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Urinary Oxalate Levels and the Enteric Bacterium Oxalobacter formigenes in Patients with Calcium Oxalate Urolithiasis
European Urology
European Urology 44 (2003) 475–481
Urinary Oxalate Levels and the Enteric Bacterium Oxalobacter formigenes in Patients with Calcium Oxalate Urolithiasis Cheol Kwaka, Hee Kyung Kimb, Eui Chong Kimc, Myung Sik Choib, Hyeon Hoe Kima,* a
Department of Urology, Seoul National University College of Medicine and Clinical Research Institute, Seoul National University Hospital, 28 Yongon Dong, Jongno Ku, Seoul 110-744, Republic of Korea b Department of Microbiology, Seoul National University College of Medicine, Seoul, Republic of Korea c Department of Clinical Pathology, Seoul National University College of Medicine and Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea First published online 9 July 2003
Abstract 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 ¼ 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 1:1 107 (04:1 108 ), and the level of 24 hours urine oxalate significantly decreased with increasing level of colony forming units of O. formigenes (r ¼ 0:356, p ¼ 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. # 2003 Elsevier B.V. All rights reserved. Keywords: Urolithiasis; Oxalate; Oxalobacter formigenes
1. Introduction The prevalence of urinary stone disease is estimated to be 2–3%, and the likelihood that a Caucasian man will develop stone disease by age 70 is about 1 in 8. The lifetime prevalence rate of urolithiasis in Korea was known to be 6.0% in men and 1.8% in women [1]. The recurrence rate without treatment for calcium oxalate renal stones is about 10% at 1 year, 35% at *
Corresponding author. Tel. þ82-2-760-2425; Fax: þ82-2-742-4665. E-mail address: [email protected] (H.H. Kim).
5 years, and 50% at 10 years [2]. Calcium oxalate stone is the most common cause of urolithiasis and accounts for 81.6% of the total number of urinary tract stones in Korea [3]. Hyperoxaluria is a risk factor for renal stones, and the urine load of oxalate plays a pivotal role in calcium oxalate stone formation even in normocalciuric patients [4]. In normal individuals, the majority of urinary oxalate is derived from the endogenous metabolism of glycine, glyoxylate, and ascorbic acid, and 10–20% is derived from oral ingestion [5]. The colon is a major site of oxalate absorption, and around 10% of dietary oxalate is absorbed under
0302-2838/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0302-2838(03)00318-X
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C. Kwak et al. / European Urology 44 (2003) 475–481
normal conditions [6]. However, the increased intestinal absorption of oxalate may lead to hyperoxaluria and a significantly enhanced risk of urinary stone formation [7,8]. Prevention of recurrence of urinary calculi is one of the biggest challenges facing the modern urologist. Oxalobacter formigenes is a recently identified oxalate degrading bacterium, which colonizes the GI tract of vertebrates, including humans [9], and it has a symbiotic relationship with its host by regulating oxalic acid absorption in the intestine and oxalate levels in the plasma and urine [10,11]. Previous studies have suggested a correlation between calcium oxalate stone activity and the lack of Oxalobacter formigenes colonization [12–16]. Although it was fascinating to learn that Oxalobacter formigenes influences the gastrointestinal absorption of oxalate, the examination of the relationship between this bacterium and urinary stone disease has been limited due to the difficult culture method. O. formigenes has shown to be a suitable target for DNA-based identification [16–18]. Using simpler DNA purification methods [16] and a rapid quantitative competitive-template PCR assay [18], we performed a prospective, controlled study by evaluating intestinal colonization by Oxalobacter formigenes and its relationship with urinary oxalate levels in patients with calcium oxalate stone disease, to confirm the pathogenic role of O. formigenes upon calcium oxalate urolithiasis. 2. Patients and methods 2.1. Patient populations This study involved 103 patients (89 males and 14 females) with calcium oxalate urolithiasis, ranging in age from 21 to 73 years (mean age, 47 years), and who were followed up from August 2000 to September 2001 at the Department of Urology, Seoul National University Hospital. Fresh stools and 24-hour urine samples were collected. Only stone-formers free from urinary tract infection for at least one year before the beginning of this study were included. Individuals who had taken antibiotics for any reason during the previous 3 months were excluded, and informed consent was obtained from all patients before participation. DNA was extracted from 250 mg of stool specimen within 3 hours of defecation for all patients. Urine samples were stored at 20 8C. 2.2. Bacterial strain and the isolation of genomic DNA Strain OxB (ATCC 35274) was used as the standard throughout this study, and was obtained from the American Type Culture Collection. The strain was grown in medium B containing 30 mM oxalate, as described elsewhere [9,16]. Isolation of genomic DNA from cultures of OxB was performed as described previously [16]. 2.3. Isolation of genomic DNA from human fecal specimens Fecal samples were collected from 103 patients with calcium oxalate urolithiasis. Whole bacterial DNA was isolated directly
