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Evaluation of Intra-Species Diversity of Oxalobacter formigenes Strains Using Pulsed-Field Gel Electrophoresis (PFGE) and Multiplex PCR
Nivedita Pareek Eastern Illinois University
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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
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Nivedita Pareek
THESIS
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science
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2019
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distribute my thes!_s. My adviser's signature indicates concurrence td rep1oduce �nd �ribute th/)hesis. 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 ...... 16
III. Materials and Methods ...... 17
• Stock Solutions forCul ture Media Preparation ...... 17
o Mineral Solution ...... 17
o Sodium Acetate Solution ...... 17
o Trace Metal Solution ...... 17
o Resazurin Solution ...... 1 7
• Standard Culture Media ...... 17
o Undefinedoxala te broth (UOB) ...... 18
o Defined Glucose Broth (DGB) ...... 18
o Anaerobic MRS broth ...... 19
• Microorganisms and Growth Conditions ...... 19
IV • Protocol for Detection of O. formigenes in Human Feces and Mice ...... 21
o Culture Media ...... 21
>- Anaerobic Dilution Solution (ADS) ...... 21
>- Calcium Oxalate Agar Roll Tubes ...... 21
>- Aerobic oxalate broth (AOB) ...... 22
>- Brain heart infusion anaerobic broth (BHI) ...... 22
>- BHI Agar Plates ...... 22
• Qualitative Detection of O.formigenes Using Calcium Oxalate Precipitation
Assay ...... 23
• Human and Animal Subjects ...... 23
o Fecal Collection and Processing ...... 24
o Isolation of 0. fo rmigenes from Enrichment
Cultures...... 24
• Multiplex Polymerase Chain Reaction (PCR) ...... 27
o DNA Extraction fromPure Bacterial Cultures and Enrichment Cultures. 27
o DNA Extraction from Human Fecal Sample ...... 28
o DNA Amplification ...... 29
o Gel Electrophoresis ...... 30
• Pulse Field Gel Electrophoresis (PFGE) ...... 30
o Preparation, Casting, and Lysis of Agarose Plugs ...... 30
o Restriction Digestion of DNA in Agarose Plugs ...... 32
o Gel Electrophoresis ...... 33
o Staining and Destaining of Agarose Gel ...... 34
v o Analysis of Gel with Bioinformatics Tool...... 34
• High Performance Liquid Chromatography (HPLC) ...... 34
o Preparation ofHPLC standards ...... 34
o Preparation of Sterile Culture Media ...... 34
o HPLC conditions ...... 35
IV. Results ...... 36
• Growth of O.formigenes Strains in UOB ...... 36
• Isolation and Characterization of 0. fo rmigenes from Human and Mouse
Feces ...... 36
• Multiplex PCR Analysis ...... 39
o Differentiation of O.formigenes Group 1 and Group 2 Strains ...... 39
o Detection ofO.jormigenes in Human Fecal Samples ...... 41
o Analysis of the Specificity of Multiplex Primer Solution in Probiotic
Strains ...... 44
o Confirmation of 0. fo rmigenes Isolates fromHuman Feces ...... 45
o Confirmation of 0. fo rmigenes Isolates from Mouse Feces ...... 4 6
• HPLC Analysis of Standards, Sterile Culture Media, and Culture ...... 47
• Nomenclature of New Isolates ...... 48
• PFGE Analysis ...... 48
V. Discussion ...... 56
VI. Conclusion ...... 63
VII. References...... 64
VI List of Figures
1. Electron micrograph of a 0. fo rmigenes isolate OxWR l in wild rat ...... 2
2. Oxalate degradation and energy conservation in 0. fo rmigenes ...... 9
3. Pathway of oxalate catabolism and anabolism in 0. fo rmigenes ...... 9
4. Setup for streaking calcium oxalate agar roll tubes ...... 26
5. Reusable plug mold for making bacterial plugs ...... 31
6. Setup for plug slicing in a sterile petri plate ...... 33
7. Pulse field gel electrophoresis assembly: Electrophoresis chamber, a pump on top,
a cooling module on bottom, and a chef mapper unit...... 33
8. A multiplex PCR gel image ofamplified genomic DNA from pure bacterial cultures
having O. formigenes ...... 40
9. A multiplex PCR gel image of amplifiedgenomic DNA frompure bacterial culture
OxWR ...... 40
10. A multiplex PCR gel image of amplified genomic DNA from two human
volunteer's fe cal samples ...... 41
11. A multiplex PCR gel image of amplified genomic DNA from human volunteer 1
fecal enrichment cultures ...... 42
12. A multiplex PCR gel image of amplified genomic DNA from human volunteer 2
fe cal enrichment cultures ...... 42
13. A multiplex PCR gel image of human fecal DNA samples mixed with pure
bacterial DNA samples showing 0. formigenes ...... 43
Vil 14. A multiplex PCR gel image of amplified genomic DNA ofprobiotic strains.... 44
15. A multiplex PCR gel image of two 0. jormigenes human fecal isolates amplified
genomic DNA from volunteer 1 ...... 45
16. A multiplex PCR gel image of three 0. .f genomic DNA from volunteer 2 ...... 45 17. A multiplex PCR gel image of amplified genomic DNA from 11 mice enrichment cultures having 0. fo rmigenes ...... 46 18. A multiplex PCR gel image of amplifiedgenomic DNA from three 0. fo rmigenes mice isolates ...... 47 19. A PFGE DNA fingerprintof pure 0. fo rmigenes strains ...... 50 20. A PFGE DNA fingerprint of 0. fo rmigenes human isolates ...... 51 21. A PFGE DNA fingerprint of 0. fo rmigenes mouse isolates ...... 51 22. Phylogenetic tree based on analysis of PFGE profile of pure 0. fo rmigenes strains ...... 53 23. Phylogenetic tree based on analysis of PFGE profileof pure 0.formigenes strains, human and mouse isolates ...... 53 Vlll List of Tables 1. Group classification and strains of 0. formigenes ...... 4 2. Additional oxalate-degrading gut microorganisms: Generalists ...... 7 3. Pure bacterial strains used in the PHiE and multiplex PCR study ...... 20 4. Summary of 0. fo rmigenes isolates fromhuman and mouse feces ...... 38 5. Summary of 0. fo rmigenes isolates fromhuman and mouse feces ...... 39 6. Number of total bands obtained in PFGE fingerprint...... 52 7. Summary of multiplex PCR detection and FGE analysis...... 55 IX I. Introduction Oxalate and Oxalate-Associated Diseases Oxalate is a naturally foundchemical substance which is highly oxidized in nature. It is richly found in plants and plants derivatives ( 1 ). Some of the main sources of oxalate are spinach, almonds, rhubarb, chocolates, wheat bread, and wheat bran (2-4). Due to its high chelating activity, it is mostly foundin salt forms. Saltsforms can be of two types: water soluble salts (sodium. potassium. and ammonium) and water-insoluble salts (calcium. magnesium, and iron) (5-7). Oxalate can bind to the insoluble salts considered as one of the essential nutrients and decrease the availability of these nutrients to the body (5. 9). Additionally, oxalate is endogenously produced in the animal body and at high levels can be detrimental to the health of mammals including humans (8). No enzymes are present in the host for oxalate degradation. Dietary and endogenously produced oxalate are removed either by fe cal and urinary excretion or by microbial degradation. If by any means oxalate is not metabolized in the human body, it tends to bind with calcium ions and formscalcium oxalate crystals and stones throughout the body, especially in the kidneys. Kidney stone disease also known as urolithiasis affects 6-10% of the worldwide population (8, 10). Higher levels of oxalate can also result in a condition of hyperoxaluria which an additional problem in the management ofurolithiasis is (9). According to a study on individuals with inflammatory bowel disease, Crohn' s disease patients showed little or no intestinal oxalate degradation with a high risk ofhyperoxaluria (13). Accumulation of oxalate in the body is associated with such pathological disorders as cardiomyopathy, cardiac conductance disorders, renal failure, Crohn's disease, steatorrhea, jejunoileal bypass surgery, cystic fibrosis, and sometimes toxic death (56-58). Oxalate-Degrading Bacteria Since mammals do not have any enzymes forthe biodegradation of oxalate, the host- microbe interaction is primarily responsible for the oxalate homeostasis and helps in reducing the risk factors forkidney stone diseases ( 10). Experimental studies have stated that bacterial degradation of oxalate in the intestine decreases oxalate absorption and urinary oxalate levels which reduces the occurrences of calcium oxalate stone formation (11, 12). Due to the strong corrosive activity of the compound, it was thought that the microorganisms will be less likely to use oxalate as its energy source, but reports have suggested that there are a wide variety of niches where oxalate is used as a source of carbon and energy by bacteria. Indeed, oxalate-degrading bacteria has been divided into two categories: the specialists and the generalists. Based on past research, 0. fo rmigenes (a specialist) has emerged as a unique oxalate- degrading bacterium, showing total dependence on oxalate for energy ( 17). 0. fo rmigenes is a gram-negative, non-motile, non-spore forming, obligately anaerobic bacterium belonging to the �-proteobacteria class and Burkholderiales order (Figure 1 ). Figure I. Electron micrograph of 0. fo rmigenes OxWRI isolated fromceca of wild rats (20). 2 0. fo rmigenes was first isolated from ruminal contents of sheep (18, 19). Later, it was isolated from the cecal contents of rats, guinea pigs, and swine (20, 21 ), human feces (22), and freshwater lake sediments (23). Other known anaerobic oxalate-degrading specialists isolated from lake sediments include Oxalobacter vibrioformis (13). To date, twenty-one strains of 0. formigenes have been isolated and, based on a comparison of l 6S rRNA sequences, membrane fatty acid and lipid profiles, and specificity of oligonucleotide probes/primers directed at the axe gene, these strains have been divided into 2 major groups: Group 1 and Group 2 (24-26) (Table 1 ). Relative to oxalate utilization, the generalists are bacteria that depend on other substrates apart from oxalate as a source of carbon and energy (13). Generalist species from the genera Lactobacillus, Bifidobacterium, Enterococcus, and Eubacterium have been recognized to ferment oxalate but oxalate alone does not growth their support ( 11, 14-16) (Table 2; I 0) although some Lactobacillus strains appear to use the same oxalate catabolism pathway. 