Antimicrobial activity of apitoxin from Apis mellifera in Salmonella enterica strains isolated from poultry and its effects on motility, biofilm formation and gene expression.

Salmonella is a major global food-borne pathogen. One of the main concerns related to Salmonella and other food-borne pathogens is their capacity to acquire antimicrobial resistance and produce biofilms. Due to the increased resistance to common antimicrobials used to treat livestock animals and human infections, the discovery of new antimicrobial substances is one of the main challenges in microbiological research. An additional challenge is the development of new methods and substances to inhibit and destruct biofilms. We determined the antimicrobial and antibiofilm activities of apitoxin in 16 Salmonella strains isolated from poultry. In addition, the effect of apitoxin on Salmonella motility and the expression of biofilm- and virulence-related genes was evaluated. The minimum inhibitory concentrations (MIC) of apitoxin ranged from 1,024-256 µg/mL, with 512 µg/mL being the most common. Sub-inhibitory concentrations of apitoxin significantly reduced biofilm formation in 14 of the 16 Salmonella strains tested, with significant increases in motility. MIC concentrations of apitoxin destroyed the pre-formed biofilm by 27.66-68.22% (47.00% ±â€¯10.91). The expression of biofilm- and virulence-related genes and small RNAs was differentially regulated according to the strain and the presence of apitoxin. The transcription of the small RNAs dsrA and csrB, related to antimicrobial resistance, was upregulated in the presence of apitoxin. We suggest that apitoxin is a potential antimicrobial substance that could be used in combination with other substances to develop new drugs and sanitizers against food-borne pathogens.

Antibacterial Activity and Antibiotic-Enhancing Effects of Honeybee Venom against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), along with other antibiotic resistant bacteria, has become a significant social and clinical problem. There is thus an urgent need to develop naturally bioactive compounds as alternatives to the few antibiotics that remain effective. Here we assessed the in vitro activities of bee venom (BV), alone or in combination with ampicillin, penicillin, gentamicin or vancomycin, on growth of MRSA strains. The antimicrobial activity of BV against MRSA strains was investigated using minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and a time-kill assay. Expression of atl which encodes murein hydrolase, a peptidoglycan-degrading enzyme involved in cell separation, was measured by reverse transcription-polymerase chain reaction. The MICs of BV were 0.085 µg/mL and 0.11 µg/mL against MRSA CCARM 3366 and MRSA CCARM 3708, respectively. The MBC of BV against MRSA 3366 was 0.106 µg/mL and that against MRSA 3708 was 0.14 µg/mL. The bactericidal activity of BV corresponded to a decrease of at least 3 log CFU/g cells. The combination of BV with ampicillin or penicillin yielded an inhibitory concentration index ranging from 0.631 to 1.002, indicating a partial and indifferent synergistic effect. Compared to ampicillin or penicillin, both MRSA strains were more susceptible to the combination of BV with gentamicin or vancomycin. The expression of atl gene was increased in MRSA 3366 treated with BV. These results suggest that BV exhibited antibacterial activity and antibiotic-enhancing effects against MRSA strains. The atl gene was increased in MRSA exposed to BV, suggesting that cell division was interrupted. BV warrants further investigation as a natural antimicrobial agent and synergist of antibiotic activity.

Evaluation of anti-inflammatory, anti-nociceptive, and anti-arthritic activities of Indian Apis dorsata bee venom in experimental animals: biochemical, histological, and radiological assessment.