from fresh stool samples obtained from individuals. Two hundredfifty mg of feces as a clean sample was suspended in 5 ml of PBS. Genomic DNA was extracted from feces as described previously [16]. 2.4. Quantitative competitive polymerase chain reaction (QC-PCR) PCR was performed as described previously [16]. The optimal reaction profile was found to be 94 8C for 5 minutes followed by 35 cycles of denaturation for 1 minute at 94 8C, annealing for 1 minute at 55 8C, and extension for 1 minute at 72 8C. Genomic DNA from OxB cultures was used as a positive control. O. formigenes expresses a unique gene, which is required for the catabolism of oxalate: oxc (encoding oxalate-coenzyme A decarboxylase). This gene has been cloned and sequenced [19], and the 50 ends of the oxc genes from numerous isolates of O. formigenes have identified unique, highly conserved regions responsible for the synthesis of a genus-specific PCR primer pair: OXFfp and OXFrp [18]. The sequence of OXFfp is 50 -AATGTAGAGTTGACTGA-30 , and the sequence of OXFrp is 50 -TTGATGCTGTTGATACG-30 . To construct a suitable competitive-DNA template to use as an internal control, a 227-bp fragment of the oxc gene flanked by sequences homologous to the OXFfp-OXFrp primer pair and containing a genus-specific probe was generated. PCR was performed with the OXFfp 50 primer and a modified 30 primer (50 -TTGATGCTGTTGATACGGTCAAGCAAACGCC-30 ), which consisted of two portions: a 50 end containing the 30 primer sequence (underlined) within the oxc gene, and a 30 end annealed with a site located approximately 200 bp downstream of the 50 primer site. The PCR with the OXFfp-modified OXFrp primer pair was used to amplify the 210-bp segment and synthesize the 17-bp OXFrp primer site at the 30 end. This PCR product was purified and ligated into PCR13.1 (Invitrogen, Inc., San Diego, CA, USA), a recombinant pCR13.1 plasmid with the proper insert (confirmed by sequencing) was selected for use as the internal competitive template. Quantitative competitive polymerase chain reaction (QC-PCR) is based on the assumption that a genomic DNA template and a competitive DNA template containing homologous primer sites will compete equally for PCR primers and that both experimentaland competitive-DNA template PCR products will subsequently be amplified collinearly. To determine the accuracy of the QC-PCR at quantifying the number of CFU in O. formigenes samples, QC-PCRs were established with an O. formigenes DNA preparation, which had a starting spectrophotometric reading of 3.043 mg of DNA/ml. Assuming that the genome of O. formigenes is similar in size to that of E. coli (4:7 103 kb), 1 mg of the genomic DNA of O. formigenes contained 2:0 108 molecules (or gene copies). Thus, the original genomic DNA preparation of O. formigenes OxB contained approximately 6:1 108 molecules/ml. Original concentration (6:1 108 molecules/ml) and twofold dilution (6:1 106 molecules/ml)of this DNA were used as template. The PCR products were sized by electrophoresis through 1.5% agarose gels and visualized with UV light. The PCR bands were scanned for intensities and normalized for differences in molecular mass, and the log ratios of O. formigenes to template band intensities were plotted against the log of the copy number of the synthetic template added per reaction to determine the log equivalence. The quantitation of oxc genes, and thereby bacteria, revealed the accuracy of this QC-PCR detection system. The log equivalence revealed that the number of molecules of O. formigenes OxB in the reaction in which original concentration and twofold dilution of
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Fig. 1. Quantification of the number of O. formigenes genomes in a sample using QC-PCR. Dilutions, ranging from 3:9 105 to 3:9 1012 molecules of purified plasmid containing the competitive template, were used either as DNA templates in PCR to establish standard curves (A) or as competitive DNA by mixing with 6:1 108 (B) or 6:1 106 (C) copies of purified O. formigenes OxB genomes. PCR band intensities were scanned and normalized for molecular mass and the log ratios of O. formigenes to template band intensities were graphed to determine log equivalence (D and E). QCT, quantitative competitive template.
OxB DNA were used were 5:9 108 and 1:2 106 , respectively (Fig. 1). 2.5. Determination of oxalate, calcium, citrate and creatinine in urine specimens Samples obtained from 24-h urine collections from patients on a self-selected diet were analyzed in our laboratory. Urine volume, pH (potentiometry) and the concentrations of creatinine (Jaffe reaction), magnesium (atomic absorption spectrophotometry), chloride (coulomb metric titration), sodium and potassium (flame emission spectrophotometry), sulfate (nephelometry), phosphate (phosphate molybdate reaction), ammonium (ion selective electrode), citrate (enzymatically, citrate lyase) and uric acid (enzymatically, uricase) were measured. Urinary calcium excretion levels were measured using a Hitachi 7170 Automatic Analyzer (Hitachi Inc., Japan), and urinary excretion levels of oxalate were measured using an oxalate kit (Sigma Diagnostics Inc., MO, USA). 2.6. Statistical analysis Differences in urinary parameters were compared using the Student’s non-paired t-test or ANOVA, and categorical comparisons were performed using the w2-test. Data are presented as mean SEM unless otherwise stated. The level of significance was set at p < 0:05.
3. Results 3.1. Relationship between urinary parameters and stone frequency Fifty patients (48.5%) had a single stone episode; 33 (32.0%) had 2 episodes and 20 (19.4%) had 3 or more stone episodes. No significant differences were observed in the distributions of age and sex in each group according to stone episodes. Urine parameters were not found to depend significantly on stone frequency (Table 1). 3.2. Relationship between stone episodes and O. formigenes detection by PCR The detection rate of Oxalobacter formigenes in stools by qualitative PCR tended to be reduced as the number of stone episodes increased ( p > 0:05) (Table 2). Intestinal Oxalobacteria were detected in 45.6% of all calcium oxalate stone patients. In stone formers who tested negative for Oxalobacteria, the average urinary
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Table 1 Results for daily urine composition according to stone frequencies (mean SEM) p valuea
Stone frequency 1 (n ¼ 50) 45.1 1.96
Age No. (%) according to sex Male Female Urine volume (ml/day) pH Calcium (mmol/day) Magnesium (mmol/day) Phosphorus (mmol/day) Uric acid (mmol/day) Oxalic acid (mmol/day) Citric acid (mmol/day) Creatinine clearance (ml/min) a
2 (n ¼ 33)
44 (88.0) 6 (12.0) 1651.5 6.24 5.79 1.71 23.41 3.59 0.30 1.70 85.5
47.1 1.8 30 (90.9) 3 (9.1) 1539.4 6.14 7.50 2.36 26.11 4.26 0.34 1.95 102.5
123.0 0.12 0.36 0.13 1.13 0.18 0.02 0.22 3.7
49.6 2.5 15 (75.0) 5 (25.0) 1913.5 6.33 7.13 2.17 28.21 3.98 0.39 1.80 104.3
172.0 0.16 0.69 0.21 1.85 0.21 0.05 0.24 5.2
0.342 0.235
0.263 0.634 0.082 0.075 0.198 0.211 0.196 0.767 0.963
ANOVA.
