Role of Oxalobacter formigenes in preventing calcium oxalate kidney stones

Objective To screen Oxalobacter formigenes (OxF) from fresh feces of healthy adults, and study its effect on the the prevention of calcium oxalate kidney stones. Methods OxF was screened and cultured from fresh feces of healthy adults. The rat model of calcium oxalate stone was established by esophageal gavage of 0.8% of ethylene glycol. Rats were divided into a control group and four groups of rats with ethylene glycol-induced calcium oxalate kidney stones according to random number table. Three groups were treated with 106 CFU, 107 CFU, 108 CFU viable OxF every day, respectively, for 4 weeks. The blood and 24-hour urine samples were collected to detect the serum creatinine, urea nitrogen, serum and urine calcium, phosphorus, magnesium and urine oxalate every week. At the end of the 4th week, the rats were sacrificed and the kidney tissues were stained with HE and Yasue. The deposition and content of calcium oxalate crystals were observed under a light microscope. Results The bacteria strain isolated from fresh feces of healthy adults was 100% as same as the known ATCC35274 bacteria strain, which means the strain screened is OxF. Among the 5 groups, there were no significant differences in body weight, Scr, BUN, serum calcium, blood magnesium, blood phosphorus, urinary magnesium and urinary phosphorus. The 24-hour urinary calcium excretion in the model group was significantly lower than that of the control group (P＜0.05). After intervention with OxF solution, the 24-hour urinary calcium excretion in the 108 CFU OxF group was significantly higher than that in the model group (P＜0.05), while there was no significant difference between the other intervention groups and the model. The oxalic acid excretion of 106 CFU OxF group and 107 CFU OxF group was lower than that of the model, but the difference did not reach statistical significance (P＞0.05). The 24 h oxalic acid excretion in the 108 CFU OxF group was significantly lower than that of the model at the end of first week (P＜0.05), and continued to decrease for the next 3 weeks. After 4 weeks of intervention, no crystal formation was observed in the control group under the deflection microscope, but a large amount of calcium oxalate crystals were formed in the renal cortex and renal medulla. The crystals were piled up and connected to each other. Yasue staining coincided with the calcium oxalate crystal in the same part of the kidneys. Compared with the model, there was no significant change in the score of calcium oxalate crystal in the kidneys of 106 CFU OxF group and 107 CFU OxF group, while the score of calcium oxalate crystal in the kidneys of 108 CFU OxF group was significantly lower (P＜0.05). Conclusions OxF are successively screened from healthy adults. Daily administration of 108 CFU OxF can safely and effectively reduce the urinary oxalic acid excretion, prevent the formation of calcium oxalate crystals and inhibit the formation of stones in kidneys of rats.     

Urinary oxalate levels and the enteric bacterium Oxalobacter formigenes in patients with calcium oxalate urolithiasis

OBJECTIVES: We performed a prospective study to evaluate the intestinal colonization of Oxalobacter formigenes and its relationship with urinary oxalate levels in patients with calcium oxalate stone disease.METHODS: One hundred and three patients with calcium oxalate urolithiasis, ranging in age from 21 to 73 years (mean age, 47 years) who were followed from August 2000 to September 2001 participated in this study. Fresh stool and 24-hour urine samples were collected. Genus specific oligonucleotide sequences corresponding to the homologous regions residing in the oxc gene were designed. In order to quantify O. formigenes in clinical specimens, a quantitative-PCR-based assay system utilizing a competitive DNA template as an internal standard was developed. Urine volume, pH, creatinine, oxalate, calcium, magnesium, phosphate, citrate and uric acid were measured. RESULTS: Intestinal Oxalobacteria were detected in 45.6% (n=47) of calcium oxalate stone patients by PCR. In stone formers who tested negative for Oxalobacteria, the average urinary oxalate level was 0.36 mmol/day, and this compared to 0.29 mmol/day for those patients that tested positive for Oxalobacteria (p<0.05). Mean colony forming units per gram of stool of all patients was 1.1 x 10(7) (0-4.1 x 10(8)), and the level of 24 hours urine oxalate significantly decreased with increasing level of colony forming units of O. formigenes (r=-0.356, p=0.021).CONCLUSION: Our results support the concept that O. formigenes is important in maintaining oxalate homeostasis and that its absence from the gut may be the risk of calcium oxalate urolithiasis.     

