• Dietary Compounds Influencing the Sensorial, Volatile and Phytochemical Properties of Bovine Milk

      Clarke, Holly J.; Griffin, Carol; Rai, Dilip K.; O’Callaghan, Tom F.; O’Sullivan, Maurice G.; Kerry, Joseph P.; Kilcawley, Kieran N.; Teagasc Walsh Fellowship Programme; 2016071 (MDPI AG, 2019-12-19)
      The main aim of this study was to evaluate the volatile profile, sensory perception, and phytochemical content of bovine milk produced from cows fed on three distinct feeding systems, namely grass (GRS), grass/clover (CLV), and total mixed ration (TMR). Previous studies have identified that feed type can influence the sensory perception of milk directly via the transfer of volatile aromatic compounds, or indirectly by the transfer of non-volatile substrates that act as precursors for volatile compounds. In the present study, significant differences were observed in the phytochemical profile of the different feed and milk samples. The isoflavone formonoetin was significantly higher in CLV feed samples, but higher in raw GRS milk, while other smaller isoflavones, such as daidzein, genistein, and apigenin were highly correlated to raw CLV milk. This suggests that changes in isoflavone content and concentration in milk relate to diet, but also to metabolism in the rumen. This study also found unique potential volatile biomarkers in milk (dimethyl sulfone) related to feeding systems, or significant differences in the concentration of others (toluene, p-cresol, ethyl and methyl esters) based on feeding systems. TMR milk scored significantly higher for hay-like flavor and white color, while GRS and CLV milk scored significantly higher for a creamy color. Milk samples were easily distinguishable by their volatile profile based on feeding system, storage time, and pasteurization.
    • Draft Genome Sequences of Macrococcus caseolyticus, Macrococcus canis, Macrococcus bohemicus, and Macrococcus goetzii

      Mazhar, Shahneela; Altermann, Eric; Hill, Colin; McAuliffe, Olivia; Teagasc Walsh Fellowship Programme; Teagasc; 2015055; 6697 (American Society for Microbiology, 2019-05-09)
      Here, we present the draft genome sequences of 14 strains of 4 species of the genus Macrococcus. These strains were isolated from bovine milk and tongue samples obtained during a screening program.
    • Draft Genome Sequences of the Type Strains of Six Macrococcus Species

      Mazhar, Shahneela; Altermann, Eric; Hill, Colin; McAuliffe, Olivia; Teagasc Walsh Fellowship Programme; Teagasc; 2015055; 6697 (American Society for Microbiology, 2019-05-09)
      We report here the draft genome sequences of Macrococcus bovicus ATCC 51825T, Macrococcus carouselicus ATCC 51828T, Macrococcus equipercicus ATCC 51831T, Macrococcus brunensis CCM4811T, Macrococcus hajekii CCM4809T, and Macrococcus lamae CCM4815T. The availability of the genome sequences of these species will enable cross-species comparison, which could lead to a more comprehensive understanding of organisms of the Macrococcus genus.
    • Draft Genome Sequences of Three Lactobacillus paracasei Strains, Members of the Nonstarter Microbiota of Mature Cheddar Cheese

      Stefanovic, Ewelina; Fitzgerald, Gerald F; McAuliffe, Olivia; Teagasc Walsh Fellowship Programme (American Society for Microbiology, 20/07/2017)
      Lactobacillus paracasei strains are common members of the nonstarter microbiota present in various types of cheeses. The draft genome sequences of three strains isolated from mature cheddar cheeses are reported here.
    • 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; Department of Agriculture, Food and the Marine; European Union; et al. (PLOS, 22/06/2016)
      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 Feeding Bt MON810 Maize to Pigs for 110 Days on Intestinal Microbiota

