Healthy Human Gastrointestinal Tract Research Articles

Evaluation of Antimicrobial Resistancein Clinical Isolates of Enterococcus spp. Obtained from Hospital Patients in Latvia.

Background and Objective: Enterococci are typically found in a healthy human gastrointestinal tract but can cause severe infections in immunocompromised patients. Such infections are treated with antibiotics. This study addresses the rising concern of antimicrobial resistance (AMR) in Enterococci, focusing on the prevalence of vancomycin-resistant enterococcus (VRE) strains. Materials and Methods: The pilot study involved 140 Enterococci isolates collected between 2021 and 2022 from two multidisciplinary hospitals (with and without local therapeutic drug monitoring protocol of vancomycin) in Latvia. Microbiological assays and whole genome sequencing were used. AMR gene prevalence with resistance profiles were determined and the genetic relationship and outbreak evaluation were made by applying core genome multi-locus sequence typing (cgMLST). Results: The acquired genes and mutations were responsible for resistance against 10 antimicrobial classes, including 25.0% of isolates expressing resistance to vancomycin, predominantly of the vanB type. Genetic diversity among E. faecalis and E. faecium isolates was observed and seven potential outbreak clusters were identified, three of them containing sequence types ST6, ST78 and ST80. The prevalence of vancomycin resistance was highest in the hospital without a therapeutic drug-monitoring protocol and in E. faecium. Notably, a case of linezolid resistance due to a mutation was documented. Conclusions: The study illustrates the concerning prevalence of multidrug-resistant Enterococci in Latvian hospitals, showcasing the rather widespread occurrence of vancomycin-resistant strains. This highlights the urgency of implementing efficient infection control mechanisms and the need for continuous VRE surveillance in Latvia to define the scope and pattern of the problem, influencing clinical decision making and planning further preventative measures.

CD25890, a conserved protein that modulates sporulation initiation in Clostridioides difficile

Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50–60% of those bacteria have the ability to produce resilient spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We report on the characterization of a signature gene, CD25890, which, as we show is involved in the control of sporulation initiation in C. difficile under certain nutritional conditions. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of CD25890 results in increased expression of spo0A per cell and increased sporulation. The effect of CD25890 on spo0A is likely indirect and mediated through repression of the sinRR´ operon. Deletion of the CD25890 gene, however, does not alter the expression of the genes coding for the cytotoxins or the genes involved in biofilm formation. Our results suggest that CD25890 acts to modulate sporulation in response to the nutrients present in the environment.

Recent advances in modulating the microbiome.

We are in the midst of “the microbiome revolution”—not a day goes by without some new revelation on the potential role of the gut microbiome in some disease or disorder. From an ever-increasing recognition of the many roles of the gut microbiome in health and disease comes the expectation that its modulation could treat or prevent these very same diseases. A variety of interventions could, at least in theory, be employed to alter the composition or functional capacity of the microbiome, ranging from diet to fecal microbiota transplantation (FMT). For some, such as antibiotics, prebiotics, and probiotics, an extensive, albeit far from consistent, literature already exists; for others, such as other dietary supplements and FMT, high-quality clinical studies are still relatively few in number. Not surprisingly, researchers have turned to the microbiome itself as a source for new entities that could be used therapeutically to manipulate the microbiome; for example, some probiotic strains currently in use were sourced from the gastrointestinal tract of healthy humans. From all of the extant studies of interventions targeted at the gut microbiome, a number of important themes have emerged. First, with relatively few exceptions, we are still a long way from a precise definition of the role of the gut microbiome in many of the diseases where a disturbed microbiome has been described—association does not prove causation. Second, while animal models can provide fascinating insights into microbiota–host interactions, they rarely recapitulate the complete human phenotype. Third, studies of several interventions have been difficult to interpret because of variations in study population, test product, and outcome measures, not to mention limitations in study design. The goal of microbiome modulation is a laudable one, but we need to define our targets, refine our interventions, and agree on outcomes.

