Coamplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities.

The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer sets targeting the V3, V3-V5, and V6-V8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting of human and fish intestinal samples. Both in silico and PCR-based analysis revealed cross-reactivity of the V3 and V3-V5 primers with the 18S rRNA gene of human and sturgeon. The consequences of this primer anomaly were illustrated by denaturing gradient gel electrophoresis (DGGE) profiling of microbial communities in human feces and mixed gut of Siberian sturgeon. DGGE profiling indicated that the cross-reactivity of 16S rRNA gene primers with nontarget eukaryotic DNA might lead to an overestimation of bacterial biodiversity. This study has confirmed previous sporadic indications in literature indicating that several commonly applied 16S rRNA gene primer sets lack specificity toward bacteria in the presence of eukaryotic DNA. The phenomenon of cross-reactivity is a potential source of systematic error in all biodiversity studies where no subsequent analysis of individual community amplicons by cloning and sequencing is performed.

Application of sequence-dependent electrophoresis fingerprinting in exploring biodiversity and population dynamics of human intestinal microbiota: what can be revealed?

Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated.

Effects of Lactobacillus casei Shirota, Bifidobacterium breve, and oligofructose-enriched inulin on colonic nitrogen-protein metabolism in healthy humans.

Pre- and/or probiotics can cause changes in the ecological balance of intestinal microbiota and hence influence microbial metabolic activities. In the present study, the influence of oligofructose-enriched inulin (OF-IN), Lactobacillus casei Shirota, and Bifidobacterium breve Yakult on the colonic fate of NH3 and p-cresol was investigated. A randomized, placebo-controlled, crossover study was performed in 20 healthy volunteers to evaluate the influence of short- and long-term administration of OF-IN, L. casei Shirota, B. breve Yakult, and the synbiotic L. casei Shirota + OF-IN. The lactose[15N,15N]ureide biomarker was used to study the colonic fate of NH3. Urine and fecal samples were analyzed for 15N content by combustion-isotope ratio mass spectrometery and for p-cresol content by gas chromatography-mass spectrometry. RT-PCR was applied to determine the levels of total bifidobacteria. Both short- and long-term administration of OF-IN resulted in significantly decreased urinary p-cresol and 15N content. The reduction of urinary 15N excretion after short-term OF-IN intake was accompanied by a significant increase in the 15N content of the fecal bacterial fraction. However, this effect was not observed after long-term OF-IN intake. In addition, RT-PCR results indicated a significant increase in total fecal bifidobacteria after long-term OF-IN intake. Long-term L. casei Shirota and B. breve Yakult intake showed a tendency to decrease urinary 15N excretion, whereas a significant decrease was noted in p-cresol excretion. In conclusion, dietary addition of OF-IN, L. casei Shirota, and B. breve Yakult results in a favorable effect on colonic NH3 and p-cresol metabolism, which, in the case of OF-IN, was accompanied by an increase in total fecal bifidobacteria.

Molecular monitoring of the fecal microbiota of healthy human subjects during administration of lactulose and Saccharomyces boulardii.

Diet is a major factor in maintaining a healthy human gastrointestinal tract, and this has triggered the development of functional foods containing a probiotic and/or prebiotic component intended to improve the host's health via modulation of the intestinal microbiota. In this study, a long-term placebo-controlled crossover feeding study in which each subject received several treatments was performed to monitor the effect of a prebiotic substrate (i.e., lactulose), a probiotic organism (i.e., Saccharomyces boulardii), and their synbiotic combination on the fecal microbiota of three groups of 10 healthy human subjects differing in prebiotic dose and/or intake of placebo versus synbiotic. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was used to detect possible changes in the overall bacterial composition using the universal V(3) primer and to detect possible changes at the subpopulation level using group-specific primers targeting the Bacteroides fragilis subgroup, the genus Bifidobacterium, the Clostridium lituseburense group (cluster XI), and the Clostridium coccoides-Eubacterium rectale group (cluster XIVa). Although these populations remained fairly stable based on DGGE profiling, one pronounced change was observed in the universal fingerprint profiles after lactulose ingestion. Band position analysis and band sequencing revealed that a band appearing or intensifying following lactulose administration could be assigned to the species Bifidobacterium adolescentis. Subsequent analysis with real-time PCR (RT-PCR) indicated a statistically significant increase (P < 0.05) in total bifidobacteria in one of the three subject groups after lactulose administration, whereas a similar but nonsignificant trend was observed in the other two groups. Combined RT-PCR results from two subject groups indicated a borderline significant increase (P = 0.074) of B. adolescentis following lactulose intake. The probiotic yeast S. boulardii did not display any detectable universal changes in the DGGE profiles, nor did it influence the bifidobacterial levels. This study highlighted the capacity of an integrated approach consisting of DGGE analysis and RT-PCR to monitor and quantify pronounced changes in the fecal microbiota of healthy subjects upon functional food administration.

Strict blood glucose control with insulin during intensive care after cardiac surgery: impact on 4-years survival, dependency on medical care, and quality-of-life.