Traditionally venoms are used from thousands of years to treat pain, inflammation, and arthritis. In Ayurveda "Suchika Voron" and "Shodhona" were practiced against pain. In the present study, venom composition of the Indian honeybee Apis florea (AF), Apis dorsata (AD), and Apis cerana indica (AC) were analyzed using electrophoresis (SDS-PAGE). This venom analysis was used to shed light upon the correlation in structure and the venom composition among the three species in Indian fields. Among the three species, Indian Apis dorsata bee venom (ADBV) is evaluated for an anti-inflammatory, anti-nociceptive activity, and antiarthritic activity in different animal models. The effect of ADBV is revealed for its anti-arthritic activity in the FCA- and CIA-induced arthritis model in male Wistar rats. The immunosuppressant action of ADBV was studied by hemagglutination antibody titer. It has been found that ADBV possesses anti-inflammatory and antinociceptive activities. In FCA- and CIA-induced arthritis, ADBV able to decrease rheumatoid factor, pain perception parameters, C-reactive protein, erythrocytes sedimentation rate, urinary hydroxyproline, serum transaminase level, and serum nitric oxide level when compared with diseased control arthritic rats. IL-6, TNF-α level was found to be decrease by ADBV treatment in collagen induced arthritis model. Thus this study confirmed the scientific validation behind utilization of venom in Indian Apis dorsata bees in arthritis and inflammatory diseases which has been not reported till date.

Structure-activity study of macropin, a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae).

A novel antimicrobial peptide, designated macropin (MAC-1) with sequence Gly-Phe-Gly-Met-Ala-Leu-Lys-Leu-Leu-Lys-Lys-Val-Leu-NH2 , was isolated from the venom of the solitary bee Macropis fulvipes. MAC-1 exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l- or d-lysine in selected positions. Furthermore, all-d analog and analogs with d-amino acid residues introduced at the N-terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α-helical secondary structure in the presence of trifluoroethanol or membrane-mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure-activity relationship for the effect of d-amino acid substitutions in MAC-1 using NMR spectroscopy.

Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells.

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic ß-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells.

Hyperalgesic and edematogenic effects of peptides isolated from the venoms of honeybee (Apis mellifera) and neotropical social wasps (Polybia paulista and Protonectarina sylveirae).

Stings by bees and wasps, including Brazilian species, are a severe public health problem. The local reactions observed after the envenoming includes typical inflammatory response and pain. Several studies have been performed to identify the substances, including peptides that are responsible for such phenomena. The aim of the present study is to characterize the possible nociceptive (hyperalgesic) and edematogenic effects of some peptides isolated from the venoms of the honeybee (Apis mellifera) and the social wasps Polybia paulista and Protonectarina sylveirae, in addition to characterize some of the mechanisms involved in these phenomena. For this purpose, different doses of the peptides mellitin (Apis mellifera), Polybia-MP-I, N-2-Polybia-MP-I (Polybia paulista), Protonectarina-MP-NH2 and Protonectarina-MP-OH (Protonectarina sylveirae) were injected into the hind paw of mice. Hyperalgesia and edema were determined after peptide application, by using an electronic von Frey apparatus and a paquimeter. Carrageenin and saline were used as controls. Results showed that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2) and Protonectarina-MP-OH peptides produced a dose- and time-related hyperalgesic and edematogenic responses. Both phenomena are detected 2 h after melittin, Polybia-MP-I, N-2-Polybia-MP-I injection; their effects lasted until 8 h. In order to evaluate the role of prostanoids and the involvement of lipidic mediators in hyperalgesia induced by the peptides, indomethacin and zileuton were used. Results showed that zileuton blocked peptide-induced hyperalgesia and induced a decrease of the edematogenic response. On the other hand, indomethacin did not interfere with these phenomena. These results indicate that melittin, Polybia-MP-I, N-2-Polybia-MP-I, Protonectarina-MP-NH(2), and Protonectarina-MP-OH peptides could contribute to inflammation and pain induced by insect venoms.

