Antibacterial effect of extracts of Hermetia illucens (Diptera: Stratiomyidae) larvae against Gram‐negative bacteria

Larvae of the black soldier fly, Hermetia illucens are well‐known fly larvae that inhabit many countries around the world. Antimicrobial agents derived from the larvae may be among the substances that are produced in the body for their survival. This study was carried out to identify the antimicrobial effects of H. illucens larvae that commonly inhabit animal waste and food waste. To evaluate the pharmacological effects of H. illucens larvae extracts, the larvae were extracted by various organic solvents, and their antibacterial effects were determined by antimicrobial methods, such as agar disk diffusion and turbidometric assays. The methanol extracts (ME) indicated antibacterial effects against the proliferation of Klebsiella pneumoniae, Neisseria gonorrhoeae and Shigella sonnei. However, antibacterial effects were not induced in Gram‐positive bacteria such as Bacillus subtilis, Streptococcus mutans and Sarcina lutea. The bacterial growth treated with ME was strongly inhibited from 20 mg/mL in a dose‐dependent manner compared with other extracts, and antibacterial activity gradually decreased after 24 h. Moreover, the minimal inhibitory concentration (MIC) values of ME against Klebsiella pneumoniae, Neisseria gonorrhoeae and Shigella sonnei for 12 h were measured as 44.74 mg/mL, 43.98 mg/mL and 43.96 mg/mL, respectively. These results demonstrate that ME of H. illucens larvae not only has antibacterial activity which strongly inhibits the growth and proliferation of the bacteria but also unique properties which effectively block the viability of the bacteria.     

In vitro antibacterial activity and physicochemical properties of a crude methanol extract of the larvae of the blow fly Lucilia cuprina

The emergence of multidrug‐resistant bacterial strains has prompted the reintroduction of maggot therapy in the treatment of chronic, infected wounds. Many previous studies have demonstrated the potent antibacterial activity of larval excretions/secretions of the blowfly Lucilia sericata (Meigen) (Diptera:Calliphoridae) against bacteria. However, the antibacterial activity of its sibling species, Lucilia cuprina (Wiedemann) (Diptera:Calliphoridae) against a wide range of pathogenic bacteria has never been determined. The aim of this study was to develop a new procedure to produce whole body extract of larvae of L. cuprina via methanol extraction as well as to demonstrate the in vitro antibacterial activity of this extract against seven selected wound pathogens (Staphylococcus aureus, methicillin‐resistant S. aureus, S. epidermidis, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli). The turbidimetric assay demonstrated that L. cuprina larval extract was significantly potent against all bacteria tested (P < 0.001). Additionally, colony‐forming unit (CFU), agar well diffusion and minimum inhibitory concentration assays have confirmed the apparent potency of larval extract against P. aeruginosa. The reconstituted larval extract was highly robust and thermally stable. These observations substantiated the feasibility of the methanol extraction method in the production of larval extract.     

Novel antibacterial peptides induced by probiotics in Hermetia illucens (Diptera: Stratiomyidae) larvae

There is a need to discover new therapeutic substances due to the emergence of deadly infectious diseases and various antibiotic resistance. We focused on the larvae that are utilized as a medical insect for the treatment of skin damage in Europe and America. This study was to investigate the pharmacological activities of novel antibacterial peptides isolated from Hermetia illucens larvae against the Klebsiella pneumoniae and Shigella dysenteriae. The larvae were immunized by probiotics (Lactobacillus casei) for 24 h. The hemolymph from the immunized larvae was fractionated through reverse‐phase chromatography. Peptides were purified using HPLC and the coomassie blue staining, and identified using Nano‐LC‐ESI‐MS/MS system. Antibacterial activities of the peptides were evaluated by turbidometric assay, liquid broth dilution assay, resazurin assay, and agar disk diffusion method. The minimum inhibitory concentrations (MICs) of the peptides were measured as 150 μg/mL through the turbidometric, liquid broth dilution, and resazurin assays. The peptides effectively inhibited their growth/proliferation as well as the survival rate of the tested bacteria. Furthermore, the immunized larvae exhibited overexpression of the peptides compared to non‐immunized larvae. These results demonstrate that the peptides induced by H. illucens exert strong antibacterial activity against Gram‐negative bacteria. The results suggest that the activation of the humoral immunity induced by immunization functioned to enhance the production of antibacterial peptides from the insect and their antibacterial properties. This study indicates the potential of the peptides produced from larvae as antibacterial peptide substance for the development of novel antibacterial drugs.     

