Stefania Silvi

Differences in Fecal Microbiota in Different European Study Populations in Relation to Age, Gender, and Country: a Cross-Sectional Study

ABSTRACT A cross-sectional study on intestinal microbiota composition was performed on 230 healthy subjects at four European locations in France, Germany, Italy, and Sweden. The study participants were assigned to two age groups: 20 to 50 years (mean age, 35 years; n = 85) and >60 years (mean age, 75 years; n = 145). A set of 14 group- and species-specific 16S rRNA-targeted oligonucleotide probes was applied to the analysis of fecal samples by fluorescence in situ hybridization coupled with flow cytometry. Marked country-age interactions were observed for the German and Italian study groups. These interactions were inverse for the predominant bacterial groups Eubacterium rectale-Clostridium coccoides and Bacteroides-Prevotella. Differences between European populations were observed for the Bifidobacterium group only. Proportions of bifidobacteria were two- to threefold higher in the Italian study population than in any other study group, and this effect was independent of age. Higher proportions of enterobacteria were found in all elderly volunteers independent of the location. Gender effects were observed for the Bacteroides-Prevotella group, with higher levels in males than in females. In summary, age-related differences in the microbiota makeup were detected but differed between the study populations from the four countries, each showing a characteristic colonization pattern.

Resistant starch modifies gut microflora and microbial metabolism in human flora-associated rats inoculated with faeces from italian and UK donors

The effect of sucrose and resistant starch (‘CrystaLean’– a retrograded, amylose starch) on human gut microflora and associated parameters was studied in human flora‐associated (HFA) rats, colonized with microfloras from UK or Italian subjects, to determine whether such floras were affected differently by dietary carbohydrates. Consumption of the resistant starch diet resulted in significant changes in four of the seven main groups of bacteria enumerated. In both the UK and Italian flora‐associated rats, numbers of lactobacilli and bifidobacteria were increased 10–100‐fold, and there was a concomitant decrease in enterobacteria when compared with sucrose‐fed rats. The induced changes in caecal microflora of both HFA rat groups were reflected in changes in bacterial enzyme activities and caecal ammonia concentration. Although it had little effect on caecal short‐chain fatty acid concentration, CrystaLean markedly increased the proportion of n‐butyric acid in both rat groups and was associated with a significant increase in cell proliferation in the proximal colon of the Italian flora‐associated rats. CrystaLean appeared to play a protective role in the colon environment, lowering caecal ammonia concentration, caecal pH and β‐glucuronidase activity.

Administration of probiotic strain to improve sea bream wellness during development

Two bacterial strains Lactobacillus fructivorans (AS17B), isolated from adult sea bream (Sparus aurata) gut, and Lactobacillus plantarum (906), isolated from human faeces, were administered contemporaneously, during sea bream development using Brachionus plicatilis and/or Artemia salina and dry feed as vectors. The probiotic treatment influenced gut colonization: at 35 days post-hatching (p.h.) L. fructivorans was not present in the gut, but the treatment induced colonization by L. plantarum. At 66 days p.h., L. fructivorans was evident also in the control; moreover, when suitable environmental conditions appeared in the post-metamorphosis gastro-intestinal tract, competition between L. plantarum and L. fructivorans occurred. At 90 days p.h., L. plantarum was displaced by L. fructivorans that became significantly higher with respect to the control. In treated groups, probiotic administration significantly decreased larvae and fry mortality.

EU project Crownalife: functional foods, gut microflora and healthy ageing isolation and identification of Lactobacillus and Bifidobacterium strains from faecal samples of elderly subjects for a possible probiotic use in functional foods

A new EU-funded project called “Crownalife” has been set up both to assess structural and functional alterations of the intestinal microflora with ageing in Europe, and to validate functional foods that promote health by improving the function of the intestinal microflora in the elderly. Species of bifidobacteria and lactobacilli were isolated from faecal samples of healthy elderly Italian subjects and identified for a possible probiotic use in functional foods. Total anaerobic and aerobic, Lactobacillus and Bifidobacterium counts were performed on the faecal samples. Colonies grown on selective media were identified using enzymatic tests. Lactobacillus fermentum and Bifidobacterium longum were the most represented species and could be considered good candidates to be further characterized and then utilized to design appropriate dietary strategies. Some of the identification results related to Bifidobacterium species were validated by using PCR procedure based on 16S rRNA gene sequences species-specific primers showing a good concordance rate with the enzymatic method.

