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  • Evaluation of Antimicrobial Activity and Anti-Quorum Sensing of Rosmarinus Methanol Extract on Pseudomonas aeruginosa
  • Samaneh Ansarinia,1,* Parisa Behshood,2 Elahe Tajbakhsh,3 Faezeh Amiri,4
    1. Master Department of Microbiology, Young Reasearchers and Eliet Club, Shahrekord Branch, Islamic Azad University Shahrekord, Iran
    2. Phd Department of Microbiology, Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University Shahrekord, Iran
    3. Associate Professor, Biology - Microbiology, Basic Biology Sciences,
    4. Biotechnology Bachelor, Naghsh Jahan Isfahan non profit organization


  • Introduction: Pseudomonas aeruginosa is a gram-negative, oxidasepositive, non-fermenting, and aerobic bacterium found on intestinal tissues of healthy individuals, various fluids, and surfaces, especially moist surfaces and even disinfectants. P. aeruginosa is an opportunistic bacterium identified as one of the most critical hospital pathogens in recent years . One of the pathogenic mechanisms of this bacterium is the quorum sensing system and high resistance to most antibiotics. Quorum sensing (QS) is the ability to detect and respond to cell population density by gene regulation. It enables bacteria to restrict the expression of specific genes to the high cell densities, at which the resulting phenotypes will be the most beneficial . Many virulence genes in P. aeruginosa are controlled and expressed by quorum sensing as biofilm growth and proliferation genes, alkaline proteases, pyocyanins, and pivorinins . A biofilm comprises any syntrophic consortiums of microorganisms in which cells stick to each other and a surface. The low densityandlow diffusion power of the matrix polysaccharide existing in the biofilm provides ideal conditions for the aggregation of signals and the induction of pathogenic factors by the coenzyme sensing phenomenon and prepares bacteria to invade the host. The growth of bacteria in biofilms can increase their resistance to the antibiotics. Onthe other hand, the overuse of antibiotics in the treatment of bacterial infections has led to the development of antibiotic-resistant strains . The process of bacterial resistance to chemical antibiotics has limited the physicians to treat some infectious diseases that are often fatal . The study of medicinal plants has become particularly critical worldwide to discover new therapies with fewer side effects and higher economic values. Rosemary extract is a compound with several antimicrobial and antioxidant properties It has been proven that there are numerous antimicrobial compounds, such as phenolic compounds. Today, over 4 billion plants worldwide are used as a source of medicines, and 25% of physicians prescribe normal herbal medicines . Plants have an unlimited ability to synthesize phenolic compounds, their derivatives, and a variety of aromatic compounds. These compounds are secondary metabolites of plants and have therapeutic effects against viruses, bacteria, and fungi . Therefore, it is necessary to investigate the active constituents of medicinal plants in different geographical areas to discover useful antimicrobial agents. Rosmarinus belongs to the mint family (Labiateae, a herb with green, aromatic, and sharp leaves) and has antinociceptive, antioxidant, antimicrobial, and anti-inflammatory effects on laboratory experiments and antimicrobial compounds . It contains phenolic compounds such as carnosol, rosmarinic acid, caffeic acid, flavonoids, including diosmin, luteolin, gencuanine, and monoterpene, such as camphor, cineole, and borneol.
  • Methods: Identification and Collection of Rosmarinus This experimental study was performed from May 2017 to January 2018 in the Microbiology Laboratory of Kashan Azad University. In spring 2017, leaves and branches of Rosmarinus from rangelands of Niassar city of Kashan were harvested and approved by Isfahan Agricultural and Natural Resources Research Center. Preparation of Bacterial Strains In this study, five clinical isolates of P. aeruginosa were isolated from clinical samples referred to Kashan hospitals. Standard bacterium P. aeruginosa (ATCC 1074) was also used. Bacterial samples were cultured in Muller-Hinton broth medium and incubated at 37°C for 24 hours. Subsequently, a few drops of the bacterial suspension were transferred to the Muller Hinton broth medium to achieve a standard McFarland 0.5 turbidity (1.5_108 cfu/mL) . Preparation of Various Dilutions and Evaluation of Antibacterial Activity of the Extract Concentrations of 500, 250, 125, 62.5, 31.25, 15.6, 7.8, and 3.9 mg/ml of extract were prepared. In this study, the antimicrobial activity of the extract was evaluated by diffusion method in wells and microdilution. After preparation of different concentrations of extract, Measurement of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by microtiter plate method . Investigation of the Anti-Quorum Sensing Activity of Ethanol and Methanol Extract of Rosmarinus We examined the anti-biofilm activity, pyocyanin production, and protease enzyme activity of P. aeruginosa to investigate the antimicrobial sensing activity of methanolic extracts of Rosmarinus. Evaluation of Anti-Biofilm Activity of Rosmarinus Methanol Extract The anti-biofilm activity of Rosmarinus methanolic extract was evaluated by crystal violet staining to investigate its anti-quorum sensing activity. Inhibition of Pyocyanin Experiment The 24-hour culture of the bacteria was prepared in the Muller Hinton broth medium and its opacity was set to half the standard McFarland opacity by spectrophotometer. 250 _l of the herbal extract was prepared at a concentration lower than the bacterial growth inhibitory, and 100 _l of microbial suspension was added. The test tube containing microbial suspension without extract was considered as control. The tubes were centrifuged at 8,000 rpm, and the supernatant was transferred to another sterile tube and added to 3 ml of chloroform. The solution was then transferred to the cuvet from the bottom of the tubes (control and extract-treated samples) and read by a spectrophotometer at 690 nm. This experiment was repeated three times, and the mean percentage reduction of pyocyanin production was calculated according to the following formula (15). Percentage reduction of pyocyanin = (Optical Absorption of extract - Treated Sample - Optical Absorption of control sample)/(optical absorption of control sample)_100 Protease Activity Test Protease activity of P. aeruginosa was evaluated in accordance with the Lori method using azocasein . Statistical Analysis The comparison of the means of this study was analyzed by two-wayANOVA and Bonferroni paired t-test using SPSS 17 software. The significance level of the test (P < 0.05) was used to interpret the data.
