The spore-associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile.

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild-type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID50 ) of spores was significantly higher (2-logs) in the bclA1(-) mutant compared to the isogenic wild-type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild-type spores. bclA1(-) spores germinated faster than wild-type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre-spore germination) stages of infection. We also show that the ID50 was higher in mice infected with R20291, a 'hypervirulent' 027 strain, that carries a truncated BclA1 protein.

Increased toxin expression in a Clostridium difficile mfd mutant.

BACKGROUND: The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY. RESULTS: During attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations. CONCLUSIONS: This study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.

Analysis of Gut Characteristics and Microbiota Changes with Maternal Supplementation in a Neural Tube Defect Mouse Model.

Adequate nutrient supply is crucial for the proper development of the embryo. Although nutrient supply is determined by maternal diet, the gut microbiota also influences nutrient availability. While currently there is no cure for neural tube defects (NTDs), their prevention is largely amenable to maternal folic acid and inositol supplementation. The gut microbiota also contributes to the production of these nutrients, which are absorbed by the host, but its role in this context remains largely unexplored. In this study, we performed a functional and morphological analysis of the intestinal tract of loop-tail mice (Vangl2 mutants), a mouse model of folate/inositol-resistant NTDs. In addition, we investigated the changes in gut microbiota using 16S rRNA gene sequencing regarding (1) the host genotype; (2) the sample source for metagenomics analysis; (3) the pregnancy status in the gestational window of neural tube closure; (4) folic acid and (5) D-chiro-inositol supplementation. We observed that Vangl2+/Lp mice showed no apparent changes in gastrointestinal transit time or fecal output, yet exhibited increased intestinal length and cecal weight and gut dysbiosis. Moreover, our results showed that the mice supplemented with folic acid and D-chiro-inositol had significant changes in their microbiota composition, which are changes that could have implications for nutrient absorption.

Lactobacillus fermentum CECT 5716 is safe and well tolerated in infants of 1-6 months of age: a randomized controlled trial.

The objective of the study was to evaluate the safety and tolerance of an infant formula supplemented with Lactobacillus fermentum CECT5716, a probiotic strain isolated from breast milk, in infants of 1-6 months of age. A randomized double blinded controlled study including healthy infants was conducted. One month aged infants received a prebiotic infant formula supplemented with L. fermentum (experimental group) or the same formula without the probiotic strain (control group) for 5 months. The primary outcome of the study was average daily weight gain between baseline and 4 months of age. Secondary outcomes were other anthropometric data (length and head circumference), formula consumption, and tolerance. Incidence of infections was also recorded by pediatricians. No significant differences in weight gain were observed between both groups, neither at 4 months of age (29.0±7.8 vs 28.9±5.7g/day) nor at 6 months (25.1±6.1 vs 24.7±5.2g/day). There were no statistically significant differences in the consumption of the formulae or symptoms related to the tolerance of the formula. The incidence rate of gastrointestinal infections in infants of the control group was 3 times higher than in the probiotic group (p=0.018). Therefore, consumption of a prebiotic infant formula enriched with the human milk probiotic strain L. fermentum CECT5716 from 1 to 6 months of life is well tolerated and safe. Furthermore, the consumption of this formula may improve the health of the infants by reducing the incidence of gastrointestinal infections.

Intestinal and immunological effects of daily oral administration of Lactobacillus salivarius CECT5713 to healthy adults.

There is an increasing interest in the intestinal and immunological effects of probiotics. The aim of the present study is to evaluate the tolerance and beneficial effects in healthy adults of the strain, Lactobacillus salivarius CECT5713 isolated from breast milk. A phase II, randomized, double-blinded, placebo-controlled human clinical trial was carried out in 40 healthy adults. The Probiotic group received a daily dose of 2 x 10(8) CFU of L. salivarius CECT5713 in capsules during 4 weeks while volunteers of the control received only a placebo. Gastrointestinal and immunological parameters were analyzed. Results showed that L. salivarius CECT5713 was well tolerated and no adverse effects were detected. Consumption of the probiotic strain increased fecal lactobacilli counts (7.9+/-0.1 vs. 7.05+/-0.2 CFU/g feces, P=0.001). Also, an improvement in the frequency of defecation (P=0.04) was observed. Probiotic treatment induced significantly the percentage of NK cells and monocytes, as well as the plasmatic levels of immunoglobulins M, A and G, and the regulatory cytokine IL-10 (72.3+/-11.7 in probiotic group vs. 27.3+/-6.4 pg/mL in control group, P<0.01). Thus, it can be concluded that daily administration of L. salivarius CECT5713 to healthy adults is safe and improve gut microbiota and different parameters related to immune response.

Microbiota and organophosphates.

