• 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.; et al. (PLOS, 20/11/2012)
      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.
    • Comparative genomics of lactic acid bacteria reveals a niche-specific gene set

      O'Sullivan, Orla; O'Callaghan, John; Sangrador-Vegas, Amaia; McAuliffe, Olivia; Slattery, Lydia; Kaleta, Pawel; Callanan, Michael J.; Fitzgerald, Gerald F; Ross, R Paul; Beresford, Tom (Biomed Central, 05/03/2009)
      Background: The recently sequenced genome of Lactobacillus helveticus DPC4571 1 revealed a dairy organism with significant homology (75% of genes are homologous) to a probiotic bacteria Lb. acidophilus NCFM 2. This led us to hypothesise that a group of genes could be determined which could define an organism's niche. Results: Taking 11 fully sequenced lactic acid bacteria (LAB) as our target, (3 dairy LAB, 5 gut LAB and 3 multi-niche LAB), we demonstrated that the presence or absence of certain genes involved in sugar metabolism, the proteolytic system, and restriction modification enzymes were pivotal in suggesting the niche of a strain. We identified 9 niche specific genes, of which 6 are dairy specific and 3 are gut specific. The dairy specific genes identified in Lactobacillus helveticus DPC4571 were lhv_1161 and lhv_1171, encoding components of the proteolytic system, lhv_1031 lhv_1152, lhv_1978 and lhv_0028 encoding restriction endonuclease genes, while bile salt hydrolase genes lba_0892 and lba_1078, and the sugar metabolism gene lba_1689 from Lb. acidophilus NCFM were identified as gut specific genes. Conclusion: Comparative analysis revealed that if an organism had homologs to the dairy specific geneset, it probably came from a dairy environment, whilst if it had homologs to gut specific genes, it was highly likely to be of intestinal origin. We propose that this "barcode" of 9 genes will be a useful initial guide to researchers in the LAB field to indicate an organism's ability to occupy a specific niche.
    • 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.; Irish Research Council for Science, Engineering and Technology; Teagasc Walsh Fellowship Programme; Science Foundation Ireland; 11/PI/1137 (Landes Bioscience, 01/05/2012)
      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.
    • 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.; Irish Research Council; Teagasc Walsh Fellowship Programme; Science Foundation Ireland; 11/PI/1137 (Biomed Central, 05/02/2014)
      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; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine (Cambridge University Press for the Institute of Food Research and the Hannah Research Institute, 29/06/2015)
      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.
    • 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.
    • 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; Enterprise Ireland; et al. (PLOS, 09/10/2013)
      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.
    • 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, 30/08/2011)
      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.
    • High-Throughput Sequencing Reveals the Incomplete, Short-Term Recovery of Infant Gut Microbiota following Parenteral Antibiotic Treatment with Ampicillin and Gentamicin

      Fouhy, Fiona; Guinane, Caitriona M.; Hussey, Seamus; Wall, Rebecca; Ryan, C. Anthony; Dempsey, Eugene; Murphy, Brendan; Ross, R Paul; Fitzgerald, Gerald F; STANTON, CATHERINE; et al. (American Society for Microbiology, 04/09/2012)
      The infant gut microbiota undergoes dramatic changes during the first 2 years of life. The acquisition and development of this population can be influenced by numerous factors, and antibiotic treatment has been suggested as one of the most significant. Despite this, however, there have been relatively few studies which have investigated the short-term recovery of the infant gut microbiota following antibiotic treatment. The aim of this study was to use high-throughput sequencing (employing both 16S rRNA and rpoB-specific primers) and quantitative PCR to compare the gut microbiota of nine infants who underwent parenteral antibiotic treatment with ampicillin and gentamicin (within 48 h of birth), 4 and 8 weeks after the conclusion of treatment, relative to that of nine matched healthy controls. The investigation revealed that the gut microbiota of the antibiotic-treated infants had significantly higher proportions of Proteobacteria (P = 0.0049) and significantly lower proportions of Actinobacteria (P = 0.00001) (and the associated genus Bifidobacterium [P = 0.0132]) as well as the genus Lactobacillus (P = 0.0182) than the untreated controls 4 weeks after the cessation of treatment. By week 8, the Proteobacteria levels remained significantly higher in the treated infants (P = 0.0049), but the Actinobacteria, Bifidobacterium, and Lactobacillus levels had recovered and were similar to those in the control samples. Despite this recovery of total Bifidobacterium numbers, rpoB-targeted pyrosequencing revealed that the number of different Bifidobacterium species present in the antibiotic-treated infants was reduced. It is thus apparent that the combined use of ampicillin and gentamicin in early life can have significant effects on the evolution of the infant gut microbiota, the long-term health implications of which remain unknown.
    • Host Specific Diversity in Lactobacillus johnsonii as Evidenced by a Major Chromosomal Inversion and Phage Resistance Mechanisms

