• Nisin in Combination with Cinnamaldehyde and EDTA to Control Growth of Escherichia coli Strains of Swine Origin

      Field, Des; Baghou, Inès; Rea, Mary; Gardiner, Gillian; Ross, R Paul; Hill, Colin; Science Foundation Ireland; 10/IN.1/B3027; SFI/12/RC/2273 (MDPI AG, 2017-12-12)
      Post-weaning diarrhoea (PWD) due to enterotoxigenic Escherichia coli (ETEC) is an economically important disease in pig production worldwide. Although antibiotics have contributed significantly to mitigate the economic losses caused by PWD, there is major concern over the increased incidence of antimicrobial resistance among bacteria isolated from pigs. Consequently, suitable alternatives that are safe and effective are urgently required. Many naturally occurring compounds, including the antimicrobial peptide nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against pathogenic microorganisms. Here, we evaluate the potential of nisin in combination with the essential oil cinnamaldehyde and ethylenediaminetetraacetic acid (EDTA) to control the growth of E. coli strains of swine origin including two characterized as ETEC. The results reveal that the use of nisin (10 μM) with low concentrations of trans-cinnamaldehyde (125 μg/mL) and EDTA (0.25–2%) resulted in extended lag phases of growth compared to when either antimicrobial is used alone. Further analysis through kill curves revealed that an approximate 1-log reduction in E. coli cell counts was observed against the majority of targets tested following 3 h incubation. These results highlight the potential benefits of combining the natural antimicrobial nisin with trans-cinnamaldehyde and EDTA as a new approach for the inhibition of E. coli strains of swine origin.
    • Nisin in Combination with Cinnamaldehyde and EDTA to Control Growth of Escherichia coli Strains of Swine Origin

      Field, Des; Baghou, Inès; Rea, Mary; Gardiner, Gillian E.; Ross, R Paul; Hill, Colin; Science Foundation Ireland; SFI/12/RC/2273; 10/IN.1/B3027 (MDPI AG, 2017-12-12)
      Post-weaning diarrhoea (PWD) due to enterotoxigenic Escherichia coli (ETEC) is an economically important disease in pig production worldwide. Although antibiotics have contributed significantly to mitigate the economic losses caused by PWD, there is major concern over the increased incidence of antimicrobial resistance among bacteria isolated from pigs. Consequently, suitable alternatives that are safe and effective are urgently required. Many naturally occurring compounds, including the antimicrobial peptide nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against pathogenic microorganisms. Here, we evaluate the potential of nisin in combination with the essential oil cinnamaldehyde and ethylenediaminetetraacetic acid (EDTA) to control the growth of E. coli strains of swine origin including two characterized as ETEC. The results reveal that the use of nisin (10 μM) with low concentrations of trans-cinnamaldehyde (125 μg/mL) and EDTA (0.25–2%) resulted in extended lag phases of growth compared to when either antimicrobial is used alone. Further analysis through kill curves revealed that an approximate 1-log reduction in E. coli cell counts was observed against the majority of targets tested following 3 h incubation. These results highlight the potential benefits of combining the natural antimicrobial nisin with trans-cinnamaldehyde and EDTA as a new approach for the inhibition of E. coli strains of swine origin.
    • Oral Delivery of Nisin in Resistant Starch Based Matrices Alters the Gut Microbiota in Mice

      Gough, Ronan; Cabrera-Rubio, Raul; O'Connor, Paula M.; Crispie, Fiona; Miao, Song; Hill, Colin; Ross, R Paul; Cotter, Paul D.; Nilaweera, Kanishka; Rea, Mary; et al. (Frontiers, 15/06/2018)
      There is a growing recognition of the role the gastrointestinal microbiota plays in health and disease. Ingested antimicrobial proteins and peptides have the potential to alter the gastrointestinal microbiota; particularly if protected from digestion. Nisin is an antimicrobial peptide that is used as a food preservative. This study examined the ability of nisin to affect the murine microbiota when fed to mice in two different starch based matrices; a starch dough comprising raw starch granules and a starch gel comprising starch that was gelatinized and retrograded. The effects of the two starch matrices by themselves on the microbiota were also examined. Following 16S rRNA compositional sequencing, beta diversity analysis highlighted a significant difference (p = 0.001, n = 10) in the murine microbiota between the four diet groups. The differences between the two nisin containing diets were mainly attributable to differences in the nisin release from the starch matrices while the differences between the carriers were mainly attributable to the type of resistant starch they possessed. Indeed, the differences in the relative abundance of several genera in the mice consuming the starch dough and starch gel diets, in particular Akkermansia, the relative abundance of which was 0.5 and 11.9%, respectively (p = 0.0002, n = 10), points to the potential value of resistance starch as a modulator of beneficial gut microbes. Intact nisin and nisin digestion products (in particular nisin fragment 22–31) were detected in the feces and the nisin was biologically active. However, despite a three-fold greater consumption of nisin in the group fed the nisin in starch dough diet, twice as much nisin was detected in the feces of the group which consumed the nisin in starch gel diet. In addition, the relative abundance of three times as many genera from the lower gastrointestinal tract (GIT) were significantly different (p < 0.001, n = 10) to the control for the group fed the nisin in starch gel diet, implying that the starch gel afforded a degree of protection from digestion to the nisin entrapped within it.
    • Oral Delivery of Nisin in Resistant Starch Based Matrices Alters the Gut Microbiota in Mice

