Marine-Derived Quorum-Sensing Inhibitory Activities Enhance the Antibacterial Efficacy of Tobramycin against Pseudomonas aeruginosa

Bacterial epiphytes isolated from marine eukaryotes were screened for the production of quorum sensing inhibitory compounds (QSIs). Marine isolate KS8, identified as a Pseudoalteromonas sp., was found to display strong quorum sensing inhibitory (QSI) activity against acyl homoserine lactone (AHL)-based reporter strains Chromobacterium violaceum ATCC 12472 and CV026. KS8 supernatant significantly reduced biofilm biomass during biofilm formation (−63%) and in pre-established, mature P. aeruginosa PAO1 biofilms (−33%). KS8 supernatant also caused a 0.97-log reduction (−89%) and a 2-log reduction (−99%) in PAO1 biofilm viable counts in the biofilm formation assay and the biofilm eradication assay respectively. The crude organic extract of KS8 had a minimum inhibitory concentration (MIC) of 2 mg/mL against PAO1 but no minimum bactericidal concentration (MBC) was observed over the concentration range tested (MBC > 16 mg/mL). Sub-MIC concentrations (1 mg/mL) of KS8 crude organic extract significantly reduced the quorum sensing (QS)-dependent production of both pyoverdin and pyocyanin in P. aeruginosa PAO1 without affecting growth. A combinatorial approach using tobramycin and the crude organic extract at 1 mg/mL against planktonic P. aeruginosa PAO1 was found to increase the efficacy of tobramycin ten-fold, decreasing the MIC from 0.75 to 0.075 µg/mL. These data support the validity of approaches combining conventional antibiotic therapy with non-antibiotic compounds to improve the efficacy of current treatments.     

5-Episinuleptolide Decreases the Expression of the Extracellular Matrix in Early Biofilm Formation of Multi-Drug Resistant Acinetobacter baumannii

Nosocomial infections and increasing multi-drug resistance caused by Acinetobacter baumannii have been recognized as emerging problems worldwide. Moreover, A. baumannii is able to colonize various abiotic materials and medical devices, making it difficult to eradicate and leading to ventilator-associated pneumonia, and bacteremia. Development of novel molecules that inhibit bacterial biofilm formation may be an alternative prophylactic option for the treatment of biofilm-associated A. baumannii infections. Marine environments, which are unlike their terrestrial counterparts, harbor an abundant biodiversity of marine organisms that produce novel bioactive natural products with pharmaceutical potential. In this study, we identified 5-episinuleptolide, which was isolated from Sinularia leptoclados, as an inhibitor of biofilm formation in ATCC 19606 and three multi-drug resistant A. baumannii strains. In addition, the anti-biofilm activities of 5-episinuleptolide were observed for Gram-negative bacteria but not for Gram-positive bacteria, indicating that the inhibition mechanism of 5-episinuleptolide is effective against only Gram-negative bacteria. The mechanism of biofilm inhibition was demonstrated to correlate to decreased gene expression from the pgaABCD locus, which encodes the extracellular polysaccharide poly-β-(1,6)-N-acetylglucosamine (PNAG). Scanning electron microscopy (SEM) indicated that extracellular matrix of the biofilm was dramatically decreased by treatment with 5-episinuleptolide. Our study showed potentially synergistic activity of combination therapy with 5-episinuleptolide and levofloxacin against biofilm formation and biofilm cells. These data indicate that inhibition of biofilm formation via 5-episinuleptolide may represent another prophylactic option for solving the persistent problem of biofilm-associated A. baumannii infections.     

