Longitudinal comparison of the developing gut virome in infants and their mothers.

The human gut virome and its early life development are poorly understood. Prior studies have captured single-point assessments with the evolution of the infant virome remaining largely unexplored. We performed viral metagenomic sequencing on stool samples collected longitudinally from a cohort of 53 infants from age 2 weeks to 3 years (80.7 billion reads), and from their mothers (9.8 billion reads) to examine and compare viromes. The asymptomatic infant virome consisted of bacteriophages, nonhuman dietary/environmental viruses, and human-host viruses, predominantly picornaviruses. In contrast, human-host viruses were largely absent from the maternal virome. Previously undescribed, sequence-divergent vertebrate viruses were detected in the maternal but not infant virome. As infants aged, the phage component evolved to resemble the maternal virome, but by age 3, the human-host component remained dissimilar from the maternal virome. Thus, early life virome development is determined predominantly by dietary, infectious, and environmental factors rather than direct maternal acquisition.

Microbiota-mediated reactivation of triclosan oxidative metabolites in colon tissues.

Triclosan (TCS) is a widespread antimicrobial agent that is associated with many adverse health outcomes. Its gut toxicity has been attributed to the molecular modifications mediated by commensal microbes, but microbial transformations of TCS derivatives in the gut lumen are still largely unknown. Aromatic hydroxylation is the predominant oxidative metabolism of TCS that linked to its toxicological effects in host tissues. Here, we aimed to reveal the biological fates of hydroxyl-TCS (OH-TCS) in the colon, where intestinal microbes mainly reside. Unlike the profiles generated via host metabolism, OH-TCS species remain unconjugated in human stools from a cohort study. Through tracking molecular compositions in mouse intestinal tract, elevated abundance of free-form OH-TCS while reduced abundance of conjugated forms was observed in the colon digesta and mucosa. Using antibiotic-treated and germ-free mice, as well as in vitro approaches, we demonstrate that gut microbiota-encoded enzymes efficiently convert glucuronide/sulfate-conjugated OH-TCS, which are generated from host metabolism, back to their bioactive free-forms in colon tissues. Thus, host-gut microbiota metabolic interactions of TCS derivatives were proposed. These results shed light on the crucial roles of microbial metabolism in TCS toxicity, and highlight the importance of incorporating gut microbial transformations in health risk assessment of environmental chemicals.

Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract.

Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial ß-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.

Metabolic fate of environmental chemical triclocarban in colon tissues: roles of gut microbiota involved.

Metabolic transformations play critical roles in the bioavailability and toxicities of environmental pollutants and toxicants. However, most previous research has focused on the metabolic reactions in host tissues, the gut microbiota-mediated biotransformation of environmental compounds is understudied. Using triclocarban (TCC) as a model environmental compound, here we study the metabolic fate of TCC in gut tissues and determine the roles of gut microbiota involved. We find that compared with other tissues, the colon tissue has a unique metabolic profile of TCC, with high abundance of the parent compound TCC and its free-form metabolites. Using a variety of approaches including antibiotic-mediated suppression of gut bacteria in vivo, germ-free mice, and in vitro culture of fecal bacteria, we found that the unique metabolic profile of TCC in the colon is mediated by the actions of gut microbiota. Overall, our findings support that gut microbiota plays important roles in colonic metabolism of TCC, highlighting the importance to consider the contributions of gut microbiota in toxicology evaluation of environmental compounds.

Frequent occurrence of triclosan hydroxylation in mammals: A combined theoretical and experimental investigation.

Triclosan (TCS) is a widespread antimicrobial agent with many adverse health risks. Its hepatoxicity invariably points to the activation of constitutive androstane receptor (CAR), which regulates cytochrome P450 (CYP) genes that are critical for oxidative metabolism. Here, we provide the theoretical and experimental evidences showing that metabolic activation of TCS frequently occurs through aromatic hydroxylation in mammals. CYP-mediated oxidation was predicted to take place at each aromatic C‒H bond. Molecular docking and in vitro approaches reveal oxidative reaction could be efficiently catalyzed by CAR-regulated CYP2B6 enzyme. Parallel reaction monitoring (PRM) high-resolution mass spectrometry was utilized to identify and profile TCS oxidative metabolites in paired mouse liver, bile, feces, plasma and urine. We found multiple hydroxylated isomers including the products generated via the NIH shift of chlorine, as well as their subsequent conjugates. These metabolites showed isomer-specific retention in mice. Glucuronide conjugates are more readily excreted than the sulfates. Moreover, for the first time, isomeric hydroxylated metabolites were detected in the urine and stool of human subjects used TCS-contained household and personal care products. Collectively, these findings suggest that hydroxylation is an important, yet often underestimated element that worth considering to fully evaluate the biological fates and health risks of TCS.

