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 past decades have seen the staggering development of molecular microbial ecology as a discipline that uses the detection of so-called biomarkers to monitor microbial communities in environment samples. A variety of molecules can be used as biomarkers, including cell-wall components, proteins, lipids, DNA or RNA. Especially, the application of small subunit ribosomal RNA (rRNA) and the corresponding genes have proven invaluable for advances in microbial ecology. Several types of fingerprinting methods have been developed for the description of microbial communities in environmental samples. Among the most commonly used approaches is denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments. DGGE allows separation of DNA fragment mixtures of equal length depending on their sequence. The separation is based on their sequence-specific melting point in a polyacrylamide gel with a gradient of a denaturant chemical (generally a combination of urea and formamide). DGGE allows for a rapid analysis and comparison of microbial communities. Compositional diversity can be visualized using DGGE where each band in principle represents a bacterial phylotype. After staining bands are visualized at each position in the gel where DNA molecules stopped migration. In principle, DGGE fingerprinting can resolve single base pair differences.

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.

Fermentable carbohydrates can lead to changes in the gut microflora, which may have positive consequences for health. However, often, ingredients are added to diets without prior investigating about their potential fermentability within the target animal. The experiment reported, was conducted to investigate the fermentation kinetics of some non-digestible carbohydrates (NDC) by the caecal microbial community of broiler chickens by an assessment of the cumulative gas production during fermentation of each substrate. It also aimed to study changes in the microbial community, following fermentation of these non-digestible carbohydrates, by use of polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Four extracted non-digestible carbohydrates (ENC): soybean meal oligo- and water-soluble polysaccharides (SMO and SMP), alfalfa meal oligo- and water-soluble polysaccharides (AMO and AMP) were studied. Two pure sugars, raffinose (RAF) and stachyose (STA) were also included. To assess the fermentability of the substrates, cumulative gas pressure was monitored continuously, for 72 h, and at the end of fermentation, pH, volatile fatty acid (VFA), and ammonia (NH3) concentrations were measured in the fermentation solution. The PCR-DGGE technique was applied to compare microbial DNA fingerprints between substrates at the end of the fermentation. The inoculum for the in vitro gas production was obtained from a mixture of caecal contents of forty 81-day-old broiler birds. Soy oligosaccharides led to significantly more butyric acid production (P<0.05) after fermentation compared with other ENC. The production of butyric acid was 155.2 mg/g DM, 100.3 mg/g DM, 84.5 mg/g DM and 71.8 mg/g DM for SMO, SMP, AMP and AMO, respectively. Soy oligosaccharides had the lowest pH (5.5) and ammonia–nitrogen concentration (199.3 mg/l) after fermentation. This was significantly different from other ENC and the pure sugars. DGGE analysis revealed that, the fingerprint of caecal bacterial communities showed some variation (C, value >60–80%) between some of the substrates. It was concluded that SMO, SMP, AMO and AMP, as well as RAF and STA were significantly different, both in terms of their fermentation kinetics and end-products using caecal contents from adult broilers. The extent to which these non-digestible, but fermentable, carbohydrates could change the microbial community of the broiler caecum, either in terms of the species detected, or its activity, needs to be investigated further, and then related to its effect on gut health in the animal itself.

To gain insight into the microorganisms involved in direct and indirect methane formation from methanol in a laboratory-scale thermophilic (55°C) methanogenic bioreactor, reactor sludge was disrupted and serial dilutions were incubated in specific growth media containing methanol and possible intermediates of methanol degradation as substrates. With methanol, growth was observed up to a dilution of 108. However, when Methanothermobacter thermoautotrophicus strain Z245 was added for H2 removal, growth was observed up to a 1010-fold dilution. With H2/CO2 and acetate, growth was observed up to dilutions of 109 and 104, respectively. Dominant microorganisms in the different dilutions were identified by 16S rRNA-gene diversity and sequence analysis. Furthermore, dilution polymerase chain reaction (PCR) revealed a similar relative abundance of Archaea and Bacteria in all investigated samples, except in enrichment with acetate, which contained 100 times less archaeal DNA than bacterial DNA. The most abundant bacteria in the culture with methanol and strain Z245 were most closely related to Moorella glycerini. Thermodesulfovibrio relatives were found with high sequence similarity in the H2/CO2 enrichment, but also in the original laboratory-scale bioreactor sludge. Methanothermobacter thermoautotrophicus strains were the most abundant hydrogenotrophic archaea in the H2/CO2 enrichment. The dominant methanol-utilizing methanogen, which was present in the 108-dilution, was most closely related to Methanomethylovorans hollandica. Compared to direct methanogenesis, results of this study indicate that syntrophic, interspecies hydrogen transfer-dependent methanol conversion is equally important in the thermophilic bioreactor, confirming previous findings with labeled substrates and specific inhibitors.

Diversity of rumen bacteria of the rumen content of Chinese white goats was analyzed by PCR amplification, denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rDNA clone libraries. DNA was extracted from rumen contents of goats fed two diets with and without the addition of daidzein. The V6-V8 region of 16S rDNA of bacteria was amplified and the amplicons were then separated based on a linear gradient of denaturants in DGGE, a fingerprinting technique. A clone library was created from complete 16S rDNA. From the library, 16 clones had their V6-V8 regions matched predominant bands on the DGGE gel and their 16S rDNAs were then sequenced and subjected to an online similarity search. Five clones showed their similarities with database sequences over 97%, with one sequence similar to Prevotella sp., the rest were similar to those unidentified rumen bacteria. From the library, eight clones with similarities in the range of 90%～96% and the remaining three clones were less than 90%.

ABSTRACT 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-μm 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 cycloheximide 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.