Probiotics, Bacteriocins and Bacteriopgahe

for limiting C. difficile infection

Gut Soutions to a Gut Problem

Gut Problem: C. difficile carriage increases with gut inflammation, aging and antibiotic usage Gut Solution: Pharmabiotics Pagebiotics: Probiotics: Bacteriocins: Prospects for managing infection using pharmabiotic solutions

Physiology

Bacterial interactions

Metabolism/ Metagenomics

Genomics

Host response

Pathogenicity

From bugs to drugs

Fergus Shanahan

Resident microbiota

• Anti-infective/Immuno-modulatory effects

• Key long term role in digestion

• Production of essential nutrients

• Production of bioactive substances

Probiotics

• Probiotics have mainly transient effects – “tourists”

• Anti-infective Immuno-modulatory effects

• Out-compete pathogens

Probiotics V Resident Microbiota

0

1

2

3

4

5

6

7

8

9

10

-2 -1 1 2 3 4 5 6 7 8 9 10 11 12 weeks

placebo

Lactobacillus

Bifidobacterium Composite symptom score

Treatment period

Probiotic IBS Treatment Study

(Double-blind placebo controlled design)

*

*

* * * *

* * P<0.05

Irritable bowel

syndrome

Align containing Bifantis™ is a daily

probiotic supplement that helps build

and maintain a healthy, balanced

digestive system when taken daily. T

easy-to-swallow capsule that you take just once a day, every day to help

even out the ups and downs of common digestive upsets such as

constipation, diarrhea, abdominal discomfort, urgency, gas, and bloating.

Mahoney et al, Gastro 2006

>1,000

species

INFANTMET

Community strata are separated by microbiota

232 subjects

LOW HIGH

Cummunity Longstay

Paul O‘Toole

J. Ryan, C. Murphy , C. Twomey, RP Ross,

MC Rea, J MacSharry B. Sheil, F. Shanahan

Eldermet study (65years +)

Healthy in community

n = 123 1.6% Out-patients

n = 43 9.5% Short or long stay hospital

n = 151 21% Ribotypes isolated:

027, 078, 014, 018, 072, 220, 026, 216,

010 308, 050, 002

IBD Study

Ulcerative colitis/Crohns

n = 122 8.2% Controls

n = 99 1.0% Ribotypes isolated

015, 005, 020, 062, 050, 003

IBS Study

IBS outpatients

n = 87 5.7% Controls

n = 88 1.1% Ribotypes isolated

050, 005, 060, 062

Cystic Fibrosis

CF patients

n = 27 55% Ribotypes isolated

001, 014, 002, 039,126,140,078, 010, 046

C. difficile strains from the elderly

100 80 60 40

EM304CD EM306CD EM011CD EM136CD EM188CD EM045CD EM112CD EM113CD EM126CD EM069CD EM139CD EM218CD EM255CD EM140CD EM148CD EM243CD EM124CD EM156CD EM321CD EM324CD EM334CD EM336CD EM149CD EM146CD EM186CD EM152CD EM012CD EM308CD

R027 R027 R027 R014 R018 R072 R072 R072 R072 R072 R072 R072 R072

R072 R072 R220 R026 R216 R216 R216 R010 Unassigned

Unassigned

R050 R002 R078

Rehab Rehab Day Long Stay Long Stay Day Long Stay Rehab Long Stay Rehab Long Stay Long Stay Long Stay Long Stay Rehab Long Stay Long Stay Long Stay Rehab Rehab Rehab Community Community Long Stay Long Stay Rehab Day Rehab

R072

R072

Mark Wilcox

252 Controls 18 asymptomatic

carriers

individual subjects with active C. difficile infection at the time of sampling

Gut microbiota different with active C. difficile infection

Gut Soutions to a Gut Problem

Gut Problem: C. difficile carriage increases with gut inflammation, aging and antibiotic usage Gut Solution: Pharmabiotics Pagebiotics: Probiotics: Bacteriocins: Prospects for managing infection using pharmabiotic solutions

Thuricin

Gut bacteria produce an almost limitless set of biopactives

Understanding these and the bacteria which produce

them is key to understanding probiotic effects

Colin Hill

Bacteriophages (bacterial viruses)

• Obligate intracellular parasites

• Minute particles (0.05 x 0.15mm)

