• 16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform

      Fouhy, Fiona; Clooney, Adam G; Stanton, Catherine; Claesson, Marcus J; Cotter, Paul D (Biomed Central, 2016-06-24)
      Background Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. Results The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. Conclusions Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.
    • Adding value to milk by increasing its protein and CLA contents

      Murphy, J.J.; Stanton, Catherine; O'Donovan, Michael; Kavanagh, S.; Maher, J.; Patton, Joe; Mohammed, Riaz (Teagasc, 2008-08-01)
      The mid-summer milk protein study was undertaken on 34 commercial dairy farms in 2005 to evaluate the influence of dietary and management variables on milk protein content in mid-season. Data on grass composition, genetic merit of the herds and milk protein content were collected and analysed by multiple regression. Both calving date and genetic merit for milk protein content were significantly associated with milk protein content and were used as adjustment factors when evaluating the association between measures of grass quality and milk protein content. Milk protein content was associated with grass OMD (P = 0.04) and NDF content (P = 0.02) but not with CP content (P = 0.80). It is concluded that herds calving earlier, with a greater genetic merit for milk protein content and consuming better quality pasture would have greater milk protein contents in mid-season.
    • Adding value to milk by increasing its protein and CLA contents.

      Murphy, J.J.; Stanton, Catherine; O'Donovan, Michael; Kavanagh, S.; Maher, J.; Patton, J.; Mohammed, R. (Teagasc, 2008-08-01)
      Five experiments were undertaken in this project; one on mid-summer milk protein and four on milk CLA content. Thus the two main objectives of this project were to determine the factors associated with milk protein concentration in mid-summer and to investigate potential further strategies to increase the CLA content of pasture produced milk.
    • The altered gut microbiota in adults with cystic fibrosis

      Burke, D.G.; Fouhy, Fiona; Harrison, M. J; Rea, Mary C; Cotter, Paul D; O’Sullivan, O.; Stanton, Catherine; Hill, C.; Shanahan, F.; Plant, B. J; Ross, R. Paul (Biomed Central, 2017-03-09)
      Background Cystic Fibrosis (CF) is an autosomal recessive disease that affects the function of a number of organs, principally the lungs, but also the gastrointestinal tract. The manifestations of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the gastrointestinal tract, as well as frequent antibiotic exposure, undoubtedly disrupts the gut microbiota. To analyse the effects of CF and its management on the microbiome, we compared the gut microbiota of 43 individuals with CF during a period of stability, to that of 69 non-CF controls using 454-pyrosequencing of the 16S rRNA gene. The impact of clinical parameters, including antibiotic therapy, on the results was also assessed. Results The CF-associated microbiome had reduced microbial diversity, an increase in Firmicutes and a reduction in Bacteroidetes compared to the non-CF controls. While the greatest number of differences in taxonomic abundances of the intestinal microbiota was observed between individuals with CF and the healthy controls, gut microbiota differences were also reported between people with CF when grouped by clinical parameters including % predicted FEV1 (measure of lung dysfunction) and the number of intravenous (IV) antibiotic courses in the previous 12 months. Notably, CF individuals presenting with severe lung dysfunction (% predicted FEV1 ≤ 40%) had significantly (p < 0.05) reduced gut microbiota diversity relative to those presenting with mild or moderate dysfunction. A significant negative correlation (−0.383, Simpson’s Diversity Index) was also observed between the number of IV antibiotic courses and gut microbiota diversity. Conclusions This is one of the largest single-centre studies on gut microbiota in stable adults with CF and demonstrates the significantly altered gut microbiota, including reduced microbial diversity seen in CF patients compared to healthy controls. The data show the impact that CF and it's management have on gut microbiota, presenting the opportunity to develop CF specific probiotics to minimise microbiota alterations.
    • The altered gut microbiota in adults with cystic fibrosis