Table 2 Stone frequencies according to PCR results for O. formigenes Stone frequency
PCR results for O. formigenesa
Total
Negative Pt. No. (%)
Positive Pt. No. (%)
1 2 3
25 (50.0) 19 (57.6) 12 (60.0)
25 (50.0) 14 (42.4) 8 (40.0)
Total
56 (54.4)
47 (45.6)
50 33 20 103 (100)
Data presented are number (%). a 2 w -test for trend, p ¼ 0:398.
oxalate level was 0.36 mmol/day, and this compared to 0.29 mmol/day in patients that tested positive ( p < 0:05) (Table 3). Table 3 Daily urine composition of calcium oxalate stone patients according to PCR results for O. formigenes (mean SEM) PCR results for O. formigenes Negative (n ¼ 56) Urine volume (ml/day) pH Calcium (mmol/day) Magnesium (mmol/day) Phosphorus (mmol/day) Uric acid (mmol/day) Oxalic acid (mmol/day) Citric acid (mmol/day) Creatinine clearance (ml/min) a
185.0 0.13 0.57 0.31 1.27 0.19 0.04 0.21 5.1
3 (n ¼ 20)
Student’s t-test.
1581.3 6.25 6.66 0.83 25.94 3.87 0.36 1.68 93.17
p valuea
3.3. Quantification of colony forming units (CFUs) by QC-PCR The mean number of colony forming units per gram of feces from all calcium oxalate urolithiasis patients was 1:1 107 5:6 107 (range 04:1 108 ) (Fig. 2). The mean number of colony forming units per gram of feces in one-time stone formers (n ¼ 50), 2-time stone formers (n ¼ 33) and 3 or more stone forming episodes (n ¼ 20) were 2:0 107 , 3:2 106 and 2:9 105 , respectively ( p > 0:05). 3.4. Correlation between colony forming units detected by QC-PCR and the clinico-biochemical parameters An inverse correlation was found between log equivalences of colony forming units per gram of feces and 24-hour urinary oxalate levels in 47 patients with calcium oxalate urolithiasis, who tested positive for Oxalobacter by PCR (r ¼ 0:356, p ¼ 0:021) (Fig. 3). However, no correlations were found between the log equivalences of colony forming units per gram of feces and the other 24-hour urinary parameters.
Positive (n ¼ 47) 128.5 0.10 0.40 0.15 1.05 0.16 0.02 0.16 3.50
1769.2 6.19 6.43 2.21 24.09 3.86 0.29 1.95 95.89
125.1 0.12 0.43 0.21 1.18 0.18 0.03 0.22 4.41
0.274 0.832 0.702 0.151 0.244 0.962 0.030 0.191 0.564
4. Discussion After the emphasis was first placed on the role of hyperoxaluria in calcium oxalate stone formation by Cochran et al. [20], urinary oxalate concentration has been accepted to be an important factor of calcium oxalate stone formation. The intestinal anaerobe O. formigenes is attracting increasing attention because of its possible role in the regulation of oxalic acid absorption in humans [10,11].
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Fig. 2. Quantification of the number of O. formigenes genomes in stool samples of patients with calcium oxalate urolithiasis by QC-PCR. Dilutions, ranging from 3:9 101 to 3:9 105 molecules of purified plasmid containing the competitive template, were used for competitive DNA by mixing with 1 ml of extracted stool DNA from patients with calcium oxalate urolithiasis (A and B). PCR band intensities were scanned and normalized for molecular mass and the log ratios of O. formigenes to template band intensities were graphed to determine log equivalence (C and D). QCT, quantitative competitive template.
Decreased intestinal colonization of O. formigenes has been reported in recurrent calcium oxalate stone formers and in enteric hyperoxaluria patients [12–14]. Kleinschmidt et al. [12] correlated the colony forming units of O. formigenes per gram of feces with the frequency of episodes of kidney stone formation, and reported a complete absence of this bacterium
Fig. 3. Correlation between the log equivalences of colony forming units per gram of feces and 24-hour urinary oxalate levels in patients with calcium oxalate urolithiasis.
in patients having four or more episodes. Sidhu and coworkers suggested that the absence of O. formigenes from the intestinal tract of cystic fibrosis patients appeared to lead to the increased absorption of oxalate, thereby increasing the risk of hyperoxaluria and its complications [13]. We also found that the colonization rate of O. formigenes in patients with urolithiasis was significantly lower than that of healthy volunteers, known to be free from urolithiasis [16]. Putting these various reports together, it is possible to hypothesize that a lack of intestinal colonization by O. formigenes is one of the pathogenic pathway of calcium oxalate urolithiasis. However, to the best of our knowledge no report has convincing described a direct correlation between colony forming units of O. formigenes and urinary oxalate levels in patients with calcium oxalate stone disease, although indirect evidence supports the role of O. formigenes in the pathogenesis of calcium oxalate stone. We undertook to evaluate the correlation between the colonization levels of O. formigenes and urinary oxalate levels in patients with calcium oxalate stone disease, to confirm the hypothesis that the inhibitory effect of O. formigenes on calcium oxalate stone formation could be induced by lowering urinary
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oxalate excretion. The present study shows that there is a significant inverse correlation between the number of colony forming units of O. formigenes and urinary oxalate levels in patients with calcium oxalate urolithiasis. Sidhu et al. [18] determined the number of colony-forming units of O. formigenes in human stool samples either by standard culture or by the QC-PCR detection system. They showed that the number of CFUs detected proved to be quite similar for all specimens. We confirmed that the determined number of OxB CFUs were nearly identical as estimated by optical densities determined by QC-PCR. Quantitative assay using QC-PCR appears to reliably quantify the number of colony forming units of O. formigenes in human feces. Several limitations of the current study should be mentioned. First, only stone formers free from urinary tract infection for at least one year and had not taken antibiotics during the previous 3 months were included in the study. Patients with more stone events have possibly received more courses of antibiotics and thus earlier antibiotics might have influenced the colonization rate even though the possibility is low. Second, our results relate to a specific group of men, and patients in a community practice setting may behave somewhat differently.