Melittin, a honeybee venom derived peptide for the treatment of chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is the most prevalent neurological complication of cancer treatment which involves sensory and motor nerve dysfunction. Severe CIPN has been reported in around 5% of patients treated with single and up to 38% of patients treated with multiple chemotherapeutic agents. Present medications available for CIPN are the use of opioids, nonsteroidal anti-inflammatory agents, and tricyclic antidepressants, which are only marginally effective in treating neuropathic symptoms. In reality, symptom reappears after these drugs are discontinued. The pathogenesis of CIPN has not been sufficiently recognized and methods for the prevention and treatment of CIPN remain vulnerable to therapeutic problems. It has witnessed that the present medicines available for the disease offer only symptomatic relief for the short term and have severe adverse side effects. There is no standard treatment protocol for preventing, reducing, and treating CIPN. Therefore, there is a need to develop curative therapy that can be used to treat this complication. Melittin is the main pharmacological active constituent of honeybee venom and has therapeutic values including in chemotherapeutic-induced peripheral neuropathy. It has been shown that melittin and whole honey bee venom are effective in treating paclitaxel and oxaliplatin-induced peripheral neuropathy. The use of melittin against peripheral neuropathy caused by chemotherapy has been limited despite having strong therapeutic efficacy against the disease. Melittin mediated haemolysis is the key reason to restrict its use. In our study, it is found that α-Crystallin (an eye lens protein) is capable of inhibiting melittin-induced haemolysis which gives hope of using an appropriate combination of melittin and α-Crystallin in the treatment of CIPN. The review summarizes the efforts made by different research groups to address the concern with melittin in the treatment of chemotherapeutic-induced neuropathy. It also focuses on the possible approaches to overcome melittin-induced haemolysis.

Effects of honeybee venom supplementation in drinking water on growth performance of broiler chickens.

The effects of water supplementation of bee venom (BV) on performance, antioxidant activity, and liver function in Arbor Acres broiler chickens were investigated. Hence, 3 experimental treatment groups (control, 0.5 mg/L of BV, and 1 mg/L of BV) were allocated to 3 replicates of 5,000 one-day-old chicks each. The control group was kept on tap water, whereas the other 2 groups were supplied water supplemented with 0.5 and 1 mg of BV, respectively, per liter of drinking water. Broilers were provided ad libitum access to feed for the experimental period of 1 to 28 d of age. Supplementing drinking water with BV significantly increased BW gain at 28 d of age (P < 0.05). The average daily weight gain from d 1 to 28 was increased for birds supplemented with BV compared with control birds. The increase in BW gain was more pronounced with supplementation of 1 mg/L of BV compared with 0.5 mg/L of BV. An improved feed intake was noted in groups supplemented with BV as compared with control chicks. Liver function enzymes, aspartate aminotransferase, and alanine aminotransferase activities including total cholesterol, total protein, albumin, and globulin were not changed by BV supplementation. Tap water supplementation of BV did not alter the number of leukocytes, erythrocytes, heterophils, and lymphocytes. However, the antioxidative activities estimated as a superoxide dismutase-like activity of broiler chicks supplemented with BV was significantly increased (P < 0.05) in comparison with those without BV supplementation. These data indicate a possibility of better broiler performance through BV supplementation under conditions of severe stressful challenges the newly born chicks encounter.

Bee or Wasp Sting.

While jogging in a local park in Hong Kong, a 55-year-old, previously healthy man was stung on the ventral aspect of his right wrist. The tiny stinger was gently removed with nail cutters and examined under a microscope at 80x magni cation; plucking the stinger is ill- advised as this may inject more venom into the wounded site. Two days after stinging, the microscopic appearance of the stinger con rmed the diagnosis to be from a bee instead of a wasp or other insect. A simple method of con rming the nature of insect stings and an overview of Hymenoptera stings and their management are provided herein.

The protein composition of honeybee venom reconsidered by a proteomic approach.

Pure honeybee venom samples were submitted to two-dimensional gel electrophoresis. A total of 49 excised spots were analyzed by mass spectrometry; 39 of them resulted in the identification of 6 different known bee venom proteins and of 3 proteins that have not been described in such samples before. The first new venom protein has a platelet-derived and vascular endothelial growth factor family domain, the second protein shows no homologies with any known protein and the third matches a hypothetical protein similar to major royal jelly protein 8.

Purification and chemical characterization of melittin and acetylated derivatives.

Melittin, the main basic and hydrophobic peptide of bee venom, displays marked detergent-like properties. At high peptide concentration, and depending on salt and pH, it forms a tetramer. This is prevented by using urea. A purification procedure in presence of 4.0 M urea was developed to prepare melittin in its monomeric form, free of other venom constituents such as N alpha-formyl melittin, degradation products of peptides and phospholipase A2. NH2-residues on the melittin molecule were modified by reaction with acetic anhydride to alter the asymmetrical charge distribution supposed to confer detergent-like properties to the molecule. This gave rise to di- and mono acetyl derivatives which could be used, once isolated, to study further the melittin structure-activity relationship.