3 Table 1. Group classification and strains of Oxalobacter formigenes: Specialists Academic Culture Possible Collection subgroup or (A, B, or Isolate Commercial Group C) Strain Source Kev Points Availabilit v References Ty(!e Strain; oxc and frc genes 18, 24 (strain Sheep OxB sequenced; ATCC, EIU isolated and Rumen cellular fatty characterized) acids; 16 rRNA Animal colonization 20 (strain Wild Rat studies; antibiotic Ox WR EIU isolated and Cecum susceptibility: characterized) cellular fatty acids; 16S rRNA (strain used but Human Cellular fatty OxDBl2 - no isolation Feces acids; 16S rRNA details given) 25 (strain used Human oxc gene HOxCC12 - but no isolation Feces sequenced details given) genome sequenced; antibiotic 33,40 Human susceptibility (strain used but HOxCC13 EIU Feces studied; no isolation proteomics study; details given) Group I cellular fatty (13 acids; 16S rRNA isolates) oxc gene 25 (strain used Human sequenced; HOxHM18 - but no isolation Feces cellular fatty details given) acids; 16S rRNA 25 (strain used Human oxc gene HOxRA - but no isolation Feces sequenced details given) 25 (strain used Human axe gene HOxUK90 - but no isolation Feces sequenced details given) oxc gene sequenced; Oxabact® OC5: 24, 40, 41 susceptibility (assumed since Human relative to HC-3 was HC-1 EIU Feces antibiotics, air, isolated and pH, bile salts characterized tested; cellular in this report): fatty acids, 16S rRNA 24 (strains Human HC-3 - isolated and Feces characterized) 4 Freshwater 23 (strain Cellular fatty SOx4 Lake ATCC, EIU isolated and acids; I 6S rRNA Sediment characterized) Freshwater 23 (strain SOx6 Lake - isolated and Sediment characterized) 24 (strains Cellular fatty POxC Pig Cecum - isolated and acids; 16S rRNA characterized) oxc gene sequenced; antibiotic 24, 40 (strain Human c OxK susceptibility EIU isolated and Feces studied; cellular characterized) fatty acids; 16S rRNA 24 (assumed oxc gene smce BA-2 Human sequenced; was isolated c BAI EIU Feces cellular fatty and acids; 16S rRNA characterized in this reoort) 24 (strain Human BA2 - DSMZ isolated and Feces characterized) 24 (assumed since BA-2 Human was isolated BA4 - - Feces and characterized Group in this reoort) II 24 (assumed (14 since BA-2 Isolates) Human was isolated BA6 - - Feces and characterized in this reoort) 21 (assumed oxc gene RW IS Human sequenced; Rockwell A HOxRW - Feces cellular fatty described in acids; 16S rRNA this oublication) genome 25, 33 (strain Human sequenced; used but no A HOxBLS EIU Feces cellular fatty isolation acids; I 6S rRNA details !!iven) 25 (used but no Human oxc gene HOxUK5 Unknown isolation Feces sequenced details given) 25 (strain used Human oxc gene but no HOxUK88 Unknown Feces sequenced isolation details !!iven) 5 25 (strain used Human axe gene but no HOxHS Unknown Feces sequenced isolation details given) Laboratory 20 (strain cellular fatty B OxCR Rat Unknown isolated and acids; l 6S rRNA Cecum characterized) Guinea cellular fatty B Ox GP EIU 21 Pig Cecum acids; 16S rRNA Antibiotic Human Va3 susceptibility ElU 40, 41 Feces tested Cellular fatty 89-112 - Unknown 85 acids 6 Table 2. Additional oxalate-degrading gut microorganisms: Generalists (10) Organism Sources Pathway Eggerthella lenta Human stool Not known Enterococcus gallinarum W oodrat fe ces Detoxification Enterococcus fa ecium Canine feces Not known Human stool. canine Enterococcusfaecalis Carbon/energy feces Provendencia rettgeri Human stool Not known Streptococcus thermophilus Pro biotic Detoxification Lactobacillus plantarum Probiotic, canine feces Detoxification Lactobacillus gasseri Probiotic, woodrat gut Detoxification Lactobacillus casei Pro biotic Detoxification Lactobacillus acidophilus Human stool Detoxification Lactobacillus rhamnosus Pro biotic Detoxification Lactobacillus salivarius Pro biotic Detoxification Lactobacillus johnsonii Woodrat gut Detoxification Bifidobacterium infantis Pro biotic Detoxification Bifidobacterium animalis Human stool Detoxification Clostridium sporogenes W oodrat fe ces Detoxification Leuconostoc lactis Canine fe ces Not known Leuconostoc mesenteroides Canine fe ces Not known 7 Physiology and Metabolism of 0.formigenes 0. jormigenes grows in a strict anaerobic culture medium with optimal growth at pH 6-7. The bicarbonate-buffered medium constitutes minerals, trace metals, oxalate, some amount of yeast extract, and acetate (24). Previously it has been mentioned that the energy yielding efficiency of oxalate is very low which results into a low yield of 0. fo rmigenes in culture (27) but the bacterial growth is proportional to the concentration of oxalate up to 111 mM (18). Oxalate is converted to formate and C02 by the action of two cytosolic enzymes: formyl-CoA transferase encoded by the fr c gene and oxalyl-CoA decarboxylase encoded by the oxc gene. Oxalyl-CoA transferase is a 45-kDa monomer and a member of a CoA transferases familywhich functions by the formation of a ternary complex between the two substrates and the enzyme (28). Oxalyl-CoA decarboxylase is a four-subunit tetramer of 265-kDa and comprises 10% of the soluble protein of the cell. In the presence of formyl-CoAtransferase, oxalate is converted to oxalyl-CoA by the transfer ofCoA from formyl-CoAto oxalate and then oxalyl-CoA decarboxylase catalyzes the formation offormyl-CoA and C02 from oxalyl-CoA in a reductive reaction by releasing C02 and consuming a proton (29). Around 99% of the carbon from oxalate is converted to formate and C02. The electrogenic exchange of oxalate (in) and formate (out) is mediated by an integral antiporter protein OX! T which generates a strong proton motive force for energy conservation (Figure 2) (27, 31). It is made up of 12 transmembrane a-helical segments with the C- and N-terminal regions located on the cytoplasmic side (30). Approximately 1 % of the oxalate carbon in 0. jormigenes enters biosynthetic pathways via 3-phosphoglycerate. To form 3-phosphoglycerate, two moles of glyoxylate 8 are converted into one mole oftartronic semialdehyde and further into glycerate. Glycerate is reduced to 3-phosphoglycerate by the glycerate pathway (Figure 3) (32). I ACl " dIC" ,------\Alkaline / Oxalate2- Formate•- �ecarboxyla�\j H+ C02 Figure 2. Oxalate degradation and energy conservation in 0. fo rmigenes. (IN) . r-Formyl-CoA�xylase CO, OX.late � � CoA: o..._ [Oxalyl CoA A1rc1 (OUT> tranaferMA - Fonnate/� OXalyl-CoA _,/ '-- H• (-1%) NAO(P (oxalyl-CoA NAOP reductase) Giyoxylate CO, ----j [glyoxylate ! carboligase] Tartronic Semialdehyde NAOH --d [tartronic semialdehyde NAO - ! reductase] Glycerate ATP - I (glycerate kinase] ADP =1 3Phosphoglycerate Figure 3. Pathway of oxalate catabolism and anabolism in 0. formigenes. 9 Prevalence and Colonization by 0. formigenes According to enumeration studies in human stool samples, 0. fo rmigenes represents a very minute proportion of the total gut microbiota (33). It has been reported to be present in healthy individual's fe cal samples at a range of 107 to 108 colony-forming units per gram wet sample ( 19). 0. fo rmigenes strains isolated from laboratory rats and wild rats cecal contents showed a viable count of less than 0.1% of the total viable population (34). Less evidence is available on how individuals are colonized and what is the source of 0. fo rmigenes, but studies suggest that colonization occurs in early childhood (e.g., children between 6-8 years old were colonized with 0. fo rmigenes) (35). However, the rate of colonization declines in older children but is regained in the adult population. The onset of the colonization by 0. fo rmigenes appears to require more time than that seen with other intestinal bacteria (35). Animal experiments suggest that bacterial transmission occurs directly from the environment rather than from the mother (36). Genome sequencing studies of 0. fo rmigenes strain OXCC 13 have shown the presence of genes that encode for proteins which are homologous to pilus 1 proteins (37). This could enable cell-to-cell transfer of genetic material or adhesion to host cells. In contrast, the genome of strain HOxBLS did not show any such genes (37). Colonization studies have reported that 38-77% of the normal human population is colonized with 0. fo rmigenes while colonization rates are roughly half that in stone formers (38). Increasing dietary oxalate by 15-foldresults in a 12-fold increase in 0. formigenes populations in the gut (39). Increased bacterial numbers are also observed when dietary calcium intake increases by 5-foldbut later the bacterial numbers decrease due to the high binding affinity of calcium and oxalate (39). 10 0. formigenes and Antibiotics Many factors such as diet, life style, and the composition of the gut microbiome may impact 0. fo rmigenes colonization and loss of colonization in humans. Various studies have demonstrated that the lack of anaerobic oxalate-degrading bacteria can be due to antibiotic exposure and may result in various diseases (38, 40, 46). In studies where 0. fo rmigenes strains was subjected to different antibiotics used in daily clinical practices, it was found that 0. fo rmigenes was resistant to ampicillin, amoxicillin, streptomycin, c!avulanic acid, ceftriaxone, and vancomycin and sensitive to doxycycline and clarithromycin ( 40, 41). The amount and route of antibiotic administration, the population of bacteria and other host factors can affect whether an antibiotic can remove 0. fo rmigenes from fe cal micro biota ( 40). Antibiotic therapies can disturb the consortium of gut bacteria so replacing these therapies with alternatives should be a consideration for future studies. 0.formigenes and Calcium Oxalate Stones In the last 30 years, according to the National Health and Nutrition Examination Surveys, USA, the prevalence of kidney stones in the United States has increased from 3.2% to 5.2% in both males and females (42). An inverse relationship is found between 0. fo rmigenes colonization and calcium oxalate stone formation. A study conducted on stone formers (with a history of 2-5 times occurrence of stones) presented 38% colonization by 0. fo rmigenes while stone formers (more than 5 times) presented 13% colonization by 0. fo rmigenes in contrast to the control subjects who were 75% positive for colonization (35). Similarly, stone former colonization studies from Korea (43), Germany and southeastern United States (44), and India (45) agree with previous studies which also saw low 11 colonization rates 0. fo rmigenes in recurrent stone formers and very high numbers and colonization rates of 0. fhrmigenes in normal individuals. Molecular Identification of 0. f ormigenes by Multiplex Polymerase Chain Reaction (PCR) Special conditions are required to grow anaerobic oxalate-degrading bacteria like 0. fo rmigenes. They are isolated from colonies surrounded by clear zones in anaerobic calcium oxalate agar ( 19) or their presence in broth culture is confirmed with the calcium chloride precipitation assay ( 18). The culturing method is always time-consuming and sometimes inaccurate which makes detection of these medically beneficial bacteria difficult.Therefore, DNA-based molecular studies for rapid identification of O. formigenes have been done in an attempt to make the process more convenient. Studies support the factthat PCR amplification is an alternative of culturing methods to detect 0. fo rmigenes from the fresh fecal samples and pure bacterial strains (35). In a study of 0. fo rmigenes colonization in children (newborn tol2-years old), out of 100 fecal samples. 13 samples were negative for oxalate degradation through the chemical-based detection but were positive for 0. formigenes colonization by PCR (35). 