PHILIPPINE ETHNOBOTANICALS DOWNREGULATE lasR EXPRESSION LINKED TO QUORUM SENSING-MEDIATED BIOFILM FORMATION IN Pseudomonas aeruginosa

The prevalence of antibacterial resistance has brought about a growing surge to develop novel approaches to control infectious diseases. Targeting Quorum-sensing (QS) controlled virulence factors in bacteria has indicated a promising strategy for antipathogenic drugs. Extracts of Philippine Ilongot-Egongot ethnobotanicals Stachytarpeta jamaicensis, Adenanthera intermedia, Mikania micrantha, Hyptis suaveolens, Premna odorata, Cymbopogan winterianus, Phyllanthus urinaria, Dillenia philippinensis, Hydrocotyle vulgaris, Senna alata, Urena lobata, Ceiba pentandra, Ficus sp., Eleusine indica, Diplazium esculentum and Talahib (no known scientific name) were screened using microtiter plate biofilm formation assay for their QS inhibition activity against biofilm formation in Pseudomonas aeruginosa clinical isolate and Pseudomonas aeruginosa PNCM 1335. Extracts of M. micrantha, H. suaveolens flowers, H. vulgaris, A. intermedia, E. indica leaves, D. esculentum and Talahib have inhibitory effect on P. aeruginosa clinical isolate biofilm formation. Decrease in biofilm formation was shown in extracts of S. jamaicensis., M. micrantha, H. suaveolens, H. vulgaris, U. lobata, C. pentandra, A. intermedia, E. indica, D. esculentum and Talahib. against P. aeruginosa PNCM 1335. RNA extracts of M. micrantha, Talahib and A. intermedia exhibited downregulation of lasR in both test bacteria using qRTPCR analysis through absolute quantification. The prospects of these ethnobotanicals to inhibit bacterial virulence avoiding antibiotic resistance is presented in this paper.


INTRODUCTION
The continuous emergence of pathogenic diseases has brought about indiscriminate use of antibiotics. This practice resulted to the development of antibiotic resistance and is now a global threat to public health (Kalia et al., 2007). With the prevalent progression of antibiotic-resistance in bacteria, interest for novel approaches to limit infectious diseases is greatly increasing. Pseudomonas aeruginosa is a multi-drug resistant (MDR) human pathogen and known for their biofilm growth making it difficult to eliminate (Driscoll et al., 2007). Biofilm formation provides great advantage to P. aeruginosa where it can degrade host tissues with proteases and toxins, while being spared from antibiotic attack . Its flagellar motility as well as type IV pilimediated twitching motility has been shown to be essential for surface attachment and colony formation in biofilms. More importantly, however, is the fact that cell-to-cell communication, or quorum-sensing (QS), is critically involved in biofilm formation, particularly in the development of its typical threedimensional architecture (Antunes et al., 2010). A number of its genes and proteins are under QS regulation, suggesting that regulation of gene expression in populations is material to its successful pathogenesis in plant and animal hosts (Bauer & Mathesius, 2004). Hence, an approach referred to as the anti-pathogenic drug principle  restricts this QS-controlled virulence and pathogenesis in bacteria in the hope that invading bacteria will fail to synchronize its activities, and prevent establishment in the host (Rasmussen & Givskov, 2006). Disabling quorumsensing systems in bacteria has become the new focal point in developing strategies to intercept bacterial pathogenicity (O'Loughlin et al., 2013). Current search for new antimicrobials and anti-virulence agents is focused on natural products, specifically, plant-derived compounds. To this day, these compounds have provided the platform for developing new sets of antipathogenic drugs. The Philippines, aside from having an immense plant diversity, hosts a number of diverse ethnic communities that have depended mostly on plants and other natural products to treat diseases (Sia et al., 2002). Among these are the plants utilized by the members of the ethnic community of Ilongot-Egongots of Maria Aurora in the province of Aurora, Philippines. A recent survey of their traditional medicine revealed a varied and huge selection of ethnobotanicals for the treatment of many diseases and ailments (Balberona et al., 2018). Their novelty has attracted researches seeking for agents that may possess quorum sensing inhibiting (QSI) compounds, and provide additional validation of the traditional medicines (Adonizio et al., 2006). Phytochemicals mimic signals in bacteria and hence, may confuse their quorum sensing regulation (Bauer & Mathesius, 2004). The potential of anti-QS compounds has paved for systematic evaluation for these agents (Adonizio, 2008). While there are worldwide ethnobotanical explorations, Philippine ethnobotanical screenings on pharmacological activities are still quite limited. This paper presents the QSI activity of Philippine Ilongot-Eǵongot ethnobotanicals against a QS-controlled virulence factor, biofilm formation, in the bioreporter bacterial strain Pseudomonas aeruginosa up to the molecular level through the quantification of the expression of lasR, a QS-linked gene responsible for many virulence factors including biofilm formation.

