Small molecular weight metabolites regulating growth and immunity as
postbiotic antibiotic alternatives
Inkyung Park
Animal Bioscience and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, MD 20705
Introduction: antibiotic alternatives
• Avian coccidiosis with Emeria spp
• Necrotic enteritis
• With AGP withdrawal, there is an urgent need to develop antibiotic alternatives. Gallucci and Matzinger, 2001; Shirley and Lillehoj, 2012
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0
20
40
60
80
100
120
1925
1945
1960
1975
1990
2005
2008
2011
2014
2017
Mar
ket
wei
ght,
kg
Mar
ket
age,
d
Years
Market age and weight since 1925 (chickens)
• Bacillus subtilis as probiotics in chicken feed
- Shows growth-promoting effect (Gadde et al. 2017)
- Shows a protective role against chicken pathogen (Park et al. 2019)
* * *
0.00E+00
5.00E-06
1.00E-05
1.50E-05
2.00E-05
2.50E-05
3.00E-05
3.50E-05
4.00E-05
CON BMD PB1 PB2 PB3
Mea
n n
orm
aliz
ed e
xpre
ssio
n
IL6 in ileum
* *
0.0E+00
2.0E-03
4.0E-03
6.0E-03
8.0E-03
1.0E-02
CON BMD PB1 PB2 PB3
Mea
n n
orm
aliz
ed e
xpre
ssio
n
Occludin in ileum
Bacillus subtilis by Nano Creative/Science Source
(Gadde et al. 2018)
* *
200
250
300
350
400
450
500
CON BMD PB1 PB2 PB3
Ave
rage
bo
dy
we
igh
t, g
Body weight (D 14)
― Con: basal diet ― BMD: antibiotics ― PB1: B. subtilis 1781 ― PB2: B. subtilis 747 ― PB3: B. subtilis 1781+747
Introduction: antibiotic alternatives
• What is the mechanisms of dietary Bacillus subtilis supplementation?
Introduction: gut microbiota and metabolites
“Postbiotics” novel materials promoting gut health as functional additive of diet
Objective of this study
• Therefore, the current study was undertaken to characterize the metabolic alterations in the chicken gut following dietary supplementation with B. subtilis DFMs with the goal of identifying potential chemical compounds that might be directly used to improve poultry growth performance without the use of AGPs.
Materials and Methods
84 male day-old Ross 708 broilers
d 0
Normal feed
d 14 d 21
• Initial body weight • Allocation to 3 treatments
Measurements: • Metabolomic profiling of
the ileal contents by mass spectrometry (Metabolon, Durham, NC)
ileal content from euthanized chciekn (2 chickens/pen = total 8 chickens/treatment)
• Body weight • Feed intake
Experimental feed
Treatments Supplements Dose Chickens/cage Replication
CON Basal diet 7 4
B. subtilis 1781 CON+B. subtilis 1781 1.5 × 105 CFU/g feed 7 4
B. subtilis 747 CON+B. subtilis 747 1.5 × 105 CFU/g feed 7 4
• B. subtilis strains were obtained from Church & Dwight Co. Inc. (Waukesha, WI).