      Buzoianu, Stefan G.; Walsh, Maria C.; Rea, Mary; O'Sullivan, Orla; Crispie, Fiona; Cotter, Paul D.; Ross, R Paul; Gardiner, Gillian E.; Lawlor, Peadar G; European Union; et al. (PLOS, 04/05/2012)
      Objective To assess the effects of feeding Bt MON810 maize to pigs for 110 days on the intestinal microbiota. Methodology/Principal Findings Forty male pigs (~40 days old) were blocked by weight and litter ancestry and assigned to one of four treatments; 1) Isogenic maize-based diet for 110 days (Isogenic); 2) Bt maize-based diet (MON810) for 110 days (Bt); 3) Isogenic maize-based diet for 30 days followed by a Bt maize-based diet for 80 days (Isogenic/Bt); 4) Bt maize-based diet for 30 days followed by an isogenic maize-based diet for 80 days (Bt/Isogenic). Enterobacteriaceae, Lactobacillus and total anaerobes were enumerated in the feces using culture-based methods on days 0, 30, 60 and 100 of the study and in ileal and cecal digesta on day 110. No differences were found between treatments for any of these counts at any time point. The relative abundance of cecal bacteria was also determined using high-throughput 16 S rRNA gene sequencing. No differences were observed in any bacterial taxa between treatments, with the exception of the genus Holdemania which was more abundant in the cecum of pigs fed the isogenic/Bt treatment compared to pigs fed the Bt treatment (0.012 vs 0.003%; P≤0.05). Conclusions/Significance Feeding pigs a Bt maize-based diet for 110 days did not affect counts of any of the culturable bacteria enumerated in the feces, ileum or cecum. Neither did it influence the composition of the cecal microbiota, with the exception of a minor increase in the genus Holdemania. As the role of Holdemania in the intestine is still under investigation and no health abnormalities were observed, this change is not likely to be of clinical significance. These results indicate that feeding Bt maize to pigs in the context of its influence on the porcine intestinal microbiota is safe.
    • Effect of milk centrifugation and incorporation of high heat-treated centrifugate on the microbial composition and levels of volatile organic compounds of Maasdam cheese

      Lamichhane, Prabin; Pietrzyk, Anna; Feehily, Conor; Cotter, Paul D.; Mannion, David T.; Kilcawley, Kieran; Kelly, Alan L.; Sheehan, Diarmuid (JJ); Dairy Levy Trust; Teagasc Walsh Fellowship Programme; et al. (Elsevier, 2018-03-15)
      Centrifugation is a common milk pretreatment method for removal of Clostridium spores which, on germination, can produce high levels of butyric acid and gas, resulting in rancid, gassy cheese. The aim of this study was to determine the effect of centrifugation of milk, as well as incorporation of high heat-treated centrifugate into cheese milk, on the microbial and volatile profile of Maasdam cheese. To facilitate this, 16S rRNA amplicon sequencing in combination with a selective media-based approach were used to study the microbial composition of cheese during maturation, and volatile organic compounds within the cheese matrix were analyzed by HPLC and solid-phase microextraction coupled with gas chromatography–mass spectrometry. Both culture-based and molecular approaches revealed major differences in microbial populations within the cheese matrix before and after warm room ripening. During warm room ripening, an increase in counts of propionic acid bacteria (by ∼101.5 cfu) and nonstarter lactic acid bacteria (by ∼108 cfu) and a decrease in the counts of Lactobacillus helveticus (by ∼102.5 cfu) were observed. Lactococcus species dominated the curd population throughout ripening, followed by Lactobacillus, Propionibacterium, and Leuconostoc, and the relative abundance of these accounted for more than 99% of the total genera, as revealed by high-throughput sequencing. Among subdominant microflora, the overall relative abundance of Clostridium sensu stricto was lower in cheeses made from centrifuged milk than control cheeses, which coincided with lower levels of butyric acid. Centrifugation as well as incorporation of high heat-treated centrifugate into cheese milk seemed to have little effect on the volatile profile of Maasdam cheese, except for butyric acid levels. Overall, this study suggests that centrifugation of milk before cheesemaking is a suitable method for controlling undesirable butyric acid fermentation without significantly altering the levels of other volatile organic compounds of Maasdam cheese.
    • Effect of pre-treatment on the generation of dipeptidyl peptidase-IV- and prolyl endopeptidase-inhibitory hydrolysates from bovine lung