Plant Sample Collection and Ethanol Extraction
Through the permission of the tribal chieftains of the Ilongot-Eǵongot community of Maria Aurora, Aurora, Philippines, plant samples were handpicked then placed in clean, properly labeled sacks, sealed and then transported to the laboratory for processing. The plants tested were: Stachytarpeta jamaicensis (Luzviminda) (leaves), Adenanthera intermedia (Kares) (leaves and seeds), Mikania micrantha (Ola-ola) (leaves), Hyptis suaveolens (Ambabangot) (flowers and leaves), Premna odorata (Asédaong) intermedia exhibited downregulation of lasR in both test bacteria using qRT-PCR analysis through absolute quantification. The prospects of these ethnobotanicals to inhibit bacterial virulence avoiding antibiotic resistance is presented in this paper.
The protocols of Tan et al., (2013) and Srisawat (2007) as followed by Velasco et al., (2020) were used for ethanol extraction with modifications. The plant samples were cleaned using distilled water and 70% (v/v) ethanol then air-dried. Fifty (50) grams of each ground plant sample was soaked in 500 ml of 80% ethanol for 72 hours. This was filtered and the solvent removed using a rotary evaporator. The resulting extracts were stored in sterile bottles at temperatures between 0-5°C. Sterilization of the The extracts was done through centrifugation at 10,000 x g for 30 minutes, then membrane filtration (Acrodisc 25mm Syringe Filter) with a pore diameter of 0.45 μm pore size. The sterile extracts were stored at 2-8°C. The sterility of the extracts was monitored by inoculating 100 μl in brain heart infusion agar (BHIA).

Preparation for Bacterial Culture
Brain-heart infusion broth (BHIB) and agar (BHIA) were used to revive and maintain cultures of P. aeruginosa. Maintenance in special culture media was done as necessary. A reference strain, P. aeruginosa PNCM 1335 and a clinical isolate obtained from the University Medical Center, De La Salle Health Sciences Institute, Dasmariñas, Cavite, Philippines were used as test bacteria.

Antibacterial Assay of Plant Extracts Against P. aeruginosa
The protocol of Rezai et al. (2011) was used with some modifications. Colonies of P. aeruginosa from each strain cultured for 16-18 hours in BHIA were transferred to sterile distilled water, the turbidity was adjusted to McFarland 0.5 standard (~ 1.5 x 108 CFU/mL). Mueller Hinton Agar (MHA) plates were inoculated and streaked in three different directions over the surface of the agar to ensure the even distribution. On empty, sterile petri plates, 20 μl of each extract was pipetted onto 6-mm sterile blank antibiotic discs and was allowed to stand for a few minutes to eliminate excess liquids. Using sterile forceps, infused discs were then transferred carefully and equidistant to each other onto 15-mm MHA previously inoculated with P. aeruginosa. Norfloxacin and sterile distilled water served as positive and negative control; Triplicate plates were prepared for all of the treatments. After 24 to 48 hrs period of incubation, the antibacterial activity of the plant extracts was noted for the appearance of zone of inhibition. Plant extracts should not exhibit zone of inhibition which was required for accuracy of the subsequent assay to rule out antibacterial-mediated decrease in virulence factor production Velasco et al., 2020).

