Tolerance and safety of the potentially probiotic strain Lactobacillus rhamnosus PRSF-L477 : a randomised, double-blind placebo-controlled trial in healthy volunteers

In Europe, the species Lactobacillus rhamnosus is currently on the Qualified Presumption of Safety list used by the European Food Safety Authority (EFSA) for internal safety assessment, but according to the EFSA the species should remain a topic of surveillance. In the present study, the safety and tolerance of the potentially probiotic strain L. rhamnosus PRSF-L477 was investigated in a placebo-controlled double-blind volunteer trial following FAO/WHO guidelines. A total of thirty-four subjects received daily doses of 1 x 10(11) colony-forming units (cfu) of L. rhamnosus PRSF-L477 (n 17) or placebo (n 17) for a period of 3 weeks, followed by a wash-out period of another 3 weeks. A questionnaire on gastrointestinal tolerance and a diary was kept daily to record compliance throughout these 6 weeks. Faecal and blood samples were collected for microbiological and haematological analysis. The recorded gastrointestinal symptoms, defecation frequency and stool consistency were not influenced indicating that L. rhamnosus PRSF-L477 was well tolerated. The species L. rhamnosus was detected in the faeces of sixteen out of seventeen subjects of the probiotic group during the intervention period. Using pulsed-field gel electrophoresis, re-isolates of L. rhamnosus PRSF-L477 were confirmed in nine of these subjects. Antibiotic susceptibility profiles of these re-isolates were unchanged compared with PRSF-L477. No clinically relevant changes in blood parameters such as liver and kidney function and no serious adverse events appeared during and after administration. Therefore, we conclude that L. rhamnosus PRSF-L477 can safely be administrated to healthy subjects at a daily dose of 1 x 10(11) cfu

Unidirectional Reconstitution into Detergent-destabilized Liposomes of the Purified Lactose Transport System of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S. thermophilus, respectively. In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter. Also a LacS deletion mutant, lacking the carboxyl-terminal regulatory domain, could be amplified to levels >20% of membrane protein. Membranes from S. thermophilus proved to be superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively low concentrations of Triton X-100, n-octyl-β-D-glucoside, n-dodecyl-β-D-maltoside, or C12E8. The LacS protein carrying a poly-histidine tag was purified in large quantities (~5 mg/liter of culture) and with a purity >98% in a two-step process involving nickel chelate affinity and anion exchange chromatography. The membrane reconstitution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E. coli phospholipid and phosphatidylcholine, and protein incorporation at the different stages of liposome solubilization. The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured. Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, whereas activity was lost irreversibly with n-octyl-β-D-glucoside. For reconstitutions mediated by n-dodecyl-β-D-maltoside, C12E8, and to a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergent (onset of liposome solubilization). Importantly, under these conditions proteoliposomes were obtained in which LacS was reconstituted in an inside-out orientation, as suggested by the outside labeling of a single cysteine mutant with a membrane impermeable biotin-maleimide. The results are consistent with a mechanism of reconstitution in which the hydrophilic regions of LacS prevent a random insertion of the protein into the membrane. Consistent with the in vivo lactose/galactose exchange catalyzed by the LacS protein, the maximal rate of lactose counterflow was almost 2 orders of magnitude higher than that of H+-lactose symport.

A Continuous Battle for Host-Derived Glycans Between a Mucus Specialist and a Glycan Generalist in vitro and in vivo

The human gastrointestinal tract is colonized by a diverse microbial community, which plays a crucial role in human health. In the gut, a protective mucus layer that consists of glycan structures separates the bacteria from the host epithelial cells. These host-derived glycans are utilized by bacteria that have adapted to this specific compound in the gastrointestinal tract. Our study investigated the close interaction between two distinct gut microbiota members known to use mucus glycans, the generalist Bacteroides thetaiotaomicron and the specialist Akkermansia muciniphila in vitro and in vivo. The in vitro study, in which mucin was the only nutrient source, indicated that B. thetaiotaomicron significantly upregulated genes coding for Glycoside Hydrolases (GHs) and mucin degradation activity when cultured in the presence of A. muciniphila. Furthermore, B. thetaiotaomicron significantly upregulated the expression of a gene encoding for membrane attack complex/perforin (MACPF) domain in co-culture. The transcriptome analysis also indicated that A. muciniphila was less affected by the environmental changes and was able to sustain its abundance in the presence of B. thetaiotaomicron while increasing the expression of LPS core biosynthesis activity encoding genes (O-antigen ligase, Lipid A and Glycosyl transferases) as well as ABC transporters. Using germ-free mice colonized with B. thetaiotaomicron and/or A. muciniphila, we observed a more general glycan degrading profile in B. thetaiotaomicron while the expression profile of A. muciniphila was not significantly affected when colonizing together, indicating that two different nutritional niches were established in mice gut. Thus, our results indicate that a mucin degrading generalist adapts to its changing environment, depending on available carbohydrates while a mucin degrading specialist adapts by coping with competing microorganism through upregulation of defense related genes.Peer reviewe

Influenza vaccination of hospital healthcare staff from the perspective of the employer:a positive balance

OBJECTIVE: To assess the annual productivity loss among hospital healthcare workers attributable to influenza and to estimate the costs and economic benefits of a vaccination programme from the perspective of the the employer.DESIGN: Cost-benefit analysis.METHODS: The percentage of work loss due to influenza was determined using monthly age and gender specific figures for productivity loss among healthcare workers of the University Medical Center Groningen (UMCG), the Netherlands over the period January 2006-June 2008. Influenza periods were determined on the basis of national surveillance data. The average increase in productivity loss in these periods was estimated by comparison with the periods outside influenza seasons. The direct costs of productivity loss from the perspective of the employer were estimated using the friction cost method. In the sensitivity analyses various modelling parameters were varied, such as the vaccination coverage.RESULTS: In the UMCG, with approximately 9,400 employees, the estimated annual costs associated with productivity loss due to influenza before the introduction of the yearly influenza vaccination program were € 675,242 or on average, € 72 per employee. The economic benefits of the current vaccination program with a vaccination coverage of 24% with a vaccine effectiveness of 71% were estimated at € 89,858 or € 10 per employee. The nett economic benefits of a vaccination program with a target vaccination coverage of 70% with a vaccine effectiveness of 71% were estimated at € 244,325 or € 26 per employee.CONCLUSION: This modelling study performed from the perspective of the employer showed that an annual influenza vaccination programme for hospital personnel can save costs.</p