Reproducibility of assessing fecal microbiota in chronic constipation.

BACKGROUND: While limited data suggest that the fecal microbiota in healthy people is stable over time, the intraindividual variability of the fecal microbiota in constipated patients is unknown. METHODS: This study evaluated the intraindividual reproducibility of fecal microbiota analyzed with 16S rRNA gene sequencing in two stool samples collected without and after a laxative, respectively, in 25 healthy people and 25 constipated women. Participants completed a food record for 3 d before the stool collection. Colonic transit was measured with scintigraphy. KEY RESULTS: The constipated patients were older (48±15 vs 39±10 y, P=.02) than healthy participants but had a similar BMI. The total daily caloric intake was less (P=.005) in constipated (1265±350 kcal) than healthy participants (1597±402 kcal). Fourteen patients but only two controls (P<.005), had delayed colonic transit. For most measures of alpha (eg, Observed OTU number, Shannon index) and beta diversity (eg, Bray-Curtis dissimilarity, UniFrac, phyla level abundance), the ICCs between two stool samples were high, indicating moderate or strong agreement, and similar in healthy people and constipated patients. The ICC for the weighted UniFrac distance, which is weighted by abundance, was lower than its unweighted counterpart, indicating that the unweighted measure is more robust and reproducible. CONCLUSIONS AND INFERENCES: The intraindividual reproducibility of fecal microbiota in constipated patients is high and comparable to healthy participants. For most purposes, evaluating the fecal microbiota in a single stool sample should generally suffice in adequately powered studies of healthy and constipated patients.

Assessing the colonic microbiome, hydrogenogenic and hydrogenotrophic genes, transit and breath methane in constipation.

BACKGROUND: Differences in the gut microbiota and breath methane production have been observed in chronic constipation, but the relationship between colonic microbiota, transit, and breath tests remains unclear. METHODS: In 25 healthy and 25 constipated females we evaluated the sigmoid colonic mucosal and fecal microbiota using 16S rRNA gene sequencing, abundance of hydrogenogenic FeFe (FeFe-hydA) and hydrogenotrophic (methyl coenzyme M reductase A [mrcA] and dissimilatory sulfite reductase A [dsrA]) genes with real-time qPCR assays, breath hydrogen and methane levels after oral lactulose, and colonic transit with scintigraphy. KEY RESULTS: Breath hydrogen and methane were not correlated with constipation, slow colon transit, or with abundance of corresponding genes. After adjusting for colonic transit, the abundance of FeFehydA, dsrA, and mcrA were greater (P<.005) in colonic mucosa, but not stool, of constipated patients. The abundance of the selected functional gene targets also correlated with that of selected taxa. The colonic mucosal abundance of FeFe-hydA, but not mcrA, correlated positively (P<.05) with breath methane production, slow colonic transit, and overall microbiome composition. In the colonic mucosa and feces, the abundance of hydrogenogenic and hydrogenotrophic genes were positively correlated (P<.05). Breath methane production was not associated with constipation or colonic transit. CONCLUSIONS & INFERENCES: Corroborating our earlier findings with 16S rRNA genes, colonic mucosal but not fecal hydrogenogenic and hydrogenotrophic genes were more abundant in constipated vs. healthy subjects independent of colonic transit. Breath gases do not directly reflect the abundance of target genes contributing to their production.

Effects of dietary clays on performance and intestinal mucus barrier of broiler chicks challenged with Salmonella enterica serovar Typhimurium and on goblet cell function in vitro.