Microtitre Plate Biofilm Formation Assay
The effect of plant extracts on the attachment phase of biofilm formation was measured by using a microtitre plate assay. 180 μl of overnight cultures of P. aeruginosa were transferred to wells in the plates added with 20 μl of each plant extract. This was incubated at 30 C for 40 hours without shaking. To remove planktonic cells, the plates were rinsed with sterile distilled water and air dried for 45 minutes. For staining, 150 μl of 1% crystal violet solution in water for 45 minutes was used Velasco et al., 2020). For quantification of biofilm, 200 μl of 95% ethanol was added to destain the wells. 100 μl from each well was then transferred to a new microtiter plate and the OD level was measured at 595 nm (Djordjevic et al., 2002). Presence and/or absence of growth inhibition was noted in biofilms and was then quantified using UV-visible Spectrophotometer (Biotek Instruments, Inc., USA) (Judan Cruz, 2018; Velasco et al., 2020).

lasR Gene Expression
Four plant extracts that showed the lowest vallues in biofilm formation in the QSI Assay were chosen for the quantification of lasR expression.

RNA Extraction
RNA extraction was done using RNeasy Minikit (Qiagen, GmbH, Germany). For each sample, 25-50 mg acid-washed glass beads (150-600 μm diameter) were weighed in a 2ml safe-lock tubes. Bacteria were collected by centrifugation at 5000 x g for five minutes at 4°C. The supernatant was decanted and aspirated to ensure removal of remaining media. Buffer RLT was added (350 μl for <5 x 108 and 700 μl for 5 x 108 -1 x 109 number of bacteria). The suspension was transferred into the 2ml safe-lock tube containing the acid-washed beads. Cells were disrupted in the TissueLyser for five minutes at maximum speed. The suspension was centrifuged for 10 seconds at maximum speed. The supernatant was transferred into a new tube and the volume of the sample was determined. An equal volume of 70% ethanol was added and mixed by pipetting. Up to 700 μl lysate was transferred to a spin column placed in a 2ml collection tube and was centrifuged for 15 seconds at ≥8000 x g. Flow-through tube was discarded. 700 μl Buffer RW1 was added to the spin column. With lid closed gently, the spin column was centrifuged for 15 seconds at ≥8000 x g to wash the spin column membrane. The spin column was placed in a new 2ml collection tube with the flow-through. Lids were closed gently and centrifuged at full speed for one minute. Spin column was then placed in a new 1.5ml collection tube. 30-50 μl RNase-free water was added directly to the spin column membrane and was centrifuged for one minute at ≥8000 x g to elute the RNA. This method followed the RNeasy Minikit protocol (Qiagen, GmbH, Germany).

qRT-PCR Analysis through Absolute Quantification
The expression of lasR in both the test bacteria was determined to evaluate QSI activity through qRT-PCR analysis. The specific primers were: lasR (F) 5' AAGTGGAAAATTGGAGTGGAG 3' and lasR (R) 5' GTAGTTGCCGACGACGATGAAG 3' (Sabharwal et al., 2014). An internal standard 16S rRNA was used with the following primers: 16S rRNA (F) 5' AGAGTRTGATCMTYGCTWAC 3' and 16S rRNA (R) 5' CGYTAMCTTWTTACGRCT 3' (Tripathi et. al., 2013). The qRT-PCR program is as follows: incubation at 42°C for 5 min for reverse transcription; 1 cycle at 95°C for 2 min; then 45 cycles at 94°C for 20 s, 60°C for 20 s and 72°C for 50 s (Wada et al., 2009). KAPA One Step RT-PCR kit (KAPA Biosystems) was used for amplifications consisting of a mixture of 2.4 HPLC water, 5.0 μl KAPA Universal Mix, 0.2 μl dUTP, 0.5 μl of reconstituted forward and reverse primers, 0.2 μl RT Mix, and 1.0 μl RNA template. Several concentrations of RNA with 16S were used as internal control to quantify RNA transcript levels. Absolute quantification of the amplified transcripts was done through Bio-Rad CFX96TM Real-Time System Thermal Cycler, which plots a standard curve from where critical threshold (Ct) values are derived. Through this, the concentration of its PCR signal (Cq) is quantified into this standard curve (Illumina Inc., 2010).