Ioniols I and II,Tetracyclic Diterpenes with Antibacterial Activity,from Sphaerococcus coronopifolius

Two naturally occurring diterpenes featuring unprecedented tetracyclic skeletons, ioniols I and II ( 1 and 2 , resp.), along with two previously reported metabolites 3 and 4 , were isolated from the organic extract of Sphaerococcus coronopifolius collected from the rocky coasts of Corfu island in the Ionian Sea. The structures of the new natural products, as well as their relative configuration, were elucidated on the basis of extensive spectral analysis, including 2D‐NMR experiments. The isolated metabolites were evaluated for their antibacterial activity against a panel of Staphylococcus aureus strains, which included multidrug‐resistant (MDR) and methicillin‐resistant Staphylococcus aureus (MRSA) strains.     

Structure and Antibacterial Activity of Brominated Diterpenes from the Red Alga Sphaerococcus coronopifolius

Four new tetracyclic brominated diterpenes, 1 – 4 , were isolated from the organic extract of Sphaerococcus coronopifolius, collected from the rocky coasts of Corfu Island. The structures of the new natural products, as well as their relative configurations, were elucidated on the basis of extensive spectral analyses, including 2D‐NMR experiments. The isolated metabolites were evaluated for their antibacterial activity against a panel of bacteria including multidrug‐resistant (MDR) and methicillin‐resistant Staphylococcus aureus (MRSA) with MIC values in the range of 16–128 μg/ml.     

Defensin‐like peptide3 from black solder fly: Identification,characterization, and key amino acids for anti‐Gram‐negative bacteria

An antibacterial peptide was isolated from a black soldier fly, Hermetia illucens. The molecular mass of this peptide was established as 4247.37 by matrix‐assisted laser desorption/ionization‐time of flight mass (MALD‐TOF MS) spectrometry. The amino acid sequence of the mature peptide was determined by N‐terminal sequencing using Edman degradation, combined with cDNA sequencing of the previously reported defensin‐like peptide (DLP) 3. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP3 had potent activity against Gram‐positive and negative bacteria, but DLP4 had only anti‐Gram‐positive activity as previously reported. Recombinant DLP3 and DLP4 were overexpressed in Escherichia coli, and antibacterial activities were identical to DLPs purified from H. illucens hemolyph. In silico analysis revealed that only six amino acid sequences were different between DLP3 and DLP4, but antibacterial activity against Gram‐negative bacteria differed. Therefore these amino acid variants may be key amino acids (Gly‐10, Val‐18, Met‐23, Arg‐25, Asp‐32, Arg‐40) related to killing Gram‐negative bacteria.     

Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

Sesquiterpenoids with Anti‐MDR Staphylococcus aureus Activities from Ferula ferulioides

Seven new sesquiterpenoids together with 21 known sesquiterpenoid derivatives were isolated from the medicinal plant Ferula ferulioides (Steud .) Korovin . Their structures were elucidated by comprehensive spectroscopic analyses and chemical transformations. The isolated compounds were evaluated for their antibacterial activities against a panel of bacteria including multidrug‐resistant (MDR) and methicillin‐resistant Staphylococcus aureus (MRSA), displaying minimum inhibitory concentration (MIC) values in the range of 0.5–128 mg/l.     

Purification,Characterization and Antibacterial Activity of l‐amino Acid Oxidase from Cerastes cerastes

Antibiotic resistance presents a real problem in which new antibacterial molecules from natural secretions could be beneficial in the development of new drugs. In this study, Cerastes cerastes venom was investigated for its antibacterial activity against Gram‐positive and Gram‐negative bacteria. The antibacterial activity was evaluated by measuring the halo inhibition and minimum inhibitory concentration (MIC). An l ‐amino acid oxidase (CcLAAO) was purified from this venom using three chromatographic steps; its homogeneity (60 kDa) was confirmed by SDS‐PAGE. LC–MS/MS analysis of CcLAAO showed similarities with other LAAO enzymes from Echis ocellatus and Viridovipera stejnegeri venoms. CcLAAO presents an antibacterial activity against three bacterial strains (Staphylococcus aureus, Methicillin‐resistant S. aureus, and Pseudomonas aeruginosa) with MIC values of 10, 10, and 20 μg/mL, respectively. However, no effect was observed against Escherichia coli and yeast strains. Kinetic parameters of CcLAAO evaluated on l ‐leucine at pH 8.0 and 20°C were K_m = 0.06 mmol and V_max = 164 mmol/min.     