Sidhu et al. demonstrated that it was possible to colonize with live O. formigenes in laboratory rats known to be noncolonized and revealed that rats receiving live O. formigenes excreted far lower levels of oxalate and did not develop the crystalluria [21]. In addition, they revealed that probiotic treatment of hyperoxaluric rats with O. formigenes might significantly reduce the level of oxalate in the urine and appeared to be safe and tolerated [22]. These observations suggest that probiotic treatment using O. formigenes may be a novel preventive treatment for patients with calcium oxalate urolithiasis. In conclusion, there is an inverse correlation between intestinal O. formigenes and urinary oxalate levels in patients with calcium oxalate urolithiasis in our study. Such a result supports the concept that O. formigenes is important in maintaining oxalate homeostasis and a lack of enteric O. formigenes increases the risk of calcium oxalate urolithiasis via increased urinary oxalate excretion.
Acknowledgements This work is supported by a Korea Research Foundation Grant (KRF 2001-041-F00216).
References [1] Kim H, Jo MK, Kwak C, Park SK, Yoo KY, Kang D, et al. Prevalence and epidemiologic characteristics of urolithiasis in Seoul, Korea. Urology 2002;59:517–21. [2] Uribarri J, Oh MS, Carroll HJ. The first kidney stone. Ann Intern Med 1989;111:1006–9. [3] Byeon SS, Kim HH, Kim SH. Analysis of the urinary stone components using chemical analysis method. Korean J Urol 1996; 37:179–86. [4] Seftel A, Resnick MI. Metabolic evaluation of urolithiasis. Urol Clin North Am 1990;17:159–69. [5] Balaji KC, Menon M. Mechanism of stone formation. Urol Clin North Am 1997;24:1–11. [6] von Unruh GE, Voss S, Sauerbruch T, Hesse A. Reference range for gastrointestinal oxalate absorption measured with a standardized [13 C2 ]oxalate absorption test. J Urol 2003;169:687–90. [7] Chadwick VS, Modha K, Dowling RH. Mechanism for hyperoxaluria in patients with ileal dysfunction. N Engl J Med 1973;289:172–6. [8] Smith LH, Fromm H, Hofmann AF. Acquired hyperoxaluria, nephrolithiasis, and intestinal disease. Description of a syndrome. N Engl J Med 1972;286:1371–5. [9] Allison MJ, Dawson KA, Mayberry WR, Foss JG. Oxalobacter formigenes gen. nov., sp. nov.: oxalate-degrading anaerobes that inhabit the gastrointestinal tract. Arch Microbiol 1985;141:1–7. [10] Doane LA, Liebman M, Caldwell DR. Microbial oxalate degradation; effects on oxalate and calcium balance in humans. Nutr Res 1989;9:957–64. [11] Hatch M, Freel RW. Oxalate transport across intestinal and renal epithelia. In: Khan S, editor. Calcium oxalate in biological systems. Boca Raton (FL): CRC Press; 1996. p. 217–38.
[12] Kleinschmidt K, Mahlmann A, Hautmann R. Anaerobic oxalatedegrading bacteria in the gut decrease faecal and urinary oxalate concentrations in stone formers. In: Ryall R, Basis R, Marshall VR, Rofe AM, Smith LH, Walker VR, editors. Urolithiasis 2. New York: Plenum; 1993. p. 439–41. [13] Allison MJ, Cook HM, Milne DB, Gallagher S, Clayman RV. Oxalate degradation by gastrointestinal bacteria from humans. J Nutr 1986;116:455–60. [14] Goldkin LD, Cave DR, Jaffin B, Robinson W, Bliss CM. A new factor in enteric hyperoxaluria: Oxalobacter formigenes. Am J Gastroenterol 1985;80:860–5. [15] Sidhu H, Hoppe B, Hesse A, Tenbrock K, Bromme S, Rietschel E, et al. Absence of Oxalobacter formigenes in cystic fibrosis patients: a risk factor for hyperoxaluria. Lancet 1998;352:1026–9. [16] Kwak C, Jeong BC, Lee JH, Kim HK, Kim EC, Kim HH. Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples. BJU Int 2001;88:627–32. [17] Sidhu H, Yenatska L, Ogden SD, Williams WN, Allison MJ, Peck AB. Natural colonization of children in the Ukraine with the intestinal bacterium, O. formigenes, using a PCR-based detection system. Mol Diagn 1997;2:89–97. [18] Sidhu H, Holmes RP, Allison MJ, Peck AB. Direct quantification of the enteric bacterium Oxalobacter formigenes in human fecal samples by quantitative competitive-template PCR. J Clin Microbiol 1999;37:1503–9. [19] Lung HY, Baetz AL, Peck AB. Molecular cloning, DNA sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes. J Bacteriol 1994; 176:2468–72.
C. Kwak et al. / European Urology 44 (2003) 475–481 [20] Cochran M, Hodgkinson A, Zarembski PM, Anderson CK. Hyperoxaluria in adults. Br J Surg 1968;55:121–8. [21] Sidhu H, Schmidt ME, Cornelius JG, Thamilselvan S, Khan SR, Hesse A, et al. Direct correlation between hyperoxaluria/oxalate stone disease and the absence of the gastrointestinal tract-dwelling bacterium
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Oxalobacter formigenes: possible prevention by gut recolonization or enzyme replacement therapy. Am Soc Nephrol 1999;10:S334–340. [22] Sidhu H, Allison MJ, Chow JM, Clark A, Peck AB. Rapid reversal of hyperoxaluria in a rat model after probiotic administration of Oxalobacter formigenes. J Urol 2001;166:1487–91.
