Antimicrobial Properties of Apis mellifera’s Bee Venom

Bee venom (BV) is a rich source of secondary metabolites from honeybees (Apis mellifera L.). It contains a variety of bioactive ingredients including peptides, proteins, enzymes, and volatile metabolites. The compounds contribute to the venom’s observed biological functions as per its anti-inflammatory and anticancer effects. The antimicrobial action of BV has been shown in vitro and in vivo experiments against bacteria, viruses, and fungi. The synergistic therapeutic interactions of BV with antibiotics has been reported. The synergistic effect contributes to a decrease in the loading and maintenance dosage, a decrease in the side effects of chemotherapy, and a decrease in drug resistance. To our knowledge, there have been no reviews on the impact of BV and its antimicrobial constituents thus far. The purpose of this review is to address the antimicrobial properties of BV and its compounds.


Introduction
According to the World Health Organization (WHO), the antimicrobial drug resistance of bacterial pathogens has reached alarming rates in several parts of the world, and few alternatives are available [1]. investigated against oral pathogens S. salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis, Lactobacillus casei, and E. faecalis. The MIC was studied for each one individually (melittin with MIC from 4 to 40 µg /mL and PLA2 with MIC values of >400 µg/mL) and in combination with each other (MIC values ranging 6-80 µg/mL) [4]. The combination of BV with ampicillin or penicillin yielded an index of inhibitory concentrations ranging from 0.631 to 1.002, indicating a partial synergistic effect. The two MRSA strains were more susceptible to the combination of BV with gentamicin or vancomycin compared to combination of BV with ampicillin or penicillin [5].  Broth microdilution MIC 0.085 µg/mL MBC 0.106 µg/ mL [5].
BV was proven effective, synergistic, and safe when combined with some conventional drugs against certain types of microbes; however, there is a slow and careful consideration towards its investigations in pre-clinical and clinical applications. For example, BV and melittin exhibited a broad-spectrum antibacterial activity against both Gram-positive (MIC values between 10 and 100 µg/mL) and Gram-negative bacteria (MIC values between 30 and >500 µg/mL). Combination of BV and melittin with other antibiotic drugs, i.e., oxacillin, vancomycin, and amikacin, using checkerboard dilution gave fractional inhibitory concentration (FIC) indices ranging between 0.24 and 0.5; the FIC index is determined by the MIC of the test material in combination with an antibiotic medication divided by the MIC of the test material individually [16]. BV increased the antibody production against formalin-killed S. gallinarum in broiler chicks [43]. BV and melittin exhibited a broad spectrum antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci at MIC values of 6-800 µg/mL, compared to vancomycin's (reference drug) MIC value of 1.6-25 µg/mL [16]. The combination of BV and melittin with oxacillin showed a bactericidal effect on MRSA ATCC 33591. The treatment with both BV and melittin led to changes in the bacterial cell membrane caused by the loss of membrane integrity and exhibition of changes in the cell morphology including cell distortion and loss of cytoplasm content [11].
The antibacterial efficiency of melittin was studied against a variety of bacteria, such as Escherichia coli, S. aureus, and B. burgdorferi [39,[44][45][46][47]. Gram-positive bacteria have sensitivity to melittin, compared to Gram-negative ones, due to the nature of the organism's cell membrane [39,46,48,49]. Melittin can penetrate the peptideoglycan layer of the Gram-positive cell membrane more easily than the Gram-negative cells, which have a layer of lipopolysaccharides protecting their membrane. The presence of proline residue in position 14 has been shown to play a central role in the antimicrobial activity of melittin. Its absence in a melittin analog significantly reduced antimicrobial activity compared to the native peptide [50]. Similarly, two synthetic melittin, serine-substituted melittin (Mel-S) and asparagine-substituted melittin (Mel-N), were capable of penetrating E. coli cell membrane. Mel-S was more efficient than Mel-N [51]. Melittin, in general, possesses a greater ability to destroy biofilms formed by S. aureus compared to E. coli (biofilm production was 56% vs. 37%, respectively) [46]. MDP1: GIGAVLKVLTTGLPALIKRKRQQ and MDP2: GIGAVLKWLPALIKRKRQQ displayed strong antibacterial activity against reference strains of S. aureus, E. coli, and Pseudomonas aeruginosa compared to the native melittin. The antibacterial effects of MDP1 and MDP2 were explained by the changes in the bacterial membrane and the destruction of the bacterial cell membrane. Furthermore, the hemolytic activity of melittin (93.5%) at the dose of 3.84 µg/mL with average MIC values showed significant reduction in MDP1 (1.46% at geometric mean (GM) of 3.01 µg/mL) and MDP2 (5.15% at GM of MICs 2.18 µg/mL) [52]. The antibacterial activity of native melittin and its two mutants, namely melittin I17K (GIGAVLKVLTTGLPALKSWIKRKRQQ) with a higher charge and lower hydrophobicity and mutant G1I (IIGAVLKVLTTGLPALISWIKRKRQQ) of higher hydrophobicity, were investigated against different strains of Listeria, as mentioned in Table 1 [53].