Theaflavin‐3,3´‐digallate increases the antibacterial activity of β‐lactam antibiotics by inhibiting metallo‐β‐lactamase activity

Metallo‐β‐lactamases (MBLs) are some of the best known β‐lactamases produced by common Gram‐positive and Gram‐negative pathogens and are crucial factors in the rise of bacterial resistance against β‐lactam antibiotics. Although many types of β‐lactamase inhibitors have been successfully developed and used in clinical settings, no MBL inhibitors have been identified to date. Nitrocefin, checkerboard and time‐kill assays were used to examine the enzyme behaviour in vitro. Molecular docking calculation, molecular dynamics simulation, calculation of the binding free energy and ligand‐residue interaction decomposition were used for mechanistic research. The behaviour of the enzymes in vivo was investigated by a mouse infection experiment. We showed that theaflavin‐3,3´‐digallate (TFDG), a natural compound lacking antibacterial activities, can inhibit the hydrolysis of MBLs. In the checkerboard and time‐kill assays, we observed a synergistic effect of TFDG with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus BAA1717. Molecular dynamics simulations were used to identify the mechanism of the inhibition of MBLs by TFDG, and we observed that the hydrolysis activity of the MBLs was restricted by the binding of TFDG to Gln242 and Ser369. Furthermore, the combination of TFDG with β‐lactam antibiotics showed effective protection in a mouse Staphylococcus aureus pneumonia model. These findings suggest that TFDG can effectively inhibit the hydrolysis activity of MBLs and enhance the antibacterial activity of β‐lactam antibiotics against pathogens in vitro and in vivo.     

A novel synthetic peptide from a tomato defensin exhibits antibacterial activities against Helicobacter pylori

Defensins are a class of cysteine‐rich proteins, which exert broad spectrum antimicrobial activity. In this work, we used a bioinformatic approach to identify putative defensins in the tomato genome. Fifteen proteins had a mature peptide that includes the well‐conserved tetradisulfide array. We selected a representative member of the tomato defensin family; we chemically synthesized its γ‐motif and tested its antimicrobial activity. Here, we demonstrate that the synthetic peptide exhibits potent antibacterial activity against Gram‐positive bacteria, such as Staphylococcus aureus A170, Staphylococcus epidermidis, and Listeria monocytogenes, and Gram‐negative bacteria, including Salmonella enterica serovar Paratyphi, Escherichia coli, and Helicobacter pylori. In addition, the synthetic peptide shows minimal (<5%) hemolytic activity and absence of cytotoxic effects against THP‐1 cells. Finally, SolyC exerts an anti‐inflammatory activity in vitro, as it downregulates the level of the proinflammatory cytokines TNF‐α and IFN‐γ. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of short cationic antimicrobial peptidomimetics containing arginine analogues

Worldwide efforts are underway to develop new antimicrobial agents against bacterial resistance. To identify new compounds with a good antimicrobial profile, we designed and synthesized two series of small cationic antimicrobial peptidomimetics (1–8) containing unusual arginine mimetics (to introduce cationic charges) and several aromatic amino acids (bulky moieties to improve lipophilicity). Both series were screened for in vitro antibacterial activity against a representative panel of Gram‐positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram‐negative (Escherichia coli and Klebsiella pneumoniae) bacterial strains, and Candida albicans. The biological screening showed that peptidomimetics containing tryptophan residues are endowed with the best antimicrobial activity against S. aureus and S. epidermidis in respect to the other synthesized derivatives (MIC values range 7.5–50 µg/ml). Moreover, small antimicrobial peptidomimetics derivatives 2 and 5 showed an appreciable activity against the tested Gram‐negative bacteria and C. albicans. The most active compounds (1–2 and 5–6) have been tested against Gram‐positive established biofilm, too. Results showed that the biofilm inhibitory concentration values of these compounds were never up to 200 µg/ml. The replacement of tryptophan with phenylalanine or tyrosine resulted in considerable loss of the antibacterial action (compounds 3–4 and 7–8) against both Gram‐positive and Gram‐negative bacterial strains. Furthermore, by evaluating hemolytic activity, the synthesized compounds did not reveal cytotoxic activities, except for compound 5. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Acute necrotic stomatitis (noma) associated with methicillin-resistant Staphylococcus aureus infection in a newly acquired rhesus macaque (Macaca mulatta)