Metabolic pathways
• Statistical Analysis
• Growth performance
- PROC MIXED in SAS (SAS Inst. Inc., Cary NC)
- P values < 0.05
- PDIFF option
• Ileal biochemicals
- Array Studio software (OmicSoft, Cary, NC)
- the programs R (R Foundation for Statistical Computing, Vienna, Austria)
- JMP (SAS Institute)
- P values < 0.05
- Random Forest Analysis (RFA) by computing the Mean Decrease Accuracy
Materials and Methods
a
b b
320
340
360
380
400
Control B. subtilis 1781 B. subtilis 747
Bo
dy
wei
gh g
ain
, g
Body weight gain of chickens
Body weight gain = final body weight – initial body weight
Control
B. subtilis 1781
B. Subtilis 747
-20 -10 0 10 20 30 40 50
Comp.1 [24.43%]
-20
-10
0
10
20
30
Co
mp
.2 [11
.99
%]
Co
mp
2 (
11.9
9)
Comp 1 (24.43)
Principal component analysis of ileal biochemicals
Result: growth effect and biochemical distribution
Result: hierarchical clustering heatmap
Control
B. subtilis 1781
B. subtilis 747
-4.00 4.00
• Total 674 biochemicals
• Con vs B. subtilis 1781 - Increased 209 (25; P < 0.05) - Decreased 461 (58; P < 0.05)
• Con vs B. subtilis 747 - Increased 265 (12; P < 0.05) - Decreased 402 (38; P < 0.05)
Bio
che
mic
al p
rofi
les
Result: after B. subtilis 1781 and 747 treatment
• 193 amino acid pathways
• 32 carbohydrate pathways
• 263 lipid pathways
• 42 cofactors and vitamins pathways
• 51 nucleotide pathways
• 12 energy pathways
• 81 unknown pathways
• 498 Human Metabolome Database (HMDB)
• 336 Kyoto Encyclopedia of Genes and Genomes (KEGG) codes
Result: pathways in this study B
ioch
em
ical
pro
file
s
CON vs B. subtilis 1781
Result: random forest analysis and plot
• 8 amino acids (26.7%) - 2-hydroxy-4-
(methylthio)butanoic acid - 3-(4-hydroyphenyl)lactate - beta-hydroxycholate - creatine - cystein s-sulfate - N-methylproline - glutamate gamma-methyl
ester - dimethylglycine
• 8 lipids (26.7%) - 2,4-dihydroxybutyrate - glycerophosphoethanolamine - 2R 3R-dihydroxybutyrate - chenodeoxycholte - 16-hydroxypalmitate - beta-sitosterol - 1-palmitoyl-
digalactosylglycerol (16:0) - octadecenedioate
• 5 vitamins and cofactors (16.7%) - carotene diol - alpha-tocopherol acetate - beta-cryptoxanthin - nicotinamide adenine dinucleotide (NAD+) - alpha-tocopherol
• 3 nucleotides (10.0%) - 2’deoxyguanosine - N6-methyladenosine - N1-methyladenosine
Result: random forest analysis and plot
CON vs B. subtilis 747 • 6 amino acids (20.0%) - glutamate gamma-methyl ester - betaine - taurine - dimethylglycine - 5-hydroxyindoleacetate - methylsuccinate
• 6 peptides (20.0%)
- glutaminylleucine - alanylleucine - valylleucine - leucylglycine - leucylalanine - valylglycine
• 10 lipids (33.0%) - 6-oxolithocholate - oleoylcholine - glycerophosphoglycerol - sebacate (C10-DC) - 2-hydroxyglutarate - palmitoylcholine - linoleoylcholine - 3-dehydrodeoxycholate - chenodeoxycholate - stearoylcarnitine (C18)
• 2 nucleotides (0.1 %) - uridine 5’-monophosphate (UMP) - guanine
Alanylleucine
Met
abo
lite
Sign
al
(Med
ium
Sca
led
Sig
nal
Inte
nsi
ty)
Control B. subtilis B. subtilis 1781 747
Glutaminylleucine
Control B. subtilis B. subtilis 1781 747
Control B. subtilis B. subtilis 1781 747
Glycylisoleucine
Valylleucine
Met
abo
lite
Sign
al
(Med
ium
Sca
led
Sig
nal
Inte
nsi
ty)
Control B. subtilis B. subtilis 1781 747
Phenylalanylalanine
Control B. subtilis B. subtilis 1781 747
Valylglutamine
Control B. subtilis B. subtilis 1781 747
Result: amino acids (dipeptides)
