RRL on Effects of bottlebrush on quorum-sensing activities of Curvibacter (Thesis Sample)

2.1. REVIEW OF RELATED LITERATURE Callistemon viminalis Callistemon viminalis is a shrub with many, long, thin, and drooping branches. Its leaves are green, narrow, and have parallel venation. Its flowers are about 7- 8 centimeters long and are spiky, arranged in a bottlebrush-like manner hence the vernacular name. Studies have proven that it has anthelmintic, anti-quorum sensing, insecticidal, anti-infective, antibacterial, molluscicidal, antioxidant, anticancer, anti-inflammatory, and anti-platelet aggregation properties. In the Philippines, plantations in Baguio cultivate this plant for ornamental purposes and weed control (Wheeler, 2005). This plant has been the subject of many studies for its properties that were proven to be preventive. Studies show that C. viminalis extracts have little to no microbicidal effects on microorganisms but exhibit a strong QSI effect towards them. This phenomenon makes the bottlebrush plant a great candidate for medical therapies and treatments aimed at microorganisms without killing them. According to Gohar et al. (2012), the genus Callistemon has a diverse chemical profile. Its members are found to have steroids and terpenes, flavonoids (Khaton et al., 2011 and Park et al., 2010), tannins and phenolic compounds (El Dib and El-Shenawy, 2008), tetradecahydroxanthenediones (Khambay et al., 1999), and various essential oils (Silva et al., 2010 and Liu et al., 2010). Callistemon’s diverse chemical profile suggests diverse biological activities, antibacterial and antifungal activities (Seyyed et al., 2010 and Dongmo et al., 2010), molluscicidal and insecticidal activities, and many more. Zubair et al.’s study studied the antioxidant potential, and oil composition of Callistemon viminalis leaves. They conducted a GC-MS analysis using a GC 6850 network gas chromatography system and separated on an HP-55 MS capillary column with 5% phenyl polysiloxane. The components were identified using mass spectrometry. They conducted this analysis of the n-hexane extract, revealing the presence of 40 compounds. The C. viminalis leaves contained high levels of total phenolic and flavonoid contents. GENUS Curvibacter The genus Curvibacter by Ding and Yokota (2004) belongs to the family Comamonadaceae. They are Gram-negative, heterotrophic, aerobic, curved rod-shaped bacteria comprising three recognized species, C. gracilis, C. delicatus, and C. lanceolatus (Ding and Yokota, 2004). The members of this genus are known to be yellow-brown as a colony, have flagella, and are curved rods shaped. Ding and Yokota’s discovery isolated them from well waters in Osaka, Japan. Fraune et al. (2015) stated that these species comprise the majority of Hydra’s colonizers. From Wein et al.’s study in 2018, the Hydra-Curvibacter host-colonizer relationship was proved to have a high dependency on the QS capabilities of the Curvibacter species and the host-mediated modifications of this QS activity. They used genetically labeled Curvibacter strains to characterize Hydra spp.’s population dynamics and quantify its carrying capacity. ETHANOLIC PLANT EXTRACTION Plant extraction is a method where undesired parts of a plant sample, including impurities, are removed from the solid plant samples obtained. A standard process of separation is by mixing the solid sample with a liquid extraction solvent. This method will facilitate the separation because the desired product will mix with the solvent and the unwanted solid parts can be easily separated either through filtration, centrifugation, or it can be done mechanically. The purity of the yielding extract will be defined by the quality of the solvent used, the quality of the plant sample itself, the equipment used, and the techniques used by the experimenter. Proper techniques should be used in order to maximize the yield and ensure purity. Extraction may also be done through alcohol extraction. It is a process where the solvent used is alcoholic, and the most popular solvents for this type of extraction are ethanol, methanol, dichloromethane, chloroform, etc. The plant sample (usually dried and in powder form) will be mixed with water and placed in an extraction flask. The solvent is then added, and slight agitation is done to avoid the forming of emulsions. Afterward, the organic layer is separated from the inorganic layer containing the undesired parts of the plant being extracted. The organic layer will be decanted and left to stand so the solvent will evaporate, leaving the desired extract. AGAR WELL DIFFUSION ASSAY Well assays are also an effective way to test for QSI activity on microorganisms. In this method, instead of treated discs, agar wells are created, and the solution being tested will be delivered into the well. In a study done by Guzman and Padilla, the production of the pigment violacein by C. violaceum through quorum sensing was tested. The results were an observation of an opaque halo around