, melittin-penicillin, and melittin-oxacillin combinations against isolates (p = 0.002 and p = 0.001, respectively), and in MBEC values of melittin alone, melittin-penicillin, and melittin-oxacillin combinations against isolates (p = 0.000 and p = 0.000, respectively).

In specifically, in MSSA 1, MRSA 2, MRSA 3, MRSA 4, MRSA 5, MRSA 6, ATCC 25923, and ATCC 29213 their MBIC values of penicillin and melittin was decreased 16, 256, 256, 16, 8, 8, 16, and 16 -fold, respectively, and for oxacillin and melittin was decreased 8, 8, 32, 16, 256, 128, 32, and 32-fold, respectively. In MSSA 1, MRSA 2, MRSA 3, MRSA 4, MRSA 5, MRSA 6, ATCC 25923 and ATCC 29213 their MBEC values of penicillin and melittin was decreased 8, 8, 4, 8, 4, 4, 4, and 4 -fold, respectively, and their MBEC values of oxacillin and melittin was decreased 16, 8, 16, 16, 8, 8, 16, and 16-fold, respectively.

The synergistic potential of melittin in eradicating or neutralizing biofilms, particularly against MDR-VRSA isolates, was assessed through the use of FE-SEM. The experimental outcomes unveiled that when melittin was combined with antibiotics at the concentration of FBICs, a remarkable and potent degradative effect was observed in biofilm structures. According to the results, melittin and antibiotics at the concentration of FBICs induced high destruction effects on the biofilm layer and interbacterial biofilm (IBB) and lysis of bacteria. Penicillin at the concentration of MBICs has less effect on biofilm destruction than melittin and oxacillin. Synergism of melittin-oxacillin at 0.156 μg/ml and 64 μg/ml induced more biofilm lysis compared to 0.312 μg/ml melittin and 128 μg/ml penicillin. Analysis of results showed that melittin not only has a significant biofilm degradation activity for removal of biofilm layers but is also able to kill them via invading their membranes (Figure 2).