u } ( ] } ] v ] } ] W u } ( Z , µ u v' µ } v v } Z u } Z P ] Z ] Z ] } o ......^ ] P v ] ( ]...

Impact of Abiotic and Biotic Parameters of the Human Gut on

Enterohemorrhagic Escherichia coli Survival and Virulence

IAFP's 13th European Symposium on Food Safety 29th March 2017, Brussels, Belgium

Stéphanie Blanquet-DiotAssociate professor, University of Clermont Auvergne, France

Université Clermont Auvergne, Clermont-Ferrand, FranceUMR UCA-INRA 454 MEDIS « Microbiota, Digestive Environment and Health »

Pr. Pierre Peyret

Introduction Conclusion

Clermont-Ferrand

Abiotic factors Biotic factors

Intestinal microbiota :main functions and impact on human and animal health

Galenicformulation

ARTIFICIAL DIGESTION /

FERMENTATION

Gnotobiotic animalsAnalytic infrastructure

Aerobic/anaerobic microbiologyBioinformatics

Molecular tools

Pr. Pierre Peyret

Introduction ConclusionAbiotic factors Biotic factors

Intestinal microbiota :main functions and impact on human and animal health

Pr. Pierre Peyret

3 main research axes

► Food and xenobiotic metabolism

► Host-microbiota interactions

► Food-borne zoonotic diseases

ENTEROHEMORRHAGIC ESCHERICHIA COLI (EHEC)(O157:H7)

Major food-borne pathogens

→ Bloody diarrhea, hemorrhagic colitis

→ Life-threatening systemic complications

Real public health problem

Tarr et al., Lancet, 2005Pennington, Lancet, 2010

Karmali et al., Lancet, 1983

RESERVOIRS

TRANSMISSION VIA CONTAMINATEDFOOD AND WATER

PHYSIOPATHOLOGY

RESERVOIRS

PHYSIOPATHOLOGY

ACID RESISTANCE

BLOODY DIARRHEA

SHIGA TOXINS

RESERVOIRS

ATTACHMENT ANDEFFACEMENT LESIONS

INTIMIN

WATERY DIARRHEA

ACID RESISTANCE

PHYSIOPATHOLOGY

BLOODY DIARRHEA

SHIGA TOXINS

BLOODSTREAM

RESERVOIRS

INTIMIN

WATERY DIARRHEA

ACID RESISTANCE B Lymphocyte

T Lymphocyte

PEYER’S PATCHES

M cellMacrophage

CROSS THE INTESTINAL BARRIER

Schüller, Toxins, 2011Etienne-Mesmin et al., PLoS One, 2011

PHYSIOPATHOLOGY

BLOODY DIARRHEA

SHIGA TOXINS

BLOODSTREAM

RESERVOIRS

INTIMIN

WATERY DIARRHEA

ACID RESISTANCE

PHYSIOPATHOLOGY

KIDNEY

HEMOLYTIC UREMIC SYNDROME (HUS)

TROMBOTIC TROMBOCYTOPENIC

PURPURA (PTT)

INTERACTIONS WITH HUMAN GUT MICROBIOTA?

HUMAN GI TRACT = A BLACK BOX…

SURVIVAL?

EXPRESSION OF VIRULENCE GENES?

Mono-compartmentalstatic in vitro models

Digestive complexity

Multi-compartmentaldynamic in vitro models

Acid pH

Isolated strains from gut microbiota

• Far from in vivo complexity• Only one digestive parameter

at the same timeHouse et al., Microbiol, 2009

Hammer et al., PLOS One, 2013

Connolly et al., Front. Microbiol., 2015

Pifer and Sperandio, Microbiol. Spectrum, 2014

OUTSTANDING QUESTION 1How EHEC survival and virulence are modulated

thoughout the human GI tract?