      Gough, Ronan; Cabrera-Rubio, Raul; O'Connor, Paula M.; Crispie, Fiona; Brodkorb, Andre; Miao, Song; Hill, Colin; Ross, R Paul; Cotter, Paul D.; Nilaweera, Kanishka; et al. (Frontiers, 2018-06-15)
      There is a growing recognition of the role the gastrointestinal microbiota plays in health and disease. Ingested antimicrobial proteins and peptides have the potential to alter the gastrointestinal microbiota; particularly if protected from digestion. Nisin is an antimicrobial peptide that is used as a food preservative. This study examined the ability of nisin to affect the murine microbiota when fed to mice in two different starch based matrices; a starch dough comprising raw starch granules and a starch gel comprising starch that was gelatinized and retrograded. The effects of the two starch matrices by themselves on the microbiota were also examined. Following 16S rRNA compositional sequencing, beta diversity analysis highlighted a significant difference (p = 0.001, n = 10) in the murine microbiota between the four diet groups. The differences between the two nisin containing diets were mainly attributable to differences in the nisin release from the starch matrices while the differences between the carriers were mainly attributable to the type of resistant starch they possessed. Indeed, the differences in the relative abundance of several genera in the mice consuming the starch dough and starch gel diets, in particular Akkermansia, the relative abundance of which was 0.5 and 11.9%, respectively (p = 0.0002, n = 10), points to the potential value of resistance starch as a modulator of beneficial gut microbes. Intact nisin and nisin digestion products (in particular nisin fragment 22–31) were detected in the feces and the nisin was biologically active. However, despite a three-fold greater consumption of nisin in the group fed the nisin in starch dough diet, twice as much nisin was detected in the feces of the group which consumed the nisin in starch gel diet. In addition, the relative abundance of three times as many genera from the lower gastrointestinal tract (GIT) were significantly different (p < 0.001, n = 10) to the control for the group fed the nisin in starch gel diet, implying that the starch gel afforded a degree of protection from digestion to the nisin entrapped within it.
    • A pilot study demonstrating the altered gut microbiota functionality in stable adults with Cystic Fibrosis

      Fouhy, Fiona; Ronan, N. J.; O'Sullivan, Orla; McCarthy, Y.; Walsh, Aaron M.; Murphy, D.M.; Daly, M.; Flanagan, E. T.; Fleming, C.; McCarthy, M.; et al. (Nature Publishing Group, 2017-07-27)
      Cystic Fibrosis (CF) and its treatment result in an altered gut microbiota composition compared to non-CF controls. However, the impact of this on gut microbiota functionality has not been extensively characterised. Our aim was to conduct a proof-of-principle study to investigate if measurable changes in gut microbiota functionality occur in adult CF patients compared to controls. Metagenomic DNA was extracted from faecal samples from six CF patients and six non-CF controls and shotgun metagenomic sequencing was performed on the MiSeq platform. Metabolomic analysis using gas chromatography-mass spectrometry was conducted on faecal water. The gut microbiota of the CF group was significantly different compared to the non-CF controls, with significantly increased Firmicutes and decreased Bacteroidetes. Functionality was altered, with higher pathway abundances and gene families involved in lipid (e.g. PWY 6284 unsaturated fatty acid biosynthesis (p = 0.016)) and xenobiotic metabolism (e.g. PWY-5430 meta-cleavage pathway of aromatic compounds (p = 0.004)) in CF patients compared to the controls. Significant differences in metabolites occurred between the two groups. This proof-of-principle study demonstrates that measurable changes in gut microbiota functionality occur in CF patients compared to controls. Larger studies are thus needed to interrogate this further.
    • Reincarnation of Bacteriocins From the Lactobacillus Pangenomic Graveyard

      Collins, Fergus W. J.; Mesa-Pereira, Beatriz; O'Connor, Paula M.; Rea, Mary; Hill, Colin; Ross, R Paul; Science Foundation Ireland; SFI/12/RC/227 (Frontiers, 02/07/2018)
      Bacteria commonly produce narrow spectrum bacteriocins as a means of inhibiting closely related species competing for similar resources in an environment. The increasing availability of genomic data means that it is becoming easier to identify bacteriocins encoded within genomes. Often, however, the presence of bacteriocin genes in a strain does not always translate into biological antimicrobial activity. For example, when analysing the Lactobacillus pangenome we identified strains encoding ten pediocin-like bacteriocin structural genes which failed to display inhibitory activity. Nine of these bacteriocins were novel whilst one was identified as the previously characterized bacteriocin “penocin A.” The composition of these bacteriocin operons varied between strains, often with key components missing which are required for bacteriocin production, such as dedicated bacteriocin transporters and accessory proteins. In an effort to functionally express these bacteriocins, the structural genes for the ten pediocin homologs were cloned alongside the dedicated pediocin PA-1 transporter in both Escherichia coli and Lactobacillus paracasei heterologous hosts. Each bacteriocin was cloned with its native leader sequence and as a fusion protein with the pediocin PA-1 leader sequence. Several of these bacteriocins displayed a broader spectrum of inhibition than the original pediocin PA-1. We show how potentially valuable bacteriocins can easily be “reincarnated” from in silico data and produced in vitro despite often lacking the necessary accompanying machinery. Moreover, the study demonstrates how genomic datasets such as the Lactobacilus pangenome harbor a potential “arsenal” of antimicrobial activity with the possibility of being activated when expressed in more genetically amenable hosts.
    • Reincarnation of Bacteriocins From the Lactobacillus Pangenomic Graveyard

