Characterization of the bacterial gut microbiota of piglets suffering from new neonatal porcine diarrhoea.

BACKGROUND: In recent years, new neonatal porcine diarrhoea (NNPD) of unknown aetiology has emerged in Denmark. NNPD affects piglets during the first week of life and results in impaired welfare, decreased weight gain, and in the worst-case scenario death. Commonly used preventative interventions such as vaccination or treatment with antibiotics, have a limited effect on NNPD. Previous studies have investigated the clinical manifestations, histopathology, and to some extent, microbiological findings; however, these studies were either inconclusive or suggested that Enterococci, possibly in interaction with Escherichia coli, contribute to the aetiology of NNPD. This study examined ileal and colonic luminal contents of 50 control piglets and 52 NNPD piglets by means of the qPCR-based Gut Microbiotassay and 16 samples by 454 sequencing to study the composition of the bacterial gut microbiota in relation to NNPD. RESULTS: NNPD was associated with a diminished quantity of bacteria from the phyla Actinobacteria and Firmicutes while genus Enterococcus was more than 24 times more abundant in diarrhoeic piglets. The number of bacteria from the phylum Fusobacteria was also doubled in piglets suffering from diarrhoea. With increasing age, the gut microbiota of NNPD affected piglet and control piglets became more diverse. Independent of diarrhoeic status, piglets from first parity sows (gilts) possessed significantly more bacteria from family Enterobacteriaceae and species E. coli, and fewer bacteria from phylum Firmicutes. Piglets born to gilts had 25 times higher odds of having NNPD compared with piglets born to multiparous sows. Finally, the co-occurrence of genus Enterococcus and species E. coli contributed to the risk of having NNPD. CONCLUSION: The results of this study support previous findings that points towards genus Enterococcus and species E. coli to be involved in the pathogenesis of NNPD. Moreover, the results indicate that NNPD is associated with a disturbed bacterial composition and larger variation between the diarrhoeic piglets.

Antibiotics modulate intestinal immunity and prevent necrotizing enterocolitis in preterm neonatal piglets.

Preterm birth, bacterial colonization, and formula feeding predispose to necrotizing enterocolitis (NEC). Antibiotics are commonly administered to prevent sepsis in preterm infants, but it is not known whether this affects intestinal immunity and NEC resistance. We hypothesized that broad-spectrum antibiotic treatment improves NEC resistance and intestinal structure, function, and immunity in neonates. Caesarean-delivered preterm pigs were fed 3 days of parenteral nutrition followed by 2 days of enteral formula. Immediately after birth, they were assigned to receive either antibiotics (oral and parenteral doses of gentamycin, ampicillin, and metronidazole, ANTI, n = 11) or saline in the control group (CON, n = 13), given twice daily. NEC lesions and intestinal structure, function, microbiology, and immunity markers were recorded. None of the ANTI but 85% of the CON pigs developed NEC lesions by day 5 (0/11 vs. 11/13, P < 0.05). ANTI pigs had higher intestinal villi (+60%), digestive enzyme activities (+53-73%), and goblet cell densities (+110%) and lower myeloperoxidase (-51%) and colonic microbial density (10(5) vs. 10(10) colony-forming units, all P < 0.05). Microarray transcriptomics showed strong downregulation of genes related to inflammation and innate immune response to microbiota and marked upregulation of genes related to amino acid metabolism, in particular threonine, glucose transport systems, and cell cycle in 5-day-old ANTI pigs. In a follow-up experiment, 5 days of antibiotics prevented NEC at least until day 10. Neonatal prophylactic antibiotics effectively reduced gut bacterial load, prevented NEC, intestinal atrophy, dysfunction, and inflammation and enhanced expression of genes related to gut metabolism and immunity in preterm pigs.

The Gut Microbiotassay: a high-throughput qPCR approach combinable with next generation sequencing to study gut microbial diversity.

