Dr Adele Costabile4cau4jsaler1zglkq3wnmje1-wpengine.netdna-ssl.com/wp... · 2019-07-30 · 3 INTRODUCTION PREVIOUS WORK ON GOINGON GOING WORK FUTUREFUTURE WORK Over the past 50 years,

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Determination of the inhibitory spectrum of pre-probiotics against Salmonella Typhimurium in in vitro studies.

DrDr AdeleAdele CostabileCostabile

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What’s Salmonella?

Facultative anaerobic, gramFacultative anaerobic, gram-- bacteria, rodsbacteria, rodsBelongs to a family; Belongs to a family; EnterobacteriaceaeEnterobacteriaceaeSalmonella (genus)Salmonella (genus)

entericaenterica (species)(species)serotype (over 2200 serotypes)serotype (over 2200 serotypes)

INTRODUCTION PREVIOUS PREVIOUS PREVIOUS WORKWORKWORK

ON GOINGON GOINGON GOINGWORKWORKWORK

FUTUREFUTUREFUTUREWORKWORKWORK

Where does Salmonella come from?

Inhabitant of intestinal tract of

• animals

• birds

• reptiles

• insects

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Over the past 50 years, there has been increasing amounts of antibiotics used prophylactically and as growth promoters.

Evidence has been accumulated to show that there is a link between risk of zoonotic disease and growth promoting antibiotics usage.




• In June of 1999: The EU banned the use of some growth promoting antibiotics in poultry.

• January 2006: The EU officially ban the usage of all antibiotics such as growth promotans in livestock. (Halfhide, 2003).

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In view of the increasing antibiotic resistence of pathogens that pose a threat for human health, alternative nutritional strategies are being actively pursued.




PIG GASTROINTESTINAL MICROBIOTA: complex and dynamic ecosystem

Provides essential products to the host

Form a key barrier against pathogens

Important roles in gut morphology

Immunity development

Digestion and even modulating gene host expression

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Several studies have investigated the species diversity of the pig intestine by anaerobic culturing (Tannock et al., 1970; Salanitro et al., 1977; Robinson et al., 1981; Varel et al., 1987).

Traditional Microbiological Techniques.

16S rDNA

PCR

Whole Bacterial Cellsin situ

Probe or primer designuse in FISH, qPCR, DGGE

Sequence16S rRNA

DGGECommunity Analysis

16S rDNA clone library

Sequence16S rRNA

Whole Bacterial

cells in situ

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h Enumeration of phylogenetically related groups of bacteria e.g. FISH. Harmsen et al., (1998) FEMS Microbiol. Ecol. 183: 125-129.

h Tracking of introduced bacterial strains in gut microflora e.g. DNA finger printing (PFGE, Ribotyping, RADP). McCartney et al., (1996) AEM 62:4608-13.

h Characterisation of gut microflora diversitye.g.Amplified 16S rRNA cloning and sequencing, D/TGGE. Suau et al., (1999) AEM 65:4799-07

…….New tools for the analysis of the gastrointestinal microbiota




GASTROINTESTINAL MICROBIOTA: complex and dynamic ecosystem

hMore than 400-500 species.

hViable counts to 1012/g of gut content in the large intestine (Eckburg et al., 2005).

hLeser and co-workers (2002) sequenced more than 4200 cloned 16S rDNA sequences from digesta samples .

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In this sense, the exploitation of dietary ingredients targeting improved intestinal function and a healthy gastrointestinal tract (GI) environment attracts a great deal of interest.




SalmonellaTyphimurium

The Alternative Approach…….