      Collins, Fergus W. J.; Mesa-Pereira, Beatriz; O'Connor, Paula M.; Rea, Mary; Hill, Colin; Ross, R Paul; Science Foundation Ireland; SFI/12/RC/227 (Frontiers, 2018-07-02)
      Bacteria commonly produce narrow spectrum bacteriocins as a means of inhibiting closely related species competing for similar resources in an environment. The increasing availability of genomic data means that it is becoming easier to identify bacteriocins encoded within genomes. Often, however, the presence of bacteriocin genes in a strain does not always translate into biological antimicrobial activity. For example, when analysing the Lactobacillus pangenome we identified strains encoding ten pediocin-like bacteriocin structural genes which failed to display inhibitory activity. Nine of these bacteriocins were novel whilst one was identified as the previously characterized bacteriocin “penocin A.” The composition of these bacteriocin operons varied between strains, often with key components missing which are required for bacteriocin production, such as dedicated bacteriocin transporters and accessory proteins. In an effort to functionally express these bacteriocins, the structural genes for the ten pediocin homologs were cloned alongside the dedicated pediocin PA-1 transporter in both Escherichia coli and Lactobacillus paracasei heterologous hosts. Each bacteriocin was cloned with its native leader sequence and as a fusion protein with the pediocin PA-1 leader sequence. Several of these bacteriocins displayed a broader spectrum of inhibition than the original pediocin PA-1. We show how potentially valuable bacteriocins can easily be “reincarnated” from in silico data and produced in vitro despite often lacking the necessary accompanying machinery. Moreover, the study demonstrates how genomic datasets such as the Lactobacilus pangenome harbor a potential “arsenal” of antimicrobial activity with the possibility of being activated when expressed in more genetically amenable hosts.
    • Simulated gastrointestinal digestion of nisin and interaction between nisin and bile

      Gough, Ronan; O'Connor, Paula M.; Rea, Mary; Gomez-Sala, Beatriz; Miao, Song; Hill, Colin; Brodkorb, Andre; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 10/RD/TMFRC/701 (Elsevier, 2017-08-14)
      Nisin, an antimicrobial peptide showing activity against many Gram positive bacteria, is widely used as a food preservative. The simulated gastrointestinal digestion of nisin (variant A) was studied using the in vitro INFOGEST digestion method. Following oral, gastric and small intestinal digestion, there was no intact nisin in the system and the nisin was primarily digested by pancreatin. After digestion, six nisin fragments (1–11, 1–12, 1–20, 1–21, 1–29 and 1–32) were identified by reversed phase high performance liquid chromatography and mass spectroscopy and four of these nisin fragments (1–20, 1–21, 1–29 and 1–32) demonstrated low antibacterial activity against Lactococcus lactis HP in agar diffusion activity assays. Additionally, it was observed that bile salts form a complex with nisin. This was examined by atomic force microscopy, turbidity and dynamic light scattering, which showed that this interaction resulted in significantly larger bile salt micelles. The presence of bile salts at physiological levels significantly altered the relative amounts of the nisin fragments 1–12, 1–20 and 1–29 produced during an in vitro digestion. This study highlights the importance of including bile in simulated digestions of antimicrobial peptides in order to obtain a more accurate simulation of the in vivo digestion products and their activity.
    • 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.
    • WS16.1 Clinical Outcomes of Real-World Kalydeco (CORK) study – Investigating the impact of CFTR potentiation on the intestinal microbiota, exocrine pancreatic function and intestinal inflammation prospectively over 12 months

      Deane, Jennifer; Ronan, N. J.; O'Callaghan, G. P.; Fouhy, Fiona; Rea, Mary; O'Sullivan, Orla; Hill, C. J.; Shanahan, F.; Ross, R Paul; McCarthy, M.; et al. (Elsevier, 2015-06-05)
      Objectives Ivacaftor is effective in the treatment of patients with CF and the G551D gating mutation. We present faecal analysis results of the CORK cohort, a single-centre, adult (n = 20), prospective, longitudinal study of G551D clinical responders (median follow-up 12 months), examining the gut microbiota, exocrine pancreatic function and intestinal inflammation on a 3 monthly basis after commencing treatment.