BACKGROUND: The intestinal microbiota is a complex and diverse ecosystem that plays a significant role in maintaining the health and well-being of the mammalian host. During the last decade focus has increased on the importance of intestinal bacteria. Several molecular methods can be applied to describe the composition of the microbiota. This study used a new approach, the Gut Microbiotassay: an assembly of 24 primer sets targeting the main phyla and taxonomically related subgroups of the intestinal microbiota, to be used with the high-throughput qPCR chip 'Access Array 48.48', AA48.48, (Fluidigm®) followed by next generation sequencing. Primers were designed if necessary and all primer sets were screened against DNA extracted from pure cultures of 15 representative bacterial species. Subsequently the setup was tested on DNA extracted from small and large intestinal content from piglets with and without diarrhoea. The PCR amplicons from the 2304 reaction chambers were harvested from the AA48.48, purified, and sequenced using 454-technology. RESULTS: The Gut Microbiotassay was able to detect significant differences in the quantity and composition of the microbiota according to gut sections and diarrhoeic status. 454-sequencing confirmed the specificity of the primer sets. Diarrhoea was associated with a reduced number of members from the genus Streptococcus, and in particular S. alactolyticus. CONCLUSION: The Gut Microbiotassay provides fast and affordable high-throughput quantification of the bacterial composition in many samples and enables further descriptive taxonomic information if combined with 454-sequencing.

Modulation of the gut microbiota with antibiotic treatment suppresses whole body urea production in neonatal pigs.

We examined whether changes in the gut microbiota induced by clinically relevant interventions would impact the bioavailability of dietary amino acids in neonates. We tested the hypothesis that modulation of the gut microbiota in neonatal pigs receiving no treatment (control), intravenously administered antibiotics, or probiotics affects whole body nitrogen and amino acid turnover. We quantified whole body urea kinetics, threonine fluxes, and threonine disposal into protein, oxidation, and tissue protein synthesis with stable isotope techniques. Compared with controls, antibiotics reduced the number and diversity of bacterial species in the distal small intestine (SI) and colon. Antibiotics decreased plasma urea concentrations via decreased urea synthesis. Antibiotics elevated threonine plasma concentrations and turnover, as well as whole body protein synthesis and proteolysis. Antibiotics decreased protein synthesis rate in the proximal SI and liver but did not affect the distal SI, colon, or muscle. Probiotics induced a bifidogenic microbiota and decreased plasma urea concentrations but did not affect whole body threonine or protein metabolism. Probiotics decreased protein synthesis in the proximal SI but not in other tissues. In conclusion, modulation of the gut microbiota by antibiotics and probiotics reduced hepatic ureagenesis and intestinal protein synthesis, but neither altered whole body net threonine balance. These findings suggest that changes in amino acid and nitrogen metabolism resulting from antibiotic- or probiotic-induced shifts in the microbiota are localized to the gut and liver and have limited impact on whole body growth and anabolism in neonatal piglets.

Postnatal amniotic fluid intake reduces gut inflammatory responses and necrotizing enterocolitis in preterm neonates.

Preterm neonates are susceptible to gastrointestinal disorders such as necrotizing enterocolitis (NEC). Maternal milk and colostrum protects against NEC via growth promoting, immunomodulatory, and antimicrobial factors. The fetal enteral diet amniotic fluid (AF), contains similar components, and we hypothesized that postnatal AF administration reduces inflammatory responses and NEC in preterm neonates. Preterm pigs (92% gestation) were delivered by caesarean section and fed parental nutrition (2 days) followed by enteral (2 days) porcine colostrum (COLOS, n = 7), infant formula (FORM, n = 13), or AF supplied before and after introduction of formula (AF, n = 10) in experiment 1, and supplied only during the enteral feeding period in experiment 2 (FORM, n = 16; AF, n = 14). The NEC score was reduced in both AF and COLOS pigs, relative to FORM, when AF was provided prior to full enteral feeding (9.9 and 7.7 compared with 17.3, P < 0.05). There was no effect of AF when provided only during enteral feeding. AF pigs showed decreased bacterial abundance in colon and intestinal inflammation-related genes (e.g., TNF-α, IL-1α, IL-6, NOS) were downregulated, relative to FORM pigs with NEC. Anti-inflammatory properties of AF were supported by delayed maturation and decreased TNF-α production in murine dendritic cells, as well as increased proliferation and migration, and downregulation of IL-6 expression in intestinal cells (IEC-6, IPEC-J2). Like colostrum, AF may reduce NEC development in preterm neonates by suppressing the proinflammatory responses to enteral formula feeding and gut colonization when provided before the onset of NEC.

