First steps towards combining faecal immunochemical testing with the gut microbiome in colorectal cancer screening.

OBJECTIVES: Many countries use faecal immunochemical testing (FIT) to screen for colorectal cancer. There is increasing evidence that faecal microbiota play a crucial role in colorectal cancer carcinogenesis. We assessed the possibility of measuring faecal microbial features in FIT as potential future biomarkers in colorectal cancer screening. METHODS: Bacterial stability over time and the possibility of bacterial contamination were evaluated using quantitative polymerase chain reaction analysis. Positive FIT samples (n = 200) of an average-risk screening cohort were subsequently analysed for universal 16S, and bacteria. Escherichia coli (E. coli), Fusobacterium nucleatum (F. nucleatum), Bacteroidetes and Faecalibacterium prausnitzii (F. prausnitzii) by qPCR. The results were compared with colonoscopy findings. RESULTS: Faecal microbiota in FIT were stably measured up to six days for E. coli (p = 0.53), F. nucleatum (p = 0.30), Bacteroidetes (p = 0.05) and F. prausnitzii (p = 0.62). Overall presence of bacterial contamination in FIT controls was low. Total bacterial load (i.e. 16S) was significantly higher in patients with colorectal cancer and high-grade dysplasia (p = 0.006). For the individual bacteria tested, no association was found with colonic lesions. CONCLUSIONS: These results show that the faecal microbial content can be measured in FIT samples and remains stable for six days. Total bacterial load was higher in colorectal cancer and high-grade dysplasia. These results pave the way for further research to determine the potential role of microbiota assessment in FIT screening.

Diversity, compositional and functional differences between gut microbiota of children and adults.

The gut microbiota has been shown to play diverse roles in human health and disease although the underlying mechanisms have not yet been fully elucidated. Large cohort studies can provide further understanding into inter-individual differences, with more precise characterization of the pathways by which the gut microbiota influences human physiology and disease processes. Here, we aimed to profile the stool microbiome of children and adults from two population-based cohort studies, comprising 2,111 children in the age-range of 9 to 12 years (the Generation R Study) and 1,427 adult individuals in the range of 46 to 88 years of age (the Rotterdam Study). For the two cohorts, 16S rRNA gene profile datasets derived from the Dutch population were generated. The comparison of the two cohorts showed that children had significantly lower gut microbiome diversity. Furthermore, we observed higher relative abundances of genus Bacteroides in children and higher relative abundances of genus Blautia in adults. Predicted functional metagenome analysis showed an overrepresentation of the glycan degradation pathways, riboflavin (vitamin B2), pyridoxine (vitamin B6) and folate (vitamin B9) biosynthesis pathways in children. In contrast, the gut microbiome of adults showed higher abundances of carbohydrate metabolism pathways, beta-lactam resistance, thiamine (vitamin B1) and pantothenic (vitamin B5) biosynthesis pathways. A predominance of catabolic pathways in children (valine, leucine and isoleucine degradation) as compared to biosynthetic pathways in adults (valine, leucine and isoleucine biosynthesis) suggests a functional microbiome switch to the latter in adult individuals. Overall, we identified compositional and functional differences in gut microbiome between children and adults in a population-based setting. These microbiome profiles can serve as reference for future studies on specific human disease susceptibility in childhood, adulthood and specific diseased populations.

Pancreatic cyst fluid harbors a unique microbiome.