Metagenomic sequencing of stool samples in Bangladeshi infants: virome association with poliovirus shedding after oral poliovirus vaccination.

The potential role of enteric viral infections and the developing infant virome in affecting immune responses to the oral poliovirus vaccine (OPV) is unknown. Here we performed viral metagenomic sequencing on 3 serially collected stool samples from 30 Bangladeshi infants following OPV vaccination and compared findings to stool samples from 16 age-matched infants in the United States (US). In 14 Bangladeshi infants, available post-vaccination serum samples were tested for polio-neutralizing antibodies. The abundance (p = 0.006) and richness (p = 0.013) of the eukaryotic virome increased with age and were higher than seen in age-matched US infants (p < 0.001). In contrast, phage diversity metrics remained stable and were similar to those in US infants. Non-poliovirus eukaryotic virus abundance (3.68 log10 vs. 2.25 log10, p = 0.002), particularly from potential viral pathogens (2.78log10 vs. 0.83log10, p = 0.002), and richness (p = 0.016) were inversely associated with poliovirus shedding. Following vaccination, 28.6% of 14 infants tested developed neutralizing antibodies to all three Sabin types and also exhibited higher rates of poliovirus shedding (p = 0.020). No vaccine-derived poliovirus variants were detected. These results reveal an inverse association between eukaryotic virome abundance and poliovirus shedding. Overall gut virome ecology and concurrent viral infections may impact oral vaccine responsiveness in Bangladeshi infants.

Author Correction: Gut microbiota plasticity is correlated with sustained weight loss on a low-carb or low-fat dietary intervention.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Gut microbiota plasticity is correlated with sustained weight loss on a low-carb or low-fat dietary intervention.

While low-carbohydrate and low-fat diets can both lead to weight-loss, a substantial variability in achieved long-term outcomes exists among obese but otherwise healthy adults. We examined the hypothesis that structural differences in the gut microbiota explain a portion of variability in weight-loss using two cohorts of obese adults enrolled in the Diet Intervention Examining The Factors Interacting with Treatment Success (DIETFITS) study. A total of 161 pre-diet fecal samples were sequenced from a discovery cohort (n = 66) and 106 from a validation cohort (n = 56). An additional 157 fecal samples were sequenced from the discovery cohort after 10 weeks of dietary intervention. We found no specific bacterial signatures associated with weight loss that were consistent across both cohorts. However, the gut microbiota plasticity (i.e. variability), was correlated with long-term (12-month) weight loss in a diet-dependent manner; on the low-fat diet subjects with higher pre-diet daily plasticity had higher sustained weight loss, whereas on the low-carbohydrate diet those with higher plasticity over 10 weeks of dieting had higher 12-month weight loss. Our findings suggest the potential importance of gut microbiota plasticity for sustained weight-loss. We highlight the advantages of evaluating kinetic trends and assessing reproducibility in studies of the gut microbiota.

Household triclosan and triclocarban effects on the infant and maternal microbiome.

In 2016, the US Food and Drug Administration banned the use of specific microbicides in some household and personal wash products due to concerns that these chemicals might induce antibiotic resistance or disrupt human microbial communities. Triclosan and triclocarban (referred to as TCs) are the most common antimicrobials in household and personal care products, but the extent to which TC exposure perturbs microbial communities in humans, particularly during infant development, was unknown. We conducted a randomized intervention of TC-containing household and personal care products during the first year following birth to characterize whether TC exposure from wash products perturbs microbial communities in mothers and their infants. Longitudinal survey of the gut microbiota using 16S ribosomal RNA amplicon sequencing showed that TC exposure from wash products did not induce global reconstruction or loss of microbial diversity of either infant or maternal gut microbiotas. Broadly antibiotic-resistant species from the phylum Proteobacteria, however, were enriched in stool samples from mothers in TC households after the introduction of triclosan-containing toothpaste. When compared by urinary triclosan level, agnostic to treatment arm, infants with higher triclosan levels also showed an enrichment of Proteobacteria species. Despite the minimal effects of TC exposure from wash products on the gut microbial community of infants and adults, detected taxonomic differences highlight the need for consumer safety testing of antimicrobial self-care products on the human microbiome and on antibiotic resistance.

