Dr Adele Costabile4cau4jsaler1zglkq3wnmje1-wpengine.netdna-ssl.com/wp... · 2019-07-30 · 3...
Transcript of Dr Adele Costabile4cau4jsaler1zglkq3wnmje1-wpengine.netdna-ssl.com/wp... · 2019-07-30 · 3...
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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)
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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.
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• 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.
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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
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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.
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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
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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
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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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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
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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
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PREBIOTIC
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-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
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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
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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)
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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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• 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%
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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
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*
Salmonella Typhimurium
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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
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SCFASCFA
GI TRACT GI TRACT TRANSITTRANSITSISTEMSISTEM
MICROBIOTAMICROBIOTA
TYPE/ CHEMICAL STRUCTURE TYPE/ CHEMICAL STRUCTURE OF POLYSACCHARIDESOF POLYSACCHARIDES
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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
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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)
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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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• 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.
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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??
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