Isolation and characterization of the 32.5 kDa protein from the venom of an endoparasitic wasp.

The major venom proteins from the endoparasitic wasp were analyzed for distribution in the venom gland. A 32.5 kDa protein was purified from the venom gland of the Chelonus near curvimaculatus wasp. The protein accounts for about 25% of the total protein content of the venom and each gland contains 3-6 pmol of this component. The protein is acidic in nature and anion-exchange chromatography facilitated the purification of the protein to apparent homogeneity. On testing the purified protein by in vivo bioassay, it was found to elicit an effect comparable with the complete venom. The protein does not appear to have any disulfide bonds of major structural importance exposed under SDS-denaturing conditions. Products of chemical partial digest of the purified protein at the methionyl residues by cyanogen bromide were analyzed by SDS-PAGE. The 27.6 kDa fragment retained an epitope to an antibody raised against total Chelonus venom proteins, whereas no epitopes were detected for 4.9 and 0.6 kDa fragments.

Water soluble fraction (<10 kDa) from bee venom reduces visceral pain behavior through spinal alpha 2-adrenergic activity in mice.

We have previously shown that subcutaneous bee venom (BV) injection reduces visceral pain behavior in mice, but it is not clear which constituent of BV is responsible for its antinociceptive effect. In the present study, we now demonstrate that a water-soluble subfraction of BV (BVA) reproduces the antinociceptive effect of BV in acetic acid-induced visceral pain model. We further evaluated three different BVA subfractions that were separated by molecular weight, and found that only the BVAF3 subfraction (a molecular weight of <10 kDa) produced a significant antinociceptive effect on abdominal stretches and suppressed visceral pain-induced spinal cord Fos expression. Injection of melittin (MEL), a major constituent of BVAF3, also produced a visceral antinociception. However, melittin's antinociception was completely blocked by boiling for 10 min at 100 degrees C, while boiling either whole BV or BVAF3 did not prevent their antinociception. The antinociceptive effect of BVAF3 was completely blocked by intrathecal pretreatment with the alpha2-adrenoceptor antagonist, yohimbine (YOH), while intrathecal pretreatment with the opioid antagonist, naloxone (NAL) or the serotonin antagonist, methysergide, had no effect. These data demonstrate that BVAF3 is responsible for the visceral antinociception of whole BV and further suggest that this effect is mediated in part by spinal alpha2-adrenergic activity.

Hornetin: the lethal protein of the hornet (Vespa flavitarsus) venom.

By gel permeation on a Fractogel TSK HW 50 column followed by ion-exchange chromatography on carboxymethylcellulose CM 52, a lethal protein, designated hornetin, was purified from the venom of Vespa flavitarsus. Hornetin is a highly basic protein (pI 10.2) with a molecular mass of about 32 kDa. Its amino acid composition is characterized by a high content of lysine, aspartic and glutamic acid, and is devoid of tryptophan and cysteine. The lack of cysteine in the molecule is distinct from other known vespid venom proteins of comparable size. The i.v. LD50 of the toxin is 0.42 microgram per g mouse. Assayed on the red blood cells of the mouse and guinea-pig as well as isolated nerve muscle preparations of the chick and mouse, hornetin showed direct hemolytic activity and presynaptic neurotoxicity at microgram level and displayed musculotropic effect at higher concentrations.

Adolapin--a newly isolated analgetic and anti-inflammatory polypeptide from bee venom.

