Forty Years of Oxalobacter formigenes, a Gutsy Oxalate-Degrading Specialist

Abstract

Oxalobacter formigenes, a unique anaerobic bacterium that relies solely on oxalate for growth, is a key oxalate-degrading bacterium in the mammalian intestinal tract. Degradation of oxalate in the gut by O. formigenes plays a critical role in preventing renal toxicity in animals that feed on oxalate-rich plants. The role of O. formigenes in reducing the risk of calcium oxalate kidney stone disease and oxalate nephropathy in humans is less clear, in part due to difficulties in culturing this organism and the lack of studies which have utilized diets in which the oxalate content is controlled. Herein, we review the literature on the 40th anniversary of the discovery of O. formigenes, with a focus on its biology, its role in gut oxalate metabolism and calcium oxalate kidney stone disease, and potential areas of future research. Results from ongoing clinical trials utilizing O. formigenes in healthy volunteers and in patients with primary hyperoxaluria type 1 (PH1), a rare but severe form of calcium oxalate kidney stone disease, are also discussed. Information has been consolidated on O. formigenes strains and best practices to culture this bacterium, which should serve as a good resource for researchers.

Keywords: Oxalobacter formigenes; culture methods; gut microbiome; kidney stone disease; oxalate degradation; probiotics.

FIG 1

(A) Oxalate crystals (arrows indicate some of the crystals present) in stained thin-sections of renal tissue from oxalate-fed laboratory rats (R. C. Cutlip and S. L. Daniel, unpublished data). (B) Dead sheep (>1,200) as a result of oxalate intoxication from consuming the high oxalate-containing plant Halogeton glomeratus (reprinted from reference 101).

FIG 2

Karl A. Dawson (A) and Milton J. Allison (B), the two microbiologists responsible for the isolation and naming of the oxalate-degrading gut bacterium Oxalobacter formigenes. Dr. Dawson (Vice President & Chief Scientific Officer of Alltech, now retired) is shown here speaking at the Alltech 30th Annual International Symposium in 2014. Dr. Allison is shown here in his laboratory at Iowa State University in 2018.

FIG 3

(A) Electron micrographs of Oxalobacter formigenes strain OxWR1 (reprinted from reference 102). (B) Gram stain of Oxalobacter formigenes strain HOxBLS (N. Pareek and S. L. Daniel, unpublished data; ×1,000 magnification) with its typical varied morphology and size.

FIG 4

Phylogenetic analysis of Oxalobacter formigenes group I and group II strains and Herbaspirillum seropedicae strain Z67 (outgroup and a member of the Betaproteobacteria class) based on 16S rRNA sequences obtained from the National Center for Biotechnology Information (NCBI). GenBank accession numbers are shown with the strain names. Evolutionary history and distances were computed using the neighbor-joining method (103) and maximum composite likelihood method, respectively (104). Bootstrap values, expressed as percentages of 1,000 replicates, are shown next to each internal node in the tree (105). The scale bar represents an estimate of the numbers of substitutions per base. Evolutionary analyses were conducted in Molecular Evolutionary Genetics Analysis (MEGA) (106).

FIG 5

Schematic of oxalate metabolism by Oxalobacter formigenes.

FIG 6

Clear zones formed around colonies by Oxalobacter formigenes after incubation in anaerobic roll tubes (A and B) and on anaerobic plates (C) containing calcium oxalate. See Table 3 for the preparation of anaerobic calcium oxalate agar in tubes and plates.

FIG 7

Genome features and composition for strains of Oxalobacter formigenes. Data and resources from the Joint Genome Institute (JGI)/Integrated Microbial Genomes (IMG) (107) and the National Center for Biotechnology Information (NCBI) websites were utilized for the preparation of this data set. Escherichia coli Nissle 1917 (34) is included for comparative purposes.

FIG 8

Distribution and abundance of genes in Clusters of Orthologous Genes (COG) categories for Oxalobacter formigenes HCI-1, Oxalobacter formigenes HOxBLS, and Escherichia coli Nissle 1917. Escherichia coli Nissle 1917 (34) is included for comparative purposes. Data and resources from the Joint Genome Institute (JGI)/Integrated Microbial Genomes (IMG) website (107) were used in the preparation of this data set. For O. formigenes HC-1, O. formigenes HOxBLS, and E. coli Nissle 1917, there are 746, 737, and 1291 genes not in COGs, respectively.

FIG 9

Amount of O. formigenes in the feces of human subjects relative to changes in dietary oxalate (red triangles) or dietary calcium (black squares). Daily calcium intake was 1,000 mg on the varied oxalate dietary phase and daily oxalate was 250 mg on the varied calcium dietary phase. Real-time quantitative PCR (qPCR) was used to quantitate O. formigenes numbers, and 5.5 × 10⁴ CFU/ng DNA was used to convert qPCR data to number of O. formigenes per g feces (108). Error bars represent SEM. (Adapted from reference with permission from Wolters Kluwer Health.)

FIG 10

(A and B) 24-h urinary oxalate excretion of healthy subjects not colonized (black squares) and colonized with O. formigenes (red triangles) on nutrient-controlled diets varying in oxalate (A) and calcium (B). Daily calcium intake was 1,000 mg on the diets varying in oxalate and daily oxalate intake was 250 mg on the diets varying in calcium. Individuals colonized with O. formigenes excreted significantly less urinary oxalate than noncolonized individuals on the 250 mg oxalate/400 mg calcium diet; indicated by *, P = 0.026. Error bars represent SD. (Adapted from reference with permission from Wolters Kluwer Health.)

FIG 11

Stool oxalate of O. formigenes colonized (black bars) and noncolonized healthy subjects (red bars) on nutrient-controlled diets varying in oxalate. Daily calcium intake was 1,000 mg. Error bars represent SEM. (Adapted from reference with permission from Wolters Kluwer Health.)