Discovery of Novel Bacterial Cell-Penetrating Phylloseptins in Defensive Skin Secretions of the South American Hylid Frogs, Phyllomedusa duellmani and Phyllomedusa coelestis

Phylloseptin (PS) peptides, derived from South American hylid frogs (subfamily Phyllomedusinae), have been found to have broad-spectrum antimicrobial activities and relatively low haemolytic activities. Although PS peptides have been identified from several well-known and widely-distributed species of the Phyllomedusinae, there remains merit in their study in additional, more obscure and specialised members of this taxon. Here, we report the discovery of two novel PS peptides, named PS-Du and PS-Co, which were respectively identified for the first time and isolated from the skin secretions of Phyllomedusa duellmani and Phyllomedusa coelestis. Their encoding cDNAs were cloned, from which it was possible to deduce the entire primary structures of their biosynthetic precursors. Reversed-phase high-performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS) analyses were employed to isolate and structurally-characterise respective encoded PS peptides from skin secretions. The peptides had molecular masses of 2049.7 Da (PS-Du) and 1972.8 Da (PS-Co). They shared typical N-terminal sequences and C-terminal amidation with other known phylloseptins. The two peptides exhibited growth inhibitory activity against E. coli (NCTC 10418), as a standard Gram-negative bacterium, S. aureus (NCTC 10788), as a standard Gram-positive bacterium and C. albicans (NCPF 1467), as a standard pathogenic yeast, all as planktonic cultures. Moreover, both peptides demonstrated the capability of eliminating S. aureus biofilm.

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7%) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7% - 49.5% identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin and spanins) and shows 29-98% homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60°C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest the AP3 phage is a promising potent agent against bacteria belonging to most common B. cenocepacia IIIA lineage strains.

Effect of time and diet change on the bacterial community structure throughout the gastrointestinal tract and in faeces of the northern brown bandicoot, Isoodon macrourus

A significant gap, in not only peramelid nutritional physiology but marsupial nutrition as a whole, is the lack of information relating to microorganisms of the gastrointestinal tract. This research is a preliminary investigation that will provide a baseline for comparisons among peramelids. The high degree of 16S rRNA gene clones identified in this research that are closely related to culturable bacteria suggests that additional research will enable a more complete description of the gastrointestinal bacteria of I. macrourus. Most identifiable clones belonged to Clostridium and Ruminococcus. This research has confirmed that the hindgut of I. macrourus, the caecum, proximal colon and distal colon, are the main sites for microbial activity.

An intimate link between antimicrobial peptide sequence diversity and binding to essential components of bacterial membranes

Antimicrobial peptides and proteins (AMPs) are widespread in the living kingdom. They are key effectors of defense reactions and mediators of competitions between organisms. They are often cationic and amphiphilic, which favors their interactions with the anionic membranes of microorganisms. Several AMP families do not directly alter membrane integrity but rather target conserved components of the bacterial membranes in a process that provides them with potent and specific antimicrobial activities. Thus, lipopolysaccharides (LPS), lipoteichoic acids (LTA) or the peptidoglycan precursor Lipid II are targeted by a broad series of AMPs. Studying the functional diversity of immune effectors tells us about the essential residues involved in AMP mechanism of action. Marine invertebrates have been found to produce a remarkable diversity of AMPs. Molluscan defensins and crustacean anti-LPS factors (ALF) are diverse in terms of amino acid sequence and show contrasted phenotypes in terms of antimicrobial activity. Their activity is directed essentially against Gram-positive or Gram-negative bacteria due their specific interactions with Lipid II or Lipid A, respectively. Through those interesting examples, we discuss here how sequence diversity generated throughout evolution informs us on residues required for essential molecular interaction at the bacterial membranes and subsequent antibacterial activity. Through the analysis of molecular variants having lost antibacterial activity or shaped novel functions, we also discuss the molecular bases of functional divergence in AMPs.