Antibacterial and Antibiofilm Activities of Tryptoquivalines and Meroditerpenes Isolated from the Marine-Derived Fungi Neosartorya paulistensis,N. laciniosa,N. tsunodae,and the Soil Fungi N. fischeri and N. siamensis

A new meroditerpene, sartorypyrone C (5), was isolated, together with the known tryptoquivalines l (1a), H (1b), F (1c), 3′-(4-oxoquinazolin-3-yl) spiro[1H-indole-3,5′]-2,2′-dione (2) and 4(3H)-quinazolinone (3), from the culture of the marine sponge-associated fungus Neosartorya paulistensis (KUFC 7897), while reexamination of the fractions remaining from a previous study of the culture of the diseased coral-derived fungus N. laciniosa (KUFC 7896) led to isolation of a new tryptoquivaline derivative tryptoquivaline T (1d). Compounds 1a–d, 2, 3, and 5, together with aszonapyrones A (4a) and B (4b), chevalones B (6) and C (7a), sartorypyrones B (7b) and A (8), were tested for their antibacterial activity against four reference strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa), as well as the environmental multidrug-resistant isolates. Only aszonapyrone A (4a) and sartorypyrone A (8) exhibited significant antibacterial activity as well as synergism with antibiotics against the Gram-positive multidrug-resistant strains. Antibiofilm assays of aszonapyrone A (4a) and sartorypyrone A (8) showed that practically no biofilm was formed in the presence of their 2× MIC and MIC. However, the presence of a sub-inhibitory concentration of ½ MIC of 4a and 8 was found to increase the biofilm production in both reference strain and the multidrug-resistant isolates of S. aureus.     

Use of Quorum Sensing Inhibition Strategies to Control Microfouling

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence^® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence^® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.     

Nature’s Lab for Derivatization: New and Revised Structures of a Variety of Streptophenazines Produced by a Sponge-Derived Streptomyces Strain

Eight streptophenazines (A–H) have been identified so far as products of Streptomyces strain HB202, which was isolated from the sponge Halichondria panicea from the Baltic Sea. The variation of bioactivities based on small structural changes initiated further studies on new derivatives. Three new streptophenazines (I–K) were identified after fermentation in the present study. In addition, revised molecular structures of streptophenazines C, D, F and H are proposed. Streptophenazines G and K exhibited moderate antibacterial activity against the facultative pathogenic bacterium Staphylococcus epidermidis and against Bacillus subtilis. All tested compounds (streptophenazines G, I–K) also showed moderate activities against PDE 4B.     

Antibacterial Molecules from Marine Microorganisms against Aquatic Pathogens: A Concise Review

Antibiotic resistance and residues in aquaculture are a growing concern worldwide and consequently identifying favorable antibacterial compounds against aquatic pathogenic bacteria are gained more attention. Active compounds derived from marine microorganisms have shown great promise in this area. This review is aimed to make a comprehensive survey of anti-aquatic pathogenic bacterial compounds that were produced by marine microorganisms. A total of 79 compounds have been reported, covering literature from 1997 to 2021. The compounds are included in different structural classes such as polyketides, terpenoids, nitrogen compounds and others, and some of them present the potential to be developed into agents for the treatment of aquatic pathogenic bacteria.     

Cultivable Alginate Lyase-Excreting Bacteria Associated with the Arctic Brown Alga Laminaria

Although some alginate lyases have been isolated from marine bacteria, alginate lyases-excreting bacteria from the Arctic alga have not yet been investigated. Here, the diversity of the bacteria associated with the brown alga Laminaria from the Arctic Ocean was investigated for the first time. Sixty five strains belonging to nine genera were recovered from six Laminaria samples, in which Psychrobacter (33/65), Psychromonas (10/65) and Polaribacter (8/65) were the predominant groups. Moreover, 21 alginate lyase-excreting strains were further screened from these Laminaria-associated bacteria. These alginate lyase-excreting strains belong to five genera. Psychromonas (8/21), Psedoalteromonas (6/21) and Polaribacter (4/21) are the predominant genera, and Psychrobacter, Winogradskyella, Psychromonas and Polaribacter were first found to produce alginate lyases. The optimal temperatures for the growth and algiante lyase production of many strains were as low as 10–20 °C, indicating that they are psychrophilic bacteria. The alginate lyases produced by 11 strains showed the highest activity at 20–30 °C, indicating that these enzymes are cold-adapted enzymes. Some strians showed high levels of extracellular alginate lyase activity around 200 U/mL. These results suggest that these algiante lyase-excreting bacteria from the Arctic alga are good materials for studying bacterial cold-adapted alginate lyases.     