      Lafarga, Tomas; Hayes, Maria; Department of Agriculture, Food and the Marine, Ireland; Teagasc Walsh Fellowship Programme; 11/F/043 (Teagasc (Agriculture and Food Development Authority), Ireland, 25/05/2017)
      The aim of this work was to study the effect of two different pre-treatments, high temperature (100 °C, 5 min) and high pressure (600 MPa, 3 min), on the potential of the enzymes papain, collagenase and Alcalase® to generate bioactive hydrolysates containing dipeptidyl peptidase-IV- (DPP-IV; EC 3.4.14.5) and prolyl endopeptidase- (PEP; EC 3.4.21.26) inhibitory peptides from bovine lung. Both pre-treatments resulted in an increase in the degree of hydrolysis over a 24 h period (P < 0.001) and significantly increased the DPP-IV- and PEP-inhibitory activities of the generated hydrolysates (P < 0.001). Generated hydrolysates included an Alcalase hydrolysate of pressure-treated bovine lung, which was the most active, and showed DPP-IV and PEP half-maximal inhibitory concentration (IC50) values of 1.43 ± 0.06 and 3.62 ± 0.07 mg/ mL, respectively. The major peptides contained in this hydrolysate were determined by liquid chromatography-tandem mass spectrometry, and results demonstrated that bovine lung is a good substrate for the release of bioactive peptides when proper pre-treatment and enzymatic treatment are applied.
    • The effect of Pulsed Electric Field as a pre-treatment step in Ultrasound Assisted Extraction of phenolic compounds from fresh rosemary and thyme by-products, Innovative Food Science and Emerging Technologies

      Tzima, Katerina; Brunton, Nigel P.; Lyng, James G.; Frontuto, Daniele; Rai, Dilip K.; Teagasc Walsh Fellowship Programme; 2016038 (Elsevier, 2021-02-22)
      Emerging extraction techniques, including pulsed electric field (PEF) and ultrasound (US), are attracting considerable interest in the recovery of bioactives. Though, limited work has focused on PEF application as pre-treatment for US assisted extraction to enhance the release of phenolics from herbs. Hence, the present study investigated the use of an optimized PEF pre-treatment to enhance the recovery of phenolics from fresh rosemary and thyme by-products in a subsequent US assisted extraction step. Total phenolic content (TPC), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and ferric reducing antioxidant power (FRAP) were assessed as an index of extraction efficacy. Qualitative and quantitative analyses were performed through liquid chromatography-mass spectrometry analyses to evaluate the influence of the methods on individual phenolic compounds and the formation of potential derivatives. The results indicated that in a number of cases PEF pre-treatment enhanced (p < 0.05) the recovery of phenolic compounds and antioxidant capacity compared to US individually.
    • Evaluation of the Potential of Lactobacillus paracasei Adjuncts for Flavor Compounds Development and Diversification in Short-Aged Cheddar Cheese

      Stefanovic, Ewelina; Kilcawley, Kieran; Roces, Clara; Rea, Mary; O'Sullivan, Maurice G.; Sheehan, Diarmuid (JJ); McAuliffe, Olivia; Teagasc Walsh Fellowship Programme; 2012040 (Frontiers, 05/07/2018)
      The non-starter microbiota of Cheddar cheese mostly comprises mesophilic lactobacilli, such as Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus plantarum. These bacteria are recognized for their potential to improve Cheddar cheese flavor when used as adjunct cultures. In this study, three strains of L. paracasei (DPC2071, DPC4206, and DPC4536) were evaluated for their contribution to the enhancement and diversification of flavor in short-aged Cheddar cheese. The strains were selected based on their previously determined genomic diversity, variability in proteolytic enzyme activities and metabolic capability in cheese model systems. The addition of adjunct cultures did not affect the gross composition or levels of lipolysis of the cheeses. The levels of free amino acids (FAA) in cheeses showed a significant increase after 28 days of ripening. However, the concentrations of individual amino acids in the cheeses did not significantly differ except for some amino acids (aspartic acid, threonine, serine, and tryptophan) at Day 14. Volatile profile analysis revealed that the main compounds that differentiated the cheeses were of lipid origin, such as long chain aldehydes, acids, ketones, and lactones. This study demonstrated that the adjunct L. paracasei strains contributed to the development and diversification of compounds related to flavor in short-aged Cheddar cheeses.
    • Evaluation of the Potential of Lactobacillus paracasei Adjuncts for Flavor Compounds Development and Diversification in Short-Aged Cheddar Cheese