Organophosphates (OPs) are important toxic compounds commonly used for a variety of purposes in agriculture, industry and household settings. Consumption of these compounds affects several central nervous system functions. Some of the most recognised consequences of organophosphate pesticide exposure in humans include neonatal developmental abnormalities, endocrine disruption, neurodegeneration, neuroinflammation and cancer. In addition, neurobehavioral and emotional deficits following OP exposure have been reported. It would be of great value to discover a therapeutic strategy which produces a protective effect against these neurotoxic compounds. Moreover, a growing body of preclinical data suggests that the microbiota may affect metabolism and neurotoxic outcomes through exposure to OPs. The human gut is colonised by a broad variety of microorganisms. This huge number of bacteria and other microorganisms which survive by colonising the gastrointestinal tract is defined as "gut microbiota". The gut microbiome plays a profound role in metabolic processing, energy production, immune and cognitive development and homeostasis. The effects are not only localized in the gut, but also influence many other organs, such as the brain through the microbiome-gut-brain axis. Therefore, given the gut microbiota's key role in host homeostasis, this microbiota may be altered or modified temporarily by factors such as antibiotics, diet and toxins such as pesticides. The aim of this review is to examine scientific articles concerning the impact of microbiota in OP toxicity. Studies focussed on the possible contribution the microbiota has on variable host pharmacokinetic responses such as absorption and biotransformation of xenobiotics will be evaluated. Microbiome manipulation by antibiotic or probiotic administration and faecal transplantation are experimental approaches recently proposed as treatments for several diseases. Finally, microbiota manipulation as a possible therapeutic strategy in order to reduce OP toxicity will be discussed.

Lactobacillus fermentum CECT 5716 Reduces Staphylococcus Load in the Breastmilk of Lactating Mothers Suffering Breast Pain: A Randomized Controlled Trial.

INTRODUCTION: Recent results indicate that human mastitis and painful breastfeeding may be characterized by a mammary bacterial dysbiosis, a process in which the population of potential pathogens increases at the expense of the normal mammary microbiota. The objective of the present study is the evaluation of three different doses of Lactobacillus fermentum CECT5716 to reduce the load of Staphylococcus in the breastmilk of women suffering from painful breastfeeding. MATERIALS AND METHODS: A randomized double-blinded controlled study with four study groups was performed. Three groups received the probiotic strain for 3 weeks at doses of 3×10(9) colony-forming units (CFU)/day, 6×10(9) CFU/day, or 9×10(9) CFU/day. The fourth group received a placebo of maltodextrin. The main outcome of the study was Staphylococcus counts in breastmilk. The secondary outcomes were Streptococcus, Lactobacillus, and total bacteria counts in breastmilk, immunoglobulin A and interleukin 8 concentrations in breastmilk, and breast pain scores. RESULTS: At the end of the study, a significant decrease in the Staphylococcus load was observed in the probiotic groups compared with the baseline loads (p=0.045), whereas the control group maintained similar levels over time. A significant difference in the pain score was observed among the groups receiving the three probiotic doses compared with the control group (p=0.035, p=0.000, and p=0.028, respectively). A dose-response effect could not be observed because the three doses tested induced similar effects, and no significant differences were detected. CONCLUSIONS: We conclude that L. fermentum CECT5716 is an efficient treatment for breast pain during lactation associated with a high level of Staphylococcus in breastmilk.

Safety and tolerance of the human milk probiotic strain Lactobacillus salivarius CECT5713 in 6-month-old children.

OBJECTIVE: Intestinal microbiota plays an important role in the prevention of certain diseases during the pediatric years. Thus, there is an increasing interest in the addition of probiotics to infant formulas. The aim of this study was to evaluate the safety of a follow-on formula with Lactobacillus salivarius CECT5713 in 6-mo-old children. METHODS: The antibiotic susceptibility of L. salivarius CECT5713 was analyzed by a dilution method. A double-blinded, randomized, placebo controlled study was performed. Children (n = 80) were distributed in two groups and consumed the formula supplemented or not with probiotics (2 × 10(6) colony-forming units [cfu]/g) during 6 mo. Fecal samples were collected at enrollment, at 3 mo, and at the end of trial. Clinical and anthropometric evaluations were performed. Depending on the variable, one-way or two-way repeated measures analysis of variance were used for the statistical analysis. RESULTS: The antibiotic susceptibility profile of the strain resulted as safe. No adverse effects associated with the consumption of the probiotic formula were reported. In addition, clinical parameters did not differ between groups. Consumption of the probiotic supplemented formula led to an increase in the fecal lactobacilli content (7.6 ± 0.2 versus 7.9 ± 0.1 log cfu/g, P < 0.05). Lactobacillus salivarius CECT5713 was detected in the feces of volunteers from the probiotic group. Probiotic consumption induced a significant increase in the fecal concentration of butyric acid at 6 mo. CONCLUSION: Thus, a follow-on formula with L. salivarius CECT5713 is safe and well tolerated in 6-mo-old infants.