Algal-Derived Halogenated Sesquiterpenes from Laurencia dendroidea as Lead Compounds in Schistosomiasis Environmental Control

Schistosomiasis has been controlled for more than 40 years with a single drug, praziquantel, and only one molluscicide, niclosamide, raising concern of the possibility of the emergence of resistant strains. However, the molecular targets for both agents are thus far unknown. Consequently, the search for lead compounds from natural sources has been encouraged due to their diverse structure and function. Our search for natural compounds with potential use in schistosomiasis control led to the identification of an algal species, Laurencia dendroidea, whose extracts demonstrated significant activity toward both Schistosoma mansoni parasites and their intermediate host snails Biomphalaria glabrata. In the present study, three seaweed-derived halogenated sesquiterpenes, (−)-elatol, rogiolol, and obtusol are proposed as potential lead compounds for the development of anthelminthic drugs for the treatment of and pesticides for the environmental control of schistosomiasis. The three compounds were screened for their antischistosomal and molluscicidal activities. The screening revealed that rogiolol exhibits significant activity toward the survival of adult worms, and that all three compounds showed activity against S. mansoni cercariae and B. glabrata embryos. Biomonitored fractioning of L. dendroidea extracts indicated elatol as the most active compound toward cercariae larvae and snail embryos.     

Flagellar Sensillar Equipment of Two Morphologically Closely Related Aphid Hyperparasitoids (Hymenoptera: Figitidae: Alloxysta)

The antennal sensillar equipment in the parasitic wasp family Figitidae was analyzed to date only in few species, despite some are associated with crop pests and can have an economic importance. It is the case of the genus Alloxysta, which includes hyperparasitoids of aphids which can potentially reduce effectiveness of primary pest parasitoids. Here we analyzed, through scanning electron microscopy, the diversity, morphology, and distribution of the antennal sensilla in males and females of Alloxysta consobrina (Zetterstedt) and Alloxysta victrix (Westwood), two species with overall very similar morphology. In both species, antennae are filiform and cylindrical, and flagellum was longer in A. victrix. Eight sensillar types have been recognized: four types of sensilla trichoidea (ST-A, ST-B, ST-C, ST-D), sensilla coeloconica, sensilla placoidea, sensilla campaniformia, and sensilla basiconica. ST-A, ST-B, ST-C, and sensilla placoidea were the most abundant types on the antennae and often increased in number and decreased in size toward the tip of antenna. The two species seem to have several differences in their sensillar equipment, possibly in accordance with the different degree of host range. On the other hand, sexual dimorphism is probably due to the different stimuli that have to be correctly processed. The comparison with the other species of Figitidae studied by far showed, at subfamily-level, that variability in sensillar equipment and phylogeny do not agree. This suggests a complex series of morphological changes during evolution of this group. The taxonomic sample should be thus substantially enlarged to disclose possible trends in sensillar equipment evolution in the family.     

Bioactive compound synthetic capacity and ecological significance of marine bacterial genus pseudoalteromonas

The genus Pseudoalteromonas is a marine group of bacteria belonging to the class Gammaproteobacteria that has come to attention in the natural product and microbial ecology science fields in the last decade. Pigmented species of the genus have been shown to produce an array of low and high molecular weight compounds with antimicrobial, anti-fouling, algicidal and various pharmaceutically-relevant activities. Compounds formed include toxic proteins, polyanionic exopolymers, substituted phenolic and pyrolle-containing alkaloids, cyclic peptides and a range of bromine-substituted compounds. Ecologically, Pseudoalteromonas appears significant and to date has been shown to influence biofilm formation in various marine econiches; involved in predator-like interactions within the microbial loop; influence settlement, germination and metamorphosis of various invertebrate and algal species; and may also be adopted by marine flora and fauna as defensive agents. Studies have been so far limited to a relatively small subset of strains compared to the known diversity of the genus suggesting that many more discoveries of novel natural products as well as ecological connections these may have in the marine ecosystem remain to be made.     

