Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples.

OBJECTIVE: To develop a simple and rapid polymerase chain reaction (PCR) method for detecting Oxalobacter formigenes (which degrades oxalate in the gut) in fecal specimens from healthy volunteers and patients with urolithiasis, and to determine whether O. formigenes can be detected in frozen or fresh fecal samples. MATERIALS AND METHODS: Whole bacterial DNA was isolated directly from fresh and frozen fecal samples obtained from 30 healthy volunteers free from urolithiasis and from fresh fecal samples obtained from 38 patients with urolithiasis. Genus-specific oligonucleotide sequences were designed, corresponding to homologous regions residing in the oxc gene that encodes for oxalyl-coenzyme A decarboxylase. A PCR-based assay was used on both fresh and frozen fecal samples, and the nucleotide sequences analysed to confirm oxc. RESULTS: A PCR product of 416 bp encoding the oxc gene was detected in 23 (77%) of 30 healthy volunteers free from urolithiasis and in 14 (37%) of 38 patients with urolithiasis. In healthy volunteers, the results of PCR for the fresh and the frozen samples were identical in each subject. The nucleotide sequence analysis showed that the sequence of the amplified product was compatible with that of oxc. CONCLUSION: O. formigenes can be identified easily and efficiently using this PCR-based detection system. The colonization rate of O. formigenes in patients with urolithiasis was significantly lower than that in healthy volunteers known to be free from urolithiasis. Furthermore, as the PCR-based assay results in the frozen fecal samples were identical to those from fresh samples in each subject, immediate processing of fecal samples may not be necessary to detect O. formigenes in the clinical setting.     

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 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.     

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.     

Effect of antibiotics on Oxalobacter formigenes colonization of human gastrointestinal tract

BACKGROUND AND PURPOSE: Oxalobacter formigenes is a bacterium residing in the human gastrointestinal tract that degrades oxalate and reduces its availability for absorption. This bacterium is assumed to be antibiotic sensitive, and repeated antibiotic therapies could eradicate it. The aim of the present study was to determine the differences in the colonization by O. formigenes of individuals who had been on antibiotics for at least 5 days at the time of sample collection and individuals who had not taken antibiotics for at least 3 months. PATIENTS AND METHODS: Stool samples were collected from 80 individuals without stone disease (35 with and 45 without antibiotic consumption) and 100 patients with stone disease (20 with and 80 without antibiotic consumption). Oxalobacter formigenes was detected by a polymerase chain reaction-based method, and the presence/absence of O. formigenes was correlated with urinary oxalate concentrations. RESULTS: Lower percentages of individuals without stone disease and with stone disease who were consuming antibiotics had O. formigenes colonization than individuals without antibiotic consumption. Urinary oxalate concentrations were higher in the individuals without O. formigenes than in colonized individuals. CONCLUSION: Our observations confirm a direct association between antibiotic consumption and absence of O. formigenes. Absence of intestinal O. formigenes could represent a pathogenic factor in calcium oxalate urolithiasis when antibiotics are prescribed generously.     

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.     

人肠道产甲酸草酸杆菌的分离培养

目的探讨产甲酸草酸杆菌分离培养及鉴定方法。方法应用选择性培养基,在37℃厌氧环境下分离培养人肠道产甲酸草酸杆菌。通过观察菌落形态、细菌涂片染色镜检、离子色谱仪及核酸序列分析,对细菌进行鉴定。结果产甲酸草酸杆菌菌落形态为白色点状或梭状,大小约(0.1~0.5)mm×(1.0~3.0)mm;菌落周边出现直径约3.5~4.5 mm的清晰透明圈。该细菌为革兰氏阴性杆菌,大小约(1.0~1.6)μm×(3.0~6.5)μm。液体培养基中草酸浓度随细菌浓度增加而逐渐下降。与文献报道核酸序列比较,该细菌分解草酸的功能基因frc、oxc的核酸同源性分别为95.8%及93.6%。结论产甲酸草酸杆菌可通过选择性培养基、在厌氧环境下从人粪便中分离出来。     

