Assessment of Lipopeptide Mixtures Produced by Bacillus subtilis as Biocontrol Products against Apple Scab (Venturia inaequalis).

Apple scab is an important disease conventionally controlled by chemical fungicides, which should be replaced by more environmentally friendly alternatives. One of these alternatives could be the use of lipopeptides produced by Bacillus subtilis. The objective of this work is to study the action of the three families of lipopeptides and different mixtures of them in vitro and in vivo against Venturia inaequalis. Firstly, the antifungal activity of mycosubtilin/surfactin and fengycin/surfactin mixtures was determined in vitro by measuring the median inhibitory concentration. Then, the best lipopeptide mixture ratio was produced using Design of Experiment (DoE) to optimize the composition of the culture medium. Finally, the lipopeptides mixtures efficiency against V. inaequalis was assessed in orchards as well as the evaluation of the persistence of lipopeptides on apple. In vitro tests show that the use of fengycin or mycosubtilin alone is as effective as a mixture, with the 50-50% fengycin/surfactin mixture being the most effective. Optimization of culture medium for the production of fengycin/surfactin mixture shows that the best composition is glycerol coupled with glutamic acid. Finally, lipopeptides showed in vivo antifungal efficiency against V. inaequalis regardless of the mixture used with a 70% reduction in the incidence of scab for both mixtures (fengycin/surfactin or mycosubtilin/surfactin). The reproducibility of the results over the two trial campaigns was significantly better with the mycosubtilin/surfactin mixture. The use of B. subtilis lipopeptides to control this disease is very promising.

Small AntiMicrobial Peptide With in Vivo Activity Against Sepsis.

Antimicrobial peptides (AMPs) are considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and alternative mechanisms of action compared to conventional antibiotics. Although AMPs present considerable advantages over conventional antibiotics, their clinical and commercial development still have some limitations, because of their potential toxicity, susceptibility to proteases, and high cost of production. To overcome these drawbacks, the use of peptides mimics is anticipated to avoid the proteolysis, while the identification of minimalist peptide sequences retaining antimicrobial activities could bring a solution for the cost issue. We describe here new polycationic ß-amino acids combining these two properties, that we used to design small dipeptides that appeared to be active against Gram-positive and Gram-negative bacteria, selective against prokaryotic versus mammalian cells, and highly stable in human plasma. Moreover, the in vivo data activity obtained in septic mice reveals that the bacterial killing effect allows the control of the infection and increases the survival rate of cecal ligature and puncture (CLP)-treated mice.

Boronic species as promising inhibitors of the Staphylococcus aureus NorA efflux pump: study of 6-substituted pyridine-3-boronic acid derivatives.

In response to the extensive use of antibiotics, bacteria have evolved numerous mechanisms of defense against antimicrobial agents. Among them, extrusion of the antimicrobial agents outside the bacterial cell through efflux pumps is a major cause of concern. At first limited to one or few structurally-related antibiotics, bacterial resistance have then progressed towards cross-resistance between different classes of antibiotics, leading to multidrug-resistant microorganisms. Emergence of these pathogens requires development of novel therapeutic strategies and inhibition of efflux pumps appears to be a promising strategy that could restore the potency of existing antibiotics. NorA is the most studied chromosomal efflux pump of Staphylococcus aureus; it is known to be implied in resistance of Methicillin-resistant S. aureus (MRSA) strains against a wide range of unrelated substrates, including hydrophilic fluoroquinolones. Starting from 6-benzyloxypyridine-3-boronic acid I that we previously identified as a potential inhibitor of the NorA efflux pump against the NorA-overexpressing S. aureus 1199B strain (SA1199B), we describe here the synthesis and biological evaluation of a series of 6-(aryl)alkoxypyridine-3-boronic acids. 6-(3-Phenylpropoxy)pyridine-3-boronic acid 3i and 6-(4-phenylbutoxy)pyridine-3-boronic acid 3j were found to potentiate ciprofloxacin activity by a 4-fold increase compared to the parent compound I. In addition, it has been shown that both compounds promote Ethidium Bromide (EtBr) accumulation in SA1199B, thus corroborating their potential mode of action as NorA inhibitors.

Novel synthetic bis-indolic derivatives with antistaphylococcal activity, including against MRSA and VISA strains.