0. fo rmigenes detection by PCR amplification showed greater sensitivity and support for the distribution of strains into further groups when compared to culturing methods and chemical-based testing. The oxc gene responsible for the catabolism of oxalate has been cloned and sequenced and used to design species-specific and strains-specific primers (35, 47). Oligonucleotide probes and primers have been used to divide 0. fo rmigenes strains into two respective groups, group 1 and group 2, but unfortunately this experiment showed less accuracy for group 2 strains 12 (25). In a subsequent PCR study with 100 human fecal samples, redesigned 0.fo rmigenes axe-based primers detected gut colonization by 0. fo rmigenes in 72 individuals and, of these, 68 individuals showed the presence of group 1 strains while the remaining 4 were positive for group 2 strains (35). These group-based studies were all done in separate PCR assays also known as uniplex PCR. However, more than one target sequence can also be amplified by using more than one primer set in a single PCR system and is defined as multiplex PCR. This technique has been effectively applied in areas of nucleic acid diagnostics (48, 49), RNA detection (50, 51), mutation and polymorphism analysis (52, 53) and identification of bacteria such as Haemophilus injluenzae, Streptococcus pneumonia, Mycoplasma catarrhalis, and Alloiococcus otitidis from upper respiratory tract and HIV and HTLV viruses in various infectious diseases (54, 55). To date, there have been no reports of a one-step differentiation of 0. fo rmigenes strains group I and group 2 using multiplex PCR. This simple DNA-based detection technique could be a novel, time saving, and a highly specificdiagnostic tool in clinical and research laboratories. Pulsed-Field Gel Electrophoresis (PFGE) Diversity in the gut microbiome at the species level has been best assessed in recent years with phylogenetic analysis of I 6S rRNA gene sequences. Bacterial species evolve into different strains due to variations in their gene content either by horizontal gene transfers or mutations. Recent studies suggest that due to sequence conservation, I 6S rRNA gene-based phylogenetic analyses may be limited in the study of intra-species diversity. Therefore, scientists have come up with alternative approaches, beyond l 6S rRNA profiling, to better resolve intra-species diversity (59-62). Bacterial strain typing or 13 genotyping methods have high resolution and characterization power and have been widely used to study the genetic content of bacterial strains (94). Whole genome sequencing of 0. formigenes strains HC-1, HOxCCl 3, and HOxBLS has been done to retrieve the complete gene content of these bacterial species yet being expensive and time demanding, this genotyping method is not affordable or do-able in every scenario (63, 64). Amid the plethora of bacterial subtyping methods (Restriction fragment length polymorphism, Amplified fragment length polymorphism, and Random amplification of polymorphic DNA), PFGE is the widely used technique to identify and characterize bacterial strains in epidemiological and microbiological studies. Escherichia coli 0157:H7, Salmonella, Shigella, and Listeria monocytogenes are extensively studied foodbome pathogens and are tracked by the world's largest PFGE database PulseNet (65, 66). PFGE is a modified version of the conventional electrophoretic technique used to separate large DNA fragments (10 kb tolO mb) digested by restriction endonucleases (RE) under alternatingele ctric fields at different angles (67, 68). Due to its high-resolving power at the genome level, PFGE is a gold standard to study genome diversity and bacterial fingerprinting (69). A standardized protocol for Escherichia coli 0 l 57:H7 published by the Centers for Disease Control and Prevention (CDC) is mostly followed for all PFGE procedure along with some modifications according to the bacterial species (95). The initial steps in the process involve making agarose plugs from active bacterial cultures, and lysis of the bacterial cells in agarose plugs. Agarose plugs are then sliced and subjected to restriction digestion by a suitable endonuclease followed by the separation of DNA fragments on a gel electrophoresis unit with a contour-clamped homogeneous electric field (CHEF).). The 14 DNA fingerprints are then compared to see if the strains are related to each other. According to Tenover's criteria, electrophoresis profiles different from each other in the position of bands can be divided into four categories: indistinguishable strains, closely related strains, possibly related strains, and unrelated strains (69). As such there are no steps for interpreting the banding patterns, but this was used initially followed for nosocomial outbreaks, are now used for all PFGE-associated studies. Studies on Serratia marcescens, an opportunistic pathogen, have reported that out of the 57 isolates examined by PFGE, 26 isolates were unrelated strains, while 27 isolates were related to the outbreak strains (70). It was concluded that PFGE could be a valuable technique to study the diversity among the strains (70). Similarly, two different studies on Salmonella isolates also presented that PFGE is one of the best and alternative method to screen, identify and distinguish the isolates from each other (71, 72). In the past, 16S rRNA profilinghas been able to provide insight into the taxonomic position of 0. fo rmigenes strains (17). However, until now, comparative studies at the genomic level of a variety of 0. fo rmigenes strains have yet to be done. Therefore, one of the objectives of the present study was to study the intra-specific diversity of different strains of 0. fo rmigenes using PFGE. This work will hopefullyhelp us to know about the genetic fingerprinting (diversity) of these strains, making it easier to know more about the strains comparatively, and to relate this information to potential therapeutic strategies linked to the prevention of kidney stone disease. 15 II. Research objectives The objectives of this study were: 1. Isolation of 0.fo rmigenes from fresh human fecal samples. 2. Isolation of 0. fo rmigenes from mouse fe cal samples. 3. One-step detection and differentiation of 0. fo rmigenes pure bacterial strains and new isolates by multiplex polymerase chain reaction. 4. Phylogenetic analysis of 0. fo rmigenes pure bacterial strains and new isolates by using pulse fieldgel electrophoresis. 16 III. Materials and Methods Culture Media, Microorganisms, and Culture Conditions Stock Solutions for Culture Media Preparation. The following solutions were prepared as described below and stored as non-sterilized solutions at 4°C in the refrigerator: Mineral Solution: The solution contained on a per 500 ml basis: 500 ml deionized water; 5 g (134 mM) KC!; 2.5 g (94 mM) NH4Cl; 2.5 g (37 mM) KH2P04; and 0.25 g (2 mM) MgS04·7H20. The mineral solution was diluted 1 :20 in culture media. Sodium Acetate Solution: The solution contained on a per 250 ml basis: 250 ml deionized water, and 17 g (500 mM) CH3C00Na·3H20. The acetate solution was diluted 1 :200 in culture media. Trace Metal Solution: The solution contained on a per 1000 ml basis: 1000 ml deionized water; 1.500 g (5,800 µM) C6H6N06Na3; 0.500 g (2,950 µM) MnS04·H20; 0.100 g (350 µM) FeS04·7H20; 0.100 g (300 µM) CO(N03)2·6H20; 0.100 g (700 µM) ZnCh; 0.050 g (200 µM) NiCh·6H20; 0.050 g (350 µM) H2Se03; 0.010 g (40 µM) CuS04·5H20; 0.010 g (20 µM) A!K(S04)2·12H20; 0.010 g (150 µM) H3B03; 0.010 g (40 µM) Na2Mo04·2H20; and 0.010 g (30 µM) Na2W04·2H20. The trace metal solution was diluted 1 :500 in culture media. Resazurin Solution: The solution contained on a per 100 ml basis: 100 ml deionized water, and 0.1 g resazurin (redox indicator). The resazurin solution was diluted 1: I 000 in culture media. Standard Culture Media. The following culture media were prepared as described below and stored as sterilized media at room temperature: 17 Undefined oxalate broth (UOB): For the cultivation of 0. fo rmigenes, UOB with final oxalate concentrations of 25, 50 or 100 mM and yeast extract concentrations of 0.1 % or 0.5% were used as indicated in this study. UOB contained on a per 200 ml basis: 210 ml deionized water (additional water was added to account for volume lost afterboiling); 0.9 g (25 mM), 1.8 g (50 mM), or 3.7 g (100 mM) potassium oxalate; 0.2 g (0.1%) or 1 g (0.5%) yeast extract (Fisher Bioreagents); 10 ml of mineral solution (see above); I ml of sodium acetate solution (see above); 0.4 ml of trace metal solution (see above); and 0.2 ml ofresazurin solution (see above). All the components were added into a flask double the volume of the amount of medium being prepared. The pH was adjusted to approximately 7. Sodium bicarbonate (1.50 g; 89.3 mM final concentration in medium) was added to the medium and mixed. The medium was boiled for approximately I 0 min while being bubbling with C02. Afterboiling, the medium was cooled down in an ice bucket to room temperature with continuous C02 bubbling. L-cysteine hydrochloride monohydrate (0.10 g; 2.8 mM final concentration in medium) was added to the medium, and the headspace was flushed with C02 until the medium was reduced (colorless). The reduced medium was dispensed ( 10 ml) into culture tubes (l 8x I 50 mm; Belko series 2048; Belko Glass Inc., Vineland, NJ; 27.2-ml approximate stoppered volume at I atm [I 01 .29 kPa]) being flushed with C02. Culture tubes were then sealed with butyl rubber stoppers and aluminum crimp seals and sterilized by autoclaving at 121°C for 15 min and fast exhaust. After autoclaving, the pH of the medium was 6.6-6.8. Defined Glucose Broth (DGB): This medium was prepared exactly as UOB with exception that the potassium oxalate and yeast extract were omitted and 0. 18 g (50 mM) ofD-glucose was added. 18 Anaerobic MRS Broth: The medium contained on a per 100 ml basis: I 05 ml deionized water; 5.5 g Lactobacilli MRS broth (BD™ Difeo™); and 0.025 g (0.025%) of L-cysteine hydrochloride monohydrate. Components were mixed, boiled, and cooled down exactly as described above using argon gas, rather than C02 as the oxygen-free gas. The medium was dispensed ( 10 ml) into culture tubes (l 8x 150 mm; Belko series 2048; Belko Glass Inc., Vineland, NJ; 27.2-ml approximate stoppered volume at 1 atm [101.29 kPa]) under argon. Culture tubes were then sealed with butyl rubber stoppers and aluminum crimp seals and sterilized by autoclaving at 121°C for 15 min and fast exhaust. After autoclaving, the pH of the medium was 6.6-6.8. Microorganisms and Growth Conditions. Nine pure cultures of 0. fo rmigenes strains were used for both PFGE and multiplex PCR studies. All strains were obtained from Dr. Steven L. Daniel's culture collection in the Department of Biological Sciences at Eastern Illinois University and were revived from -80°C glycerol stocks. 