Backgroud A newly acquired rhesus macaque was suffering from rapid destruction of the left cheek caused by necrotizing stomatitis. Methods To restore reconstructive surgery and intensive care with antibiotics, wound protection, wound healing agents, and debridement were applied. Results Staphylococcus aureus and Enterococcus faecalis were isolated from the culture of the lesion, and the antibiotic susceptibility test revealed methicillin‐resistant Staphylococcus aureus infection. Vancomycin and ampicillin‐sulbactam effectively treated the bacterial infections, and reconstructive surgery was performed once the infection was cleared. Topical application of recombinant human epidermal growth factor (rhEGF) was useful to treat exposed wound of the noma lesion. Conclusions Simian noma associated with methicillin‐resistant Staphylococcus aureus (MRSA) had not previously been reported in non‐human primates. Although noma associated with MRSA is hard to cure because of its rapid and destructive progress, the aggressive therapy used in this study led to the successful resolution of an acute necrotic stomatitis lesion in a rhesus macaque.     

Hexanedioic acid from Hermetia illucens larvae (Diptera: Stratiomyidae) protects mice against Klebsiella pneumoniae infection

The antibacterial effects of larval extract from Hermetia illucens, commonly known as the black soldier fly, have been demonstrated in vitro. In this study, gas chromatography–mass spectrometry analysis identified the active compound within this larval extract as hexanedioic acid. The antibacterial effects of hexanedioic acid were investigated in mice infected with Klebsiella pneumoniae. After administration of hexanedioic acid, infected mice showed decreased lung bacterial loads and lower rates of body weight loss compared to those in the infection‐only control group. Based on lung bacterial loads, oral hexanedioic acid treatment showed better protection than intraperitoneal treatment. Histopathology confirmed that daily administration of hexanedioic acid for 10 days showed zero toxicity to the kidneys or livers of mice. Therefore, hexanedioic acid may be a novel antibacterial agent.     

Synergistic activity of 1-(1-naphthylmethyl)-piperazine with ciprofloxacin against clinically resistant Staphylococcus aureus, as determined by different methods

Aims: To evaluate the interaction of 1‐(1‐naphthylmethyl)‐piperazine (NMP) and ciprofloxacin (CPFX) in vitro against fluoroquinolone (FQ)‐resistant clinical isolates of methicillin‐resistant Staphylococcus aureus (MRSA). Methods and Results: The in vitro interaction of NMP and CPFX in 12 FQ‐resistant clinical isolates of MRSA was assessed using a checkerboard microdilution method. In the study, a synergistic antimicrobial effect between NMP and CPFX was observed in all 12 FQ‐resistant strains tested, as determined by the fractional inhibitory concentration index (FICI), and in 10 strains using ΔE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed using the time–killing test and agar diffusion assay for the selected strain, MRSA 1862; synergistic activity was observed when NMP was combined with the first‐line antimicrobial agent CPFX against Staph. aureus. Conclusions: Synergistic activity between NMP and CPFX against clinical isolates of FQ‐resistant Staph. aureus was observed in vitro. Significance and Impact of the Study: This report might provide alternative methods to reduce the resistance of Staph. aureus to CPFX.     

Surface disinfection properties of the combination of an antimicrobial peptide,ranalexin, with an endopeptidase,lysostaphin, against methicillin‐resistant Staphylococcus aureus (MRSA)

Aims: To characterize the antibacterial synergy of the antimicrobial peptide, ranalexin, used in combination with the anti‐staphylococcal endopeptidase, lysostaphin, against methicillin‐resistant Staphylococcus aureus (MRSA), and to assess the combination’s potential as a topical disinfectant or decolonizing agent for MRSA. MRSA causes potentially lethal infections, and pre‐operative patients colonized with MRSA are often treated with chlorhexidine digluconate and mupirocin cream to eradicate carriage. However, chlorhexidine is unsuitable for some patients, and mupirocin resistance is increasingly encountered, indicating new agents are required. Methods and Results: Using an ex vivo assay, ranalexin and lysostaphin tested in combination reduced viable MRSA on human skin to a greater extent than either compound individually. The combination killed bacteria within 5 min and remained effective and synergistic even in high salt and low pH conditions. Conclusions: The combination is active against MRSA on human skin and under conditions that may be encountered in sweat. Significance and Impact of the Study: Although the exact mechanism of activity remains unresolved, considering its specific spectrum of activity, fast killing kinetics and low likelihood of resistance arising, the combination of ranalexin with lysostaphin warrants consideration as a new agent to eradicate nasal and skin carriage of Staph. aureus, including MRSA.     