Pre- orProbiotics

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….Probiotics might modulate the intestinal ecosystem by

Probiotics

Competition with pathogens for epithelial receptors

Production of organic acids

acetate, lactate

Competition for nutrients in the gut

Production of anti-microbial agents

Immuno-modulation

Sucrose

----

Laevan

√√√√√√√√

Melezitose

√√√√√√√√

FOS

--√√-

IMO

--√√-

Xylan

√√---

Lactose

---√√

Maltose

√√√√√√√√

Cellobiose

----

Inulin

√√--√

XOS

----

Pathogen

E. coli 0157 VT -S. TyphimuriumC. jejuni 4731E. coli HE 320

PathogenE. coli 0157 VT -S. TyphimuriumC. jejuni 4731E. coli HE 320

Anti-pathogen Activity of Probiotics can be Mediated by Substrate

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DIETARY MODIFICATIONS

TO CHANGE STRUCTURE OF

THE MICROBIAL COMMUNITY

MAKE IT RESISTANT TO THE ESTABILISHMENT

OF OPPORTUNISTIC PATHOGENS

Prebiotics1. Must be stable under acidic conditionsand small gut secretions

2. Must get tothe colon intact

3. Must beselectivelymetabolised

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How Can Prebiotics Help?

• Fahnwort et al. (1992): numerical increases in total anaerobes, aerobes and bifidobacteria when the pigs were fed with inulin.

• Houdijk et al. (1997): decreases in total aerobes in the ileum in response to feeding oligofructose and …….

How Can Prebiotics Help?

• Krueger et al. (2002): Lactulose with good results in the productive performance of the piglets.

• Kostantinov et al. (2004): 20g/Kg feed of lactulose as syrup …….other study have shown improvements in pig growth performance after administration of different prebiotics.

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Combining Probiotics and Prebiotics

SYNBIOTICS• Nemcova et al. 1999: The administration to weanling

pigs of Lactobacillus paracasei + FOS resulted in higher numbers of total anerobes, aerobes and lactobacilli.

• Estrada et al. 1999: Feeding early-weaned pigs with FOS and Bifidobacterium longum found an improvement in feed efficiency.

PREBIOTICS

INTRODUCTIONINTRODUCTIONINTRODUCTION ON GOINGON GOINGON GOINGWORKWORKWORK


PREVIOUS WORK

Gal β(1-4)Fru99% Lactulose

Lactulose

95% FructoligosaccharidesActilight

50%-Raftilose-P95(FOS)50%-Inulin

Synergy 1

{Fru β(2-1)Fru}1-9-78%{Fru β(2-1)Fru}1-9- Fru β(2-1) α-Glu}-22%

95% oligosaccharidesRaftilose-P95 (FOS)

95%oligosaccharidesGentioligosaccharides (GeOS)

Xyl β(1-4)Xyl2-595% oligosaccharides

Xyloligosaccharide (XOS)

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PROBIOTICS

BIFIDOBACTERIUMB. bifidumB. longumB. breveB. infantis

LACTOBACILLUSL. acidophilusL. rhamnosusL. caseiL. plantarum

Salmonella

Typhimurium

Enterococcus faecium



PREVIOUS WORK



OD (660nm)

GROWTH CURVES (pure cultures)

PREVIOUS WORK

24h 37ºC

BACTERIA

PREBIOTIC

Microbial growth: arithmetic scale

0

50

100

150

200

250

300

0 2 4 6 8

Number of divisions

Cel

l num

ber

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PREVIOUS WORK

0.001630.00690.00470.01530.00530.00620.00660.0096SEM

<0.0001<0.0001<0.0001<0.0001<0.0001<0.00010.0002<0.0001p-treatment

0.0375b0.0374d0.1092e0.0388c0.0461d0.0330d0.0730a0.0610dXos

0.0377b0.0328d0.1183de0.1321b0.0492d0.0352d0.0730a0.1536bSynergy1

0.0326c0.0265d0.125d0.1840a0.0592d0.0390cd0.0472b0.1737abRaftilose P95

0.0156d0.2054a0.2694a0.1946a0.1921b0.1069a0.0531b0.1753abMRS

0.0306c0.1609b0.1971b0.1879a0.2180a0.0858b0.0889a0.1763aLactulose

0.0475a0.0917c0.1746c0.1404b0.1585c0.0520c0.0548b0.1079cGentio

S. TyphimuriumL. rhammosusL. plantarumL. caseiL. acidophilusB. longumB. infantisB. breve

Prebiotic



PREVIOUS WORK

Growth rates S.1344

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07

S.1344

units

h-1

ActilightGentioLactuloseMRSRaftilose P95Synergy1XosBPW

d

b

d

e

dc c

a

- Values are the means of units ml-1

- Different letters within the same time point indicates significant differences between treatments (p<0.05)