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs.

BACKGROUND: Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). RESULTS: A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, P<0.0001) and in non-cloned control pigs (r=0.9, P<0.0001). Shannon Weaver and principal component analysis (PCA) of the terminal restriction fragments (T-RFs) revealed no differences in the bacterial composition or variability of the fecal microbiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; P<0.02) and non cloned-control pigs (r=0.45; P<0.006), and negatively with the abundance of Bacteroidetes in cloned pigs (r=-0.33, P<0.04), but not in the non-cloned control pigs. CONCLUSION: The cloned pigs did not have reduced inter-individual variation as compared to non-cloned pigs in regard to their gut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

The influence of the cage system and colonisation of Salmonella Enteritidis on the microbial gut flora of laying hens studied by T-RFLP and 454 pyrosequencing.

BACKGROUND: In the EU conventional cages for laying hens are forbidden beginning in January 2012, however concerns about a higher transmission rate of Salmonella in alternative cages systems have been raised. The extent to which cage systems may affect the intestinal microbiota of laying hens is not known, and different microbiota may demonstrate different resistance towards colonization with Salmonella. To investigate this, ileal and caecal samples from two experimental studies where laying hens were inoculated with Salmonella Enteritidis and housed in different systems (conventional cage, furnished cage or aviary), were compared using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The distribution of genera in the microbiota in caecum was furthermore described by next generation sequencing of 16S rDNA libraries. RESULTS: Hens in the same cage type developed similar T-RFLP fingerprints of the ileal and caecal microbiota, and these could be separated from layers in the other cages types. No significant difference in the fingerprint profiles could be observed between Salmonella positive and negative samples from same cage. By deep sequencing of 16S rDNA libraries from caecum, 197 different Operational Taxonomic Units (OTU) were identified, and 195 and 196 OTU respectively, were found in hens in aviary and furnished cages, but only 178 OTU of these were recovered from conventional cages. The ratio between the dominating phyla or families and genera in the microbiota remained fairly constant throughout the study. Faecalibacterium and Butyricimonas were the most prevalent genera found in the caecal microbiota of layers irrespective of the cage type. CONCLUSIONS: Hens confined in the same cage group tend to develop similar microbiota in their ileum and caecum possibly due to isolation, while differences in the microbiota between cages may be caused by environmental or individual bird factors. Although the cages type had influence on composition of the microbiota in the layers by promoting higher diversity in furnished and aviary systems, we did not observe differences in colonization and excretion pattern of Salmonella from these groups. We suggest, that differences in group size and exposure to a more faecally contaminated environment in the alternative systems may explain the observed differences in diversity of the caecal microbiota.

Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in newborn neonates. Bacteria are believed to be important in the pathogenesis of NEC but bacterial characterization has only been done on human faecal samples and experimental animal studies. The aim of this study was to investigate the microbial composition and the relative number of bacteria in inflamed intestinal tissue surgically removed from neonates diagnosed with NEC (n=24). The bacterial populations in the specimens were characterized by laser capture microdissection and subsequent sequencing combined with fluorescent in situ hybridization (FISH), using bacterial rRNA-targeting oligonucleotide probes. RESULTS: Bacteria were detected in 22 of the 24 specimens, 71% had moderate to high densities of bacteria. The phyla detected by 16S rRNA gene sequencing were: Proteobacteria (49.0%), Firmicutes (30.4%), Actinobacteria (17.1%) and Bacteroidetes (3.6%). A major detected class of the phylum Proteobacteria belonged to δ-proteobacteria. Surprisingly, Clostridium species were only detected in 4 of the specimens by FISH, but two of these specimens exhibited histological pneumatosis intestinalis and both specimens had a moderate to a high density of C. butyricum and C. parputrificum detected by using species specific FISH probes. A 16S rRNA gene sequence tag similar to Ralstonia species was detected in most of the neonatal tissues and members of this genus have been reported to be opportunistic pathogens but their role in NEC has still to be clarified. CONCLUSION: In this study, in situ identification and community analysis of bacteria found in tissue specimens from neonates with NEC, were analysed for the first time. Although a large variability of bacteria was found in most of the analyzed specimens, no single or combination of known potential pathogenic bacteria species was dominating the samples suggestive NEC as non-infectious syndrome. However there was a significant correlation between the presence of C. butyricum & C. parputrificum and histological pneumatosis intestinalis. Finally this study emphasizes the possibility to examine the microbial composition directly on excised human tissues to avoid biases from faecal samples or culturing.