High alkaline phosphatase activity in phosphate replete waters: The case of two macrotidal estuaries

The occurrence of alkaline phosphatase activity (APA) that hydrolyses organic phosphorus into phosphate (PO4) is commonly related to PO4 deficiency of oceanic, coastal and fresh waters. APA is almost never investigated in PO4-rich estuaries, since very low activities are expected to occur. As a consequence, microbial mineralization of organic phosphorus into PO4 has often been ignored in estuaries. In this study, we examined the importance of potential APA and the associated microbial dynamics in two estuaries, the Aulne and the Elorn (Northwestern France), presenting two different levels of PO4 concentrations. Unexpected high potential APA was observed in both estuaries. Values ranged from 50 to 506 nmol L−1 h−1, which range is usually found in very phosphorus-limited environments. High potential APA values were observed in the oligohaline zone (salinity 5–15) in spring and summer, corresponding to a PO4 peak and a maximum bacterial production of particle-attached bacteria. In all cases, high potential APA was associated with high suspended particulate matter and total particulate phosphorus. The low contribution of the 0.2–1 μm fraction to total APA, the strong correlation between particulate APA and bacterial biomass, and the close relationship between the production of particle-attached bacteria and APA, suggested that high potential APA is mainly due to particle-attached bacteria. These results suggest that the microbial mineralization of organic phosphorus may contribute to an estuarine PO4 production in spring and summer besides physicochemical processes.

Demonstration of the interactions between aromatic compound-loaded lipid nanocapsules and Acinetobacter baumannii bacterial membrane

International audience

Bacterial communities of the specialty crop phyllosphere: response to biological soil amendment use, rainfall, and insect visitation

Microorganisms in the plant rhizosphere, the zone under the influence of roots, and phyllosphere, the aboveground plant habitat, exert a strong influence on plant growth, health, and protection. Tomatoes and cucumbers are important players in produce safety, and the microbial life on their surfaces may contribute to their fitness as hosts for foodborne pathogens such as Salmonella enterica and Listeria monocytogenes. External factors such as agricultural inputs and environmental conditions likely also play a major role. However, the relative contributions of the various factors at play concerning the plant surface microbiome remain obscure, although this knowledge could be applied to crop protection from plant and human pathogens. Recent advances in genomic technology have made investigations into the diversity and structure of microbial communities possible in many systems and at multiple scales. Using Illumina sequencing to profile particular regions of the 16S rRNA gene, this study investigates the influences of climate and crop management practices on the field-grown tomato and cucumber microbiome. The first research chapter (Chapter 3) involved application of 4 different soil amendments to a tomato field and profiling of harvest-time phyllosphere and rhizosphere microbial communities. Factors such as water activity, soil texture, and field location influenced microbial community structure more than soil amendment use, indicating that field conditions may exert more influence on the tomato microbiome than certain agricultural inputs. In Chapter 4, the impact of rain on tomato and cucumber-associated microbial community structures was evaluated. Shifts in bacterial community composition and structure were recorded immediately following rain events, an effect which was partially reversed after 4 days and was strongest on cucumber fruit surfaces. Chapter 5 focused on the contribution of insect visitors to the tomato microbiota, finding that insects introduced diverse bacterial taxa to the blossom and green tomato fruit microbiome. This study advances our understanding of the factors that influence the microbiomes of tomato and cucumber. Farms are complex environments, and untangling the interactions between farming practices, the environment, and microbial diversity will help us develop a comprehensive understanding of how microbial life, including foodborne pathogens, may be influenced by agricultural conditions.

The potential of Asparagopsis armata to control the bacterial load associated to live feed to improve Seabream (Sparus aurata) larvae performance

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Preliminary structure-activity relationship studies on some novel s-substituted aliphatic analogues of 5-{1-[(4- chlorophenyl) sulfonyl]-3-piperidinyl}-1, 3, 4-oxadiazol-2-yl sulfide