BACKGROUND: It is clear that specific intestinal bacteria are involved in the development of different premalignant conditions along the gastrointestinal tract. An analysis of the microbial constituents in the context of pancreatic cystic lesions has, however, as yet not been performed. This consideration prompted us to explore whether endoscopically obtained pancreatic cyst fluids (PCF) contain bacterial DNA and to determine the genera of bacteria present in such material. METHODS: Total DNA was isolated from 69 PCF samples. Bacterial 16S rRNA gene-specific PCR was performed followed by Sanger sequencing and de novo deep sequencing for the V3-V4 variable region of 16S rRNA gene. RESULTS: We observed that 98.2% of the samples were positive in conventional PCR, and that 100% of selected PCF samples (n = 33) were positive for bacterial microbiota as determined by next generation sequencing (NGS). Comprehensive NGS data analysis of PCF showed the presence of 408 genera of bacteria, of which 17 bacterial genera were uniquely abundant to PCF, when compared to the Human Microbiome Project (HMP) database and 15 bacterial microbiota were uniquely abundant in HMP only. Bacteroides spp., Escherichia/Shigella spp., and Acidaminococcus spp. which were predominant in PCF, while also a substantial Staphylococcus spp. and Fusobacterium spp. component was detected. CONCLUSION: These results reveal and characterize an apparently specific bacterial ecosystem in pancreatic cyst fluid samples and may reflect the local microbiota in the pancreas. Some taxa with potential deleterious functions are present in the bacterial abundance profiles, suggesting that the unique microbiome in this specific niche may contribute to neoplastic processes in the pancreas. Further studies are needed to explore the intricate relationship between pathophysiological status in the host pancreas and its microbiota.

Depletion of Saccharomyces cerevisiae in psoriasis patients, restored by Dimethylfumarate therapy (DMF).

BACKGROUND: Psoriasis and inflammatory bowel disease (IBD) are chronic inflammatory diseases sharing similar pathogenic pathways. Intestinal microbial changes such as a decrease of bakers' yeast Saccharomyces cerevisiae have been reported in IBD, suggesting the presence of a gut-skin axis. OBJECTIVE: To investigate whether the S. cerevisiae abundance was altered in psoriasis patients versus healthy controls, and whether dimethylfumarate (DMF) interacted with this yeast. METHODS: Using qPCR, faecal samples were compared between psoriasis patients without DMF (n = 30), psoriasis patients with DMF (n = 28), and healthy controls (n = 32). RESULTS: Faecal S. cerevisiae abundance was decreased in psoriasis compared to healthy controls (p<0.001). Interestingly, DMF use raised S. cerevisiae levels (p<0.001). Gastrointestinal adverse-effects of DMF were correlated with a higher S. cerevisiae abundance (p = 0.010). In vitro, a direct effect of DMF on S. cerevisiae growth was observed. In addition, anti-Saccharomyces cerevisiae antibodies were not elevated in psoriasis. CONCLUSION: The abundance of baker's yeast S. cerevisiae is decreased in psoriasis patients, but appears to be restored upon DMF use. S. cerevisiae is generally classified as a yeast with beneficial immunomodulatory properties, but may also be involved in the occurrence of DMF's gastrointestinal adverse-effects. Potentially, DMF might be a new therapy for IBD.

Bacterial Biofilms in Colorectal Cancer Initiation and Progression.

Intestinal microbiota have emerged as an important factor in colorectal cancer (CRC) initiation and progression. The currently prominent view on bacterial tumorigenesis is that CRC initiation is triggered by local mucosal colonization with specific pathogens (drivers), and that subsequent changes in the peritumoral environment allow colonization by opportunistic (passenger) microbes, further facilitating disease progression. Screening for CRC 'driver-passenger' microorganisms might thus allow early CRC diagnosis or preventive intervention. Such efforts are now being revolutionized by the notion that CRC initiation and progression require organization of bacterial communities into higher-order structures termed biofilms. We explore here the concept that a polymicrobial biofilm promotes pro-carcinogenic activities that may partially underlie progression along the adenoma-CRC axis.

Diet, microbiome, and colorectal cancer.

The scientific interests in the colorectal cancer (CRC) associated microbiome have increased significantly in the past decade. Mechanistically, several members of the human microbiome and products thereof have been implicated as inductors of the pathogenic inflammation related to CRC. Conversely, the activities of the human intestinal microbial community influenced by specific diet might confer a protective effect against the CRC risks and progression. As the microbiome is both a key contributor and one of the tools to prevent CRC, the current review gives a summary of the CRC-associated microbiome and the dietary strategies relevant to CRC. As more evidences become available, new microbiome-based treatments and specific diets may emerge to reduce the CRC risk and improve CRC patients' quality of life.

