The host genotype affects the bacterial community in the human gastrointestinal tract

The gastrointestinal (GI) tract is one of the most complex ecosystems consisting of microbial and host cells. It is suggested that the host genotype, the physiology of the host and environmental factors affect the composition and function of the bacterial community in the intestine. However, the relative impact of these factors is unknown. In this study, we used a culture-independent approach to analyze the bacterial composition in the GI tract. Denaturing gradient gel electrophoresis (DGGE) profiles of fecal bacterial 16S rDNA amplicons from adult humans with varying degrees of genetic relatedness were compared by determining the similarity indices of the profiles compared. The similarity between fecal DGGE profiles of monozygotic twins were significantly higher than those for unrelated individuals (ts = 2.73, p1-tail = 0.0063, df=21). In addition, a positive relationship (F1, 30 = 8.63, p = 0.0063) between the similarity indices and the genetic relatedness of the hosts was observed. In contrast, fecal DGGE profiles of marital partners, which are living in the same environment and which have comparable feeding habits, showed low similarity which was not significantly different from that of unrelated individuals (ts = 1.03, p1-tail = 0.1561, df=27). Our data indicate that factors related to the host genotype have an important effect on determining the bacterial composition in the GI tract

Victivallis vadensis gen. nov., sp. nov., a sugar-fermenting anaerobe from human faeces

A novel strictly anaerobic, cellobiose-degrading bacterium, strain Cello, was isolated from a human faecal sample by combining enrichments in liquid and soft-agar basal media. A noteworthy characteristic was its inability to grow on normal agar plates and in roll tubes. The cells were coccus shaped and non-motile, with an extracellular slime layer. Growth of strain Cello T occurred between 20 and 40 degreesC, with optimal growth at 37 degreesC. The pH range for growth was 5-7-5 with an optimum at 6-5. In pure culture, strain Cello T could only grow on a variety of sugars. Glucose was converted to acetate, ethanol and H-2. The doubling time on glucose was 0.5 h. In a syntrophic co-culture with Methanospirillum hungatei strain JF-1(T), strain Cello(T) converted glucose to acetate and H-2. The G+C content was 59.2 mol%. 16S rDNA analysis revealed that the closest relatives of strain Cello(T) were two uncultured bacteria from anaerobic digesters, both with 94% 16S rDNA sequence similarity. The closest cultured representatives belong to genera of the bacterial division 'Verrucomicrobia'. The name Victivallis vadensis gen. nov., sp. nov. is proposed for strain Cello(T) (=DSM 14823(T) =ATCC BAA-548(T))

On-farm impact of cattle slurry manure management on biological soil quality

The effects of dairy cattle slurry management on soil biota, soil respiration and nitrogen (N) mineralization were evaluated in a farm trial across 12 farms and a field experiment on 2 farms located in a dairy farming area in the north of the Netherlands. The slurry management consisted of slit injection or surface application of slurry; the use or no use of additives [Euromestmix® (MX) and Effective Microbes® (EM)] and the type and level of inorganic N fertilization. Slit injection negatively affected epigeic earthworms whereas its effect on anecic and endogeic earthworms was absent or even positive. Enchytraeids were not affected in a consistent way, whereas numbers of nematodes indicative of nutrient- enriched conditions increased. Inorganic N fertilizer had similar effects. Bacterial diversity was not different among the treatments. Nitrifier diversity, however, was high at one of the farms in the field experiment, and was negatively affected by inorganic N fertilizer. The use of MX was usually associated with higher numbers of earthworms. EM affected numbers of earthworms and numbers of bacterial and plant-feeding nematodes, but only in specific combinations of field history, slurry type and slurry application method. We found no effects of EM on the composition of the microbial community. Soil respiration was increased when slurry was surface-applied. The calculated N mineralization by earthworms was in the order of 70–200 kg N ha −¹ year −¹. It was highest under farm-characteristic surface application of slurry with MX and lowest under farm-characteristic slit injection of slurry without additives. Compared with the N mineralization by earthworms, that by enchytraeids and nematodes was quantitatively insignificant. Negative treatment effects on earthworms led to corresponding reductions in calculated N mineralization

On-farm impact of cattle slurry manure management on biological soil quality

The effects of dairy cattle slurry management on soil biota, soil respiration and nitrogen (N) mineralization were evaluated in a farm trial across 12 farms and a field experiment on 2 farms located in a dairy farming area in the north of the Netherlands. The slurry management consisted of slit injection or surface application of slurry; the use or no use of additives [Euromestmix® (MX) and Effective Microbes® (EM)] and the type and level of inorganic N fertilization. Slit injection negatively affected epigeic earthworms whereas its effect on anecic and endogeic earthworms was absent or even positive. Enchytraeids were not affected in a consistent way, whereas numbers of nematodes indicative of nutrient- enriched conditions increased. Inorganic N fertilizer had similar effects. Bacterial diversity was not different among the treatments. Nitrifier diversity, however, was high at one of the farms in the field experiment, and was negatively affected by inorganic N fertilizer. The use of MX was usually associated with higher numbers of earthworms. EM affected numbers of earthworms and numbers of bacterial and plant-feeding nematodes, but only in specific combinations of field history, slurry type and slurry application method. We found no effects of EM on the composition of the microbial community. Soil respiration was increased when slurry was surface-applied. The calculated N mineralization by earthworms was in the order of 70–200 kg N ha -¹ year -¹. It was highest under farm-characteristic surface application of slurry with MX and lowest under farm-characteristic slit injection of slurry without additives. Compared with the N mineralization by earthworms, that by enchytraeids and nematodes was quantitatively insignificant. Negative treatment effects on earthworms led to corresponding reductions in calculated N mineralization