The increased frequency of multi-drug resistant (MDR) bacteria is a major challenge to antimicrobial treatment. Melittin shows broad antibacterial activity toward different types of bacteria such as methicillin-susceptible S. aureus (MSSA), MRSA, and Enterococcus spp at MICs 0.5-4, 0.5-4, and 1-8 µg/mL, respectively. Furthermore, synergetic action between melittin and some antibiotics, i.e., daptomycin, vancomycin, linezolid, ampicillin, and erythromycin, against the previously mentioned bacteria were investigated by Dosler et al. [54]. Melittin's antibacterial and synergistic effects with β-lactam antibiotics to Acinetobacter baumannii was reported using broth microdilution method. The MIC values of melittin, ciprofloxacin, co-amoxiclav, imipenem, netilmicin, ceftazidime, and piperacillin are 4,8,16,16,16,32, and 128 µg/mL, respectively. However, FIC indices for combinations of melittin with the same antibacterial drugs are 0.750, 0.312, 0.250, 1.25, 0.187, and 0.375 µg/mL, respectively [55]. The application of melittin-doripenem has resulted in a significant decrease in the MIC of MDR baumannii strains. When the combinations of melittin-doripenem and melittin-ceftazidime were administrated to strains of MDR P. aeruginosa, the dose of melittin was significantly reduced. The combination of melittin with doripenem and ceftazidime against MDR microbial pathogens could be of great therapeutic value [56].
Furthermore, the combination between melittin and PLA2 (0.5 mg of each compound) has been investigated against oral pathogens S. salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis, Lactobacillus casei, and E. faecalis. The MIC was studied for each one individually (melittin with MIC from 4 to 40 µg /mL and PLA2 with MIC values of >400 µg/mL) and in combination with each other (MIC values ranging 6-80 µg/mL) [4]. The combination of BV with ampicillin or penicillin yielded an index of inhibitory concentrations ranging from 0.631 to 1.002, indicating a partial synergistic effect. The two MRSA strains were more susceptible to the combination of BV with gentamicin or vancomycin compared to combination of BV with ampicillin or penicillin [5].

Anti-Viral
During the last decade, viral diseases such as hepatitis C, smallpox, polio, rubella, and AIDS have threatened the lives of millions worldwide, especially immunocompromised patients [71]. Water contamination (waterborne diseases) represents a major health problem in regards to the spread of many viral diseases like hepatitis viral disease, poliomyelitis, gastroenteritis, diarrhea, etc. [72][73][74]. Searching for anti-viral substitutes that are low or completely free of diverse effects is an urgent need. In this context, natural products, in particular BV, embody a variable of exotic constituents, suggesting an immeasurable source of anti-viral agents [75].
BV and its constituents show prominent anti-viral activities against various enveloped and non-enveloped viruses such as Vesicular Stomatitis (VSV), Herpes Simplex (HSV), Enterovirus-71 (EV-71), Coxsackie (H3), Respiratory Syncytial Influenza A (A/PuertoRico/8/34) (in vitro study), and influenza A subtype (H1N1) (in vivo study) ( Table 2 and Figure 2) [76]. Papillomaviruses (HPVs) are considered the most common agents responsible for cervical carcinoma. BV was able to inhibit the growth of cervical cancer cells by the downregulation of E6/E7 proteins of HPV viruses ( Table 2) [77]. BV and its constituent melittin (Figure 1) can induce the immunity against porcine reproductive and respiratory syndrome viruses (PRRSV) via significant up-regulation of Th1 cytokines (IFN-γ and IL-12) and several types of immune cells, including CD3 + CD8 + , CD4 + CD8 + , and γδ T cells, leading to reduction of the viral load and decrease of the severity of interstitial pneumonia in PRRSV-infected pigs [78].