In vivo and in vitro experiments were conducted to test for beneficial effects of dietary clays on broiler chicks challenged with Salmonella enterica serovar Typhimurium and to explore potential mechanisms. First, two hundred forty 1-d-old male broilers (initial BW: 41.6 ± 0.4 g) were allotted in a 2 × 4 factorial arrangement in a randomized complete block design. There were 2 infection treatments (with or without Salmonella) and 4 diets: basal (BAS), 0.3% smectite A (SMA), 0.3% smectite B, and 0.3% zeolite. The Salmonella reduced (P < 0.05) the growth rate of chicks fed the BAS, and feeding clay largely restored it (challenge × diet interaction, P < 0.05). Goblet cell number and size were increased (P < 0.05) by Salmonella in chicks fed the BAS and were reduced (P < 0.05) in Salmonella-challenged chicks by feeding SMA. Villus height was reduced by the Salmonella challenge in the chicks fed dietary clays (P < 0.01) but not in chicks fed the BAS (interaction P < 0.05). A human adenocarcinoma cell line (LS174T) was cultured in vitro in 3 separate experiments in the absence or presence of 3 concentrations (0.05, 0.10, and 0.50%) of SMA. Expression of mucin 2 (MUC2), resistin-like molecule ß (RELMß), and trefoil factor 3 (TFF3) were determined by real-time reverse-transcription PCR. The expression of RELMß was increased and expression of MUC2 was reduced (P < 0.05) by 0.10% SMA. Also, LS174T cells were cultured without or with SMA (0.05 and 0.10%) and the medium and cell lysate were analyzed for RELMß using an immunoblot assay. Protein expression of RELMß in the cell lysate was reduced (P < 0.05) by SMA addition but increased in the medium, indicating that SMA increased secretion of RELMß, thus depleting the cell and concentrating this protein in the medium. In conclusion, the dietary clays restored the growth depression caused by Salmonella, and changes in goblet cell function may contribute to the benefits of one of the clays, specifically SMA.

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species.

BACKGROUND: Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species. METHODS: Rectal swabs were collected from 10 sooty mangabeys (Cercocebus atys) and 10 baboons (Papio hamadryas). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR). RESULTS: The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB. CONCLUSIONS: Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth.

This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.

A combined in vitro/bioinformatic investigation of redox regulatory mechanisms governing cell cycle progression.

The intracellular reduction-oxidation (redox) environment influences cell cycle progression; however, underlying mechanisms are poorly understood. To examine potential mechanisms, the intracellular redox environment was characterized per cell cycle phase in Chinese hamster ovary fibroblasts via flow cytometry by measuring reduced glutathione (GSH), reactive oxygen species (ROS), and DNA content with monochlorobimane, 2',7'-dichlorohydrofluorescein diacetate (H2DCFDA), and DRAQ5, respectively. GSH content was significantly greater in G2/M compared with G1 phase cells, whereas GSH was intermediate in S phase cells. ROS content was similar among phases. Together, these data demonstrate that G2/M cells are more reduced than G1 cells. Conventional approaches to define regulatory mechanisms are subjective in nature and focus on single proteins/pathways. Proteome databases provide a means to overcome these inherent limitations. Therefore, a novel bioinformatic approach was developed to exhaustively identify putative redox-regulated cell cycle proteins containing redox-sensitive protein motifs. Using the InterPro (http://www.ebi.ac.uk/interpro/) database, we categorized 536 redox-sensitive motifs as: 1) active/functional-site cysteines, 2) electron transport, 3) heme, 4) iron binding, 5) zinc binding, 6) metal binding (non-Fe/Zn), and 7) disulfides. Comparing this list with 1,634 cell cycle-associated proteins from Swiss-Prot and SpTrEMBL (http://us.expasy.org/sprot/) revealed 92 candidate proteins. Three-fourths (69 of 92) of the candidate proteins function in the central cell cycle processes of transcription, nucleotide metabolism, (de)phosphorylation, and (de)ubiquitinylation. The majority of oxidant-sensitive candidate proteins (68.9%) function during G2/M phase. As the G2/M phase is more reduced than the G1 phase, oxidant-sensitive proteins may be temporally regulated by oscillation of the intracellular redox environment. Combined with evidence of intracellular redox compartmentalization, we propose a spatiotemporal mechanism that functionally links an oscillating intracellular redox environment with cell cycle progression.

Molecular ecological analysis of porcine ileal microbiota responses to antimicrobial growth promoters.