0. fo rmigenes strains were grown anaerobically and maintained at 37°C in 25, 50, and 100 mM UOB. Three pure cultures of probiotic strains were used additionally for the multiplex PCR experiments. These strains were grown anaerobically at 37°C in anaerobic MRS broth. Escherichia coli Nissie was used as a control culture and maintained in DGB (Table 3). All cultures were anaerobically transferred (0.5 ml) using sterile I-ml syringes (Becton Dickinson [BD]) and 23-gauge BD needles. When cells were needed for multiplex PCR or PFGE protocols, bacterial culture (10 ml) upon reaching late exponential growth were transferred to multiple 1.5-ml microfugetubes and centrifuged (AccuSpin Micro 17, Fisher Scientific) at 14,000 x g for 5 min. The supernatant was discarded, and the pellet was 19 resuspended and washed in sterile water and centrifuged again at 14,000 x g for 5 min. The supernatant was discarded, and the bacterial cell pellet was processed further according to multiplex PCR to PFGE and protocols. T a ble -3 . P ure b actcna• 1 strams use d.Ill t h c PFCE• an d mu 1 tip • I ex PCR stu d y (A) O.formir.:enes Strains Culture Group Strain Isolate Source Collection HCI Human Feces EIU HOxCCl3 Human Feces Ell.I OxB Sheep Rumen Ell I Freshwater Lake SOx4 EIU Sediment Group I Ox WR Wild Rat EIU OxK Human Feces EIU HOxBLS Human Feces EllJ Group 2 BAI Human Feces EIU Ox GP Guinea Pig Cecum EIU (B) Probiotic Strains Control Escherichia coli Nissie Human r:n; Lactobacillus - Fermented fo od [JU acidophilus NCFM Lactobacillus - Fermented food EIU acidophilus LA-14 Bifidobacterium lactis - Fermented milk nu BL-07 20 Protocol for Detection of O.formigenes in Human and Mouse Feces Culture Media. For detection of 0. fo rmigenes, UOB with 25 mM/50 mM/100 mM oxalate and 0.5% yeast extract concentrations as indicated was required. Apart from UOB, anaerobic dilution solution (ADS), calcium oxalate agar roll tubes, aerobic oxalate broth, anaerobic BHI medium, and BHI plates were also used in this part of the study. Anaerobic Dilution Solution (ADS). ADS contained on a per 200 ml basis: 210 ml deionized water, 10 ml of mineral solution, 0.20 ml of resazurin solution, and 0.10 g (3 .4 mM) of L-cysteine hydrochloride monohydrate. All the components were mixed, and pH was adjusted approximately to 7 by adding 10 N NaOH. The medium was boiled and flushed with argon. Once the medium was pink or colorless, it was cooled down to room temperature in an ice bucket while flushing with argon. The reduced medium was dispensed (7 ml) into culture tubes (l8x 150 mm; Bellco series 2048; Bellco Glass Inc., Vineland, NJ) while flushing with argon. Culture tubes were then sealed and autoclaved as done for standard culture media. Calcium Oxalate Agar Roll Tubes. The medium contained on a per 200 ml basis: 150 ml deionized water; 0. 74 g (20 mM) potassium oxalate; 0.20 g (0.1 % ) yeast extract; 10 ml of mineral solution (see above); 1 ml of sodium acetate solution (see above); 0.40 ml of trace metal solution (see above); 0.20 ml ofresazurin solution (see above); 40 ml of a 1% CaCh·2H20 solution (14 mM final concentration of CaCh·2H20 in culture medium; 0.10 g (2.8 mM) of L-cysteine hydrochloride monohydrate; 1.50 g (89.3 mM) sodium bicarbonate; and 3.50 g agar (Difeo). All the components were added into a flask double the volume of the medium prepared. The medium was mixed and brought to a boil on a hot plate with C02 bubbling. Once the agar was melted, the flask was transferred to a hot water bucket ( �50°C), and the 21 medium was continuously stirred with C02 bubbling. The hot agar medium was tempered for 5 min and then dispensed (7 ml per tube); tubes were sealed and sterilized as mentioned earlier. The tubes were stored in upright position at room temperature. Aerobic Oxalate Broth (AOB). This aerobically prepared medium was UOB but lacked sodium bicarbonate, resazurin, L-cysteine hydrochloride monohydrate, and C02. The remaining components were mixed, dispensed, and autoclaved in 15x100 mm screw cap culture tubes as described above. Brain Heart Infusion(BHI) Broth. Anaerobic BHI broth contained on a per 200 ml basis: 200 ml deionized water; 7.4 g brain heart infusion (BD BBL™); 1.0 g (0.5%) yeast extract; 0.4 g (1 1 mM) glucose; 0.2 ml of resazurin solution (see above); and 1.5 g (89.3 mM) sodium bicarbonate. All the components were mixed and boiled for 10 min and bubbled with C02. After boiling, the medium was cooled down in an ice bucket to room temperature with continuous C02 bubbling. L-cysteine hydrochloride monohydrate (0.10 g with a final concentration of 2.8 mM in the medium) was added to the medium, and the headspace was flushed with C02. When the medium was reduced (colorless), it was dispensed (10 ml), sealed and autoclaved as described above. BHI Agar Plates. Brain heart infusion broth (9.25 g, BD BBL™) and agar (3.75 g, Difeo ™) was mixed in 250 ml of deionized water in a flask and placed on a stirring hot plate. The medium was boiled and stirred till all the agar was melted. The medium was autoclaved for 15 min at 121 °C with slow exhaust. After autoclaving, the medium was tempered in a water bath (approximately 50°C) for 25-30 min and then poured (15-20 ml) into sterile 100 x 15 mm plastic Petri plates. This was all done in a sterile bench. The lids 22 were left slightly open for 10-15 min to prevent condensation while agar was solidifying. The plates were stored at 4°C in cold room. Qualitative Detection of 0. {Ormigenes Using Calcium Oxalate Precipitation Assay. This assay was used to test for the presence of 0. fo rmigenes based on the loss of oxalate from UOB cultures. Calcium chloride solution (!%) was used for the assay. The solution contained 1 g of CaC'2·2H20 and 100 ml of deionized water. Cultures were tested after 3 days of incubation and on day 5, 7, and 10 if the previous test results were negative. For test samples, 0.5 ml of culture and for controls, 0.5 ml of uninoculated UOB media were transferredinto 1.5-ml microfugetubes and centrifuged at 14,000 x g for 5 min. From both test and control tubes, 0.1 ml of supernatant was transferred to two culture tubes (12 x 75-mm disposable culture tubes, Fisherbrand®) and were labeled as Tcalcium oxalate, Twater, and Ccaicium oxalate and Cwater respectively. Later, 0. 1 ml of 1 % calcium chloride solution was added to Tcalcium oxalate and Ccalcium oxalate tubes, 0.1 ml of water was added to Twater and Cwater, and mixed. Finally, 1 ml of water was added to all the tubes and observed forcalcium oxalate precipitation (i.e., the presence of a white, milky precipitate). To obtain numbers for comparison, an additional 2 ml of water was added to all the tubes and the OD was taken at 600 nm vs water as a blank. Human and Animal Subjects Human fecal samples were collected, processed, and tested from two healthy human volunteers at Eastern Illinois University using an internal review board (IRB)-approved protocol (Daniel, #12-102). Volunteers were healthy, non-kidney stone formers, free of 23 gastrointestinal diseases or disorders, and without antimicrobial agent treatment prior (3 months) to the fecal collection. The animal study was approved by the University of Florida Institutional Animal Care and Use Committee. Sample collection and processing was conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals approved protocols (protocol no. 4003). At the University of Florida, eleven healthy C57BL mice were obtained from Charles River Laboratories and were fed a standardized rodent chow (diet 7912; Harlan Teklad, Indianapolis, IN). Fecal Collection and Processing. Culturette EZ collection swabs from BD were used forcollecting human fecal samples. Date and time of fe cal collections were noted on the swab container. Prior to and after collecting samples, the swabs were weighed to determine the approximate wet weight of fecal sample collected. After collection, 2 ml of UOB, containing 25 mM oxalate and 0.1 % yeast extract, or ADS was added to the swab system along with 3-4 3-mm sterile glass beads. The swab system was vortexed repeatedly to mix the fe ces into the added liquid. Mouse fecal samples were collected, added to 1 ml of UOB (20 mM oxalate and 0.1 % yeast extract) and mixed by vortexing for 5 min. Fecal sample mixtures were transferred to serum vials with 8 ml ofUOB (20 mM oxalate and 0.1% yeast extract). Isolation of 0. formigenes from Enrichment Cultures. Two different approaches were used for isolation of 0. fo rmigenes from human fecal samples. With the firstvol unteer, 0.5 ml of vortexed fecal sample was transferred to tubes of UOB (25 mM oxalate and 0.1 % yeast extract) and incubated at 37°C. This enrichment culture was tested for oxalate degradation after 5, 7, and 10 days of incubation by performing the calcium oxalate 24 precipitation test. When cultures were positive for oxalate degradation, they were transferred to secondary and tertiary passages accordingly. Afterthe tertiary passage, serial dilutions of enrichment cultures were prepared. The remainder of the fe cal sample was spun down by centrifugation at 14000 x g for 5 min and used for PCR confirmatory test. For serial dilutions, 0.1 ml of an actively growing enrichment culture was anaerobically transferred to ADS using sterile !-ml syringes and 23-gauge needles and subsequent dilutions of 10-2, 10-4, and 10-6 were prepared. Calcium oxalate agar tubes were heated in an autoclave for5 min and placed in a water bath ( �50°C) for I 0 - 1 5 min to temper the agar medium. Once tempered, 0.2 ml of each dilution series was added to the agar tubes via sterile syringes and needles and mixed gently by inverting the tubes. The tubes were placed horizontally in an ice bucket and rolled on the ice to allow the agar to solidify on the inside walls of the tubes. Final dilutions of enrichment cultures were 2x10-3, 2x10-5, and 2x10-7, respectively. Inoculated tubes were kept upright to avoid contact between colonies and condensation water and incubated at 3 7°C. Duplicate tubes were made for all the dilutions. With the second volunteer, the vortexed fecal sample was directly used to make serial dilutions. The vortexed sample (0.5 ml) was transferred to ADS, and four dilutions 10-2, 10-4, 10-6, and 10-s were prepared. The remaining steps were the same as discussed above for the first volunteer. A portion of the fecal sample (0.5 ml) was also transferred to sterile UOB (25 mM oxalate and 0.1 % yeast extract) and incubated at 37°C. Both volunteers' enrichment cultures were maintained for further PCR studies. With mouse fecal samples, the same approach for isolation was used as for human volunteer 1. The primary enrichment cultures were tested for oxalate degradation by calcium oxalate precipitation test and transferred to fresh UOB (secondary enrichment 25 cultures). Dilutions were prepared in ADS, and 0.2 ml of each dilution was added to tempered calcium oxalate agar tubes and rolled in ice. Tubes were incubated at 37°C until any clear zones were seen. The agar roll tubes were checked daily forthe presence of clear zones. The colonies surrounded by clear zones were streaked onto calcium oxalate agar roll tubes (primary streak). After 5-15 days of incubation, secondary agar roll tubes were streaked from primary streaked agar roll tubes. A special setup was done on a sterile bench to observe, transfer, and streak the colonies with clear zones onto agar roll tubes with continuous flushing of C02 (Figure 4). Figure 4. Setup for streaking calcium oxalate agar roll tubes. Colonies with clear zones were transferred anaerobically to UOB (25 mM oxalate and 0.5% yeast extract) and incubated at 37°C. The growth was measured as optical density 26 ® ™ (OD) at 600 nm with a Spectronic 20 Genesys spectrophotometer. The cultures were checked every day for oxalate degradation with the calcium chloride precipitation test and, when positive for complete degradation, they were transferred to UOB (25 mM oxalate and 0.5% yeast extract) and then adapted to higher oxalate concentrations by transferred to UOB (50 mM oxalate and 0.5% yeast extract) and finally to UOB (100 mM oxalate and 0.5% yeast extract). If UOB was positive for oxalate degradation, gram staining was performedand, if the organisms were identified as gram-negative rods or curved rods, they were subjected to further confirmatory tests. Active UOB cultures were transferred (0.5 ml) to aerobic oxalate broth and anaerobic BHI broth and streaked onto aerobic BHI plates. Broth cultures and streak plates were incubated at 3 7°C for five days. If all confirmatory tests were negative for growth, PCR was performed as a species confirmatory test. If the PCR results were positive for 0. fo