Crossover Control Study of the Effect of Personal Care Products Containing Triclosan on the Microbiome.

Commonly prescribed antibiotics are known to alter human microbiota. We hypothesized that triclosan and triclocarban, components of many household and personal care products (HPCPs), may alter the oral and gut microbiota, with potential consequences for metabolic function and weight. In a double-blind, randomized, crossover study, participants were given triclosan- and triclocarban (TCS)-containing or non-triclosan/triclocarban (nTCS)-containing HPCPs for 4 months and then switched to the other products for an additional 4 months. Blood, stool, gingival plaque, and urine samples and weight data were obtained at baseline and at regular intervals throughout the study period. Blood samples were analyzed for metabolic and endocrine markers and urine samples for triclosan. The microbiome in stool and oral samples was then analyzed. Although there was a significant difference in the amount of triclosan in the urine between the TCS and nTCS phases, no differences were found in microbiome composition, metabolic or endocrine markers, or weight. Though this study was limited by the small sample size and imprecise administration of HPCPs, triclosan at physiologic levels from exposure to HPCPs does not appear to have a significant or important impact on human oral or gut microbiome structure or on a panel of metabolic markers. IMPORTANCE Triclosan and triclocarban are commonly used commercial microbicides found in toothpastes and soaps. It is unknown what effects these chemicals have on the human microbiome or on endocrine function. From this randomized crossover study, it appears that routine personal care use of triclosan and triclocarban neither exerts a major influence on microbial communities in the gut and mouth nor alters markers of endocrine function in humans.

Region-specific tauopathy and synucleinopathy in brain of the alpha-synuclein overexpressing mouse model of Parkinson's disease.

BACKGROUND: α-synuclein [α-Syn]-mediated activation of GSK-3ß leading to increases in hyperphosphorylated Tau has been shown by us to occur in striata of Parkinson's diseased [PD] patients and in animal models of PD. In Alzheimer's disease, tauopathy exists in several brain regions; however, the pattern of distribution of tauopathy in other brain regions of PD or in animal models of PD is not known. The current studies were undertaken to analyze the distribution of tauopathy in different brain regions in a widely used mouse model of PD, the α-Syn overexpressing mouse. RESULTS: High levels of α-Syn levels were seen in the brain stem, with a much smaller increase in the frontal cortex; neither cerebellum nor hippocampus showed any overexpression of α-Syn. Elevated levels of p-Tau, hyperphosphorylated at Ser202, Ser262 and Ser396/404, were seen in brain stem, with lower levels seen in hippocampus. In both frontal cortex and cerebellum, increases were seen only in p-Ser396/404 Tau, but not in p-Ser202 and p-Ser262. p-GSK-3ß levels were not elevated in any of the brain regions, although total GSK-3ß was elevated in brain stem. p-p38MAPK levels were unchanged in all brain regions examined, while p-ERK levels were elevated in brain stem, hippocampus and cerebellum, but not the frontal cortex. p-JNK levels were increased in brain stem and cerebellum but not in the frontal cortex or hippocampus. Elevated levels of free tubulin, indicating microtubule destabilization, were seen only in the brain stem. CONCLUSION: Our combined data suggest that in this animal model of PD, tauopathy, along with microtubule destabilization, exists primarily in the brain stem and striatum, which are also the two major brain regions known to express high levels of α-Syn and undergo the highest levels of degeneration in human PD. Thus, tauopathy in PD may have a very restricted pattern of distribution.

Hyperphosphorylated Tau in an α-synuclein-overexpressing transgenic model of Parkinson's disease.