ANTIBIOTIC THERAPY IS CONTROVERSIAL↑ Toxin synthesis

↑ Risk of developing HUS

SYMPTOMATIC TREATMENT

Wong et al., N. Eng. J. Med., 2000CDC, 2011

TREATMENT OF EHEC INFECTIONS

NO MARKETED VACCINEGarcia-Angulo et al., Vaccine, 2013

ALTERNATIVE STRATEGIES

PROBIOTICS

“Live microorganisms which, when administered in adequate amounts, confer a health benefit on the host”

FAO/WHO, 2002Hill et al., Nat Rev Gastroenterol

Hepatol, 2014

In vitro In vivo

Acid pH, bile, SCFA…

ANTAGONISTIC POTENTIAL OF PROBIOTICSON EHEC PATHOGEN

Eaton et al., 2011Medellin-Pena et al., 2007

Carey et al., 2008

Dalmasso et al., 2006

Tsai et al., 2010

Gagnon et al., 2006

Arena et al., 2014

Fukuda et al., 2011

Kim et al., 2008

BACTERIA

- Lactobacillus

- Bifidobacterium

- Saccharomyces cerevisiae var boulardii

Cordonnier et al., Crit. Rev. Microbiol., 2015

Effect of probiotic on human gut microbiota modulationin the context of EHEC infection ?

- Saccharomyces cerevisiae var boulardii

- Saccharomyces cerevisiae CNCM I-3856

AIEC/miceSivignon et al., Infl. Bowel Dis., 2015

Pineton de Chambrun et al., Dig. Liver Dis., 2015 ; Cayzeele-Decherf et al., World J. Gastroenterol., 2017

Anti-inflammatoryproperties

Reduction of digestivediscomfort and abdominal pain in IBS patients

Improvement of Intestinal barrier function

AIEC/miceSivignon et al., Infl. Bowel Dis., 2015

ETEC/ intestinal epithelial cellsZanello et al., PLoS One, 2011

chemically-induced colitis/miceFoligné et al., World J. Gastroenterol., 2010

OUTSTANDING QUESTION 2Can Saccharomyces cerevisiae CNCM I-3856 be useful

in the control of EHEC infections ?

PHYSICO-CHEMICALPARAMETERS

INTESTINALMICROBIOTA

ABIOTIC FACTORS BIOTIC FACTORS

Impact of Abiotic and Biotic Parameters of the Human Gut

on EHEC Survival and Virulence

ConclusionBiotic factors

Introduction Abiotic factors

STOMACH

SMALL INTESTINE

Multi-compartmentaldynamic gastro-intestinal model

SIMULATED PARAMETERS

Body temperature

Temporal and longitunalchanges in gastric and

intestinal pH

Chyme mixing

Transit time

Gastric and ileal deliveries

Digestive secretions(pepsin, lipase,

pancreatic juice, bile)

Passive absorption(dialysis)Minekus et al., Atla, 1995

Guerra et al., Trends Biotechnol., 2012Cordonnier et al., Microorganisms, 2015

Stomach

Jejunum

Duodenum

TIMTNO gastroIntestinal Model

ConclusionBiotic factorsIntroduction Abiotic factors

Experimental plan

Roussel et al., Ped. Res., 2016

CHILD ADULT

EHEC O157:H7 EDL 933 (107 CFU/mL)

Digestive compartmentsn = 4

NumerationLB medium

SURVIVAL

VIRULENCE

Propidiumiodide

SYTO 9

Flow cytometryLife/dead analysis

Viable Altered

qRT-PCR (stx, shigatoxin)(eae, intimin)

ELISA (Stx)

Experimental plan

Healthy childHealthy adult

Results : effect of « age » on EHEC survival 0157:H7Transit marker

Adult Child

EHEC survival unaffected in the stomach and duodenum

under both adult and child digestive conditions

Adult Child

Stomach Duodenum

Adult Child Adult Child

Jejunum Ileum

At the middle of digestion :

Significant bacterial mortality in the jejunum and ileumbut only in adult

Results : effect of « age » on EHEC survival

0157:H7Transit marker

At the end of digestion :