Diversity of Peptides Produced by Nodularia spumigena from Various Geographical Regions

Cyanobacteria produce a great variety of non-ribosomal peptides. Among these compounds, both acute toxins and potential drug candidates have been reported. The profile of the peptides, as a stable and specific feature of an individual strain, can be used to discriminate cyanobacteria at sub-population levels. In our work, liquid chromatography-tandem mass spectrometry was used to elucidate the structures of non-ribosomal peptides produced by Nodularia spumigena from the Baltic Sea, the coastal waters of southern Australia and Lake Iznik in Turkey. In addition to known structures, 9 new congeners of spumigins, 4 aeruginosins and 12 anabaenopeptins (nodulapeptins) were identified. The production of aeruginosins by N. spumigena was revealed in this work for the first time. The isolates from the Baltic Sea appeared to be the richest source of the peptides; they also showed a higher diversity in peptide profiles. The Australian strains were characterized by similar peptide patterns, but distinct from those represented by the Baltic and Lake Iznik isolates. The results obtained with the application of the peptidomic approach were consistent with the published data on the genetic diversity of the Baltic and Australian populations.     

Lipopolysaccharides from Commensal and Opportunistic Bacteria: Characterization and Response of the Immune System of the Host Sponge Suberites domuncula

Marine sponges harbor a rich bacterioflora with which they maintain close relationships. However, the way these animals make the distinction between bacteria which are consumed to meet their metabolic needs and opportunistic and commensal bacteria which are hosted is not elucidated. Among the elements participating in this discrimination, bacterial cell wall components such as lipopolysaccharides (LPS) could play a role. In the present study, we investigated the LPS chemical structure of two bacteria associated with the sponge Suberites domuncula: a commensal Endozoicomonas sp. and an opportunistic Pseudoalteromonas sp. Electrophoretic patterns indicated different LPS structures for these bacteria. The immunomodulatory lipid A was isolated after mild acetic acid hydrolysis. The electrospray ionization ion-trap mass spectra revealed monophosphorylated molecules corresponding to tetra- and pentaacylated structures with common structural features between the two strains. Despite peculiar structural characteristics, none of these two LPS influenced the expression of the macrophage-expressed gene S. domuncula unlike the Escherichia coli ones. Further research will have to include a larger number of genes to understand how this animal can distinguish between LPS with resembling structures and discriminate between bacteria associated with it.     

Alterins Produced by Oyster-Associated Pseudoalteromonas Are Antibacterial Cyclolipopeptides with LPS-Binding Activity

Discovery after discovery, host-associated microbiota reveal a growing list of positive effects on host homeostasis by contributing to host nutrition, improving hosts’ immune systems and protecting hosts against pathogens. In that context, a collection of oyster associated bacteria producing antibacterial compounds have been established to evaluate their role in non-host-derived immunity. Here, we described alterins; potent anti-Gram negative compounds produced by Pseudoalteromonas hCg-6 and hCg-42 isolated from different healthy oyster hemolymph. The strains hCg-6 and hCg-42 produce a set of at least seven antibacterial compounds, ranging from 926 to 982 Da structurally characterized as cyclolipopeptides (CLPs). Alterins share the same cationic heptapeptidic cycle connected via an amido bond to different hydrophobic hydrocarbon tails. Their MICs disclosed a potent antibacterial activity directed against Gram-negative bacteria including oyster and human pathogens that may confer a beneficial defense mechanism to the host but also represents an untapped source of new antibiotics. The alterins’ mechanisms of action have been deciphered: after binding to lipopolysaccharides (LPS), alterins provoke a membrane depolarization and permeabilization leading to bacterial lysis. As hCg-6 and hCg-42 produced a set of natural derivatives, the structure/activity relationship linked to the carbon tail is clarified. We showed that the hydrocarbon tail determines the LPS-binding properties of alterins and consequently their antibacterial activities. Its length and saturation seem to play a major role in this interaction.     