Complete Genome Sequence of Collinsella aerofaciens Isolated from the Gut of a Healthy Indian Subject.

ABSTRACTCollinsella aerofaciens, a rod-shaped nonmotile obligate anaerobe, is the most abundant actinobacterium in the gastrointestinal tract of healthy humans. An altered abundance of C. aerofaciens may be linked with several health disorders, including irritable bowel syndrome. In the present study, we report the complete genome sequence of C. aerofaciens strain indica.

Selective method for identification and quantification of Bifidobacterium animalis subspecies lactis BB-12 (BB-12) from the gastrointestinal tract of healthy volunteers ingesting a combination probiotic of BB-12 and Lactobacillus rhamnosus GG.

To develop a novel validated method for the isolation of Bifidobacterium animalis ssp. lactis BB-12 (BB-12) from faecal specimens and apply it to studies of BB-12 and Lactobacillus rhamnosus GG (LGG) recovered from the healthy human gastrointestinal (GI) tract. A novel method for isolating and enumerating BB-12 was developed based on its morphologic features of growth on tetracycline-containing agar. The method identified BB-12 correctly from spiked stool close to 100% of the time as validated by PCR confirmation of identity, and resulted in 97-104% recovery of BB-12. The method was then applied in a study of the recovery of BB-12 and LGG from the GI tract of healthy humans consuming ProNutrients® Probiotic powder sachet containing BB-12 and LGG. Viable BB-12 and LGG were recovered from stool after 21days of probiotic ingestion compared to baseline. In contrast, no organisms were recovered 21days after baseline in the nonsupplemented control group. We demonstrated recovery of viable BB-12, using a validated novel method specific for the isolation of BB-12, and LGG from the GI tract of healthy humans who consumed the probiotic supplement. This method will enable more detailed and specific studies of BB-12 in probiotic supplements, including when in combination with LGG.

Multilocus sequence typing analyses of Clostridium perfringens type A strains harboring tpeL and netB genes

Clostridium perfringens is an anaerobic bacterium ubiquitous in various environments, especially in soil and the gastrointestinal tract of healthy humans and animals. In this study, multilocus sequence typing protocol was used to investigate genotypic relationships among 40 C. perfringens strains isolated from humans and broiler chicken with necrotic enteritis [NE]. The results indicated a few clonal populations, mainly observed in human strains, with 32.5% of all strains associated with one of three clonal complexes and 30 sequences types. The CC-1 cluster showed an interesting and unexpected result because it contained seven strains [six from animals and one of human origin]. Detection assays for toxin genes tpeL and netB were also performed. The netB gene was only observed in 7.5% of the strains from healthy human. The toxin gene tpeL was detected in 22.5% of the C. perfringens strains isolated from three individuals and in six broilers with NE. Our study describes the role of some C. perfringens strains of human origin acting as reservoirs of virulence genes and sources of infection. In addition, the strains of human and animal origin were found to be genetically distinct but phylogenetically close, and the human strains showed more diversity than the animal strains.

Microbial Ecology along the Gastrointestinal Tract.

The ecosystem of the human gastrointestinal (GI) tract traverses a number of environmental, chemical, and physical conditions because it runs from the oral cavity to the anus. These differences in conditions along with food or other ingested substrates affect the composition and density of the microbiota as well as their functional roles by selecting those that are the most suitable for that environment. Previous studies have mostly focused on Bacteria, with the number of studies conducted on Archaea, Eukarya, and Viruses being limited despite their important roles in this ecosystem. Furthermore, due to the challenges associated with collecting samples directly from the inside of humans, many studies are still exploratory, with a primary focus on the composition of microbiomes. Thus, mechanistic studies to investigate functions are conducted using animal models. However, differences in physiology and microbiomes need to be clarified in order to aid in the translation of animal model findings into the context of humans. This review will highlight Bacteria, Archaea, Fungi, and Viruses, discuss differences along the GI tract of healthy humans, and perform comparisons with three common animal models: rats, mice, and pigs.

Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines.

Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A–E) according to the toxins that the bacterium produces: alpha, beta, epsilon, or iota. Each of these toxinotypes is associated with myriad different, frequently fatal, illnesses that affect a range of farm animals and humans. Alpha, beta, and epsilon toxins are the main causes of disease. Vaccinations that generate neutralizing antibodies are the most common prophylactic measures that are currently in use. These vaccines consist of toxoids that are obtained from C. perfringens cultures. Recombinant vaccines offer several advantages over conventional toxoids, especially in terms of the production process. As such, they are steadily gaining ground as a promising vaccination solution. This review discusses the main strategies that are currently used to produce recombinant vaccines containing alpha, beta, and epsilon toxins of C. perfringens, as well as the potential application of these molecules as vaccines for mammalian livestock animals.

Fungi inhabiting the healthy human gastrointestinal tract: a diverse and dynamic community

Fungal DNA was selectively amplified, and the ITS region sequenced, from fecal samples taken from 45 healthy human volunteers at one (21 volunteers) or two (24 volunteers) time points. Seventy-two operational taxonomic units, representing two phyla and ten classes of fungi, were recovered. Candida yeasts, notably Candida tropicalis (present in 51 samples), and yeasts in the Dipodascaceae (39 samples), dominated, while 38 OTUs were detected in a single sample each. Fungi included known human symbionts (Candida, Cryptococcus, Malassezia and Trichosporon spp.), common airborne fungi (Cladosporium sp.) and fungi known to be associated with food (Debaryomyces hansenii and high salt fermented foods; Penicillium roqueforti and blue cheese). In contrast with gut-associated bacteria, fungi occurred in much lower abundance and diversity, and fungal composition appeared unstable over time.

Distribution of CD4(pos) -, CD8(pos) - and regulatory T cells in the upper and lower gastrointestinal tract in healthy young subjects.

The gastrointestinal immune system is involved in the development of several autoimmune-mediated diseases, including inflammatory bowel disease, multiple sclerosis, and type 1 diabetes mellitus. Alterations in T-cell populations, especially regulatory T cells (Tregs), are often evident in patients suffering from these diseases. To be able to detect changes in T-cell populations in diseased tissue, it is crucial to investigate T-cell populations in healthy individuals, and to characterize their variation among different regions of the gastrointestinal (GI) tract. While limited data exist, quantitative data on biopsies systematically drawn from various regions of the GI tract are lacking, particularly in healthy young humans. In this report, we present the first systematic assessment of how T cells—including Tregs—are distributed in the gastrointestinal mucosa throughout the GI tract of healthy young humans by means of multi-parameter FACS analysis. Gastroduodenoscopy and colonoscopy were performed on 16 healthy volunteers aged between 18 and 32. Biopsies were drawn from seven GI regions, and were used to determine the frequencies of CD8+-, CD4+- and Tregs in the gastrointestinal mucosa by means of multi-parameter FACS analysis. Our data show that there is significant variation in the baseline T-cell landscape along the healthy human gastrointestinal tract, and that mucosal T-cell analyses from a single region should not be taken as representative of the entire gastrointestinal tract. We show that certain T-cell subsets in the gastrointestinal mucosa vary significantly among regions; most notably, that Tregs are enriched in the appendiceal orifice region and the ascending colon, and that CD8pos T cells are enriched in the gastric mucosa.