Significant bacterial growth in the jejunum and ileum, mostly in child

Adult Child Adult Child

Jejunum Ileum

Results : effect of « age » on EHEC survival

0157:H7Transit marker

Results : EHEC physiological state

Ileal effluents : flow cytometry analysis

→ Higher number of altered cells under adult digestive conditions

viable

altereddead

Green fluorescence

37% 38%

14% 15%

viable

altereddead

Green fluorescence

→ Higher number of viable cells under child digestive conditions

→ Bacterial growth as previously observed by numeration

22% 34%TIM

Results : EHEC physiological state

A higher number of cells, in a better physiological state,

is likely to reach the terminal ileum and colon under child conditions

→ main sites of EHEC pathogenesis

Results : EHEC survival and physiological state

Chong et al., Microbiol., 2007Lewis et al., Infect. Immun., 2015

Results : effect on « age » on stx expression

stx1 stx2

Gastric effluents

In the gastric effluents :

stx1 and stx2 encoding Shigatoxins are significantly overexpressed

under child conditions compared to adult ones

RTqPCR(stx)

stx1 stx2

Ileal effluents

In the ileal effluents :

similar trends are observed for stx but less obvious than in the stomach

Results : effect on « age » on stx expressionadult

RTqPCR(stx)

Stx production

Ileal effluents

Adult : no toxin was detected

Child: regular increase in the amount of Stx produced

Results : effect of « age » on Stx production

ELISA (Stx)

Results : effect on « age » of stx expression and production

stx expression and production higher under child vs adult digestive conditions

→ Stx : main virulence factor of EHEC, responsible for systemic complications

eae gene encoding intimin overexpressed :

→ under adult conditions at the beginning of digestion

RTqPCR(eae)

Results : effect of « age » on eae expression

Ileal effluentsGastric effluents

eae gene encoding intimin overexpressed :

→ under child conditions at the end of digestion

RTqPCR(eae)

Overexpression of adhesin under child conditions

→ most of bacteria have reached the distal parts of the small intestine→ main site of EHEC colonization

RTqPCR(eae)

Differences in the physico-chemical parameters of the upper human GIT

→ partially explain why child are more susceptible to EHEC infections than adults

Concluding remarks

StomachShiga toxin production

Small Intestine

Bacterial growth

+++Shiga toxin production

Small Intestine

Bacterial growth

M cell

Shiga toxin production

Intestinal epithelium

Adhesin expression

Small Intestine

Bacterial growth

M cell

Shiga toxin production

Intestinal epithelium

Adhesin expression

++Gb 3

KidneysGb3 expression

Chaisri et al., Microb. Pathog., 2001

ConclusionAbiotic factors

Introduction Biotic factors

IN VITRO AND IN VIVO COMPLEMENTARYAPPROACHES

TIM : humanupper GIT

ARCOL : humanlower GIT

ConclusionAbiotic factorsIntroduction Biotic factors

IN VITRO IN VIVO

MICE ILEAL LOOPS : interactions with

intestinal epithelium

GNOTOBIOTIC RATS : interactions with gut

microbiota

IN VITRO IN VIVO

microbiota

Experimental plan1

TIMhealthy adult

Western diet

O157:H7 EDL 933(2.105 CFU/mL)

+ S. cerevisiae CNCM I-3856 (107 CFU/mL)

O157:H7 EDL 933(2.105 CFU/mL)

Digestive compartmentsn = 4

LB medium

SURVIVAL

Bacterial mortality in the stomach and duodenumProbiotic → EHEC survival unchanged

E. coli O157:H7

E. coli O157:H7 + S. cerevisiae

Transit marker

0 30 60 90 120 150

Time of digestion (min)

Stomach

††

30 60 120 180 240

Duodenum

††

Etienne-Mesmin et al., Appl. Environ. Microbiol., 2011

Results : EHEC survival1

Significant reduction of bacterial growth in the jejunum and ileum→ competition for nutrients?

→ production of inhibitory compounds?

60 120 180 240 300

Jejunum

** †

60 120 180 240 300

Ileum*

1 Results : EHEC survivalE. coli O157:H7

Transit marker

Significant higher concentrations of ethanol with probiotic yeasts→ toxic for cell membrane? Chiou et al., Appl. Environ. Microbiol., 2004

→ synergetic action with other inhibitory compounds?

