Characteristics and Activity Anti Qourum Sensing Bacillus spp. Isolated from Penaeus vannamei Shrimp Ponds

Certain strains of V. parahaemolyticus carry a gene that encodes a toxin that causes Acute hepatopancreatic necrosis disease (AHPND) in P. vannamei. AHPND attacks shrimp post larvae within 20-30 days after stocking causing up to 100% mortality. The expression of these virulent genes is controlled by the quorum sensing system. This system is inhibited by an anti-quorum sensing (AQS) mechanism. Several Bacillus strains have AQS mechanism by producing AHL-Lactonase enzyme. Therefore, this study aimed to obtain Bacillus spp. having AQS activity for controlling AHPND. The study was conducted from isolation and selection of Bacillus isolates, as well as determination of AQS activity. From 22 samples consisting of shrimp intestines, water and pond sediment samples, a total of 151 isolates of Bacillus spp. were isolated. The screening test for AQS activity obtained 11 isolates that showed AQS activity on Cromobacterium violaceum. Determination of violacein pigment in liquid cultures of C. violaceum showed the index value of the pigment formation was between 0.025-0.166 and 0.026-0.567 at 24-hour and between 48-hour incubations, respectively. The quantitative analysis of violacein production showed that there were six isolates of Bacillus could inhibit the pigment production more than 75%. The isolates were identified as Bacillus cereus (four isolates), Bacillus thuringiensis (one isolate), and Bacillus velezensis (one isolate), respectively. The molecular analysis had confirmed that the isolates have aiiA genes encoding AHL-lactonase enzyme. These Bacillus isolates have potential application for controlling AHNPD disease.

Chemical Composition, Anti-Quorum Sensing, Enzyme Inhibitory, and Antioxidant Properties of Phenolic Extracts of Clinopodium nepeta L. Kuntze

Phenolic extracts of Clinopodium nepeta were prepared and their preliminary phenolic profiles determined using HPLC-DAD with 26 phenolic standards. Apigenin (21.75 ± 0.41 µg/g), myricetin (72.58 ± 0.57 µg/g), and rosmarinic acid (88.51 ± 0.55 µg/g) were the most abundant compounds in DCM (dichloromethane), AcOEt (ethyl acetate), and BuOH (butanol) extracts, respectively. The DCM and AcOEt extracts inhibited quorum-sensing mediated violacein production by C. violaceum CV12472. Anti-quorum-sensing zones on C. violaceum CV026 at MIC (minimal inhibitory concentration) were 10.3 ± 0.8 mm for DCM extract and 12.0 ± 0.5 mm for AcOEt extract. Extracts showed concentration-dependent inhibition of swarming motility on flagellated P. aeruginosa PA01 and at the highest test concentration of 100 μg/mL, AcOEt (35.42 ± 1.00%) extract displayed the best activity. FRAP assay indicated that the BuOH extract (A0.50 = 17.42 ± 0.25 µg/mL) was more active than standard α-tocopherol (A0.50 = 34.93 ± 2.38 µg/mL). BuOH extract was more active than other extracts except in the ABTS●+, where the DCM extract was most active. This antioxidant activity could be attributed to the phenolic compounds detected. C. nepeta extracts showed moderate inhibition on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, and α-amylase. The results indicate that C. nepeta is a potent source of natural antioxidants that could be used in managing microbial resistance and Alzheimer′s disease.

Evaluation of Enzyme Inhibition and Anti-Quorum Sensing Potentials of Melaleuca alternifolia and Citrus sinensis Essential Oils

In this study, Melaleuca alternifolia (Maiden and Betche) Cheel (tea tree) oil and C. sinensis (L.) Osbeck (orange) oil was procured, and their chemical compositions were characterized by gas chromatography coupled with mass spectrometry, with co-injection using authentic samples. The oils were rich in monoterpene hydrocarbons making up 84.5% of C. sinensis and 48.9% of M. alternifolia oils. High amounts of oxygenated monoterpenoids were also identified in C. sinensis (9.6%) and M. alternifolia (49.3%) oils. The three most abundant compounds were limonene (71.2%), linalool (5.5%), and β-myrcene (5.1%) in C. sinensis, and terpinen-4-ol (45.6%), γ-terpinene (19.4%) and α-terpinene (9.3%) in M. alternifolia. Enzyme inhibitions (anticholinesterase, antiurease, antityrosinase) of both essential oils were evaluated. In acetylcholinesterase assay, M. alternifolia and C. sinensis had inhibition concentration (IC50) values of 153.7 ± 1.25 and 96.4 ± 1.0 µg/mL, respectively as compared to 5.42 ± 0.11 µg/mL for galantamine, while in butyrylcholinesterase assay, M. alternifolia (IC50 = 85.6 ± 0.7 µg/mL) and C. sinensis (IC50 = 127.8 ± 0.6 µg/mL) exhibited moderate activities compared to galantamine (IC50 = 45.8 ± 0.8 µg/mL). In the urease inhibitory activity, essential oils of C. sinensis and M. alternifolia showed 48.4% ± 0.9% and 30.6% ± 0.7% inhibitions at 200 µg/mL concentration, respectively. Quorum sensing (QS) mediated violacein production in Chromobacterium violaceum CV12472 was inhibited by 100% at minimum inhibitory concentration (MIC) values for both oils while showing QS inhibition diameter zones in C. violaceum of 22.5 ± 0.4 mm and 14.3 ± 0.5 mm for C. sinensis and M. alternifolia oils, respectively at MIC concentration. The good quorum-sensing potential indicates that these oils can suppress microbial resistance and severity of infections.