Highlighting new phylogenetic specificities of Crohnʼs disease microbiota

Recent studies suggest that gastrointestinal (GI) microbes play a part in the pathogenesis of Crohn's disease (CD). Fecal samples were collected from 16 healthy individuals and 16 CD patients (age- and sex-matched). The DNA extracted from these samples were subjected to two different methods of microbiome analysis. Specific bacterial groups were quantified by real-time polymerase chain reaction (PCR) methods using primers designed using a high-throughput in-house bioinformatics pipeline. The same DNA extracts were also used to produce fluorescently labeled cRNA amplicons to interrogate a custom-designed phylogenetic microarray for intestinal bacteria. Even though the intersubject variability was high, differences in the fecal microbiomes of healthy and CD patients were detected. Faecalibacterium prausnitzii and Escherichia coli were more represented in healthy and ileal CD patients, respectively. Additionally, probes specific for Ruminococcus bromii, Oscillibacter valericigenes, Bifidobacterium bifidum, and Eubacterium rectale produced stronger hybridization signals with the DNA samples from healthy subjects. Conversely, species overrepresented in CD patients were E. coli, Enterococcus faecium, and species from the Proteobacteria not normally found in the healthy human GI tract. Furthermore, we detected "healthy specific" molecular species or operational taxonomic units (OTUs) that are not closely related to any known species (Faecalibacterium, Subdoligranulum, and Oscillospora species), indicating that the phylogenetic dysbiosis is broader than at strain or species level. These two techniques of microbiome analysis provided a statistically robust new picture of the dysbiosis in fecal microbiota from ileal CD patients. Specifically, we identified a set of six species discriminant for CD, which provides a preliminary diagnostic tool.

High-quality draft genome sequences of 28 Enterococcus sp. isolates.

The enterococci are low-GC Gram-positive bacteria that have emerged as leading causes of hospital-acquired infection. They are also commensals of the gastrointestinal tract of healthy humans and most other animals with gastrointestinal flora and are important for food fermentations. Here we report the availability of draft genome sequences for 28 enterococcal strains of diverse origin, including the species Enterococcus faecalis, E. faecium, E. casseliflavus, and E. gallinarum.

Biomarkers of human gastrointestinal tract regions

Dysregulation of intestinal epithelial cell performance is associated with an array of pathologies whose onset mechanisms are incompletely understood. While whole-genomics approaches have been valuable for studying the molecular basis of several intestinal diseases, a thorough analysis of gene expression along the healthy gastrointestinal tract is still lacking. The aim of this study was to map gene expression in gastrointestinal regions of healthy human adults and to implement a procedure for microarray data analysis that would allow its use as a reference when screening for pathological deviations. We analyzed the gene expression signature of antrum, duodenum, jejunum, ileum, and transverse colon biopsies using a biostatistical method based on a multivariate and univariate approach to identify region-selective genes. One hundred sixty-six genes were found responsible for distinguishing the five regions considered. Nineteen had never been described in the GI tract, including a semaphorin probably implicated in pathogen invasion and six novel genes. Moreover, by crossing these genes with those retrieved from an existing data set of gene expression in the intestine of ulcerative colitis and Crohn's disease patients, we identified genes that might be biomarkers of Crohn's and/or ulcerative colitis in ileum and/or colon. These include CLCA4 and SLC26A2, both implicated in ion transport. This study furnishes the first map of gene expression along the healthy human gastrointestinal tract. Furthermore, the approach implemented here, and validated by retrieving known gene profiles, allowed the identification of promising new leads in both healthy and disease states.