Chenodeoxycholate
Control B. subtilis B. subtilis 1781 747
3-dehydrodeoxycholate
Control B. subtilis B. subtilis 1781 747
Cholesterol
Control B. subtilis B. subtilis 1781 747
Sebacate (C10-DC)
Control B. subtilis B. subtilis 1781 747
Linoleoylcholine
Control B. subtilis B. subtilis 1781 747
Valerylglycine (C5)
Control B. subtilis B. subtilis 1781 747
Met
abo
lite
Sign
al
(Med
ium
Sca
led
Sig
nal
Inte
nsi
ty)
M
etab
olit
e Si
gnal
(M
ediu
m S
cale
d S
ign
al In
ten
sity
)
Result: lipids
Miyamoto et al., 2015
Met
abo
lite
Sign
al
(Med
ium
Sca
led
Sig
nal
Inte
nsi
ty)
Control B. subtilis B. subtilis 1781 747
N1-methyladenosine N6-methyladenosine
Control B. subtilis B. subtilis 1781 747
Guanine
Control B. subtilis B. subtilis 1781 747
Control B. subtilis B. subtilis 1781 747
Met
abo
lite
Sign
al
(Med
ium
Sca
led
Sig
nal
Inte
nsi
ty)
2-deoxyguanosine Uridine 5-monophosphate
Control B. subtilis B. subtilis 1781 747
Control B. subtilis B. subtilis 1781 747
Cytidine
Result: nucleotides
N1-methyladenosine
N6-methyladenosine
Guanine
2-deoxyguanosine
Donia and Fischbach, Science, 2015
2-(4-hydroxyphenyl)propionate
Met
abo
lite
Sign
al
(Med
ium
Sca
led
Sig
nal
In
ten
sity
)
Control B. subtilis B. subtilis 1781 747
Salicylate-glucoside
Control B. subtilis B. subtilis 1781 747
Nicotinamide ribonucleotide (NMN)
Met
abo
lite
Sign
al
(Med
ium
Sca
led
Sig
nal
In
ten
sity
)
Nicotinamide adenine dinucleotide (NAD+)
Fructose Lactate
Met
abo
lite
Sign
al
(Med
ium
Sca
led
Sig
nal
In
ten
sity
)
Control B. subtilis B. subtilis 1781 747
Control B. subtilis B. subtilis 1781 747
Control B. subtilis B. subtilis 1781 747
Control B. subtilis B. subtilis 1781 747
Result: others
Polyphenol
Fructose
Fructose feeding negatively affects antioxidant
capacity in the blood of hypertensive rats but
improves this capacity in the liver (Girard et al., 2006, Nutrition).
Lactate ion may prevent lipid peroxidation by
scavenging free radicals such as O2- and -OH
but not lipid radicals (Groussard et al., 2000, J Appl Physiol).
epithelial barrier
Strong antioxidant
Antioxidant effects in the gut or
organs
Conclusions
• Dietary supplementation with B. subtilis has profound effects on the levels of a wide variety of chemical metabolites in the chicken gut.
• These altered metabolite levels provide a biochemical signature unique to each B. subtilis supplementation group.
• Future studies are warranted to assess the growth promoting properties, if any, of the identified chemical compounds in lieu of antibiotics.
Further study: extra data
Chicken macrophage cells (HD11)
40 metabolites of Up-regulated common metabolites between B. subtilis 1781 and 747
Proinflammatory responses
Chicken epithelial cells (8E11)
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0.05
0.10
0.15
0.20
0.25
CON LPS 0.1 ug/mL 1.0 ug/mL 10.0 ug/mL
Mea
n n
orm
aliz
ed e
xpre
ssio
n
IL6
0
0.1
0.2
0.3
0.4
0.5
CON LPS 0.1 ug/mL 1.0 ug/mL 10.0 ug/mL
IL1β
0.E+00
5.E-05
1.E-04
2.E-04
2.E-04
3.E-04
3.E-04
4.E-04
4.E-04
CON LPS 0.1 ug/mL 1.0 ug/mL 10.0 ug/mL
IL6
-20
0
20
40
60
80
100
120
CON LPS 0.1 ug/mL 1.0 ug/mL 10.0 ug/mL
Mea
n n
orm
aliz
ed e
xpre
ssio
n IL8
N-acetylglucosamine Syringic acid
2'-deoxyadenosine β-alanine
Acknowledgment
USDA-ARS • Dr. Hyun S. Lillehoj
Church & Dwight • Dr. N. P. Zimmerman • Dr. A. H. Smith • Dr. T. Rehberger
Thank you !
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