Similar Depletion of Protective Faecalibacterium prausnitzii in Psoriasis and Inflammatory Bowel Disease, but not in Hidradenitis Suppurativa.

BACKGROUND AND AIMS: Psoriasis and hidradenitis suppurativa [HS] co-occur more often with inflammatory bowel disease [IBD] than expected, due to shared pathogenic and genetic features. It is known that IBD patients harbour an altered intestinal microbiome characterised by a depletion of Faecalibacterium prausnitzii and increase of Escherichia coli. At present, it is unclear whether a similar intestinal microbiome trend can be identified in IBD-associated skin disorders. We therefore investigated the F. prausnitzii and E. coli abundance in psoriasis and HS, with and without concomitant IBD. METHODS: Using quantitative polymerase chain reaction , we compared the F. prausnitzii and E. coli abundances in faecal samples from healthy controls [n = 33] with samples from patients with psoriasis [n = 29], IBD [n = 31], and concomitant IBD and psoriasis [n = 13]. Likewise, we analysed samples from patients with HS [n = 17], and concomitant IBD and HS [n = 17]. RESULTS: Psoriasis patients harboured a significantly lower abundance of F. prausnitzii in their stool than healthy controls [p < 0.001], which was similar to IBD patients. Together with the reduced F. prausnitzii levels, the psoriasis patients had a significantly higher abundance of E. coli [p < 0.001]. No significant difference in F. prausnitzii or E. coli abundance was found in HS. It was apparent that patients with concomitant IBD and associated skin disorder had the greatest decrease of F. prausnitzii and increase of E. coli. CONCLUSIONS: The study demonstrates, for the first time, an IBD-like decrease of F. prausnitzii together with an increase of E.coli in psoriasis, supporting the presence of a gut-microbiome-skin axis in psoriasis and IBD.

Human fecal microbiome-based biomarkers for colorectal cancer.

Colorectal cancer may develop slowly over years from precursor lesions, and thus screening combined with early diagnosis is the key to disease prevention. Recent studies have elucidated specific traits in the gut microbiome associated with colorectal cancer and suggested that the microbiome may be useful in screening for colorectal cancer purposes but failed to provide protocols that can be applied in a practical situation. A recent study by Zackular and colleagues, presented on page 1112, provides an important way forward here in showing that specific analysis of multiple aspects of the microbiome composition in toto provides reliable detection of both precancerous and cancerous lesions. This important achievement when combined with other noninvasive techniques promises to provide highly effective tools for early colorectal cancer diagnosis and its prevention.

The microbiome and psoriatic arthritis.

Psoriatic arthritis is a chronic inflammatory joint disease, seen in combination with the chronic inflammatory skin disease psoriasis and belonging to the family of spondylarthritides (SpA). A link is recognized between psoriatic arthritis and inflammatory bowel disease (IBD). Environmental factors seem to induce inflammatory disease in individuals with underlying genetic susceptibility. The microbiome is a subject of increasing interest in the etiology of these inflammatory immune-mediated diseases. The intestinal microbiome is able to affect extra-intestinal distant sites, including the joints, through immunomodulation. At this point, evidence regarding a relationship between the microbiome and psoriatic arthritis is scarce. However, we hypothesize that common immune-mediated inflammatory pathways seen in the "skin-joint-gut axis" in psoriatic arthritis are induced or at least mediated by the microbiome. Th17 has a crucial function in this mechanism. Further establishment of this connection may lead to novel therapeutic approaches for psoriatic arthritis.

Genomic ATG16L1 risk allele-restricted Paneth cell ER stress in quiescent Crohn's disease.