• Protein and DNA

Activity against drug-resistant microorganisms.

high specificity.

self-replicating and self-limiting

low cost

ADVANTAGES

Temperate phages ,

phiC6356 and phiC6365,

induced from clinical

C. difficile isolated from

ulcerative colitis patient

phiC6356 phiC6365

Siphoviridae family

C. difficile phages induced.....

C.difficile isolate

phiC

6356

phiC

6365

DPC6218 +

DPC6219 + +

DPC6220 + +

DPC6221 + +

DPC6353 +

DPC6355 +

DPC6359 + +

DPC6365 +

DPC6366 + +

DPC6534 +

DPC6538 +

56 + +

DPC6506 +

DPC6508 +

DPC6511 +

DPC6512 +

DPC6515 +

Total 13 10

No plaques on

C. spongenes,

C. histolyticum,

C. perfringens,

Lb. paracasei,

B. cereus or

S. aureus.

Phage CD6356 in faecal fermentation

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

0 10 20 30

Time (h)

(P/C

) FU

/ml C. difficile control

C. difficile + Phage

Phage count

Phage therapy

Good evidence for AAD

Evidence for CDAD equivocal

Need good clinical studies

May be other strains with better efficacy

Probiotics and CDAD

‘Prescribed

probiotic’ in a

Dublin Hospital

Hickson et al., BMJ 335:80

Many gut bacteria produce Bacteriocins

Antimicrobial peptides produced by one bacterium which can kill other

bacteria

Bacteriocins are heat stable, active at nanomolar range, producers are immune to their own bacteriocin

Bacteriocin production is widespread among bacteria, including gut

bacteria

Colin Hill

Microbiota

Epithelium

Immune cells

Lumen

Probiotic

Bacteriocin

Probiotic Probiotic

Colonising peptide

Bacteriocin

Bacteriocin

Pathogen

Killing peptide

Signalling peptide

From: Bacteriocin Production as a Probiotic Trait? Dobson et al, AEM minireview, in Press, 2011

Bacteriocin production as a probiotic trait?

Bacteriocin production mediates Lb. salivarius UCC118 protection against L. monocytogenes infection of A/J mice

Corr S. C. et.al. PNAS 2007;104:7617-7621

Placebo Bac+ Bac-

Lux tagged Listeria

Corr S. C. et.al. PNAS 2007;104:7617-7621

Trn-

Trn-

Thuricin CD Selectively Kills C. difficile

Thuricin CD

Overlaid with Clostridium difficile

Bacillus thuringiensis

Thuricin CD; a two

component

bacteriocin

Rea et al., unpublished

30,000 sporeformers

Rea et al. PNAS 2010 (1)

Mary Rea

clostridia

bacilli

Thurici

n S

pect

rum

listeria Rea et al. PNAS 2010 (1)

0.0

0.5

1.0 80

70

60

50

% aceto

nitrile

Vo

lts

2763 2861

G W V A C V G A C G T V C L A S G G V G T E F A A A S Y F L

G N A A C V I G C I G S C V I S E G I G S L V G T A F T L G

G W V A C V G A C G T V C L A S G G V G X E F A X A S X F L

G N A A C V I G C I G S C V I S E G I G X L V G X A F X L G

-2 -2 -2

-2 -2 -2

Radical SAM proteins Protease ABC transporter

2769 (-6)

2867 (-6)

A new class of antimicrobial

peptides called sactibiotics

Thuricin operon

Rea et al., unpublished

Thuricin contains unusual post-translational modifications

H2NNH

HN

NH

HN

NH

HN

NH

HN

NH

O

O

O

O

O

O

O

O

O

HN

HN

NH

O

OHN

OOH

NH

OHN

O

HN

NH

OHN

NH

HN

NH

HN

NH

HN

NH

O

O

O

O

O

O

OO

HOOC

HO

HO

OHO

NH

NH

HN

O

O

OH

O

OHN

O

NH

O

S S S

Tyr 28

Ala 25

Thr 21

Gly 18

Gly 17

Cys 13Cys 9Cys 5

OH

H2NNH

HN

NH

HN

NH

HN

NH

HN

NH

O

O

O

O

O

O

O

O

OHN

NH

O

OHN

O

OH

NH

OHN

O

HN

OH

NH

OHN

NH

HN

NH

HN

NH

HN

NH

O

O

O

O

O

O

OO

OHO

NH

NH

HN

O

O

OH

O

OHN

O

NH

O

Cys 5 Cys 9 Cys 13

Thr 28

Thr 25 Ser 21

S S S

OH

O

HO

HO

H2N

O

Trn

Trn

Vederas Group

Rea et al. PNAS 2010 (1)