Preterm birth and necrotizing enterocolitis alter gut colonization in pigs.

Necrotizing enterocolitis (NEC) in preterm neonates is dependent on bacterial colonization, but it remains unclear whether a particular microbiota or specific pathogens are involved. We hypothesized that gut colonization differs between preterm and term neonates and that overgrowth of Clostridium perfringens predisposes to NEC. By using terminal-RFLP and FISH, we characterized the gut microbiota of preterm, caesarean-delivered, formula-fed pigs (n = 44) with or without NEC and of formula- or colostrum-fed term, and vaginally born pigs (n = 13). A different microbiota with high C. perfringens abundance was observed in preterm pigs with NEC compared with healthy individuals. However, immunization against C. perfringens toxins did not prevent NEC, and C. perfringens inoculation (3.6 × 10 cfu/d) failed to induce NEC (n = 16), whereas prophylactic broad-spectrum antibiotics treatment prevented NEC (n = 24). Colonization in both groups of term pigs differed from preterm pigs and was dominated by Lactobacilli spp. In conclusion, gestational age (GA) and NEC influence neonatal gut colonization, whereas diet has minor effects. C. perfringens is more abundant in pigs with NEC but rather as a consequence than a cause of disease. The general bacterial load and underdeveloped gut immune responses in preterm neonates seem more important for NEC development than specific pathogens.

Molecular fingerprints of the human fecal microbiota from 9 to 18 months old and the effect of fish oil supplementation.

OBJECTIVES: The aim of this study was to monitor changes in the fecal microbiota from 9 to 18 months and to investigate the effect of increasing dietary n-3 polyunsaturated fatty acids on the fecal microbiota. PATIENTS AND METHODS: In a double-blind controlled trial with random allocation to daily supplementation with 5 mL of fish oil (FO) or sunflower oil (SO) from 9 to 18 months of age, stool samples were collected from 132 healthy Danish infants. Molecular fingerprints of the bacterial DNA were obtained by terminal restriction fragment length polymorphism (T-RFLP). RESULTS: The T-RFLP profiles indicated that a few T-RFs became dominant with age (bp100 and 102, both presumed to be Bacteroidetes) concomitantly with an overall increase in the microbial diversity (P = 0.04). Breast-feeding influenced both the T-RFLP profiles at 9 months and the changes from 9 to 18 months, and breast-feeding cessation during the trial modified the response to the dietary oils. In the FO group, the increase in bp102 was significantly reduced among children weaned before compared with those weaned during the trial (P = 0.027), whereas the increase in bp100 was reduced in the preweaned children of the SO group relative to those weaned during the trial (P = 0.004). This was supported by intervention group differences in the changes in bp102 and bp100 among the earlier weaned children (P = 0.06 and P = 0.09, respectively). CONCLUSIONS: Cessation of breast-feeding played a dominant role relative to developmental changes in the fecal microbiota from 9 to 18 months. FO compared with SO supplementation affected changes in large bacterial groups, but only among children who had stopped breast-feeding before 9 months of age.

Examination of equine glandular stomach lesions for bacteria, including Helicobacter spp by fluorescence in situ hybridisation.

