Oxalobacter formigenes reduce the risk of kidney stones in patients exposed to oral antibiotics: a case-control study.

This is the first prospective study to investigate the association between kidney stones, bone mineral density, serum testosterone, colon cancer and O. formigenes colonization. 40 kidney stone patients and 85 controls were enrolled. O. formigenes colonization was established. BMD was examined from T- and Z-scores using dual energy absorptiometry. O. formigenes was found in 28 of 40 cases and 80 of 85 controls. BMD was significantly reduced in patients (p < 0.05). The evaluation revealed a significant association between lowered O. formigenes colonization and low testosterone. Urinary calcium and oxalates levels were greater in patient. Serum testosterone and urinary citrate concentrations was reduced in patients with a significant difference. Also an association between O. formigenes and colon cancer was noted. Absence of O. formigenes might stand for a pathogenic factor in calcium oxalate stone, low bone mineral density, low testosterone levels and also colon cancer, when antibiotics are prescribed generously.

Induction of enteric oxalate secretion by Oxalobacter formigenes in mice does not require the presence of either apical oxalate transport proteins Slc26A3 or Slc26A6.

Oxalobacter sp. promotion of enteric oxalate excretion, correlating with reductions in urinary oxalate excretion, was previously reported in rats and mice, but the mechanistic basis for this affect has not been described. The main objective of the present study was to determine whether the apical oxalate transport proteins, PAT1 (slc26a6) and DRA (slc26a3), are involved in mediating the Oxalobacter-induced net secretory flux across colonized mouse cecum and distal colon. We measured unidirectional and net fluxes of oxalate across tissues removed from colonized PAT1 and DRA knockout (KO) mice and also across two double knockout (dKO) mouse models with primary hyperoxaluria, type 1 (i.e., deficient in alanine-glyoxylate aminotransferase; AGT KO), including PAT1/AGT dKO and DRA/AGT dKO mice compared to non-colonized mice. In addition, urinary oxalate excretion was measured before and after the colonization procedure. The results demonstrate that Oxalobacter can induce enteric oxalate excretion in the absence of either apical oxalate transporter and urinary oxalate excretion was reduced in all colonized genotypes fed a 1.5% oxalate-supplemented diet. We conclude that there are other, as yet unidentified, oxalate transporters involved in mediating the directional changes in oxalate transport across the Oxalobacter-colonized mouse large intestine.

Inhibition of urinary stone disease by a multi-species bacterial network ensures healthy oxalate homeostasis.

The incidence of urinary stone disease is rapidly increasing, with oxalate being a primary constituent of approximately 80% of all kidney stones. Despite the high dietary exposure to oxalate by many individuals and its potential nephrotoxicity, mammals do not produce enzymes to metabolize this compound, instead relying in part on bacteria within the gut to reduce oxalate absorption and urinary excretion. While considerable research has focused on isolated species of oxalate-degrading bacteria, particularly those with an absolute requirement for oxalate, recent studies have pointed to broader roles for microbiota both in oxalate metabolism and inhibition of urinary stone disease. Here we examined gut microbiota from patients with and live-in individuals without urinary stone disease to determine if healthy individuals harbored a more extensive microbial network associated with oxalate metabolism. We found a gender-specific association between the gut microbiota composition and urinary stone disease. Bacteria enriched in healthy individuals largely overlapped with those that exhibited a significant, positive correlation with Oxalobacter formigenes, a species presumed to be at the center of an oxalate-metabolizing microbial network. Furthermore, differential abundance analyses identified multiple taxa known to also be stimulated by oxalate in rodent models. Interestingly, the presence of these taxa distinguished patients from healthy individuals better than either the relative abundance or colonization of O. formigenes. Thus, our work shows that bacteria stimulated by the presence of oxalate in rodents may, in addition to obligate oxalate users, play a role in the inhibition of urinary stone disease in man.

Comparative prevalence of Oxalobacter formigenes in three human populations.