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CO-CULTURES

24h 37ºC

BGA+NalSalmonella

MRSProbiotics

OD (660nm), pH

INTRODUCTIONINTRODUCTIONINTRODUCTION PREVIOUS PREVIOUS PREVIOUS WORKWORKWORK


ON GOINGWORK

SALMONELLAPROBIOTIC

PREBIOTIC



ON GOINGWORK

-0.0312.2382.207LactuloseL. casei

1.6432.2623.905LactuloseL. rhamnosus

1.6092.2713.879LactuloseB. longum

0.0992.1642.262LactuloseB. breve

µProbiotic-µSalmonellaµSalmonellaµProbioticPREBIOTICPROBIOTIC

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E.faecium+S1344+lact

4

5

6

7

8

9

10

0 5 9 12 24

Time (h)

Log

FCU

/mL P2

S2

pH

µP=2,366/hµS=2,295/h∆µ=0,071

B.breve+S1344+lact

4

5

6

7

8

9

10

0 5 9 12 24

Time (h)

Log

FCU

/mL P5

S5pH

µP=2,262/hµS=3,164/h∆µ=0,099

B.longum+S1344+lact

4

5

6

7

8

9

10

0 5 9 12 24

Time (h)

Log

FCU

/mL P8

S8pH

µP=3,879/hµS=2,271/h∆µ=1,609

L.rhamnosus+S1344+lact

4

5

6

7

8

9

10

0 5 9 12 24

Time (h)Lo

g FC

U/m

L P18

S18

pH

µP=3,905/hµS=2,262/h∆µ=1,643

INTRODUCTIONINTRODUCTIONINTRODUCTION ON GOINGWORK


PREVIOUS PREVIOUS PREVIOUS WORKWORKWORK

BATCH CULTURES (mixed cultures)

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BATCH CULTURES (mixed cultures)

•pig faecal slurry

•A nutrient basal medium capable of supporting the gut microflora.

• 1% prebiotics

• pH setting 6.4-6.5 (distal colon)

• fermentations ran for 24h.

• Supernatant samples collected at time

0, 5, 10 and 24 hours.

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Microbial enumeration

by FISHWhole Bacterial Whole Bacterial

cells cells in situin situ

1. Bacteroides spp. (Bac303, Manz et al., 1996)

2. Bifidobacterium spp. (Bif164, Langendijk et al., 1995)

3. Clostridium perfringens/histolyticum sub. grp. (CHis150, Franks et al., 1998)

4. Clostridium Cluster XIVa (Erec482, Franks et al., 1998)

5. Clostridium Cluster IX (Prop853 Walker et al., 2005)

6. Lactobacillus/Enterococcus spp. (Lab158, Harmsen et al., 1999)

7. Ruminococcus bromii- flavefaciens (Rbro730-729,Harmsen et al., 2000)

8. Atopobobium group (Ato291, Harmsen et al., 2000)

9. Salmonella serovars (Sal303, Nordentoft et al., 1997)

10. Total bacterial counts (nucleic acid stain DAPI)


ON GOINGWORK


Autochthonous microbiota in the pig adult

• In the distal small intestine, the environmental conditions slightly differ form the upper sections.

• The cecum and colon are the major sites for bacterial fermentation in the pig gut, characterized by a high diverse population.

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ON GOINGWORK


• Gram – cover only about 10% of total culturablebacteria, most isolates belonging to the Bacteroides and Prevotella groups.

• The Group targeted by Erec482 represents Firmicute bacteria that belong Clostridium cluster XIVa is significant mainly in the hind gut;

• Bacteroides/Prevotella 5–10%


ON GOINGWORK


Proximal colon/ rectum• Prop853 (Clostridial cluster IX) In the stomach is

abundant (14–41% of total bacterial count). ~ less 5% in the colon. The targeted group includes a diverse collection of anaerobes, including Selenomanas, Veionella, Megasphaera and Mitsuokella, most produce propionate as end product.

