Recruitment and subject enrollment. Participants were recruited for the ESSAY study (Eradication Study in Stable Adults/Youths) evaluating the effect of the standard-of-care practice of H. pylori eradication on metabolic profile and anthropometric measures of healthy adults. Participants were identified from the Bellevue Hospital primary care clinic, and community. Healthy young adults who were 18–40 years old were screened by research coordinators for eligibility criteria and then signed an informed consent if meeting those criteria. In total, 139 participants were screened for participation. Of the 87 participants who met the eligibility requirements and provided informed consent, 69 completed the baseline visit while 18 were lost to follow-up (Fig. 1). The clinical study was conducted between April 2012 and July 2016. We excluded participants with diabetes, hyper- or hypothyroidism, prior gastric or bariatric surgery, prior H. pylori treatment, steroid or other immunomodulatory drug use within 4 weeks of the first visit, and antibiotic use within the prior 6 months. Participants completed baseline questionnaires to provide their demographic information, medical history, and current medication use. The study was approved by the Institutional Review Board (IRB) at NYU Langone Health. All research was performed in accordance with the Declaration of Helsinki and our local IRB guidelines. Informed consent was obtained from all participants.
Determination of H. pylori status at baseline and at follow-up and antibiotics regimen. Subjects underwent a non-radioactive 13C Urea Breath Test (Meretek Diagnostics, New York NY) to determine H.pylori status. Subjects (n = 23) who tested H. pylori-positive were offered treatment with a 14-day twice-daily regimen [amoxicillin 1000 mg, clarithromycin 500 mg, and proton pump inhibitor (PPI; omeprazole 20 mg, rabeprazole or esomeprazole 40 mg)], per the then-current standard of care [24]. In total, 19 subjects received a course of antibiotics, including three who received a second antibiotic course because they failed eradication with the first course, and four other subjects withdrew from the study before receiving antibiotics (Fig. 1). The 46 subjects who were H.pylori negative at baseline did not receive antibiotics and were followed serially as controls.
Study time points and assessments. Subjects fasted overnight at home and then underwent basic assessment at the NYU Clinical and Translational Science Institute (CTSI) at Bellevue Hospital at the baseline and 6, 12, and 24-week timepoints. And height and weight obtained. Stool was collected at home the day prior to the assessments using a stool collection kit, brought to NYU, and stored immediately at -80oC. In total, we obtained fecal samples from 65 subjects at baseline (Fig. 1).
Test meal and urine collections. To assess the differences in urinary electrolytes before and after H. pylori eradication, we collected a fasting urine sample and then administered a standard 16-oz liquid meal totaling 700 calories (2 cans of Ensure Plus®) (Table S1). Urine was collected for three hours after the meal. In total, we collected urine samples (pre and post) from 32 controls and 14 treated subjects, (Fig. 1).
DNA isolation and PCR. DNA was extracted from fecal samples and PCR was performed as described[21].
Quantitative PCR. qPCR was performed using the LightCycler 480 SYBR Green І Master Mix, primers targeting oxc, in the LightCycler 480 system (Roche, Pleasanton CA). The oligonucleotide primers were: forward, 5’-GTGTTGTCGGCATTCCTATC-3’ and reverse 5’-GAAGCAGTTGGTGGTTGC-3’. Melting peak analysis was performed from 65oC to 95oC to confirm amplicon specificity. A positive result was defined by amplification greater than 1.0E2, with melting peak between 86-87oC.
16S rRNA sequencing. For amplicon library generation, the V4 region of the 16S rRNA gene was amplified with gene-specific primers, as described[21]. The 254 bp V4 region was sequenced using the Illumina MiSeq 2 × 150 bp platform at NYU Langone Health. Operational taxonomic units (OTUs) were picked using GreenGenes 13_8 for reference, using the DADA2 pipeline [25].
Sample diversity analyses. Intra-sample α-diversity was calculated using QIIME2, using phylogenetic diversity, observed OTU number, Chao1, and Shannon indices at rarefaction depths of 8,000 sequences/sample. Beta-diversity was assessed using the unweighted UniFrac distance metric [26].
Oxalobacter formigenes status determination. We tested each fecal sample for the presence of O. formigenes using PCR in duplicate, qPCR, and 16S rRNA sequencing. Based on the known several log-fold biological variations in O. formigenes abundance in human fecal specimens[26, 27], given the ascertainment variability using different methods, we developed a positivity score based on the 42 control subjects who did not receive antibiotics and who were tested at baseline and at least once in follow-up. The maximum number of assessments for each test subject was 16 if the participant was tested at all 4 time points (baseline, weeks 6, 12, 24) in all four assays; and a minimum of 8 if only tested at two time points. For the 42 control participants, the mean ± SD number of determinations was 14.7 ± 2.6. We computed the positivity score by dividing the number of positive assessments by the total number of assessments for that subject. Since the results were bimodal, as expected (Suppl. Figure 1) we assigned baseline O. formigenes status as negative with score ≤ 0.2 and positive with score ≥ 0.4. Only one subject had a score of 0.3, which we considered indeterminate and we removed this participant from further analysis (Suppl. Figure 1).
Urine testing. Urine aliquots were mixed with either HCl (for oxalate measurements) or with thymol (for all other assessments) and then stored at -80oC. Pre-meal (fasting) and the 3-hour post-meal urinary samples were analyzed by Litholink Corporation (Chicago IL). In each urine sample, we measured calcium, chloride, creatinine, magnesium, sodium, potassium, phosphate, and ammonium concentrations by standard techniques by Beckman Synchron AU680 (Beckman Instruments, Brea CA), as described [28]; pH was measured by glass electrode. Oxalate was measured by enzyme assay using oxalate oxidase (Trinity Biotech, Bray, Ireland). Citrate was measured by enzyme assay using citrate lyase (Mannheim Bohringer, Mannheim, Germany). All urine parameters except pH were normalized by dividing by creatinine concentration to account for hydration status.
Longitudinal analysis on urine parameters. We compared urine parameters and their changing rates according to the subjects’ baseline treatment status (control, treatment group) and O. formigenes colonization status during the period of 0–24 weeks. A linear mixed model was fitted to data over that time interval in which the changing rates in urine parameters were compared. The covariates age, sex, and body mass index (BMI) were adjusted in the model.
Association between Uox/Cr and bacterial taxa. We used the linear mixed effect model to identify which microbial taxa were associated with pre/post-meal urinary oxalate level, adjusted for age, gender, ethnicity, BMI, and calcium level. We included data for the participants in (i) the control group, we analyzed a total of 108 pairs of microbial measurements and urinary oxalate level (pre-/post- dietary) at baseline, 6, 12 and 24 weeks; and (ii), in the antibiotic-treatment group, we analyzed a total of 51 pairs of microbial measurements and urinary oxalate level (pre-/post- dietary) at 6, 12, and 24 weeks. In the mixed effects model, intercept and slope of the linear time trend for each subject are regarded as random effects. ANOVA results indicate that no correlation exists between random intercept and slope. We used the pre- and post-meal urinary oxalate levels as a dependent variable, respectively, while the relative abundance of individual microbial taxa after centered log-ratio (CLR) transformation as the independent variable. To calculate adjusted p-values, the Benjamini-Hochberg procedure was applied for each taxonomic level. Unclassified microbial taxa were not included in the analyses.