Although clinically distinct diseases, tauopathies and synucleinopathies share a common genesis and mechanisms, leading to overlapping degenerative changes within neurons. In human postmortem striatum of Parkinson's disease (PD) and PD with dementia, we have recently described elevated levels of tauopathy, indexed as increased hyperphosphorylated Tau (p-Tau). Here we assessed tauopathy in striatum of a transgenic animal model of PD, overexpressing human α-synuclein under the platelet-derived growth factor promoter. At 11 months of age, large and progressive increases in p-Tau in transgenic mice, hyperphosphorylated at sites reminiscent of Alzheimer's disease, were noted, along with elevated levels of α-synuclein and glycogen synthase kinase 3ß phosphorylated at Tyr216 (p-GSK-3ß), a major kinase involved in the hyperphosphorylation of Tau. Differential Triton X-100 extraction of striata showed the presence of aggregated α-synuclein in the transgenic mice, along with p-Tau and p-GSK-3ß, which was also confirmed through immunohistochemistry. After p-Tau formation, both Tau and microtubule-associated protein 1 (MAP1) dissociated from the cytoskeleton, consistent with the diminished ability of these cytoskeleton-binding proteins to bind microtubules. Increases in free tubulin and actin were also noted, indicative of cytoskeleton remodeling and destabilization. In vivo magnetic resonance imaging of the transgenic animals showed a reduction in brain volume of transgenic mice, indicating substantial atrophy. From immunohistochemical studies, α-synuclein, p-Tau and p-GSK-3ß were found to be overexpressed and co-localized in large inclusion bodies, reminiscent of Lewy bodies. The elevated state of tauopathy seen in these platelet-derived growth factor-α-synuclein mice provides further confirmation that PD may be a tauopathic disease.

Tauopathic changes in the striatum of A53T α-synuclein mutant mouse model of Parkinson's disease.

Tauopathic pathways lead to degenerative changes in Alzheimer's disease and there is evidence that they are also involved in the neurodegenerative pathology of Parkinson's disease [PD]. We have examined tauopathic changes in striatum of the α-synuclein (α-Syn) A53T mutant mouse. Elevated levels of α-Syn were observed in striatum of the adult A53T α-Syn mice. This was accompanied by increases in hyperphosphorylated Tau [p-Tau], phosphorylated at Ser202, Ser262 and Ser396/404, which are the same toxic sites also seen in Alzheimer's disease. There was an increase in active p-GSK-3ß, hyperphosphorylated at Tyr216, a major and primary kinase known to phosphorylate Tau at multiple sites. The sites of hyperphosphorylation of Tau in the A53T mutant mice were similar to those seen in post-mortem striata from PD patients, attesting to their pathophysiological relevance. Increases in p-Tau were not due to alterations on protein phosphatases in either A53T mice or in human PD, suggesting lack of involvement of these proteins in tauopathy. Extraction of striata with Triton X-100 showed large increases in oligomeric forms of α-Syn suggesting that α-Syn had formed aggregates the mutant mice. In addition, increased levels of p-GSK-3ß and pSer396/404 were also found associated with aggregated α-Syn. Differential solubilization to measure protein binding to cytoskeletal proteins demonstrated that p-Tau in the A53T mutant mouse were unbound to cytoskeletal proteins, consistent with dissociation of p-Tau from the microtubules upon hyperphosphorylation. Interestingly, α-Syn remained tightly bound to the cytoskeleton, while p-GSK-3ß was seen in the cytoskeleton-free fractions. Immunohistochemical studies showed that α-Syn, pSer396/404 Tau and p-GSK-3ß co-localized with one another and was aggregated and accumulated into large inclusion bodies, leading to cell death of Substantia nigral neurons. Together, these data demonstrate an elevated state of tauopathy in striata of the A53T α-Syn mutant mice, suggesting that tauopathy is a common feature of synucleinopathies.

Effect of Helicobacter pylori infection on symptoms of gastroenteritis due to enteropathogenic Escherichia coli in adults.