OBJECTIVE: Although genome wide association studies have partly uncovered the genetic basis of Crohn's disease (CD), it remains a challenge to link genetic polymorphisms to functional intestinal phenotypes. Paneth cells are specialised antimicrobial epithelial cells localised to the small-intestinal crypt-base. Here, we investigate whether genomic variations in ATG16L1 affect Paneth cell function. DESIGN: Genomic variation of ATG16L1 (T300A, rs2241880) was determined in DNA from 78 patients with CD and 12 healthy controls. Paraffin-embedded ileal biopsies from patients with genotype AA (n=17), GA (n=38) and patients with the GG allele (n=23) were stained for GRP78, phospho-EIF2α, lysozyme, cleaved-caspase 3, phosphohistone H3, phospho-IκB, p65, phospho-p38MAPK and PHLDA1. Microbial composition of biopsies was assessed by PCR. Disease phenotype was scored. RESULTS: In patients with quiescent disease but with an ATG16L1 risk allele, the endoplasmic reticulum (ER) stress markers GRP78 and pEIF2α were highly expressed in Paneth cells. Other CD risk gene variations did not correlate with Paneth cell ER stress. Functionally, patients with ER-stressed Paneth cells showed no changes in intestinal epithelial cells proliferation or apoptosis, Paneth cell or stem cell numbers, p65, phospho-IκB and phospho-p38 staining. However, a significantly increased presence of adherent-invasive Escherichia coli was observed in biopsies from patients with ER-stressed Paneth cells. Phenotypically, patients with GRP78 positive Paneth cells have relatively less colonic disease over ileal disease (-21%, p=0.04), more fistulas (+21%, p=0.05) and an increased need for intestinal surgery (+38%, p=0.002). CONCLUSIONS: The ATG16L1 T300A polymorphism defines a specific subtype of patients with CD, characterised by Paneth cell ER stress even during quiescent disease. Paneth cell ER stress correlates with bacterial persistence, and is thus likely to modulate antimicrobial functionality of this cell type in patients with CD.

Whole genome analysis of epidemiologically closely related Staphylococcus aureus isolates.

The change of the bacteria from colonizers to pathogens is accompanied by a drastic change in expression profiles. These changes may be due to environmental signals or to mutational changes. We therefore compared the whole genome sequences of four sets of S. aureus isolates. Three sets were from the same patients. The isolates of each pair (S1800/S1805, S2396/S2395, S2398/S2397, an isolate from colonization and an isolate from infection, respectively) were obtained within <30 days of each other and the isolate from infection caused skin infections. The isolates were then compared for differences in gene content and SNPs. In addition, a set of isolates from a colonized pig and a farmer from the same farm at the same time (S0462 and S0460) were analyzed. The isolates pair S1800/S1805 showed a difference in a prophage, but these are easily lost or acquired. However, S1805 contained an integrative conjugative element not present in S1800. In addition, 92 SNPs were present in a variety of genes and the isolates S1800 and S1805 were not considered a pair. Between S2395/S2396 two SNPs were present: one was in an intergenic region and one was a synonymous mutation in a putative membrane protein. Between S2397/S2398 only one synonymous mutation in a putative lipoprotein was found. The two farm isolates were very similar and showed 12 SNPs in genes that belong to a number of different functional categories. However, we cannot pinpoint any gene that explains the change from carrier status to infection. The data indicate that differences between the isolate from infection and the colonizing isolate for S2395/S2396 and S2397/S2398 exist as well as between isolates from different hosts, but S1800/S1805 are not clonal.

Functional genomic analyses of the gut microbiota for CRC screening.

The evidence for a strong correlation between the gut microbiota and colorectal carcinogenesis is quickly gathering pace. This correlation raises important questions, such as whether analysis of the microbiota can be used for screening purposes, and whether targeted intervention can influence the risk of development and progression of neoplasia. The recovery of several pathobionts-such as members of the different bacterial phyla Proteobacteria, Bacteroidetes and Fusobacteria-from the tumour microenvironment of patients with colorectal cancer (CRC) now provides a link between specific microbial colonization and cancer. However, other intestinal bacteria belonging to another major intestinal phylum, Firmicutes, might be effective in the treatment of pathogenic inflammation related to CRC. Future approaches based on the analysis of the gut microbiota of patients with CRC combined with large human cohort studies might open up new possibilities for further prophylactic, screening and treatment strategies.

Do pregnancy-related changes in the microbiome stimulate innate immunity?