There has been increasing interest in the human anaerobic colonic bacterium Oxalobacter formigenes because of its ability to metabolize oxalate, and its potential contribution to protection from calcium oxalate kidney stones. Prior studies examining the prevalence of this organism have focused on subjects in developed countries and on adults. Now using O. formigenes-specific PCR, we have compared the prevalence of these organisms among subjects in two remote areas in which modern medical practices have hardly been present with a USA group of mothers and their infants for the first three years of life. Among the Amerindians of the Yanomami-Sanema and Yekwana ethnic groups in Venezuela and the Hadza in Tanzania, O. formigenes was detected in 60-80% of the adult subjects, higher than found in adults from USA in this and prior studies. In young children, the prevalence was much lower in USA than in either tribal village. These data extend our understanding of the epidemiology of O. formigenes carriage, and are consistent with the hypothesis that the rising incidence of kidney stones is associated with the progressive loss of O. formigenes colonization in populations that have been highly impacted by modern medical practices.

Oxalobacter formigenes-associated host features and microbial community structures examined using the American Gut Project.

BACKGROUND: Increasing evidence shows the importance of the commensal microbe Oxalobacter formigenes in regulating host oxalate homeostasis, with effects against calcium oxalate kidney stone formation, and other oxalate-associated pathological conditions. However, limited understanding of O. formigenes in humans poses difficulties for designing targeted experiments to assess its definitive effects and sustainable interventions in clinical settings. We exploited the large-scale dataset from the American Gut Project (AGP) to study O. formigenes colonization in the human gastrointestinal (GI) tract and to explore O. formigenes-associated ecology and the underlying host-microbe relationships. RESULTS: In >8000 AGP samples, we detected two dominant, co-colonizing O. formigenes operational taxonomic units (OTUs) in fecal specimens. Multivariate analysis suggested that O. formigenes abundance was associated with particular host demographic and clinical features, including age, sex, race, geographical location, BMI, and antibiotic history. Furthermore, we found that O. formigenes presence was an indicator of altered host gut microbiota structure, including higher community diversity, global network connectivity, and stronger resilience to simulated disturbances. CONCLUSIONS: Through this study, we identified O. formigenes colonizing patterns in the human GI tract, potential underlying host-microbe relationships, and associated microbial community structures. These insights suggest hypotheses to be tested in future experiments. Additionally, we proposed a systematic framework to study any bacterial taxa of interest to computational biologists, using large-scale public data to yield novel biological insights.

Hyperoxaluria leads to dysbiosis and drives selective enrichment of oxalate metabolizing bacterial species in recurrent kidney stone endures.

Hyperoxaluria due to endogenously synthesized and exogenously ingested oxalates is a leading cause of recurrent oxalate stone formations. Even though, humans largely rely on gut microbiota for oxalate homeostasis, hyperoxaluria associated gut microbiota features remain largely unknown. Based on 16S rRNA gene amplicons, targeted metagenomic sequencing of formyl-CoA transferase (frc) gene and qPCR assay, we demonstrate a selective enrichment of Oxalate Metabolizing Bacterial Species (OMBS) in hyperoxaluria condition. Interestingly, higher than usual concentration of oxalate was found inhibitory to many gut microbes, including Oxalobacter formigenes, a well-characterized OMBS. In addition a concomitant enrichment of acid tolerant pathobionts in recurrent stone sufferers is observed. Further, specific enzymes participating in oxalate metabolism are found augmented in stone endures. Additionally, hyperoxaluria driven dysbiosis was found to be associated with oxalate content, stone episodes and colonization pattern of Oxalobacter formigenes. Thus, we rationalize the first in-depth surveillance of OMBS in the human gut and their association with hyperoxaluria. Our findings can be utilized in the treatment of hyperoxaluria associated recurrent stone episodes.

Microbial Community Transplant Results in Increased and Long-Term Oxalate Degradation.