60 120 180 240 300

Ileum*

E. coli O157:H7

Transit marker

60 120 180 240 300

Jejunum

** †

1 Results : ethanol production

Ethanol

IN VITRO IN VIVO

microbiota

Human colonic model

ARCOLARtificial COLon

Blanquet-Diot et al., Pharm. Res., 2012Cordonnier et al., Microorganisms, 2015

SIMULATED PARAMETERS

Inoculation with freshhuman fecal samples :

complex and metabolicallyactive microbiota

Body temperature

Mean colonic pH

Retention time

Anaerobiosis

(sustained by the sole activityof the microbiota)

Passive absorption(dialysis)

Redox potential

Experimental plan2

n = 3 with three different healthy donors

1) Control experiment2) S. cerevisiae3) O157:H74) S. cerevisiae + O157:H7

Experimental plan2

D0 D4 D8 D12

STABILISATION

Inoculation of the bioreactorwith fecal sample

Experimental plan2

D0 D4 D8

STABILISATION S. cerevisiae CNCM-I 3856

S. cerevisiae CNCM I-3856107 CFU/mLTwice daily administration

Experimental plan2

D0 D4 D8

STABILISATION

Gastric and smallintestinal stresses

O157:H7 EDL 933107 UFC/mL

Experimental plan2

D0 D4 D8

STABILISATION S. cerevisiae CNCM-I 3856

S. cerevisiae CNCM I-3856107 CFU/mL

Twice daily administration

Gastric and smallintestinal stresses

O157:H7 EDL 933107 UFC/mL

Experimental plan2

SURVIVAL VIRULENCE INTERACTION WITHGUT MICROBIOTA

Composition of gut microbiotaqPCR

Metabolic activitySCFA (GC)

qPCR qRT-PCRstx / eae

Time post-administration (days)

Transit markerO157:H7

No colonization by O157:H7 of the human simulated colonic environment→ in accordance with the rapid elimination

from feces of HUS patients Tarr et al., J. Infect. Dis., 1990

Thévenot et al., Appl. Microbiol. Biotechnol., 2015

qPCREHEC

Results : EHEC survival

No influence of the probiotic yeaston the survival of the pathogen in the in vitro colon

Time post-administration (days)

Transit markerO157:H70157:H7 (with probiotic)

Thévenot et al., Appl. Microbiol. Biotechnol., 2015Thévenot et al., Appl. Environ. Microbiol., 2013