OBJECTIVES: We report the synthesis, antibacterial activity and toxicity of 24 bis-indolic derivatives obtained during the development of new ways of synthesis of marine bis-indole alkaloids from the spongotine, topsentin and hamacanthin classes. METHODS: Innovative ways of synthesis and further structural optimizations led to bis-indoles presenting either the 1-(1H-indol-3'-yl)-1,2-diaminoethane unit or the 1-(1H-indol-3-yl)ethanamine unit. MIC determination was performed for reference and clinical strains of Staphylococcus aureus and CoNS species. MBC, time-kill kinetics, solubility, hydrophobicity index, plasma protein-binding and cytotoxicity assays were performed for lead compounds. Inhibition of the S. aureus NorA efflux pump was also tested for bis-indoles with no antistaphylococcal activity. RESULTS: Lead compounds were active against both S. aureus and CoNS species, with MICs between 1 and 4 mg/L. Importantly, the same MICs were found for MRSA and vancomycin-intermediate S. aureus strains. Early concentration-dependent bactericidal activity was observed for lead derivatives. Compounds with no intrinsic antibacterial activity could inhibit the S. aureus NorA efflux pump, which is involved in resistance to fluoroquinolones. At 0.5 mg/L, the most effective compound led to an 8-fold reduction of the ciprofloxacin MIC for the SA-1199B S. aureus strain, which overexpresses NorA. However, the bis-indole compounds displayed a high hydrophobicity index and high plasma protein binding, which significantly reduced antibacterial activity. CONCLUSIONS: We have synthesized and characterized novel bis-indole derivatives as promising candidates for the development of new antistaphylococcal treatments, with preserved activity against MDR S. aureus strains.

1-(1H-indol-3-yl)ethanamine derivatives as potent Staphylococcus aureus NorA efflux pump inhibitors.

The synthesis of 37 1-(1H-indol-3-yl)ethanamine derivatives, including 12 new compounds, was achieved through a series of simple and efficient chemical modifications. These indole derivatives displayed modest or no intrinsic anti-staphylococcal activity. By contrast, several of the compounds restored, in a concentration-dependent manner, the antibacterial activity of ciprofloxacin against Staphylococcus aureus strains that were resistant to fluoroquinolones due to overexpression of the NorA efflux pump. Structure-activity relationships studies revealed that the indolic aldonitrones halogenated at position 5 of the indole core were the most efficient inhibitors of the S. aureus NorA efflux pump. Among the compounds, (Z)-N-benzylidene-2-(tert-butoxycarbonylamino)-1-(5-iodo-1H-indol-3-yl)ethanamine oxide led to a fourfold decrease of the ciprofloxacin minimum inhibitory concentration against the SA-1199B strain when used at a concentration of 0.5 mg L(-1) . To the best of our knowledge, this activity is the highest reported to date for an indolic NorA inhibitor. In addition, a new antibacterial compound, tert-butyl (2-(3-hydroxyureido)-2-(1H-indol-3-yl)ethyl)carbamate, which is not toxic for human cells, was also found.

First identification of boronic species as novel potential inhibitors of the Staphylococcus aureus NorA efflux pump.

Overexpression of efflux pumps is an important mechanism of bacterial resistance that results in the extrusion of antimicrobial agents outside the bacterial cell. Inhibition of such pumps appears to be a promising strategy that could restore the potency of existing antibiotics. The NorA efflux pump of Staphylococcus aureus confers resistance to a wide range of unrelated substrates, such as hydrophilic fluoroquinolones, leading to a multidrug-resistance phenotype. In this work, approximately 150 heterocyclic boronic species were evaluated for their activity against susceptible and resistant strains of S. aureus. Twenty-four hit compounds, although inactive when tested alone, were found to potentiate ciprofloxacin activity by a 4-fold increase at concentrations ranging from 0.5 to 8 µg/mL against S. aureus 1199B, which overexpresses NorA. Boron-free analogues showed no biological activity, thus revealing that the boron atom is crucial for biological activity. This work describes the first reported efflux pump inhibitory activity of boronic acid derivatives.

In vitro inhibitory properties of ferrocene-substituted chalcones and aurones on bacterial and human cell cultures.

Two series of ten chalcones and ten aurones, where ferrocene replaces the C ring and with diverse substituents on the A ring were synthesized. The compounds were tested against two antibiotic-sensitive bacterial strains, E. coli ATCC 25922 and S. aureus ATCC 25923, and two antibiotic-resistant strains, S. aureus SA-1199B and S. epidermidis IPF896. The unsubstituted compound and those with methoxy substitution showed an inhibitory effect on all bacterial strains at minimum inhibitory concentrations ranging between 2 and 32 mg L(-1). For four of these compounds, the effect was bactericidal, as opposed to bacteriostatic. The corresponding organic aurones did not show growth inhibition, underscoring the role of the ferrocene group. The methoxy-substituted aurones and the unsubstituted aurone also showed low micromolar (IC(50)) activity against MRC-5 non-tumoral lung cells and MDA-MB-231 breast cancer cells, suggesting non-specific toxicity.