      Burke, D.G.; Fouhy, Fiona; Harrison, M. J; Rea, Mary C; Cotter, Paul D; O’Sullivan, Orla; Stanton, Catherine; Hill, C.; Shanahan, F.; Plant, B. J; Ross, R. Paul (Biomed Central, 2017-03-09)
      Background Cystic Fibrosis (CF) is an autosomal recessive disease that affects the function of a number of organs, principally the lungs, but also the gastrointestinal tract. The manifestations of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in the gastrointestinal tract, as well as frequent antibiotic exposure, undoubtedly disrupts the gut microbiota. To analyse the effects of CF and its management on the microbiome, we compared the gut microbiota of 43 individuals with CF during a period of stability, to that of 69 non-CF controls using 454-pyrosequencing of the 16S rRNA gene. The impact of clinical parameters, including antibiotic therapy, on the results was also assessed. Results The CF-associated microbiome had reduced microbial diversity, an increase in Firmicutes and a reduction in Bacteroidetes compared to the non-CF controls. While the greatest number of differences in taxonomic abundances of the intestinal microbiota was observed between individuals with CF and the healthy controls, gut microbiota differences were also reported between people with CF when grouped by clinical parameters including % predicted FEV1 (measure of lung dysfunction) and the number of intravenous (IV) antibiotic courses in the previous 12 months. Notably, CF individuals presenting with severe lung dysfunction (% predicted FEV1 ≤ 40%) had significantly (p < 0.05) reduced gut microbiota diversity relative to those presenting with mild or moderate dysfunction. A significant negative correlation (−0.383, Simpson’s Diversity Index) was also observed between the number of IV antibiotic courses and gut microbiota diversity. Conclusions This is one of the largest single-centre studies on gut microbiota in stable adults with CF and demonstrates the significantly altered gut microbiota, including reduced microbial diversity seen in CF patients compared to healthy controls. The data show the impact that CF and it's management have on gut microbiota, presenting the opportunity to develop CF specific probiotics to minimise microbiota alterations.
    • Application of Probiotic Bacteria to Functional Foods

      Stanton, Catherine; Ross, R.Paul; Fitzgerald, G.; Collins, K.; McBrearty, S.; Gardiner, G.; Desmond, C.; Kelly, J.; Bouchier, P.; Lawless, F.; Auty, Mark; Corcoran, M.; Mullins, C.; Mullholand, E.; Eason, D. (Teagasc, 2001-05-01)
      Probiotic cultures are described as live microbial feed supplements that improve intestinal microbial balance and are intended for maintenance of health or prevention, rather than the curing of disease. The demand for probiotic foods is increasing in Europe, Japan and the U.S. reflecting the heightened awareness among the public of the relationship between diet and health. Traditionally, the most popular food delivery systems for these cultures have been freshly fermented dairy foods, such as yogurts and fermented milks, as well as unfermented milks with cultures added. However, in the development of functional foods, the technological suitability of probiotic strains poses a serious challenge since their survival and viability may be adversely affected by processing conditions as well as by the product environment and storage conditions. This is a particular concern, given that high levels (at least 107 per gram or ml) of live micro-organisms are recommended for probiotic products. In previous studies (see DPRC No. 29) the successful manufacture of probiotic Cheddar cheese harbouring high levels (>108 cfu/g) of the probiotic Lactobacillus paracasei NFBC 338 strain was reported. Hence, the overall objective of these studies was to continue the development and evaluation of Functional Foods containing high levels of viable probiotic bacteria, with particular emphasis on overcoming the technological barriers and the identification of strains suited to particular applications, such as incorporation into Cheddar cheese and spray-dried powders.
    • Bifidobacterium breve with α-Linolenic Acid and Linoleic Acid Alters Fatty Acid Metabolism in the Maternal Separation Model of Irritable Bowel Syndrome