2 Results : effect of S. cerevisiae on EHEC survival

qPCREHEC

BacteroidetesFirmicutesActinobacteria

BacteroidesLactococcus/Pediococcus/LeuconostocEnterobacteriaceae

Yeast_2_12Yeast_2_10

Yeast_2_11

Yeast_2_9Yeast_3_12

Yeast_1_9Yeast_3_11

Yeast_3_10Yeast_3_9

Yeast and EHEC_2_11Yeast and EHEC_2_9

EHEC_1_12EHEC_1_9

EHEC_1_10EHEC_1_11

Yeast and EHEC_3_9Yeast and EHEC_3_10Yeast and EHEC_3_11

Yeast_1_11

Yeast and EHEC_3_12Control_1_12

Control_2_11Control_2_12

Control_3_10

EHEC_3_10

EHEC_3_11EHEC_3_12

EHEC_2_12EHEC_2_10EHEC_2_11

EHEC_2_9EHEC_3_9

0.0 0.5 1.0 1.5

Agglomerativehierarchical clustering

qPCRgut microbiota

Results : composition of gut microbiota

Control= 3 donors

Yeast_2_11

Yeast_2_9Yeast_3_12

Yeast_1_9Yeast_3_11

Yeast_3_10Yeast_3_9

EHEC_1_12EHEC_1_9

EHEC_1_10EHEC_1_11

Yeast_1_11

Control_3_10

EHEC_3_10

EHEC_3_11EHEC_3_12

EHEC_2_9EHEC_3_9

0.0 0.5 1.0 1.5

qPCRgut microbiota

Yeast_2_11

Yeast_2_9Yeast_3_12

Yeast_1_9Yeast_3_11

Yeast_3_10Yeast_3_9

EHEC_1_12EHEC_1_9

EHEC_1_10EHEC_1_11

Yeast_1_11

Control_3_10

EHEC_3_10

EHEC_3_11EHEC_3_12

EHEC_2_9EHEC_3_9

0.0 0.5 1.0 1.5

O157:H7 1

S. cerevisiae

O157:H7 + S. cerevisiae

O157:H7 2

Control= 3 donors

qPCRgut microbiota

Individual-dependant effect of pathogen and probioticon the human gut microbiota

→ clinical course of EHEC infections? Pifer and Sperandio, Microbiol. Spectrum, 2014

→ efficacy of probiotic treatment? Thevenot et al., Appl. Microbiol. Biotechnol., 2015

qPCRgut microbiota

During in vitro EHEC infectionProbiotic → ↑ acetate and ↓ butyrate

GCSCFA

Results : metabolic activity of gut microbiota

↑ Acetate↓ Shiga toxin gene expressionAshara et al., Infect. Immun., 2004

Fukuda et al., Nature, 2011

↑ Butyrate↑ A/E gene expression

Tobe et al., Infect. Immun., 2011

During in vitro EHEC infectionProbiotic → ↑ acetate and ↓ butyrate

→ Beneficial modulation of SCFA production

GCSCFA

Results : effect of S. cerevisiae on metabolic activity

x 3 x 3

Shiga toxins

Time post-administration (hours)

Intimin

Until 12h post-infectionOverexpression of major virulence genes : Shiga toxins, intimin

→ even if the in vitro colonic environment is devoid of epithelial cells

stx1 stx2 eae

RT-qPCRstx, eae

Results : EHEC virulence

Shiga toxins

Time post-administration (hours)

Intimin

At 9h and 12h post-infectionProbiotic → significant reduction in stx1 and stx2 mRNA levels

→ due to the increase in acetate production?

stx1 stx2 eae

RT-qPCRstx, eae

Results : effect of S. cerevisiae on EHEC virulence

IN VITRO IN VIVO

microbiota

Euthanasia

Axenic ratsHuman microbiota-associated rats

CAECAL CONTENTS CENTRIFUGED/FILTRATED

Inoculation with O157:H7 EDL 933

TRANSCRIPTOMIC ANALYSIS (DNA MICROARRAY)

n = 8 n = 8

Experimental plan3

Caecal contents

Humanized ratsAxenic rats

Results : effect of microbiota on EHEC gene expression

669 repressed genes 408 activated genes

Human intestinal microbiota : modulation of ~ 20 % of O157:H7 EDL 933 genes

Le Bihan et al., Microb. Infect., 2015

VIRULENCE GENES

Intestinal microbiota : repression of LEE encoding-genes involved in the intimate adhesion

of EHEC to the intestinal epithelium

-8-6-4-202468

espB tir ea

Validation by qRT-PCR

Le Bihan et al., Microb. Infect., 2015

Caecal contents

Humanized ratsAxenic rats

Results : effect of microbiota on EHEC gene expression3

IN VITRO IN VIVO

microbiota

Daily oral administration ofS. cerevisiae CNCM I-3856 (2.106 CFU)

or saline water (control group)

Sacrifice of mice5 h post-infection

MICE ILEAL LOOPS

Injection of O157:H7 EDL 933Gastric and smallintestinal stresses

Intracellular bacteria

Peyer’s patches Mucosa

(107 CFU/mL)

Experimental plan4

n = 26

Mice ileal loops

Oral administration of S. cerevisiae

Peyer’s PatchesIn

yer’

Mucosa

Specific inhibition of EHEC O157:H7 interactionswith Peyer’s Patches

→ Physical interactions between EHEC and probiotic→ Competition for cell receptor?