the well, indicating a QSI effect of the concerned extract to C. violaceum. In addition, they tested for the inhibitory growth by the same plant extracts, and results show that growth inhibition occurred in wells tested with T. scandens, A. moluccana, and P. guajava. The exact observations were: a transparent halo around the well followed by an opaque halo. This translates to an inhibitory effect close to the extract and a strong anti-quorum sensing activity on the outer sides. The diffusion of the plant extracts along with the observed results showed that the QSI effect is concentration-dependent. DISC ASSAY Antibiosis is a phenomenon commonly tested in the laboratory. The antibiotic capabilities of solutions are usually tested by performing disc assays, a method where small discs are placed in Petri plates inoculated with microorganisms. Varying methods of performing disc assays are accepted in practice. They differ in the way they prepare a microbial culture where the solutions will be tested. Some methods require growing a culture overnight on plates using spreading techniques to have a ready lawn of microorganisms for testing. After the lawn is ready, the discs will be soaked in the solution to be tested and placed in areas of the plates. This method aims to test for the microbicidal effects of the solution. Another method requires the inoculation of the culture onto the agar itself for the growth of surface to sub-surface microorganisms. After the inoculated agar has hardened, the discs will be applied prior to incubation. This method will test for the antibacterial properties of the concerned solution where zones of inhibition would mean that the solution killed the cells present in the area and prevented the growth of colonies around that area. Disc assays may also be applied to test for QSI effects among microorganisms. Microorganisms are subjected conditions where normally a QS event will take place. To test for the QSI activity of samples, and in this study, plants, the experiment could include disc assays to introduce plant extracts in a controlled manner to see if there will be significant effects for QSI activity. QSI SCREENING Controlling infectious diseases is considered a big problem nowadays. Bacterial tolerance and resistance to methods killing them are present, but they keep evolving to combat them to live. The discovery of Quorum Sensing among bacteria has given us an opportunity for another method of control of antimicrobial treatments. Virulence and biofilm formation is highly dependent on bacterial quorum sensing. McLean, Pearson, and Fuqua developed a screening protocol in 2004 based on pigmentation inhibition through QSI. They used soft agar overlays to screen for the presence of potential quorum sensing inhibition by bacteria. The test organisms are streaked onto the appropriate agar medium and were incubated overnight. For plant samples, an additional step of covering the agar with an overlay of soft agar containing an inoculum of either Pseudomonas aureofaciens or C. violaceum. The lack of pigment production in the indicator culture indicates quorum sensing inhibition. The growth inhibition of the indicator culture suggests antibiotic production. Another study by Rasmussen and his colleagues (2005) developed a few ways to screen for quorum sensing inhibitor selectors which identified new quorum-sensing inhibitors among natural and synthetic compound collections. In their study, they found that Allium sativum (garlic) extract and 4-nitro-pyridine-N-oxide (4-NPO) were the most active among others for QSI selectors. They conducted a GeneChip transcriptome analysis, and from that, they found out that garlic extract and 4-NPO had quorum sensing-controlled virulence gene specificity in P. aeruginosa. The QSI selectors significantly reduced P. aeruginosa biofilm tolerance to the antibiotic tobramycin and virulence in Caenorhabditis elegans. They created two general types of QSI selector (QSIS) systems. The first basic design comprised a gene encoding a lethal protein which causes growth inhibition and cell death fused to a quorum sensing-controlled promoter. To test this system, they cast QSIS bacteria with AHL signals in agar plates and applied the test samples in the wells of the agar. This created a concentration gradient making it easy to test for concentration-dependent tests. This first system was unable to grow in the presence of AHL molecules without a high concentration of a nontoxic QSI compound. The second system was based on the P. aeruginosa quorum-sensing systems. It employed an antibiotic resistance gene controlled by a repressor gene in which expression was controlled by a quorum sensing-regulated promoter. In the presence of AHL, production of the repressor quenched the expression of the antibiotic resistance gene, causing the growth inhibition with the presence of a certain antibiotic. However, the downregulation of the repressor enables the growth of the bacteria when the QSI compound is presen...