European Urology 44 (2003) 475–481
Urinary Oxalate Levels and the Enteric Bacterium Oxalobacter formigenes in Patients with Calcium Oxalate Urolithiasis Cheol Kwaka, Hee Kyung Kimb, Eui Chong Kimc, Myung Sik Choib, Hyeon Hoe Kima,* a
Department of Urology, Seoul National University College of Medicine and Clinical Research Institute, Seoul National University Hospital, 28 Yongon Dong, Jongno Ku, Seoul 110-744, Republic of Korea b Department of Microbiology, Seoul National University College of Medicine, Seoul, Republic of Korea c Department of Clinical Pathology, Seoul National University College of Medicine and Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea First published online 9 July 2003
Abstract 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 ¼ 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 1:1 107 (04:1 108 ), and the level of 24 hours urine oxalate significantly decreased with increasing level of colony forming units of O. formigenes (r ¼ 0:356, p ¼ 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. # 2003 Elsevier B.V. All rights reserved. Keywords: Urolithiasis; Oxalate; Oxalobacter formigenes
1. Introduction The prevalence of urinary stone disease is estimated to be 2–3%, and the likelihood that a Caucasian man will develop stone disease by age 70 is about 1 in 8. The lifetime prevalence rate of urolithiasis in Korea was known to be 6.0% in men and 1.8% in women [1]. The recurrence rate without treatment for calcium oxalate renal stones is about 10% at 1 year, 35% at *
Corresponding author. Tel. þ82-2-760-2425; Fax: þ82-2-742-4665. E-mail address: [email protected] (H.H. Kim).
5 years, and 50% at 10 years [2]. Calcium oxalate stone is the most common cause of urolithiasis and accounts for 81.6% of the total number of urinary tract stones in Korea [3]. Hyperoxaluria is a risk factor for renal stones, and the urine load of oxalate plays a pivotal role in calcium oxalate stone formation even in normocalciuric patients [4]. In normal individuals, the majority of urinary oxalate is derived from the endogenous metabolism of glycine, glyoxylate, and ascorbic acid, and 10–20% is derived from oral ingestion [5]. The colon is a major site of oxalate absorption, and around 10% of dietary oxalate is absorbed under
0302-2838/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0302-2838(03)00318-X
476
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normal conditions [6]. However, the increased intestinal absorption of oxalate may lead to hyperoxaluria and a significantly enhanced risk of urinary stone formation [7,8]. Prevention of recurrence of urinary calculi is one of the biggest challenges facing the modern urologist. Oxalobacter formigenes is a recently identified oxalate degrading bacterium, which colonizes the GI tract of vertebrates, including humans [9], and it has a symbiotic relationship with its host by regulating oxalic acid absorption in the intestine and oxalate levels in the plasma and urine [10,11]. Previous studies have suggested a correlation between calcium oxalate stone activity and the lack of Oxalobacter formigenes colonization [12–16]. Although it was fascinating to learn that Oxalobacter formigenes influences the gastrointestinal absorption of oxalate, the examination of the relationship between this bacterium and urinary stone disease has been limited due to the difficult culture method. O. formigenes has shown to be a suitable target for DNA-based identification [16–18]. Using simpler DNA purification methods [16] and a rapid quantitative competitive-template PCR assay [18], we performed a prospective, controlled study by evaluating intestinal colonization by Oxalobacter formigenes and its relationship with urinary oxalate levels in patients with calcium oxalate stone disease, to confirm the pathogenic role of O. formigenes upon calcium oxalate urolithiasis. 2. Patients and methods 2.1. Patient populations This study involved 103 patients (89 males and 14 females) with calcium oxalate urolithiasis, ranging in age from 21 to 73 years (mean age, 47 years), and who were followed up from August 2000 to September 2001 at the Department of Urology, Seoul National University Hospital. Fresh stools and 24-hour urine samples were collected. Only stone-formers free from urinary tract infection for at least one year before the beginning of this study were included. Individuals who had taken antibiotics for any reason during the previous 3 months were excluded, and informed consent was obtained from all patients before participation. DNA was extracted from 250 mg of stool specimen within 3 hours of defecation for all patients. Urine samples were stored at 20 8C. 2.2. Bacterial strain and the isolation of genomic DNA Strain OxB (ATCC 35274) was used as the standard throughout this study, and was obtained from the American Type Culture Collection. The strain was grown in medium B containing 30 mM oxalate, as described elsewhere [9,16]. Isolation of genomic DNA from cultures of OxB was performed as described previously [16]. 2.3. Isolation of genomic DNA from human fecal specimens Fecal samples were collected from 103 patients with calcium oxalate urolithiasis. Whole bacterial DNA was isolated directly
from fresh stool samples obtained from individuals. Two hundredfifty mg of feces as a clean sample was suspended in 5 ml of PBS. Genomic DNA was extracted from feces as described previously [16]. 