4 Results : effect of S. cerevisiae on interactions with PPs

Significant decreasein the number of

hemorrhagic Peyer’s Patches

Mice ileal loops

Oral administration of S. cerevisiae

Non hemorrhagic Hemorrhagic

Results : effect of S. cerevisiae on hemorrhagic PPs

Concluding remarks (1)

Individual-dependant effect of EHEC on human gut microbiota compositionInfluence of intestinal microbiota on EHEC virulence gene expression

Role of gut microbiota in clinical course of EHEC infections ?

Concluding remarks (2)

Complementary in vitro and in vivo approaches

→ S. cerevisiae CNCM I-3856: promising antagonistic effects against EHEC O157:H7

Small Intestine

Bacterial growth

S. cerevisiae CNCM-I 3856

Small Intestine

Bacterial growth

Peyer’s Patches

Tropism to Peyer’s Patches

Small Intestine

Bacterial growth

Shiga toxin expression

Peyer’s Patches

Large intestine

Small Intestine

Bacterial growth

Peyer’s Patches

Large intestine

↑ ACETATE

↓ BUTYRATEa

Small Intestine

Bacterial growth

Peyer’s Patches

Large intestine

↑ ACETATE

↓ BUTYRATE

ABIOTIC FACTORSpHBile et digestive enzymesTransit time…

BIOTIC FACTORSIntestinal Microbiota

ProbioticsSURVIVAL / PHYSIOLOGICAL STATE

VIRULENCE

INTERACTION WITH INTESTINAL EPITHELIUM

EHEC AND DIGESTIVE ENVIRONMENT

Conclusion

Temporal/spacial modulations of food-borne pathogen survival and virulence

in the human GIT

Abiotic factorsIntroduction Biotic factors

Dynamic multi-compartmental in vitro models

GI parameters

ABIOTICpH, enzymes, bile

fluid shearoxygenation

BIOTICresident microbiota

probiotics

Bacteria

strainculture conditions

growth phase

Other parameters

food matrixfood process…

Dynamic multi-compartmental in vitro models

Temporal/spacial modulations of food-borne pathogen survival and virulence

in the human GIT

Thank you for your attention !

Alric MoniqueBernalier-Donadille AnnickChalancon SandrineCordonnier CharlotteDenis Sylvain Jubelin GrégoryLe Goff OlivierRoussel CharlèneThévenot Jonathan

Galia WessamLeriche FrançoiseDelphine Thevenot-Sergentet

Tom Van de Wiele

Cordonnier CharlotteLivrelli ValérieNguyen HangRougeron AmandineThévenot Jonathan

Harel JoséeLe Bihan Guillaume

Ballet NathalieVandekerckove Pascal

Valérie Livrelli

Delphine Thévenot-Sergentet

Grégory Jubelin

u } ( ] } ] v ] } ] W u } ( Z , µ u v' µ } v v } Z u } Z P ] Z ] Z ] } o ......^ ] P v ] ( ]...

Documents

o ] ( } v ] & } z } µ Z µ ] } v > Á & ^ Z · 2019-09-06 · ^ } v } u } µ v Ç K ( ( ] } ( µ ] } v U & } , } u o z } µ Z µ ] } v ^ À ] ^ ( ] Z o o ] v d Z u Ç W } i d Z ]

BEETHOVEN’S FIFTH - BernaertsMusic.comBEETHOVEN’S FIFTH E } } ( Z ] µ o ] } v u Ç } µ ] v v Ç ( } u } ( ] v Á ] Z } µ ] } v Á ] © v u ] ] } v } ( Z µ o ] Z BEETHOVEN’S

SC-CompassÂ®: Stray Current Corrosion - Identification, … · 2019. 5. 3. · Z } µ P Z Z } Z Z v Z ] v v ] µ ] X / v Z } } ] } v o ] µ U v } v r v u µ Ç } u µ Z } ] v o

Brochure€¦ · /(Ç}µ vv} Z µ i Ç}µÁ] Z } µ Ç]v Z] o] U o } }v µ XdZ ] v Æ oo v Zv Z Á Á]oo o } vP }µ (} Ç}µ]v Ç}µ Z} v. o } ( µ ÇX u] W }P uu t Z}À Á v Ç ]+