rmigenes species and strains, bacterial subtyping by PFGE was also performed to study the intra-species diversity of the strains. Multiplex Polymerase Chain Reaction (PCR) DNA Extraction from Pure Bacterial Cultures and Enrichment Cultures. DNA was extracted using a ZR Fungal/Bacterial DNA Miniprep™ kit (Zymo Research). Pelleted bacterial cells (50-100 mg) from pure and enrichment cultures were resuspended in 200 µl of sterile water and added to a bashing bead lysis tube containing 750 µl of lysis solution. The lysis tube was placed into the bead beater machine at maximum speed for 5 min followed by centrifugation at 10,000 rpm for 1 min. Supernatantfluid was transferred (400 µl) to a Zymo spin filter (orange cap) with a collection tube 1 and centrifuged at 7,000 rpm for 1 min. The Zymo spin filter was discarded, and 1,200 µl of bacteria-binding buffer was 27 added to collection tube I. The mixture was transferred (800 µI) to a Zymo spin column with a new collection tube 2 and centrifuged at I 0,000 rpm for I min. The flow-through was discarded and the process repeated. DNA pre-wash bufferwas added (200 µl) to the same Zymo spin column with a new collection tube 3 and centrifuged at I 0,000 rpm for I min. Next, 500 µI of DNA-wash buffer was added to the same Zymo spin column followed by centrifugation at I 0,000 rpm for I min. Finally, I 00 µI of DNA-elution buffer was added to the Zymo spin column and placed in a 1.5-ml microfuge tube. The system was centrifuged at I 0,000 rpm for 30 sec, and DNA was eluted out in the microfugetube. The Zymo spin column was discarded, and the DNA sample was stored at 4°C. DNA Extraction from Human Fecal Sample. Fecal DNA was extracted using the PureLink™ Microbiome DNA purification kit (Invitrogen). Fecal samples (150-250 mg per sample) were added to 600 µI oflysis buffer in bead-beating tubes and vortexed briefly. Next, I 00 µl of lysis enhancer was added to the same bead-beating tubes and vortexed. This mixture was incubated at 65°C in a water bath for I 0 min. After incubation, the mixture was homogenized in bead beater machine at maximum speed for I 0 min followed by centrifugation at 14,000 x g for 5 min. Supernatant fluid was transferred (400 µI) to a clean microcentrifuge tube and 250 µI of the cleanup buffer added. The mixture was vortexed immediately to ensure uniform precipitation of inhibitors and centrifuged at 14,000 x g for 2 min. Binding buffer (900 µI) was added to 500 µl of the supernatant. From this sample mixture, 700 µl was loaded onto a spin column and centrifuged at 14,000 x g for I min. This step was repeated to make sure the sample mixture passed through the spin column. The spin column was placed in a clean collection tube to which 500 µl of wash bufferwas added and then centrifuged at 14,000 x g for I min. The flow through was 28 discarded, and the spin column was recentrifuged at 14,000 x g for 30 sec to optimize the removal of wash buffer. Lastly, the spin column was placed into a clean microfuge tube with 100 µl of elution bufferand incubated at room temperature for 1 min. The assembly was centrifuged at 14,000 x g for 1 min. The DNA was stored in a microfuge tube at4°C. DNA was quantified using an Epoch microplate spectrophotometer (Biotek). Sterile elution buffer (2 µl) was used as a blank and 2 µl of DNA sample was used for quantification. Duplicates of each sample were analyzed, and the average used as the DNA concentration (ng/µl). DNA Amplification. PCR was performed using PuReTaq1m Ready-To-Galm PCR Beads (GE Healthcare). For each PCR reaction, one Ready-To-Galm PCR Bead, multiplex primer solution (5 µl; 5 pnmoles), DNA template (1-5 µl) based on the DNA concentration (0.25-500 ng/µl), and sterile water was used to make up the final volume (25 µl) of the reaction. The multiplex primer solution consisted of primer sets for species-specific axe gene (4 16 bp) mmFp-Oxf is 5'- CGACGACAATGTAGAGTTGACTGATGGC-3'; mmRp-Oxf is 5'-GATGCTGTTGATACGGAAAGAAGCTTTGC-3', group 1 specific gene (201 bp) Fp-Group I 5'-GACAATGTAGAGTTGACTGATGGCTTTCATG-3'; Rp Group I 5'-TCCTTCGATGTAACCGGCGATAGA-3', and group 2 specific gene (140 bp) Fp-Group II 5'-GACAATGTAGAGTTGACTGATGGCTTTCATG-3'; Rp-Group II 5' TAGAACTTCTGACCATCCTGTT-3' (Integrated DNA Technologies, Inc.). The multiplex primer solution was prepared by adding 190 µl of sterile water to a microfuge tube containing 1 µI of 100 µM primer stock solution of mmFp-Oxf (I :200 dilution; 0.5 µM), 1 µI of 100 µM primer stock solution of mmRp-Oxf ( 1 :200 dilution; 0.5 µM), 2 µI of 100 µM primer stock solution of Fp-Group I/II ( 1:10 0 dilution; 1.0 µM), 1 µI of 100 µM 29 primer stock solution of Rp-Group I ( 1 :200 dilution; 0.5 µM), and 5 µI of 100 µM primer stock solution of Rp-Group II (1 :40 dilution; 2.5 µM). Optimal reaction conditions for thermocycler were initial denaturation at 94°C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 sec, annealing at 60°C at 15 sec, primer extension at 72°C for 20 sec, and a final extension at 72°C for 30 sec. Gel Electrophoresis. Agarose gel (1.5%) was prepared in 0.5X TBE buffer (5X TBE- 10.8 g Tris base; 5.5 g boric acid; 4 ml 500 mM EDTA, pH 8; 200 ml of deionized water; filtered through a 0.2 µm filter unit). GelRed (Biotium, Inc.) was added to each gel (5 µI of GelRed per 100 ml of gel). PCR products (1-2 µI) were loaded with 5 µl of loading buffer which consisted of 4 µl 0.5X TBE and 1 µl gel loading dye (Purple 6X, BioLabs). A low-scale DNA ladder (5 µl; 100 bp; Fisher Scientific) was used. The gel was run for2 hat 100 volts and analyzed with the UV gel doc system (Gel docTM 135 XR+ imaging system, BioRad). Pulse Field Gel Electrophoresis (PFGE) A standard operating procedure forPulseNet PFGE of Escherichia coli 0157:H7 from CDC (Centers forDisease Control and Prevention) was followed for Oformigenes strains. Preparation, Casting, and Lysis of Agarose Plugs. As soon as the 0. fo rmigenes strains reached the exponential growth phase (i.e., an OD at 600 nm of0.25-0.30) in UOB media (I 0 ml; 50 mM or 100 mM oxalate; 0.5% yeast extract), bacterial cells were harvested by centrifugation at 14,000 x g for 5 min. The supernatant was discarded, and the pellet was resuspended in sterile water and centrifuged at 14,000 x g for 5 min. This cell paste was used for plug preparation. 30 Pelleted cells were resuspended in 500-600 µl of cell suspension buffer (50 ml of 1 M Tris, pH 8.0; 100 ml of 0.5 M EDTA, pH 8.0; 500 ml of sterile ultrapure water) to make the optical density (600 nm) of the cell suspension between 1.5-2.0. In a microcentrifuge tube, 400 µl of bacterial cell suspension, 20 µl of proteinase K (20 mg/ml stock solution), and 400 µl of melted 1 % Seakem gold agarose (melted agarose was maintained in a water bath at 50-55°C) were gently mixed and pipetted into plug molds (Figure 5). The plugs were allowed to solidify atroom temperature for 10-15 min. Figure 5. Reusable plug mold formaking bacterial plugs. The plugs were transferred from the plug mold to 50-ml Beckman centrifuge tubes. The centrifuge tubes contained 5 ml of cell lysis buffer (25 ml of 1 M Tris, pH 8.0; 50 ml of 0.5 M EDTA, pH 8.0; 50 ml of 10% N-Lauroylsarcosine sodium salt; 500 ml of sterile Ultrapure water) and 25 µl of proteinase K (20 mg/ml stock solution). The tubes were incubated at 54-55°C with constant mixing for 1.5-2 h. After lysis, the plugs were washed with pre-heated sterile distilled water (2-3 times) followed by preheated sterile TE buffer (3-4 times; 10 ml of 1 M Tris, pH 8.0; 2 ml of 0.5 M EDTA, pH 8.0; 1000 ml of sterile 31 ultrapure water) with incubation and agitation at 54-55°C for 10-15 min. After washing, the plugs were stored in freshTE buffer at 4°C in refrigerator until used fornext step. Restriction Digestion of DNA in Agarose Plugs. This included two steps: pre restriction incubation and restriction enzyme master mix incubation. The pre-restriction master mix was prepared by diluting the 1 OX restriction buffer (Thermo Scientific) 1 : 10 with sterile distilled water. Per plug slice, 20 µl of 1 OX restriction buffer was diluted in 180 µl of sterile distilled water. The plugs were sliced 2-2.5 mm wide with a sterile scalpel (Figure 6) and added to 200 µl of the pre-restriction master mix. The plug slices were incubated in an Eppendorfthermomixer at 300 rpm at 37°C for 5-10 min. The enzyme Xbal ( 10 U/µl; 1500 U; Thermo Scientific) was used for restriction digestion of cell DNA in agarose plugs. Per plug slice, restriction enzyme master mix was prepared by diluting 5 µl Xbal, 20 µl of 10 X restriction buffer in 173 µl of sterile distilled water. After incubation, the plug slices were transferred to 200 µl of restriction enzyme master mix and incubated at 37°C for 1.5-2 h in an Eppendorf thermomixer at 300 rpm. Afterdigestion, the buffermixture was removed and 200 µl of0.5X TBE buffer (5X TBE- 10.8 g tris base; 5.5 g boric acid; 4 ml 500 mM EDTA, pH 8; 200 ml deionized water; filtered through 0.2 µm filterunit) was added to the plug slices. If plug slices were not used on the same day, they were stored at 4°C until the next step. Gel Electrophoresis. Electrophoresis consisted of a Chef mapper, pump, cooling module, and electrophoresis chamber (Figure 7). Prior to running the gel (30 min), the Chef mapper unit was turned on, the pump was set to a flow rate of 1/1 min (i.e., 70), and the cooling module was turned on and set to 14°C. It was made sure that the buffer was flowing through the system uniformly without any bubbles or debris. 32 Figure 6. Setup forplug slicing in a sterile petri plate. Figure 7. From leftto right showing an electrophoresis chamber, a pump on top, a cooling module on bottom, and a Chef mapper unit (all from BioRad). Seakem gold agarose (1%) was prepared in 0.5X TBE buffe r and cast in a gel box. The plug slices were loaded at the bottom and gently pushed towards the front of wells with the help of a sterile spatula. The wells were sealed with I% Seakem gold agarose and allowed to solidify for 5 min. Freshly prepared 0.5X TBE buffer(2-2.2 1) was added to the chamber. The cast and loaded gel was taken out of the gel box and placed into the 33 electrophoresis chamber on a black gel frame. The chamber was closed, and the following electrophoresis conditions were set on the Chef mapper: auto algorithm; low MW, 50 kb; high MW, 400 kb; run time, 18 h; initial switch time, 6.76 s; and final switch time, 35.38 s. The gel was processed further after 18 h. Staining and Destaining of Agarose Gel. The gel was placed into a staining box containing 40 µl of GelRed (Biotium, Inc.) and 400 µl of 0.5X TBE buffer. The gel was stained for20-25 min. After staining, the gel was destained in 500 µl of 0.5X TBE buffer for 5-10 min. The gel was visualized under the UV gel doc system (Gel doc™ 135 XR+ imaging system, BioRad). Analysis of Gel with Bioinformatics Tool. Banding patterns observed on gels were analyzed by the Bionumerics Applied Maths software. High-PerformanceLiquid Chromatography (HPLC) Preparation of HPLC standards. Stock solutions of 20 mM potassium oxalate and 20 mM potassium formate were prepared in 25 ml volumetric flasks with deionized water. The 20 mM stock solutions were serially diluted I :2 in deionized water to yield final concentrations of 10 mM and 5 mM oxalate or formate for calibration of the HPLC unit. Preparation of Sterile Culture Media. For HPLC analysis, I-ml samples were taken from culture media and transferred to 1.5-ml microfuge tubes. The samples were centrifuged at 14,000 x g for 5 min. Supernatant fluids were collected and dispensed into HPLC vials via microfiltration through 0.2 µm, 13-mm-disposable-syringe filters (Fisher Scientific).Samples were stored at -20°C until analyzed. 