BACKGROUND: Helicobacter pylori can cause hypochlorhydria in some hosts and predispose to diarrheal infections. AIMS: We tested the hypothesis that chronic H. pylori infection increases the risk of diarrheal illness due to an acid-sensitive organism: enteropathogenic Escherichia coli (EPEC). METHODS: After testing healthy adult volunteers for H. pylori, 19 infected and 26 uninfected subjects had gastric pH probes placed and were given 5-10 × 10(9) EPEC organisms; six had previously received a proton pump inhibitor. We measured diarrhea and created a composite gastroenteritis severity score based on symptoms in the 48 h following exposure. Outcomes were compared using logistic regression and analysis of covariance. RESULTS: More H. pylori-infected (36.8%) than H. pylori-uninfected subjects (7.7%) were hypochlorhydric (P = 0.02). Six (31.6%) H. pylori-infected and five H. pylori-uninfected subjects (19.2%) developed diarrhea (P = 0.34). Hypochlorhydria was a strong risk factor for diarrhea [odds ratio (OR) 6.25, confidence interval (CI): 1.29-30.35]. After adjusting for hypochlorhydria and EPEC dose, H. pylori was not associated with diarrhea (OR 0.89, CI: 0.17-4.58). Among those with symptoms, H. pylori-infected subjects had lower gastroenteritis severity score than did H. pylori-uninfected subjects (2.6, CI: 1.9-3.4 versus 1.5, CI: 1.1-1.9, P = 0.01), particularly if they were also hypochlorhydric (3.8, CI: 2.3-5.3 versus 1.9, CI: 1.3-2.5, P = 0.02). CONCLUSIONS: In adults, H. pylori infection was associated with hypochlorhydria but had no detectable effect on occurrence of diarrhea. Among symptomatic subjects, H. pylori infection decreased severity of gastroenteritis.

Elevated tauopathy and alpha-synuclein pathology in postmortem Parkinson's disease brains with and without dementia.

Parkinson's disease (PD), a progressive neurodegenerative disease, results in abnormal accumulation of insoluble alpha-synuclein (alpha-Syn) in dopaminergic neurons. Here we examined tauopathic changes and the alpha-Syn/p-GSK-3beta/proteasome pathway in postmortem striata and inferior frontal gyri (IFG) from patients with PD and PD with dementia (PDD). In both PD and PDD, alpha-Syn levels were high, especially the insoluble form of this protein; in PDD, insoluble alpha-Syn levels were persistently higher than PD across both brain regions. Levels of p-GSK-3beta phosphorylated at Tyr 216, which hyperphosphorylates Tau to produce toxic pathological forms of p-Tau, were higher in striata of both PD and PDD compared to controls, but were unaltered in IFG. While proteasomal activity was unchanged in striatum of PD and PDD, such activity was diminished in the IFG of both PD and PDD. A decrease in 19S subunit of the proteasomes was seen in IFG of PDD, while lower levels of 20S subunits were seen in striatum and IFG of both PD and PDD patients. Parkin levels were similar in PD and PDD, suggesting lack of involvement of this protein. Most interestingly, tauopathic changes were noted only in striatum of PD and PDD, with increased hyperphosphorylation seen at Ser262 and Ser396/404; increases in Ser202 levels were seen only in PD but not in PDD striatum. We were unable to detect any tauopathy in IFG in either PD or PDD despite increased levels of alpha-Syn, and decreased proteasomal activity, and is probably due to lack of increase in p-GSK-3beta in IFG. Unlike Alzheimer's disease where tauopathy is more globally observed in diverse brain regions, our data demonstrates restricted expression of tauopathy in brains of PD and PDD, probably limited to dopaminergic neurons of the nigrostriatal region.

The complete genome of klassevirus - a novel picornavirus in pediatric stool.

BACKGROUND: Diarrhea kills 2 million children worldwide each year, yet an etiological agent is not found in approximately 30-50% of cases. Picornaviral genera such as enterovirus, kobuvirus, cosavirus, parechovirus, hepatovirus, teschovirus, and cardiovirus have all been found in human and animal diarrhea. Modern technologies, especially deep sequencing, allow rapid, high-throughput screening of clinical samples such as stool for new infectious agents associated with human disease. RESULTS: A pool of 141 pediatric gastroenteritis samples that were previously found to be negative for known diarrheal viruses was subjected to pyrosequencing. From a total of 937,935 sequence reads, a collection of 849 reads distantly related to Aichi virus were assembled and found to comprise 75% of a novel picornavirus genome. The complete genome was subsequently cloned and found to share 52.3% nucleotide pairwise identity and 38.9% amino acid identity to Aichi virus. The low level of sequence identity suggests a novel picornavirus genus which we have designated klassevirus. Blinded screening of 751 stool specimens from both symptomatic and asymptomatic individuals revealed a second positive case of klassevirus infection, which was subsequently found to be from the index case's 11-month old twin. CONCLUSION: We report the discovery of human klassevirus 1, a member of a novel picornavirus genus, in stool from two infants from Northern California. Further characterization and epidemiological studies will be required to establish whether klasseviruses are significant causes of human infection.

