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G Cooke Bugs, Babies & Vitamin K
 

G Cooke Bugs, Babies & Vitamin K

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G Cooke Bugs, Babies & Vitamin K G Cooke Bugs, Babies & Vitamin K Presentation Transcript

  • Bugs,Bugs, BabiesBabies & Vitamin K& Vitamin K Gordon Cooke, Dept of AppliedGordon Cooke, Dept of Applied Science, Institute of TechnologyScience, Institute of Technology Tallaght.Tallaght.
  • gordon.cooke@it-tallaght.ie ObjectivesObjectives • To examine, by enumeration, the development of the major groups of bacteria within the faecal flora of new born infants over three different time points. • To determine any statistically significant differences between the faecal flora of breast and formula fed infants at each of the different time points. • To identify, to species level, selected representative isolates from the enumerated faecal flora and evaluate the production of vitamin K by these selected isolates. • To examine the toxicity and absorption of various forms of Vitamin K across human intestinal cells.
  • gordon.cooke@it-tallaght.ie Introduction-Vitamin KIntroduction-Vitamin K • Vitamin K – Phylloquinone (MW: 450) – Menaquinone (MW: 444) – Menadione (MW: 172) • Roles – Blood Clotting Process – Bone Health – Possible Neurological Growth Factor
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  • gordon.cooke@it-tallaght.ie Introduction-Vitamin KIntroduction-Vitamin K • Haemorrhagic Disease of the Newborn (HDN) • Deficiency in Vitamin K – Low levels present in Breast Milk – Supplemented in Formula Milk • 3 Types – Early: 0-24hrs – Classical: 2-7 days – Late: up to 1 year after birth • Occurrences • Vitamin K Prophylaxis – No Consensus
  • gordon.cooke@it-tallaght.ie Methods-Gut FloraMethods-Gut Flora • Full Term Healthy Neonates • 3 Sampling Periods – Birth to 1 day old Neonates – 2-5 day old Neonates – 6 week old Neonates • Bacteria Enumerated and Identified – Selective Media – Gram Stains – API Tests
  • gordon.cooke@it-tallaght.ie Bacterial Groups EnumeratedBacterial Groups Enumerated Media: Bacteria Isolated Rogosa –O2: Lactobacilli sp. RCA –O2: Clostridia sp. RCA & Analine Blue –O2: Bifidobacteria sp. BBAA –O2: Bacteroides sp. EMB +O2: E.coli and Enterobacteriaceae sp. Baird Parker +O2: Staphylococci sp. Slanetz and Bartley +O2: Enterococci sp.
  • gordon.cooke@it-tallaght.ie BifidobacteriaBifidobacteria spsp.. 100x100x
  • gordon.cooke@it-tallaght.ie LactobacilliLactobacilli spsp.. 100x100x
  • gordon.cooke@it-tallaght.ie Reinforced Clostridial agarReinforced Clostridial agar with Analine Bluewith Analine Blue
  • gordon.cooke@it-tallaght.ie Rogosa AgarRogosa Agar
  • 0%50%56%0%43%26%25%46%% Dominance 7%30%33%7%26%85%44%48%% of Samples testing positive 2727272727272727Total number of samples tested Bacteroides sp.Enterobacteriaceae sp. E. coliClostridia sp.Lactobacilli sp.Staphylococci sp.Enterococci sp.Bifidobacteria sp.Bacteria Formula 33%17%33%0%0%15%38%60%% Dominance 21%43%43%7%29%93%57%71%% of Samples testing positive 1414141414141414Total number of samples tested Bacteroides sp. Enterobacteriaceae sp. E. coliClostridia sp.Lactobacilli sp.Staphylococci sp.Enterococci sp.Bifidobacteria sp.Bacteria Breast Table 1. Percentage of Positive Samples and Dominance in Breast and Formula FedTable 1. Percentage of Positive Samples and Dominance in Breast and Formula Fed Neonates Aged 2-5 Days OldNeonates Aged 2-5 Days Old
  • 10%36%40%0%17%0%17%67%% Dominance 59%65%59%6%71%88%94%35%% of Samples testing positive 1717171717171717 Total number of samples tested Bacteroides sp. Enterobacteriaceae sp. E. coli Clostridia sp. Lactobacilli sp.Staphylococci sp.Enterococci sp. Bifidobacteria sp. Bacteria Formula 33%13%0%0%45%6%0%80%% Dominance 75%75%38%6%69%100%75%31%% of Samples testing positive 1616161616161616 Total number of samples Tested Bacteroides sp. Enterobacteriaceae sp. E. coli Clostridia sp. Lactobacilli sp.Staphylococci sp.Enterococci sp.Bifidobacteria sp.Bacteria Breast Table 2.Percentage of Positive Samples and Dominance in 6 Week Old Breast and Formula Fed NeonatesTable 2.Percentage of Positive Samples and Dominance in 6 Week Old Breast and Formula Fed Neonates
  • gordon.cooke@it-tallaght.ie Statistically significant results in the gut flora of neonatesStatistically significant results in the gut flora of neonates aged 2-5 Days Oldaged 2-5 Days Old In the gut flora of breast fed neonates aged 2-5 days old, on average, Bifidobacteria sp. accounted for 36.35% of all bacteria enumerated whereas in formula fed neonates they accounted for 12.81% of all bacteria enumerated. (p=0.03).