34 HPLC conditions. The following HPLC equipment and conditions were used for analysis of standards and cultures: Beckman Gold System (125 solvent module, model 166 variable wavelength detector, Jasco RI-1530 refractive index [RI] detector, Eppendorf CH-30 column heater, model 508 autosampler, and 32 Karat software,version 5); Aminex HPX-87H (300-mm long) analytical column (BioRad); 0.01 N H2S04 solvent; solvent flow rate of 0.6 ml/min; column temperature of 55°C; injection size of 10 µl; and oxalate and formate were detected by UV detector at 210 nm at a retention time of 6.35 and 13.65 min, respectively, with a total run time of 45 min per sample. 35 IV. Results Growth of O.formigenes Strains in UOB 0. fo rmigenes strains were grown in UOB and adapted to oxalate by growing cells in the presence of increasing concentrations of oxalate from 25 mM to 50 mM to 100 mM. This avoids nutritional and environmental stress upon the microorganisms. Like previous reports, these organisms showed increased growth and biomass with increasing oxalate concentrations ( 18). 0. fo rmigenes strains examined achieved late exponential phase and entered the early stationary phase in less than 24 h. Growth (final OD at 600 nm) ranged between 0.20-0.30. Qualitative analysis of the culture was done before each transfer using the calcium oxalate precipitation test to ensure oxalate degradation and thus the presence of 0. fo rmigenes. Afterthe third transfer, cultures were used foreither PFGE or multiplex PCR studies. In general, strains specific to group 1 grew more efficiently and higher in biomass rather than strains specific to group 2. The latter strains were also foundto be more difficult to maintain over time. Isolation and Characterization of O.formigenes from Human and Mouse Feces For isolation. calcium oxalate agar plates in an anaerobic pouch system were initially used but due to the lack of a proper anaerobic environment these proved to be unreliable. Instead, calcium oxalate agar roll tubes were used as described previously (20). Dilutions in ADS were prepared, and, from each dilution, duplicate calcium oxalate agar roll tubes (melted and tempered agar) were inoculated, rolled in ice, and incubated upright at 37°C. With human volunteer 1, colonies with clear zones were seen in all three-dilution 4 series agar roll tubes containing 10-2• 10- • and 10-6 ml of enrichment culture after 10-15 36 days of incubation. The absence of other bacterial growth and the presence of obvious clear zones were the criteria used to select 1 o-6 roll tube clear zone colonies for streaking onto primary and secondary agar tubes. Two colonies from secondary streaked agar tubes were picked and inoculated into UOB (25 mM oxalate and 0.5% yeast extract). Both isolates grew well in UOB containing 25 mM, 50 mM, and 100 mM oxalate and reached stationary phase in less than 24 h. Isolates were gram-negative rods, sometimes slightly curved. Two isolates VIA and VlB were obtained, and both were positive for oxalate degradation and formate production (Table 4). Neither isolate grew in anaerobic BHI broth or aerobic oxalate broth or on aerobic BHI plates as a part of confirmatory tests (Table 5). With human volunteer 2, the same isolation approach as with volunteer 1 was used except with volunteer 2 fe ces rather than enrichment cultures were diluted and transferred to calcium oxalate roll tubes. Three isolates V2A (10-4 g of feces),V2B (10-4 g of feces), and V2C (10-6 g of fe ces) were isolated from volunteer 2 (Table 4). Unlike isolates from volunteer 1, the 3 isolates from volunteer 2 were not fast growers in UOB containing 25 or 50 mM oxalate. However, isolates were gram-negative rods and positive for oxalate degradation and formate production but took �48 h to reach stationary phase. Surprisingly, these 3 isolates did not grow in UOB containing 100 mM oxalate. All isolates failed to grow in anaerobic BHI broth or aerobic oxalate broth or on aerobic BHI plates (Table 5). Mouse isolates proved more difficultto grow in the calcium agar roll tubes. Out of 11 mouse enrichment cultures tested (all positive for oxalate degradation), only two enrichment cultures yielded three isolates C3 86-1 (10-2 ml of enrichment culture), C3 86-2 ( 10-2 ml of enrichment culture), and C405 (I0- 4 ml of enrichment culture). Colonies with clear zones appeared after �2 months of incubation. These colonies were streaked onto 37 primary and then secondary calcium oxalate agar rolls; streaked tubes took significantly less time to yield colonies with clear zones (7-12 days). Isolates were gram-negative rods and positive for growth, oxalate degradation, and formate production in UOB containing 25 mM, 50 mM, and 100 mM oxalate (Table 4). Isolates were negative for growth in aerobic oxalate broth or anaerobic BHI broth or on aerobic BHI plates (Table 5). Table 4. Summarv of 0. formi2enes isolates from human and mouse feces Enrichment culture No. of Oxalate Formate Stud' subject dilution isolates degradation production Volunteer 10-6 2 Yes Yes 1 Human 10-4 2 Yes Yes Volunteer 2 10-6 1 Yes Yes C386 10-2 2 Yes Yes Mouse C405 10-4 1 Yes Yes Table 5. Summary of 0.form igenes isolates from human and mouse feces Enrichment Gram Aerobic Aerobic Study culture or No. of stain Anaerobic oxalate BUI subject fecal isolates and cell BUI broth broth plate dilution shape Volunteer Gram (-) No No 10-6 2 No growth H I rod growth growth u Gram (-) No No m 10-4 2 No growth rod growth growth a Volunteer n 2 Gram (-) No No 10-6 1 No growth rod growth growth M Gram (-) No No C386 10-2 2 No growth 0 rod growth growth u Gram (-) No No s C405 10-4 1 No growth e rod growth growth 38 Multiplex PCR Analysis Previous studies suggested that strain groupings have been done based on the axe gene using different sets of primers in separate PCR assays (25). The aim of the study was to generate a multiplex PCR system, consisting of a multi-primer mix directed at the axe gene in a single PCR assay, which allows the simultaneous detection of 0. fa rmigenes as well as the differentiation between group 1 and group 2 strains. Differentiation of 0. fOrmigenes Group 1 and Group 2 Strains. Genomic DNA from nine pure cultures of 0. fa rmigenes: group 1 strains HCl, HOxCC13, OxB, and SOx4, and group 2 strains HOxBLS, OxK, BAI, OxGP, and OxWR (18, 20-23) was amplifiedby multiplex PCR system. All the strains showed an amplicon product of 416 bp for 0. fa rmigenes (species-specific); HCl, HOxCC13, OxB, SOx4, and OxWR showed an ampIi con product of 210 bp for 0. fa rmigenes group 1 specificstrains and HOxBLS, OxK, BA 1, and OxGP showed an amplicon product of 140 bp for0. fa rmigenes group 2 specific strains (Figure 8 and 9). These results agreed with previous studies relative to the groupings of these strains of 0. fa rmigenes (24-26). 39 oxc (416 bp) oxc (416 bp) Grp 1 (201 bp) --.;,. Grp 1 (201 bp) --.;,. Grp 2 (140 bp) �igure 8. A multiplex PCR gel Figure 9. A multiplex PCR image of amplified genomic DNA gel image of amplified from pure bacterial cultures having genomic DNA from pure 0. fo rmigenes species-specific oxc bacterial culture OxWR gene ( 416 bp) , group I strain having 0. fo rmigenes specific gene (201 bp), and group 2 species-specific oxc gene strain-specific gene (140 bp). L= ( 416 bp ), group I strain 100-bp ladder; I= E.coli nissle specific gene (201 bp). L= (control); 2= HC l; 3= HOxCC13; 100-bp ladder; I= OxWR 4= OxB; 5= SOx4 (group I strains); (group I strain). 6= HOxBLS; 7= OxK; 8= BA 1; and 9= OxGP (group 2 strains). 40 Detection of 0. formigenes in Human Fecal Samples. Both volunteers were checked for colonization by 0. fo rmigenes with the newly developed multiplex PCR assay. Amplified genomic DNA of volunteer 1 showed amplicon products of 416 bp for 0. fo rmigenes (species-specific) and 210 bp for 0. fo rmigenes group 1 (group 1 specific); amplifiedgenomic DNA of volunteer 2 was positive for 416 bp for O. formigenes (species- specific) but exhibited a 140-bp amplicon for 0. fo rmigenes group 2 (group 2 specific) (Figure 10). The fecal enrichment cultures of volunteer 1 and 2, which were positive for oxalate degradation, were confirmed with the multiplex PCR assay forthe presence of 0. fo rmigenes (species-specific)am plicon 416 bp, 210 bp for0. fo rmigenes group 1 (group 1 strain specific) and 0. fo rmigenes (species-specific) amplicon 416 bp, 140 bp for 0. fo rmigenes group 2 (group 2 strain specific),respectively (Figure 11 and 12). ' � y � � --7 oxc, 416 bp - - .. --7 Grp I, 201 bp --7 Grp 2, 140 bp Figure 10. A multiplex PCR gel image of amplified genomic DNA from two human volunteers' fecal samples with 0. fo rmigenes species-specific oxc gene ( 416 bp), group 1 strain-specific gene (201 bp ), and group 2 strain specific gene (140 bp ). L= 100-bp ladder; VI= volunteer 1; and V2= volunteer 2. 41 -7 oxc, 416 bp -7 OXC, 416 bp Grp 2, 140 bp Figure 11. A multiplex PCR gel Figure 12. A multiplex PCR gel image of amplified genomic DNA image of amplified genomic DNA from human volunteers' fecal from human volunteers' fecal enrichment cultures with 0. enrichment cultures with 0. fo rmigenes species-specific oxc fo rmigenes species-specific oxc gene ( 416 bp ), group 1 strain gene ( 416 bp ), group 2 strain specific gene (201 bp ). L = 100-bp specific gene (140 bp). L = 100-bp ladder; V 1 = volunteer 1. ladder; V2 = volunteer 2. The next approach to optimize the multiplex PCR system was to see how the system performed when human fecal DNA samples and O. formigenes DNA samples were mixed with each other at high and low concentrations. PCR reaction mixtures containing volunteer 1 fecal DNA sample (250 ng/µl; positive for group 1) with DNA from a 0. fo rmigenes group 2 strain (25 ng/µl) and volunteer 2 fecal DNA sample (250 ng/µl; positive for group 2) was mixed with DNA from a 0. fo rmigenes group 1 strain (25 ng/µl) were prepared and amplified. Unexpectedly, the strain-specific amplicons from fecal DNA did not appear in the presence of 0. fo rmigenes DNA (results not shown). The reasons forthis 42 absence of strain-specificamplicons are unclear. The experiment was repeated multiple times with different multi-primer solution concentrations, different thermocycler conditions, a new DNA extraction kit, different high and low DNA concentrations but none of the conditions used permitted the formation of the strain-specific amplicons from fecal DNA. However, a slight modification was done before the DNA extraction step. Rather than combining extracted DNA from fecal samples and 0. fo rmigenes cultures, the diluted human fecal sample (1 ml) and 0. fo rmigenes cultures (100 µl) were combined and then the DNA was extracted. When amplification with the multiplex primer system was performed, the expected outcomes were yielded in all the lanes as shown in Figure 13. oxc, 416 bp ------7 Grp I, 201 bp ------7 Grp 2, 140 bp Figure 13. Multiplex PCR gel image of human fecal DNA ixed with pure 0. fo rmigenes DNA showing 0. fo r mi genes species-specific oxc gene ( 416 bp ), group 1 strain-specific gene (201 bp), and group 2 strain-specific gene (140 bp). L = 100-bp ladder; I = Volunteer 2 fecal DNA; 2 = Volunteer 2 fecal DNA + HOxCC13 (group 1); 3= Volunteer 1 fecal DNA; 4 =Volunteer I fecal DNA + HOxBLS (group 2); 5 = Volunteer I fecal DNA + Volunteer 2 fecal DNA; group I specific strains (red color); and group 2 specific strains (green color). 