Toxins 2020, 12, x FOR PEER REVIEW 9 of 17 mechanism behind the inhibition of HIV replication is different for the two enzymes. PLA2 is presumably linked to a high-affinity binding receptor of the host cells but P3bv peptide is linked with a CXCR4 chemokine receptor [79,80]. Another type of BV phospholipases A2 called sPLA2 was evaluated using plaque assay and proven to suppress the activity of Japanese encephalitis virus (JEV), Hepatitis C virus (HCV), and Dengue virus (DENV) with IC50 values of 49, 117, and 183 ng/mL, respectively [81]. Furthermore, melittin represents an agent against Arenavirus Junin (JV) and Herpes Simplex Types 1 (HSV-1) and 2 (HSV-2) via inhibition of virus multiplication, adsorption, and penetration, as well as Na + and K + pumps of the host cell. Utilizing plaque and viral penetration assays, melittin at a multiplicity of infections (m.i.o) of 0.02 and 0.05 µM inhibited plaque formation giving 37 plaque formation units (PFU) compared to 220 PFU observed in the absence of melittin [82,83]. In another in vitro study, melittin was evaluated using the plaque assay against different viruses, namely Respiratory syncytial virus (RSV), EV-71, HSV, H3, Fused Influenza A virus (PR8), and VSV, with EC50 values of 0.35 ± 0.08, 0.76 ± 0.03, 0.94 ± 0.07, 0.99 ± 0.09, 1.15 ± 0.09, and 1.18 ± 0.09 µg/mL, respectively [76]. Furthermore, melittin could also be used against HIV-1, as it can inhibit the replication of HIV-1 by interfering with host cell-directed viral gene expression [84]. In an in vivo study, melittin was examined against lethal doses of the pathogenic H1N1 virus in mice; the results show that melittin can inhibit the replication of the virus, as the Log10 50% tissue culture infectious dose of a virus (TCID50) was 1.53 ± 0.25, compared to phosphate-buffered saline (PBS) at Log10 TCID50 4.22 ± 0.2 [76]. Taken together, these results suggest that BV and its constituents have the potential to become therapeutic agents to combat infectious viral diseases.   Based on the HIV tropism, honeybee PLA2 and its derivatives p3bv (containing 21-35 amino acids of PLA2) possess potent anti-human immunodeficiency virus (HIV) activity. The p3bv peptide showed anti-HIV activity via the prevention of the cell-cell fusion process and inhibition of the replication of T-tropic viruses in contrast to PLA2 that inhibited both M-and T-tropic HIV viruses but was unable to inhibit cell-cell fusion under the same condition. The authors illustrated that the mechanism behind the inhibition of HIV replication is different for the two enzymes. PLA2 is presumably linked to a high-affinity binding receptor of the host cells but P3bv peptide is linked with a CXCR4 chemokine receptor [79,80]. Another type of BV phospholipases A2 called sPLA2 was evaluated using plaque assay and proven to suppress the activity of Japanese encephalitis virus (JEV), Hepatitis C virus (HCV), and Dengue virus (DENV) with IC 50 values of 49, 117, and 183 ng/mL, respectively [81]. Furthermore, melittin represents an agent against Arenavirus Junin (JV) and Herpes Simplex Types 1 (HSV-1) and 2 (HSV-2) via inhibition of virus multiplication, adsorption, and penetration, as well as Na + and K + pumps of the host cell. Utilizing plaque and viral penetration assays, melittin at a multiplicity of infections (m.i.o) of 0.02 and 0.05 µM inhibited plaque formation giving 37 plaque formation units (PFU) compared to 220 PFU observed in the absence of melittin [82,83]. In another in vitro study, melittin was evaluated using the plaque assay against different viruses, namely Respiratory syncytial virus (RSV), EV-71, HSV, H3, Fused Influenza A virus (PR8), and VSV, with EC 50 values of 0.35 ± 0.08, 0.76 ± 0.03, 0.94 ± 0.07, 0.99 ± 0.09, 1.15 ± 0.09, and 1.18 ± 0.09 µg/mL, respectively [76]. Furthermore, melittin could also be used against HIV-1, as it can inhibit the replication of HIV-1 by interfering with host cell-directed viral gene expression [84]. In an in vivo study, melittin was examined against lethal doses of the pathogenic H1N1 virus in mice; the results show that melittin can inhibit the replication of the virus, as the Log 10 50% tissue culture infectious dose of a virus (TCID 50 ) was 1.53 ± 0.25, compared to phosphate-buffered saline (PBS) at Log 10 TCID 50 4.22 ± 0.2 [76]. Taken together, these results suggest that BV and its constituents have the potential to become therapeutic agents to combat infectious viral diseases.