2.4. Quantitative competitive polymerase chain reaction (QC-PCR) PCR was performed as described previously [16]. The optimal reaction profile was found to be 94 8C for 5 minutes followed by 35 cycles of denaturation for 1 minute at 94 8C, annealing for 1 minute at 55 8C, and extension for 1 minute at 72 8C. Genomic DNA from OxB cultures was used as a positive control. O. formigenes expresses a unique gene, which is required for the catabolism of oxalate: oxc (encoding oxalate-coenzyme A decarboxylase). This gene has been cloned and sequenced [19], and the 50 ends of the oxc genes from numerous isolates of O. formigenes have identified unique, highly conserved regions responsible for the synthesis of a genus-specific PCR primer pair: OXFfp and OXFrp [18]. The sequence of OXFfp is 50 -AATGTAGAGTTGACTGA-30 , and the sequence of OXFrp is 50 -TTGATGCTGTTGATACG-30 . To construct a suitable competitive-DNA template to use as an internal control, a 227-bp fragment of the oxc gene flanked by sequences homologous to the OXFfp-OXFrp primer pair and containing a genus-specific probe was generated. PCR was performed with the OXFfp 50 primer and a modified 30 primer (50 -TTGATGCTGTTGATACGGTCAAGCAAACGCC-30 ), which consisted of two portions: a 50 end containing the 30 primer sequence (underlined) within the oxc gene, and a 30 end annealed with a site located approximately 200 bp downstream of the 50 primer site. The PCR with the OXFfp-modified OXFrp primer pair was used to amplify the 210-bp segment and synthesize the 17-bp OXFrp primer site at the 30 end. This PCR product was purified and ligated into PCR13.1 (Invitrogen, Inc., San Diego, CA, USA), a recombinant pCR13.1 plasmid with the proper insert (confirmed by sequencing) was selected for use as the internal competitive template. Quantitative competitive polymerase chain reaction (QC-PCR) is based on the assumption that a genomic DNA template and a competitive DNA template containing homologous primer sites will compete equally for PCR primers and that both experimentaland competitive-DNA template PCR products will subsequently be amplified collinearly. To determine the accuracy of the QC-PCR at quantifying the number of CFU in O. formigenes samples, QC-PCRs were established with an O. formigenes DNA preparation, which had a starting spectrophotometric reading of 3.043 mg of DNA/ml. Assuming that the genome of O. formigenes is similar in size to that of E. coli (4:7 103 kb), 1 mg of the genomic DNA of O. formigenes contained 2:0 108 molecules (or gene copies). Thus, the original genomic DNA preparation of O. formigenes OxB contained approximately 6:1 108 molecules/ml. Original concentration (6:1 108 molecules/ml) and twofold dilution (6:1 106 molecules/ml)of this DNA were used as template. The PCR products were sized by electrophoresis through 1.5% agarose gels and visualized with UV light. The PCR bands were scanned for intensities and normalized for differences in molecular mass, and the log ratios of O. formigenes to template band intensities were plotted against the log of the copy number of the synthetic template added per reaction to determine the log equivalence. The quantitation of oxc genes, and thereby bacteria, revealed the accuracy of this QC-PCR detection system. The log equivalence revealed that the number of molecules of O. formigenes OxB in the reaction in which original concentration and twofold dilution of
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Fig. 1. Quantification of the number of O. formigenes genomes in a sample using QC-PCR. Dilutions, ranging from 3:9 105 to 3:9 1012 molecules of purified plasmid containing the competitive template, were used either as DNA templates in PCR to establish standard curves (A) or as competitive DNA by mixing with 6:1 108 (B) or 6:1 106 (C) copies of purified O. formigenes OxB genomes. PCR band intensities were scanned and normalized for molecular mass and the log ratios of O. formigenes to template band intensities were graphed to determine log equivalence (D and E). QCT, quantitative competitive template.
OxB DNA were used were 5:9 108 and 1:2 106 , respectively (Fig. 1). 2.5. Determination of oxalate, calcium, citrate and creatinine in urine specimens Samples obtained from 24-h urine collections from patients on a self-selected diet were analyzed in our laboratory. Urine volume, pH (potentiometry) and the concentrations of creatinine (Jaffe reaction), magnesium (atomic absorption spectrophotometry), chloride (coulomb metric titration), sodium and potassium (flame emission spectrophotometry), sulfate (nephelometry), phosphate (phosphate molybdate reaction), ammonium (ion selective electrode), citrate (enzymatically, citrate lyase) and uric acid (enzymatically, uricase) were measured. Urinary calcium excretion levels were measured using a Hitachi 7170 Automatic Analyzer (Hitachi Inc., Japan), and urinary excretion levels of oxalate were measured using an oxalate kit (Sigma Diagnostics Inc., MO, USA). 2.6. Statistical analysis Differences in urinary parameters were compared using the Student’s non-paired t-test or ANOVA, and categorical comparisons were performed using the w2-test. Data are presented as mean SEM unless otherwise stated. The level of significance was set at p < 0:05.
3. Results 3.1. Relationship between urinary parameters and stone frequency Fifty patients (48.5%) had a single stone episode; 33 (32.0%) had 2 episodes and 20 (19.4%) had 3 or more stone episodes. No significant differences were observed in the distributions of age and sex in each group according to stone episodes. Urine parameters were not found to depend significantly on stone frequency (Table 1). 3.2. Relationship between stone episodes and O. formigenes detection by PCR The detection rate of Oxalobacter formigenes in stools by qualitative PCR tended to be reduced as the number of stone episodes increased ( p > 0:05) (Table 2). Intestinal Oxalobacteria were detected in 45.6% of all calcium oxalate stone patients. In stone formers who tested negative for Oxalobacteria, the average urinary
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Table 1 Results for daily urine composition according to stone frequencies (mean SEM) p valuea
Stone frequency 1 (n ¼ 50) 45.1 1.96
Age No. (%) according to sex Male Female Urine volume (ml/day) pH Calcium (mmol/day) Magnesium (mmol/day) Phosphorus (mmol/day) Uric acid (mmol/day) Oxalic acid (mmol/day) Citric acid (mmol/day) Creatinine clearance (ml/min) a
2 (n ¼ 33)
44 (88.0) 6 (12.0) 1651.5 6.24 5.79 1.71 23.41 3.59 0.30 1.70 85.5
47.1 1.8 30 (90.9) 3 (9.1) 1539.4 6.14 7.50 2.36 26.11 4.26 0.34 1.95 102.5
123.0 0.12 0.36 0.13 1.13 0.18 0.02 0.22 3.7
49.6 2.5 15 (75.0) 5 (25.0) 1913.5 6.33 7.13 2.17 28.21 3.98 0.39 1.80 104.3
172.0 0.16 0.69 0.21 1.85 0.21 0.05 0.24 5.2
0.342 0.235
0.263 0.634 0.082 0.075 0.198 0.211 0.196 0.767 0.963
ANOVA.