      Barrett, Eoin; Fitzgerald, Patrick; Dinan, Timothy G.; Cryan, John F.; Ross, R Paul; Quigley, Eamonn M.; Shanahan, Fergus; Kiely, Barry; Fitzgerald, Gerald F.; O'Toole, Paul W.; Stanton, Catherine (PLOS, 2012-11-20)
      The aim of this study was to compare the impact of dietary supplementation with a Bifidobacterium breve strain together with linoleic acid & α-linolenic acid, for 7 weeks, on colonic sensitivity and fatty acid metabolism in rats. Maternally separated and non-maternally separated Sprague Dawley rats (n = 15) were orally gavaged with either B. breve DPC6330 (109 microorganisms/day) alone or in combination with 0.5% (w/w) linoleic acid & 0.5% (w/w) α-linolenic acid, daily for 7 weeks and compared with trehalose and bovine serum albumin. Tissue fatty acid composition was assessed by gas-liquid chromatography and visceral hypersensitivity was assessed by colorectal distension. Significant differences in the fatty acid profiles of the non-separated controls and maternally separated controls were observed for α-linolenic acid and arachidonic acid in the liver, oleic acid and eicosenoic acid (c11) in adipose tissue, and for palmitoleic acid and docosahexaenoic acid in serum (p<0.05). Administration of B. breve DPC6330 to MS rats significantly increased palmitoleic acid, arachidonic acid and docosahexaenoic acid in the liver, eicosenoic acid (c11) in adipose tissue and palmitoleic acid in the prefrontal cortex (p<0.05), whereas feeding B. breve DPC6330 to non separated rats significantly increased eicosapentaenoic acid and docosapentaenoic acid in serum (p<0.05) compared with the NS un-supplemented controls. Administration of B. breve DPC6330 in combination with linoleic acid and α-linolenic acid to maternally separated rats significantly increased docosapentaenoic acid in the serum (p<0.01) and α-linolenic acid in adipose tissue (p<0.001), whereas feeding B. breve DPC6330 with fatty acid supplementation to non-separated rats significantly increased liver and serum docosapentaenoic acid (p<0.05), and α-linolenic acid in adipose tissue (p<0.001). B. breve DPC6330 influenced host fatty acid metabolism. Administration of B. breve DPC6330 to maternally separated rats significantly modified the palmitoleic acid, arachidonic acid and docosahexaenoic acid contents in tissues. The effect was not observed in non-separated animals.
    • Bile acids at the cross-roads of gut microbiome–host cardiometabolic interactions

      Ryan, Paul M; Stanton, Catherine; Caplice, Noel M; Science Foundation Ireland; Enterprise Ireland Commercialization Fund; SFI/12/RC/2273; CF/2013/3030A/B (Biomed Central, 2017-12-28)
      While basic and clinical research over the last several decades has recognized a number of modifiable risk factors associated with cardiometabolic disease progression, additional and alternative biological perspectives may offer novel targets for prevention and treatment of this disease set. There is mounting preclinical and emerging clinical evidence indicating that the mass of metabolically diverse microorganisms which inhabit the human gastrointestinal tract may be implicated in initiation and modulation of cardiovascular and metabolic disease outcomes. The following review will discuss this gut microbiome–host metabolism axis and address newly proposed bile-mediated signaling pathways through which dysregulation of this homeostatic axis may influence host cardiovascular risk. With a central focus on the major nuclear and membrane-bound bile acid receptor ligands, we aim to review the putative impact of microbial bile acid modification on several major phenotypes of metabolic syndrome, from obesity to heart failure. Finally, attempting to synthesize several separate but complementary hypotheses, we will review current directions in preclinical and clinical investigation in this evolving field.
    • Comparing Apples and Oranges?: Next Generation Sequencing and Its Impact on Microbiome Analysis

      Clooney, Adam G.; Fouhy, Fiona; Sleator, Roy D.; O'Driscoll, Aisling; Stanton, Catherine; Cotter, Paul D; Claesson, Marcus J. (PLOS, 2016-02-05)
      Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number ofmethodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance inmicrobiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced withMiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.
    • Composition of the early intestinal microbiota: Knowledge, knowledge gaps and the use of high-throughput sequencing to address these gaps

      Fouhy, Fiona; Ross, R Paul; Fitzgerald, Gerald F; Stanton, Catherine; Cotter, Paul D. (Landes Bioscience, 2012-05-01)
      The colonization, development and maturation of the newborn gastrointestinal tract that begins immediately at birth and continues for two years, is modulated by numerous factors including mode of delivery, feeding regime, maternal diet/weight, probiotic and prebiotic use and antibiotic exposure pre-, peri- and post-natally. While in the past, culture-based approaches were used to assess the impact of these factors on the gut microbiota, these have now largely been replaced by culture-independent DNA-based approaches and most recently, high-throughput sequencing-based forms thereof. The aim of this review is to summarize recent research into the modulatory factors that impact on the acquisition and development of the infant gut microbiota, to outline the knowledge recently gained through the use of culture-independent techniques and, in particular, highlight advances in high-throughput sequencing and how these technologies have, and will continue to, fill gaps in our knowledge with respect to the human intestinal microbiota.
    • Dairy Ingredients for the Baking Industry.