      Guinane, Caitriona M.; Kent, Robert M.; Norberg, Sarah; Hill, Colin; Fitzgerald, Gerald F; STANTON, CATHERINE; Ross, R Paul; Enterprise Ireland; CFTD/07/116 (PLOS, 20/04/2011)
      Genetic diversity and genomic rearrangements are a driving force in bacterial evolution and niche adaptation. We sequenced and annotated the genome of Lactobacillus johnsonii DPC6026, a strain isolated from the porcine intestinal tract. Although the genome of DPC6026 is similar in size (1.97mbp) and GC content (34.8%) to the sequenced human isolate L. johnsonii NCC 533, a large symmetrical inversion of approximately 750 kb differentiated the two strains. Comparative analysis among 12 other strains of L. johnsonii including 8 porcine, 3 human and 1 poultry isolate indicated that the genome architecture found in DPC6026 is more common within the species than that of NCC 533. Furthermore a number of unique features were annotated in DPC6026, some of which are likely to have been acquired by horizontal gene transfer (HGT) and contribute to protection against phage infection. A putative type III restriction-modification system was identified, as were novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) elements. Interestingly, these particular elements are not widely distributed among L. johnsonii strains. Taken together these data suggest intra-species genomic rearrangements and significant genetic diversity within the L. johnsonii species and indicate towards a host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure.
    • In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

      Fouhy, Fiona; O'Connell-Motherway, Mary; Fitzgerald, Gerald F; Ross, R Paul; STANTON, CATHERINE; van Sinderen, Douwe; Cotter, Paul D.; Irish Research Council for Science, Engineering and Technology; Science Foundation Ireland; Health Research Board; et al. (PLoS, 06/12/2013)
      Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.
    • Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model

      Marques, T. M.; Patterson, E.; Wall, Rebecca; O'Sullivan, Orla; Fitzgerald, Gerald F; Cotter, Paul D.; Dinan, Timothy G.; Cryan, John F.; Ross, R Paul; STANTON, CATHERINE; et al. (Wageningen Academic Publishers, 26/05/2016)
      The aim of this study was to investigate if dietary administration of γ-aminobutyric acid (GABA)-producing Lactobacillus brevis DPC 6108 and pure GABA exert protective effects against the development of diabetes in streptozotocin (STZ)-induced diabetic Sprague Dawley rats. In a first experiment, healthy rats were divided in 3 groups (n=10/group) receiving placebo, 2.6 mg/kg body weight (bw) pure GABA or L. brevis DPC 6108 (~109microorganisms). In a second experiment, rats (n=15/group) were randomised to five groups and four of these received an injection of STZ to induce type 1 diabetes. Diabetic and non-diabetic controls received placebo [4% (w/v) yeast extract in dH2O], while the other three diabetic groups received one of the following dietary supplements: 2.6 mg/kg bw GABA (low GABA), 200 mg/kg bw GABA (high GABA) or ~109 L. brevis DPC 6108. L. brevis DPC 6108 supplementation was associated with increased serum insulin levels (P<0.05), but did not alter other metabolic markers in healthy rats. Diabetes induced by STZ injection decreased body weight (P<0.05), increased intestinal length (P<0.05) and stimulated water and food intake. Insulin was decreased (P<0.05), whereas glucose was increased (P<0.001) in all diabetic groups, compared with non-diabetic controls. A decrease (P<0.01) in glucose levels was observed in diabetic rats receiving L. brevis DPC 6108, compared with diabetic-controls. Both the composition and diversity of the intestinal microbiota were affected by diabetes. Microbial diversity in diabetic rats supplemented with low GABA was not reduced (P>0.05), compared with non-diabetic controls while all other diabetic groups displayed reduced diversity (P<0.05). L. brevis DPC 6108 attenuated hyperglycaemia induced by diabetes but additional studies are needed to understand the mechanisms involved in this reduction.
    • Microbiome and metabolome modifying effects of several cardiovascular disease interventions in apo-E−/− mice

