THE EFFECT OF ALKALOIDS, SAPONINSAND THYMOQUINONE OF NIGELLA SATIVA SEEDS ON BIOFILM PRODUCTION, MOTILITY, OUTER MEMBRANE PROTEINS AND LIPOPOLYSACCHARIDE OF SOME BACTERIA

Mohamed Shohayeb 1 and Eman Halawani 2 . 1. Taif University, Faculty of pharmacy, Department of microbiology, Taif, Saudi Arabia. 2. Taif University, Faculty of Science, Department of Biology , Taif, Saudi Arabia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Results:-
Biofilm formation by S. aureus, P. aeruginosa, E. coli, K. pneumoniae, Sh. flexneri and Sal. Typhimurium, was investigated in presence and absence of the thymoquinone, saponins and alkaloids (Table 1). Biofilm production by S. aureus was not very much affected by the three active constituents of N. sativa. On the other hand, while biofilm production by E. coli, was reduced, its production by P. aeruginosa and Sh. flexneri was enhanced ( Table 1). The percentage of production of biofilm by E. coli was 19-85% compared to the control. On the other hand, biofilm production was 210-235% and 117-240% increased as compared with the control in the case of P. aeruginosa and Sh. flexneri respectively (Table 1). In the cases of K. pneumoniae and Sal. Typhimurium there was an enhancement of biofilm production by thymoquinone and a reduction of production by both alkaloids and saponins at ½ MIC (Table 5).   The changes of the apparent level of expression of some outer membrane proteins of E. coli and P. aeruginosa exposed to 1/2 MICs are shown in figure3 and table 3. Changes in the levels of expression of seven proteins in E. coli and three proteins in P. aeruginosa were observed and Thymoquinone caused an increase in the expression of 3 proteins in E. coli and a decrease in 3 others. On the other hand, it caused an apparent decrease in the amount of a protein which has a molecular weight of 80 kDa in P. aeruginosa (Table 3). Alkaloids cause an increase in the level of 4 proteins and a reduction in one protein in E. coli and an increase in the level of expression of two proteinsone of which was OprFin P. aeruginosa (Table 3). Saponins increased the level of expression of 4 proteins and reduced two in E. coli and reduced the level of only one protein in P. aeruginosa (Table 3).
Thymoquinone, alkaloids and saponins affected the expression of lipopolysaccharides in E. coliand not P. aeruginosa. As shown in figure4, there was a decrease in the amount of high molecular weight LPS in E. coli treated with thymoquinone, alkaloids and saponins. S. aureus, E. coli and P. aeruginosa as treated with thymoquinone, saponins and alkaloids, were examined for changes in their morphology. While no apparent changes in the morphology of E. coli, (data not shown), some changes were observed in both P. aeruginosa and S. aureus. Thymoquinone and alkaloids caused P. aeruginosa cells to become more elongated and thinner compared to the control (Fig. 1). This was more apparent in the case of thymoquinone. On the other hand, S. aureus exposed to saponins at sub-inhibitory concentration suffered from protoplasting and some cells suffered from the retraction of their cytoplasmic contents away from the cell wall as shown in Fig 5B.