Investigations on ileal microbial flora in weaning piglets

To characterize ileal microbial flora in weaning piglets a slaughter trial was conducted. 224 German Landrace piglets of both genders were allocated to four different feeding regimes (with or without avilamycin, 3 resp. 8 % crude fibre content). At predefined times pre- and postweaning piglets were sacrificed, the whole intestinal tract was removed and its content collected separately for each section. The microbial community was examined applying classical plate counting (selective agar plates) and molecular techniques (DGGE, 16SrDNA-sequencing). Furthermore a range of microbial metabolites was determined. Changes in ileal microflora ¿ rather due to age than to diet - were observed pre- and postweaning

Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride

The need for protozoa for the proliferation of Legionella pneumophila in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of L. pneumophila in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous L. pneumophila originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-mum pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable L. pneumophila, and for their concentrations of protozoa. L. pneumophila grew to high numbers (6.3 log CFU/cm(2)) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of Hartmannella vermiformis in all flasks in which L. pneumophila multiplied and also when cyclobeximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of L. pneumophila in trophozoites of H. vermiformis, with 25.9% +/- 10.5% of the trophozoites containing L. pneumophila on day 10 and >90% containing L. pneumophila on day 14. Calculations confirmed that intracellular growth was most likely the only way for L. pneumophila to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher L. pneumophila concentrations, and therefore the growth of L. pneumophila within engineered water systems can be limited by controlling biofilm formation

Response of a soil bacterial community to grassland succession as monitored by 16S rRNA levels of the predominant ribotypes

The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the subclass of the class Proteobacteria. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes

ß-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria.

A series of transposons are described which contain the gusA gene, encoding β-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out

Development and application of a selective pcr-denaturing gradient gel electrophoresis approach to detect a recently cultivated Bacillus group predominant in soil

The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance and cultivability of at least some of these microorganisms makes them an appropriate subject for studies on their biogeographical dissemination and diversity. Since cultivability can vary significantly with the physiological state and even between closely related strains, we developed a culture-independent 16S rRNA gene-targeted DGGE fingerprinting protocol for the detection of these bacteria from soil samples. The composition of the B. benzoevorans relatives in the soil samples from The Netherlands, Bulgaria, Russia, Pakistan, and Portugal showed remarkable differences between the different countries. Differences in the DGGE profiles of these communities in archived soil samples from the Dutch Wieringermeer polder were observed over time during which a shift from anaerobic to aerobic and from saline to freshwater conditions occurred. To complement the molecular methods, we additionally cultivated B. benzoevorans-related strains from all of the soil samples. The highest number of B. benzoevorans relatives was found in the soils from the northern part of The Netherlands. The present study contributes to our knowledge of the diversity and abundance of this interesting group of microbes in soils throughout the world

Postnatal development of intestinal immune system in piglets: implications for the process of weaning

European-wide directives are in place to establish a sustainable production of pigs without using production enhancers and chemotherapeutics. Thus, an economically-viable pig production is now only possible when the physiological mechanisms of defense against pathogens and tolerance against nutrients and commensal bacteria in the intestinal immune system are taken into account. During the postnatal period the piglet is facing first the time large amounts of new antigens and at weaning a second wave of nutritional antigens is entering the intestinal tract. The appropriate development of humoral and cellular functions of the intestinal immune system is essential for optimum growth and performance of the piglets. The integrity of the intestinal surfaces is a prerequisite of intestinal immunity and tolerance. Secretory IgA serves to exclude harmful antigens from uptake. The induction of intestinal immune reactions starts with antigen presentation by professional antigen presenting cells of Peyer's patches and mesenteric lymph nodes. In addition, the intestinal lamina propria serves as a mucosal compartment for regulation of immune responses. Here especially T regulatory cells (CD4(+) CD25(+)) have their function for maintaining intestinal homeostasis. The network of mucosal T and B cells develops after birth in a programmed sequence; it is almost completed at week 7 after birth. Weaning is associated with changes in the regulation of the lymphoid cells in the mucosa. In small and large intestine increases in pro- and anti-inflammatory cytokines were observed after weaning in lymphocytes. Epithelial cells were studied both in intestinal samples and in vitro. Here the cytokine patterns provide evidence that weaning is inducing a transient inflammation of the mucosa. Piglets weaned under conventional conditions have a thicker mucosa than pigs weaned from isolators. Cells of isolator-reared pigs show slightly higher levels of activation markers - probably reflecting the interaction of the foreign protein derived from bovine milk. The results presented in this overview demonstrate that further effort is necessary to elucidate the function of the porcine intestinal immune system in the postnatal period and at the time of weaning to provide criteria for porcine intestinal health