Statistical Analysis
The non-parametric Mann-Whitney U Test with 0.05 level of significance using SPSS 13.0 program was used for the analysis of quantified biofilm as affected by the plant extracts. ct values was determined using qPCR delta delta ct (ddCt) method. Kruskal-Wallis test (non-parametric ANOVA) where means between the control and experimental set-up were compared and the significance determined if the F-values was greater than the F-crit at 0.05 level of significance was used. Statistical analysis of the critical threshold value was done through ANOVA (Velasco et al., 2020).

Antibacterial Activity of Plant Extracts Against P. aeruginosa
Three (3) ethanolic extracts (A. intermedia seeds, C. winterianus leaves, H. suaveolens leaves) have antibacterial activity against the clinical isolate and two ethanolic extracts (A. intermedia seeds; C. winterianus leaves) have antibacterial activity against the reference strain P. aeruginosa PNCM 1335. Sixteen (16) ethanolic extracts tested in clinical isolate while seventeen (17) extracts tested in reference strain did not exhibit antibacterial activity making them qualified for the biofilm virulence assay. Results are shown in Table 1.

DISCUSSION
QS-linked genes often control the production of virulence and gene products essential for bacterial host connections (Pirhonen et al., 1993;Parsek & Greenberg, 2000;Pearson et al., 2000). P. aeruginosa QS system consists of a transcriptional activator, lasR or rhlR, and an autoinducer synthase, LasI or RhlI which manages the autoinducer PAI-1, N-(3-oxododecanoyl)-L-homoserine lactone; , RhlI on the other hand, controls the synthesis of the autoinducer PAI-2, N-butyryl-L-homoserine lactone (Pesci et al., 1999). Blocking the path to the LasR receptor may have an effect on virulence productions synthesized by other signal receptors due to their overlapping mechanics. As such in the case of P. aeruginosa, QS molecules C4-HSL and 3-oxo-C12-HSL, synthesized by LasI and RhlI, respectively, are identified by their corresponding receptors, LasR and RhlR, when threshold quorum concentration is attained. The las and rhl systems in combination, with two interconnected acyl-HSL signal-receptor pairs, 3 oxododecanoyl-HSL-LasR and butanoyl-HSL-RhlR, control as much as 353 genes that comprise around six percent of the P. aeruginosa genome (Tay & Yew, 2013). These virulence system receptors can be blocked from synthesizing signals that mimic their complexes thereby confusing the bacterial QS, as shown when these plant extracts were applied (Livorsi et al., 2011). lasR as affected by the plant extracts was significantly downregulated. This antagonistic effect may mean that the compounds in the plant extracts may have blocked the lasR pathway which control several QS virulence factors, particularly biofilm formation. As a consequence, blocking the LasR receptor may mean decrease in virulence factor production, e.g. biofilms. In other QS researches, specifically in P. aeruginosa biofilms, strains showing deficient production of the las signal molecule, 3O-C12-HSL, produced significantly reduced biofilms without their typical three-dimensional structure (Davies et al., 1998), confirming that the las QS system is critical in biofilm formation (De Kievit et al., 2001). LasR coordinates pathogenicity in P. aeruginosa  and activates virulence genes lasB, lasA, apr, and toxA (Gambello et al., 1993;(Pearson et al.,1997;). Therefore, if QSI compounds can block las expression, then all of the other QS-linked genes would be regulated as well, especially those involved in biofilm formation (De Kievit et al., 2001). It can be observed that the plant extracts showed more antivirulence activity in biofilm formation in the reference strain P. aeruginosa PNCM 1335 than the clinical isolate. Mutations in lasR have been reported from clinical isolates of P. aeruginosa in cystic fibrosis patients (Hoffman et al., 2009) suggesting more possible complications in controlling its virulence system compared to the wildtype