  • gordon.cooke@it-tallaght.ie Statistically significant results in the gut flora of neonatesStatistically significant results in the gut flora of neonates aged 6 weeks oldaged 6 weeks old In the gut flora of breast fed neonates aged 6 weeks old, on average, Lactobacilli sp. accounted for 25.04% of all bacteria enumerated whereas in formula fed neonates they accounted for 2.85% of all bacteria enumerated. (p=0.03)
  • gordon.cooke@it-tallaght.ie Statistically significant results in the gut flora of neonatesStatistically significant results in the gut flora of neonates aged 6 weeks oldaged 6 weeks old In the gut flora of breast fed neonates aged 6 weeks old, on average, Enterococci sp. accounted for 5.44% of all bacteria enumerated whereas in formula fed neonates they accounted for 16.60% of all bacteria enumerated. (p=0.04)
  • gordon.cooke@it-tallaght.ie Methods-Vitamin K2Methods-Vitamin K2 • Total lipid content isolated from selected identified species • Suspect menaquinones purified and visualised on TLC and detected on HPLC • Confirmatory identification of menaquinones carried out using LC-MS
  • gordon.cooke@it-tallaght.ie Results of Thin Layer ChromatographyResults of Thin Layer Chromatography Purification of Bacterial Lipid ExtractPurification of Bacterial Lipid Extract A TLC plate with lipid extract of E.coli and B.bifidum showing that E.coli produces Menaquinone whereas B.bifidum does not. Bacterial Menaquinones Menaquinone Standard
  • gordon.cooke@it-tallaght.ie Results of LC-MS Analysis of Purified SuspectResults of LC-MS Analysis of Purified Suspect Menaquinone Bands from TLCMenaquinone Bands from TLC Peak A 647.5 Peak A LC-MS scan of Bacteroides sp. Peak A had a mass of 647.5 and MK7 has a mass of 648.