Discussion:-
The saponins, alkaloids, and thymoquinone were previously reported to be inhibitory to different types of bacteria even though they might be multi-drug resistant clinical isolates (15,32).In this study, we investigated the effect of subinhibitory concentrations of saponins, alkaloids, and thymoquinone of N. sativa on some pathogenesis mechanisms of some Gram-positive and Gram-negative.
Is this study, S. aureus, P. aeruginosa, E. coli, K. pneumonia, Sh. flexneri and Sal. Typhimurium, were grown at ½ and ¼ the MICs of thymoquinone, saponins and alkaloids and were tested for their capability of forming biofilms.
Biofilms are formed on surfaces of living tissues, medical devices and contact lenses, etc. (43). Biofilms help bacteria to survive and withstand hostile conditions on surfaces and contribute to the persistence of chronic infections (44). Several studies have been performed to find natural antimicrobial agents that influence microbial biofilm formation (45-48).
While there was a reduction in the ability of E. coli to produce biofilms by the three tested constituents, only saponins and alkaloids were capable of reducing biofilm formation by K. pneumoniae and Sal. Typhimurium. On the other hand, biofilm formation was enhanced in P. aeruginosa and Sh. flexneri and was indifferent in the case of S. aureus.
The induction of biofilm formation, in P. aeruginosa, and the failure to reduce biofilm formation in S. aureus, contradicts with Chaieb, et al., (49), who reported a reduction in biofilm formation by both S. aureus and P. aeruginosa treated with thymoquinone. However, it should be mentioned that the inhibition detected was at concentrations higher than their reported MICs. Therefore, while thymoquinone caused inhibition of biofilm formation at concentrations 22 and >512, for S. aureus and P. aeruginosa respectively, the MICs reported were 8 and >512 respectively (49).
Motility plays a key role in the colonisation of surfaces by bacteria (50,51). In this study, thymoquinone inhibited the motility and swarming of Escherichia coli, P. aeruginosa and Sal. Typhimurium. In a previous study,tannins of cranberry fruit and the hydrolysable tannin in pomegranate were reported to inhibit swarming motility but did not block swimming or twitching motilities (51). Sub-inhibitory concentrations of alkaloids like piperine of black pepper and reserpine of snakeroot, decreased bacterial swarming and swimming motilities (52). Tannic acid and epigallocathecingallate were found to block swarming motility in Pseudomonas aeruginosa (53).
Motility plays a key role in the colonisation of surfaces by bacteria (51, 54. In this study, thymoquinone inhibited the motility and swarming of Escherichia coli, P. aeruginosa and Sal. Typhimurium. In a previous study,tannins of cranberry fruit and the hydrolysable tannin in pomegranate were reported to inhibit swarming motility but did not block swimming or twitching motilities (51).
Escherichia coli, P. aeruginosa and S. aureus treated with a sub-inhibitory concentration of thymoquinone, alkaloids and saponins were examined under transmission electron microscope for morphological changes. Thymoquinone and alkaloids caused cells of P. aeruginosa to become thinner and elongated, compared to the 568 control. Though, the morphological changes might be attributed, to their effect on penicillin-binding proteins, however, this is not necessary because quinolone antibiotics, which do not bind to penicillin-binding proteins, affect the morphology of E. coli (55).
S. aureus exposed to saponins at a sub-inhibitory concentration suffered from protoplasting and retraction of the cytoplasmic contents away from the cell wall. Morphological changes of S. aureus exposed to aqueous extracts of green tea (56) or extracted catechingallates (57) have been reported in S. aureus. Methanolic extract of a soft sponge, Haliclona sp., caused internal shrinkage of methicillin-resistant S. aureus and B. subtiliscells which finally collapsed after prolonged exposure to the extract (58).
There is evidence that several outer membrane proteins are involved in adherence of bacteria to mammalian cells (59)(60)(61). In this study, the effect of thymoquinone, alkaloids and saponins at 1/2 MICs on the outer membrane proteins was examined. There were indeed apparent changes in the levels of expression of seven and three proteins in E. coli and P. aeruginosa respectively. While in E. coliOmpF outer membrane protein apparently decreased in amounts by treatment with the three constituents, OmpA increased.
On the other hand, three outer membrane proteins of P. aeruginosa were affected by the tested phytochemicals one of which was OprF. The expression of the latter was increased by the treatment with alkaloids. Recent studies have shown that the expression of 5% of bacterial promoters may be affected by sub-inhibitory concentrations of antibiotics (62). Likewise, it seems that phytochemicals like thymoquinone, alkaloids and saponins affect the expression of bacterial promoters.
The effect of phytochemicals on the expression of some bacterial proteins has been previously documented.Bioactive fraction 9EA-FC-B ofAcalyphawilkesianainhibited the production of MRSA by reducing the amount PBP2a in the matrix (63). Proteomic analysis of bacterial expression profiles following exposure to flower extracts of Melastomacandidum affected the expression of four proteins in E. coli and one protein in S. aureus (64).
In this study, alkaloids and saponins were found to reduce the amounts of high molecular weight Lipopolysaccharide (LPS) in E. coli.LPS is a main outer membrane component of Gram-negative bacteria (65).It causes pathophysiological effects such as fever, leucopenia, leucocytosis and Shwartzman reactivity (65). It is also involved in the attachment to host cells and it is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli (66)(67)(68).
LPS is associated closely with the OmpF protein of the outermembrane (69). The low expression of OmpF caused by alkaloids and saponins might have a role in the decreased in the detected amounts of high molecular weight LPS. LPS are essential for the pathogenesis of bacteria. It is an important mechanism to evade complement activation (70). Thisimplies that alkaloids and saponins could affect the pathogenesis of E. coli.

Conclusion:-
Thymoquinone, saponins and alkaloids of N. sativaat subinhibitory concentrationsaffect motility, biofilm formation and the expression of some proteins and LPS of theouter membrane of bacteria. This presumably would affectthe pathogenesis of bacteria.