Behavioral Effects and Retention of Protein Immunomarkers on Plum Curculio Conotrachelus nenuphar (Coleoptera: Curculionidae)

Protein immunomarking can be used to track the dispersal of insects in the field or identify plant–insect interactions. By marking insects with known proteins and recapturing them, their movement or host use can be quantified with Enzyme Linked Immunosorbent Assay (ELISA). Before using this technique, retention and behavioral effects of these markers should be evaluated to ensure that the insect’s natural behaviors are conserved. Here, we tested the effects of protein markers on the plum curculio, Conotrachelus nenuphar (Coleoptera: Curculionidae) using two different application methods. This weevil is native to North American and a pest of tree fruit and blueberry in the United States and causes damage resulting in near complete crop loss if left untreated. We tested the effects of marking adult C. nenuphar with two inexpensive food-based immunoprotein markers, bovine casein (cow’s milk) and chicken albumin (egg whites) on climbing distance (total cm), lateral movement (total cm), and lateral movement speed (cm/s), as well as retention time of protein immunomarkers. Neither protein immunomarker affected C. nenuphar movement or climbing, although females climbed significantly greater distances than males. ELISA assays detected 37.5–56.2% of milk protein and 56.2–59.3% of egg on the insect 7 d after application depending on application method. Our findings indicate that food-based protein immunomarkers can be used in future studies to test C. nenuphar movement within host plants without impacting behavior. The use of protein immunomarking will allow studies that will lead to behaviorally based management tactics.     

Phaseolus vulgaris cultivar and Rhizobium strain variation in acid-pH tolerance and nodulation under acid conditions

Acid-soil pH is a major factor limiting nodulation and N₂ fixation in beans. The experiments reported here sought strains of Rhizobium and cultivars of Phaseolus vulgaris L. tolerant of acid pH, and to study the interaction of strains and cultivars differing in pH tolerance. Seventy-eight of 217 isolates of R. leguminosarum bv. phaseoli grew on modified Keyser-Munns medium, pH 4.5. Of these, 33 grew at pH 4.5 in the presence of 100 μM aluminum and 40 tolerated pH 4.5 medium containing 200 μM manganese. Twenty isolates were both highly efficient in nitrogen (N₂) fixation and acid-pH-tolerant.Marked variation was also evident in the growth and nodulation at pH 4.5 of 126 cultivars of Phaseolus vulgaris. Thus ‘Capixaba precoce’, ‘Preto 143’ and ‘Bico de Ouro’ had high nodule number and nodule mass plant⁻¹ when grown at this pH, whereas BAT115 and BAT165 were essentially without nodules. When host cultivars and Rhizobium strains which differed in acid pH tolerance were used in inoculation experiments in soil of pH 4.5, nodulation and plant growth were most affected when both host and Rhizobium were intolerant of low pH. this was associated with reduced multiplication of Rhizobium in the rhizosphere 2 days after planting and with lack of attachment of the Rhizobium to the host root. Competition for nodule sites at acid pH was also affected. When either the Rhizobium strain or the cultivar was acid-tolerant, nodulation and plant development were similar to that achieved when both host cultivars and Rhizobium strain were acid-pH-tolerant.     

