Faculty Bibliography

Background: Oxalobacter formigenes (O.f.) are symbiotic bacteria in the human gut that degrade oxalate, a component of most kidney stones. Observational studies suggest that O.f. colonization reduces the risk for kidney stones. Given the importance of dietary oxalate and calcium levels, studies in mice are more practical than in humans; however, O.f. do not naturally colonize laboratory rodents. Our objective was to develop a humanized murine model to investigate the therapeutic potential of O. formigenes in its native microbiome.
Method(s): To humanize mice, we transplanted feces from a pool of healthy human donors who were O.f.-negative (confirmed by PCR, qPCR, and oxalate degradation assay), supplemented with a human O.f. strain: OXCC13 (108 CFU/mL). The inoculum was introduced to C57BL/6J mice via esophageal gavage three times over six days. We compared two methods of humanization, transplanting inocula into mice that were (i) germ free; or (ii) treated with high-dose, broad-spectrum antibiotics (0.5g/L vancomycin, 1g/L ampicillin, 1g/L neomycin, 1g/L metronidazole in drinking water for 6 days) to suppress their native microbiome. As controls, one group received humanization with no pre-treatment and another received a sham gavage.
Result(s): Based on oxc qPCR and 16S rRNA sequencing, all humanized groups were stably colonized with O.f. through 8 weeks post-gavage, whereas mice that received sham gavage remained uncolonized (p<0.001). Humanization significantly changed microbial community structure as measured by unweighted UniFrac distances (p<0.001) and humanized germ-free and antibiotic-treated groups were highly similar in beta-diversity. We also assessed humanization by the number of shared OTUs between treatment groups and donor inoculum over time. Both germ-free and antibiotic-treated mice had a significant increase in shared OTUs compared to sham (p=0.024, p=0.036). The number of shared OTUs was stable in each group through 8 weeks post-gavage without significant difference between germ-free and antibiotic-treated mice.
Conclusion(s): Our method of transplanting human feces and O.f. conferred a new microbial phenotype in mice that resembled a human microbiome and was stable over time. Antibiotic pre-treatment, a simpler alternative to germ-free mice, provided comparable results. This model may allow insights to O.f.'s role in preventing calcium oxalate stones

Background: Multiple bacterial species are capable of degrading oxalate in vitro. Certain taxa degrade oxalate as their sole source of energy and carbon (e.g. Oxalobacter formigenes), whereas others use oxalate as an auxiliary carbon source. For oxalate metabolism, it is not yet well-understood how genomic potential relates to transcriptional regulation. We asked whether the human gut could have a community of oxalatedegrading taxa working synergistically to diminish the effects of this toxic metabolite. Our hypothesis is that oxalate metabolism is regulated by a multi-organism oxalatedegrading community (oxalobiome) that is dominated by specialist oxalate degraders.
Method(s): We used data from 2 public databases: (i)8 healthy subjects in the USA; and (ii)471 healthy subjects in the Netherlands as part of the Human Functional Genomic Project (HFGP). Both collected fecal samples for metagenomic and/or metatranscriptomic high throughput sequencing. Using HUMAnN2 with customized settings, we profiled the metabolic activity of oxalate-degrading bacterial species. Output from these analyses was expressed as Reads per Kilobase per Million mapped reads (RPKM).
Result(s): We identified the oxalate degradation pathway (ODP) in the metagenome and metatranscriptome of all 8 subjects. Mean ODP is 35.3+/-28.1 and 90.1+/-43.5 RPKM in the metagenome and the metatranscriptome, respectively, indicating active expression. O. formigenes, E. coli, and unclassified bacteria were present in metagenomic and metatranscriptomic reads. B. dentium had detectable ODP in its genome but was not transcribing it. In the HFGP database, we identified ODP in 328 subjects of the 471 tested (70%) (Mean=18.1+/-2.1 RPKM). ODP was detected in B. animalis, B. dentium, B. pseudocatenulatum, E.coli/Shigella, L. acidophilus, L. gasseri, L. mucosae, O. formigenes and unclassified bacteria. ODP was examined in the metagenome of 265 females (Mean ODP= 21.7+/-3.3) and 200 males (Mean ODP=13.3+/-1.9 RPKM; p=0.04 by unpaired t test).
Conclusion(s): We have identified a community of bacteria with the potential to degrade oxalate in healthy humans and species actively transcribing ODP. These include E.coli, which might be a common contributor of oxalate degradation in humans. The sex differences in ODP is consistent with the ~ 2:1 male/female incidence and prevalence of calcium oxalate stones

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.