Table 2 Stone frequencies according to PCR results for O. formigenes Stone frequency
PCR results for O. formigenesa
Total
Negative Pt. No. (%)
Positive Pt. No. (%)
1 2 3
25 (50.0) 19 (57.6) 12 (60.0)
25 (50.0) 14 (42.4) 8 (40.0)
Total
56 (54.4)
47 (45.6)
50 33 20 103 (100)
Data presented are number (%). a 2 w -test for trend, p ¼ 0:398.
oxalate level was 0.36 mmol/day, and this compared to 0.29 mmol/day in patients that tested positive ( p < 0:05) (Table 3). Table 3 Daily urine composition of calcium oxalate stone patients according to PCR results for O. formigenes (mean SEM) PCR results for O. formigenes Negative (n ¼ 56) Urine volume (ml/day) pH Calcium (mmol/day) Magnesium (mmol/day) Phosphorus (mmol/day) Uric acid (mmol/day) Oxalic acid (mmol/day) Citric acid (mmol/day) Creatinine clearance (ml/min) a
185.0 0.13 0.57 0.31 1.27 0.19 0.04 0.21 5.1
3 (n ¼ 20)
Student’s t-test.
1581.3 6.25 6.66 0.83 25.94 3.87 0.36 1.68 93.17
p valuea
3.3. Quantification of colony forming units (CFUs) by QC-PCR The mean number of colony forming units per gram of feces from all calcium oxalate urolithiasis patients was 1:1 107 5:6 107 (range 04:1 108 ) (Fig. 2). The mean number of colony forming units per gram of feces in one-time stone formers (n ¼ 50), 2-time stone formers (n ¼ 33) and 3 or more stone forming episodes (n ¼ 20) were 2:0 107 , 3:2 106 and 2:9 105 , respectively ( p > 0:05). 3.4. Correlation between colony forming units detected by QC-PCR and the clinico-biochemical parameters An inverse correlation was found between log equivalences of colony forming units per gram of feces and 24-hour urinary oxalate levels in 47 patients with calcium oxalate urolithiasis, who tested positive for Oxalobacter by PCR (r ¼ 0:356, p ¼ 0:021) (Fig. 3). However, no correlations were found between the log equivalences of colony forming units per gram of feces and the other 24-hour urinary parameters.
Positive (n ¼ 47) 128.5 0.10 0.40 0.15 1.05 0.16 0.02 0.16 3.50
1769.2 6.19 6.43 2.21 24.09 3.86 0.29 1.95 95.89
125.1 0.12 0.43 0.21 1.18 0.18 0.03 0.22 4.41
0.274 0.832 0.702 0.151 0.244 0.962 0.030 0.191 0.564
4. Discussion After the emphasis was first placed on the role of hyperoxaluria in calcium oxalate stone formation by Cochran et al. [20], urinary oxalate concentration has been accepted to be an important factor of calcium oxalate stone formation. The intestinal anaerobe O. formigenes is attracting increasing attention because of its possible role in the regulation of oxalic acid absorption in humans [10,11].
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Fig. 2. Quantification of the number of O. formigenes genomes in stool samples of patients with calcium oxalate urolithiasis by QC-PCR. Dilutions, ranging from 3:9 101 to 3:9 105 molecules of purified plasmid containing the competitive template, were used for competitive DNA by mixing with 1 ml of extracted stool DNA from patients with calcium oxalate urolithiasis (A and B). PCR band intensities were scanned and normalized for molecular mass and the log ratios of O. formigenes to template band intensities were graphed to determine log equivalence (C and D). QCT, quantitative competitive template.
Decreased intestinal colonization of O. formigenes has been reported in recurrent calcium oxalate stone formers and in enteric hyperoxaluria patients [12–14]. Kleinschmidt et al. [12] correlated the colony forming units of O. formigenes per gram of feces with the frequency of episodes of kidney stone formation, and reported a complete absence of this bacterium
Fig. 3. Correlation between the log equivalences of colony forming units per gram of feces and 24-hour urinary oxalate levels in patients with calcium oxalate urolithiasis.
in patients having four or more episodes. Sidhu and coworkers suggested that the absence of O. formigenes from the intestinal tract of cystic fibrosis patients appeared to lead to the increased absorption of oxalate, thereby increasing the risk of hyperoxaluria and its complications [13]. We also found that the colonization rate of O. formigenes in patients with urolithiasis was significantly lower than that of healthy volunteers, known to be free from urolithiasis [16]. Putting these various reports together, it is possible to hypothesize that a lack of intestinal colonization by O. formigenes is one of the pathogenic pathway of calcium oxalate urolithiasis. However, to the best of our knowledge no report has convincing described a direct correlation between colony forming units of O. formigenes and urinary oxalate levels in patients with calcium oxalate stone disease, although indirect evidence supports the role of O. formigenes in the pathogenesis of calcium oxalate stone. We undertook to evaluate the correlation between the colonization levels of O. formigenes and urinary oxalate levels in patients with calcium oxalate stone disease, to confirm the hypothesis that the inhibitory effect of O. formigenes on calcium oxalate stone formation could be induced by lowering urinary
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oxalate excretion. The present study shows that there is a significant inverse correlation between the number of colony forming units of O. formigenes and urinary oxalate levels in patients with calcium oxalate urolithiasis. Sidhu et al. [18] determined the number of colony-forming units of O. formigenes in human stool samples either by standard culture or by the QC-PCR detection system. They showed that the number of CFUs detected proved to be quite similar for all specimens. We confirmed that the determined number of OxB CFUs were nearly identical as estimated by optical densities determined by QC-PCR. Quantitative assay using QC-PCR appears to reliably quantify the number of colony forming units of O. formigenes in human feces. Several limitations of the current study should be mentioned. First, only stone formers free from urinary tract infection for at least one year and had not taken antibiotics during the previous 3 months were included in the study. Patients with more stone events have possibly received more courses of antibiotics and thus earlier antibiotics might have influenced the colonization rate even though the possibility is low. Second, our results relate to a specific group of men, and patients in a community practice setting may behave somewhat differently.