  • Results: In this study, the strains of P. aeruginosa were identified by hot staining and conventional biochemical tests. All isolates were Gram-negative bacilli, catalase-positive, and oxidase-positive cultured in oxidation-fermentation (OF) medium. These isolates produced pyocyanin were mobile and grew at 42°C. The susceptibility of clinical isolates and standard strains of P. aeruginosa to methanolic extracts of Rosmarinus was investigated by the well diffusion method (Table 1). Table 1 shows the inhibition zone mean of methanolic extract of Rosmarinus plant on different isolates of P. aeruginosa (in mm). As the two-way ANOVA test shows, the size of the inhibition zone diameter is directly proportional to different concentrations (P < 0.001). The minimum inhibitory concentration of methanolic extract of Rosmarinus was 125 mg / ml, and minimum bactericidal concentration was 250 mg/ml. Results of Pyocyanin Pigment Reduction in P. aeruginosa at Different Concentrations of Methanolic Extract of Rosmarinus As presented in Figure 1, we evaluated the efficacy of methanolic extract of Rosmarinus on the reduction of pyocyanin pigment production of different P. aeruginosa isolates. Figure 1 shows a decrease in pyocyanin pigment production in the presence of different concentrations of Rosmarinus methanol on different P. aeruginosa isolates. Twoway analysis of variance showed that the percentage of reduced pigment production of piocyanin was directly correlated with different concentrations of the extract (P < 0.001). In other words, with increasing methanol concentration of Rosmarinus, pyocyanin pigment production can decrease in standard P. aeruginosa and clinical isolates, which means that methanolic extract of Rosmarinus can strongly significantreduce pyocyanin pigment production in Pseudomonas aeruginosa. Results of Percentage Reduction of P.aeruginosa Protease Activity at Different Concentrations of Methanolic Extract of Rosmarinus We assessed the efficiency of methanolic extract of Rosmarinus on reducing the protease production of different isolates of P. aeruginosa used by Lorry method, as presented in Figure 2. Figure 2 shows a decrease in protease production in the presence of different concentrations of methanolic extract of Rosmarinus plant on different isolates of P. aeruginosa. Two-way analysis of variance showed that the percentage of reduced protease production was directly correlated with different concentrations of extract (P< 0.001). In other words, as the concentration of methanolic extract of Rosmarinus increases, the production of protease in standard P. aeruginosa and clinical isolates has decreased, which means that methanolic extract of Rosmarinus has a significant effect in reducing the production of protease in P. aeruginosa. Results of Anti-Biofilm Activity of Methanolic Extract of Rosmarinus In the present study, we examined the anti-biofilm activity of methanolic extract of Rosmarinus via crystal violet staining on P. aeruginosa (Figure 3). Figure 3 shows the reduction of biofilm production in the presence of different concentrations of methanolic extract of Rosmarinus plant on different P. aeruginosa isolates. Two-way analysis of variance showed that the percentage of reduction in biofilm production was directly correlated with different concentrations of extract (P < 0.001). In other words, with increasing concentration of methanolic extract of Rosmarinus, biofilm production in P. aeruginosa decreased, and clinical isolates has decreased, whichmeansthat methanolic extract of Rosmarinus has a significant effect on reducing biofilm production in P. aeruginosa.
  • Conclusion: Mentioning the above bacteria, the present research tended to confirm the efficacy of the methanol extract. The results could verify the ability of Rosmarinus methanol extract to reduce microbial growth, biofilm, elastase, protease, and pyocyanin of P. aeruginosa. Given the high potential of P. aeruginosa in biofilm formation and the microbial and biofilm growth inhibitory activity of Rosmarinus extracts, it can be concluded that Rosmarinus extract can be used in different compounds for the elimination of infection with pathogenic bacteria such as P. aeruginosa. Also, it can be a substitute for chemical drugs to treat infections, although more thorough investigation on all the effects of this plant extract. Currently, one of the major problems in the treatment of infections and the use of antibiotics is the development of antibiotic resistance, which requires special attention for treatment. Since the antibacterial effects of rosemary extract have been verified in various studies on numerous species of bacteria, it can be employed in the treatment of infections caused by resistant bacteria. To sum up, the effects of plant extracts on inhibition of biofilm formation can be attributed to the those of constituents on bacterial growth and ultimately on the reduction of biofilm formation. Therefore, further studies are needed to evaluate the variety and composition of essential oils and extracts of medicinal plants and to compare different herbs in terms of their constituents in indigenous regions and identify the superior breeds.
  • Keywords: Methanol, Rosmarinus, Biofilms, Diffusion, Pseudomonas