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Pseudomonas aeruginosa is one of important opportunistic pathogen. That cases serious infections. It produces many virulence factors. This bacterium usually is resistance against antimicrobial agents.
Objective:The aim of this study was evaluate the effects of sub-MICs of essential oils of Mentha spicata and Cumminum cyminum on alginate production, biofilm formation, swimming, twitching and adhesion in P. aeruginosa 8821M.
Methods: Minimal inhibitory concentrations (MIC) of essential oils of Mentha spicata and Cumminum cyminum were determined by macrodilution method. Alginate production, biofilm formation, swimming, twitching and adhesion in the present of sub-MICs (1/2, 1/4 and 1/8 MIC) of essential oils were determined in mucoid P. aeruginosa 8821M and compared with controls.
Results: The MICs of essential oils against P. aeruginosa for M. spicata and C. cyminum oils were obtained 16 and 32μg/ml respectively. The results show that all oils at 1/2 and 1/4 MICs were significantly reduced all tested virulence factors. At 1/8 MICs, M.spicata had effect just on adhesion but C. cyminum had effect on Alginate production, biofilm formation, swimming and twitching.
Conclusion: This study showed that sub-MIC levels of M. spicata and C. cyminum essential oils affected alginate production, biofilm formation, swimming, twitching and adhesion in P. aeruginosa 8821M and it is probable to use of these medicinal plants for treating.
Keywords: Alginate, Biofilm, Cumminum cyminum, Mentha spicata, Pseudomonas aeruginosa.
The opportunistic pathogen Pseudomonas aeruginosa, a ubiquitous environmental bacterium showing great adaptability and metabolic versatility, is the leading cause of morbidity and premature mortality in patients with cystic fibrosis. Pseudomonas aeruginosa is remarkable in that it can cause both very acute and very chronic infections. Progress in understanding the pathogenesis of acute P. aeruginosa infections has implicated virulence factors. This opportunistic pathogen produces a number of unique virulence factors. Extracellular toxins, proteases, hemolysins, and exopolysaccharides are some of the types of virulence factors that have been implicated in the pathogenicity of P. aeruginosa [1,2]. Its pathological effects are attributed to various characteristics, including the elaboration of many cell-associated virulence/survival factors , such as fimbriation, interaction with host defences and, most importantly, their adhesive and biofilm formation abilities . Treatment with sub-inhibitory concentrations (sub-MIC) of some antimicrobial agents may influence bacterial virulence factors, such as adherence , cell surface hydrophobicity , biofilm formation , sensitivity to oxidative stress and motility . Together with cell-surface structures, the polar flagellum is responsible for one type of motility in aqueous environments. Another cell-surface structure acting as a virulence factor is the type IV fimbria. These mediate adherence to biotic and abiotic surfaces and are responsible for surface translocation or twitching motility . Some plant-derived compounds inhibit peptidoglycan synthesis , damage microbial membrane structures , modify bacterial membrane surface hydrophobicity , and modulate quorum sensing , all of which could influence biofilm formation. Terrestrial plants also support populations of surface-attached bacteria and could potentially produce phytochemicals that attenuate biofilm development through specific mechanisms . However, many plant essential oils, which are mixtures of numerous organic chemicals, contain compounds that inhibit microbial growth . To date, there is no information in the literature concerning the influence of sub-MIC essential oils on P. aeruginosa virulence factors. The present study describes specific inhibition of virulence factors production by activity of Cumminum cyminum and Mentha spicata essential oils against P. aeruginosa.
Materials and Methods
Mucoid P. aeruginosa 8821M was kindly donated by Dr. Isabel Sa-Corria, Instituto Superior Tecnico, Lisboa, Portugal . The strain was maintained in 10% skimmed milk (Difco Laboratories, Ditroit, MI) at -80°C and was subcultured on muller-Hinton agar (Difco Laboratories, Ditroit, MI).
Plants and oil isolation
The plants (Mentha spicata and Cumminum cyminum) were identified and provided by Research Institute of Medicinal Plants (Tehran, Iran). The shadow dried plants were hydro-distilled for 90 minutes in full glass apparatus. The oil was isolated using a Clevenger type apparatus. The extraction was carried out for 2 hours after 4-hours maceration in 500 ml of water. The essential oils were dissolved in dimethylsulfoxide (with volume ratio of 1:1) and sterilized by filtration through a 0.45μm membrane filter.
The oils so extracted were stored in dark glass bottles in a refrigerator until they were used.
Determination of MICs was carried out using a macrobroth dilution method according to the NCCLS procedure. An adjusted inoculum of the test organism was inoculated into Mueller-Hinton broth (Merck) containing twofold dilutions of an initial antibiotic solution so that each well contained approximately 1.0 - 105 CFU/ml. Results were observed after overnight incubation at 37°C. MIC was defined as the lowest concentration that inhibited visible growth .
In the adhesion assay, bacterium was grown overnight (37oC, 150 rpm) in Luria-Bertani (LB) broth (Difco) in the presence or absence of oils at a subinhibitory concentration (1/2, 1/4 and 1/8 MIC).Then bacteria were washed twice in PBS, pH 7.4, by centrifugation for 20 min at 1000 g and resuspended in PBS in a standard inoculum corresponding to approximately 107 CFU/ml. This inoculum concentration was determined by making serial dilutions in PBS and performing colony counts in duplicate on LB agar after overnight incubation. Two hundred microlitres of this inoculum was added to the wells of sterile 96-well polystyrene microtitre plates containing medium with or without oils and incubated for 1 h at 37 °C. After incubation, microtitre plates were rinsed twice with PBS to remove non-adherent bacteria for bacterial quantification. Plates were then air-dried in an inverted position, a solution of 0.1% safranin was added for 30 s and the wells were rinsed again to remove excess dye. The bound dye was extracted using acetone/ethanol and the A492 was measured using a micro-ELISA plate reader .