Early Diagnosis of Disseminated Mycobacterium genavense Infection

To the Editor: Nontuberculous mycobacteria are environmental organisms that cause life-threatening diseases, particularly in immunocompromised hosts. They are increasingly recognized for causing problems in the management of solid-organ transplant recipients, due to improved diagnostic methods as well as increasing numbers and life expectancy of these patients (1). The slow-growing Mycobacterium genavense is a ubiquitous nontuberculous mycobacterium; it is reportedly isolated from tap water, pets, and the gastrointestinal tract of healthy humans (1,2). It was first recognized as a human pathogen in a patient with AIDS but has not yet been found in heart transplant recipients (3). We report early diagnosis of disseminated M. genavense infection in a heart transplant recipient. A 37-year-old man was hospitalized in September 2001 for abdominal pain, sweats, and weight loss; he had received a heart transplant 3 years earlier. Immunosuppressive treatment, which began immediately after transplantation, consisted of tacrolimus (5 mg) and mycophenolate mofetil (2 g) daily; concurrent steroid therapy was tapered off over the next 6 months. A computed tomographic (CT) scan showed numerous large lymph nodes in his abdomen (Figure). Endoscopic examinations showed diffuse inflammation of the mucosa of the duodenum, ileum, and colon. Multiple biopsy samples were submitted for histologic analysis, culture, and molecular biological analysis. Immediate 16S rRNA gene amplification that used universal primers (4) and sequencing of samples taken directly from the biopsy material led to the identification of M. genavense. A 475-bp fragment was sequenced, and 99% homology with the gene of type strain ATCC 51234 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:X60070,term_id:44288}}X60070) was found. PCR results were positive for 2 of the 4 samples tested. The molecular identification was compatible with the subsequent histologic finding of profound macrophage infiltration without granuloma and the presence of Ziehl-Neelsen–positive bacilli. Five weeks later, the molecular diagnosis was confirmed by blood cultures and cultures of the intestinal mucosa samples (Inno-LiPA Mycobacteria test, version 2, Innogenetics, Courtaboeuf, France). The direct molecular diagnosis of M. genavense enabled immediate treatment of the patient with the combination of moxifloxacin, ethambutol, clarithromycin, and amikacin; mycophenolate mofetil was discontinued. Clofazimine was added to the treatment regimen 3 months later, when a control CT scan showed that some of the enlarged mesenteric lymph nodes had increased further. After 5 months, the clinical signs resolved, and after 9 months, the lymph nodes were substantially smaller. CT scan results were within normal limits after 12 months of treatment; only ethambutol and clarithromycin were continued for an additional 6 months. There was no sign of M. genavense infection relapse 3 years after the diagnosis had been made. Figure Initial computed tomographic scan of the abdomen, performed after intravenous injection of contrast dye, showing numerous enlarged para-aortic lymph nodes (arrow). Nontuberculous mycobacteria in persons who have received heart or other solid-organ transplants remain a rare cause of late infectious complications and occur ≈3.5 years after transplantation (1,4). In the subgroup of heart transplant recipients, skin disease is the most common clinical manifestation, followed by pulmonary and disseminated disease; M. kansasii, M. avium complex, and M. haemophilum infections are most frequently encountered (1,5). M. genavense causes up to 12.8% of all nontuberculous mycobacteria infections in AIDS patients; these infections are clinically similar to those caused by the M. avium complex (1,6). M. genavense infections occur only rarely in persons other than AIDS patients (as in the present case), but they always occur in immunocompromised persons (7,8). To date, only 1 case of disseminated infection has been reported in a solid-organ (kidney) transplant recipient; the diagnosis was made by molecular identification in isolates from blood and marrow cultures. That patient died of complications from M. genavense infection (9). Because M. genavense is a fastidious organism, the infections it causes are difficult to diagnose and their frequency is probably underestimated, which may change with increased use of direct molecular biological methods. Optimal treatment of M. genavense infections has not been established (10). Experience with M. genavense infections in AIDS patients and with other nontuberculous mycobacteria infections in solid-organ transplant recipients suggests that at least 2 antimicrobial drugs should be used for a prolonged period; when possible, immunosuppressive drugs should be concurrently reduced (1,3,6,10). Outcome of nontuberculous mycobacteria infections in transplant patients is highly variable (1,5) but was satisfactory in the present patient, who was treated with quintuple antimicrobial-drug therapy and reduced immunosuppressive therapy. This case of a disseminated infection due to M. genavense in a heart transplant recipient was diagnosed early. Universal 16S rRNA gene sequencing after amplification directly from intestinal biopsy specimens enabled fast diagnosis and appropriate management.