Gut-inhabiting bacterium Oxalobacter formigenes: role in calcium oxalate urolithiasis

Oxalate plays a crucial role in the formation of most renal stones. Oxalate is a common constituent of most diets and a byproduct of metabolism, and if it is not sufficiently degraded, it may accumulate. In humans, gut bacteria degrade 70 to 100 mg of oxalate per day. Oxalobacter formigenes is a gram-negative, obligately anaerobic, rod-shaped bacterium with an absolute requirement for oxalate. Although not present in the gut at birth, it quickly colonizes most children, and there is epidemiologic evidence that its absence is a risk factor in calcium oxalate stone formation. We review the metabolism, genetics, and identification of this organism and its possible therapeutic role in recurrent stone-forming patients.     

Oxalobacter formigenes may reduce the risk of calcium oxalate kidney stones

Most kidney stones are composed primarily of calcium oxalate. Oxalobacter formigenes is a Gram-negative, anaerobic bacterium that metabolizes oxalate in the intestinal tract and is present in a large proportion of the normal adult population. It was hypothesized that the absence of O. formigenes could lead to increased colonic absorption of oxalate, and the subsequent increase in urinary oxalate could favor the development of stones. To test this hypothesis, a case-control study involving 247 adult patients with recurrent calcium oxalate stones and 259 age-, gender-, and region-matched control subjects was performed. The prevalence of O. formigenes, determined by stool culture, was 17% among case patients and 38% among control subjects; on the basis of multivariate analysis controlling demographic factors, dietary oxalate, and antibiotic use, the odds ratio for colonization was 0.3 (95% confidence interval 0.2 to 0.5). The inverse association was consistently present within strata of age, gender, race/ethnicity, region, and antibiotic use. Among the subset of participants who completed a 24-h urine collection, the risk for kidney stones was directly proportional to urinary oxalate, but when urinary factors were included in the multivariable model, the odds ratio for O. formigenes remained 0.3 (95% confidence interval 0.1 to 0.7). Surprisingly, median urinary oxalate excretion did not differ with the presence or absence of O. formigenes colonization. In conclusion, these results suggest that colonization with O. formigenes is associated with a 70% reduction in the risk for being a recurrent calcium oxalate stone former.     

Intestinal Oxalobacter formigenes colonization in calcium oxalate stone formers and its relation to urinary oxalate

BACKGROUND AND PURPOSE: Oxalobacter formigenes is an anaerobic commensal colonic bacterium capable of degrading oxalate through the enzyme oxalyl-CoA decarboxylase. It has been theorized that individuals who lack this bacterium have higher intestinal oxalate absorption, leading to a higher urinary oxalate concentration and an increased risk of calcium oxalate urolithiasis. We performed a prospective, controlled study to evaluate O. formigenes colonization in calcium oxalate stone formers and to correlate colonization with urinary oxalate and other standard urinary stone risk factors. PATIENTS AND METHODS: Thirty-five first-time calcium oxalate stone formers were compared with 10 control subjects having no history of urolithiasis and a normal renal ultrasound scan. All subjects underwent standard metabolic testing by submitting serum and 24-hour urine specimens. In addition, all subjects submitted stool samples for culture and detection of O. formigenes by Xentr(ix) O. formigenes Monitor. RESULTS: Intestinal Oxalobacter was detected in only 26% of the stone formers compared with 60% of the controls (p < 0.05). Overall, the average urinary oxalate excretion by the two groups was similar (38.6 mg/day v 40.8 mg/day). Among stone formers, however, there were statistically higher urinary oxalate concentrations in O. formigenes-negative patients compared with those testing positive (41.7 mg/day v 29.4 mg/day) (p = 0.03). Furthermore, all 10 stone formers with hyperoxaluria (>44 mg/day) tested negative for O. formigenes (p < 0.05). CONCLUSIONS: Calcium oxalate stone formers have a low rate of colonization with O. formigenes. Among stone formers, absence of intestinal Oxalobacter correlates with higher urinary oxalate concentration and an increased risk of hyperoxaluria. Introduction of the Oxalobacter bacterium or an analog of its enzyme oxalyl-CoA decarboxylase into the intestinal tract may be a treatment for calcium oxalate stone disease.     

