Use of batch culture and a two-stage continuous culture system to study the effect of supplemental a-lactalbumin and glycomacropeptide on mixed populations of human gut bacteria

Certain milk factors can promote the growth of a host-friendly gastrointestinal microflora. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts. The effect of formula supplementation with two such factors was investigated in this study. Infant faecal specimens were used to ferment formulas supplemented with glycomacropeptide and α-lactalbumin in a two-stage compound continuous culture model. Bacteriology was determined by fluorescence in situ hybridisation. Vessels that contained breast milk as well as α-lactalbumin and glycomacropeptide had stable counts of bifidobacteria while lactobacilli increased significantly only in vessels with breast milk. Bacteroides, clostridia and Escherichia coli decreased significantly in all runs. Acetate was the principal acid found along with high amounts of propionate and lactate. Supplementation of infant formulas with appropriate milk proteins may be useful in simulating the beneficial bacteriological effects of breast milk.

An assessment of the prebiotic potential of single and blended substrates in anaerobic in vitro batch culture fermentations using canine faecal samples as inocula

Previously, using an in vitro static batch culture system, it was found that rice bran (RB), inulin, fibersol, mannanoligosaccharides (MOS), larch arabinogalactan and citrus pectin elicited prebiotic effects (in terms of increased numbers of bifidobacteria and lactic acid bacteria) on the faecal microbiota of a dog. The aim of the present study was to confirm the prebiotic potential of each individual substrate using multiple faecal donors, as well as assessing the prebiotic potential of 15 substrate blends made from them. Anaerobic static and stirred, pH-controlled batch culture systems inoculated with faecal samples from healthy dogs were used for this purpose. Fluorescence in situ hybridization (FISH) analysis using seven oligonucleotide probes targeting selected bacterial groups and DAPI (total bacteria) was used to monitor bacterial populations during fermentation runs. High-performance liquid chromatography was used to measure butyrate produced as a result of bacterial fermentation of the substrates. RB and a MOS/RB blend (1:1, w/w) were shown to elicit prebiotic and butyrogenic effects on the canine microbiota in static batch culture fermentations. Further testing of these substrates in stirred, pH-controlled batch culture fermentation systems confirmed the prebiotic and butyrogenic effects of MOS/RB, with no enhancement of Clostridium clusters I and II and Escherichia coli populations.

In vitro batch cultures of gut microbiota from healthy and ulcerative colitis (UC) subjects suggest that sulphate-reducing bacteria levels are raised in UC and by a protein-rich diet.

Imbalances in gut microbiota composition during ulcerative colitis (UC) indicate a role for the microbiota in propagating the disorder. Such effects were investigated using in vitro batch cultures (with/without mucin, peptone or starch) inoculated with faecal slurries from healthy or UC patients; the growth of five bacterial groups was monitored along with short-chain fatty acid (SCFA) production. Healthy cultures gave two-fold higher growth and SCFA levels with up to ten-fold higher butyrate production. Starch gave the highest growth and SCFA production (particularly butyrate), indicating starch-enhanced saccharolytic activity. Sulphate-reducing bacteria (SRB) were the predominant bacterial group (of five examined) for UC inocula whereas they were the minority group for the healthy inocula. Furthermore, SRB growth was stimulated by peptone presumably due to the presence of sulphur-rich amino acids. The results suggest raised SRB levels in UC, which could contribute to the condition through release of toxic sulphide.

Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing

Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism’s phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

Tagged phosphine reagents to assist reaction work-up by phase-switched scavenging using a modular flow reactor

The use of three orthogonally tagged phosphine reagents to assist chemical work-up via phase-switch scavenging in conjunction with a modular flow reactor is described. These techniques (acidic, basic and Click chemistry) are used to prepare various amides and tri-substituted guanidines from in situ generated iminophosphoranes.

[3 + 2] Cycloaddition of acetylenes with azides to give 1,4-disubstituted 1,2,3-triazoles in a modular flow reactor

The cycloaddition of acetylenes with azides to give the corresponding 1,4-disubstituted 1,2,3-triazoles is reported using immobilised reagents and scavengers in pre-packed glass tubes in a modular flow reactor.

A modular flow reactor for performing Curtius rearrangements as a continuous flow process

The use of a mesofluidic flow reactor is described for performing Curtius rearrangement reactions of carboxylic acids in the presence of diphenylphosphoryl azide and trapping of the intermediate isocyanates with various nucleophiles.

A flow reactor process for the synthesis of peptides utilizing immobilized reagents, scavengers and catch and release protocols

A general flow process for the multi-step assembly of peptides has been developed and this procedure has been used to successfully construct a series of Boc, Cbz and Fmoc N-protected dipeptides in excellent yields and purities, including an extension of the method to enable the preparation of a tripeptide derivative.

