Anti-quorum Sensing Potential of Ageratum Conyzoides L. (Asteraceae) Extracts


 Background: Pseudomonas aeruginosa causes infections in human particularly immunocompromised patients with cystic fibrosis, severe burns and HIV, resulting in high morbidity and mortality. The pathogenic bacteria P aeruginosa produces virulence factors regulated by the mechanism called quorum sensing system. Objective: The aim of this study was to assess the anti-quorum sensing activity of Ageratum conyzoides extracts Method: Chromobacterium violaceum reporter strain CV026 was used to highlight any interference with bacterium QS and strains derived from P. aeruginosa PAO1 were used to reveal any interference with the expression of quorum sensing genes, and to assess any impact of extract on the kinetics of the production of pyocyanin, elastases and biofilm formation. Results: Hydro-methanolic extract at the sub-inhibitory concentration of 100 μg/mL reduced quorum sensing virulence factors production such as, pyocyanin, elastases, rhamnolipids and biofilm formation in P. aeruginosa PAO1 after 18 hours monitoring. Extract showed significant inhibition in HSL-mediated violacein production on C. violaceum CV026 after 48 hours monitoring. Biofilm formation was inhibited up to 32%. It affected QS gene expression in PAO1. The regulatory genes lasR / rhlR and the lasI synthases were most affected. At 8hours, hydro-methanolic extract reduced both QS gene to more than 30% (lasI/lasR and rhlI/R respectively 33.8% /30.2% and 36% /33.2%). RhlA and lasB genes have been relatively affected (13.4% and 28.9%). After 18 h, this extract reduced significantly the expression of regulatory 30 genes lasR (31%) and rhlR (39.6%) although synthases genes seemed to be less affected (lasI/21.2% and rhlI/11.6%). A limited impact was observed on the downstream genes (lasB /20.0% and rhlA /15.3%). No negative impact was observed on CV026 and PAO1 growth and cell viability. Our study also showed that A. conyzoides having ample amount of phenolics, flavonoids and triterpenoids. This phytochemical content could be one of the factors for showing anti quorum potential. Conclusion: Results indicate that hydro methanol 80 % extract from A. conyzoides could be a source of potential QS inhibition compounds.

biofilm formation in P. aeruginosa PAO1 after 18 hours monitoring. Extract showed significant 24 inhibition in HSL-mediated violacein production on C. violaceum CV026 after 48 hours 25 monitoring. Biofilm formation was inhibited up to 32%. It affected QS gene expression in 26 PAO1. The regulatory genes lasR / rhlR and the lasI synthases were most affected. At 8hours, 27 hydro-methanolic extract reduced both QS gene to more than 30% (lasI/lasR and rhlI/R 28 respectively 33.8% /30.2% and 36% /33.2%). RhlA and lasB genes have been relatively affected 29 (13.4% and 28.9%). After 18 h, this extract reduced significantly the expression of regulatory 30 genes lasR (31%) and rhlR (39.6%) although synthases genes seemed to be less affected 31 (lasI/21.2% and rhlI/11.6%). A limited impact was observed on the downstream genes (lasB 32 /20.0% and rhlA /15.3%). No negative impact was observed on CV026 and PAO1 growth and 33 cell viability. Our study also showed that A. conyzoides having ample amount of phenolics, 34 flavonoids and triterpenoids. This phytochemical content could be one of the factors for 35 showing anti quorum potential. 36 Introduction 41 Conventional infectious diseases treatment used antibiotics for killing or inhibiting the bacteria 42 growth [1]. A major consequence was the development of drug resistance pathogens and the 43 emergence of multi-resistant strains resulting from antibiotics overuse [2]. It has become 44 essential to search for new effective antibacterial molecules. One of promoting strategies was 45 the founding of compounds reducing the virulence of bacteria without killing them in quorum quorum sensing pathway. Therefore, the present study, was initiated to investigate the anti-71 quorum sensing properties of hydro methanolic extract of A. conyzoides extracts. 72 Material  The minimal inhibitory concentrations (MIC)of the extract were determined using 96 well 104 microplates [18]. An overnight bacterial culture was diluted with LB broth to obtain a starting 105 inoculum (10 6 CFU/mL). Each inoculum (180 µL) was incubated with a serial concentration of 106 extracts ranged from 5 mg/mL to 0.049 mg/mL. Bacteria growth were studied by using p-107 iodonitroterazolium coloration and the MIC and MBC were determined. 108 Growth kinetics of C. violaceum CV026 and P. aeruginosa PAO1 109 The growth kinetics were assessed for 48 hours for CVO26 and 18 hours for PAO1. At regular 110 time intervals (6h for CVO26, and 3h for PAO1), a bacterial colony count was performed to 111 evaluate the impact of the plant extract on bacterial viability. The number of bacteria at baseline 112 was 10 6 CFU m/L. 113