      Stefanovic, Ewelina; Kilcawley, Kieran; Roces, Clara; Rea, Mary; O'Sullivan, Maurice G.; Sheehan, Diarmuid (JJ); McAuliffe, Olivia; Teagasc Walsh Fellowship Programme; 2012040 (Frontiers, 2018-07-05)
      The non-starter microbiota of Cheddar cheese mostly comprises mesophilic lactobacilli, such as Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus plantarum. These bacteria are recognized for their potential to improve Cheddar cheese flavor when used as adjunct cultures. In this study, three strains of L. paracasei (DPC2071, DPC4206, and DPC4536) were evaluated for their contribution to the enhancement and diversification of flavor in short-aged Cheddar cheese. The strains were selected based on their previously determined genomic diversity, variability in proteolytic enzyme activities and metabolic capability in cheese model systems. The addition of adjunct cultures did not affect the gross composition or levels of lipolysis of the cheeses. The levels of free amino acids (FAA) in cheeses showed a significant increase after 28 days of ripening. However, the concentrations of individual amino acids in the cheeses did not significantly differ except for some amino acids (aspartic acid, threonine, serine, and tryptophan) at Day 14. Volatile profile analysis revealed that the main compounds that differentiated the cheeses were of lipid origin, such as long chain aldehydes, acids, ketones, and lactones. This study demonstrated that the adjunct L. paracasei strains contributed to the development and diversification of compounds related to flavor in short-aged Cheddar cheeses.
    • Exploitation of SPR to Investigate the Importance of Glycan Chains in the Interaction between Lactoferrin and Bacteria

      O'Riordan, Noelle; Kilcoyne, Michelle; Joshi, Lokesh; Hickey, Rita M.; Teagasc Walsh Fellowship Programme (MDPI, 2017-06-27)
      Bovine lactoferrin (LF) has been shown to prevent adhesion to and invasion of mammalian cell lines by pathogenic bacteria, with evidence for direct bacterial binding by the milk glycoprotein. However, the glycosylation pattern of LF changes over the lactation cycle. In this study, we aim to investigate the effect that this variation has on the milk glycoprotein’s ability to interact with pathogens. Surface plasmon resonance technology was employed to compare the binding of LF from colostrum (early lactation) and mature milk (late lactation) to a panel of pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Cronobacter sakazakii, Streptococcus pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes and Salmonella typhimurium). Novel interactions with LF were identified for C. sakazakii, S. pneumoniae and P. aeruginosa with the highest binding ability observed for mature milk LF in all cases, with the exception of S. typhimurium. The difference in bacterial binding observed may be as a result of the varying glycosylation profiles. This work demonstrates the potential of LF as a functional food ingredient to prevent bacterial infection
    • Extensive bacteriocin gene shuffling in the Streptococcus bovis/Streptococcus equinus complex reveals gallocin D with activity against vancomycin resistant enterococci

      Hill, Daragh; O’Connor, Paula M.; Altermann, Eric; Day, Li; Hill, Colin; STANTON, CATHERINE; Ross, Paul; Teagasc Walsh Fellowship Programme; Science Foundation Ireland; SFI/12/RC/2273 (Springer Science and Business Media LLC, 2020-08-10)
      Streptococcus gallolyticus LL009 produces gallocin D, a narrow spectrum two component bacteriocin with potent activity against vancomycin-resistant enterococci. Gallocin D is distinct from gallocin A, a separate two component bacteriocin produced by S. gallolyticus. Although the gene clusters encoding gallocin A and gallocin D have a high degree of gene synteny, the structural genes are highly variable and appear to have undergone gene shufing with other streptococcal species. Gallocin D was analysed in laboratory-based experiments. The mature peptides are 3,343± 1 Da and 3,019 ± 1 Da and could be readily synthesized and display activity against a vancomycin resistant Enterococcus strain EC300 with a MIC value of 1.56 µM. Importantly, these bacteriocins could contribute to the ability of S. gallolyticus to colonize the colon where they have been associated with colorectal cancer.
    • Gamma-aminobutyric acid-producing lactobacilli positively affect metabolism and depressive-like behaviour in a mouse model of metabolic syndrome