Gut microbes are essential for the degradation of dietary oxalate, and this function may play a role in decreasing the incidence of kidney stones. However, many oxalate-degrading bacteria are susceptible to antibiotics and the use of oxalate-degrading probiotics has only led to an ephemeral reduction in urinary oxalate. The objective of the current study was to determine the efficacy of using whole-community microbial transplants from a wild mammalian herbivore, Neotoma albigula, to increase oxalate degradation over the long term in the laboratory rat, Rattus norvegicus. We quantified the change in total oxalate degradation in lab rats immediately after microbial transplants and at 2- and 9-month intervals following microbial transplants. Additionally, we tracked the fecal microbiota of the lab rats, with and without microbial transplants, using high-throughput Illumina sequencing of a hyper-variable region of the 16S rRNA gene. Microbial transplants resulted in a significant increase in oxalate degradation, an effect that persisted 9 months after the initial transplants. Functional persistence was corroborated by the transfer, and persistence of a group of bacteria previously correlated with oxalate consumption in N. albigula, including an anaerobic bacterium from the genus Oxalobacter known for its ability to use oxalate as a sole carbon source. The results of this study indicate that whole-community microbial transplants are an effective means for the persistent colonization of oxalate-degrading bacteria in the mammalian gut.

The Presence of Oxalobacter formigenes in the Microbiome of Healthy Young Adults.

PURPOSE: Oxalobacter formigenes, a member of the human colonic microbiota with a major role in net colonic oxalate transport and secretion, is protective against the formation of calcium oxalate kidney stones. We describe the prevalence, relative abundance and stability of O. formigenes in healthy young adults in the United States. MATERIALS AND METHODS: We used HMP (Human Microbiome Project) data on fecal samples from 242 healthy young adults who had 1 to 3 study visits. Samples underwent whole genomic shotgun sequencing and/or 16S rRNA sequencing. Three data sets available from the processed sequence data were studied, including whole genomic shotgun metagenomic analysis by alignment to reference genomes using shotgun community profiling, or MetaPhlAn (http://huttenhower.sph.harvard.edu/metaphlan) or QIIME (http://qiime.org/) analysis of the V1-3 or V3-5 16S sequences. RESULTS: O. formigenes was detected in fecal samples using whole genomic shotgun and 16S rRNA data. Analysis of the whole genomic shotgun data set using shotgun community profiling showed that 29 of 94 subjects (31%) were O. formigenes positive. V1-3 and V3-5 analyses were less sensitive for O. formigenes detection. When present, O. formigenes relative abundance varied over 3 log10 and was normally distributed. All assays agreed in 58 of 66 samples (88%) studied by all 3 methods. Of 14 subjects who were O. formigenes positive at baseline 13 (93%) were positive at the followup visit, indicating the stability of colonization. CONCLUSIONS: O. formigenes appears to be stably present in fewer than half of healthy young adults in the United States. It is most sensitively detected by whole genomic shotgun.

Sensitivity of human strains of Oxalobacter formigenes to commonly prescribed antibiotics.

OBJECTIVE: To determine the sensitivity of 4 strains of Oxalobacter formigenes (Oxf) found in humans--HC1, Va3, CC13, and OxK--to varying concentrations of commonly prescribed antibiotics. Oxf gut colonization has been associated with a decreased risk of forming recurrent calcium oxalate kidney stones. METHODS: For each strain and each antibiotic concentration, 100 µL of an overnight culture and 100 µL of the appropriate antibiotic were added to a 7-mL vial of oxalate culture medium containing 20 mM oxalate. On the fourth day, vials were visually examined for growth, and a calcium oxalate precipitation test was performed to determine whether Oxf grew in the presence of the antibiotic. RESULTS: All 4 Oxf strains were resistant to amoxicillin, amoxicillin/clavulanate, ceftriaxone, cephalexin, and vancomycin, and they were all sensitive to azithromycin, ciprofloxacin, clarithromycin, clindamycin, doxycycline, gentamicin, levofloxacin, metronidazole, and tetracycline. One strain, CC13, was resistant to nitrofurantoin, and the others were sensitive. Differences in minimum inhibitory concentration between strains were demonstrated. CONCLUSION: Four human strains of Oxf are sensitive to a number of antibiotics commonly used in clinical practice; however, minimum inhibitory concentrations differ between strains.

