g cooke bugs, babies & vitamin k

BugsBugs, ,

BabiesBabies & Vitamin K& Vitamin K

Gordon Cooke, Dept of Applied Gordon Cooke, Dept of Applied Science, Institute of Science, Institute of Technology Tallaght.Technology 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.

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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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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

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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

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Bacterial Groups Bacterial Groups EnumeratedEnumerated

Media: Bacteria IsolatedRogosa –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.

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Bifidobacteria Bifidobacteria spsp. . 100x100x

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Lactobacilli Lactobacilli spsp. . 100x100x

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Reinforced Clostridial agar Reinforced Clostridial agar with Analine Bluewith Analine Blue

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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 Table 1. Percentage of Positive Samples and Dominance in Breast and Formula Fed Neonates Aged 2-5 Days Old and Formula Fed Neonates 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

1717171717171717Total number of

samples tested

Bacteroides sp.

Enterobacteriaceae sp.

E. coliClostridia

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

1616161616161616Total 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

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Statistically significant results in the gut flora of Statistically significant results in the gut flora of neonates aged 2-5 Days Oldneonates aged 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).

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Statistically significant results in the gut flora of Statistically significant results in the gut flora of neonates aged 6 weeks oldneonates aged 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)

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Statistically significant results in the gut flora of Statistically significant results in the gut flora of neonates aged 6 weeks oldneonates aged 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)

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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

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Results of Thin Layer Chromatography Results 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

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Results of LC-MS Analysis of Purified Results of LC-MS Analysis of Purified Suspect Menaquinone Bands from TLCSuspect Menaquinone 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.

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Vitamin K ProductionVitamin K ProductionMenaquinone Producers Non Menaquinone ProducersStaphylococcus warneri Lactobacilli sp.Staphylococcus warneri Clostridia sp.

Staphylococcus haemolyticus Propionibacterium acnesEnterobacter agglomerans Bifidobacterium bifidum

Serratia marcescens Lactobacilli sp.Staphylococcus capitis

Escherichia coliPrevotella buccaeBacteroides sp.Bacteroides sp.

Enterococcus faecalisEnterococcus faecium

Staphylococcus epidermidisBacteroides ovatus

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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

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% Survival of HT-29 Cells dosed with various concentrations of Vitamin K2

0

20

40

60

80

100

120

140

Concentration of Vitamin K2 in ug/ml

% S

urv

ival

% Survival of Caco-2 Cells dosed with various concentrations of Vitamin K3

0

20

40

60

80

100

120

Concentration of Vitamin K3 in ug/ml

% S

urvi

val

% Survival of Caco-2 Cells dosed with various concentrations of Vitamin K2

0

20

40

60

80

100

120

140

160

Concentration of Vitamin K2 in ug/ml

% S

urv

ival

% Survival of Caco-2 Cells dosed with various concentrations of Vitamin K1

0

20

40

60

80

100

120

140

160

Concentration of vitamin K1

% S

urv

ival

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

Concentration of vitamin K1

% S

urv

ival

% Survival of HT-29 Cells dosed with various concentrations of Vitamin K3

0

20

40

60

80

100

120

Concentration of Vitamin K3 in ug/ml

% S

urvi

val

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Transport StudiesTransport Studies

• Transwells– CACO-2 cells– Apical to Basolateral movement– Basolateral to Apical Movement

• Monitored TEER values • HPLC-UV analysis

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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

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TEER values for CACO-2 with 100ug/ml of Phylloquinone

0102030405060708090

100110120130

0 1 2 3 4 5 6 7

Time after Drug addition (hrs)

TE

ER

val

ue

as a

% o

f co

ntr

olw

ith

n

o d

rug

TEER values for CACO-2 with 100ug/ml of Menaquinone

0102030405060708090

100110120

0 1 2 3 4 5 6 7

Time after Drug addition (hrs)

TE

ER

val

ue

as a

% o

f co

ntr

olw

ith

n

o d

rug

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)

TE

ER

val

ue

as a

% o

f co

ntr

olw

ith n

o

dru

g

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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

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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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CACO-2 cells in a MTP with WST-1 only

CACO-2 cells in a MTP with WST-1 and 25μg/ml

of Menadione