AIMS: To document the impact of intensive insulin therapy during intensive care on long-term (4 years) outcome of high-risk cardiac surgery patients. METHODS AND RESULTS: In this pre-planned sub-analysis and follow-up study of a large, randomized controlled trial on the effects of intensive insulin therapy during critical illness, we assessed long-term outcome in the 970 patients who had been admitted after high-risk cardiac surgery (mean+/-SD EuroSCORE of 6.0+/-3.7; EuroSCORE-predicted hospital mortality of 9.9%; observed hospital mortality of 7.5% in the conventional insulin group and 3.4% in the intensive insulin group). Long-term outcome was quantified as: (a) 4 years survival; (b) incidence of hospital re-admission; (c) level of activity and medical care requirements at 4 years as assessed by the Karnofsky score; and (d) perceived health-related quality-of-life at 4 years as assessed by the Nottingham Health Profile. Four years after ICU admission, the number of post-hospital discharge deaths was similar in the two study groups, reflecting maintenance of the acute survival benefit with intensive insulin therapy. Survivors who had been treated with intensive insulin during ICU stay revealed a similar risk for hospital re-admission and a comparable level of dependency on medical care. There was no effect on quality-of-life in the total group, whereas the increased survival of sicker patients with at least 3 days of insulin therapy evoked a more compromised perceived quality-of-life, in particular regarding social and family life. CONCLUSION: The short-term survival benefit obtained with insulin-titrated glycaemic control during intensive care after cardiac surgery was maintained after 4 years, without inducing increased medical care requirements but possibly at the expense of compromised perceived quality of social and family life.

Molecular monitoring and characterization of the faecal microbiota of healthy dogs during fructan supplementation.

The large intestine of dogs contains a complex microbial ecosystem with predominance of streptococci, bifidobacteria, lactobacilli, Bacteroides and Clostridium. Generally, this predominant microbiota in dogs is relatively stable in time but much less is known about its taxonomic composition. Moreover, almost no studies have been conducted to investigate this stability of the faecal microbial population in dogs upon prebiotic administration. The objective of the present study was to monitor possible changes in faecal microbiota of seven healthy adult dogs related to the administration of two fructans, oligofructose and inulin. For this purpose, population fingerprints generated by denaturing gradient gel electrophoresis (DGGE) analysis of universal V3 16 S rRNA gene PCR amplicons were compared between control (baseline) samples and samples collected after prebiotic feeding. From these DGGE gels, marked changes were observed in the faecal microbiota between subjects and before and after fructan administration. One DGGE band that appeared or intensified after fructan intake was further analyzed. Sequence analysis could attribute this band to a member of the Streptococcus bovis-equinus group. Following cultivation on MRS medium, a set of faecal isolates that most likely represent the stimulated streptococci were allocated to the species Streptococcus lutetiensis by (GTG)(5)-PCR fingerprinting and partial 16 S rRNA and sodA gene sequencing. The data provided in this study demonstrate the ability of fructans to influence the bacterial composition of the gut microbiota in healthy dogs. More work is needed to unravel the relevance of S. lutetiensis or other autochthonous organisms of the dog gut as target groups for prebiotic supplementation.

Anoxybacillus contaminans sp. nov. and Bacillus gelatini sp. nov., isolated from contaminated gelatin batches.

Aerobic, endospore-forming bacteria that are attributed to the genus Bacillus or related genera constitute a hazard to the quality of gelatin. During repetitive extragenic palindromic DNA (rep)-PCR screening of gelatin isolates, a group of five isolates (group 1) and a group of 66 isolates (group 2) that did not match any pattern in our database were found. On the basis of 16S rDNA sequence analysis, representative strains of the different rep-PCR fingerprint types of group 1 were shown to be related most closely to Anoxybacillus species, but with sequence similarity of <97 %. Likewise, representative strains of group 2 were shown to be related most closely to Bacillus species, with 16S rDNA sequence similarity of <97 %. DNA-DNA reassociation values of isolates that displayed the most divergent rep-PCR profiles revealed that strains within each group belonged to a single species, according to recommendations for species delineation. A mean fatty acid profile could be calculated for each group. Isolates within a single group had similar patterns of results in API and other phenotypic tests; no correlation of patterns of results with rep-PCR groups was seen. Physiological characterization of group 1 isolates allows their distinction from other Anoxybacillus species. Despite the weak reaction of group 2 isolates in API tests, physiological characterization allows distinction between Bacillus species that react weakly in API tests. Two novel species are therefore proposed, with the names Anoxybacillus contaminans sp. nov. (type strain, LMG 21881(T)=DSM 15866(T)) and Bacillus gelatini sp. nov. (type strain, LMG 21880(T)=DSM 15865(T)).

Temporal stability analysis of the microbiota in human feces by denaturing gradient gel electrophoresis using universal and group-specific 16S rRNA gene primers.

According to the current insights, the predominant bacterial community in human feces is considered to be stable and unique for each individual over a prolonged period of time. In this study, the temporal stability of both the predominant population and a number of specific subpopulations of the fecal microbiota of four healthy volunteers was monitored for 6-12 weeks. For this purpose, a combination of different universal (V(3) and V(6)-V(8)) and genus- or group-specific (targeting the Bacteroides fragilis subgroup, the genera Bifidobacterium and Enterococcus and the Lactobacillus group, which also comprises the genera Leuconostoc, Pediococcus and Weisella) 16S rRNA gene primers was used. Denaturing gradient gel electrophoresis (DGGE) was used to analyze the 16S rRNA gene amplicons generating population fingerprints which were compared visually and by numerical analysis. DGGE profiles generated by universal primers were relatively stable over a three-month period and these profiles were grouped by numerical analysis in subject-specific clusters. In contrast, the genus- and group-specific primers yielded profiles with varying degrees of temporal stability. The Bacteroides fragilis subgroup and Bifidobacterium populations remained relatively stable which was also reflected by subject-specific profile clustering. The Lactobacillus group showed considerable variation even within a two-week period and resulted in complete loss of subject-grouping. The Enterococcus population was detectable by DGGE analysis in only half of the samples. In conclusion, numerical analysis of 16S rRNA gene-DGGE profiles clearly indicates that the predominant fecal microbiota is host-specific and relatively stable over a prolonged time period. However, some subpopulations tended to show temporal variations (e.g., the Lactobacillus group) whereas other autochthonous groups (e.g., the bifidobacteria and the Bacteroides fragilis subgroup) did not undergo major population shifts in time.