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Harald BrüssowNutrition and Health Department
Nestlé Research Center LausanneSwitzerland
Gut Microbiome Modulation:Alternative to Antibiotics?
OIE Headquarters, 2nd International Symposium ATAParis, December 12-15, 2016
Our background: Diarrhea in pediatrics
20 years of scientific collaboration with NRC
E. coli diarrhea is largely antibiotic-resistant:
we studied alternative treatment options
n1
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n4 n
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….nm
Diet, fibres, prebiotics, milkoligosaccharides
n1, n2, N3, n4, n5, N6, n7, n8, ….nmFecaltransplantation, probiotics
n1, n2, n3, Px, n4, n5, Py, n6, ….nm
Introducingbeneficialbacteria by:
Modifying nutrientinput:
Phages, bacteriocins, antibodies
n1, n2, n3, n4, n5, n6, n7, n8, ….nmX
Eliminatingundesiredbacteria:
Targeting →pathogen or →microbiota or →host with nutritional interventions
Pathogen-Microbiota-Host Interaction determines Infection Outcome
Microb Biotech 9:553
Passive immunization with bovine milk antibodies
Dairy cows as bioreactors: modified concept of P. Ehrlich & E.v. Bering (1890)
°We vaccinated cows with rotaviruses and E. coli
°Dam immunization stimulates broadly cross-reacting antibodies
°Cows transfer antibodies to the calf via colostrum
°We collected tons of colostral milk
°We isolated 10 kg immunoglobulins per 1 t of milk
°Clinical trials showed treatment effects against RV diarrhea
J.Clin. Micro. 25, 982;J. Gen. Virol. 69, 1647; J. Infect. Dis. 156, 158;
Pediatr. Infect. Dis. J. 17,1149;Scand. J. Gastro. 35, 711;
BMC Microbiol 7, 86
-1 0 1 2 3 4
25
50
75
100 HBC 100 µg/dose (32)HBC 10 µg/dose (24)untreated (36)imulin 100 µg/dose (7)
*** ***
days post infection
% d
iarr
hea
Preclinical work: dose effect in mice
BMC Microb 7: 86
Preclinical work: intestinal histology (mouse)
RRV only 100 µg HBC+ RRV Non infectedRV control RV + bov. Ig Control
BMC Microb 7: 86
Clinical trials in Germany, USA and Bangladesh showed treatment effects against RV diarrhea…
Attenuated fluid loss in g/kg/day:
RV neutralization titer in stool
day 1
day4
Accelerated clinical recovery
…but it did not work against E. coli diarrhea
JID 156:158
PIDJ 17: 1149
SJG 35: 711
Probiotic bacteria: another concept of a grand old man …
Yogurt: lactic streptococci and lactobacilli
Health-promoting lactobacilli: Modified concept of E. Metchnikoff (1905)
J. Bacteriol. 189, 1311; 189, 8109; 190, 3161Pediatrics 116, e221
Isolation of Lactobacillus paracasei from infant feces→safety studies→RCT
Patient enrolment scheme of probiotic trial at icddr,b
no clinical effect
treatment effect
NIH selected dietary trial: RCT, WHO diarrhea criteria, pathogen-specific
Ped 116: e221
Clinical outcome in non-rotavirus patients
Low amounts of probiotic were detected in stool, but fecal microbiota was not investigated
Ped 116: e221
p=0.04p=0.029
p=0.001
VDBreastfed
VDMixfed
CSMixfed
0%
20%
40%
60%
80%
100% Bifidobacterium longumgroupBifidobacteriumpseudocatenulatum groupBacteroidesStreptococcus thermophilusgroup
Klebsiella
Escherichia
Granulicatella
Streptococcus agalactiaegroup
Lachnospiraceae
Clostridium butyricum
Remaining
VD: vaginal deliveryCS: C-section
Feeding and delivery mode influence gut microbiota composition
Is it possible to modify the gut microbiota composition in C-section delivered infants with a probiotic?