Subtilomycin: A New Lantibiotic from Bacillus subtilis Strain MMA7 Isolated from the Marine Sponge Haliclona simulans

Bacteriocins are attracting increased attention as an alternative to classic antibiotics in the fight against infectious disease and multidrug resistant pathogens. Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans displays a broad spectrum antimicrobial activity, which includes Gram-positive and Gram-negative pathogens, as well as several pathogenic Candida species. This activity is in part associated with a newly identified lantibiotic, herein named as subtilomycin. The proposed biosynthetic cluster is composed of six genes, including protein-coding genes for LanB-like dehydratase and LanC-like cyclase modification enzymes, characteristic of the class I lantibiotics. The subtilomycin biosynthetic cluster in B. subtilis strain MMA7 is found in place of the sporulation killing factor (skf) operon, reported in many B. subtilis isolates and involved in a bacterial cannibalistic behaviour intended to delay sporulation. The presence of the subtilomycin biosynthetic cluster appears to be widespread amongst B. subtilis strains isolated from different shallow and deep water marine sponges. Subtilomycin possesses several desirable industrial and pharmaceutical physicochemical properties, including activity over a wide pH range, thermal resistance and water solubility. Additionally, the production of the lantibiotic subtilomycin could be a desirable property should B. subtilis strain MMA7 be employed as a probiotic in aquaculture applications.     

Bioactive Polyoxygenated Steroids from the South China Sea Soft Coral,Sarcophyton sp

Seven new polyoxygenated steroids (1–7) were isolated together with seven known analogues (8–14) from the South China Sea soft coral, Sarcophyton sp. The structures of the new compounds were identified on the basis of extensive spectroscopic analysis and comparison with reported data. All the steroids are characterized with 3β,5α,6β-hydroxy moiety, displaying carbon skeletons of cholestane, ergostane, gorgostane and 23,24-dimethyl cholestane. In the in vitro bioassay, metabolites exhibited different levels of antimicrobial activity against bacterial species Escherichia coli and Bacillus megaterium, and fungal species Microbotryum violaceum and Septoria tritici. No inhibition was detected towards microalga Chlorella fusca. Preliminary structure-activity analysis suggests that the 11α-acetoxy group may increase both antibacterial and antifungal activities. The terminal-double bond and the cyclopropane moiety at the side chain may also contribute to the bioactivity.     

Tanjungides A and B: New Antitumoral Bromoindole Derived Compounds from Diazona cf formosa. Isolation and Total Synthesis

Tanjungides A (1) (Z isomer) and B (2) (E isomer), two novel dibrominated indole enamides, have been isolated from the tunicate Diazona cf formosa. Their structures were determined by spectroscopic methods including HRMS, and extensive 1D and 2D NMR. The stereochemistry of the cyclised cystine present in both compounds was determined by Marfey’s analysis after chemical degradation and hydrolysis. We also report the first total synthesis of these compounds using methyl 1H-indole-3-carboxylate as starting material and a linear sequence of 11 chemical steps. Tanjungides A and B exhibit significant cytotoxicity against human tumor cell lines.     

Black fungi in lichens from seasonally arid habitats

We present a phylogenetic study of black fungi in lichens, primarily focusing on saxicolous samples from seasonally arid habitats in Armenia, but also with examples from other sites. Culturable strains of lichen-associated black fungi were obtained by isolation from surface-washed lichen material. Determination is based on ITS rDNA sequence data and comparison with published sequences from other sources. The genera Capnobotryella, Cladophialophora, Coniosporium, Mycosphaerella, and Rhinocladiella were found in different lichen species, which showed no pathogenic symptoms. A clade of predominantly lichen-associated strains is present only in Rhinocladiella, whereas samples of the remaining genera were grouped more clearly in clades with species from other sources. The ecology of most-closely related strains indicates that Capnobotryella and Coniosporium, and perhaps also Rhinocladiella strains opportunistically colonise lichens. In contrast, high sequence divergence in strains assigned to Mycosphaerella could indicate the presence of several lichen-specific species with unknown range of hosts or habitats, which are distantly related to plant-inhabitants. Similar applies to Cladophialophora strains, where the closest relatives of the strains from lichens are serious human pathogens.