• Cluster IV classified as ruminococci 4–10%, include specialist fibre-degrading, such as R. bromii (starch degrading) and flavefaciens (cellulolytic)

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Bac303

Chis150

Lab158

Sal 303

Rbro730-729 DAPI

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Lactulose had a stimulatory effect upon colonic lactobacilli at concentrations of 1%. This prebiotic showed an increase of 2 log in lactobacilli between 10 and 24 h of fermentation




*

Salmonella Typhimurium

6

6.5

7

7.5

8

8.5

0 5 10 15 20 25Time (h)

Log1

0 C

FU/m

l

Gentio Lactulose RaftiloseP95Synergy1 Xos

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SCFASCFAAcetateAcetatePropionatePropionateButyrateButyrateBranchedBranched

VFAVFA

LactateLactate

SuccinateSuccinate

EthanolEthanol

HH22 / CO/ CO22 / CH/ CH44

Metabolic products of CHOMetabolic products of CHOcolonic fermentationcolonic fermentation

FERMENTATION PRODUCT ANALYSIS


ON GOINGWORK


SCFASCFA

GI TRACT GI TRACT TRANSITTRANSITSISTEMSISTEM

MICROBIOTAMICROBIOTA

TYPE/ CHEMICAL STRUCTURE TYPE/ CHEMICAL STRUCTURE OF POLYSACCHARIDESOF POLYSACCHARIDES


ON GOINGWORK


SUBSTRATESUBSTRATE

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……..IN THE PIG COLON..IN THE PIG COLON

The fermentation of CHO results in the

production ofLactic acidLactic acidand gasesand gases

SCFASCFA(70 to 100 (70 to 100 mMmM)) Varying in concentration Varying in concentration

and relative proportions and relative proportions depending of GI sectiondepending of GI section



PREVIOUS WORK

Total SCFA

010203040506070

5 10 24

Time (h)

SCFA

con

cent

ratio

n (m

M)

ActilightGentioLactuloseRaftilose P95Synergy1Xosc

abc

aba

a c

a

cbc

a ab

ab

dcd

a

bc

- Values are means of total concentration (mM)

- Different letters within the same time point indicates significant differences between treatments (p<0.05)

23



PREVIOUS WORK

Total SCFA

010203040506070

5 10 24

Time (h)

SCFA

con

cent

ratio

n (m

M)

ActilightGentioLactuloseRaftilose P95Synergy1Xosc

abc

aba

a c

a

cbc

a ab

ab

dcd

a

bc

- Values are lsmeans of total concentration (mM)

- Diferent letters within the same time point indicates significant differrences between treatments (p<0.05)

Principal Bacterial Groups Involved in Metabolite Formation

•Acetate: bacteroides, bifidobacteria, eubacteria, lactobacilli, clostridia, ruminococci, peptococci, veillonella, peptostreptococci, fusobacteria, butyrivibrio•Propionate: bacteroides, propionibacteria, veillonella•Butyrate: clostridia, fusobacteria, eubacteria, peptostreptococci•Lactate: bacteroides, bifidobacteria, lactobacilli, eubacteria•Ethanol: bacteroides, bifidobacteria, clostridia, lactobacilli•Succinate: bacteroides, ruminococci, prevotella•Hydrogen: clostridia, ruminococci, fusobacteria

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ON GOINGWORK


• Higher lactic acid concentration were expected as lactic acid is the major end product in the metabolism of lactic acid bacteria

• However lactate may be converted into butyrate • Prop853 are capable of utilizing lactate and

converting it largely to acetate and propionate in GI of pigs.


ON GOINGWORK


Pure and batch cultures results show a inhibition in Salmonella growth in presence of Lactulose. This effect could be due to the stimulation in the lactic acid bacteria growth (Pascual et al., 1999) in presence of Lactulose, as it was manifested in the growth curves.

.........CONCLUSIONS

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Thank you for your kind attention…

……QUESTIONSQUESTIONS??


ON GOINGWORK


Documents

Dr Adele Costabile4cau4jsaler1zglkq3wnmje1-wpengine.netdna-ssl.com/wp... · 2019-07-30 · 3 INTRODUCTION PREVIOUS WORK ON GOINGON GOING WORK FUTUREFUTURE WORK Over the past 50 years,