NARUC Risk management paper · ï v X Z P µ o } v u l Z } ] } µ Z ( µ µ µ ] v P } o o Z ] l

K v ^ Z ] } Z µ o d Z u o o W W µ µ ] v P Z s ] ] } v

d Z v ] o ' µ ] o ] v ( } Z } v µ ] } v U Z Z ] o ] ] } v ... · d Z v ] o ' µ ] o ] v ( } Z } v µ ] } v U Z Z ] o ] ] } v } ( ] o o î ' o } Ç v Z } } P v ] t u v r u µ ] (

Z µ o } ( Z µ À Ç } v Z µ } ( Z } ] v Á v : µ v î ì U î ì ... · Z µ o } ( Z µ À Ç } v Z µ } ( Z ] } o ] v / v v } v o } ] v Á v : µ v î ì U î ì í ò v u î

Z } E µ } Z h í h d ] v P Z } · 2020. 12. 14. · d Z ] v ( } u ] } v } v ] v ] v Z ] } µ u v ] ] o Ç } v ( ] v ] o v ] ] v v ( } Z } v o Ç X d Z µ v µ Z } ] µ U d Z ] v

%XFKVWDUW.DIIHH LP 6200(5 · lp 6200(5 t ] + v v µ v ' Z ] Z v^ Z Ìd µ Z u ] u ] o ] } Z l u µ v µ v > v ] u } ] v W ]

u u v ] P o o Z ( · • v l W ] Ì µ u ] µ v l Z o µ v P ] ' o o Z ( À u P v u ] u v µ Z µ Z ] v v ' o o Z ( µ ó ï ô / ï ' o

, À Ç } µ ~ } Z Ç ] Ç } µ ~ } Æ ] v , Z Z v P } Z , Z v ......, o Z } Ç } µ ( ] o o ] v P Z ] µ À Ç ] v } µ , o Z } Ç } µ ( ] o o ] v P Z ] µ À Ç ] v } µ

This electronic thesis or dissertation has been downloaded ... · ^Á ] Z } µ ] _ µ Ç } ( Z ] o v v P [ u } ] o Z } v µ ] v Z } } o Ç W W µ o ] v v Z µ ] Ì o o }

o v d µ ] v P / v ] µ t ^ } ] Z v ] v o Ç ] v P , µ u v ... · o v d µ ] v P / v ] µ t ^ } ] Z v ] v o Ç ] v P , µ u v ] ] µ ] v P Ç ï X d Z U Z } Z h v ] À ] Ç } ( ]

NEW APPLICATION GUIDANCE CORRECT - Gwent Police · 2020-01-10 · u µ ] ( ] Z Z o ] v v u ] } } P µ v Z Á ] Z } µ v P } Z µ o ] ( Ç } Z

] v µ P Z Z ] o v [ , } ] o Z ] Ç d µ [ Z } v } µ v z ... · Z ] o v v Ç } µ v P } o [ o ] À o ] v µ Ç ] o o v X

dZ s > dZ /> Z^ î ñ ì ì ^ Z/ 2019 25B eBrochure.pdf · /'&KKd dZ s > dZ /> Z^ î ñ ì ì ^ Z/ ^ Z ] P ( } } Zs ] µ ] o µ ] v P } µ µ v ] µ } v µ } v ] v µ o } v Ç u Á

^dZ ] v}(µ µ ]vv Çi} ---- Bµ Z (µ µ o] ]v Z }vÁZ}Z }o Z i}

K t Z Z/ &/E' W W Z d Z ^ } ( Z u Ç µ v Z K ' µ ] o ] v · 2020. 6. 16. · d Z ^ } ( Z u Ç µ v Z K ' µ ] o ] v r h v v ] v P E W } v o µ ] v î ì í õ Z } µ P Z Z o v }

µ } v } ] } v ( } Z ] µ o µ } v µ o ] } v G } v o Ç Z µ Z ... · Rodica Crisan Donatella Fiorani Loughlin Kealy Stefano Francesco Musso ^ µ } } Z } P v ] } v Marta Acierno,