Pregnancy has a beneficial influence on the course of certain autoimmune diseases such as inflammatory bowel disease (IBD). It was recently reported that during pregnancy the microbiome undergoes profound changes that are associated with host physiological and immunological adaptations. Here we propose that microbiome remodeling during pregnancy is an active response of the mother, possibly to alter immune system status, and to facilitate metabolic and immunological adaptations that are needed for a successful pregnancy. Furthermore, these changes in the microbiome may ensure the transfer of specific traits into the neonatal gut. As the underlying mechanisms are not well understood, elucidating how pregnancy-related changes in the microbiome influence IBD would be of obvious value for designing rational therapy.

Correlation between protection against sepsis by probiotic therapy and stimulation of a novel bacterial phylotype.

Prophylactic probiotic therapy has shown beneficial effects in an experimental rat model for acute pancreatitis on the health status of the animals. Mechanisms by which probiotic therapy interferes with severity of acute pancreatitis and associated sepsis, however, are poorly understood. The aims of this study were to identify the probiotic-induced changes in the gut microbiota and to correlate these changes to disease outcome. Duodenum and ileum samples were obtained from healthy and diseased rats subjected to pancreatitis for 7 days and prophylactically treated with either a multispecies probiotic mixture or a placebo. Intestinal microbiota was characterized by terminal-restriction fragment length polymorphism (T-RFLP) analyses of PCR-amplified 16S rRNA gene fragments. These analyses showed that during acute pancreatitis the host-specific ileal microbiota was replaced by an "acute pancreatitis-associated microbiota." This replacement was not reversed by administration of the probiotic mixture. An increase, however, was observed in the relative abundance of a novel bacterial phylotype most closely related to Clostridium lituseburense and referred to as commensal rat ileum bacterium (CRIB). Specific primers targeting the CRIB 16S rRNA gene sequence were developed to detect this phylotype by quantitative PCR. An ileal abundance of CRIB 16S rRNA genes of more than 7.5% of the total bacterial 16S rRNA gene pool was correlated with reduced duodenal bacterial overgrowth, reduced bacterial translocation to remote organs, improved pancreas pathology, and reduced proinflammatory cytokine levels in plasma. Our current findings and future studies involving this uncharacterized bacterial phylotype will contribute to unraveling one of the potential mechanisms of probiotic therapy.

S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions.

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFalpha, and IL-1beta were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA-DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.

Feeding of Lactobacillus sobrius reduces Escherichia coli F4 levels in the gut and promotes growth of infected piglets.

The microbial community in the guts of mammals is often seen as an important potential target in therapeutic and preventive interventions. The aim of the present study was to determine whether enterotoxigenic Escherichia coli (ETEC) F4 infection in young animals might be counteracted by a probiotic treatment with Lactobacillus sobrius DSM 16698. The experiment was conducted in three randomized consecutive replications, each consisting of 16 piglets, and including a control group and an L. sobrius fed group, both experimentally challenged with ETEC. During the entire trial, the animals' health status, body weight, and microbial parameters were monitored periodically. Probiotic supplementation containing L. sobrius significantly reduced the levels of ETEC in the ileum when fed directly to piglets after weaning. In contrast, the number of days when the piglets had an increased faecal water content was significantly higher in the probiotic group. Nevertheless, an improved daily weight gain was also observed in the animals that received probiotic L. sobrius relative to the control fed group. The data indicate that L. sobrius may be effective in the reduction of the E. coli F4 colonization and may improve the weight gain of infected piglets.

The novel porcine Lactobacillus sobrius strain protects intestinal cells from enterotoxigenic Escherichia coli K88 infection and prevents membrane barrier damage.