      Keogh, M.K.; Neville, D.; Stanton, Catherine; Auty, Mark; Kennedy, R.; Arendt, E. (Teagasc, 2001-08-01)
      Shortenings (baking fats), microencapsulated using dairy ingredients and milk protein hydrolysates, were produced for testing in a variety of baked products. The powders were evaluated for their functionality as powdered baking fats, as potential replacers of synthetic emulsifiers, as ingredients capable of improving baking performance or as potential health-enhancing ingredients. These studies provide the technology for the dairy industry to enter the specialised food ingredients sector with a siftable, non-greasy, free-flowing powdered fat for the baking industry.
    • A degenerate PCR-based strategy as a means of identifying homologues of aminoglycoside and ß-lactam resistance genes in the gut microbiota

      Fouhy, Fiona; Ross, R Paul; Fitzgerald, Gerald F; Stanton, Catherine; Cotter, Paul D. (Biomed Central, 2014-02-05)
      Background: The potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and β-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would determine if the gut microbiota of healthy adults, lacking recent antibiotic exposure, is a reservoir for resistance genes. Results: The strategy employed resulted in the identification of numerous aminoglycoside (acetylation, adenylation and phosphorylation) and β-lactam (including bla OXA, bla TEM, bla SHV and bla CTX-M) resistance gene homologues. On the basis of homology, it would appear that these genes originated from different bacterial taxa, with members of the Enterobacteriaceae being a particularly rich source. The results demonstrate that, even in the absence of recent antibiotic exposure, the human gut microbiota is a considerable reservoir for antibiotic resistance genes. Conclusions: This study has demonstrated that the gut can be a significant source of aminoglycoside and β-lactam resistance genes, even in the absence of recent antibiotic exposure. The results also demonstrate that PCR-based approaches can be successfully applied to detect antibiotic resistance genes in the human gut microbiota, without the need to isolate resistant strains. This approach could also be used to rapidly screen other complex environments for target genes.
    • Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay

      Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F.; Kelly, Philip M. (Cambridge University Press for the Institute of Food Research and the Hannah Research Institute, 2015-06-29)
      While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6·20 and 6·06 log CFU/ml, for raw bovine and human milks, respectively – the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.
    • Development of Technologies for Separation and Functional Improvement of Individual Milk Protein Fractions

      Stanton, Catherine; Fitzgerald, R.J.; Donnelly, W.J.; O'Connor, Paula M. (Teagasc, 1999-02-01)
      Milk proteins can be hydrolysed (i.e. fragmented) using proteolytic enzymes to give enhanced functional and nutritional properties. There is an increasing demand for hydrolysed protein ingredients with specific properties for nutrition of individuals with specialised dietary requirements including infants, the critically ill, the immuno-compromised and athletes. Such hydrolysed proteins can be specifically designed to provide distinctive tailor-made solutions to meet customer needs in these areas. This project explored the technologies for the production of two types of hydrolysates i.e. acid-soluble and glutamine-rich. Acid-soluble protein hydrolysates have potential in the fortification of acidic beverages, including soft drinks. Glutamine-rich hydrolysates are suggested as an optimal glutamine source for administration during periods of stress, such as recovery from strenuous exercise, or from surgery. Casein was selected as the protein for development of acid-soluble product and cereal protein for the glutamine-rich product. The main conclusions were as follows: A number of protein hydrolysate products with value added properties and the processes required for their manufacture have been developed and are available for uptake by the food industry. Laboratory investigations identified conditions for the generation of two casein hydrolysates with desirable functional properties. Scale-up conditions for the manufacture of these hydrolysates in the pilot plant were successfully developed. Both hydrolystates were 100% soluble at pH 4.6, exhibited clarity in solution at low pH in clear soft drinks and in caramelised beverages and were stable in solution over a wide temperature range (from 4 to 30ºC) for extended periods. Solutions containing these hydrolysates exhibited no foaming properties and had acceptable sensory properties, being considered as weakly bitter compared to unsupplemented solutions. These performance characteristics make the acid-soluble hydrolysates useful supplements for caramelised beverages, such as colas, and clear soft drinks. Six glutamine-enriched peptide products were produced at laboratory scale using two commercially available enzyme preparations. These products had desirable characteristics such as increased levels of peptide bound glutamine, low free amino acid and free pyroglutamate levels. Pilot plant processes were developed for manufacture of the two glutamine-rich hydrolysates with most suitable compositional properties and these were fully characterised chemically. The manufacturing process was modified to enable industrial scale batches (5,000 litres) to be produced.
    • Early Gut Microbiota Perturbations Following Intrapartum Antibiotic Prophylaxis to Prevent Group B Streptococcal Disease