Optimized grafting of antimicrobial peptides on stainless steel surface and biofilm resistance tests.

Antibacterial peptides, magainin I and nisin were covalently bound to stainless steel surfaces. Several procedures of surface functionalisation processes have been investigated and optimized, each step being characterized by polarization modulation reflection absorption infrared spectroscopy (PM-RAIRS) and X-ray photoemission spectroscopy (XPS). Grafting of antibacterial peptides was successfully achieved by a 3 steps functionalisation process on a chitosan polymeric layer. The antibacterial activity of the anchored magainin and nisin was tested against a gram-positive bacteria, Listeria ivanovii, i.e., the possible survival and attachment of this bacteria, was characterized on modified stainless steel surfaces. The results revealed that the adsorbed peptides reduced the adhesion of bacteria on the functionalised stainless steel surface.

Elaboration of antibiofilm materials by chemical grafting of an antimicrobial peptide.

A peptide antibiotic, gramicidin A, was covalently bound to cystamine self-assembled monolayers on gold surfaces. Each step of the surface functionalization was characterized by polarization modulation infrared reflection absorption spectroscopy and X-ray photoelectron spectroscopy. The antimicrobial activity of the anchored gramicidin was tested against three Gram-positive bacteria (Listeria ivanovii, Enterococcus faecalis, and Staphylococcus aureus), the Gram-negative bacterium Escherichia coli and the yeast Candida albicans. The results revealed that the adsorbed gramicidin reduced, from 60% for E. coli to 90% for C. albicans, the number of culturable microorganisms attached to the surface. The activity was proven to be persistent overtime, up to 6 months after the first use. The bacteria attached to the functionalized surfaces were permeabilized as shown by confocal microscopy. Taken together, these results indicate a bacteriostatic mode of action of the immobilized peptide. Finally, using green fluorescent protein-expressing bacteria, it was shown that the development of a bacterial biofilm was delayed on peptide-grafted surfaces for at least 24 h.

The antibacterial activity of Magainin I immobilized onto mixed thiols Self-Assembled Monolayers.

An antibacterial peptide, Magainin I, was covalently bound to a mixed 11-mercaptoundecanoïc acid (MUA) and 6-mercaptohexanol (C6OH) (ratio 1:3) Self-Assembled Monolayer (SAM) on gold surfaces. Each step of the surface functionalization was characterized by Polarization Modulation Reflection Absorption InfraRed Spectroscopy (PM-RAIRS) and X-ray Photoelectron Spectroscopy (XPS). The antibacterial activity of the anchored Magainin was tested against three Gram-positive bacteria (Listeria ivanovii, Enterococcus faecalis and Staphylococcus aureus), and the results revealed that the adsorbed Magainin I reduced by more than 50% the adhesion of bacteria at the surface, together with the killing of the bacteria that nonetheless adhered to the surface. No release of the peptide was observed upon contact with the bacterial suspension; the activity has proven to be persistent overtime, up to six months after the first use.

Characterization of new bacteriocinogenic lactic acid bacteria isolated using a medium designed to simulate inhibition of Listeria by Lactobacillus sakei 2512 on meat.

Bacteriocinogenic bacteria have been proposed to protect food products from Listeria contamination as bioprotective cultures. Lactobacillus sakei 2512 was demonstrated to inhibit the growth of Listeria on sliced cooked ham by challenge test. A liquid medium simulating ham, BHI5L200, was designed in order to select bioprotective strains for meat protection. Two strains were selected, from the 201 lactic acid bacteria screened, that produced bacteriocins at pH 5.8 in BHI5L200. The first one, Leuconostoc pseudomesenteroides 2733, produced a new bacteriocin which was purified and partially characterized. The second, Lactobacillus curvatus 2711, produced sakacin X and was shown to contain sakacin T and sakacin P structural genes. Co-culture experiments in BHI5L200 demonstrated that growth of Listeria was inhibited by L. sakei 2512 as well as by L. curvatus 2711.

Influence of amino acid substitutions in the leader peptide on maturation and secretion of mesentericin Y105 by Leuconostoc mesenteroides.

By site-specific mutagenesis, the hydrophobic conserved amino acids and the C-terminal GG doublet of the leader peptide of pre-mesentericin Y105 were demonstrated to be critical for optimal secretion of mesentericin Y105, as well as for the maturation of the pre-bacteriocin by the protease portion of the ABC transporter MesD.