The use of culture-independent tools to characterize bacteria in endo-tracheal aspirates from pre-term infants at risk of bronchopulmonary dysplasia

Although premature infants are increasingly surviving the neonatal period, up to one-third develop bronchopulmonary dysplasia (BPD). Despite evidence that bacterial colonization of the neonatal respiratory tract by certain bacteria may be a risk factor in BPD development, little is known about the role these bacteria play. The aim of this study was to investigate the use of culture-independent molecular profiling methodologies to identify potential etiological agents in neonatal airway secretions. This study used terminal restriction fragment length polymorphism (T-RFLP) and clone sequence analyses to characterize bacterial species in endo-tracheal (ET) aspirates from eight intubated pre-term infants. A wide range of different bacteria was identified in the samples. Forty-seven T-RF band lengths were resolved in the sample set, with a range of 0-15 separate species in each patient. Clone sequence analyses confirmed the identity of individual species detected by T-RFLP. We speculate that the identification of known opportunistic pathogens including S. aureus, Enterobacter sp., Moraxella catarrhalis, Pseudomonas aeruginosa and Streptococcus sp., within the airways of pre-term infants, might be causally related to the subsequent development of BPD. Further, we suggest that culture-independent techniques, such as T-RFLP, hold important potential for the characterization of neonatal conditions, such as BPD.

Improving survival of probiotic bacteria using bacterial poly-γ-glutamic acid

A major hurdle in producing a useful probiotic food product is bacterial survival during storage and ingestion. The aim of this study was to test the effect of γ-PGA immobilisation on the survival of probiotic bacteria when stored in acidic fruit juice. Fruit juices provide an alternative means of probiotic delivery, especially to lactose intolerant individuals. In addition, the survival of γ-PGA-immobilised cells in simulated gastric juice was also assessed. Bifidobacteria strains (B. longum, B. breve), immobilised on 2.5 % γ-PGA, survived significantly better (P < 0.05) in orange and pomegranate juice for 39 and 11 days respectively, compared to free cells. However, cells survived significantly better (P < 0.05) when stored in orange juice compared to pomegranate juice. Moreover, both strains, when protected with 2.5 % γ-PGA, survived in simulated gastric juice (pH 2.0) with a marginal reduction (<0.47 log CFU/ml) or no significant reduction in viable cells after four hours, whereas free cells died within two hours. In conclusion, this research indicates that γ-PGA can be used to protect Bifidobacteria cells in fruit juice, and could also help improve the survival of cells as they pass through the harsh conditions of the gastrointestinal tract (GIT). Following our previous report on the use of γ-PGA as a cryoprotectant for probiotic bacteria, this research further suggests that γ-PGA could be used to improve probiotic survival during the various stages of preparation, storage and ingestion of probiotic cells.

The endophytic fungus Piriformospora indica protects wheat from Fusarium crown rot disease in simulated UK autumn conditions

The root endophytic fungus Piriformospora indica (Sebacinacea) forms mutualistic symbioses with a broad range of host plants, increasing their biomass production and resistance to fungal pathogens. We evaluated the effect of P. indica on Fusarium crown rot disease of wheat, under in vitro and glasshouse conditions. Interaction of P. indica and Fusarium isolates under axenic culture conditions indicated no direct antagonistic activity of P. indica against Fusarium isolates. Seedlings of wheat were inoculated with P. indica and pathogenic Fusarium culmorum or F. graminearum and grown in sterilised soil-free medium or in a non-sterilised mix of soil and sand. Fusarium alone reduced emergence and led to visible browning and reduced root growth. Roots of seedlings in pots inoculated with both Fusarium isolates and P. indica were free of visible symptoms; seed emergence and root biomass were equivalent to the uninoculated. DNA was quantified by real-time polymerase chain reaction (qPCR). The ratio of Fusarium DNA to wheat DNA rose rapidly in the plants inoculated with Fusarium alone; isolates and species were not significantly different. P. indica inoculation reduced the ratio of Fusarium to host DNA in the root systems. The reduction increased with time. The ratio of P. indica to wheat DNA initially rose but then declined in root systems without Fusarium. With Fusarium, the ratio rose throughout the experiment. The absolute amount of Fusarium DNA in root systems increased in the absence of P. indica but was static in plants co-inoculated with P. indica.