Variability of Oxalobacter formigenes and oxalate in stool samples

PURPOSE: The intestinal organism Oxalobacter formigenes is unique in using oxalate as its primary carbon and energy source. Intestinal colonization with O. formigenes may have clinical significance by decreasing intestinal oxalate and its absorption, thereby influencing the concentration of oxalate in plasma and urine, and the development of calcium oxalate stone disease. Because the oxalate content of the diet varies considerably, we hypothesized that the number of O. formigenes and amount of oxalate would vary in feces. MATERIALS AND METHODS: To enumerate the number of O. formigenes in feces an accurate and reproducible real-time polymerase chain reaction assay was developed to quantify O. formigenes DNA. Stool samples were obtained from 10 colonized individuals to determine the levels of O. formigenes by this assay and the oxalate content by ion chromatography. RESULTS: Concentrations of O. formigenes ranged from lower than the limit of detection of 5 x 10(3) to 1.04 x 10(9) cells per gm stool. The total oxalate content of stool samples varied from 0.1 to 1.8 mg/gm and fecal water oxalate varied from 60 to 600 microM. All parameters measured varied within each stool collection, among stool collections on different days and among individuals. Notably in 7 of 10 individuals at least 1 stool sample contained no detectable O. formigenes. In addition, 7 of 10 subjects had a fecal colonization of less than 4 x 10(4) per gm stool. CONCLUSIONS: This study demonstrates that there is intrastool and interstool sample variability in the amount of O. formigenes measured by real-time polymerase chain reaction that did not correlate with the quantity of oxalate in stool. Most subjects had a fecal colonization of less than 4 x 10(4) per gm stool.     

Role of Oxalobacter formigenes in calcium oxalate stone disease: a study from North India

OBJECTIVE: The present study was performed to detect the presence of an oxalate degrading bacteria Oxalobacter formigenes in the GI tract of calcium oxalate stone patients and normal individuals from North India. Furthermore, the possible relationship of this bacterium with number of stone episodes in this part of the world was also studied. The correlation of the presence or absence of O. formigenes with the urinary oxalate levels was evaluated. METHODS: DNA was extracted from the stool samples of 63 calcium oxalate stone formers and 40 normal individuals. Polymerase chain reaction (PCR) was performed using genus specific primers for O. formigenes. The presence of which was confirmed by Southern blotting. Urinary oxalate levels were tested in each patient. RESULTS: As shown by PCR and Southern blotting, O. formigenes was present in 65% of normal individuals and in 30% of calcium oxalate stone formers. In patients with three or greater than three stone episodes colonies were present only in 5.6% of patients. Oxalate excretion was less in patients colonized with O. formigenes as compared to those with no colonization. CONCLUSION: In North Indian population the absence of O. formigenes can lead to a significant increase in the risk of absorptive hyperoxaluria and resultant recurrent calcium oxalate stone episodes.     

Association of absence of intestinal oxalate degrading bacteria with urinary calcium oxalate stone formation

AIM: Urinary concentration of oxalate is considered an important factor in the formation of renal stones. Dietary oxalate is a major contributor to urinary oxalate excretion in most individuals. Furthermore, oxalate degrading bacteria have been isolated from human feces. We investigated the significance of oxalate degrading bacteria for urinary oxalate excretion and urinary stone formation. METHODS: Twenty-two known calcium oxalate stone-forming patients (stone formers) and 34 healthy volunteers (non-stone formers) were included in the study. Stool specimens were inoculated into pepton yeast glucose (PYG) medium supplemented with oxalate under anaerobic condition at 37 C for one week. After the incubation period, each colony was checked for the loss of oxalate from the culture medium. A 24-h urine sample was collected in 43 individuals and analyzed for oxalate excretion. RESULTS: Twenty-eight of 34 (82%) healthy volunteers and 10 of 22 (45%) calcium oxalate stone formers were colonized with oxalate degrading bacteria. Calcium oxalate stone formers were more frequently free of oxalate degrading bacteria (P < 0.01). Urinary excretion of oxalate in those with oxalate degrading bacteria was significantly less than in those without oxalate degrading bacteria (P < 0.05). Hyperoxaluria (> 40 mg/day) was found in four of 27 individuals (15%) with oxalate degrading bacteria compared to seven of 16 (44%) without oxalate degrading bacteria (P < 0.05), suggesting an association between the absence of oxalate degrading bacteria and the presence of hyperoxaluria. CONCLUSION: The absence of oxalate degrading bacteria in the gut could promote the absorption of oxalate, thereby increasing the level of urinary oxalate excretion. The absence of oxalate degrading bacteria from the gut appears to be a risk factor for the presence of absorptive hyperoxaluria and an increased likelihood of urolithiasis.     