BACKGROUND: The equine glandular stomach is commonly affected by erosion and ulceration. The aim of this study was to assess whether bacteria, including Helicobacter, could be involved in the aetiology of gastric glandular lesions seen in horses. RESULTS: Stomach lesions, as well as normal appearing mucosa were obtained from horses slaughtered for human consumption. All samples were tested for urease activity using the Pyloritek assay, while mucosal bacterial content was evaluated using Fluorescence In Situ Hybridisation. In selected sub samples, bacteria characterisation was pursued further by cloning and sequencing. Mucosal lesions were found in 36/63 stomachs and included hyperplastic rugae, polypoid structures and focal erosions. None of the samples were tested positive for urease activity or for FISH using the Helicobacter genus specific probe. In samples of lesions, as well as normal samples, clones with 99% similarities to Lactobacillus salivarius and Sarcina ventriculi were found. Escherichia like bacterium clones and Enterococcus clones were demonstrated in one focal erosion. Based on a phylogenetic tree these clones had 100% similarity to Escherichia fergusonii and Enterococcus faecium. The Enterococcus were found colonising the mucosal surface, while E. fergusonii organisms were also demonstrated intraepithelial. CONCLUSION: Gastric Helicobacter spp. could not be verified as being involved in lesions of the glandular stomach of the horse. Since E. fergusonii has been described as an emerging pathogen in both humans and animals, the finding of this bacterium in gastric erosion warrants further clarification to whether gastric infection with this type bacterium is important for horses.

Carbohydrate maldigestion induces necrotizing enterocolitis in preterm pigs.

Necrotizing enterocolitis (NEC) remains the most severe gastrointestinal disorder in preterm infants. It is associated with the initiation of enteral nutrition and may be related to immature carbohydrate digestive capacity. We tested the hypothesis that a formula containing maltodextrin vs. a formula containing lactose as the principal source of carbohydrate would predispose preterm pigs to a higher NEC incidence. Cesarean-derived preterm pigs were given total parenteral nutrition for 48 h followed by total enteral nutrition with a lactose-based (n = 11) or maltodextrin-based (n = 11) formula for 36 h. A higher incidence (91% vs. 27%) and severity (score of 3.3 vs. 1.8) of NEC were observed in the maltodextrin than in the lactose group. This higher incidence of NEC in the maltodextrin group was associated with significantly lower activities of lactase, maltase, and aminopeptidase; reduced villus height; transiently reduced in vivo aldohexose uptake; and reduced ex vivo aldohexose uptake capacity in the middle region of the small intestine. Bacterial diversity was low for both diets, but alterations in bacterial composition and luminal concentrations of short-chain fatty acids were observed in the maltodextrin group. In a second study, we quantified net portal absorption of aldohexoses (glucose and galactose) during acute jejunal infusion of a maltodextrin- or a lactose-based formula (n = 8) into preterm pigs. We found lower net portal aldohexose absorption (4% vs. 42%) and greater intestinal recovery of undigested carbohydrate (68% vs. 27%) in pigs acutely perfused with the maltodextrin-based formula than those perfused with the lactose-based formula. The higher digestibility of the lactose than the maltodextrin in the formulas can be attributed to a 5- to 20-fold higher hydrolytic activity of tissue-specific lactase than maltases. We conclude that carbohydrate maldigestion is sufficient to increase the incidence and severity of NEC in preterm pigs.

Better living through microbial action: the benefits of the mammalian gastrointestinal microbiota on the host.

Mammals live in a homeostatic symbiosis with their gastrointestinal microbiota. The mammalian host provides the microbiota with nutrients and a stable environment; whereas the microbiota helps shaping the host's gut mucosa and provides nutritional contributions. Microorganisms start colonizing the gut immediately after birth followed by a succession of populations until a stable, adult microbiota has been established. However, physiological conditions differ substantially among locations in the gut and determine bacterial density and diversity. While Firmicutes and Bacteroidetes dominate the gut microbiota in all mammals, the bacterial genera and species diversity is huge and reflects mammalian phylogeny. The main function of the gastrointestinal epithelium is to absorb nutrients and to retain water and electrolytes, yet at the same time it is an efficient barrier against harmful compounds and microorganisms, and is able to neutralize antagonists coincidentally breaching the barrier. These processes are influenced by the microbiota, which modify epithelial expression of genes involved in nutrient uptake and metabolism, mucosal barrier function, xenobiotic metabolism, enteric nervous system and motility, hormonal and maturational responses, angiogenesis, cytoskeleton and extracellular matrix, signal transduction, and general cellular functions. Whereas such effects are local at the gut epithelium they may eventually have systemic consequences, e.g. on body weight and composition.

Early administration of probiotics alters bacterial colonization and limits diet-induced gut dysfunction and severity of necrotizing enterocolitis in preterm pigs.