Assays were performed on bacterium grown overnight with 1/2, 1/4 and 1/8 MIC oils and inoculated on to plates with medium containing 1/2, 1/4 and 1/8 MIC oils. Control plates contained no oils.
Swimming- The medium for swimming assays was composed of 1% tryptone (Difco), 0.5% NaCl and 0.3% agar. Briefly, the plates were inoculated in the centre with a sterile toothpick and incubated for 16 h at 25°C. Motility was assessed by observation of the circular turbid zone formed by bacteria migrating away from the point of inoculation .
Twitching- Cells were stab-inoculated with a toothpick through a thin (approx. 3 mm) 1% LB agar layer to the bottom of the Petri dish. After incubation for 24-48 h at 30 °C, a hazy zone of growth at the interface between the agar and the polystyrene surface was observed. The ability of bacteria to twitch strongly on the polystyrene surface of petri dish was examined by removing the agar, washing away unattached cells with a stream of tap water and staining the attached cells with a 1% (w/v) crystal violet solution .
Biofilm formation was examined in microtitre plates. Briefly, bacteria was grown overnight in LB broth (37 °C, 150 rpm.) in the presence or absence of 1/2, 1/4 and 1/8 MIC oils and added to wells in a standard inoculum corresponding to approximately 107 CFU/ ml, as described for the adhesion assay. After 16 h incubation at 37°C, bacteria were quantified as for the adhesion assay, with the exception that a solution of 0.025% safranin was used .
Alginate production assay
500µl of bacterial suspension corresponding to 0.5 McFarland standard solution was added to each of 50ml flasks each containing 20ml sterile LB broth. Test flasks contained 1/2, 1/4 and 1/8 MIC of essential oil while flasks without oil served as control. The flasks were then placed on an incubator shaker for 24 hours at 37°C. The samples were subjected to quantitative assay of alginate. Alginate production was estimated as follows: 70 μl of the sample was slowly added to 600 μl of boric acid- H2SO4 solution in a test tube placed in an ice bath. The mixture was vortexed for about 4 seconds and was placed back in the ice bath. 20 μl of 0.2% carbazole solution in ethanol was added to the test tube and was then immediately vortexed for about 4 seconds. The mixture was placed in a water bath at 55°C for 30 minutes. The absorbance was measured spectrophotometrically at 530 nm .
Each assay was repeated 6 times. Data obtained from the experiments were presented as differences between controls and tests and were analyzed by the paired t-test.
The MICs of essential oils against Pseudomonas aeruginosa for Mentha spicata and Cumminum cyminum oils were 16 and 32μg/ml respectively.
The results of sub-MICs of essential oils on swimming, twitching, adhesion, alginate production and biofilm formation in P. aeruginosa were summarized in Table 1. The results show that all oils at 1/2 and 1/4 MICs were significantly reduced swimming, twitching, adhesion, alginate production and biofilm formation in P. aeruginosa. At 1/8 MICs, M. spicata oil had not significantly effects on swimming, twitching and biofilm formation, but had significantly effect on adhesion. Also, 1/8 MIC of C. cyminum oil had significantly affected on all virulence factors except adhesion.
Motility (Swimming and twitching), adhesion, alginate production and biofilm formation, has several roles in P. aeruginosa pathogenesis. Consequently, any treatment which reduces production of these virulence factors might be effective in counteracting the pathogenesis of P. aeruginosa especially in patients with suppressed immune system .
It has been shown that sub -MIC values of antibiotics, reduce the level of alginate and proteases in-vitro . Fonseca et al., had shown that effects of sub-inhibitory concentration of piperacillin/tazobactam, on bacterial characteristics was different for the various strains of P. aeruginosa. There was a change in bacterial morphology and hydrophobicity that could explain a significant decrease in adhesion values in all clinical isolates and controls tested, a decrease in biofilm formation, a significant increase in sensitivity to oxidative stress, a significant decrease in flagellum-mediated swimming and a decrease in type IV fimbriae-mediated twitching . However, a few reports regarding the effects of essential oils on virulence factors production were reported. Owlia et al. had shown that Matricaria chamomilla essential oil, reduced alginate production and biofilm formation in P. aeruginosa .
An important determinant of virulence is the ability to adhere. The exposure of bacteria to sub-MICs of antibiotics generally weakens this ability . We showed that sub-MICs of mentioned essential oils reduced the adherence potential of P. aeruginosa. The motility (swimming and twitching) has very important role in pathogenicity .The results indicate that some essential oils can reduce motility. The establishment of biofilms by alginate-producing P. aeruginosa strains is the most common mode of growth in infections, with the biofilms providing a protected environment against the host immune system and a number of antibiotics .
The antibacterial activity of essential oils has been consider as a substitution for antibiotics, because, resistance to antibiotics has been developing in the world and Iran [22, 23]. In this study, the sub-MIC levels of M. spicata and C. cyminum essential oils affected adhesion, swimming, twitching, alginate production and biofilm formation. New antimicrobial agents against P. aeruginosa are very valuable, especially in multi drug resistant strains. We believe that the present investigation together with previous studies provide support to the antibacterial properties of M. spicata and C. cyminum essential oils. It can be used as antibacterial supplement towards the development of new therapeutic agents. Additional in vivo studies and clinical trials will also be needed to justify and further evaluate the potential of this oil as an antibacterial agent in topical or oral applications.