Serum ghrelin levels and risk of subsequent adenocarcinoma of the esophagus.

OBJECTIVE: Several large studies have shown a negative association between Helicobacter pylori (H. pylori) infection and esophageal adenocarcinoma. Diminution of gastric ghrelin secretion by H. pylori could protect against esophageal malignancy by decreasing appetite, food intake, and acid production, thereby decreasing weight and gastroesophageal reflux. METHODS: We evaluated the association of ghrelin with esophageal adenocarcinoma using a population from a previous nested case-control study. Among 128,992 enrolled in a multiphasic health checkup (MHC) between 1964 and 1969, 52 patients developed esophageal adenocarcinoma by the year 2000. Three random controls from the MHC cohort were matched to each case by age, sex, race, and the date and site of their MHC. Serum samples collected at the MHC had been previously tested for IgG antibodies against H. pylori and the CagA protein. Serum ghrelin concentrations were determined by a commercial EIA on 52% of the initial subjects (31 cases and 79 controls). RESULTS: A concentration of ghrelin greater than 3,200 pg/mL at MHC (fourth quartile) was associated with a lower risk of esophageal cancer (H. pylori and body mass index [BMI] adjusted OR=0.18 [CI 0.04-0.78]). This inverse association was seen only in overweight subjects (BMI>or=25, P value for interaction=0.09). The effects of H. pylori and ghrelin were independent. CONCLUSION: Contrary to the original hypothesis, high rather than low serum ghrelin was associated with protection against esophageal adenocarcinoma but only among overweight subjects.

Gastroenteritis and transmission of Helicobacter pylori infection in households.

The mode of transmission of Helicobacter pylori infection is poorly characterized. In northern California, 2,752 household members were tested for H. pylori infection in serum or stool at a baseline visit and 3 months later. Among 1,752 person considered uninfected at baseline, 30 new infections (7 definite, 7 probable, and 16 possible) occurred, for an annual incidence of 7% overall and 21% in children <2 years of age. Exposure to an infected household member with gastroenteritis was associated with a 4.8-fold (95% confidence interval [CI] 1.4-17.1) increased risk for definite or probable new infection, with vomiting a greater risk factor (adjusted odds ratio [AOR] 6.3, CI 1.6-24.5) than diarrhea only (AOR 3.0, p = 0.65). Of probable or definite new infections, 75% were attributable to exposure to an infected person with gastroenteritis. Exposure to an H. pylori-infected person with gastroenteritis, particularly vomiting, markedly increased risk for new infection.

Significance of transiently positive enzyme-linked immunosorbent assay results in detection of Helicobacter pylori in stool samples from children.

In young children, the significance of stool samples transiently positive for Helicobacter pylori antigen is unknown. As part of a larger prospective study on enteric infections, stool samples were obtained from 323 children at two time points 3 months apart and tested for H. pylori antigen using a commercially available enzyme-linked immunosorbent assay (ELISA) test. Seminested PCR for a Helicobacter-specific 16S rRNA gene was performed on all 26 pairs reverting from positive to negative (transient positives), all 4 persistent antigen-positive pairs, and 10 randomly selected persistent antigen-negative pairs. Helicobacter species were amplified from the first stool samples of 15/26 (58%) of the transient positives and 1 (25%) of 4 persistent positives. No Helicobacter species were amplified from the 10 persistent negatives. Among the 15 amplicons from transient-positive stool, H. pylori was sequenced and identified from 12 (80%; 95% confidence interval, 52% to 96%) and other Helicobacter spp. were identified from three (Helicobacter canis, Helicobacter winghamensis, and MIT 99-5504). Four of the 15 remained positive by PCR for the second (antigen-negative) stool sample, including all 3 initially identified as non-H. pylori. Helicobacter bilis was amplified from the second sample of a persistent positive. Two of eight transient positives from whom serum was available had accompanying transient elevations in anti-H. pylori antibodies. Transiently positive stool ELISAs for H. pylori are common and represent H. pylori in the majority of cases where sequences can be obtained. A not-insignificant percentage of antigen-positive stools, however, may represent other Helicobacter species.