Cultivation-independent microbial molecular ecology approaches were used to examine the effects of antibiotic growth promoters on the pig ileal microbiota. Five-week-old barrows were fitted with a simple T-cannula at the distal ileum. Three diets meeting or exceeding the minimum nutrient requirements were fed for 5 wk and supplemented as follows: 1) negative control (no antibiotic; n = 5), 2) continuous tylosin administration (n = 5), and 3) an antibiotic rotation sequence (wk 1, chlorotetracycline sulfathiazole penicillin; wk 2, bacitracin and roxarsone; wk 3, lincomycin; wk 4, carbadox; wk 5, virginiamycin; n = 5). Ileal luminal contents were collected for DNA isolation at the end of each of the 5 wk of the testing period. The V3 region of 16S rDNA was amplified by PCR and analyzed via denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR). Resulting PCR-DGGE band numbers (bacterial species) were counted, and the banding patterns analyzed by calculating Sorenson's pairwise similarity coefficients (C(S)), an index measuring bacterial species in common among samples. Band numbers and total bacterial DNA concentrations decreased (P < 0.05) temporally in antibiotic-treated pigs compared with controls. Comparisons between treatments yielded low intertreatment C(S) indices, indicating treatment-dependent alterations in banding patterns, whereas intratreatment comparisons revealed increased homogeneity in antibiotic-treated vs. control pigs. Sequence analysis of treatment-specific bands identified three Lactobacillus, one Streptococcus, and one Bacillus species that were diminished with antibiotic rotation treatment, whereas tylosin selected for the presence of L. gasseri. Lactobacillus-specific qPCR was performed and analyzed as a percentage of total bacteria to further evaluate the effects of antibiotic administration on this genus. Total bacteria were decreased (P < 0.05) by tylosin and rotation treatments, whereas the percentage of lactobacilli increased (P < 0.05) by d 14 and through d 28 in tylosin-treated pigs. The decrease in total bacteria by antibiotics may reduce host-related intestinal or immune responses, which would divert energy that could otherwise be used for growth. Conversely, the ability of tylosin to improve animal growth may relate to its apparent selection for lactobacilli, commensals known to competitively exclude potentially pathogenic species from colonizing the intestine.

Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis.

Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats. Outbreaks of NE can be prevented or treated by the use of in-feed antibiotics. However, the current debate regarding the prophylactic use of antibiotics in animal diets necessitates a better understanding of factors that influence intestinal colonization by C. perfringens as well as the pathophysiological consequences of its growth. We report a study with a chick model of NE, which used molecular (16S rRNA gene [16S rDNA]) and culture-based microbiological techniques to investigate the impact of the macrolide antibiotic tylosin phosphate (100 ppm) and a dietary NSP (pectin) on the community structure of the small intestinal microbiota relative to colonization by C. perfringens. The effects of tylosin and pectin on mucolytic activity of the microbiota and C. perfringens colonization and their relationship to pathological indices of NE were of particular interest. The data demonstrate that tylosin reduced the percentage of mucolytic bacteria in general and the concentration of C. perfringens in particular, and these responses correlated in a temporal fashion with a reduction in the occurrence of NE lesions and an improvement in barrier function. The presence of pectin did not significantly affect the variables measured. Thus, it appears that tylosin can control NE through its modulation of C. perfringens colonization and the mucolytic activity of the intestinal microbiota.

Acidomucin goblet cell expansion induced by parenteral nutrition in the small intestine of piglets.

Total parenteral nutrition (TPN) impairs small intestine development and is associated with barrier failure, inflammation, and acidomucin goblet cell expansion in neonatal piglets. We examined the relationship between intestinal goblet cell expansion and molecular and cellular indices of inflammation in neonatal piglets receiving TPN, 80% parenteral + 20% enteral nutrition (PEN), or 100% enteral nutrition (control) for 3 or 7 days. Epithelial permeability, T cell numbers, TNF-alpha and IFN-gamma mRNA expression, and epithelial proliferation and apoptosis were compared with goblet cell numbers over time. Epithelial permeability was similar to control in the TPN and PEN jejunum at day 3 but increased in the TPN jejunum by day 7. By day 3, intestinal T cell numbers were increased in TPN but not in PEN piglets. However, goblet cell expansion was established by day 3 in both the TPN and PEN ileum. Neither TNF-alpha nor IFN-gamma mRNA expression in the TPN and PEN ileum correlated with goblet cell expansion. Thus goblet cell expansion occurred independently of overt inflammation but in association with parenteral feeding. These data support the hypothesis that goblet cell expansion represents an initial defense triggered by reduced epithelial renewal to prevent intestinal barrier failure.