Anti‐infectious activity of synbiotics in a novel mouse model of methicillin‐resistant Staphylococcus aureus infection

The anti‐infectious activity of synbiotics against methicillin‐resistant Staphylococcus aureus (MRSA) infection was evaluated using a novel lethal mouse model. Groups of 12 mice treated with multiple antibiotics were infected orally with a clinical isolate of MRSA at an inoculum of 10⁸ CFU on day 7 after starting the antibiotics. A dose of 400 mg/kg 5‐fluorouracil (5‐FU) was injected intraperitoneally on day 7 after the infection. A dose of 10⁸ CFU Bifidobacterium breve strain Yakult and 10 mg of galactooligosaccharides (GOS) were given orally to mice daily with the antibiotic treatment until day 28. The intestinal population levels of MRSA in the mice on multiple antibiotics were maintained stably at 10⁸ CFU/g of intestinal contents after oral MRSA infection and the subsequent 5‐FU treatment killed all the mice in the group within 14 days. B. breve administration saved most of the mice, but the synbiotic treatment saved all of the mice from lethal MRSA infection. The synbiotic treatment was effective for the treatment of intestinal infection caused by four MRSA strains with different toxin productions. There was a large difference among the six Bifidobacteria strains that were naturally resistant to the antibacterial drugs used. B. breve in combination with GOS is demonstrated to have valuable preventive and curative effects against even fatal MRSA infections.     

Brominated Diphenyl Ethers Including a New Tribromoiododiphenyl Ether from the Vietnamese Marine Sponge Arenosclera sp. and Their Antibacterial Activities

A new tribromoiododiphenyl ether ( 1 ) and eight known brominated diphenyl ethers ( 2 – 9 ) were isolated from the MeOH extract of the sponge Arenosclera sp. collected in Vietnam, using repeated open column chromatography and preparative thin layer chromatography. The chemical structure of the new compound 1 was determined by analyses of spectroscopic (1D‐ and 2D‐NMR, and MS) data and by comparison of our data with those reported in the literature. Compounds 1 , 3 , and 8 exhibited strong antibacterial activities against the Gram‐positive bacteria Bacillus subtilis and Staphylococcus aureus and the Gram‐negative bacterium Klebsiella pneumoniae with MIC values ranging from 0.8 to 6.3 μm , while compounds 5 and 7 only displayed activities against Gram‐positive bacteria with MIC values from 0.5 to 3.1 μm . Compound 2 showed activities against the four tested bacteria with MIC values ranging from 0.5 to 6.3 μm .     

Development,growth and metabolic rate of Hermetia illucens larvae

The larvae of Hermetia illucens are known to successfully bio‐convert a vast range of organic substrates into high protein and fat biomass, but little is known about the larval instars. During this research, larval head capsules and biomass growth were measured daily and the specific metabolic rate of larger instars were considered. The head capsule measurements revealed that H. illucens pass through 6 actively feeding larval stadia before entering the last nonfeeding but migrating 7th stadium. Larval growth follows a sigmoid curve with slowly accelerating growth in the earlier stadia and decelerating growth in the latest stadia. In contrast, development was fast until reaching stadium 6 and then slowed down. Accordingly, the specific metabolic rate was high in instars 3, 4 and 5 and reduced in instars 6 and 7.     

Synthesis and Biological Activity of New 4‐tert‐Butylcyclohexanone Derivatives

In the synthesis performed in this study, derivatives of 4‐tert‐butylcyclohexanone 1 were obtained using typical reactions of organic synthesis. The bioactivity of the selected compounds was evaluated. 1‐(Bromomethyl)‐8‐tert‐butyl‐2‐oxaspiro[4.5]decan‐3‐one ( 5 ) was characterized by attractant properties against larvae and a weak feeding deterrent activity against adults of Alphitobius diaperinus Panzer . This bromolactone was a moderate antifeedant towards Myzus persicae Sulzer . In addition, ethyl (4‐tert‐butylcyclohexylidene)acetate ( 2 ) and bromolactone 5 displayed antibacterial activity. The strongest bacteriostatic effect was observed against Gram‐positive strains: Bacillus subtilis and Staphylococcus aureus. The bromolactone 5 also limited the growth of Escherichia coli strain.