Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2019

Thesis Director

Steven L. Daniel

Thesis Committee Member

Kai Hung

Thesis Committee Member

Gary A. Bulla

Abstract

Oxalobacter formigenes 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 O. 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 O. formigenes strains using pulsed-field gel electrophoresis (PFGE) and multiplex PCR.

Nine pure cultures of O. formigenes strains (5 strains group 1 specific; 4 strains group 2 specific), five O. formigenes human isolates (2 isolates group 1 specific; 3 isolates group 2 specific), and three O. formigenes mouse isolates (group 1 specific) were grown anaerobically at 37°C in undefined oxalate 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 feces, 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 feces and 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 O. formigenes strains and isolates by multiplex PCR showed the following products (amplicons): 416 bp for O. formigenes groups 1 and 2 (species specific); 210 bp for O. formigenes group 1 (group 1 specific); and 140 bp for O. formigenes 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 O. formigenes strains. The multiplex PCR system for the one step detection and differentiation of O. formigenes strains is novel and optimized at every possible step asserting it as a unique and time saving technique in clinical field.

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