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.

Presence of Oxalobacter formigenes in the intestinal tract is associated with the absence of calcium oxalate urolith formation in dogs.

The incidence of calcium oxalate (CaOx) urolithiasis in dogs has increased steadily over the last two decades. A potential mechanism to minimize CaOx urolithiasis is to reduce enteric absorption of dietary oxalate by oxalate-metabolizing enteric bacteria. Enteric colonization of Oxalobacter formigenes, an anaerobe which exclusively relies on oxalate metabolism for energy, is correlated with absence of hyperoxaluria or CaOx urolithiasis or both in humans and laboratory animals. We thus hypothesized that decreased enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis in dogs. Fecal samples from dogs with CaOx uroliths, clinically healthy, age-, breed- and gender-matched dogs, and healthy non-stone forming breed dogs were screened for the presence of O. formigenes by quantitative PCR to detect the oxalyl CoA decarboxylase (oxc) gene, and by oxalate degrading biochemical activity in fecal cultures. Prevalence of O. formigenes in dogs with CaOx uroliths was 25%, compared to 50% in clinically healthy, age-, breed- and gender-matched dogs, and 75% in healthy non-stone forming breeds. The presence of oxc genes of O. formigenes was significantly higher in healthy non-stone forming breed dogs than in the dogs with CaOx stones. Further, dogs with calcium oxalate stones and the stone-forming breed-matched controls showed comparable levels of biochemical oxalate degrading activity. We conclude that the absence of enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis.

The construction of an oxalate-degrading intestinal stem cell population in mice: a potential new treatment option for patients with calcium oxalate calculus.

About 80% of all urological stones are calcium oxalate, mainly caused by idiopathic hyperoxaluria (IH). The increased absorption of oxalate from the intestine is the major factor underlying IH. The continuous self-renewal of the intestinal epithelium is due to the vigorous proliferation and differentiation of intestinal stem cells. If the intestinal stem cell population can acquire the ability to metabolize calcium oxalate by means of oxc and frc transgenes, this will prove a promising new therapy option for IH. In our research, the oxalate-degrading genes of Oxalobacter formigenes (Oxf)-the frc gene and oxc gene-were cloned and transfected into a cultured mouse-derived intestinal SC population to give the latter an oxalate-degrading function. Oxf was isolated and cultivated and the oxalate-degrading genes-frc and oxc-were cloned. The dicistronic eukaryotic expression vector pIRES-oxc-frc was constructed and transferred into the mouse stem cell population. After selection with G418, the expression of the genes was identified. The oxalate-degrading function of transfected cells was determined by transfection into the intestinal stem cell population of the mouse. The change in oxalate concentration was determined with an ion chromatograph. The recombinant plasmid containing oxc and frc genes was transfected into the stem cell population of the mouse and the expression of the genes found normal. The cell population had acquired an oxalate-degrading function. The oxc and frc genes could be transfected into the intestinal stem cell population of the mouse and the cells acquired an oxalate-degrading function.

Reduction of plasma oxalate levels by oral application of Oxalobacter formigenes in 2 patients with infantile oxalosis.

