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.

Factors affecting hydrogen uptake by bacteria growing in the human large intestine

The human large intestine is a highly complex ecosystem that contains somewhere in the region of 400 different species of bacterial1.The vast majority of these bacteria are strict anaerobes and grow on a wide variety of substrates that have either escaped digestion in the small bowel or have been produced by the host2. In Western populations, between 10–60g of carbohydrate and 6–18g of proteinaceous material are potentially available for fermentation each day, producing a total bacterial mass of approximately 90g3.

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.

Protection of dried probiotic bacteria from bile using bile adsorbent resins

Enteric coated oral tablets or capsules can deliver dried live cells directly into the intestine. Previously, we found that a live attenuated bacterial vaccine acquired sensitivity to intestinal bile when dried, raising the possibility that although gastric acid can be bypassed, significant loss of viability might occur on release from an enteric coated oral formulations. Here we demonstrate that some food-grade lyophilised preparations of Lactobacillus casei and Lactobacillus salivarius also show temporary bile sensitivity that can be rapidly reversed by rehydration. To protect dried bacterial cells from temporary bile sensitivity, we propose using bile acid adsorbing resins, such as cholestyramine, which are bile acid binding agents, historically used to lower cholesterol levels. Vcaps™ HPMC capsules alone provided up to 830-fold protection from bile. The inclusion of 50% w/w cholestyramine in Vcaps™ HPMC capsules resulted in release of up to 1700-fold more live Lactobacillus casei into simulated intestinal fluid containing 1% bile, when compared to dried cells added directly to bile. We conclude that delivery of dried live probiotic organisms to the intestine may be improved by providing protection from bile by addition of bile adsorbing resins and the use of HPMC capsules.

Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.

The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Array based detection of antibiotic resistance genes in Gram negative bacteria isolated from retail poultry meat in the UK and Ireland

The use of antibiotics in birds and animals intended for human consumption within the European Union (EU) and elsewhere has been subject to regulation prohibiting the use of antimicrobials as growth promoters and the use of last resort antibiotics in an attempt to reduce the spread of multi-resistant Gram negative bacteria. Given the inexorable spread of antibiotic resistance there is an increasing need for improved monitoring of our food. Using selective media, Gram negative bacteria were isolated from retail chicken of UK-Intensively reared (n = 27), Irish-Intensively reared (n = 19) and UK-Free range (n = 30) origin and subjected to an oligonucleotide based array system for the detection of 47 clinically relevant antibiotic resistance genes (ARGs) and two integrase genes. High incidences of β-lactamase genes were noted in all sample types, acc (67%), cmy (80%), fox (55%) and tem (40%) while chloramphenicol resistant determinants were detected in bacteria from the UK poultry portions and were absent in bacteria from the Irish samples. Denaturing Gradient Gel Electrophoresis (DGGE) was used to qualitatively analyse the Gram negative population in the samples and showed the expected diversity based on band stabbing and DNA sequencing. The array system proved to be a quick method for the detection of antibiotic resistance gene (ARG) burden within a mixed Gram negative bacterial population.

A laminated polymer film formulation for enteric delivery of live vaccine and probiotic bacteria

Live bacterial cells (LBC) are administered orally as attenuated vaccines, to deliver biopharmaceutical agents, and as probiotics to improve gastrointestinal health. However, LBC present unique formulation challenges and must survive gastrointestinal antimicrobial defenses including gastric acid after administration. We present a simple new formulation concept, termed Polymer Film Laminate (PFL). LBC are ambient dried onto cast acid-resistant enteric polymer films that are then laminated together to produce a solid oral dosage form. LBC of a model live bacterial vaccine and a probiotic were dried directly onto a cast film of enteric polymer. The effectiveness at protecting dried cells in a simulated gastric fluid (pH 2.0) depended on the composition of enteric polymer film used, with a blend of ethylcellulose plus Eudragit L100 55 providing greater protection from acid than Eudragit alone. However, although PFL made from blended polymers films completely released low molecular weight dye into intestinal conditions (pH 7.0), they failed to release LBC. In contrast, PFL made from Eudragit alone successfully protected dried probiotic or vaccine LBC from simulated gastric fluid for 2h, and subsequently released all viable cells within 60min of transfer into simulated intestinal fluid. Release kinetics could be controlled by modifying the lamination method.