Sidhu et al. demonstrated that it was possible to colonize with live O. formigenes in laboratory rats known to be noncolonized and revealed that rats receiving live O. formigenes excreted far lower levels of oxalate and did not develop the crystalluria [21]. In addition, they revealed that probiotic treatment of hyperoxaluric rats with O. formigenes might significantly reduce the level of oxalate in the urine and appeared to be safe and tolerated [22]. These observations suggest that probiotic treatment using O. formigenes may be a novel preventive treatment for patients with calcium oxalate urolithiasis. In conclusion, there is an inverse correlation between intestinal O. formigenes and urinary oxalate levels in patients with calcium oxalate urolithiasis in our study. Such a result supports the concept that O. formigenes is important in maintaining oxalate homeostasis and a lack of enteric O. formigenes increases the risk of calcium oxalate urolithiasis via increased urinary oxalate excretion.
Acknowledgements This work is supported by a Korea Research Foundation Grant (KRF 2001-041-F00216).
References [1] Kim H, Jo MK, Kwak C, Park SK, Yoo KY, Kang D, et al. Prevalence and epidemiologic characteristics of urolithiasis in Seoul, Korea. Urology 2002;59:517–21. [2] Uribarri J, Oh MS, Carroll HJ. The first kidney stone. Ann Intern Med 1989;111:1006–9. [3] Byeon SS, Kim HH, Kim SH. Analysis of the urinary stone components using chemical analysis method. Korean J Urol 1996; 37:179–86. [4] Seftel A, Resnick MI. Metabolic evaluation of urolithiasis. Urol Clin North Am 1990;17:159–69. [5] Balaji KC, Menon M. Mechanism of stone formation. Urol Clin North Am 1997;24:1–11. [6] von Unruh GE, Voss S, Sauerbruch T, Hesse A. Reference range for gastrointestinal oxalate absorption measured with a standardized [13 C2 ]oxalate absorption test. J Urol 2003;169:687–90. [7] Chadwick VS, Modha K, Dowling RH. Mechanism for hyperoxaluria in patients with ileal dysfunction. N Engl J Med 1973;289:172–6. [8] Smith LH, Fromm H, Hofmann AF. Acquired hyperoxaluria, nephrolithiasis, and intestinal disease. Description of a syndrome. N Engl J Med 1972;286:1371–5. [9] Allison MJ, Dawson KA, Mayberry WR, Foss JG. Oxalobacter formigenes gen. nov., sp. nov.: oxalate-degrading anaerobes that inhabit the gastrointestinal tract. Arch Microbiol 1985;141:1–7. [10] Doane LA, Liebman M, Caldwell DR. Microbial oxalate degradation; effects on oxalate and calcium balance in humans. Nutr Res 1989;9:957–64. [11] Hatch M, Freel RW. Oxalate transport across intestinal and renal epithelia. In: Khan S, editor. Calcium oxalate in biological systems. Boca Raton (FL): CRC Press; 1996. p. 217–38.
[12] Kleinschmidt K, Mahlmann A, Hautmann R. Anaerobic oxalatedegrading bacteria in the gut decrease faecal and urinary oxalate concentrations in stone formers. In: Ryall R, Basis R, Marshall VR, Rofe AM, Smith LH, Walker VR, editors. Urolithiasis 2. New York: Plenum; 1993. p. 439–41. [13] Allison MJ, Cook HM, Milne DB, Gallagher S, Clayman RV. Oxalate degradation by gastrointestinal bacteria from humans. J Nutr 1986;116:455–60. [14] Goldkin LD, Cave DR, Jaffin B, Robinson W, Bliss CM. A new factor in enteric hyperoxaluria: Oxalobacter formigenes. Am J Gastroenterol 1985;80:860–5. [15] Sidhu H, Hoppe B, Hesse A, Tenbrock K, Bromme S, Rietschel E, et al. Absence of Oxalobacter formigenes in cystic fibrosis patients: a risk factor for hyperoxaluria. Lancet 1998;352:1026–9. [16] Kwak C, Jeong BC, Lee JH, Kim HK, Kim EC, Kim HH. Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples. BJU Int 2001;88:627–32. [17] Sidhu H, Yenatska L, Ogden SD, Williams WN, Allison MJ, Peck AB. Natural colonization of children in the Ukraine with the intestinal bacterium, O. formigenes, using a PCR-based detection system. Mol Diagn 1997;2:89–97. [18] Sidhu H, Holmes RP, Allison MJ, Peck AB. Direct quantification of the enteric bacterium Oxalobacter formigenes in human fecal samples by quantitative competitive-template PCR. J Clin Microbiol 1999;37:1503–9. [19] Lung HY, Baetz AL, Peck AB. Molecular cloning, DNA sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes. J Bacteriol 1994; 176:2468–72.
C. Kwak et al. / European Urology 44 (2003) 475–481 [20] Cochran M, Hodgkinson A, Zarembski PM, Anderson CK. Hyperoxaluria in adults. Br J Surg 1968;55:121–8. [21] Sidhu H, Schmidt ME, Cornelius JG, Thamilselvan S, Khan SR, Hesse A, et al. Direct correlation between hyperoxaluria/oxalate stone disease and the absence of the gastrointestinal tract-dwelling bacterium
481
Oxalobacter formigenes: possible prevention by gut recolonization or enzyme replacement therapy. Am Soc Nephrol 1999;10:S334–340. [22] Sidhu H, Allison MJ, Chow JM, Clark A, Peck AB. Rapid reversal of hyperoxaluria in a rat model after probiotic administration of Oxalobacter formigenes. J Urol 2001;166:1487–91.