Antibiotics as growth promotants: mode of action.

Recent concerns about the use of growth-promoting antibiotics in pig diets have renewed interest in the immunologic and growth-regulating functions of the gastrointestinal (GI) tract. The numerically dense and metabolically active microbiota ofthe pig GI tract represents a key focal point for such questions. The intestinal microbiota is viewed typically as a beneficial entity for the host. Intestinal bacteria provide both nutritional and defensive functions for their host. However, the host animal invests substantially in defensive efforts to first sequester gut microbes away from the epithelial surface, and second to quickly mount immune responses against those organisms that breach epithelial defenses. The impact of host responses to gut bacteria and their metabolic activities require special consideration when viewed in the context of pig production in which efficiency of animal growth is a primary objective. Here, we summarize the working hypothesis that antibiotics improve the efficiency of animal growth via their inhibition of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs ofthe GI system. In addition, novel molecular ecology techniques are described that can serve as tools to uncover the relationship between intestinal microbiology and growth efficiency.

Intestinal transport of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg through a Caco-2 Bbe monolayer.

ACE inhibitory peptides are biologically active peptides that play a role in blood pressure regulation. When derived from food proteins during food processing or gastrointestinal digestion, these peptides could function as efficient agents in treating and preventing hypertension. However, in order to exert an antihypertensive effect by inhibition of the ACE enzyme, they have to reach the bloodstream intact. The aim of this research was to assess if the known ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, derived from a tryptic digest of beta-lactoglobulin, could be absorbed through a Caco-2 Bbe cell monolayer in an Ussing chamber and reach the serosal side undegraded. Samples of the mucosal compartment showed high ACE inhibitory activity. No or only little ACE inhibitory activity was detected in the serosal compartment. However, when the serosal sample was concentrated three-fold, a substantial ACE inhibitory activity was registered. Concomitantly, HPLC and MS clearly showed the presence of Ala-Leu-Pro-Met-His-Ile-Arg in the mucosal compartment, whereas in the serosal compartment only MS was able to detect the heptapeptide. In conclusion. under the observed experimental conditions, the ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg was transported intact through the Caco-2 Bbe monolayer, but in concentrations too low to exert an ACE inhibitory activity.

Molecular ecological analysis of dietary and antibiotic-induced alterations of the mouse intestinal microbiota.

A cultivation-independent approach, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterize changes in fecal bacterial populations resulting from consumption of a low residue diet or oral administration of a broad-spectrum antibiotic. C57BL/6NHsd mice were weaned to either a standard nonpurified diet (LC-diet) or a low residue diet (LR-diet) and at 17 wk of age were randomly assigned to receive drinking water with or without 25 ppm cefoxitin for 14 d. On d 1, 2, 7 and 14, microbial DNA was extracted from feces, and the V3 region of the 16S rDNA gene was amplified by PCR and analyzed by DGGE. The diversity of fecal microbial populations, assessed using Shannon's index (H'), which incorporates species richness (number of species, or in this case, PCR-DGGE bands) and evenness (the relative distribution of species), was not affected by cefoxitin. However, use of Sorenson's pairwise similarity coefficient (C(s)), an index that measures the species in common between different habitats, indicated that the species composition of fecal bacterial communities was altered by cefoxitin in mice fed either diet. Dietary effects on fecal microbial communities were more pronounced, with greater H' values (P < 0.05) in mice fed the LR-diet (1.9 +/- 0.1) compared with the LC-diet (1.6 +/- 0.1). The C(s) values were also greater (P < 0.05) in fecal bacterial populations from mice fed the LR-diet (C(s) = 69.8 +/- 2.0%) compared with mice fed the LC-diet (C(s) = 50.1 +/- 3.8%), indicating greater homogeneity of fecal bacterial communities in mice fed the LR-diet. These results demonstrate the utility of cultivation-independent PCR-DGGE analysis combined with measurements of ecological diversity for monitoring diet- and antibiotic-induced alterations of the complex intestinal microbial ecosystem.