Endophytic bacterial community composition in wheat (Triticum aestivum) is determined by plant tissue type, developmental stage and soil nutrient availability

Aims: To understand effects of tissue type, growth stage and soil fertilisers on bacterial endophyte communities of winter wheat (Triticum aestivum cv. Hereward). Methods: Endophytes were isolated from wheat grown under six fertiliser conditions in the long term Broadbalk Experiment at Rothamsted Research, UK. Samples were taken in May and July from root and leaf tissues. Results: Root and leaf communities differed in abundance and composition of endophytes. Endophytes were most abundant in roots and the Proteobacteria were most prevalent. In contrast, Firmicutes and Actinobacteria, the Gram positive phyla, were most prevalent in the leaves. Both fertiliser treatment and sample time influenced abundance and relative proportions of each phylum and genus in the endosphere. A higher density of endophytes was found in the Nil input treatment plants. Conclusions: Robust isolation techniques and stringent controls are critical for accurate recovery of endophytes. The plant tissue type, plant growth stage, and soil fertiliser treatment all contribute to the composition of the endophytic bacterial community in wheat. These results should help facilitate targeted development of endophytes for beneficial applications in agriculture.

Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2 h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid.

New insights into the impact of Lactobacillus population on host-bacteria metabolic interplay

We aimed at evaluating the association between intestinal Lactobacillus sp. composition and their metabolic activity with the host metabolism in adult and elderly individuals. Faecal and plasma metabolites were measured and correlated to the Lactobacillus species distribution in healthy Estonian cohorts of adult (n=16; <48 y) and elderly (n=33; >65 y). Total cholesterol, LDL, C-reactive protein and glycated hemoglobin were statistically higher in elderly, while platelets, white blood cells and urinary creatinine were higher in adults. Aging was associated with the presence of L. paracasei and L. plantarum and the absence of L. salivarius and L. helveticus. High levels of intestinal Lactobacillus sp. were positively associated with increased concentrations of faecal short chain fatty acids, lactate and essential amino acids. In adults, high red blood cell distribution width was positively associated with presence of L. helveticus and absence of L. ruminis. L. helveticus was correlated to lactate and butyrate in faecal waters. This indicates a strong relationship between the composition of the gut Lactobacillus sp. and host metabolism. Our results confirm that aging is associated with modulations of blood biomarkers and intestinal Lactobacillus species composition. We identified specific Lactobacillus contributions to gut metabolic environment and related those to blood biomarkers. Such associations may prove useful to decipher the biological mechanisms underlying host-gut microbial metabolic interactions in an ageing population.

Culex pipiens development is greatly influenced by native bacteria and exogenous yeast

Culex pipiens is the most cosmopolitan mosquito of the Pipiens Assemblage. By studying the nature of interactions between this species and microorganisms common to its breeding environment we can unravel important pitfalls encountered during development. We tested the survival rate of larval stages, pupae and adults of a Cx. pipiens colony exposed to a variety of microorganisms in laboratory conditions and assessed the transmission to offspring (F1) by those organisms that secured development up to adulthood. Three complementary experiments were designed to: 1) explore the nutritional value of yeasts and other microorganisms during Cx. pipiens development; 2) elucidate the transstadial transmission of yeast to the host offspring; and 3) to examine the relevance of all these microorganisms in female choice for oviposition-substratum. The yeast Saccharomyces cerevisiae proved to be the most nutritional diet, but despite showing the highest survival rates, vertical transmission to F1 was never confirmed. In addition, during the oviposition trials, none of the gravid females was attracted to the yeast substratum. Notably, the two native bacterial strains, Klebsiella sp. and Aeromonas sp., were the preferred oviposition media, the same two bacteria that managed to feed neonates until molting into 2nd instar larvae. Our results not only suggest that Klebsiella sp. or Aeromonas sp. serve as attractants for oviposition habitat selection, but also nurture the most fragile instar, L1, to assure molting into a more resilient stage, L2, while yeast proves to be the most supportive diet for completing development. These experiments unearthed survival traits that might be considered in the future development of strategies of Cx. pipiens control. These studies can be extended to other members of the Pipiens Assemblage

Wheat seed embryo excision enables the creation of axenic seedlings and Koch’s postulates testing of putative bacterial endophytes

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.