Biofilm formation 130
Biofilm formation in P. aeruginosa PAO1 was performed according to protocol described by 131 [22]. After 18 hours growth, the culture of PAO1 was washed three times, then fixed with 132 methanol 99% for 15 min. Crystal violet (0.1% in water) was added to each well after removing 133 of methanol and incubated for 30 min. Biofilm were stained coloured with crystal violet and 134 dissolved in acetic acid (33%) then the absorbance was read at 590 nm. 135 Genes Expression and β-Galactosidase assay

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All the reporter strains of PAO1 were incubated in LB-MOPS-Carbenicillin for 8hours and 137 18hours (50 µL, 37 ℃, 175 rpm) supplemented with samples (Extract and DMSO). After 138 incubation, the absorbances were read at 600 nm. Then samples were used to perform the β-139 galactosidase assay with O-nitrophenyl-β-D-galactopyranoside. β-Galactosidase activity has 140 been expressed in Miller units [23]. 141 Determination of total polyphenol, total flavonoid and total triterpenoid content 142 Plant extract (10mg/mL in methanol) was use for the quantitative tests. Total polyphenolic 143 contents in extract were determined by spectrometric method as described by Singleton and al 144 [24], total flavonoids as described by Arvouet-Grand and al [25] and total terpenoids as 145 described by Fan and He [26] was carried out. total polyphenolics were expressed as mg/g gallic 146 acid equivalents (GAE). Total flavonoid as mg/g quercetin equivalent (QE) and total 147 triterpenoids as mg/g ursolic acid equivalent (UAE). 148