      Ryan, Paul M; London, Lis E E; Bjorndahl, Trent C; Mandal, Rupasri; Murphy, Kiera; Fitzgerald, Gerald F; Shanahan, Fergus; Ross, R Paul; Wishart, David S; Caplice, Noel M; et al. (Biomed Central, 13/03/2017)
      Background There is strong evidence indicating that gut microbiota have the potential to modify, or be modified by the drugs and nutritional interventions that we rely upon. This study aims to characterize the compositional and functional effects of several nutritional, neutraceutical, and pharmaceutical cardiovascular disease interventions on the gut microbiome, through metagenomic and metabolomic approaches. Apolipoprotein-E-deficient mice were fed for 24 weeks either high-fat/cholesterol diet alone (control, HFC) or high-fat/cholesterol in conjunction with one of three dietary interventions, as follows: plant sterol ester (PSE), oat β-glucan (OBG) and bile salt hydrolase-active Lactobacillus reuteri APC 2587 (BSH), or the drug atorvastatin (STAT). The gut microbiome composition was then investigated, in addition to the host fecal and serum metabolome. Results We observed major shifts in the composition of the gut microbiome of PSE mice, while OBG and BSH mice displayed more modest fluctuations, and STAT showed relatively few alterations. Interestingly, these compositional effects imparted by PSE were coupled with an increase in acetate and reduction in isovalerate (p < 0.05), while OBG promoted n-butyrate synthesis (p < 0.01). In addition, PSE significantly dampened the microbial production of the proatherogenic precursor compound, trimethylamine (p < 0.05), attenuated cholesterol accumulation, and nearly abolished atherogenesis in the model (p < 0.05). However, PSE supplementation produced the heaviest mice with the greatest degree of adiposity (p < 0.05). Finally, PSE, OBG, and STAT all appeared to have considerable impact on the host serum metabolome, including alterations in several acylcarnitines previously associated with a state of metabolic dysfunction (p < 0.05). Conclusions We observed functional alterations in microbial and host-derived metabolites, which may have important implications for systemic metabolic health, suggesting that cardiovascular disease interventions may have a significant impact on the microbiome composition and functionality. This study indicates that the gut microbiome-modifying effects of novel therapeutics should be considered, in addition to the direct host effects.
    • Molecular Characterisation of Bacteriophage K Towards Applications for the Biocontrol of Pathogenic Staphylococci

