BACKGROUND:Dietary intervention with prebiotics can cause changes in the colonic microbiota and their metabolic activities.AIM:To investigate whether the response to prebiotic dosing is influenced by the baseline metabolic activity of the colonic flora and bifidobacteria counts.METHODS:The 4-week effect of lactulose (10 g bid.; n = 29) and oligofructose-enriched inulin (10 g bid.; n = 19) was evaluated in healthy human volunteers. Lactose-[(15)N, (15)N]-ureide was used to study the colonic NH(3)-metabolism. Urine (48 h) and faeces (72 h) were collected and analysed for p-cresol and (15)N-content by gas chromatography-mass spectrometry and isotope ratio mass spectrometer, respectively. Faecal bifidobacteria were quantified by real-time polymerase chain reaction.RESULTS:After the 4-week prebiotic administration period, the urinary excretion of p-cresol and (15)N was significantly decreased in both groups (P < 0.05) corresponding to a significantly higher faecal excretion of (15)N (P < 0.05). The decrease in urinary (15)N and p-cresol excretion significantly correlated with baseline (15)N and p-cresol levels (P < 0.05), indicating that subjects with higher baseline levels showed a higher response to prebiotic dosing. Furthermore, a significant correlation was seen between baseline bifidobacteria counts and the effect of prebiotic intake (P < 0.05).CONCLUSION:The response to prebiotic dosing, as indicated by the fate of NH(3), p-cresol and bifidobacteria, is determined by the initial colonic conditions.

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.

BACKGROUND:Protein fermentation products, especially ammonia, are implicated in the pathogenesis of certain diseases.AIM:To investigate the influence of lactulose and Saccharomyces boulardii cells on the composition of the intestinal microbiota and on the metabolic fate of ammonia by means of lactose-[(15)N, (15)N]-ureide.METHODS:An at random, placebo-controlled, crossover study was performed in 43 healthy volunteers to evaluate the influence of lactulose and/or S. boulardii cells either administered as a single dose or after a 4-week intake period. Urine and faeces were collected. All samples were analysed for (15)N-content by combustion-isotope ratio mass spectrometry. Real-time polymerase chain reaction was applied to determine the composition of the predominant faecal microbiota.RESULTS:A single administration of lactulose significantly decreased urinary (15)N-excretion in a dose-dependent way. After long-term administration of lactulose, a significant reduction of the urinary (15)N-excretion was observed, which was accompanied with a significant increase in the faecal (15)N-output, more specifically more (15)N was found in the bacterial fraction. A significant rise in the Bifidobacterium population was found after lactulose intake. No significant effects were observed after S. boulardii intake.CONCLUSION:Dietary addition of lactulose can exert a bifidogenic effect accompanied by a favourable effect on the colonic NH(3)-metabolism.

The application of real-time PCR targeting the multicopy 16S rRNA gene and the single copy recA gene was evaluated for the enumeration of bifidobacteria in 29 probiotic products claimed to contain these organisms. Both assays relied on the use of genus-specific primers and the non-specific SYBR Green I chemistry. For both applications, the calibration curve was constructed using the type strain of Bifidobacterium animalis subsp. lactis. Upon correction with a factor corresponding to the 16S rRNA gene copy number, both assays generally produced comparable enumeration results. Only in exceptional cases, differences between both gene targets were found in probiotic products containing low amounts of bifidobacteria in which case the quantification of the multicopy 16S rRNA gene turned out to be more sensitive than the recA-based assay. On the other hand, the use of the latter single copy gene in real-time PCR quantification offers the advantage that no prior knowledge of bacterial content is required when using genus-specific primers, since no correction for multiple gene copies has to be performed. Only 11 of the analysed products (38%), including one dairy based product and ten dried products, contained a minimal Bifidobacterium concentration of 10(6) CFU per ml or g of product. Depending on the application, both assays proved to be rapid and reproducible alternatives for culture-based detection and quantification of bifidobacteria in probiotic products.

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.

Amongst the various claimed beneficial effects of pro- and prebiotics for the human host, it has been hypothesised that functional foods are able to suppress the generation and accumulation of toxic fermentation metabolites (NH3, p-cresol). Direct evidence supporting this hypothesis is lacking mainly because of the unavailability of reliable biomarkers. Preliminary data indicate that lactose-[15N]ureide and [2H4]tyrosine may be potential biomarker candidates. The aim of the present study was to evaluate the effect of pro- and prebiotics on the colonic fate of these biomarkers in a randomised, placebo-controlled, cross-over study with nineteen healthy volunteers. At the start of the study and at the end of each 2-week study period, during which they were administered either a probiotic (n 10; 6·5×109Lactobacillus casei Shirota cells twice daily) or a prebiotic (n 9; lactulose 10 g twice daily), the volunteers consumed a test meal containing the two biomarkers. Urine was collected during 48 h. Results were expressed as percentage of the administered dose. As compared with the placebo, the decrease in the percentage dose of p-[2H4]cresol in the 24–48 h urine fraction was significantly higher after probiotic intake (P=0·042). Similar changes were observed for the 15N tracer (P=0·016). After prebiotic intake, a significantly higher decrease in the percentage dose of p-[2H4]cresol (P=0·005) and 15N tracer (P=0·029) was found in the 0–24 h urine collection. The present results demonstrate that suppression of the generation and accumulation of potentially toxic fermentation metabolites by pro- and prebiotics can reliably be monitored in vivo by the use of stable isotope-labelled biomarkers.

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)).

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 (V3 and V6–V8) 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.

ABSTRACT Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25: 611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus , Bacillus coagulans , Bacillus fumarioli , Bacillus amyloliquefaciens , Bacillus licheniformis , Bacillus pumilus , Bacillus sonorensis , Bacillus subtilis , Bacillus gelatini , Bacillus thermoamylovorans , Anoxybacillus contaminans , Anoxybacillus flavithermus , Brevibacillus agri , Brevibacillus borstelensis , and Geobacillus stearothermophilus . The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium -specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum , all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.