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Evaluation of Intra-Species Diversity of Oxalobacter Formigenes Strains Using Pulsed-Field Gel Electrophoresis (PFGE) and Multiplex PCR

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Evaluation of Intra-Species Diversity of Oxalobacter Formigenes Strains Using Pulsed-Field Gel Electrophoresis (PFGE) and Multiplex PCR Eastern Illinois University The Keep Masters Theses Student Theses & Publications 2019 Evaluation of Intra-Species Diversity of Oxalobacter formigenes Strains Using Pulsed-Field Gel Electrophoresis (PFGE) and Multiplex PCR Nivedita Pareek Eastern Illinois University Follow this and additional works at: https://thekeep.eiu.edu/theses Part of the Bacteriology Commons, and the Biodiversity Commons Recommended Citation Pareek, Nivedita, "Evaluation of Intra-Species Diversity of Oxalobacter formigenes Strains Using Pulsed- Field Gel Electrophoresis (PFGE) and Multiplex PCR" (2019). Masters Theses. 4580. https://thekeep.eiu.edu/theses/4580 This Dissertation/Thesis is brought to you for free and open access by the Student Theses & Publications at The Keep. It has been accepted for inclusion in Masters Theses by an authorized administrator of The Keep. For more information, please contact [email protected]. Evaluation of Intra-Species Diversity of Oxalobacter formigenes Strains using Pulsed-Field Gel Electrophoresis (PFGE) and Multiplex PCR (TITLE) BY Nivedita Pareek THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science IN THE GRADUATE SCHOOL, EASTERN ILLINOIS UNIVERSITY CHARLESTON, ILLINOIS 2019 YEAR I HEREBY RECOMMEND THAT THIS THESIS BE ACCEPTED AS FULFILLING THIS PART OF THE GRADUATE DEGREE CITED ABOVE TheGraduate School� EA�mlll'J lwNOJsl.NwERS1Tr- Thesis Maintenance and Reproduction Certificate FOR: Graduate Candidates CompletingTheses in Partial Fulfillment of the Degree Graduate Faculty Advisors Directing the Theses RE: Preservation, Reproduction, and Distribution ofThesis Research Preserving, reproducing, and distributing thesis research is an important part of Booth Library's responsibility to provide access to scholarship. In order to further this goal, Booth Library makes all graduate theses completed as part of a degree program at Eastern Illinois University available for personal study, research, and other not-for­ profit educational purposes. Under 17 U.S.C. § 108, the library may reproduce and distribute a copy without infringing on copyright; however, professional courtesy dictates that permission be requested from the author before doing so. Your signatures affirm the following: •The graduate candidate is the author of this thesis. •The graduate candidate retains the copyright and intellectual property rights associated with the original research, creative activity, and intellectual or artistic content of the thesis. •The graduate candidate certifies her/his compliance with federal copyright law (Title 17 of the U. S. Code) and her/his right to authorize reproduction and distribution of all copyrighted materials included in this thesis. •The graduate candidate in consultation with the faculty advisor grants Booth Library the nonexclusive, perpetual right to make copies of the thesis freely and publicly available without restriction, by means of any current or successive technology, including but not limited to photocopying, microfilm, digitization, or internet. •The graduate candidate acknowledges that by depositing her/his thesis with Booth Library, her/his work is available for viewing by the public and may be borrowed through the library's circulation and interlibrary loan departments, or accessed electronically. 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Abstract Oxalobacter fo rmigenes is a beneficial, strict anaerobic gut bacterium that plays an important role in the prevention of kidney stone disease with a unique characteristic of using only oxalate as a carbon and energy source. To date, twenty-one strains of 0. formigenes have been isolated which are further divided into two groups based on differences in the patterns of cell membrane lipids, cellular proteins, and nucleic acid fragments. The objective of the study was to evaluate intra-species diversity of 0. fo rmigenes strains using pulsed-field gel electrophoresis (PFGE) and multiplex PCR. Nine pure cultures of 0. fo rmigenes strains (5 strains group 1 specific;4 strains group 2 specific), five 0. fo rmigenes human isolates (2 isolates group 1 specific;3 isolates group 2 specific), and three 0. fo rmigenes mouse isolates (group 1 