Purpose: To study the structure-activity relationships of synthetic multifunctional sulfides through evaluation of lipoxygenase and anti-bacterial activities. Methods: S-substituted derivatives of the parent compound 5-(1-(4-chlorophenylsulfonyl) piperidin-3- yl)-1, 3, 4-oxadiazole-2-thiol were synthesized through reaction with different saturated and unsaturated alkyl halides in DMF medium, with NaH catalyst. Spectral characterization of each derivative was carried out with respect to IR, 1H - NMR, 13C - NMR and EI - MS. The lipoxygenase inhibitory and antibacterial activities of the derivatives were determined using standard procedures. Results: Compound 5e exhibited higher lipoxygenase inhibitory potential than the standard (Baicalein®), with % inhibition of 94.71 ± 0.45 and IC50 of 20.72 ± 0.34 μmoles/L. Compound 5b showed significant antibacterial potential against all the bacterial strains with % inhibition ranging from 62.04 ± 2.78, 69.49 ± 0.41, 63.38 ± 1.97 and 59.70 ± 3.70 to 78.32 ± 0.41, while MIC ranged from 8.18 ± 2.00, 10.60 ± 1.83, 10.84 ± 3.00, 9.81 ± 1.86 and 11.73 ± 5.00 μmoles/L for S. typhi, E. coli, P. aeruginosa, B. subtilis and S. aureus, respectively. Compounds 5d, 5e and 5g showed good antibacterial activity against S. typhi and B. subtilis bacterial strains. Conclusion: The results suggest that compound 5e bearing n-pentyl group is a potent lipoxygenase inhibitor, while compound 5b with n-propyl substitution is a strong antibacterial agent. In addition, compounds 5d, 5e and 5g bearing n-butyl, n-pentyl and n-octyl groups, respectively, are good antibacterial agents against S. typhi and B. subtilis.

S-Alkylated/aralkylated 2-(1H-indol-3-yl-methyl)-1,3,4- oxadiazole-5-thiol derivatives. 2. Anti-bacterial, enzymeinhibitory and hemolytic activities

Purpose: To evaluate the antibacterial, enzyme-inhibitory and hemolytic activities of Salkylated/ aralkylated 2-(1H-indol-3-ylmethyl)-1,3,4-oxadiazole-5-thiol derivatives. Methods: Antibacterial activities of the compounds were evaluated using broth dilution method in 96 well plates. Enzyme inhibitory activities assays were investigated against α-glucosidase, butyrylcholinesterase (BchE) and lipoxygenase (LOX) using acarbose, eserine and baicalien as reference standards, respectively. A mixture of enzyme, test compound and the substrate was incubated and variation in absorbance noted before and after incubation. In tests for hemolytic activities, the compounds were incubated with red blood cells and variations in absorbance were used as indices their hemolytic activities. Results: The compounds were potent antibacterial agents. Five of them exhibited very good antibacterial potential similar to ciprofloxacin, and had minimum inhibitory concentrations (MIC) of at least 9.00 ± 4.12 μM against S. aureus, E.coli, and B. subtilis. One of the compounds had strong enzyme inhibitory potential against α-glucosidase, with IC50 of 17.11 ± 0.02 μg/mL which was better than that of standard acarbose (IC50 38.25 ± 0.12 μg/mL). Another compound had 1.5 % hemolytic activity. Conclusion: S-Alkylated/aralkylated 2-(1H-indol-3-ylmethyl)-1,3,4-oxadiazole-5-thiol deviratives with valuable antibacterial, anti-enzymatic and hemolytic activities have been successfully synthesized. These compounds may be useful in the development of pharmaceutical products.

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

Investigation of the enhanced antimicrobial activity of combination dry powder inhaler formulations of lactoferrin

The airways of most people with cystic fibrosis are colonized with biofilms of the Gram-negative, opportunistic pathogen Pseudomonas aeruginosa. Delivery of antibiotics directly to the lung in the form of dry powder aerosols offers the potential to achieve high local concentrations directly to the biofilms. Unfortunately, current aerosolised antibiotic regimes are unable to efficiently eradicate these biofilms from the airways. We investigated the ability of the innate antimicrobial, lactoferrin, to enhance the activity of two aminoglycoside antibiotics (tobramycin and gentamicin) against biofilms of P. aeruginosa strain PAO1. Biofilms were prepared in 96 well polystyrene plates. Combinations of the antibiotics and various lactoferrin preparations were spray dried. The bacterial cell viability of the various spray dried combinations was determined. Iron-free lactoferrin (apo lactoferrin) induced a 3-log reduction in the killing of planktonic cell by the aminoglycoside antibiotics (p < 0.01) and also reduced both the formation and persistence of P. aeruginosa biofilms (p < 0.01). Combinations of lactoferrin and an aminoglycoside displays potential as an effective new therapeutic strategy in the treatment of P. aeruginosa biofilms infections such as those typical of the CF lungs.