Quantitative analysis of colonization with real-time PCR to identify the role of Oxalobacter formigenes in calcium oxalate urolithiasis

The objective of the study was to quantitatively measure the number of Oxalobacter formigenes (O. formigenes) colonizations in the gastrointestinal tract in calcium oxalate-forming patients with real-time polymerase chain reaction (PCR). Calcium oxalate-forming patients (n: 27) were included in the study. Serum calcium, sodium, potassium, urea and creatinine levels, as well as 24?h urine levels of calcium and oxalate were measured. The numbers of O. formigenes colonies in stool samples were detected by real-time PCR. One or two metabolic abnormalities were detected in 15 of 27 patients. The O. formigenes levels in patients with metabolic disturbance were significantly decreased when compared to the patients with no metabolic abnormalities (p: 0.038). The undetectable levels of O. formigenes were encountered in one of five patients with hypercalciuria, in three of four patients with hyperoxaluria and in four of six patients with both hypercalciuria and hyperoxaluria. In nine patients with a history of stone recurrence, O. formigenes colonization was significantly lower than the patients with the first stone attack (p: 0.001). O. formigenes formation ceased or significantly diminished in patients with calcium oxalate stones with a coexistence of both hyperoxaluria and hypercalciuria. The measurement of O. formigenes colonies by real-time PCR seemed to be an inconvenient and expensive method. For this reason, the real-time PCR measurements can be spared for the patients with stone recurrences and with metabolic abnormalities like hypercalciuria and hyperoxaluria. The exact measurement of O. formigenes may also help more accurate programming of O. formigenes-based treatments.     

The genetic composition of Oxalobacter formigenes and its relationship to colonization and calcium oxalate stone disease

Oxalobacter formigenes is a unique intestinal organism that relies on oxalate degradation to meet most of its energy and carbon needs. A lack of colonization is a risk factor for calcium oxalate stone disease. Protection against calcium oxalate stone disease appears to be due to the oxalate degradation that occurs in the gut on low calcium diets with a possible further contribution from intestinal oxalate secretion. Much remains to be learned about how the organism establishes and maintains gut colonization and the precise mechanisms by which it modifies stone risk. The sequencing and annotation of the genomes of a Group 1 and a Group 2 strain of O. formigenes should provide the informatic tools required for the identification of the genes and pathways associated with colonization and survival. In this review we have identified genes that may be involved and where appropriate suggested how they may be important in calcium oxalate stone disease. Elaborating the functional roles of these genes should accelerate our understanding of the organism and clarify its role in preventing stone formation.     

A critical analysis of the role of gut Oxalobacter formigenes in oxalate stone disease

Hyperoxaluria is a major risk factor for the formation of calcium oxalate stones, but dietary restriction of oxalate intake might not be a reliable approach to prevent recurrence of stones. Hence, other approaches to reduce urinary oxalate to manage stone disease have been explored. The gut‐dwelling obligate anaerobe Oxalobacter formigenes (OF) has attracted attention for its oxalate‐degrading property. In this review we critically evaluate published studies and identify major gaps in knowledge. Recurrent stone‐formers are significantly less likely to be colonized with OF than controls, but this appears to be due to antibiotic use. Studies in animals and human subjects show that colonization of the gut with OF can decrease urinary oxalate levels. However, it remains to be determined whether colonization with OF can affect stone disease. Reliable methods are needed to detect and quantify colonization status and to achieve durable colonization. New information about oxalate transport mechanisms raises hope for pharmacological manipulation to decrease urinary oxalate levels. In addition, probiotic use of lactic acid bacteria that metabolize oxalate might provide a valid alternative to OF.     

Role of agglomeration in calcium oxalate monohydrate urolith development.

Formation of agglomerates of calcium oxalate monohydrate (COM) crystals on semi-batch precipitation performed at conditions relevant to urolithiasis (37 degrees C, pH = 6, initial ratio [Ca]/[Ox] = 10), but without any specific admixture, was followed by both optical and electron microscopy. COM crystals formed on precipitation developed into large agglomerates consisting of intergrown crystals by a mechanism of primary agglomeration. Primary agglomeration of COM crystals represents an important mechanism of COM renal calculi growth.     