149
Experiment was performed in triplicate and data were expressed as mean ± SD. GraphPad prism 150 Software was used for statistical analysis (GraphPad software Inc., San Diego, CA, USA) the 151 One-way or two-way ANOVA followed by the Tukey or Bonferonni test on GraphPad at the 152 value ≤ 0.05 was considered significant.  Effect on violacein kinetic production 183 Significant inhibition of HHL-induced violacein production was recorded during the 184 exponential phase ranging from 3% (6h) to 38% (24h) (figure 1 curve). In the stationary phase, 185 there was a more or less constant reduction running from 35% to 32% for 30h-48h period. 186 Highest rate was reached after 12h (42%). The results showed the time-dependent inhibitory 187 effect of hydro-methanolic 80% extract on violacein kinetics production by C. violaceum violacein production (data not shown). This was an indication that the extract did not contain 190 any HHL-type compounds but contained some compounds with QS system deactivating ability. 191 On this basis, A. conyzoides extract has been further investigates for its impact on the QS 192 mechanism in P. aeruginosa PAO1. 193 Effect on pyocyanin kinetic production 194 As shown in figure 2 (curve), a negative impact was found on the time-dependent kinetics of 195 pyocyanin production. A significant reduction at the exponential phase of PAO1 growth was 196 induced by the extract. Thereafter, a lag phase in pyocyanin production was observed (about 197 3h). Pyocyanin production reduction was appeared at 9hour (exponential phase). The reported 198 reduction rates were 45% and 42% at 9 hours and 12 hours respectively. In the stationary phase, 199 there was a more or less constant reduction running from 40%, to 37% at 15h and 18h. 200 Maximum reduction (45%) was reached after 9 hours of growth. The results clearly showed 201 that pyocyanin production inhibition occurred during the exponential phase. The hydro-202 methanolic extract was further tested for its impact on elastase production and biofilm 203 formation. 204 Effect on elastase production 205 The effect of methanolic 80 % extract (100 µg/mL) on the elastase production in PAO1 strain 206 has been studied after 8h and 18h of growth according to previously described procedures in 207 method section. Elastase amounts time depending for this experiment were given in figure 3a. 208 As shown in figure 3a, no significant negative effect was observed on PAO1 growth at 8hours 209 and 18 hours. However, hydro-methanolic extract decreased significantly (P<0.05) the activity 210 of elastases as salicylic acid. At 8 hours, the effect of extract (35% inhibition) was relatively 211 greater than salicylic acid (31% inhibition). In contrast after 18 hours, a low activity of the 212 extract (31% inhibition) was observed compared to salicylic acid (37% inhibition), but significant compared to DMSO. In both time points, no significant difference was found 214 between the activity of extract and that of salicylic acid. 215 Effect on rhamnolipids production 216 Figure 3b shows the effect of the extract on the production of rhamnolipids. After 8h as at 18h, 217 the amount of rhamnolipids was significantly reduced by 24% and 18% respectively compared 218 to DMSO (negative control). After 8h of growth (exponential phase, figure 2 histogram) the 219 effect of the extract was slightly increased over salicylic acid (21%) before settling at 18% 220 (against 24% for salicylic acid). 221 Anti-biofilm formation in P aeruginosa PAO1 222 Given the link between QS and biofilm formation, methanolic 80% extract from A. conyzoides 223 was assessed against QS-mediated biofilm formation by P. aeruginosa PAO1 (Figure 4). 224 Biofilm is a matrix of polysaccharides which protect P. aeruginosa from environment. As 225 shown in figure 4, the number of cellular polysaccharides produced by P. aeruginosa PA01 226 over 18 h growth was reduced by the hydro-methanolic extract (100µg/mL) and salicylic acid. 227 A moderate anti-biofilm activity was observed for extract (32%) and salicylic acid (44%). As 228 shown in figure 1 and 2, the extract had no significant impact on the P. aeruginosa PAO1 229 growth. Based on these preliminary screenings, the effects of hydro-methanolic on QS 230 regulation in PAO1 was further analysed. 231 Effect on QS genes in P. aeruginosa PAO1 232 In order to highlight any interference with the QS genes expression in P. aeruginosa PAO1, it 233 was followed the transcription rate over 8h and 18h of growth ( Table 1). The expression of 234 synthetases genes (lasI and rhlI), regulatory genes (lasR and rhlR) and genes controlling 235 virulence factors (lasB and rhlA) was investigated. Similarly, the expression of the aceA gene positive control. With a final concentration of 100 μg/mL, no negative impact on PAO1end-238 point cell density was recorded but most of the QS genes were affected by extract when 239 compared with salicylic acid and DMSO treatment ( Table 1). As shown in this table, at 8h,  240 hydro-methanolic 80% extract reduced both QS gene to more than 30% (lasI/lasR and rhlI/R 241 respectively 33.8%/30.2% and 36% /33.2%). RhlA and lasB genes have been relatively affected 242 (13.4% and 28.9%). After 18 h, hydro-methanolic 80% extract significantly reduced the 243 expression of regulatory genes lasR (31%) and rhlR (39.6%) although synthetases genes 244 seemed to be less affected (lasI/21.2% and rhlI/11.6%). A limited impact was observed on the 245 downstream genes (lasB /20.0% and rhlA /15.3%). In general, for most of QS-related genes (i.e 246 RhlI, rhlR and lasI and lasB) significant decreases were recorded after 8h. As shown in figure  247 2, no negative impact was observed on PAO1 viability; suggesting that the decrease in QS genes 248 expression, were not due to a drop in cell viability. Moreover, it is interesting to point out that 249 plant extract has no negative effect on aceA gene transcription. This is proof of its specific 250 action on rhlI/R, lasI/B and las genes without disturbing the transcriptional machinery of PAO1. medicines [27,28]. A great deal of plant-directed research has been carried out to discover 260 compounds to control multidrug resistant pathogens [29,30]. This study is the first in vitro 261 investigation on the anti-QS properties of A. conyzoides from Burkina Faso. Susceptibility test 262 of hydro methanolic 80% extract showed an inhibition of quorum sensing rather than anti-263 bacterial effect. 264 Quorum sensing is a complex regulatory network that modulates the expression of multiple 265 virulence factors such elastase, pyocyanin and rhamnolipids [31,32]. C. violaceum CV026, has 266 a low human health impact, but widely used as a reporter strain in QS screening [33]. A. 267 conyzoides extract were found to affect significantly (time dependent) HHL-induced violacein 268 production by CV026, and inhibits the production of pyocyanin. The extract also decreased the 269 production of elastases as much as biofilm formation by wild type strain PAO1. Virulence 270 factors and the biofilm formation examined in this study are under QS control [34] . As observed 271 with C. violaceum CVO26, methanolic 80% extract had no negative impact on P. aeruginosa 272 PAO1 growth and cell viability. Thus, at a concentration (100 µg/mL) below the MIC (5 273 mg/mL), no bacteriostatic or bactericidal effects were detected. This observation supports the 274 findings of Chah and al [12] who reported the lack of inhibition of the PAO1 growth by the 275 methanolic extract and those of Odeleye and al [10] whose results indicted a sensitivity of 276 160mg/mL well above the MIC value recorded in this study. 277 In P. aeruginosa, the QS regulates the expression of lasI/R and rhlI/R genes as well as the 278 production of virulence factors such as elastase (lasB), LasA protease (lasA), alkaline protease 279 (aprA), rhamnolipids (rhlA/B) and pyocyanin [35,36]. Methanolic 80% plant extract used in 280 this study showed significant effect on QS genes expression in P. aeruginosa. In general, the 281 regulatory genes lasR / rhlR and the lasI synthase gene were most affected. Indeed. Plant extract 282 caused inhibition of QS-controlled virulence factors genes (rhlA and lasB) which confirm the 283 significant reduction in pyocyanin production, elastases production, rhamnolipids and biofilm 284 formation [35]. The expression of the QS-independent gene, aceA gene (isocitrate lyase gene expression regulator) was analysed. As shown in 1 the A. conyzoides extract has no impact on 286