Following preterm birth, bacterial colonization and enteral formula feeding predispose neonates to gut dysfunction and necrotizing enterocolitis (NEC), a serious gastrointestinal inflammatory disease. We hypothesized that administration of probiotics would beneficially influence early bacterial colonization, thereby reducing the susceptibility to formula-induced gut atrophy, dysfunction, and NEC. Caesarean-delivered preterm pigs were provided total parenteral nutrition (1.5 d) followed by enteral feeding (2 d) with porcine colostrum (COLOS; n = 5), formula (FORM; n = 9), or formula with probiotics (FORM-P; Bifidobacterium animalis and Lactobacillus: L. acidophilus, L. casei, L. pentosus, L. plantarum; n = 13). Clinical NEC scores were reduced (P < 0.05) in FORM-P (2.0 +/- 0.2) and COLOS groups (1.7 +/- 0.5) compared with FORM pigs (3.4 +/- 0.6). Lower NEC scores were associated with elevated intestinal weight, mucosa proportion, villus height, RNA integrity, and brush border aminopeptidase A and N activities, and lower gastric organic acid concentration in the FORM-P and COLOS groups (P < 0.05). Diversity of the mucosa-associated bacteria in the distal small intestine was similar among formula-fed pigs, yet the abundance of specific bacterial groups differed between FORM-P and FORM pigs. FORM-P pigs had lower colonization density of a potential pathogen, Clostridium perfringens, and had commensal Lactobacillus bacteria more closely associated with enterocytes along the villus-crypt axis relative to FORM pigs. These results suggest that probiotic administration immediately after birth promotes the colonization of a beneficial commensal microbiota capable of limiting the formula-induced mucosal atrophy, dysfunction, and pathogen load in preterm neonates, thereby reducing the incidence and severity of NEC.

The microbiota of pigs influenced by diet texture and severity of Lawsonia intracellularis infection.

Pigs with and without naturally occurring Lawsonia intracellularis infection were fed diets with different texture. In a previous study from 79 pig herds using a similar feeding on pelleted or non-pelleted form showed that the non-pelleted diet was associated with a reduced prevalence of L. intracellularis. In this study a mechanistic approach was taken for explaining and testing this observation by studying the microbiota and the occurrence of L. intracellularis in the distal ileum of 54 pigs by terminal restriction fragment length polymorphism (T-RFLP) analysis, Real-Time PCR and in situ hybridization. The texture of the diet influenced the microbiota, and from a quantitative discriminative analysis of the terminal restriction fragments (T-RFs) of ileum samples it was deduced that Clostridium spp. and Lactobacillus spp. were associated with the non-pelleted diet and Streptococcus spp. with the pelleted diet. In experimentally infected pigs it was verified that 89bp and 90bp sized T-RFs (HhaI) from ileum represented L. intracellularis. The non-pelleted diet seemed to reduce the relative amount of L. intracellularis in the total microbiota of the ileum, but the number of pigs detected positive with L. intracellularis by Real-Time PCR was not influenced. The five pigs with highest L. intracellularis content showed T-RFs that were not present in profiles from less or non-infected pigs, which may indicate that some bacterial species were associated with L. intracellularis infection.

Root growth and exudate production define the frequency of horizontal plasmid transfer in the Rhizosphere.

To identify the main drivers of plasmid transfer in the rhizosphere, conjugal transfer was studied in the rhizospheres of pea and barley. The donor Pseudomonas putida KT2442, containing plasmid pKJK5::gfp, was coated onto the seeds, while the recipient P. putida LM24, having a chromosomal insertion of dsRed, was inoculated into the growth medium. Mean transconjugant-to-donor ratios in vermiculite were 4.0+/-0.8 x 10(-2) in the pea and 5.9+/-1.4 x 10(-3) in the barley rhizospheres. In soil, transfer ratios were about 10 times lower. As a result of a 2-times higher root exudation rate in pea, donor densities in pea (1 x 10(6)-2 x 10(9) CFU g(-1) root) were about 10 times higher than in barley. No difference in recipient densities was observed. In situ visualization of single cells on the rhizoplane and macroscopic visualization of the colonization pattern showed that donors and transconjugants were ubiquitously distributed in the pea rhizosphere, while they were only located on the upper parts of the barley roots. Because the barley root elongated about 10 times faster than the pea root, donors were probably outgrown by the elongating barley root. Thus by affecting the cell density and distribution, exudation and root growth appear to be key parameters controlling plasmid transfer in the rhizosphere.