Lactobacilli have a potential to overcome intestinal disorders; however, the exact mode of action is still largely unknown. In this study, we have used the intestinal porcine intestinal IPEC-1 epithelial cells as a model to investigate a possible protective activity of a new Lactobacillus species, the L. sobrius DSM 16698(T), against intestinal injury induced by enterotoxigenic Escherichia coli (ETEC) K88 infection and the underlying mechanisms. Treatment of infected cells with L. sobrius strongly reduced the pathogen adhesion. L. sobrius was also able to prevent the ETEC-induced membrane damage by inhibiting delocalization of zonula occludens (ZO)-1, reduction of occludin amount, rearrangement of F-actin, and dephosphorylation of occludin caused by ETEC. RT-PCR and ELISA experiments showed that L. sobrius counteracted the ETEC-induced increase of IL-8 and upregulated the IL-10 expression. The involvement of IL-8 in the deleterious effects of ETEC was proven by neutralization of IL-8 with a specific antibody. A crucial role of IL-10 was indicated by blockage of IL-10 production with neutralizing anti-IL-10 antibody that fully abrogated the L. sobrius protection. L. sobrius was also able to inhibit the internalization of ETEC, which was likely favored by the leaking barrier. The protective effects were not found with L. amylovorus DSM 20531(T) treatment, a strain derived from cattle waste but phylogenetically closely related to L. sobrius. Together, the data indicate that L. sobrius exerts protection against the harmful effects of ETEC by different mechanisms, including pathogen adhesion inhibition and maintenance of membrane barrier integrity through IL-10 regulation.

Modification of intestinal flora with multispecies probiotics reduces bacterial translocation and improves clinical course in a rat model of acute pancreatitis.

BACKGROUND: Infection of pancreatic necrosis by gut bacteria is a major cause of morbidity and mortality in patients with severe acute pancreatitis. Use of prophylactic antibiotics remains controversial. The aim of this experiment was assess if modification of intestinal flora with specifically designed multispecies probiotics reduces bacterial translocation or improves outcome in a rat model of acute pancreatitis. METHODS: Male Sprague-Dawley rats were allocated into 3 groups: (1) controls (sham-operated, no treatment), (2) pancreatitis and placebo, and (3) pancreatitis and probiotics. Acute pancreatitis was induced by intraductal glycodeoxycholate and intravenous cerulein infusion. Daily probiotics or placebo was administered intragastrically from 5 days prior until 7 days after induction of pancreatitis. Tissue and fluid samples were collected for microbiologic and quantitative real-time PCR analysis of bacterial translocation. RESULTS: Probiotics reduced duodenal bacterial overgrowth of potential pathogens (Log(10) colony-forming units [CFU]/g 5.0 +/- 0.7 [placebo] vs 3.5 +/- 0.3 CFU/g [probiotics], P < .05), resulting in reduced bacterial translocation to extraintestinal sites, including the pancreas (5.38 +/- 1.0 CFU/g [placebo] vs 3.1 +/- 0.5 CFU/g [probiotics], P < .05). Accordingly, health scores were better and late phase mortality was reduced: 27% (4/15, placebo) versus 0% (0/13, probiotics), respectively, P < .05. CONCLUSIONS: This experiment supports the hypothesis that modification of intestinal flora with multispecies probiotics results in reduced bacterial translocation, morbidity, and mortality in the course of experimental acute pancreatitis.

Post-natal development of the porcine microbiota composition and activities.

The current study describes the development of the porcine microbiota and its metabolic activities during the neonatal and weaning period. Using 16S rRNA-based approaches, we first analysed the ileal and colonic microbiota of neonatal piglets at days 2, 5 and 12 after birth. To further investigate the effect of weaning at 3 weeks of age, 19-day-old piglets (n = 64) were randomly allocated into two groups. Half of the piglets remained with their sows throughout the study, while the remaining piglets were weaned. As revealed by sequence analysis of 16S rRNA gene amplicons, the samples of 2-day-old piglets harboured a consortium of bacteria related to Escherichia coli, Shigella flexneri, Lactobacillus sobrius, Lactobacillus reuteri and Lactobacillus acidophilus. Moreover, species-specific real-time polymerase chain reaction assays unveiled that L. sobrius and L. reuteri predominated in the ileal samples of the neonatal and unweaned piglets with population levels up to 7 x 10(8) cells per gram of lumen content. Following weaning, however, these two lactobacilli were detected at significantly lower levels (< 10(3)) in the ileal samples. Furthermore, a shift in composition and metabolic activities of the predominant microbiota, and emergence of clostridia and E. coli, were encountered in the intestinal samples of the piglets after the early post-weaning period.