表达产甲酸草酸杆菌草酸分解基因的人肝细胞系的构建

目的：构建表达产甲酸草酸杆菌（Ox．F）草酸分解基因的人肝细胞系,探索高草酸尿的治疗方法。方法：分离培养人肠道Ox．F并克隆其功能基因OXC和fre,构建同时表达OXC和fre的真核双表达载体质粒pIRES—oxc-frc,将pIRES-oxe—fre转染人正常肝细胞系L—02。用RT—PCR和Western blot技术了解转基因细胞的目的基因表达状况;用离子色谱法测定转基因后细胞培养液中草酸浓度,了解其草酸分解功能。结果：转基因后人肝细胞系L-02在mRNA和蛋白质水平均成功表达OXC和fre基因,其草酸分解功能较转染前明显增强（P〈0．01）。结论：Ox．F分解草酸的两个重要功能基因OXC和frc能在体外转入人正常肝细胞（L-02）中,并使后者草酸分解能力明显增强。转草酸分解基因的人肝细胞系的构建,有望用于高草酸尿,尤其是PH的病因治疗,值得进一步研究．     

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.     

Rapid reversal of hyperoxaluria in a rat model after probiotic administration of Oxalobacter formigenes

PURPOSE: The gut inhabiting bacterium Oxalobacter formigenes may be a negative risk factor in recurrent calcium oxalate kidney stone disease that apparently maintains oxalic acid homeostasis in its host via the degradation of dietary oxalate. The possibility of using this bacterium as probiotic treatment to reduce urinary oxalate was investigated in a rat model. MATERIALS AND METHODS: Male Sprague-Dawley rats were placed on a diet supplemented with ammonium oxalate to induce a state of severe hyperoxaluria. Subgroups of these rats received an esophageal gavage of 1 x 10(3), 10(5), 10(7) or 10(9) O. formigenes per feeding for a 2-week period. Each rat was followed for general health and changes in urinary oxalate. RESULTS: Rats with chronic hyperoxaluria resulting from high dietary oxalate that were treated with O. formigenes showed decreased urinary oxalate within 2 days of initiating probiotic supplementation. The amount of the decrease in a 2-week period proved directly proportional to the dose of bacteria. Urinary oxalate in rats receiving higher amounts of O. formigenes returned to almost normal. Throughout the study the rats remained healthy with no signs of toxicity, antibody development or a histopathological condition. CONCLUSIONS: Probiotic treatment of hyperoxaluric rats with O. formigenes may significantly and rapidly reduce the level of oxalate in the urine. This probiotic treatment appears to be safe and well tolerated. The approach may be feasible for treating calcium oxalate kidney stone disease.     

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.     

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

International Urology and Nephrology -     

Oxalobacter formigenes: a potential tool for the treatment of primary hyperoxaluria type 1

Primary hyperoxaluria is characterized by severe urolithiasis, nephrocalcinosis, and early renal failure. As treatment options are scarce, we aimed for a new therapeutic tool using colonic degradation of endogenous oxalate by Oxalobactor formigenes. Oxalobacter was orally administered for 4 weeks as frozen paste (IxOC-2) or as enteric-coated capsules (IxOC-3). Nine patients (five with normal renal function, one after liver-kidney transplantation, and three with renal failure) completed the IxOC-2 study. Seven patients (six with normal renal function and one after liver-kidney transplantation) completed the IxOC-3 study. Urinary oxalate or plasma oxalate in renal failure was determined at baseline, weekly during treatment and for a 2-week follow-up. The patients who showed >20% reduction both at the end of weeks 3 and 4 were considered as responders. Under IxOC-2, three out of five patients with normal renal function showed a 22-48% reduction of urinary oxalate. In addition, two renal failure patients experienced a significant reduction in plasma oxalate and amelioration of clinical symptoms. Under IxOC-3 treatment, four out of six patients with normal renal function responded with a reduction of urinary oxalate ranging from 38.5 to 92%. Although all subjects under IxOC-2 and 4 patients under IxOC-3 showed detectable levels of O. formigenes in stool during treatment, fecal recovery dropped directly at follow up, indicating only transient gastrointestinal-tract colonization. The preliminary data indicate that O. formigenes is safe, leads to a significant reduction of either urinary or plasma oxalate, and is a potential new treatment option for primary hyperoxaluria.     

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.