  • gordon.cooke@it-tallaght.ie Vitamin K ProductionVitamin K Production Menaquinone Producers Non Menaquinone Producers Staphylococcus warneri Lactobacilli sp. Staphylococcus warneri Clostridia sp. Staphylococcus haemolyticus Propionibacterium acnes Enterobacter agglomerans Bifidobacterium bifidum Serratia marcescens Lactobacilli sp. Staphylococcus capitis Escherichia coli Prevotella buccae Bacteroides sp. Bacteroides sp. Enterococcus faecalis Enterococcus faecium Staphylococcus epidermidis Bacteroides ovatus
  • gordon.cooke@it-tallaght.ie Toxicity StudiesToxicity Studies • WST-1 (Tetrazolium Salt) – One Step Reaction – Concentration Range 0-1000ug/ml – 24hr incubation – n=3 • Cells – CACO-2 Non Mucosal Human Intestinal Cells – HT-29 Mucosal Human Intestinal Cells
  • gordon.cooke@it-tallaght.ie % Survival of HT-29 Cells dosed with various concentrations of Vitamin K2 0 20 40 60 80 100 120 140 Cells O nly 1 10 25 50 100 250 500 1000 Ethanol Concentration of Vitamin K2 in ug/ml %Survival % Survival of Caco-2 Cells dosed with various concentrations of Vitamin K3 0 20 40 60 80 100 120 Cells Only 1 10 25 50 100 250 500 1000 Ethanol Concentration of Vitamin K3 in ug/ml %Survival % Survival of Caco-2 Cells dosed with various concentrations of Vitamin K2 0 20 40 60 80 100 120 140 160 Cells O nly 1 10 25 50 100 250 500 1000 Ethanol Concentration of Vitamin K2 in ug/ml %Survival % Survival of Caco-2 Cells dosed with various concentrations of Vitamin K1 0 20 40 60 80 100 120 140 160 Cells O nly 1 10 25 50 100 250 500 1000 Ethanol Concentration of vitamin K1 %Survival IC50 Values For Menadione μg/ml μM HT-29 45 261 CACO-2 12 70 % Survival of HT-29 Cells dosed with various concentrations of Vitamin K1 0 20 40 60 80 100 120 140 Cells O nly 1 10 25 50 100 250 500 1000 Ethanol Concentration of vitamin K1 %Survival % Survival of HT-29 Cells dosed with various concentrations of Vitamin K3 0 20 40 60 80 100 120 Cells Only 1 10 25 50 100 250 500 1000 Ethanol Concentration of Vitamin K3 in ug/ml %Survival
  • gordon.cooke@it-tallaght.ie Transport StudiesTransport Studies • Transwells – CACO-2 cells – Apical to Basolateral movement – Basolateral to Apical Movement • Monitored TEER values • HPLC-UV analysis
  • gordon.cooke@it-tallaght.ie Transport StudiesTransport Studies • Phylloquinone and Menaquinone – No Absorption across CACO-2 intestinal cells – TEER values remained high • Menadione – Papp= 1.27x10-7 cm/s Apical-Basolateral (+/-0.68) – Papp= 2.05x10-7 cm/s Basolateral-Apical (+/-1.4) – TEER values dropped 50% after 2 hours • Preliminary work with Cytochalasin D – No absorption of Phylloquinone and Menaquinone – Adhere to the cell
  • gordon.cooke@it-tallaght.ie TEER values for CACO-2 with 100ug/ml of Phylloquinone 0 10 20 30 40 50 60 70 80 90 100 110 120 130 0 1 2 3 4 5 6 7 Time after Drug addition (hrs) TEERvalueasa%ofcontrolwith nodrug TEER values for CACO-2 with 100ug/ml of Menaquinone 0 10 20 30 40 50 60 70 80 90 100 110 120 0 1 2 3 4 5 6 7 Time after Drug addition (hrs) TEERvalueasa%ofcontrolwith nodrug TEER values for CACO-2 with 100ug/ml of Menadione 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 Time after Drug Addition (hrs) TEERvalueasa%ofcontrolwithno drug
  • gordon.cooke@it-tallaght.ie ConclusionsConclusions • Breast-fed Neonates – Higher levels of Bifidobacteria and Lactobacilli • Formula-fed Neonates – Higher levels of E.coli and Enterococci • Menaquinone production in gut flora – Confirmed – New previously unknown producers of menaquinones identified • Phylloquinone and Menaquinone – Non-toxic – Does not absorb across intestinal cells in-vitro over a 6 hour period • Menadione – Toxic – Absorbs across intestinal cells in-vitro over a 6 hour period • Hospital recommendations
  • gordon.cooke@it-tallaght.ie AcknowledgementsAcknowledgements • Dr. Mary Costello & John Behan • Sr. Nicola Clarke CMS (Clinical Midwife Specialist N.M.H) • Dr. Winifred Gorman (Paediatric Consultant N.M.H) • Brian Carr, Dr Mary Deasy, Dr Siobhán McClean, Dr Brian Murray, James Reilly and Dr Maureen Walsh • Dr Brett Paul, Leon Bannon and DCU • PDRSP for funding, IT Tallaght Seed fund and Ph.D. continuance fund. • The mothers and their newborns of the National Maternity Hospital Holles Street
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  • gordon.cooke@it-tallaght.ie CACO-2 cells in a MTP with WST-1 only CACO-2 cells in a MTP with WST-1 and 25μg/ml of Menadione