Enea Rezzonico

Effect of Lactobacillus rhamnosus CGMCC1.3724 supplementation on weight loss and maintenance in obese men and women.

The present study investigated the impact of a Lactobacillus rhamnosus CGMCC1.3724 (LPR) supplementation on weight loss and maintenance in obese men and women over 24 weeks. In a double-blind, placebo-controlled, randomised trial, each subject consumed two capsules per d of either a placebo or a LPR formulation (1.6 × 10(8) colony-forming units of LPR/capsule with oligofructose and inulin). Each group was submitted to moderate energy restriction for the first 12 weeks followed by 12 weeks of weight maintenance. Body weight and composition were measured at baseline, at week 12 and at week 24. The intention-to-treat analysis showed that after the first 12 weeks and after 24 weeks, mean weight loss was not significantly different between the LPR and placebo groups when all the subjects were considered. However, a significant treatment × sex interaction was observed. The mean weight loss in women in the LPR group was significantly higher than that in women in the placebo group (P = 0.02) after the first 12 weeks, whereas it was similar in men in the two groups (P= 0.53). Women in the LPR group continued to lose body weight and fat mass during the weight-maintenance period, whereas opposite changes were observed in the placebo group. Changes in body weight and fat mass during the weight-maintenance period were similar in men in both the groups. LPR-induced weight loss in women was associated not only with significant reductions in fat mass and circulating leptin concentrations but also with the relative abundance of bacteria of the Lachnospiraceae family in faeces. The present study shows that the Lactobacillus rhamnosus CGMCC1.3724 formulation helps obese women to achieve sustainable weight loss.

Lactose-over-Glucose Preference in Bifidobacterium longum NCC2705: glcP, Encoding a Glucose Transporter, Is Subject to Lactose Repression

Analysis of culture supernatants obtained from Bifidobacterium longum NCC2705 grown on glucose and lactose revealed that glucose utilization is impaired until depletion of lactose. Thus, unlike many other bacteria, B. longum preferentially uses lactose rather than glucose as the primary carbon source. Glucose uptake experiments with B. longum cells showed that glucose transport was repressed in the presence of lactose. A comparative analysis of global gene expression profiling using DNA arrays led to the identification of only one gene repressed by lactose, the putative glucose transporter gene glcP. The functionality of GlcP as glucose transporter was demonstrated by heterologous complementation of a glucose transport-deficient Escherichia coli strain. Additionally, GlcP exhibited the highest substrate specificity for glucose. Primer extension and real-time PCR analyses confirmed that expression of glcP was mediated by lactose. Hence, our data demonstrate that the presence of lactose in culture medium leads to the repression of glucose transport and transcriptional down-regulation of the glucose transporter gene glcP. This may reflect the highly adapted life-style of B. longum in the gastrointestinal tract of mammals.

Sugar Transport Systems of Bifidobacterium longum NCC2705

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.

Development of a real-time RT-PCR method for enumeration of viable Bifidobacterium longum cells in different morphologies.

Viability of probiotic bacteria is traditionally assessed by plate counting which has several limitations, including underestimation of cells in aggregates or chains morphology. We describe a quantitative PCR (qPCR)-based method for an accurate enumeration of viable cells of Bifidobacterium longum NCC2705 exhibiting different morphologies by measuring the mRNA levels of cysB and purB, two constitutively expressed housekeeping genes. Three primer-sets targeting short fragments of 57-bp of cysS and purB and one 400-bp fragment of purB were used. Cell quantification of serially diluted samples showed a good correlation coefficient of R(2) 0.984 +/- 0.003 between plate counts and qRT-PCR for all tested primer sets. Loss of viable cells exposed to a lethal heat stress (56 degrees C, 10, 20 and 30 min) was estimated by qRT-PCR and plate counts. No significant difference was observed using qRT-PCR targeting the 400-bp fragment of purB compared to plate counts indicating that this fragment is a suitable marker of cell viability. In contrast, the use of the 57-bp fragments led to a significant overestimation of viable cell counts (18 +/- 3 and 7 +/- 2 fold for cysB and purB, respectively). Decay of the mRNA fragments was studied by treatment of growing cells with rifampicin prior qRT-PCR. The 400-bp fragment of purB was faster degraded than the 57-bp fragments of cysB and purB. The 400-bp fragment of purB was further used to enumerate viable cells in aggregate state. Cell counts were more than 2 log(10) higher using the qRT-PCR method compared to plate counts. Growing interest in probiotic characteristics of aggregating bacteria cells make this technique a valuable tool to accurately quantify viable probiotic bacteria exhibiting heterogeneous morphology.