Molecular Monitoring of the Fecal Microbiota of Healthy Human Subjects during Administration of Lactulose and Saccharomyces boulardii

Diet is a major factor in maintaining a healthy human gastrointestinal tract, and this has triggered the development of functional foods containing a probiotic and/or prebiotic component intended to improve the host's health via modulation of the intestinal microbiota. In this study, a long-term placebo-controlled crossover feeding study in which each subject received several treatments was performed to monitor the effect of a prebiotic substrate (i.e., lactulose), a probiotic organism (i.e., Saccharomyces boulardii), and their synbiotic combination on the fecal microbiota of three groups of 10 healthy human subjects differing in prebiotic dose and/or intake of placebo versus synbiotic. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was used to detect possible changes in the overall bacterial composition using the universal V(3) primer and to detect possible changes at the subpopulation level using group-specific primers targeting the Bacteroides fragilis subgroup, the genus Bifidobacterium, the Clostridium lituseburense group (cluster XI), and the Clostridium coccoides-Eubacterium rectale group (cluster XIVa). Although these populations remained fairly stable based on DGGE profiling, one pronounced change was observed in the universal fingerprint profiles after lactulose ingestion. Band position analysis and band sequencing revealed that a band appearing or intensifying following lactulose administration could be assigned to the species Bifidobacterium adolescentis. Subsequent analysis with real-time PCR (RT-PCR) indicated a statistically significant increase (P < 0.05) in total bifidobacteria in one of the three subject groups after lactulose administration, whereas a similar but nonsignificant trend was observed in the other two groups. Combined RT-PCR results from two subject groups indicated a borderline significant increase (P = 0.074) of B. adolescentis following lactulose intake. The probiotic yeast S. boulardii did not display any detectable universal changes in the DGGE profiles, nor did it influence the bifidobacterial levels. This study highlighted the capacity of an integrated approach consisting of DGGE analysis and RT-PCR to monitor and quantify pronounced changes in the fecal microbiota of healthy subjects upon functional food administration.

Dependence of Oxalate Absorption on the Daily Calcium Intake

Two to 20% of ingested oxalate is absorbed in the gastrointestinal tract of healthy humans with a daily 800 mg calcium intake. Calcium is the most potent modifier of the oxalate absorption. Although this has been found repeatedly, the exact correlation between calcium intake and oxalate absorption has not been assessed to date. Investigated was oxalate absorption in healthy volunteers applying 0.37 mmol of the soluble salt sodium [(13)C(2)]oxalate in the calcium intake range from 5 mmol (200 mg) calcium to 45 mmol (1800 mg) calcium. Within the range of 200 to 1200 mg calcium per day, oxalate absorption depended linearly on the calcium intake. With 200 mg calcium per day, the mean absorption (+/- SD) was 17% +/- 8.3%; with 1200 mg calcium per day, the mean absorption was 2.6% +/- 1.5%. Within this range, reduction of the calcium supply by 70 mg increased the oxalate absorption by 1% and vice versa. Calcium addition beyond 1200 mg/d reduced the oxalate absorption only one-tenth as effectively. With 1800 mg calcium per day, the mean absorption was 1.7% +/- 0.9%. The findings may explain why a low-calcium diet increases the risk of calcium oxalate stone formation.

16S rRNA gene sequences of 'Candidatus Campylobacter hominis', a novel uncultivated species, are found in the gastrointestinal tract of healthy humans.