Adolapin was isolated by a two-step procedure: gel filtration and chromatography on CM cellulose. The molecular mass of the polypeptide as determined by SDS electrophoresis and amino acid composition proved to be 11500 and 11092 respectively. Adolapin exhibited a potent analgesic effect demonstrated by the "writhing" test (ED50-0,016mg/kg) and by the Randall-Sellito's test (ED50-0,013 mg/kg). The anti-inflammatory activity of adolapin was most marked with regard to carrageenin, prostaglandin and adjuvant rat hind paw edemas and adjuvant polyarthritis. The adolapin effects are presumably due to its capacity to inhibit the prostaglandin synthase system, following a biphasic dose-response relationship. It is likely that central mechanisms are also involved in the analgetic action of adolapin.

[Orientotoxin--a new presynaptic neurotoxin from the venom of the giant hornet Vespa orientalis]. / Orientotoksin--novyi presinapticheskii neirotoksin iz iada bol'shogo shershnia Vespa orientalis.

Orientotoxin, a novel presynaptically acting neurotoxin from the venom of giant hornet Vespa orientalis, has been isolated by gel filtration and ion exchange chromatography and characterized. The toxin has a molecular mass of 18,000. Highly purified preparations of orientotoxin possessed clearly manifested lysophospholipase activity and can block both induced and spontaneous release of neurotransmitter from the presynaptic nerve membrane.

[Histamine liberation (or histamine release HR): study of the positivity threshold in hymenoptera venom allergy]. / Histamino-libération (ou histamine release HR): étude au seuil de positivité dans l'allergie aux venins d'hyménoptères.

Dialysis of Hymenoptera venoms for measurement of HR by the radioimmunoassay technique (RIA, Immunotech) lowers the threshold of positivity of the HR titre from 30% to 12%. This modification of dialysis of the venoms significantly improves the sensitivity and specificity of the technique.

[Preparation of highly-purified melittin from bee venom by an affinity chromatography method]. / Otrimaniia visokoochishchenogo preparatu melitinu z otruti bdzholi metodom afinnoï khromatografiï.

The method of melittin (a lytic peptide from bee venom) purification is described. The method is based on application of immunosorbent with antibodies against phospholipase A2 and permits obtaining peptide without the residual phospholipase activity. It can be also used for the phospholipase A2 purification from melittin admixtures.

[Development of a highly efficient method for separating bee venom components]. / Razrabotka vysokoéffektivnogo metoda razdeleniia komponentov pchelinogo iada.

Factors preventing complete separation of melittin and phospholipase A2 under fractionation of the bee venom were studied. The methods of electrophoresis in PAAG, gel-filtration, ion-exchange and reverse-phase chromatography were used to show that the intermolecular interactions of suppositionally hydrophobic character prevent from complete separation of melittin and phospholipase A2. Conditions and methods are chosen which permit isolating melittin and phospholipase in a pure form.

Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom.

Honeybee stings are a severe public health problem. Bee venom contains a series of active components, including enzymes, peptides, and biogenic amines. The local reactions observed after envenoming include a typical inflammatory response and pain. Honeybee venom contains some well-known polycationic peptides, such as Melittin, Apamin, MCD peptide, Cardiopep, and Tertiapin. Secapin in honeybee venom was described 38 years ago, yet almost nothing is known about its action. A novel, variant form of this peptide was isolated from the venom of Africanized honeybees (Apis mellifera). This novel peptide, named Secapin-2, is 25 amino acid residues long. Conformational analyses using circular dichroism and molecular dynamics simulations revealed a secondary structure rich in strands and turns, stabilized by an intramolecular disulfide bridge. Biological assays indicated that Secapin-2 did not induce hemolysis, mast cell degranulation or chemotactic activities. However, Secapin-2 caused potent dose-related hyperalgesic and edematogenic responses in experimental animals. To evaluate the roles of prostanoids and lipid mediators in the hyperalgesia and edema induced by this peptide, Indomethacin and Zileuton were used to inhibit the cyclooxygenase and lipoxygenase pathways, respectively. The results showed that Zileuton partially blocked the hyperalgesia induced by Secapin-2 and decreased the edematogenic response. In contrast, Indomethacin did not interfere with these phenomena. Zafirlukast, a leukotriene receptor antagonist, blocked the Secapin-2 induced hyperalgesia and edematogenic response. These results indicate that Secapin-2 induces inflammation and pain through the lipoxygenase pathway in both phenomena.