      Mazzola, Giuseppe; Murphy, Kiera; Ross, R. Paul; Di Gioia, Diana; Biavati, Bruno; Corvaglia, Luigi T.; Faldella, Giacomo; Stanton, Catherine (PLOS, 2016-06-22)
      The faecal microbiota composition of infants born to mothers receiving intrapartum antibiotic prophylaxis with ampicillin against group B Streptococcus was compared with that of control infants, at day 7 and 30 of life. Recruited newborns were both exclusive breastfed and mixed fed, in order to also study the effect of dietary factors on the microbiota composition. Massive parallel sequencing of the V3-V4 region of the 16S rRNA gene and qPCR analysis were performed. Antibiotic prophylaxis caused the most marked changes on the microbiota in breastfed infants, mainly resulting in a higher relative abundance of Enterobacteriaceae, compared with control infants (52% vs. 14%, p = 0.044) and mixed-fed infants (52% vs. 16%, p = 0.13 NS) at day 7 and in a lower bacterial diversity compared to mixed-fed infants and controls. Bifidobacteria were also particularly vulnerable and abundances were reduced in breastfed (p = 0.001) and mixed-fed antibiotic treated groups compared to non-treated groups. Reductions in bifidobacteria in antibiotic treated infants were also confirmed by qPCR. By day 30, the bifidobacterial population recovered and abundances significantly increased in both breastfed (p = 0.025) and mixed-fed (p = 0.013) antibiotic treated groups, whereas Enterobacteriaceae abundances remained highest in the breastfed antibiotic treated group (44%), compared with control infants (16%) and mixed-fed antibiotic treated group (28%). This study has therefore demonstrated the short term consequences of maternal intrapartum antibiotic prophylaxis on the infant faecal microbial population, particularly in that of breastfed infants.
    • The Effect of Dietary Supplementation with Spent Cider Yeast on the Swine Distal Gut Microbiome

      Upadrasta, Aditya; O'Sulivan, Lisa; O'Sullivan, Orla; Sexton, Noel; Lawlor, Peadar G; Hill, Colin; Fitzgerald, Gerald F.; Stanton, Catherine; Ross, R Paul (PLOS, 2013-10-09)
      Background: There is an increasing need for alternatives to antibiotics for promoting animal health, given the increasing problems associated with antibiotic resistance. In this regard, we evaluated spent cider yeast as a potential probiotic for modifying the gut microbiota in weanling pigs using pyrosequencing of 16S rRNA gene libraries. Methodology and Principal Findings: Piglets aged 24–26 days were assigned to one of two study groups; control (n = 12) and treatment (n = 12). The control animals were fed with a basal diet and the treatment animals were fed with basal diet in combination with cider yeast supplement (500 ml cider yeast containing ,7.6 log CFU/ml) for 21 days. Faecal samples were collected for 16s rRNA gene compositional analysis. 16S rRNA compositional sequencing analysis of the faecal samples collected from day 0 and day 21 revealed marked differences in microbial diversity at both the phylum and genus levels between the control and treatment groups. This analysis confirmed that levels of Salmonella and Escherichia were significantly decreased in the treatment group, compared with the control (P,0.001). This data suggest a positive influence of dietary supplementation with live cider yeast on the microbial diversity of the pig distal gut. Conclusions/Significance: The effect of dietary cider yeast on porcine gut microbial communities was characterized for the first time using 16S rRNA gene compositional sequencing. Dietary cider yeast can potentially alter the gut microbiota, however such changes depend on their endogenous microbiota that causes a divergence in relative response to that given diet.
    • The Effects of Freezing on Faecal Microbiota as Determined Using MiSeq Sequencing and Culture-Based Investigations