Diagnosis of intestinal oxalate hyperabsorption in patients with idiopathic recurrent calcium oxalate urinary calculi

By oral administration of 14C-labelled oxalic acid (2.2 microns Ci; 2 mg), the mean enteral oxalate absorption in 24-h urines (collecting intervals 3, 3, 6, 12 h) of 19 healthy control subjects was determined to be 8.3%. It was 14.6% (alpha less than 1%) in 20 patients with recurrent idiopathic calcium-oxalate lithiasis. The differences in absorption were most marked in the first two 3-h urines. Under the test conditions described, maximum excretion of 14C oxalate had already occurred after 3 h (55%-57%); after 6 h it was about 85%, and after 12 h 95% of the total activity in the 24-h urine. In 68% of the control subjects, the oxalate absorption values were below 10%; in 32% they were less than or equal to 15%. For the group of lithiasis patients, these findings suggest a metabolic disorder of gastrointestinal origin. In 40%, the oxalate absorption rates were clearly above 15%. The test method described is simple to carry out and can be recommended for the assessment of idiopathic calcium-oxalate lithiasis.     

Vascular endothelial growth factor gene polymorphism is associated with calcium oxalate stone disease

Growth factor-related genes regulate cell growth, differentiation and apoptosis in the kidney in response to cellular injury. One of the theories of stone formation is that cellular injury, and thus growth factors, play a role. We therefore investigated the association between growth factor genes and calcium oxalate stone disease. The most frequently seen polymorphism of the vascular endothelial growth factor (VEGF) gene is Bst U I C/T, which is located upstream at the -460th nucleotide. Other growth factor-related gene polymorphisms include the cytochrome P450c17alpha enzyme (CYP17) gene MspA I C/T polymorphism at the 5'-UTR promoter region, the epidermal growth factor receptor (EGFR) gene Bsr I polymorphism (A to T) at position 2,073, and the insulin-like growth factor-2 (IGF-2) gene Apa I A/G at exon 9. All four polymorphisms were used as genetic markers in this study in the search for an association between stone disease and growth factor related genes. A normal control group of 230 healthy people, and 230 patients with calcium oxalate stone, were examined. The polymorphism was seen following polymerase chain reaction based restriction analysis. The result revealed a significant difference between normal individuals and stone patients (P=0.0003, Fisher's exact test) in the distribution of the VEGF gene polymorphism as well as an odds ratio of 1.30 (95% confidence interval=0.993-1.715) per copy of the "T" allele. Whereas, the IGF-2, EGFR and CYP17 gene polymorphisms did not reveal a significant association with stone disease. We conclude that the VEGF gene Bst U I polymorphism is a suitable genetic marker of urolithiasis.     

Crystal agglomeration is a major element in calcium oxalate urinary stone formation

The effects of urines from 36 healthy subjects and 86 calcium oxalate renal stone formers on calcium oxalate monohydrate crystallization kinetics were studied using a seeded crystal growth method in which the solubility, the growth and the agglomeration of the crystals are measured as three separate and system-independent parameters. The urines of healthy subjects were found to increase the solubility and to strongly inhibit the growth and the agglomeration of calcium oxalate monohydrate crystals. The urines of stone formers had a similar effect on the solubility, but a significantly lower ability to inhibit the crystal growth and the crystal agglomeration. Of these two kinetic processes the inhibition of crystal agglomeration was more clearly affected, with 55% of the stone formers having abnormally low values, while the changes in crystal growth inhibition occurred within the normal range. The defect in crystal agglomeration inhibition was related to stone frequency, and urines from patients with very high stone frequency rates had also the most severely impaired ability to inhibit the agglomeration of the calcium oxalate monohydrate crystals. The inhibitory effect of urines on crystal agglomeration was found to be related to its citrate content (r = 0.68, P less than 0.001). All patients with hypocitraturia, except two, had also abnormally low values for crystal agglomeration inhibition. In a group of 15 hypocitraturic stone formers, alkali treatment for a mean period of 18 months resulted in a parallel increase in urinary citrate excretion and in the ability of urines to inhibit crystal agglomeration (r = 0.77, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

The importance of Oxalobacter formigenes and oxalic acid in the pathogenesis of chronic kidney disease

International Urology and Nephrology -