Influence of apitoxin and melittin from Apis mellifera bee on Staphylococcus aureus strains.

The antibacterial activities of apitoxin, a venom produced by Apis mellifera bee, and melittin, an antimicrobial peptide from apitoxin, were tested against planktonic and biofilm states of Staphylococcus aureus methicillin-resistant (MRSA), including clinical, and enterotoxin-producing isolates. Also, the synergism of apitoxin and melittin in combination with oxacillin were evaluated as well. The induced morphological changes on S. aureus cells of both products were detected by transmission electronic microscopy (TEM). The minimum inhibitory concentration (MIC) values were 7.2 µg/mL, and 6.7 µg/mL, for apitoxin and melittin, respectively. The minimum bactericidal concentration (MBC) values were 28.7 µg/mL, and 26 µg/mL for apitoxin and melittin, respectively. The time-kill curve assays of apitoxin or melittin with oxacillin exhibited bactericidal synergism against MRSA isolates. TEM images showed cell distortion, cell disintegration with leakage of cytoplasmic content and loss of cytoplasm content. However, apitoxin and melittin did not interfere with staphylococcal enterotoxin production or release. Thus, apitoxin and melittin are potential agents against MRSA that can serve as possible models for new antibacterial drugs.

Antibacterial and anti-biofilm activities of cinnamaldehyde against S. epidermidis.

The search for new antimicrobial drugs has been necessary due to the increased bacterial resistance to antibiotics currently in use, and natural products play an important role in this field. The aim of this study was to evaluate the in vitro effect of cinnamaldehyde on S. epidermidis strains, biofilm set-up prevention, as well as its effect on pre-established biofilms. The minimum inhibitory concentration (MIC) ranged from 300 to 500 µg/mL, and the minimum bactericidal concentration (MBC) from 400 to 600 µg/mL. The biofilm inhibitory concentration and biofilm eradication concentration values were four-fold (clinical isolate) and eight-fold (ATCC strain) greater than the concentration required to inhibit planktonic growth. Sub-inhibitory concentrations of cinnamaldehyde attenuated biofilm formation of S. epidermidis strains on polystyrene microtiter plates. The combination of cinnamaldehyde and linezolid was able to inhibit S. epidermidis with a bactericidal effect. Further investigation of the mechanism of action of cinnamaldehyde revealed its effect on the cell membrane permeability, and confocal laser scanning microscopy (CLSM) images illustrated the impact of cinnamaldehyde in the detachment and killing of existing biofilms. Thereby, our data confirmed the ability of cinnamaldehyde to reduce bacterial planktonic growth of S. epidermidis, inhibiting biofilm formation and eradicating pre-formed biofilm.

Inhibitory activities of propolis, nisin, melittin and essential oil compounds on Paenibacillus alvei and Bacillus subtilis.

Background: Natural products represent important sources of antimicrobial compounds. Propolis and compounds from essential oils comprise good examples of such substances because of their inhibitory effects on bacterial spores, including bee pathogens. Methods: Ethanol extracts of propolis (EEP) from Apis mellifera were prepared using different methods: double ultrasonication, double maceration and maceration associated with ultrasonication. Together with the antimicrobial peptides nisin and melittin, and compounds present in the essential oils of clove (Syzygium aromaticum) and cinnamon (Cinnamomum zeylanicum), assays were carried out on one Bacillus subtilis isolate and Paenibacillus alvei (ATCC 6344) against vegetative and sporulated forms, using the resazurin microtiter assay. Synergism with all the antimicrobials in association with tetracycline was verified by the time-kill curve method. Potassium and phosphate efflux, release of proteins and nucleic acids were investigated. Results: EEPs showed the same MIC, 156.25 µg/mL against B. subtilis and 78.12 µg/mL against P. alvei. The peptides showed better activities against B. subtilis (MIC of 12 µg/mL for melittin and 37.50 µg/mL for nisin). Antimicrobials showed similar inhibitory effects, but cinnamaldehyde (39.06 µg/mL) showed the best action against P. alvei. Melittin and nisin showed the greatest capacity to reduce spores, regarding B. subtilis there was a 100% reduction at 6.25 and 0.78 µg/mL, respectively. Concerning P. alvei, the reduction was 93 and 98% at concentrations of 80 µg/mL of melittin and 15 µg/mL of nisin. EEPs showed the highest effects on the protein release against B. subtilis and P. alvei. Nucleic acid release, phosphate and potassium efflux assays indicated bacterial cell membrane damage. Synergism between antimicrobials and tetracycline was demonstrated against both bacteria. Conclusion: All antimicrobials tested showed antibacterial activities against vegetative and sporulated forms of P. alvei and B. subtilis, especially nisin and melittin. Synergism with tetracycline and damage on bacterial cell membrane also occurred.

Comparative Proteomics of Methicillin-Resistant Staphylococcus aureus Subjected to Synergistic Effects of the Lantibiotic Nisin and Oxacillin.

We investigated the responses and mechanisms of action of methicillin-resistant Staphylococcus aureus (MRSA) metabolism when exposed under sublethal concentrations of the synergistic antibacterial combination of nisin + oxacillin (» of maximum sublethal concentration) and sublethal concentrations of oxacillin only and nisin only. A total of 135 proteins were identified, showing an alteration in the expression of 85 proteins when treatment was compared with untreated bacteria (control). When the bacteria were treated using the combination, there was an increase in the expression of proteins related to resistance (e.g., beta-lactamase) and also in the ones involved in protein synthesis, and there was a decrease in the expression of proteins related to stress and alterations in proteins related to bacterial energy metabolism. Bacterial oxidative stress showed that the combination was able to induce oxidative stress (p < 0.05) and increase enzyme activities and lipid hydroperoxide levels compared with individual treatments. The analysis of cell ultrastructure showed damage in MRSA, especially on the bacterial wall and the plasma membrane, with cell lysis and death. Thus, the changes caused by these treatments affected different proteins related to the bacterial biological processes and signaling pathways such as cell division, structure, stress, regulation, bacterial resistance, protein synthesis, gene expression, energetic metabolism, and virulence. It was observed that synergism among antimicrobials has high potential in therapeutic use and may reduce the required amounts of antibacterial substances in addition to being effective on different targets in bacterial cells.