Microbial modulation of innate defense: goblet cells and the intestinal mucus layer.

The gastrointestinal epithelium is covered by a protective mucus gel composed predominantly of mucin glycoproteins that are synthesized and secreted by goblet cells. Changes in goblet cell functions and in the chemical composition of intestinal mucus are detected in response to a broad range of luminal insults, including alterations of the normal microbiota. However, the regulatory networks that mediate goblet cell responses to intestinal insults are poorly defined. The present review summarizes the results of developmental, gnotobiotic, and in vitro studies that showed alterations in mucin gene expression, mucus composition, or mucus secretion in response to intestinal microbes or host-derived inflammatory mediators. The dynamic nature of the mucus layer is shown. Available data indicate that intestinal microbes may affect goblet cell dynamics and the mucus layer directly via the local release of bioactive factors or indirectly via activation of host immune cells. A precise definition of the regulatory networks that interface with goblet cells may have broad biomedical applications because mucus alterations appear to characterize most diseases of mucosal tissues.

Small intestine epithelial barrier function is compromised in pigs with low feed intake at weaning.

Compromising alterations in gastrointestinal architecture are common during the weaning transition of pigs. The relation between villous atrophy and epithelial barrier function at weaning is not well understood. This study evaluated in vitro transepithelial transport by Ussing metabolic chambers, local alterations in T-cell subsets and villous architecture at low energy intake level and their relation with lactose/protein ratios in the diet. Pigs (n = 66, 26 d old) were sampled either at weaning (d 0), d 1, 2 or 4 postweaning. Piglets received one of three diets at a low energy intake level, which differed in lactose and protein ratio as follows: low lactose/high protein (LL/HP), control (C), or high lactose/low protein (HL/LP). Mean digestible energy intake was 648 kJ/pig on d 1, 1668 kJ/pig on d 2, 1995 kJ/pig on d 3 and 1990 kJ/pig on d 4 postweaning. The CD4(+)/CD8(+) T-lymphocytes ratio decreased after weaning (P < 0.05). Decreased paracellular transport (P < 0.01), greater villous height (P < 0.01), shallower crypts and lower villus/crypt ratios (P < 0.01) were observed on d 2 compared with d 0. Piglets consuming the HL/LP diet tended to have less paracellular transport (P < 0.10) and greater villous height (P < 0.10) compared with piglets fed the other diets. During the first 4 d postweaning, the effect of diet composition on mucosal integrity was not as important as the sequential effects of low energy intake at weaning. Stress and diminished enteral stimulation seem to compromise mucosal integrity as indicated by increased paracellular transport and altered T-cell subsets.

Phylogenomic analysis of the alpha proteasome gene family from early-diverging eukaryotes.

We employed a phylogenomic approach to study the evolution of alpha subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven alpha proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple alpha subunit sequences generated single alpha proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct alpha proteasome genes each. The presence of seven distinct alpha proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the alpha proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the alpha proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented.

Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53.

The diversity and stability of the fecal bacterial microbiota in weaning pigs was studied after introduction of an exogenous Lactobacillus reuteri strain, MM53, using a combination of cultivation and techniques based on genes encoding 16S rRNA (16S rDNA). Piglets (n = 9) were assigned to three treatment groups (control, daily dosed, and 4th-day dosed), and fresh fecal samples were collected daily. Dosed animals received 2.5 x 10(10) CFU of antibiotic-resistant L. reuteri MM53 daily or every 4th day. Mean Lactobacillus counts for the three groups ranged from 1 x 10(9) to 4 x 10(9) CFU/g of feces. Enumeration of strain L. reuteri MM53 on MRS agar (Difco) plates containing streptomycin and rifampin showed that the introduced strain fluctuated between 8 x 10(3) and 5 x 10(6) CFU/g of feces in the two dosed groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments, with primers specific for variable regions 1 and 3 (V1 and V3), was used to profile complexity of fecal bacterial populations. Analysis of DGGE banding profiles indicated that each individual maintained a unique fecal bacterial population that was stable over time, suggesting a strong host influence. In addition, individual DGGE patterns could be separated into distinct time-dependent clusters. Primers designed specifically to restrict DGGE analysis to a select group of lactobacilli allowed examination of interspecies relationships and abundance. Based on relative band migration distance and sequence determination, L. reuteri was distinguishable within the V1 region 16S rDNA gene patterns. Daily fluctuations in specific bands within these profiles were observed, which revealed an antagonistic relationship between L. reuteri MM53 (band V1-3) and another indigenous Lactobacillus assemblage (band V1-6).