The spectrum of primary hyperoxaluria type I is extremely heterogeneous, ranging from singular to recurrent urolithiasis and early end-stage renal disease (ESRD). In infantile oxalosis, the most devastating form, ESRD occurs as early as within the first weeks of life. No kidney replacement therapy sufficiently removes endogenously overproduced oxalate. However, curative combined liver-kidney transplant often is impracticable in small infants. Oxalobacter formigenes (O formigenes), an anaerobic oxalate-degrading bacterium, is a colonizer of the healthy human colon. Oral administration of O formigenes has been shown to significantly decrease urine and plasma oxalate levels in patients with primary hyperoxaluria. We report compassionate use of O formigenes in two 11-month-old girls with infantile oxalosis and ESRD. They received O formigenes twice a day for 4 weeks (or until transplant). Dialysis regimens were unchanged. Plasma oxalate levels decreased from >110 µmol/L before to 71.53 µmol/L under treatment in patient 1 and from >90 to 68.56 µmol/L (first treatment period) and 50.05 µmol/L (second treatment period) in patient 2. O formigenes was well tolerated. No serious side effects were reported. Extremely increased plasma oxalate levels in patients with infantile oxalosis may enable intestinal elimination of endogenous oxalate in the presence of O formigenes. Therefore, O formigenes therapy may be helpful as a bridging procedure until transplant in such patients.

Identification of Oxalobacter formigenes in the faeces of healthy cats.

AIMS: Oxalobacter formigenes is an oxalate-degrading intestinal bacterium that has been found in humans, cattle, sheep, rats and dogs. Its presence in the intestinal tract may be a protective factor against calcium oxalate urolithiasis because of its ability to degrade oxalate. The objective of this study was to determine whether O. formigenes could be detected in the faeces of healthy cats. METHODS AND RESULTS: A convenience sample of 28 cats was enrolled. Faecal samples were tested for oxc, a gene specific for O. formigenes, by real-time PCR. This gene was detected in 5/28 (18%) cats; however, the prevalence increased to 86% (24/28) with a modification of the methodology. CONCLUSIONS: Demonstrating the presence of O. formigenes in the faeces of healthy cats for the first time in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Future investigation of the role of this organism in the pathophysiology of calcium oxalate urolithiasis in cats is indicated.

Oxalate balance in fat sand rats feeding on high and low calcium diets.

Oxalate reduces calcium availability of food because it chelates calcium, forming the sparingly soluble salt calcium-oxalate. Nevertheless, fat sand rats (Psammomys obesus; Gerbillinae) feed exclusively on plants containing much oxalate. We measured the effects of calcium intake on oxalate balance by comparing oxalate intake and excretion in wild fat sand rats feeding on their natural, oxalate-rich, calcium-poor diet with commercially-bred fat sand rats feeding on an artificial, calcium-rich, oxalate-poor diet of rodent pellets. We also tested for the presence of the oxalate degrading bacterium Oxalobacter sp. in the faeces of both groups. Fat sand rats feeding on saltbush ingested significantly more oxalate than fat sand rats feeding on pellets (P < 0.001) and excreted significantly more oxalate in urine and faeces (P < 0.01 for both). However the fraction of oxalate recovered in excreta [(oxalate excreted in urine + oxalate excreted in faeces)/oxalate ingested] was significantly higher in pellet-fed fat sand rats (61%) than saltbush-fed fat sand rats (27%). We found O. sp. in the faeces of both groups indicating that fat sand rats harbor oxalate degrading bacteria, and these are able, to some extent, to degrade oxalate in its insoluble form.

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.

Cloning and identification of frc gene from Oxalobacter frmigenes.

The cloning and identification of frc gene from Oxalobacter formigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacter formigenes was extracted. frc gene fragment was amplified by polymerase chain reaction (PCR) and linked with pEGFP-C1. The recombinant plasmid was designated pEGFP-frc and was identified by restriction-enzyme digestion and sequencing. Human embryo kidney 293 cells were transfected with pEGFP-frc, then RT-PCR and Western blotting were performed to detect the expression of frc gene. The length of frc gene was found to be 1287 bp, and the homology of nucleotides and amino-acid residue with the sequence in GenBank was 95.88% and 99.07%. Bright green fluorescent light could be observed in 293 cells transfected with the pEGFP-frc. frc mRNA and fusion protein FCoAT-EGFP were detected in the cells. It is concluded that frc gene cloned from the Oxalobacter formigenes in the intestines of Chinese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.

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.