43 Analysis of the Specificity of Multiplex Primers with Probiotic Strains. Previous studies have identified that probiotic strains of lactic acid bacteria are capable of oxalate consumption and have a pathway for oxalate metabolism similar to that of 0. fo rmigenes (76-78). The aim of this experiment was to see if the newly developed multiplex primers for detection of oxc in 0. fo rmigenes exhibited any specificity (i.e., cross-reactivity) with oxc present in lactic acid bacterial strains. Three probiotic strains, Lactobacillus acidophilus NCFM, Lactobacillus acidophilus LA-14, and Bifidobacterium lactis Bl-07 were used. Only amplified genomic DNA from L. acidophilus LA-14 showed a very faint 201-bp amplicon band (Figure 14). None of the other two organisms showed any amplicon bands. ------;:. Grp 1, 201 bp Figure 14. Multiplex PCR gel image of amplified genomic DNA of probiotic strains having very faint band for group 1 strain-specific gene (201 bp ). L = 100-bp ladder. 44 Confirmation of 0. formigenes isolates from human feces. 0. fo rmigenes new isolates VIA, VIB, V2A, V2B, and V2C from human feces were also confirmed by the newly developed multiplex PCR system. Similar to fecal and enrichment samples, all the 0. fo rmigenes isolates were positive for the species-specific 4 I 6-bp amplicon (Figure I 5). As anticipated, 0. fo rmigenes isolates from volunteer I were group I specific with amplicon size of 2 I 0 hp (Figure I 5) and isolates from volunteer 2 were group 2 specific with amplicon size of I 40 hp (Figure I 6). - - - � oxc, 416 bp - -7 oxc, 416 bp - - � Grp I, 201 bp -7 Grp 2, 140 bp Figure 15. A multiplex PCR gel Figure 16. A multiplex PCR gel image image of two 0. fo rmigenes human of three 0. fo rmigenes human fecal fecal isolates amplified genomic isolates amplified genomic DNA from DNA from volunteer I with species volunteer 2 with species-specific oxc specific oxc gene ( 4 I 6 hp), group I gene ( 416 hp), group I strain-specific strain-specific gene (20 I hp), and gene (201 hp), and group 2 strain group 2 strain-specific gene (I 40 hp). specific gene (I40 hp). L = 100- hp L = 100-bp ladder; VIA = volunteer ladder; V2A = volunteer 2, isolate A; I, isolate A; and VIB = volunteer I, V2B = volunteer 2, isolate B; and V2C isolate B. = volunteer 2, isolate C. 45 Confirmation of 0. for migenes isolates from mouse feces. Eleven mouse enrichment cultures were tested for the presence of 0. fo rmigenes and group 1 or group 2 strains with the newly developed multiplex PCR assay. Amplified genomic DNA from all mouse enrichment cultures showed amplicon products of 416 hp for 0. fo rmigenes (species- specific)and 210 hp for 0. fo rmigenes group 1 (group 1 specific) (Figure 17). The results demonstrated that the mice were colonized by 0. fo rmigenes group 1 strain(s). Three new 0. fo rmi genes isolates C3 86-1, C3 86-2, and C405 obtained frommice were also confirmed by the multiplex PCR system. As observed with the enrichment cultures, all isolates were positive for the presence of 0. /ormigenes with an amplicon size of 416 hp and a group 1 specificamp Iicon product size of 210 hp (Figure 18). - ----;;. oxc, 416 hp - Grp I, 201 hp Figure 17. A multiplex PCR gel image of amplified genomic DNA from 11 mice enrichment cultures having 0. fo rmigenes species-specific oxc gene ( 416 hp), group 1 strain-specificgene (201 hp). L = 100-bp ladder. 46 oxc, 416 bp Grp I, 201 bp Figure 18. A multiplex PCR gel image of amplified genomic DNA from mouse isolates having 0. fo rmigenes species-specificoxc gene ( 416 bp ), group 1 strain-specificgene (201 bp ). L= 100-bp ladder. HPLC Analysis of Standards, Sterile Culture Media, and Cultures Three different standards (20 mM, 10 mM, and 5 mM) of oxalate and formate were used to calibrate the HPLC unit. In all the three concentrations, oxalate showed a retention time near to 6.9 min and formate showed a retention time near to 14.25 min. Two pure O. formigenes cultures HC-1 and HOxBLS, O. formigenes isolate HOxSDl from volunteer 1 , 0. fo rmigenes isolate HOxNPl from volunteer 2, and 0. fo rmigenes 47 isolates MOxC386-l, MOxC386-2, and MOxC405 from mice were subjected for the quantification of oxalate consumption and formate production. All strains and isolates completely consumed the oxalate and converted it into formate. Nomenclature of New Isolates With the help of confirmatory tests and the multiplex PCR system, the study was able to generate five new 0. fo rmigenes human isolates from two healthy volunteers. From volunteer 1, VIA and VlB were designated as HOxSDl and HOxSD2 respectively. From volunteer 2, V2A, V2B, and V2C were designated as HOxNPl, HOxNP2, and HOxNP3 respectively. Isolates from mice C386-1, C386-2, and C405 were named MOxC386-l, MOxC386-2, and MOxC405 respectively. PFGE Analysis Characterization of bacteria fromspecies to subspecies level is an important aspect in the study of the intra-species diversity among organisms. In the past, this was best achieved by bacterial subtyping methods such as PFGE which is considered one of the gold standard methods (84). The present study was directed towards understanding the phylogenetic relatedness of 0.fo rmi genes strains using PFGE. The results of the CHEF Mapper profiles were visualized by GelDoc imaging system and further analyzed by BioNumerics Applied Maths software forstrain comparisons. All 0. fo rmigenes strains (stock cultures from the EIU culture collection) and new isolates from humans and mice (this study) produced reproducible DNA banding patterns (Figure 19, 20, and 21) with each yielding between 12-20 bands (Table 6). All group 1 48 specific strains HC 1 (isolated from human feces) and HOxCC 13 (isolated from human feces), OxB (isolated from sheep rumen), and SOx4 (isolated from fresh water lake sediment) showed a tight clustering with each other (Figure 22) while the other group 1 specific strain Ox WR (isolated from the wild rat cecum) was tightly associated with group 1 specific human isolates HOxSDl and HOxSD2 (Figure 23). Relative to band differences, HCl and HOxCC 13 were different by two bands while OxB showed a 4-band difference with HC l and a 7-band difference with HOxCC13. OxWR showed an eight and ten band difference with HOxSDl and HOxSD2, respectively, whereas HOxSDl and HOxSD2 were different from each other by only three bands (Figure 20 and Table 7). Group 2 specific strains OxK (isolated from human feces), HOxBLS (isolated from human feces),and OxGP (isolated from the guinea pig cecum) showed high similarity with each other but strain BAI (Group 2 strain; isolated from human feces) was closer to the group 1 specific strain SOx4 (Figure 22). All four of the group 2 specific strains tested were different from each other by more than 10 bands (Figure 19). In contrast, Group 2 specific human isolates HOxNPl, HOxNP2, and HOxNP3 presented 100% similarity with each other and were very similar to group 1 specific human isolates (HOxSDl and HOxSD2) and the wild rat strain Ox WR (Figure 23 and Table 7). Group 1 specific mouse isolates were compactly grouped with each other where MOx-C386-1 and MOx-C386-2 displayed identical PFGE profiles. Mouse isolates did not show closeness with any of the strains examined (Figure 21, 23 and Table 7). 49 Figure 19. A PFGE DNA fingerprint of 0. fo rmigenes pure strains where E. coli Nissle (ECN = control); green color represents group 1 specific strains and red color represents group 2 specific strains. 50 Figure 20. A PFGE DNA Figure 21. A PFGE DNA fingerprint of 0. fo rmigenes fingerprint of 0. human isolates green color fo rmigenes mouse isolates represents volunteer 1, group 1 green color represents specific isolates and red color group 1 specific isolates represents volunteer 2, group 2 specific isolates. 51 T a ble 6 . N um b er o f to ta I b an d so b tained in PFGE fim!!erorint Strains No. of bands ECN3 24 HC l b 17 HOxCC 13b 17 OxBb 17 SOx4b 17 Ox Kc 14 HOxBLSc 16 BA ie 18 OxGPc 12 OxWRb 15 HOxSDlb 16 HOxSD2b 17 HOx Pl c 16 HOxNP2c 16 HOxNP3c 16 C386-l b 20 C386-2b 20 C405b 21 a E.coli Nissie as a control b 0. fo rmigenes strains belonging to group 1 (In red) c 0. fo rmif!enes strains belonging to group 2 (In green) 52 I/) 8 � ... I I T . I I �. I 88.589.� 85.7 864 89.� 80.3 88.5 � 98f[ 97.0 E.coli nissle Figure 22. Phylogenetic tree based on analysis of PFGE profile showing 0. fo rmigenes group 1 specific strains in green and group 2 specific strains in red. Bootstrap values are shown at the branch points in percentage of 1000 replications. 53 ,,, I !fl Iljll I ,f, I 1¥1 I !er! I !flI! '! I If! I I� 88.5 856 91 1 87.5 970 93.5 89.9 846 97.0 81 9 MOxC388-1 ����������������...... o MOxC388-2 MOxC405 E.coli nissle Figure 23. Phylogenetic tree based on analysis of PFGE profile of 0. fo rmigenes pure strains and isolates showing 0. fo rmigenes group 1 specific strains and isolates in green and group 2 specific strains and isolates in red. Bootstrap values are shown at the branch points in percentage of 1000 replications. 54 Table 7. Summary of Multiplex PCR detection and PFGE analysis Fecal Fecal PFGE Subject enrichment Isolates samples profile cultures Group HoxSDl 1 Vo lunteer 97% Group 1 Group 1 1 similarity Group HoxSD2 1 Group Human HoxNPl 2 Vo lunteer Group 100% Group 2 Group 2 HoxNP2 2 2 similarity Group HoxNP3 2 Group MOxC386-1 1 100% C386 Group 1 Group 1 Group similarity MOxC386-2 Mouse 1 Group 99% C405 Group 1 Group 1 MOxC405 1 similarity 55 V. Discussion 0. fo rrnigenes is responsible for sustaining oxalate homeostasis by maintaining a healthy symbiotic relationship with the host via oxalate degradation and the prevention of intestinal oxalate absorption (73, 74). With its exceptional capability of metabolizing only oxalate as a source of carbon and energy, 0. fo rmigenes is distinct from other oxalate degrading gut bacteria (18). In the past, several 0. fo rrnigenes strains have been isolated from different sources (18-23) but isolation is difficult. 0.fo rmigenes is a strictly anaerobic bacterium and requires special conditions for culturing, isolation, and identification. 