Anti-fungal
Fungal related diseases cause colonization, superficial skin infections, and allergies, representing a devastating health problem worldwide. Additionally, the toxicity and resistance to antifungal drugs are major challenges. Natural products from plants, marine life, microorganisms, and bee products could be considered promising antifungal agents with fewer side effects [88].
Recently, BV was reported as an effective agent against many of the fungal related diseases, as mentioned in Table 3. BV can inhibit dermatophytosis, which occurs via Trichophyton mentagrophytes and Trichophyton rubrum fungi. BV reduced all populations of T. mentagrophytes at 15 and 30 ppm within 5 min, while, at the same dose of BV, T. rubrum growth inhibition was observed within 5 min. On the other hand, fluconazole did not prevent the development of the same pathogens. The study proved that the BV was more potent than fluconazole (commercial antifungal drugs) [89]. The anti-fungal action of BV on 10 clinical isolates of Candida albicans was studied, with MIC values ranging from 62.5 to 125 µg/mL [88]. In another study, melittin showed antimicrobial activity against various strains of fungi with MIC values between 30 and 300 µg/mL [16]. Melittin produced oxygen free radicals (OH) . that could induce apoptosis of C. albicans. The fungal cell death was explained by the disrupted mitochondrial membrane via the Ca 2+ release [90,91].
Alternaria alternate sp. and Aspergillus pillows are common pathogens that grow in the nasal cavity. The irritation/inflammation caused by the fungus induces the production of chemical mediators from nasal epithelial cells and fibroblasts. Melittin and apamin ( Figure 1) were able to inhibit the growth of A. alternate sp. and A. pillows causing upper airway inflammatory diseases. The mechanism of action was shown to be via the inhibition of chemical mediators production, i.e., interleukin (IL)-6, IL-8, and ECM, as well as induction of the phosphorylation of Smad 2/3 and p38 MAPK [3]. At 1 µg/mL inhibits 38.7% of interleukin (IL)-8 production. [3] NR, Not reported; ppm, Parts per million.

Concluding Remarks
Microbial diseases are problematic, particularly with the emergence of drug resistance; therefore, researchers are looking for new sources of bioactive candidates. Natural products are considered a renewable source with fewer complications that could provide a wide number of active compounds. BV is a complex mixture of proteins, peptides, and low molecular weight components including melittin, PLA2, apamin, adolapin, and MCDP. BV contains a variety of bioactive components including melittin, apamin, and PLA2, which play a vital role as antimicrobials through various mechanisms against bacteria, viruses, and fungi. The synergistic effect of BV and melittin through the combination of chemotherapy drugs leads to a reduction in dosage, side effects, and greater efficacy of the treatment strategy against microbial ailments. BV established its role as antimicrobial.
BV and its constituents in combination with antibiotic drugs emerge as a plausible approach to overcome drug resistance of current antibiotics treatment in a controlled manner. Another promising and feasible implication is to test BV to combat microbes causing skin diseases. Interestingly, BV can be useful as a topical agent for encouraging skin regeneration or treatment of certain epidermal conditions [5,94]. Therefore, BV has contributed to some formulations against bacteria that cause acne [95,96]. Therefore, BV and melittin are attractive therapeutic candidates for microbial diseases. However, using BV and melittin induces extensive hemolysis and toxicity of the cells, a severe side effect that limits their future development and clinical application. Ongoing research is addressing practical issues including standardization, toxicity, and stability [97][98][99][100].