Although some Campylobacter species are agents of gastroenteritis and periodontal disease in humans, little is known of the variety of campylobacters in the gastrointestinal tract of healthy individuals. This paper provides evidence for the existence of a previously undescribed, uncultivated Campylobacter species that may be a commensal in the healthy human gut. Saliva and faeces from 20 healthy individuals were examined by PCR assays specific for nine species of campylobacter (C. sputorum, C. concisus, C. upsaliensis, C. helveticus, C. lari, C. fetus, C. hyointestinalis, C. jejuni and C. coli) and for the genus as a whole. Genus-specific amplicons were produced from 19 of 20 saliva samples and from 18 of 20 faecal samples. C. concisus species-specific amplicons were produced from 19 of 20 saliva samples and 3 of 20 faecal samples. The faecal samples were all PCR-negative for other Campylobacter species. Three unidentified 16S rRNA Campylobacter genus-specific amplicons of faecal origin were sequenced. Phylogenetic analysis showed that these sequences were 99% similar, and clustered within the genus as a novel group which was termed HS (HS = healthy subject). A PCR primer pair specific for the HS group was designed from the sequence data and used to reexamine the original samples. Although it was not possible to culture the organism from faeces, specific PCR assay detected it in 10 of the 20 faecal samples, but not in any corresponding saliva samples. The authors propose that the source of the amplicons is a previously undescribed and so far uncultivated species, which they term 'Candidatus Campylobacter hominis'.

Biosynthesis of mucins (MUC2-6) along the longitudinal axis of the human gastrointestinal tract

Little is known about the biosynthesis of mucin molecules in humans. Our aim was to examine the mucin biosynthesis (MUC2-6) along the longitudinal axis of the healthy human gastrointestinal tract. Biopsies of human stomach and small and large intestine were metabolically labeled with 35S-labeled amino acids, [35S]sulfate, or[3H]galactose, immunoprecipitated with antibodies against MUC2-6, and analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), MUC5AC [apparent molecular weight (M(r)) 500,000] and MUC6 (apparent M(r) 400,000) were detected in the stomach but not in the small or large intestine, MUC3 (apparent M(r) 550,000) was detected in duodenum and jejunum, MUC2 (apparent M(r)600,000) was detected throughout the small and large intestine, and MUC4 (apparent M(r) > 900,000) was detected predominantly in the large intestine. Interestingly, some individuals displayed double bands of MUC2 and MUC3 precursors, suggesting allelic variation within the respective genes. Between small and large intestine mature secreted MUC2 showed differences in mobility on SDS-PAGE, suggesting differences in glycosylation. Each of the MUC2, MUC3, MUC4, MUC5AC, and MUC6 precursors could be distinguished electrophoretically, and each showed region-specific expression along the gastrointestinal tract.

Differentiation of Ingested and Endogenous Bifidobacteria by DNA Fingerprinting Demonstrates the Survival of an Unmodified Strain in the Gastrointestinal Tract of Humans , , ,

Consumption of bifidobacteria as a dietary adjunct has received considerable attention for its possible role in the maintenance of gastrointestinal health. However, speculation exists about these presumed health benefits because of an inability to assess the fate and mechanism of action of ingested bifidobacteria. Thus, our objective was to examine the fate of ingested bifidobacteria through the gastrointestinal tract. Variations in the highly conserved 16S ribosomal DNA (rDNA) of bifidobacteria from six male subjects (18 to 35 y old) were assessed by restriction fragment length polymorphism (RFLP) analysis. During the 16-d study, 1010 colony-forming units (CFU) of a commercially available bifidobacteria were delivered to subjects in fluid milk for each of 8 d. During the remaining 8 d, subjects consumed milk without bifidobacteria. Feces were collected at 4-d intervals and plated on selective media. For each subject, 10–15 colonies were randomly selected and used as template for PCR-amplification of 16S rDNA. 16S rDNA was restriction digested and resolved by electrophoresis. The 16S rDNA-RFLP of the ingested bifidobacteria was unique compared with bifidobacteria found in subjects prior to the feeding study. When subjects consumed bifidobacteria, a 16S rDNA-RFLP identical to that of the ingested bifidobacteria was observed in feces. The concentration of the ingested bifidobacteria in feces increased to 67.2 ± 8.5% (mean ± SEM) of total bifidobacteria. After feeding stopped, the ingested bifidobacteria diminished and became undetectable. Using this molecular approach to monitor ingested bifidobacteria, we demonstrate the kinetics of passage of this organism through the gastrointestinal tract of healthy humans.