Biosensor-Assisted Adaptive Laboratory Evolution for Violacein Production

Violacein is a naturally occurring purple pigment, widely used in cosmetics and has potent antibacterial and antiviral properties. Violacein can be produced from tryptophan, consequently sufficient tryptophan biosynthesis is the key to violacein production. However, the complicated biosynthetic pathways and regulatory mechanisms often make the tryptophan overproduction challenging in Escherichia coli. In this study, we used the adaptive laboratory evolution (ALE) strategy to improve violacein production using galactose as a carbon source. During the ALE, a tryptophan-responsive biosensor was employed to provide selection pressure to enrich tryptophan-producing cells. From the biosensor-assisted ALE, we obtained an evolved population of cells capable of effectively catabolizing galactose to tryptophan and subsequently used the population to obtain the best violacein producer. In addition, whole-genome sequencing of the evolved strain identified point mutations beneficial to the overproduction. Overall, we demonstrated that the biosensor-assisted ALE strategy could be used to rapidly and selectively evolve the producers to yield high violacein production.

Clitoria ternatea ethanolic extract prevents dental caries via inhibiting Streptococcus mutans growth and quorum sensing

Our previous study demonstrated that the blue butterfly pea flower (Clitoria ternatea) had several pharmacological effects to treat inflammatory-related diseases, including edema and diabetes. However, its benefit for preventing dental caries and protecting the tooth has not been explored yet. Here, we investigated whether C. ternatea ethanolic extract (CTEE) prevented dental caries through antibacterial and antiquorum sensing activities toward oral pathogen Streptococcus mutans in vitro. CTEE was made by using kinetic maceration in ethanol. Antibacterial activity of CTEE against S. mutans was tested using disk diffusion agar and microdilution assays. Quorum sensing system employed Chromobacterium violaceum bacteria to produce violacein, and CTEE at various concentrations was tested for its antiquorum sensing activity to inhibit the violacein production. Our results demonstrated that CTEE at 1 mg/mL showed a significant inhibition >90% against S. mutans, indicating its MIC value. For the quorum sensing system, CTEE at the lowest concentration (0.25 mg/mL) significantly inhibit up to 68% of violacein produced by C. violaceum. These data indicate that CTEE may act as a natural oral functional food with antibacterial and antiquorum sensing activities for the prevention of dental caries.

Coumarin’s Anti-Quorum Sensing Activity Can Be Enhanced When Combined with Other Plant-Derived Small Molecules

Coumarins are class of natural aromatic compounds based on benzopyrones (2H-1-benzopyran-2-ones). They are identified as secondary metabolites in about 150 different plant species. The ability of coumarins to inhibit cell-to-cell communication in bacterial communities (quorum sensing; QS) has been previously described. Coumarin and its derivatives in plant extracts are often found together with other small molecules that show anti-QS properties too. The aim of this study was to find the most effective combinations of coumarins and small plant-derived molecules identified in various plants extracts that inhibit QS in Chromobacterium violaceum ATCC 31532 violacein production bioassay. The coumarin and its derivatives: 7-hydroxycoumarin, 7.8-dihydroxy-4-methylcoumarin, were included in the study. Combinations of coumarins with gamma-octalactone, 4-hexyl-1.3-benzenediol, 3.4.5-trimethoxyphenol and vanillin, previously identified in oak bark (Quercus cortex), and eucalyptus leaves (Eucalyptus viminalis) extracts, were analyzed in a bioassay. When testing two-component compositions, it was shown that 7.8-dihydroxy-4-methylcoumarin, 4-hexyl-1.3-benzendiol, and gamma-octalactone showed a supra-additive anti-QS effect. Combinations of all three molecules resulted in a three- to five-fold reduction in the concentration of each compound needed to achieve EC50 (half maximal effective concentration) against QS in C. violaceum ATCC 31532.