      O'Flaherty, Sarah; Flynn, James; Coffey, Aidan; Fitzgerald, Gerald F; Meaney, William J; Ross, R Paul (Teagasc, 01/01/2006)
      The aim of this work was to characterise staphylococcal bacteriophage (a bacterial virus) and to assess their potential as therapeutic agents against pathogenic strains of Staphylococcus aureus, particularly mastitis-causing strains. The project included the use of two newly isolated phage CS1 and DW2, and an existing polyvalent phage. The new phage were isolated from the farmyard and characterised by electron microscopy and restriction analysis. Both phage were shown to belong to the Siphoviridae family and were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. A cocktail of three phage (CS1, DW2 and K) at 108 (plaque forming units) PFU/ml was infused into cows teats in animal trials. The lack of an increase in somatic cell counts in milks indicated strongly that the phage did not irritate the animal. In addition, the most potent phage used in this study, phage K, was further studied by genome sequencing, which revealed a linear DNA genome of 127,395 base pairs, which encodes 118 putative ORFs (open reading frames).
    • Myosin-cross-reactive antigen (MCRA) protein from Bifidobacterium breve is a FAD-dependent fatty acid hydratase which has a function in stress protection

      Rosberg-Cody, Eva; Liavonchanka, Alena; Gobel, Cornelia; Ross, R Paul; O'Sullivan, Orla; Fitzgerald, Gerald F; Feussner, Ivo; STANTON, CATHERINE (Biomed Central, 17/02/2011)
      Background The aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates. Results MCRA catalysed the conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but shorter chain fatty acids were not used as substrates, while the presence of trans-double bonds and double bonds beyond the position C12 abolished hydratase activity. The hydroxy fatty acids produced were not metabolised further. We also found that heterologous Lactococcus and Corynebacterium expressing MCRA accumulated increasing amounts of 10-HOA and 10-HOE in the culture medium. Furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls. Conclusions MCRA protein in B. breve can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, which may be catalysing the first step in conjugated linoleic acid (CLA) production, and this protein has an additional function in bacterial stress protection.
    • Phages of non-dairy lactococci: isolation and characterization of ΦL47, a phage infecting the grass isolate Lactococcus lactis ssp. cremoris DPC6860

      Cavanagh, Daniel; Guinane, Caitriona M.; Neve, Horst; Coffey, Aidan; Ross, R Paul; Fitzgerald, Gerald F; McAuliffe, Olivia; Irish Dairy Levy Research Trust; Teagasc Walsh Fellowship Programme (Frontiers, 13/01/2014)
      Lactococci isolated from non-dairy sources have been found to possess enhanced metabolic activity when compared to dairy strains. These capabilities may be harnessed through the use of these strains as starter or adjunct cultures to produce more diverse flavor profiles in cheese and other dairy products. To understand the interactions between these organisms and the phages that infect them, a number of phages were isolated against lactococcal strains of non-dairy origin. One such phage, ΦL47, was isolated from a sewage sample using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. Visualization of phage virions by transmission electron microscopy established that this phage belongs to the family Siphoviridae and possesses a long tail fiber, previously unseen in dairy lactococcal phages. Determination of the lytic spectrum revealed a broader than expected host range, with ΦL47 capable of infecting 4 industrial dairy strains, including ML8, HP and 310, and 3 additional non-dairy isolates. Whole genome sequencing of ΦL47 revealed a dsDNA genome of 128, 546 bp, making it the largest sequenced lactococcal phage to date. In total, 190 open reading frames (ORFs) were identified, and comparative analysis revealed that the predicted products of 117 of these ORFs shared greater than 50% amino acid identity with those of L. lactis phage Φ949, a phage isolated from cheese whey. Despite their different ecological niches, the genomic content and organization of ΦL47 and Φ949 are quite similar, with both containing 4 gene clusters oriented in different transcriptional directions. Other features that distinguish ΦL47 from Φ949 and other lactococcal phages, in addition to the presence of the tail fiber and the genome length, include a low GC content (32.5%) and a high number of predicted tRNA genes (8). Comparative genome analysis supports the conclusion that ΦL47 is a new member of the 949 lactococcal phage group which currently includes the dairy Φ949.
    • Protein Quality and the Protein to Carbohydrate Ratio within a High Fat Diet Influences Energy Balance and the Gut Microbiota In C57BL/6J Mice