Nephrolithiasis is a complex disease of worldwide prevalence that is influenced by both genetic and environmental factors. About 75% of kidney stones are predominantly composed of calcium oxalate and urinary oxalate is considered a crucial risk factor. Microorganisms may have a role in the pathogenesis and prevention of kidney stones and the involvement of the intestinal microbiome in this renal disease has been a recent area of interest. Oxalobacter formigenes is a gram negative bacteria that degrades oxalate in the gut decreasing urinary oxalate excretion. In this review, we examine the data studying the role of Oxalobacter formigenes kidney stone disease in humans and animals, the effect of antibiotics on its colonization, and the potential role of probiotics and whole microbial communities as therapeutic interventions.

Kidney stones are a disease of worldwide prevalence with significant public health implications. About 60-80 % of stones are composed of calcium oxalate (CaOx). Hyperoxaluria is a major risk factor. Oxalobacter formigenes (OF), a member of the human colonic microbiota, plays a major role in net colonic oxalate absorption and secretion. We now report OF colonization rates in a young healthy population, the stability of colonization, the effects of antibiotic treatment, and OF colonization on urinary oxalate (Uox) excretion. We followed 64 healthy subjects tested for Helicobacter pylori (HP), who were treated with antibiotics (amoxicillin and clarithromycin for 2 weeks) for HP eradication. Using species-specific PCR, we tested for OF colonization at baseline and at follow-up. Urine samples 3 h after a low oxalate standard meal were analyzed for Uox, factored for urine creatinine (Cr). Of the 65 subjects (M/F: 23/42; mean age 25.2 +/- 5.7 years) tested for OF, 28 (43 %) were positive at baseline. Of 7 OF + subjects at baseline, subject to HP elimination, 6 became OF-negative at 12 wks, only 2 reverted to positive at week 24, and 4 patients remained negative at follow up (Mean 22.5 +/- 4.2 weeks). Of 18 untreated positive people with follow assessments, 16 (89 %) remained positive at follow up (Mean 23.0 +/- 4.2 week), but of 24 untreated negative subjects, only 3 (12 %) were positive at follow up (mean 20.2 +/- 6.8 weeks), significantly fewer than the untreated positives (p = 0.001 by Fisher exact test). We tested Uox/Cr in 137 samples from 46 subjects with no antibiotic exposure at different time points. We found that the presence of OF was associated with 14 % lower Uox/cr as compared with its absence (17.0 +/- 0.0 vs 19.4 +/- 0.1 mg/g, p = 0.04). We conclude that OF colonization status remains stable over a follow- up period of several months, with antibiotics suppressing colonization in the majority of people in the short term. The differences in urinary oxalate levels with respect to OF status is consistent with its protective effects for the prevention of calcium oxalate kidney stones

Hyperoxaluria is a frequent complication of inflammatory bowel diseases, ileal resection and Roux-en-Y gastric bypass and is well-known to cause nephrolithiasis and nephrocalcinosis. The associated prevalence of chronic kidney disease and end-stage kidney disease (ESKD) is less clear but may be more consequential than recognized. In this review, we highlight three cases of ESKD due to enteric hyperoxaluria following small bowel resections. We review current information on the pathophysiology, complications and treatment of this complex disease.

Oxalobacter formigenes, a member of the human colonic microbiota that plays a major role in net colonic oxalate transport and secretion, is protective against formation of calcium oxalate kidney stones. We now describe the prevalence, relative abundance and stability of O. formigenes in healthy young adults in the United States, as revealed by Human Microbiome Project (HMP) data from fecal samples from 242 healthy young adults who had 1-3 study visits. Samples underwent whole-genomic shotgun (WGS) sequencing, and/or 16S rRNA sequencing. Three datasets available from the processed sequence data were studied: WGS metagenomic analysis by alignment to reference genomes (HMSCP) or using MetaPhlAn, or QIIME analysis of the V1-3 or V3-5 16S sequences. O. formigenes was detected in fecal samples using both the WGS and 16S rRNA data. Analysis of the WGS dataset, using HMSCP, showed that 29 (31%) of 94 subjects were O. formigenes-positive while the 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. For 66 samples studied by all three methods, all assays agreed in 58 (88%). Of 14 subjects who were O. formigenes-positive at baseline, 13 (93%) were positive on follow-up visit, indicating the stability of colonization. O. formigenes appears to be stably present in fewer than half of healthy young USA adults and is most sensitively detected by WGS.