Effect of dietary probiotics on clownfish: a molecular approach to define how lactic acid bacteria modulate development in a marine fish

We set out to determine whether probiotic addition would improve larval development in the false percula clownfish Amphiprion ocellaris and to determine what molecular responses could be observed in the larvae following probiotic exposure. We supplied the probiotic bacterial strain Lactobacillus rhamnosus IMC 501 to clownfish larvae from the first day posthatch simultaneously by live prey and with addition to rearing water (group 2) and exclusively by live prey (group 3). We observed twofold higher body weight in both clownfish larvae and juveniles when probiotics were supplied via live prey and added to the rearing water. In addition, development was accelerated with metamorphosis occurring 3 days earlier in fingerlings treated with probiotic. Alteration in molecular biomarkers supported the faster growth observation. There was significantly increased gene expression of factors involved in growth and development (insulin-like growth factors I and II, myostatin, peroxisome proliferator-activated receptors alpha and beta, vitamin D receptor alpha, and retinoic acid receptor gamma) when probiotics were delivered via live prey and added to the rearing water. Moreover, probiotic treatment lessened the severity of the general stress response as exhibited by lower levels of glucocorticoid receptor and 70-kDa heat shock protein gene expression. Furthermore, an improvement of skeletal head development was observed, with a 10-20% reduction in deformities for juveniles treated with probiotic. All data suggest a potent effect on development resulting from the administration of lactic acid bacteria to larval clownfish, and this study provides a preliminary molecular entry path into the investigation of mechanisms responsible for probiotic enhancement in fish development.

Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

Endogenous microbiota play essential roles in the host’s immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host’s development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

Pilot-scale production and viability analysis of freeze-dried probiotic bacteria using different protective agents.

The functional food industry requires an improvement of probiotic strain stability during storage, especially when they are stored at room temperature. In this study, the viability of freeze-dried Lactobacillus rhamnosus IMC 501(®) and Lactobacillus paracasei IMC 502(®) using different protective agents (i.e., glycerine, mannitol, sorbitol, inulin, dextrin, Crystalean(®)) was determined and compared with semi skimmed milk (SSM) control. No significant differences were observed between the tested protectants and the control (SSM) during storage at refrigerated conditions. During storage at room temperature, only glycerine was found to stabilize viability better than other tested substances.

The effect of sucrose or starch‐based diet on short‐chain fatty acids and faecal microflora in rats

An investigation was carried out to determine whether variations of dietary carbohydrates could modify the colonic flora in rats. Sprague‐Dawley rats were fed with two equicaloric diets based on the AIN‐76 diet ( American Institute of Nutrition 1977 ) but differing from that diet in content of carbohydrates, i.e. high sucrose (64%) or high corn starch (64%). Feeding was continued for 9 months ad libitum and no variation in weight gain was recorded among the different diets. A prevalence of aerobes, and a significant reduction in the ratio anaerobes/aerobes in the faeces of rats on the high starch diet compared with the high sucrose diet, was observed. The anaerobe genera identified included Actinomyces, Bacteroides, Bifidobacterium, Clostridium, Eubacterium, Lactobacillus and Propionibacterium. Bacteroides was the most prevalent genus in both dietary groups (51·2 and 29·5% in the faeces of rats fed the sucrose and starch diets, respectively). In contrast, clostridia were prevalent in the starch‐fed group (23·8%) and less so in the sucrose diet (11·5%), as propionibacteria were prevalent in faeces of rats fed the starch diet (15·5%), and low in the sucrose diet (3·9%). The remaining genera were scarce in faeces from rats on either diet. Total short‐chain fatty acids (SCFA) were significantly higher in the faeces of animals fed the starch diet compared with those fed the sucrose diet. The relative concentrations of acetic, propionic and butyric acids were not significantly different between the two dietary groups. In conclusion, high starch diet can markedly modify the composition of faecal flora and alter considerably the faecal concentration of SCFAs, compounds which might have a health‐promoting effect.

In vitro evaluation of antimicrobial activity of Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and SYNBIO® against pathogens

Probiotic lactobacilli have a great potential to produce antimicrobial compounds that inhibit and control the microbial pathogen growth. The antimicrobial and antifungal activities of two probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502®, and their 1 : 1 combination, named SYNBIO®, were studied using four different methods.

Evaluation of antipathogenic activity and adherence properties of human Lactobacillus strains for vaginal formulations

To test different Lactobacillus strains for their antipathogenic activity towards Candida strains and their adhesion properties for the preparation of vaginal ovules and douches to be used in vaginal candidiasis prevention.