Lebanese American University the Inhibitory Effect of the Methanolic Extracts of Origanum Syriacum and Rosmarinus Officinalis An
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Lebanese American University The inhibitory effect of the methanolic extracts of Origanum syriacum and Rosmarinus officinalis and their major components on the biofilm formation of clinical isolates of Staphylococcus aureus By Reeda Shawkat Harb A thesis Submitted in partial fulfillment of the requirements for the degree of Master of Science in Molecular Biology School of Arts and Sciences May 2019 ii iii iv Dedication All my work, this thesis and every other achievement I did or will ever do in my life will always be dedicated to: My father, Shawkat Harb who supported me by all means all through out my career and was always present to motivate me, guid me and provide me with his immense knowledge in life and career. The most kindhearted and tender woman, my beloved mother, Nada Harb who taught me from her patience and perseverance the way to overcome all the obstacles I might face throughout my life and career. The person who was always present by my side to guard me, encourage me and support me all throughout my path. v Acknowledgment First, I would like to extensively thank my advisor, Dr. Tarek Nawas for his continuous support and guidance all through out my research project. I would like to thank him for his patience, his encouragement, his faith in his students and his wide knowledge from which I learned a lot. I am very glad and I was very lucky to have had the chance to work under his supervision throughout my years of research. Besides my advisor, I would like to thank my committee members, Dr. Michella Ghassibe-Sabbagh and Dr. Roy Khalaf for taking the time to review my thesis and provide me with their helpful comments. My sincere thanks goes, as well, to my labmates and my friends in the masters program for all their support, their motivation and for all the memories we shared throughout these two years. Further, I would like to thank all the faculty members in the department of Natural Sciences for their deep knowledge provided through the courses taught to us in the masters program. They allowed me to gain a lot of new information in the field of science. Last but not least, I would like to thank the Lebanese American University for funding my project throughout these two years. vi The inhibitory effect of the methanolic extracts of Origanum syriacum and Rosmarinus officinalis and their major components on the biofilm formation of clinical isolates of Staphylococcus aureus Reeda Shawkat Harb Abstract Control of bacterial pathogenesis is becoming more difficult by the day. The emergence of bacterial resistance has made treatment of infections more difficult, a reason why a twist towards traditional medication was made as part of the efforts to control such infections. It is well known that bacterial biofilm formation is directly associated with initiation of bacterial infections, so a new trend in research is to try to inhibit biofilm formation and thus prevent infections. This study aimed at detecting whether the methanolic extract of two natural herbs, Rosmarinus Officinalis (Rosemary) and Origanum syriacum (Zaatar) have the ability to inhibit the biofilms that are produced by a serious pathogen, Staphylococcus aureus, using standard methods. The 21 clinical isolates S. aureus, included in the study, were defined phenotypically and genotypically. Six isolates were found to be methicillin resistant (MRSA) while the remaining 15 were methicillin susceptible (MSSA). The PFGE pulsotypes of the strains determined, allowed for segregation of the isolates into one major clade that was subdivided into two subclades, each, of which, contained a set of more closely related isolates. The rest of the isolates were each individually related to others. Notably, the methicillin susceptible S. aureus strains were more closely related to each other as compared to the methicillin resistant strains, which were either grouped in pairs or individually. Interestingly, few methicillin susceptible vii isolates obtained from one hospital were more closely related to isolates obtained from a health center, than to other methicillin susceptible isolates from the same hospital. The methanolic extract of each of the two plants (0.1 g/ml) revealed an antibacterial effect at volumes equal or higher than 200 µL for Rosmarinus Officinalis and 150 µL for Origanum syriacum against the S. aureus isolates. It was, however, found that the volumes of 100 and 150 µL of the Rosmarinus Officinalis extracts and 50 and 100 µL of the Origanum syriacum extracts at the lower concentrations of 0.02 g/ml (five times diluted extract) and 0.01 g/ml (ten times diluted extract), of the two plants respectively, significantly inhibited the S. aureus biofilm formation. The contribution of the major components of each of the two herbs to that effect was then determined. The two major components of Rosmarinus Officinalis: α- pinene and camphor at different concentrations inhibited the biofilm formation of all the tested isolates. On the other hand, the two major components of Origanum syriacum: Thymol and Carvacrol at different concentrations did not affect the S. aureus biofilm formation. The results of this study revealed the previously undetermined ability of the tested plants to inhibit the S. aureus biofilm formation and demonstrated the significance of α-pinene and camphor as the effective inhibitors and suggested their use to prevent the serious infections that may be caused by the organism. Keywords: Biofilm formation, Rosmarinus Officinalis, Origanum syriacum, Staphylococcus aureus, Prevention of biofilm viii Table of content Abstract ..................................................................................................................... vii List of Tables ........................................................................................................... xvi List of Figures .......................................................................................................... xxi List of Abbreviations ............................................................................................ xxxi I- Introduction ............................................................................................................ 1 1.1. Staphylococcus aureus ................................................................................ 1 1.1.1. Staphylococcus aureus characteristics ....................................................... 1 1.1.2. Staphylococcus aureus pathogenesis ......................................................... 2 1.1.3. Staphylococcus aureus toxins and extracellular enzymes ......................... 3 1.1.3.1. Toxins targeting the host cell membrane ............................................ 3 1.1.3.2 Toxins interacting with host cell receptors .......................................... 5 1.1.3.3. Proteolytic Enzymes of Staphylococcus aureus ................................. 6 1.1.4. Staphylococcus aureus drug resistant strains ............................................. 8 1.2. Bacterial biofilm ............................................................................................ 11 1.2.1. Definition of a biofilm ............................................................................. 11 1.2.2. Stages of biofilm formation ..................................................................... 12 1.2.2.1. Stage 1: Reversible Bacterial Adhesion ............................................ 12 1.2.2.3. Stages 3 and 4: Initiation and maturation of a structured biofilm..... 13 1.2.2.4. Stage 5: Detachment and dispersal of bacterial cells from the biofilm matrix ............................................................................................................. 15 1.2.3. Sites of biofilm formation: non-biological and biological surfaces......... 15 1.2.4. Mechanical and biological factors involved in S. aureus Biofilm formation ............................................................................................................ 16 1.2.5. Contribution of biofilms to pathogenesis ................................................. 22 1.3. Origanum syriacum ....................................................................................... 24 1.3.1. Taxonomy and distribution of Origanum syriacum ................................. 24 1.3.2. Description of Origanum syriacum morphology ..................................... 25 1.3.3. Origanum syriacum chemical components .............................................. 25 1.3.3.1 The Origanum syriacum essential oils ............................................... 25 ix 1.3.3.2. The Origanum syriacum fatty acids .................................................. 25 1.3.3.3. The Origanum syriacum minerals .................................................... 26 1.3.4. Pharmacological significance of Origanum syriacum ............................. 26 1.3.4.1. Antimicrobial effect of Origanum syriacum ..................................... 26 1.3.4.2. Anti-inflammatory effect of Origanum syriacum ............................. 28 1.3.4.3. Anti-oxidant role of Origanum syriacum ......................................... 28 1.3.4.4. Anti-tumor effect of Origanum syriacum ......................................... 29 1.3.4.5. Other pharmacological benefits of Origanum syriacum ................... 29 1.4. Rosmarinus officinalis ................................................................................... 29 1.4.1. Taxonomy and distribution of Rosmarinus officinalis............................. 29 1.4.2. Description of Rosmarinus officinalis morphology