A Mesocosm of Lactobacillus johnsonii, Bifidobacterium longum, and Escherichia coli in the Mouse Gut

The relative contribution of competition and cooperation at the microbe-microbe level is not well understood for the bacteria constituting the gut microbiota. The high number and variability of human gut commensals have hampered the analysis. To get some insight into the question how so many different bacterial species can coexist in the mammalian gut, we studied the interaction between three human gut commensals (Escherichia coli K-12, Lactobacillus johnsonii NCC533, and Bifidobacterium longum NCC2705) in the intestine of gnotobiotic mice. The bacterial titers and their anatomical distribution were studied in the colonized mice. L. johnsonii achieved the highest cell counts in the stomach, while B. longum dominated the colon. The colon was also the intestinal location in which B. longum displayed the highest number of expressed genes, followed by the cecum and the small intestine. Addition of further bacterial strains led to strikingly different results. A Lactobacillus paracasei strain coexisted, while a second B. longum strain was excluded from the system. Notably, this strain lacked an operon involved in the degradation, import, and metabolism of mannosylated glycans. Subsequent introduction of the E. coli Nissle strain resulted in the elimination of L. johnsonii NCC533 and E. coli K-12, while B. longum NCC2705 showed a transient decrease in population size, demonstrating the dynamic nature of microbe-microbe interactions. The study of such simple interacting bacterial systems might help to derive some basic rules governing microbial ecology within the mammalian gut.

Microbiota in Breast Milk of Chinese Lactating Mothers

The microbiota of breast milk from Chinese lactating mothers at different stages of lactation was examined in the framework of a Maternal Infant Nutrition Growth (MING) study investigating the dietary habits and breast milk composition in Chinese urban mothers. We used microbiota profiling based on the sequencing of fragments of 16S rRNA gene and specific qPCR for bifidobacteria, lactobacilli and total bacteria to study microbiota of the entire breast milk collected using standard protocol without aseptic cleansing (n = 60), and the microbiota of the milk collected aseptically (n = 30). We have also investigated the impact of the delivery mode and the stage of lactation on the microbiota composition. The microbiota of breast milk was dominated by streptococci and staphylococci for both collection protocols and, in the case of standard collection protocol, Acinetobacter sp. While the predominance of streptococci and staphylococci was consistently reported previously for other populations, the abundance of Acinetobacter sp. was reported only once before in a study where milk collection was done without aseptic cleansing of the breast and rejection of foremilk. Higher bacterial counts were found in the milk collected using standard protocol. Bifidobacteria and lactobacilli were present in few samples with low abundance. We observed no effect of the stage of lactation or the delivery mode on microbiota composition. Methodological and geographical differences likely explain the variability in microbiota composition reported to date.

Improved tolerance to bile salts of aggregated Bifidobacterium longum produced during continuous culture with immobilized cells.

The effect of cell immobilization and continuous culture was studied on selected physiological and technological characteristics of Bifidobacterium longum NCC2705 cultivated for 20 days in a two stage continuous fermentation system. Continuous immobilized cell (IC) cultures with and without glucose limitation exhibited formation of macroscopic cell aggregates after 12 and 9 days, respectively. Auto-aggregation resulted in underestimation of viable cell counts by plate counts by more than 2 log units CFU/ml compared with qPCR method. Modifications of cell membrane composition might partially explain aggregate formation in IC cultures. Decreases in the ratio of unsaturated to saturated fatty acid content from 1.74 to 0.58 might also contribute to the enhanced tolerance of IC cells to porcine bile salts and aminoglycosidic antibiotics compared with free cells from batch cultures. The enhanced resistance against bile salts in combination with auto-aggregation may confer an advantage to probiotic bacteria produced by IC technology.