      Patterson, E.; Ryan, P. M.; Wiley, N.; Carafa, I.; Sherwin, E.; Moloney, G.; Franciosi, E.; Mandal, R.; Wishart, D. S.; Tuohy, K.; et al. (Springer Science and Business Media LLC, 2019-11-08)
      Metabolic and neuroactive metabolite production represents one of the mechanisms through which the gut microbiota can impact health. One such metabolite, gamma-aminobutyric acid (GABA), can modulate glucose homeostasis and alter behavioural patterns in the host. We previously demonstrated that oral administration of GABA-producing Lactobacillus brevis DPC6108 has the potential to increase levels of circulating insulin in healthy rats. Therefore, the objective of this study was to assess the efcacy of endogenous microbial GABA production in improving metabolic and behavioural outcomes in a mouse model of metabolic dysfunction. Diet-induced obese and metabolically dysfunctional mice received one of two GABA-producing strains, L. brevis DPC6108 or L. brevis DSM32386, daily for 12 weeks. After 8 and 10 weeks of intervention, the behavioural and metabolic profles of the mice were respectively assessed. Intervention with both L. brevis strains attenuated several abnormalities associated with metabolic dysfunction, causing a reduction in the accumulation of mesenteric adipose tissue, increased insulin secretion following glucose challenge, improved plasma cholesterol clearance and reduced despair-like behaviour and basal corticosterone production during the forced swim test. Taken together, this exploratory dataset indicates that intervention with GABA-producing lactobacilli has the potential to improve metabolic and depressive- like behavioural abnormalities associated with metabolic syndrome in mice.
    • Generation of Bioactive Hydrolysates and Peptides from Bovine Hemoglobin with In Vitro Renin, Angiotensin-I-Converting Enzyme and Dipeptidyl Peptidase-IV Inhibitory Activities

      Lafarga, Tomas; Rai, Dilip K.; O'Connor, Paula M.; Hayes, Maria; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 11/F/043 (Wiley, 02/03/2016)
      Bovine hemoglobin was selected for use in the generation of bioactive hydrolysates with potential for use as functional food ingredients for prevention of disorders such as hypertension, obesity and diabetes. Bovine hemoglobin was isolated and hydrolyzed with papain, which was selected using in silico analysis. The generated hydrolysate was enriched by ultrafiltration and further purified by high performance liquid chromatography. A number of peptides were identified using de novo peptide sequencing and these peptides were chemically synthesized to confirm their bioactivity in vitro. Three multifunctional peptides with both, ACE-I and renin-inhibitory properties and one peptide with ACE-I-inhibiting properties were identified. These included the di-peptide HR with ACE-I and renin IC50 values of 0.19 and 7.09 mM, respectively. The generated papain hydrolysate of bovine hemoglobin not only inhibited the enzymes ACE-I and renin but also the enzyme DPP-IV, which has been linked to type-2 diabetes.
    • Genome Sequence of Staphylococcus saprophyticus DPC5671, a Strain Isolated from Cheddar Cheese

      Bertuzzi, Andrea; Guinane, Caitriona M.; Crispie, Fiona; Kilcawley, Kieran; McSweeney, Paul L. H.; Rea, Mary; Teagasc Walsh Fellowship Programme (American Society for Microbiology, 20/04/2017)
      The draft genome sequence of Staphylococcus saprophyticus DPC5671, isolated from cheddar cheese, was determined. S. saprophyticus is a common Gram-positive bacterium detected on the surface of smear-ripened cheese and other fermented foods.
    • Genomic Characterization of Sulphite Reducing Bacteria Isolated From the Dairy Production Chain

