High-throughput sequencing of 16S rRNA gene amplicons : effects of extraction procedure, primer length and annealing temperature

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers

Identification and Characterisation of an Iron-Responsive Candidate Probiotic

Background: Iron is an essential cofactor in almost all biological systems. The lactic acid bacteria (LAB), frequently employed as probiotics, are unusual in having little or no requirement for iron. Iron in the human body is sequestered by transferrins and lactoferrin, limiting bacterial growth. An increase in the availability of iron in the intestine by bleeding, surgery, or under stress leads to an increase in the growth and virulence of many pathogens. Under these high iron conditions, LAB are rapidly out-competed; for the levels of probiotic bacteria to be maintained under high iron conditions they must be able to respond by increasing growth rate to compete with the normal flora. Despite this, iron-responsive genera are poorly characterised as probiotics. Methodology/Principal Findings: Here, we show that a panel of probiotics are not able to respond to increased iron availability, and identify an isolate of Streptococcus thermophilus that can increase growth rate in response to increased iron availability. The isolate of S. thermophilus selected was able to reduce epithelial cell death as well as NF-kB signalling and IL-8 production triggered by pathogens. It was capable of crossing an epithelial cell barrier in conjunction with E. coli and downregulating Th1 and Th17 responses in primary human intestinal leukocytes. Conclusions/Significance: We propose that an inability to compete with potential pathogens under conditions of high iron availability such as stress and trauma may contribute to the lack of efficacy of many LAB-based probiotics in treatin

<i>Streptococcus thermophilus</i> NCIMB 41856 ameliorates signs of colitis in an animal model of inflammatory bowel disease

Treatment of inflammatory bowel disease (IBD) is mainly based on suppression of symptoms, often with numerous side effects. Trials of probiotics in IBD have frequently produced disappointing results. The majority of probiotics are unusual, since they do not require iron for growth, unlike many bacteria resident in the intestine. The IBD intestine is iron-rich due to bleeding and use of oral iron supplements; conventional probiotics would be rapidly outcompeted. We have evaluated an iron-responsive Streptococcus thermophilus strain for its potential to reduce signs of colitis. Efficacy of S. thermophilus was evaluated in the dextran sodium sulphate mouse model of colitis. Treated animals were given 1×108 cfu S. thermophilus per day and clinical observations were taken daily. At termination, gross and histopathological signs of disease, cellular infiltration, location of bacteria, and cytokine expression in the intestine were determined. S. thermophilus delayed onset of colitis and reduced clinical signs of disease, including bodyweight loss and gastrointestinal bleeding. It reduced bacterial translocation into the colonic tissue. Increased numbers of CD8+ intraepithelial lymphocytes were seen in control animals treated with S. thermophilus. S. thermophilus had no effect on gross pathology, histopathology or cytokine production in either colitic or control animals. We propose that S. thermophilus promotes maintenance of mucosal barrier function which reduces bacterial translocation, thereby reducing immune stimulation and associated inflammation. This allows mucosal healing, reducing gastrointestinal bleeding and weight loss. This could be studied as a locally-acting adjunct or alternative to current IBD treatments. </jats:p

Enrichment of Lactic Acid Silage Bacteria with Selenium by Growing Cultures in Modified MRS Broth Supplemented with Sodium Selenite

Selenium (Se) is a trace element essential for normal cellular function, which has been linked with reduced risk of cancer, cardiovascular disease, cognitive decline and thyroid disease in humans. Se deficiency in livestock is associated with white muscle disease, retained placenta, ill-thrift and mastitis. Where Se status or bioavailability from the soil for plants is poor, livestock rely on supplemental Se in their diets either as sodium selenite (inorganic form) or seleno-yeast (organic form). As lactic acid bacteria have been shown to incorporate Se as either organic or elemental (Nano-Se), (Eszenyi et al., 2011) there may be potential to use silage inoculant bacteria to improve the Se status of feed to provide Se requirements to livestock. The aim of this experiment was to examine the growth, uptake of selenium as organic (selenocysteine and selenomethionine), inorganic (selenite and selenate) and Nano-Se by lactic acid bacterial isolates (LAB), which could then be used in future ensiling studies (Lee et al., 2015) to produce high Se silage

Efficacy and dermal tolerance of a novel alcohol-based skin antiseptic in horses

Objective: To determine the efficacy and dermal tolerance of a novel alcohol‐based skin antiseptic (ABSA) in horses. Study Design: Experimental study. Animal Population Systemically healthy horses (n = 25) with no history or clinical signs of skin disease. Methods: Four clipped sites on the abdomen were randomly assigned to a skin preparation protocol: saline (negative control; NC), chlorhexidine gluconate followed by isopropyl alcohol (positive control; PC), saline followed by the ABSA (ABSA A), or a commercially available horse shampoo followed by the ABSA (ABSA B). Microbiological swabs were obtained from each site and cultured on MacConkey and mannitol salt agar plates. Colony‐forming units were counted 18‐24 hours later. All sites were scored for signs of skin reaction before, immediately after, 1 hour after, and 24 hours after skin preparation. Results: The PC, ABSA A, and ABSA B methods reduced skin microbial burden compared with the NC method (P &lt; .001), but no difference was detected between antiseptic products. Preparation time did not differ between ABSA A and ABSA B methods (P = 0.108); both were faster than the PC method (P &lt; 0.001 for both). Skin reactions were most abundant 24 hours after skin preparation (30.5%), but there was no significant association with antiseptic used, and no horses required veterinary treatment. Conclusion: The ABSA preparations tested in this study were as effective and well tolerated as a chlorhexidine gluconate‐based method, but required less time in healthy horses