Antiviral, Antibacterial and Anti-Quorum Sensing Activities of a Lyophilized Aqueous Pine Resin Extract

This work aimed to evaluate antiviral, antibacterial and anti-quorum sensing effects of a lyophilized aqueous pine resin extract. The pine resin toxicity was evaluated by a crystal violet assay at 492 nm for Hep-2 cells and the trypan blue method for P3HR1. The cytotoxicity expressed in CC50 was 12.5 mg/ml and 0.23 mg/ml on Hep-2 and P3HR1 cells, respectively. The antiviral effect was obtained at 4 mg/ml against coxsackievirus B4 (CVB4). Using the diffusion method discs for the antibacterial activity, the extract was more active against Gram+ than Gram−. Inhibition zones of 22 mm were obtained at a 275 mg/ml concentration. Inhibition of violacein production by pine resin extract was tested using Chomobacterium violaceum 026 (CV026) in a medium supplemented with acyl-homoserine lactone (AHL); cytotoxicity was evaluated toward Artemia and its protecting effect against Vibrio harveyi BB120 and then AHL degradation was studied. Results showed that pine resin inhibited violacein production by CV026, was not toxic for Artemia and protected it against Vibrio harveyi BB120; AHL was degraded with inhibition areas from 9.6, 11.03 and 10.26 to 12.34 mm at the contact times of the mixtures “resin-AHL” respectively at 0 h, 4 h 24 h and 72 h. This study constitutes a confirmation of aqueous extract pine resin as antibacterial, by anti-quorum sensing pathway and antiviral efficacy.

Ethanol Extract of Campsis grandiflora Flower and Its Organic Acid Components Have Inhibitory Effects on Autoinducer Type 1 Quorum Sensing

Chinese herbs are a useful resource bank for natural drug development, and have attracted considerable attention to exploit quorum sensing inhibitors (QSIs). This study was designed to screen QSIs from raw Chinese herb materials. Of the 38 common herbs examined, the ethanol extract of Campsis grandiflora flower had the strongest QSI activity. The C. grandiflora flower ethanol extract (CFEE) was purified by HPD600, and the QSI activities were examined in further detail. CFEE inhibited violacein production of Chromobacterium violaceum 026 in a dose-dependent manner, and inhibit the swarming abilities of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Furthermore, CFEE could inhibited biofilm formation and destroyed mature biofilms of E. coli K-12 and P. aeruginosa PAO1. The composition of CFEE was determined by UPLC-MS/MS to distinguish active QSI compounds, and 21 compounds were identified. In addition to gallic acid and caffeic acid, two organic acids, malic acid and succinic acid, were confirmed for the first time to have autoinducer type 1 QSI activities. Therefore, CFEE is a potential QSI that could be used as a novel antimicrobial agent and should be considered for medicinal development.

Pullulan Films Containing Rockrose Essential Oil for Potential Food Packaging Applications

Active packaging is designed to control the development of decay- and disease-causing microorganisms and is emerging as a promising technology for extending shelf-life, maintaining food safety, reducing waste, and minimizing the risks for foodborne diseases. The goal of this work was to develop and characterize bioactive pullulan-based films, containing rockrose (Cistus ladanifer) essential oil. Among other abundant compounds (camphene, bornyl acetate and trans-pinocarveol), α-pinene was identified as the major compound of rockrose essential oil (39.25%). The essential oil presented stronger antibacterial activity against Gram-positive than against Gram-negative bacteria. The antioxidant results indicate the potential of the developed films to be used to package foods susceptible to oxidation and rancification, thus improving their shelf-life. Also, this study reflects the potential of rockrose essential oil, free or incorporated in pullulan, as a promising quorum sensing inhibitor, since it was able to interrupt intercellular communication, inhibiting violacein production. Electronic microscopy images showed the antibiofilm activity of the films with rockrose essential oil that were able to influence bacterial adhesion, which may be explained by the differences in the surface free energy of the films, as also determined.

Rapid Characterization of Quorum Sensing Inhibitory Molecules from Garcinia indica Choisy Seed Methanol Extract by GC-MS Analysis

Background: Bacteria communicate by producing small signaling molecules to maintain threshold cell density. Quorum sensing inhibition is an effective and most potent approach for the prevention of cell to cell communication. Methods: In the present study, the methanol extract of Garcinia indica was tested for anti-quorum sensing activity using Chromobacterium violaceum as a biosensor. The extract inhibited violacein production in biosensor strain without affecting the growth of the bacteria and this was corroborated by bioautography assay. Results: The bioactive fraction was analysed by the aid of GC-MS revealed that, the presence of coumarin (7-hydroxy-3-(1,1-dimethylprop-2-enyl) coumarin) and eugenol (1,2-dimethoxy-4-(1-methoxy-1- propenyl) benzene) derivatives as anti-quorum sensing molecules. Conclusion: A detailed investigation is required to study the mechanism of action involved in control of quorum sensing signals and possible applications.