Comparison of methods and animal models commonly used for investigation of fecal microbiota: effects of time, host and gender.

Denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and plating on selective agars were used to study variation in the fecal microbiota of rats over time as well as variation between individuals. Investigated rats were either conventional and specific pathogen free (SPF), or human flora associated (HFA). A higher variation (p<0.05) in fecal microbiota over time was observed for HFA than for SPF animals. Analysis of DGGE and T-RFLP profiles of fecal microbiota from SPF and HFA rats revealed that variation over time was less significant than variation between individuals, and that phylogenetic profiles clustered according to gender. These observations should be taken into account when designing future research addressing changes in fecal microbiota.

Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization.

Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells. By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S rRNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria within the tissue with molecular analysis of 16S rRNA gene or other genes of interest. This method is widely applicable for the identification of noncultivable bacteria and their gene pool from formalin-fixed paraffin-embedded tissue samples.

Necrotizing Enterocolitis in Preterm Pigs Is Associated with Increased Density of Intestinal Mucosa-Associated Bacteria Including Clostridium perfringens.

BACKGROUND: Necrotizing enterocolitis (NEC) is associated with changes in the luminal gut microbiota. It is not known whether the mucosa-associated microbiota is affected by NEC and stimulates inflammatory lesions. OBJECTIVE: We hypothesized that the density of the mucosa-associated microbiota correlates with NEC severity in preterm pigs and that in vitro infection with increasing densities of Clostridium perfringens, which has been associated with NEC in preterm infants, would lead to a transcriptional response related to the inflammatory conditions of NEC. METHODS: First, we determined the density of total bacteria and C. perfringens in the distal small intestinal mucosa of 58 NEC and healthy preterm pigs using quantitative PCR. Next, we analyzed in IPEC-J2 cells the effect of different infection densities of C. perfringens type A on the expression of genes related to intestinal function and immune response. RESULTS: Total bacterial and C. perfringens densities were higher in NEC versus healthy pigs and correlated positively with NEC severity. In IPEC-J2 cells expression levels of inflammation-related genes (CCL5, NFKBIA, IL8, IL1RN, and TNFAIP3) increased, while the expression of the sodium/glucose co-transporter (SLC5A1) decreased, with increasing density of C. perfringens. CONCLUSIONS: Total bacterial and C. perfringens densities were higher in NEC versus healthy pigs and correlated positively with NEC severity. In IPEC-J2 cells expression levels of inflammation-related genes (CCL5, NFKBIA, IL8, IL1RN, and TNFAIP3) increased, while the expression of the sodium/glucose co-transporter (SLC5A1) decreased, with increasing density of C. perfringens.

Impact of probiotics on colonic microflora in patients with colitis: a prospective double blind randomised crossover study.

BACKGROUND & AIMS: The aim of this study was to investigate the spectrum of colonic microflora in patients with colitis and if this could be altered with one month's treatment with synbiotics. METHODS: This was a pilot study in which patients were randomised to either receive a synbiotics preparation for a month and then "crossed over" to receive a placebo, or alternatively to receive the placebo first followed in the second month by synbiotic. Stool samples were collected on entry into the study and then at the end of first and second months respectively. Colonic microflora was measured by terminal restriction fragment length polymorphism technique. Quantitative PCR was used to determine the concentration of individual species. RESULTS: Sixteen patients completed the study of whom 8 had Crohn's colitis and 8 had ulcerative colitis. Their median age was 62 (IQR 50-65) years. An average of 22 terminal restriction fragments (T-RF's) was identified in each patient. Dice cluster analysis showed that each patient had a unique microbial composition which did not change significantly at different time points in the study, irrespective of whether they had probiotics or the placebo. Probiotic organisms were identified in stool samples but did not alter overall spectrum of microflora. In this pilot study we were unable to identify any specific characteristics related to nature of colitis. CONCLUSIONS: This study suggests that there is no difference in colonic microflora between patients with Crohn's or Ulcerative colitis and that the spectrum of bacteria was not altered by synbiotic administration.