Trn-

Trn-

Rea et al. PNAS 2010 (2)

G N A

A C

V I G C

I G S C

V I S

G L T s

F A T

s s

G V

L S

G I G E

G W V

A C

V G A C

G T V C

L A S

L F Y s

S A A

s s

A F

E T

G V G G

Distal Colon Model

20% human faecal slurry

control Thuricin CD (90uM)

control Vancomycin (90uM)

Metranidazole (90uM)

106 Clostridium

difficile

24h 24h 24h 24h 24h

Total DNA purified, amplified V4 region of 16S rRNA, pyrosequencing, MEGAN

Rea et al. PNAS 2010 (2)

Thuricin CD (90 uM) Vancomycin (90 uM) Metranidazole (90 uM)

6

7

8

4

5

3

C. diff

log

0 4 8 12 16 20 24 h

0 4 8 12 16 20 24 h 0 4 8 12 16 20 24 h 0 4 8 12 16 20 24 h

6

7

8

4

5

3

C. diff

log

0 4 8 12 16 20 24 h

6

7

8

4

5

3

C. diff

log

0 4 8 12 16 20 24 h

Faecal fermentations

Rea et al. PNAS 2010 (2)

Phylum

Collateral

damage 16S profiling

Rea et al. PNAS 2010 (2)

Rectally in conjunction with C. difficile ribotype 027

3

4

5

6

Control mice Test miceControl Test

Lo

g C

. d

iffi

cile C

FU

ml-

1 c

olo

n c

on

ten

ts

3

4

5

6

0

10

20

30

40

50

60

70

80

90

100

%Int.

2500 2550 2600 2650 2700 2750 2800 2850 2900 2950 3000 3050 3100 3150 3200

m/z

1[c].I6

38 mV[sum= 6885 mV] Prof iles 1-182 Smooth Av 30 -Baseline 100

AXIMA-TOF2

Data: 2012\january 2012\100112\mary rea\T1.0001.I6[c] 10 Jan 2012 20:19 Cal: Brady -Ins B Lin 1702 10 Jan 2012 20:19

Shimadzu Biotech Axima ToF² 2.8.4.20081127: Mode linear, Power: 91, Blanked, P.Ext. @ 3000 (bin 85)

2786.99

2764.702804.88

2902.31

2641.57 3003.202862.31

2720.482544.552616.08 2659.45 2918.96 2984.762846.29

2748.182521.92 2566.60 2700.62 2935.262896.402606.68 3145.823089.133043.352994.832816.29 3196.39

trn-α trn-β

Gut Soutions to a Gut Problem

Conclusions: Carriage up to 10 fold higher in elderly and IBD Gut microbiota radically changed in active infection Pharmabiotics: both bacteriocins and bacteriophage hold promise to selective target infection Probiotics is still at an early stage for C. diff treatment

Thanks

Mary Rea Eileen O’Shea Paula O’Connor Orla O’Sullivan Gillian Gardiner Sinead Corr Evelyn Clayton Alleson Dobson Fiona Crispie Sheila Morgan

Paul O’Toole

Catherine Stanton

Ger Fitzgerald

Fergus Shanahan

arry Kiely

Colin Hill

Paul Cotter

John Vederas

ANTIMICROBIALS

MICROBIOME

Mark Wilcox for Ribotyping

Fidaxomicin was shown to have a lesser effect on the microbiome as measured by

qPCR. Vancomycin treated patients showed a 2-4 log reduction in the

Bacteroides/Provetella group which persisted up to 28 days which was absent in

the Fidaxomicin treated group. In addition reappearance of the toxin in the culture

filtrates was apparent in 28% of the vancomycin treated patients compared to 14%

of the Fidaxomicin treated group post treatment and also there was decreased

incidences of reoccurrences of CDAD in the Fidaxomicin treated group.