Figure credit: O. Sakwinska, NRC
VG: Vaginal delivery; CtCS: control C-section; LrCS: L. reuteri supplemented C-section
The probiotic does not dominate the gut microbiota, but shifts the microbiota in supplemented C-section infants to that of vaginal-delivered infants
Clinical trial in Greek infants with Lactobacillus reuteri probiotic
Keystone species concept JPGN 63: 681
Figure credit: B. Berger, NRC
Phage therapy: Renaissance of another old concept
Collection of phagesEcological survey in Bangladesh
In vivo tests in miceLytic activity of oral phage in the gut
In silico safety evaluationGenome sequencing and risk assessment
Phase I trials in humansSafety and pharmacokinetics in volunteers
Phase II efficacy trial in humansAcute E. coli diarrhea in children at icddr,b
Antimicrobial Agents Chemother. 48, 2558; 49, 2874 J. Bacteriol. 186, 8287; 186, 8276; 189, 8206
Virology 388, 21; 393, 16; EBioMedicine 4,124Ann Rev Virol 2, 599
Bacteriophages as anti-microbial agents: concept of F. d’Herelle (1920)
Phage therapy: An appealing concept that workswell in the laboratory
T4 phage on E. coli
Time scale: all is done in 20 min
CurOpMicrob 16:500
T4 Phage goes into the fermentation unit
MALDI-TOF
Metagenomics:
AEM 80: 1469Virol 434:222
http://www.apsnet.org/publications/apsnetfeatures/Article%20Images/Phages_Fig03.jpghttp://www.apsnet.org/publications/apsnetfeatures/Article%20Images/Phages_Fig03.jpg
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0 1 2 3 4 5 6 7Days
%
HighLowPlacebo
oral phage
Healthy Humans: Safety test of T4 phage in Switzerland and Bangladesh
Followed by three safety trialsat icddr,b (healthy adults, older, then younger children):no adverse events
AAC 49,2874Kinetics of oral phage appearance in stool
Stool microbiota composition is not affected by oral phageEMI, in press
Chart1
-2-2-2
-1-1-1
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52.6
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89.4
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53.3
0
0
37.5
0
0
10.5
0
0
23sept
-1-1-1
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222
333
444
555
666
Days
% positive
0
0
0
17.6
5.2
0
80
50
0
100
52.6
0
89.4
10
0
53.3
0
0
37.5
0
0
10.5
0
0
Chart3
-1-1-1
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111
222
333
444
555
666
days
% positive
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0
0
17.6
5.2
0
80
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0
100
52.6
0
89.4
10
0
53.3
0
0
37.5
0
0
10.5
0
0
Chart4
-1-1-1
000
111
222
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444
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666
days
% positive
0
0
0
17.6
5.2
0
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50
0
100
52.6
0
89.4
10
0
53.3
0
0
37.5
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ChartAAC
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111
222
333
444
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777
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100
52.6
1
89.4
10
1
53.3
1
1
37.5
1
1
10.5
1
1
Chart2
000
111
222
333
444
555
666
777
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Days
%
0
0
0
17.6
5.2
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52.6
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1
Sheet1
Days1235678910111213141516TotalTotal +
High titerdaysHighLowPlacebodaysHighLowPlacebo
-2+++++++++-+-++---+1300-20200-1000
-1+++++++++++---++1300-1000017.65.20
0+++++++++++++++++17317,6017.65.20180500
1++++++++++++++++++++201680180500210052.60
2+++++++++++++++++1717100210052.60389.4100
3+++++++++++++++++++191789,4389.4100453.300
4++++++++++-+++++15853,3453.300537.500
5-+-+++++++++++++++16637,5537.500610.500
6+++++++++++++++++++19210.5610.500
Low titer1235678910111213141516TotalTotal +%0000
-2--++++++++++10220-220117.65.21
-1+++++++++++++1300-10280501
0+++++++++++++++++++1915.205.2310052.61
1+++++++++++-+++++++18950150489.4101
2++++++++-+++++++++++191052.6252.6553.311
3++++++++++++++++++++20210310637.511
4++++++++++++++++++180040710.511
5++++-+++++++-+++140050
6++++++++++++++++++++-200060
-20
-10
00
10
20
30
40
50
60
Sheet2