In vitro activity of commercial probiotic Lactobacillus strains against uropathogenic Escherichia coli

Urinary tract infection (UTI) is one of the most prevalent infections in humans. In ≥80% of cases, the etiologic agents are strains of uropathogenic Escherichia coli (UPEC), which commonly reside in the gastrointestinal tract. Lactobacilli have been shown to prevent UTI reoccurrence by restoring the urogenital microbiota when administered vaginally or orally. The goal of this study was to determine if commercial probiotic Lactobacillus spp. reduce or clear UPEC in vitro. Results show that it is likely that lactobacilli may, in addition to restoring a healthy urogenital microbiota through acidification of their environment, also displace adhering UPEC and cause a reduction of infection.

New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

BackgroundOxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H2O2 from 0 to 130 ppm.ResultsAt the beginning of the culture, high cell density of 1013 CFU per litre of reactor was tested. The continuous culture gradually adapted to increasing H2O2 concentrations. However, after increasing the H2O2 concentration to 130 ppm the OD of the culture decreased to 0. Full wash out was prevented by the immobilization of the cells in gel matrix. Hence after stopping the stress, it was possible to re-grow the cells that survived the highest lethal dose of H2O2 and to select two adapted colonies (HPR1 and HPR2) after plating of the culture effluent. In contrast to HPR1, HPR2 showed stable characteristics over at least 70 generations and exhibited also higher tolerance to O2 than non adapted wild type cells. Preliminary characterization of HPR2 was carried out by global genome expression profile analysis. Two genes coding for a protein with unknown function and possessing trans-membrane domains and an ABC-type transporter protein were overexpressed in HPR2 cells compared to wild type cells.ConclusionsOur study showed that continuous culture with cell immobilization is a valid approach for selecting cells adapted to hydrogen peroxide. Elucidation of H2O2 adaptation mechanisms in HPR2 could be helpful to develop oxygen resistant bifidobacteria.

Fast screening of Bifidobacterium longum sublethal stress conditions in a novel two-stage continuous culture strategy

A central issue in the application of probiotics as food additives is their fastidious production and their sensitivity to many environmental stresses. The importance of inducible cell-protective mechanisms triggered by application of sublethal stresses for survival under stress conditions has been demonstrated. Continuous cultures could be a suitable and more efficient method to test stress factors on one culture instead of several repeated batch cultures. In this study, the application of a two-stage continuous culture of Bifidobacterium longum NCC2705 was investigated. The first reactor was operated under fixed conditions at 37 °C and pH 6.0 and used to produce cells with controlled physiology, mimicking cells in the late exponential growth phase. Stress pretreatment combinations of pH (6.0, 5.0 and 4.0), temperature (37, 45 and 47 °C) and NaCl (0, 5 and 10%) were tested in the second reactor. Of all tested combinations, only those of pH 4.0 significantly decreased cell viability in the second reactor compared to control conditions (37 °C, pH 6.0, 0% NaCl) and, therefore, could not be considered as sublethal stresses. Pretreatments with 5 or 10% NaCl had a negative effect on cell viability after gastric lethal stress. A significant improvement in cell resistance to heat lethal stress (56 °C, 5 min) was observed for cells pretreated at 47 °C. In contrast, heat pretreatment negatively affected cell viability after freeze drying and osmotic lethal stresses. The two-stage continuous culture allowed for efficient screening of several stress pretreatments during the same experiment with up to four different conditions tested per day. Optimal sublethal stress conditions can also be applied for producing cells with traditional batch cultures.