P. aeruginosa in which additional required autoinducer is normally provided by the las system (Papenfort & Bassler, 2016). Although no confirmation was done, it is hypothesized that the plant extract H. vulgaris downregulates another gene/s involved in biofilm formation, hence, its slightly upregulated expression. P. aeruginosa, possess numerous virulence systems aside from las, which in turn, could have been the one affected by the plant extract. These systems are rhl, qsc and pqs (Gilbert et al., 2009;Chugani et al., 2001;Pesci et al., 1999;Dubern & Diggle, 2008;Tay & Yew, 2013). Possible targeting of these other systems by the plant extract might have occurred by blocking their receptors. QS in P. aeruginosa consists of an intricately organized hierarchical system, where substantial communications between pathways often lead to an organization of a notable number of genes (Wilder et al., 2011). As such, H. vulgaris may have affected other virulence factors such as pyocyanin production, DNAse and swarming motility. This result may also mean that other bacterial pathogens in which QS is controlled by lasR may benefit from the potential antagonistic or inhibitory effect of the plant extracts.
The plants that showed QSI activities are known to possess phytochemicals with known QSI activities such as phenols, tannins flavonoids, alkaloids and other secondary metabolites such as saponins (da Gama et al., 2014). It has been found that compounds from plants have already proven promising candidates for inhibiting QSI Pawar & Arumugan, 2011;Tan et al., 2014;Zhou et al., 2013;Husain et al., 2013). Understanding QS gene systems crucial for virulence factors will present better insights of the connected genetic and phenotypic aspects, particularly biofilm development, and this information may ultimately pave the discovery of novel approaches for preventing and controlling complex and resistant biofilms. Since the discovery of QSI, plants have been a basis of medicines and presently continues to contribute significantly to pharmaceutical development (Cragg et al., 1997 as cited by Adonizio et al., 2006) and ironically, only around 5-15% of the higher plants have been scientifcally explored for their bioactive molecules (Pieters & Vlietinck, 2005). Likewise, research on the QSI activities of herbal plants is limited and it is highly probable that with in-depth studies, antimicrobial efficiency mediated by QS control will be thoroughly deliberated (Adonizio et al., 2008). The ethnobotanical extracts found to have QSI activity against a human pathogen multi-drug resistant P. aeruginosa can be one of the possible means of treating infections and alleviating the emerging antibiotic-resistance of many bacterial species of today. In P. aeruginosa, a switch to the biofilm development is connected to increased antibacterial resistance and thereby creates a distinctly more severe infection . Hence, it is now becoming more crucial to discover QSI compounds to control pathogenicity. Strategies and approaches designed to block biofilm formation in clinical and industrial cases is currently the target of researches on drug development. The confirmation of QSI of these plants is one of these strategies. The reduction of lasR expression and its effect on biofilm formation provide some understanding on how these can be used in the future to combat P. aeruginosa, a multi-drug resistant (MDR) human pathogen, and other bacterial infections.

CONCLUSION
The ethnobotanical extracts of the Ilongot-Eǵongot community showed antibacterial as well as QSI activity through inhibition of the biofilm formation in P. aeruginosa which was molecularly confirmed through gene expression analysis of lasR. This indicates the prospects of these ethnobotanicals for therapeutic approach to control bacterial virulence without developing resistance. It is recommended for future researches to test the extracts' QSI activity against other the P. aeruginosa virulence system rhl and analyze its coregulation with the other QS systems.