In vitro evaluation of the fermentation properties and potential probiotic activity of Lactobacillus plantarum C4 in batch culture systems

Lactobacillus plantarum C4 has been tested in in vitro pH-controlled anaerobic faecal batch cultures as compared to Lactobacillus rhamnosus GG to determine changes caused to the composition of faecal bacteria. Effects upon major groups of the microbiota and levels of short-chain fatty acids (SCFA) were assessed over 24 h. Concomitantly, hydrophobic character and ability of both bacterial cells to adhere in vitro to Caco-2 cells were investigated. Quantitative analysis of bacterial populations revealed that there was a significant increase in Lactobacillus/Enterococcus numbers in vessels with probiotic supplemented with fructooligosaccharides (FOS), compared to the negative control. L. plantarum C4 showed to have more hydrophilic behaviour and fulfilled better adhesive properties, compared to L. rhamnosus GG. Thus, L. plantarum C4 can modulate the intestinal microbiota in vitro, promoting changes in some numerically and metabolically relevant microbial populations and shifts in the production of SCFA.

Effect of enzymatic pretreatment and increasing the organic loading rate of lipid-rich wastewater treated in a hybrid UASB reactor

This study aimed at evaluating the effect of increasing organic loading rates and of enzyme pretreatment on the stability and efficiency of a hybrid upflow anaerobic sludge blanket reactor (UASBh) treating dairy effluent. The UASBh was submitted to the following average organic loading rates (OLR) 0.98 Kg.m(-3).d(-1), 4.58 Kg.m(-3).d(-1), 8.89 Kg.m(-3).d(-1) and 15.73 Kg.m(-3).d(-1), and with the higher value, the reactor was fed with effluent with and without an enzymatic pretreatment to hydrolyze fats. The hydraulic detention time was 24 h, and the temperature was 30 +/- 2 degrees C. The reactor was equipped with a superior foam bed and showed good efficiency and stability until an OLR of 8.89 Kg.m(-3).d(-1). The foam bed was efficient for solid retention and residual volatile acid concentration consumption. The enzymatic pretreatment did not contribute to the process stability, propitiating loss in both biomass and system efficiency. Specific methanogenic activity tests indicated the presence of inhibition after the sludge had been submitted to the pretreated effluent It was concluded that continuous exposure to the hydrolysis products or to the enzyme caused a dramatic drop in the efficiency and stability of the process, and the single exposure of the biomass to this condition did not inhibit methane formation. (C) 2011 Elsevier B.V. All rights reserved.

Next Generation Sequencing of Pooled Samples Reveals New SNRNP200 Mutations Associated with Retinitis Pigmentosa

The gene SNRNP200 is composed of 45 exons and encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Two mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease, in two families from China. In this work we analyzed the entire 35-Kb SNRNP200 genomic region in a cohort of 96 unrelated North American patients with adRP. To complete this large-scale sequencing project, we performed ultra high-throughput sequencing of pooled, untagged PCR products. We then validated the detected DNA changes by Sanger sequencing of individual samples from this cohort and from an additional one of 95 patients. One of the two previously known mutations (p.S1087L) was identified in 3 patients, while 4 new missense changes (p.R681C, p.R681H, p.V683L, p.Y689C) affecting highly conserved codons were identified in 6 unrelated individuals, indicating that the prevalence of SNRNP200-associated adRP is relatively high. We also took advantage of this research to evaluate the pool-and-sequence method, especially with respect to the generation of false positive and negative results. We conclude that, although this strategy can be adopted for rapid discovery of new disease-associated variants, it still requires extensive validation to be used in routine DNA screenings. (C) 2011 Wiley-Liss, Inc.

Exploring Bacterial Diversity of Endodontic Microbiota by Cloning and Sequencing 16S rRNA

Introduction: The characterization of microbial communities infecting the endodontic system in each clinical condition may help on the establishment of a correct prognosis and distinct strategies of treatment. The purpose of this study was to determine the bacterial diversity in primary endodontic infections by 16S ribosomal-RNA (rRNA) sequence analysis. Methods: Samples from root canals of untreated asymptomatic teeth (n = 12) exhibiting periapical lesions were obtained, 165 rRNA bacterial genomic libraries were constructed and sequenced, and bacterial diversity was estimated. Results: A total of 489 clones were analyzed (mean, 40.7 +/- 8.0 clones per sample). Seventy phylotypes were identified of which six were novel phylotypes belonging to the family Ruminococcaceae. The mean number of taxa per canal was 10.0, ranging from 3 to 21 per sample; 65.7% of the cloned sequences represented phylotypes for which no cultivated isolates have been reported. The most prevalent taxa were Atopobium rimae (50.0%), Dialister invisus, Pre-votella oris, Pseudoramibacter alactolyticus, and Tannerella forsythia (33.3%). Conclusions: Although several key species predominate in endodontic samples of asymptomatic cases with periapical lesions, the primary endodontic infection is characterized by a wide bacterial diversity, which is mostly represented by members of the phylum Firmicutes belonging to the class Clostridia followed by the phylum Bacteroidetes. (J Ended 2011;37:922-926)