Molecular ecological analysis of the succession and diversity of sulfate-reducing bacteria in the mouse gastrointestinal tract.

Intestinal sulfate-reducing bacteria (SRB) growth and resultant hydrogen sulfide production may damage the gastrointestinal epithelium and thereby contribute to chronic intestinal disorders. However, the ecology and phylogenetic diversity of intestinal dissimilatory SRB populations are poorly understood, and endogenous or exogenous sources of available sulfate are not well defined. The succession of intestinal SRB was therefore compared in inbred C57BL/6J mice using a PCR-based metabolic molecular ecology (MME) approach that targets a conserved region of subunit A of the adenosine-5'-phosphosulfate (APS) reductase gene. The APS reductase-based MME strategy revealed intestinal SRB in the stomach and small intestine of 1-, 4-, and 7-day-old mice and throughout the gastrointestinal tract of 14-, 21-, 30-, 60-, and 90-day-old mice. Phylogenetic analysis of APS reductase amplicons obtained from the stomach, middle small intestine, and cecum of neonatal mice revealed that Desulfotomaculum spp. may be a predominant SRB group in the neonatal mouse intestine. Dot blot hybridizations with SRB-specific 16S ribosomal DNA (rDNA) probes demonstrated SRB colonization of the cecum and colon pre- and postweaning and colonization of the stomach and small intestine of mature mice only. The 16S rDNA hybridization data further demonstrated that SRB populations were most numerous in intestinal regions harboring sulfomucin-containing goblet cells, regardless of age. Reverse transcriptase PCR analysis demonstrated APS reductase mRNA expression in all intestinal segments of 30-day-old mice, including the stomach. These results demonstrate for the first time widespread colonization of the mouse intestine by dissimilatory SRB and evidence of spatial-specific SRB populations and sulfomucin patterns along the gastrointestinal tract.

Desulfotomaculum genus- and subgenus-specific 16S rRNA hybridization probes for environmental studies.

Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum, a set of phylogenetically nested hybridization probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans), and five specific probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' using samples obtained from four different environments. Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized. The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3-2.1% of the total rRNA in the digesters, 2.6-6.6% in soil, 1.5-3.3% in human faeces and 2.5-6.2% in pig colon samples.

Total parenteral nutrition alters molecular and cellular indices of intestinal inflammation in neonatal piglets.

BACKGROUND: The adverse effects of TPN on systemic immunity are well-documented; however, the impact of IV feeding on neonatal intestinal immunity is unknown. METHODS: A piglet TPN model was used to compare immune cell composition within the intestinal epithelium and lamina propria of parenterally and orally fed piglets. RESULTS: Small intestinal weight of piglets maintained intravenously was reduced 50% after 7 days. Intestinal atrophy in piglets fed parenterally was evidenced by decreased width of intestinal villi and colon cuffs and reduced intestinal crypt depth. The numbers of CD4+ and CD8+ T lymphocytes were threefold greater within the lamina propria of jejunal and ileal villi of piglets supported intravenously. Inverse correlations were observed between villus height or width and T-lymphocyte numbers (r = -.80; p < .05). Major histocompatibility complex class II mRNA expression, an indicator of localized inflammation, was increased in the ileum and colon of piglets receiving parenteral nutrition. Goblet cell numbers were two-fold greater in jejunal and ileal villi, and mast cells were more abundant in the colon of piglets fed parenterally. Furthermore, jejunal T-lymphocyte numbers were correlated with goblet cell numbers (r = .80; p = .01). CONCLUSIONS: These data identify molecular and cellular indices of intestinal inflammation that are responsive to IV feeding in neonates and provide a novel framework to investigate mechanisms underlying gut atrophy during TPN.