      Fouhy, Fiona; Deane, Jennifer; Rea, Mary C.; O’Sullivan, Orla; Ross, R. Paul; O’Callaghan, Grace; Plant, Barry J.; Stanton, Catherine (PLoS, 2015-03-06)
      Background High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed. Methods Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed. Results No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples. Conclusions Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.
    • Effects of lipid-encapsulated conjugated linoleic acid supplementation on milk production, bioenergetic status and indicators of reproductive performance in lactating dairy cows

      Hutchinson, Ian A.; de Veth, Michael J.; Stanton, Catherine; Dewhurst, Richard J.; Lonergan, P.; Evans, A.C.O.; Butler, Stephen T. (Cambridge University Press: Published for the Institute of Food Research and the Hannah Research Institute, 2011-07)
      Conjugated linoleic acid (CLA) reduces mammary milk fat synthesis in a dose-dependent manner. Our objective was to determine the effects of lipid-encapsulated CLA (LE-CLA) supplementation on milk production, reproductive performance and metabolic responses in lactating dairy cows fed a grass silage-based diet. Seventy-two Holstein-Friesian cows (32 primiparous and 40 multiparous) were used in a completely randomized block design. Cows received either 80 g of LE-CLA daily or 60 g of calcium salts of palm fatty acids daily (control) from parturition until 60 days in milk. LE-CLA contained a 50:50 mix of cis-9,trans-11 CLA and trans-10,cis-12 CLA, resulting in a daily intake of 6 g of each isomer. Milk production and dry matter intake were recorded daily, and blood samples were collected 3-times a week. Blood samples were analysed for circulating concentrations of glucose, non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), insulin and insulin-like growth factor-I (IGF-I). Progesterone was measured in blood samples collected after the first post-partum insemination. Ovarian ultrasound examinations commenced at 8–10 d post partum and were carried out 3-times a week until first ovulation. LE-CLA treatment resulted in decreased milk fat concentration, with consequent improvements in energy balance and body condition score (BCS). The peak concentration of NEFA in blood was reduced by LE-CLA, but circulating concentrations of insulin, glucose, IGF-I, BHBA and progesterone were not affected. There was no effect of LE-CLA supplementation on the post-partum interval to first ovulation. Services per conception tended to be reduced. The reduction in milk energy output and improvement in energy status and BCS in LE-CLA-supplemented cows provides a strong rationale for further studies with greater cow numbers to test effects on reproductive performance.
    • Enhancing the stress responses of probiotics for a lifestyle from gut to product and back again

      Mills, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Ross, R Paul (Biomed Central, 2011-08-30)
      Before a probiotic bacterium can even begin to fulfill its biological role, it must survive a battery of environmental stresses imposed during food processing and passage through the gastrointestinal tract (GIT). Food processing stresses include extremes in temperature, as well as osmotic, oxidative and food matrix stresses. Passage through the GIT is a hazardous journey for any bacteria with deleterious lows in pH encountered in the stomach to the detergent-like properties of bile in the duodenum. However, bacteria are equipped with an array of defense mechanisms to counteract intracellular damage or to enhance the robustness of the cell to withstand lethal external environments. Understanding these mechanisms in probiotic bacteria and indeed other bacterial groups has resulted in the development of a molecular toolbox to augment the technological and gastrointestinal performance of probiotics. This has been greatly aided by studies which examine the global cellular responses to stress highlighting distinct regulatory networks and which also identify novel mechanisms used by cells to cope with hazardous environments. This review highlights the latest studies which have exploited the bacterial stress response with a view to producing next-generation probiotic cultures and highlights the significance of studies which view the global bacterial stress response from an integrative systems biology perspective.
    • Erratum to: Evolution of gut microbiota composition from birth to 24 weeks in the INFANTMET Cohort

      Hill, Cian J; Lynch, Denise B; Murphy, Kiera; Ulaszewska, Marynka; Jeffery, Ian B; O’Shea, Carol A; Watkins, Claire; Dempsey, Eugene; Mattivi, Fulvio; Tuohy, Kieran; Ross, R. P; Ryan, C. A; O’Toole, Paul W; Stanton, Catherine (Biomed Central, 2017-02-14)
      Erratum Following publication of this article [1], it has come to our attention that the name of the author Kieran Tuohy’s name was captured incorrectly as “Touhy” and instead should be Kieran Tuohy.