      McAllan, Liam; Skuse, Peter; Cotter, Paul D.; O'Connor, Paula M.; Cryan, John F.; Ross, R Paul; Fitzgerald, Gerald F; Roche, Helen M.; Nilaweera, Kanishka; Teagasc Vision Programme on Obesity; et al. (PLOS, 10/02/2014)
      Macronutrient quality and composition are important determinants of energy balance and the gut microbiota. Here, we investigated how changes to protein quality (casein versus whey protein isolate; WPI) and the protein to carbohydrate (P/C) ratio within a high fat diet (HFD) impacts on these parameters. Mice were fed a low fat diet (10% kJ) or a high fat diet (HFD; 45% kJ) for 21 weeks with either casein (20% kJ, HFD) or WPI at 20%, 30% or 40% kJ. In comparison to casein, WPI at a similar energy content normalised energy intake, increased lean mass and caused a trend towards a reduction in fat mass (P = 0.08), but the protein challenge did not alter oxygen consumption or locomotor activity. WPI reduced HFD-induced plasma leptin and liver triacylglycerol, and partially attenuated the reduction in adipose FASN mRNA in HFD-fed mice. High throughput sequence-based analysis of faecal microbial populations revealed microbiota in the HFD-20% WPI group clustering closely with HFD controls, although WPI specifically increased Lactobacillaceae/Lactobacillus and decreased Clostridiaceae/Clostridium in HFD-fed mice. There was no effect of increasing the P/C ratio on energy intake, but the highest ratio reduced HFD-induced weight gain, fat mass and plasma triacylglycerol, non-esterified fatty acids, glucose and leptin levels, while it increased lean mass and oxygen consumption. Similar effects were observed on adipose mRNA expression, where the highest ratio reduced HFD-associated expression of UCP-2, TNFa and CD68 and increased the diet-associated expression of b3-AR, LPL, IR, IRS-1 and GLUT4. The P/C ratio also impacted on gut microbiota, with populations in the 30/ 40% WPI groups clustering together and away from the 20% WPI group. Taken together, our data show that increasing the P/C ratio has a dramatic effect on energy balance and the composition of gut microbiota, which is distinct from that caused by changes to protein quality.
    • Strains of the Lactobacillus casei group show diverse abilities for the production of flavor compounds in 2 model systems

      Stefanovic, Ewelina; Thierry, Anne; Maillard, Marie-Bernadette; Bertuzzi, Andrea; Rea, Mary; Fitzgerald, Gerald F; McAuliffe, Olivia; Kilcawley, Kieran; Teagasc Walsh Fellowship Programme (Elsevier, 2017-07-12)
      Cheese flavor development is directly connected to the metabolic activity of microorganisms used during its manufacture, and the selection of metabolically diverse strains represents a potential tool for the production of cheese with novel and distinct flavor characteristics. Strains of Lactobacillus have been proven to promote the development of important cheese flavor compounds. As cheese production and ripening are long-lasting and expensive, model systems have been developed with the purpose of rapidly screening lactic acid bacteria for their flavor potential. The biodiversity of 10 strains of the Lactobacillus casei group was evaluated in 2 model systems and their volatile profiles were determined by gas chromatography-mass spectrometry. In model system 1, which represented a mixture of free AA, inoculated cells did not grow. In total, 66 compounds considered as flavor contributors were successfully identified, most of which were aldehydes, acids, and alcohols produced via AA metabolism by selected strains. Three strains (DPC2071, DPC3990, and DPC4206) had the most diverse metabolic capacities in model system 1. In model system 2, which was based on processed cheese curd, inoculated cells increased in numbers over incubation time. A total of 47 compounds were identified, and they originated not only from proteolysis, but also from glycolytic and lipolytic processes. Tested strains produced ketones, acids, and esters. Although strains produced different abundances of volatiles, diversity was less evident in model system 2, and only one strain (DPC4206) was distinguished from the others. Strains identified as the most dissimilar in both of the model systems could be more useful for cheese flavor diversification.