specific) were grown anaerobically at 37°C in undefinedoxala te broth. Upon reaching late exponential growth, bacterial cells were harvested and added to melted agarose to make plugs which were incubated (55°C) in proteinase K solution to lyse cells, sliced, and then digested with Xbal 1 at 37°C. Digested agarose slices were loaded onto 1 % Seakem Gold agarose gels, loaded onto PFGE for 18-19 h, and analyzed by BioNumerics software to generate a dendrogram. For multiplex PCR, bacterial cells were subjected to DNA extraction followed by amplification with suitable species and strain specific primers. In case of PFGE, four group 1 specific strains (isolated from human fe ces, sheep rumen, and fresh water lake sediment) showed a tight clustering with each other while other group 1 specific strain (isolated from wild rat) was tightly associated with group 1 specific human isolates. Three group 2 specific strains (isolated from human fecesand Guinea pig) showed high similarity with each other but one group 2 specific strain (isolated from human feces) was in proximity with group 1 specific strains. Mouse isolates were compactly grouped with each other but did not show any closeness with rest of the strains. Amplification of genomic DNA obtained from 0. fo rmigenes strains and isolates by multiplex PCR showed the following products (amplicons): 416 bp for 0. fo rmigenes groups 1and 2 (species specific);210 bp for O. formigenes group 1 (group 1 specific); and 140 bp for 0. fo rmigenes group 2 (group 2 specific). Multiplex PCR of mixed bacterial DNA samples, containing high or low DNA concentrations of each group, yielded the expected 3 amplicons. This study was in overall agreement with the grouping of the strains with some exceptions indicating PFGE is a useful tool to study the phylogenetic relationship among 0. fo rmigenes strains. The multiplex PCR system for the one step detection and differentiation of 0. fo rmigenes strains is novel and optimized at every possible step asserting it as a unique and time saving technique in clinical field. II Acknowledgment I would like to express my gratitude to Dr. Steven Daniel, forhis constant support and guidance during my time at Eastern Illinois University. His enthusiasm, positive attitude, and motivation towards work and life has helped me to grow a better individual. I would like to extend my appreciation to my committee members Dr. Kai Hung and Dr. Gary Bulla fortheir valuable suggestions to complete my project. I would also like to thank Dr. Nazzal (NYU), and Dr. Hatch (UFL) for providing me wonderful opportunities to enhance by project work. I am gratefulto Department of Biological Sciences for providing the financial aids to complete the project. I appreciate the help provided by lab members Matt, Lina, Rachana and Sunil. They were a great joy to be with. I would like to thank my friends Tajdar, Ibrahim, Hasni, Israt, Iffat, and Nadh to make this journey more beautiful. I would not have been able to complete my studies in the United States without my brother's support. He has been a great source of inspiration throughout this journey. I would like to thank my parents for incessant love to me and my dreams. I would also want to appreciate my American host family Mrs. Victoria Norton & familyfor making me fe el home away fromhome. At last but not the least. special thanks to a special person added in my life: My husband Kapish. Thank you for respecting and believing in whatever I do. Ill Table of Contents I. Introduction .... ...... ................................... ..... ................... .... ....... 1 • Oxalate and Oxalate Associated Diseases ...... ............. ..... ...... .................. 1 • Oxalate Degrading Bacteria ..... .................. ...... .................................. 2 • Physiology and Metabolism of 0. fo rmigenes .. ............ ............ ................8 • Prevalence and Colonization by 0.fo rmigenes ........ .................. .............. 10 • Oxalobacter fo rmigenes and Antibiotics ............... .................. ............... 11 • Oxalobacter fo rmigenes and Calcium Oxalate Stones ................................. 11 • Molecular Identification of Oxalobacter fo rmigenes by Multiplex Polymerase Chain Reaction (PCR) ........ ......................... ............ ........................ 12 • Pulse Field Gel Electrophoresis (PFGE) ................................................ 13 II. Research Objectives ......... ...... ................ ..................
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