      Doyle, Conor J.; O'Toole, Paul W.; Cotter, Paul D.; Teagasc Walsh Fellowship Programme; 2013030 (Frontiers, 2018-07-05)
      Anaerobic sporeformers, specifically spoilage and pathogenic members of the genus Clostridium, are a concern for producers of dairy products, and of powdered dairy products in particular. As an alternative to testing for individual species, the traditional, and still current, approach to detecting these sporeformers, including non-spoilage/non-pathogenic species, in dairy products has involved testing for a sulphite reducing phenotype [Sulphite reducing Clostridia (SRCs)] under anaerobic conditions. This phenotype is conserved throughout the Order Clostridia. Unfortunately, however, this phenotype is exhibited by other sulphite reducing bacteria (SRBs) also, potentially leading to potential for false positives. Here, this risk was borne out through the identification of several SRBs from industry samples that were identified as Proteus mirabilis and various Bacillus/Paenibacillus sp. Genome wide comparison of a number of representative SRCs and SRBs was employed to determine phylogenetic relationships, especially among SRCs, and to characterize the genes responsible for the sulphite reducing phenotype. This screen identified two associated operons, i.e., asrABC in SRCs, and cysJI in Bacillus/Paenibacillus spp. and P. mirabilis. This screen identified spp. belonging to sensu stricto, Lachnospiraceae and Cluster XIV of the Clostridia all producing the SRC phenotype. This study highlights the inaccuracy of the industry standard SRC test but highlights the potential to generate an equivalent molecular test designed to detect the genes responsible for this phenotype in clostridia.
    • The Gut Microbiota Composition in Dichorionic Triplet Sets Suggests a Role for Host Genetic Factors

      Murphy, Kiera; O'Shea, Carol A; Ryan, C. Anthony; Dempsey, Eugene; O'Toole, Paul W.; STANTON, CATHERINE; Ross, R Paul; Department of Agriculture, Food and the Marine; Science Foundation Ireland; Teagasc Walsh Fellowship Programme; et al. (PLoS, 14/04/2015)
      Monozygotic and dizygotic twin studies investigating the relative roles of host genetics and environmental factors in shaping gut microbiota composition have produced conflicting results. In this study, we investigated the gut microbiota composition of a healthy dichorionic triplet set. The dichorionic triplet set contained a pair of monozygotic twins and a fraternal sibling, with similar pre- and post-natal environmental conditions including feeding regime. V4 16S rRNA and rpoB amplicon pyrosequencing was employed to investigate microbiota composition, and the species and strain diversity of the culturable bifidobacterial population was also examined. At month 1, the monozygotic pair shared a similar microbiota distinct to the fraternal sibling. By month 12 however, the profile was more uniform between the three infants. Principal coordinate analysis (PCoA) of the microbiota composition revealed strong clustering of the monozygotic pair at month 1 and a separation of the fraternal infant. At months 2 and 3 the phylogenetic distance between the monozygotic pair and the fraternal sibling has greatly reduced and by month 12 the monozygotic pair no longer clustered separately from the fraternal infant. Pulse field gel electrophoresis (PFGE) analysis of the bifidobacterial population revealed a lack of strain diversity, with identical strains identified in all three infants at month 1 and 12. The microbiota of two antibiotic-treated dichorionic triplet sets was also investigated. Not surprisingly, in both triplet sets early life antibiotic administration appeared to be a major determinant of microbiota composition at month 1, irrespective of zygosity. By month 12, early antibiotic administration appeared to no longer exert such a strong influence on gut microbiota composition. We hypothesize that initially host genetics play a significant role in the composition of an individual’s gut microbiota, unless an antibiotic intervention is given, but by month 12 environmental factors are the major determinant.
    • High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel; Fallico, Vincenzo; O'Sullivan, Orla; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Giblin, Linda; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 2012205 (Biomed Central, 17/11/2015)
      Background The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.
    • High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel; Fallico, Vincenzo; O'Sullivan, Orla; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Giblin, Linda; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 2012205 (Biomed Central, 17/11/2015)
      Background: The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results: Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions: In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.