0. fo rmigenes is typically isolated as bacterial colonies surrounded by clear zones in a calcium oxalate-rich medium (19) or detected in broth cultures by the calcium oxalate precipitation assay (18). This study was able to produce eight isolates of 0. fo rrnigenes from fecal samples: two isolates from human volunteer 1 (HOxSD 1 and HOxSD2), three isolates from volunteer 2 (HOxNPl, HOxNP2, and HOxNP3), and three anaerobic oxalate degrading isolates from laboratory mice (MOxC386-1, MOxC386-2, and MOxC405. All these isolates grew better in undefined media (with yeast extract) rather than in defined media (no yeast extract) as previously observed (24). Isolates were gram-negative rods and were morphologically the same (24) but contrasted in growth potentials in undefined media. Identification and classification (grouping) of 0. fo rmigenes isolates were further confirmed by a multiplex PCR system which emerged as a novel technique in this study. Anomalies in oxalate metabolism can lead to several clinical conditions, and some are linked with less 0. fo rrnigenes colonization rates in the human intestinal tract (9, 13, 56- 58). For rapid diagnosis of 0. fo rmigenes in clinical conditions, it is not always possible to use anaerobic culture techniques (75). In addition, culturing techniques are not efficacious 56 hence, many times failed to confirm the existence of 0. fo rmigenes. To overcome these culture-dependent limitations, the oxc gene responsible for oxalate metabolism in 0. fo rmigenes has been cloned, sequenced, and used to develop a rapid, culture-independent, DNA-based PCR assay for the identification and classification of 0. fo rmigenes (25, 4 7). A study based on the combined PCR detection of both 16S rRNA and oxc gene were able to detect the 0. fo rmigenes colonization fromthe human fecal samples of Japanese subjects (96). Another study focused on quantification of 0. fo rmigenes by quantitative competitive-template PCR (QC-PCR) was also significantly faster and sensitive in quantifyingthe 0. fo rmigenes genomes in human fecal samples (75). A study conducted to check the colonization of 0. fo rmigenes in Ukrainian children with the help of genus and group-specific oligonucleotide sequences were also able to detect and classify the bacterium in human fecal samples (35). In this study, a multiplex primer mix consisting of three sets of primers targeting different portions of the oxc gene in 0. fo rmigenes was designed. This multiplex system was rapid and reliable, yielding a species-specific amplicon ( 416 bp ), a strain-specific group 1 amplicon (201 bp), and a strain-specific group 2 amplicon (140 bp). When tested with fresh human fecal samples, human and mouse enrichment cultures, and stock cultures of 0. fo rmigenes, the multiplex assay produced results that were in accordance with previous reports (35, 43). For example, in a previous study (75), the specificity of the primers constructed for PCR detection of the oxc gene in 0. fo rmigenes has been cross-checked with other 24 bacterial strains including E. coli, and L. acidophilus found in gut microbiota. Not a single bacterial strain other than 0. fo rmigenes was found to produce the desired amplicon 57 product, indicating that the primers used were distinctively specificfor the target organism (75). In the present study, the multiplex primer specificity was tested against L. acidophilus NCFM, L. acidophilus LA-14, and B. lactis BI-07. L. acidophilus NCFM possesses a gene encoding oxalyl-CoA decarboxylase which is extremely homologous to 0.fo rmigenes oxc gene (76). The oxc gene in L. acidophilus LA-14 shows 100% homology with L. acidophilus NCFM (77) while B. lactis shares a 56% nucleotide sequence similarity with 0. fo rmigenes oxc gene (78). In the present study, only a very faint 201-bp band was detected with L. acidophilus LA-14, suggesting a common nucleotide sequence between this organism and 0.fo rmigenes. In a multiplex PCR system, it is difficult to optimize the conditions perfectly to enable all the primer sets to work equally as it increases the chances of obtaining spurious bands (79). Spurious bands may result from variations in the interaction of PCR reagents (80), inappropriate template concentrations (81 ), variations in the thermocycler profile, faultin primer designing, or human experimental errors (82). By targeting all these possibilities, the non-specific interactions can be reduced. This study showed that the primer sets were compatible with each other with no interference and highly specificfor the target organism. This robust, automated, quick, and sensitive system can be very convenient for the one step identificationof 0. fo rmigenes species and strains for diagnostic purposes. The second goal of this study was to understand the genomic diversity of nine 0. fo rmigenes strains (stock cultures from the culture collection), five new isolates of 0. fo rmigenes from two human volunteers, and three new mouse isolates of O. formigenes by using PFGE. A control strain E. coli Nissle was also included in the study. A control strain is necessary to process along with the other isolates to make sure that each step (e.g., cell 58 lysis, restriction digestion, and electrophoretic run parameters) is working appropriately and that the banding patternsobserved are reproducible and consistent with results obtained from previous studies for the same strain (69). In this regard, banding patterns for E. coli Nissie were reproducible and matched those in previous reports (83), thus validating the PFGE procedure used in the present study. According to reports, the PFGE profilesshould be reproducible within and between the laboratories to ensure the consistency and reliability of the data (84). The PFGE profiles of all the strains and new isolates in the present study consisted of 12-21 distinct bands, all which were reproducible each time the assay was performed. Group 1 specific strains HC-1 and HOxCC13 showed a tight clustering in the dendrogram. This high degree of relatedness correlated to only a two-band position difference in the profiles may be related to the loss or gain of nucleotide bases through such genetic events as point mutations, deletions, and insertions. Another possibility for these differences could be that the fragments generated through genetic events are so small that they migrate out of the gel causing loss of bands (69). Whole genome sequencing ofHC-1 and HOxCC13 also proved the close proximity ofHC-1 and HOxCC13 strains observed in the dendrogram. At the protein level, about 97% of coding sequences of HC-1 genome showed greater than 99% similarity with the HOxCC 13 coding sequences. The remaining dissimilarity in the coding sequences was stated as hypothetical and phage-related proteins (63). Strains having difference in profiles up to three bands position can be considered as closely related strains while profiles having six bands differences are considered as possibly related strains (69). Profiles completely identical to each other are considered as 59 similar strains. Three-band differences can be a result of a single genetic event while six band differences can be due to two genetic events (69). OxB is clustered with the strains HC-1 and HOxCC13 posing 4-to7-band differences. In addition, three strains (HC-1, HOxCC 13, and OxB) isolated from human feces belong to group 1 with a clustering in the dendrograrn proves the previous group classification and validating them as closely related strains. The restriction fragment patterns in the PFGE profile of group 1 specific strain SOx4 varied from group 2 specific strain HOxBLS by more than ten bands, suggesting they are unrelated strains which is in agreement with previous results where they are placed in different groups (24, 25). The phylogenetic analysis based on 16S rRNA gene sequences demonstrated a tight proximity (close relatedness) of strain SOx4 and HC-1 which the data from the present study supports (85). New human fecal isolates HOxSDl and HOxSD2 from volunteer 1 were characterized as group 1 specific strains based on the multiplex PCR detection system. The PFGE profile showed tight clustering with 3 bands difference with each other and concluded that these two isolates are closely related strains. Concurrently, human fecal isolates HOxNPl, HOxNP2, and HOxNP3 from volunteer 2 were assigned as group 2 specific strains by multiplex PCR assay. Upon viewing the PFGE banding pattern profiles, they presented 100% similarity indicating they were indistinguishable (69). Based on these observations, of the five isolates generated from the two human volunteers, a total oftwo differentstrains were associated with volunteer 1 and one strain from volunteer 2. To date, there have been 0. fo rmigenes strains isolated from various animals but not from mice. In the present study, all eleven mice were colonized with 0. fo rrnigenes, with 60 two mice yielding 0. fo rmigenes isolates. In comparison to pure cultures of 0. fo rmigenes from different animal subjects, mouse isolates were similar in morphology, nutrition, and ability to degrade oxalate to formate, but growth potentials appeared limited in culture media which may explain the difficulties in the isolation of 0. fo rmigenes from mice (24 ). According to PFGE, the mouse isolates were segregated from other strains showing less similarity. PFGE profile bands of OxM-C386-l and OxM-C386-2 were identical suggesting these two isolates were indistinguishable from each other (69). The genus Bacteroides, Gram-negative anaerobic bacteria, was reclassified using PFGE technique as several questions were imposed on the taxonomy classification constructed based on l 6S rRNA analysis (86). Bacteroides species underwent restriction digestion with three restriction endonucleases simultaneously. It was hypothesized that closely related bacteria will have same number of restriction fragments, but instead four to seven fragment differences were observed. A new phylogenetic tree was proposed on the basis of genome size, restriction patterns, and number of restriction fragments. With a few exceptions, the results agreed with the 16S rRNA classification and the authors concluded that PFGE is a usefultechnique forphylog enetic analysis (86, 87). On the other side, He licobacter py lori has been typed by various molecular methods including PFGE, and, interestingly, all the typing systems do not essentially correlate with each other as different systems assess diverse traits (88, 89). However, studies on this bacterium demonstrated that PFGE is highly reproducible and possesses good discriminatory power with low ease of interpretation (90). Results generated with restriction enzymes Notl and Nrul showed a significant, but rare, heterogeneity in the genome of H. pylori associated with gastritis in patients (91 -93). 61 According to the evidences (Table 1) and present study, animal and human 0. fo rrnigenes strains classify in either group 1 or group 2. There are no facts available about the grouping patternrelativity between animals and humans. The present study also did not show any group relatedness among animal and human 0. fo rmigenes strains (Figure 23). Past studies have shown that individuals not colonized with 0. fo rmigenes have increased chances of kidney stones (35, 43-45) but there is no experimental evidence showing the potential relationship between which 0. fo rmigenes group the individual is colonized with and the chances of kidney stones. 62 VI. Conclusion Results from the present study were in accord with previously published research with a few exceptions, but it has equally raised questions on the categorization of strains of 0. fo rrnigenes. Work presented here suggested that to identifyand differentiate bacteria at the species and sub-species levels multiplex PCR detection system and PFGE technique together can provide an immense value to taxonomic characterization. Since the above standards used to interpret the PFGE profilewere generated primarily for epidemiological and foodbome outbreaks, it would be a further area of research to study the sizes of the similar restriction fragments via gene sequencing. 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