aceA. 287
Overall, the data indicated that the anti-QS activity of A. conyzoides extract could be caused by 288 the interference with the target genes. The phytochemicals such as polyphenols, flavonoid and 289 triterpenoids are able to inhibit the QS genes expression and the production of virulence factors 290 [37,38]. 291 Altogether, these non-bactericidal anti-virulence properties, the ample amount of phenolic, 292 flavonoid, triterpenoid and the reported antimicrobial activities, provides additional evidence 293 and support the wide anti-infectious use of this plant in traditional medicine [7,16]  Availability of data and materials 312 All data generated or analysed during this study are included in this article. 313

Competing interests 314
The authors declare that they have no competing interests 315 Funding 316 This study was carried out with funding from the "Centre national de l'Information, de     aeruginosa PAO1after 18 hours compared to DMSO 1% used as negative control. Salicylic acid was used as positive control. P value of p<0.05 was considered as significant. *** Data that are statistically different (p<0.05). Figure 1 Effect on bacterial C. violaceum CV026: Left y axis: kinetics growth UFC/ml histogram. Right y axis: Kinetics of violacein production curve graph.  Methanol 80% extract from A. conyzoides reduce elastase (a) and rhamnolipids (b) production in P. aeruginosa PAO1 growth at 8 hours and 18 hours compared to DMSO 1% used as negative control.

Figures
Salicylic acid was used as positive control. P value of p<0.05 Effect of methanol 80% extract from A. conyzoides on Bio lm formation in P. aeruginosa PAO1after 18 hours compared to DMSO 1% used as negative control. Salicylic acid was used as positive control. P value of p<0.05 was considered as signi cant. *** Data that are statistically different (p<0.05).