Gout is a common crystal-induced arthritis, in which monosodium urate (MSU) crystals precipitate within joints and soft tissues and elicit an inflammatory response. The causes of elevated serum urate and the inflammatory pathways activated by MSU crystals have been well studied, but less is known about the processes leading to crystal formation and growth. Uric acid, the final product of purine metabolism, is a weak acid that circulates as the deprotonated urate anion under physiologic conditions, and combines with sodium ions to form MSU. MSU crystals are known to have a triclinic structure, in which stacked sheets of purine rings form the needle-shaped crystals that are observed microscopically. Exposed, charged crystal surfaces are thought to allow for interaction with phospholipid membranes and serum factors, playing a role in the crystal-mediated inflammatory response. While hyperuricemia is a clear risk factor for gout, local factors have been hypothesized to play a role in crystal formation, such as temperature, pH, mechanical stress, cartilage components, and other synovial and serum factors. Interestingly, several studies suggest that MSU crystals may drive the generation of crystal-specific antibodies that facilitate future MSU crystallization. Here, we review MSU crystal biology, including a discussion of crystal structure, effector function, and factors thought to play a role in crystal formation. We also briefly compare MSU biology to that of uric acid stones causing nephrolithasis, and consider the potential treatment implications of MSU crystal biology.

PURPOSE: To determine the sensitivity, specificity, and accuracy of contrast-enhanced magnetic resonance (MR) imaging performed 1 month after localized chemotherapy as a measure of tumor response, before detectable changes in size. MATERIALS AND METHODS: This trial was approved by the authors' institutional review board and was compliant with the Health Insurance Portability and Accountability Act (HIPAA). Inclusion criteria selected patients receiving chemoembolization for hepatocellular carcinoma (HCC) with MR imaging within 2 months before treatment, in addition to MR imaging after treatment at 1 month and 6 months. Pathology was used as a surrogate for 6-month follow-up if the patient underwent interval transplantation. The final population consisted of 23 tumors (occurring within 21 patients). MR imaging studies were evaluated separately by two radiologists. Tumors were scored as showing complete loss of enhancement or as showing some residual tissue enhancement. Changes in T1 and T2 signal and perilesional enhancement were tabulated and recorded. Lesion size was also measured on all MR imaging studies by using a one-dimensional measure of the longest dimension. Increase in tumor size from 1-6 months of 20% or greater was used as confirmation of residual disease. In 5 of 23 tumors, review of pathology served as the surrogate standard. Sensitivity, specificity and accuracy were computed for each rater. RESULTS: The sensitivity, specificity, and accuracy of 1-month follow-up MR imaging were 71.4-85.7%, 100%, and 91.3-95.7%. There was a high degree of agreement between the two readers for both the 1-month (kappa = 0.88) and 6-month (kappa = 1.0) MR imaging studies. CONCLUSIONS: This investigation shows high accuracy for using tumor enhancement features on 1-month posttherapy MR imaging to predict residual disease after chemoembolization of HCC.

PURPOSE: To evaluate the efficacy of thrombolysis with the EndoWave peripheral infusion system in the treatment of patients with massive pulmonary embolism (PE) as compared to patients treated with catheter-directed thrombolysis. MATERIALS AND METHODS: Ten patients (five men and five women; age range, 31-85 years; mean age, 54.20 years) with massive acute PE (17 lesions) were treated with ultrasonography (US)-assisted catheter-directed thrombolysis with the Endowave system. All patients had hypoxia and dyspnea. No patient had contraindication for thrombolysis. Angiographic findings, duration of lysis, dose of thrombolytics used, and procedural complications were recorded. Thrombolytics used were urokinase, tissue-type plasminogen activator (tPA), and Reteplase. RESULTS: Complete thrombus removal was achieved in 13 of the 17 lesions (76%), near complete thrombolysis was achieved in three lesions (18%), and partial thrombolysis was achieved in one lesion (6%). The mean time of thrombolysis was 24.76 hours +/- 8.44 (median, 24 hours). The mean dose of tPA used for the Endowave group was 0.88 mg/h +/- 0.19 (13 lesions). CONCLUSIONS: US-assisted catheter-directed thrombolysis is an effective method for treating massive thrombolysis. It has the potential to shorten the time of lysis and lower the dose of thrombolytics.