Harnessing bacterial signals for suppression of biofilm formation in the nosocomial fungal pathogen Aspergillus fumigatus

Faced with the continued emergence of antibiotic resistance to all known classes of antibiotics, a paradigm shift in approaches toward antifungal therapeutics is required. Well characterized in a broad spectrum of bacterial and fungal pathogens, biofilms are a key factor in limiting the effectiveness of conventional antibiotics. Therefore, therapeutics such as small molecules that prevent or disrupt biofilm formation would render pathogens susceptible to clearance by existing drugs. This is the first report describing the effect of the Pseudomonas aeruginosa alkylhydroxyquinolone interkingdom signal molecules 2-heptyl-3-hydroxy-4-quinolone and 2-heptyl-4-quinolone on biofilm formation in the important fungal pathogen Aspergillus fumigatus. Decoration of the anthranilate ring on the quinolone framework resulted in significant changes in the capacity of these chemical messages to suppress biofilm formation. Addition of methoxy or methyl groups at the C5–C7 positions led to retention of anti-biofilm activity, in some cases dependent on the alkyl chain length at position C2. In contrast, halogenation at either the C3 or C6 positions led to loss of activity, with one notable exception. Microscopic staining provided key insights into the structural impact of the parent and modified molecules, identifying lead compounds for further development.

Green mitigation strategy for cultural heritage: bacterial potential for biocide production

Several biosurfactants with antagonistic activity are produced by a variety of microorganisms. Lipopeptides (LPPs) produced by some Bacillus strains, including surfactin, fengycin and iturin are synthesized nonribosomally by mega-peptide synthetase (NRPS) units and they are particularly relevant as antifungal agents. Characterisation, identification and evaluation of the potentials of several bacterial isolates were undertaken in order to establish the production of active lipopeptides against biodeteriogenic fungi from heritage assets. Analysis of the iturin operon revealed four open reading frames (ORFs) with the structural organisation of the peptide synthetases. Therefore, this work adopted a molecular procedure to access antifungal potential of LPP production by Bacillus strains in order to exploit the bioactive compounds synthesis as a green natural approach to be applied in biodegraded cultural heritage context. The results reveal that the bacterial strains with higher antifungal potential exhibit the same morphological and biochemical characteristics, belonging to the genera Bacillus. On the other hand, the higher iturinic genetic expression, for Bacillus sp. 3 and Bacillus sp. 4, is in accordance with the culture antifungal spectra. Accordingly, the adopted methodology combining antifungal screening and molecular data is represent a valuable tool for quick identification of iturin-producing strains, constituting an effective approach for confirming the selection of lipopeptides producer strains.

Essential oils and hydrolates as potential tools for integrated prevention of bacterial diseases in plants

The present work aims to investigate the potential use of natural substances against bacterial plant pathogens. Microdilution tests were therefore carried out in vitro to identify the minimum inhibitory and bactericidal concentrations (MIC and MBC) of several EOs and Hys against selected bacterial pathogens. Commercial products based on a mixture of EOs were in addition assayed with macrodilution experiments against Erwinia amylovora (Ea-causal agent of fire blight). Subsequently, using selected EOs, Hys, and commercial products, ex vivo tests on disease incidence and Ea population dynamics were carried out; the latter experiment was followed by SEM observations. In addition, in vivo resistance induction test was carried out against bacterial leaf of tomato, caused by Xanthomonas vesicatoria (Xv). EOs and Hys showed high bactericidal activity in vitro (MBC <0.1 and <10% for the most active EOs and Hys: Origanum compactum and Thymus vulgaris EOs and Citrus aurantium var. amara Hy, respectively), but they were not effective ex vivo, while resulted very active when used in vivo as resistance inducers in the tomato-Xv pathosystem (relative protection >40%). Differently, commercial products resulted active in all tests, but not as resistance inducers against Xv. An open field trial with commercial products was carried out on strawberry plants naturally infected with Xanthomonas fragariae; the results showed discrete relative protection, concerning that provided by the conventional products based on copper; mostly, the disease severity reduction on those plants treated with EOs commercial products was significant when disease severity resulted high. The papers already published described in the present work investigate (1)the activity of Hys in comparison to EOs with respect to their active volatile content; (2) the potential use of EOs and Hys in cultural heritage; for the restoration of paintings; (3) the induction of resistance caused by plasma-activated water-based root treatments.