volunteerday1day2day3day4day5day6day7day8day9day10day11day12day13day14day15day16day17day18day19day20day21
10.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+001.00E+048.80E+036.80E+023.00E+01NT0.00E+000.00E+003.00E+012.00E+010.00E+000.00E+000.00E+000.00E+00
20.00E+000.00E+001.00E+034.00E+014.00E+010.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+008.00E+012.00E+012.20E+029.00E+020.00E+00
30.00E+004.00E+028.00E+034.40E+022.00E+01NT0.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+00
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100.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+008.00E+010.00E+000.00E+000.00E+000.00E+00
110.00E+000.00E+001.00E+010.00E+002.00E+010.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+00NT0.00E+000.00E+005.00E+017.10E+021.60E+03NT4.70E+026.00E+01
12NT0.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+009.20E+021.00E+043.50E+010.00E+000.00E+000.00E+000.00E+002.00E+011.00E+010.00E+000.00E+000.00E+000.00E+00
130.00E+006.00E+022.00E+024.80E+034.20E+020.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+00NT0.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+00
146.00E+012.36E+022.60E+031.00E+026.00E+012.00E+010.00E+000.00E+000.00E+002.00E+010.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+00
150.00E+003.60E+022.00E+044.00E+020.00E+000.00E+000.00E+000.00E+000.00E+004.00E+010.00E+00NT0.00E+000.00E+000.00E+00NT0.00E+000.00E+000.00E+000.00E+000.00E+00
160.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+001.20E+021.10E+034.00E+010.00E+000.00E+000.00E+001.60E+020.00E+000.00E+000.00E+000.00E+00NT
T4 phage 3.6 x 108/ml
Placebo
A double blinded placebo controlled randomized phage therapy trial at icddr,b
Planned: Efficacy evaluation after 375 patients
Microgenphage1.4 x 109/ml
Safety evaluation after 75 patients
Outcome: Oral phage is safe in diarrhea patients
The trial was sponsored by:
Interim analysis after 120 patients
Lack of phage amplification and clinical efficacy
EBioMed 4: 124
Stool microbiota: more streptococci than E. coli in ETEC
EBioMed 4: 124
The failure of the phage therapy trial:*Is not an argument against phage therapy, *but against using ETEC diarrhea as target
Indeed, when investigating other forms of childhood diarrhea, we identified patients with endogenous T4 phages replicating to high titers on outgrowing intestinal E. coli *killing the winning hypothesis*importance of target bacteria above phage replication threshold
Interpretation
Figure credit: N. Sprenger, NRC
Prebiotics: milk oligosaccharides
Conceptual framework The oligosaccharides:
Proteo
Firmicutes
Bacteroides
Bifido
BF FF BMOEnv. Microbiol. 18: 2185
BF FF BMO
Bifidobacterium E. coli
PCA of stool microbiota compositional shifts
Streptococcus
*There is hope for intervention into the pathogen-microbiota-host interaction network,*These treatment options need still to be developed, * …and adapted to veterinary use,*There are currently more descriptive than interventional microbiota studies*These interventions need a case by case development, don’t expect universal solutions
Conclusions
…issue was also intensively discussed in childhood malnutrition & obesity→ interdisciplinary cross-talk
Growth promotion by antibiotics: many unanswered questions
EMI 17: 2216
Acknowledgements
…and the children of Bangladesh participating in the clinical trials
At icddr,b:S. A. Sarker (PI), S. Sultana, F. Qadri, A. Cravioto
At NRC:B. Berger, N. Sprenger, J. Sidoti, A. Bruttin, S. Chibani-Chennoufi, M. Weiss, G. Bourdin, G. Reuteler, S. McCallin, S. Kieser, C. Barretto, C. Ngom-Bru, M. Delley, A.-C. Pittet
Other Nestlé units: D. Moine, P. Decombes (NIHS); L. Philippe (CDU); N. Conus, T. v.d. Weid (NN); M. Kuslys (NHS)
Gut Microbiome Modulation:�Alternative to Antibiotics? Slide Number 2Slide Number 3Passive immunization with bovine milk antibodiesSlide Number 5Preclinical work: intestinal histology (mouse)Slide Number 7Probiotic bacteria: another concept of a grand old man …Slide Number 9Slide Number 10Slide Number 11Slide Number 12Phage therapy: Renaissance of another old conceptPhage therapy: An appealing concept that works well in the laboratoryT4 Phage goes into the fermentation unitHealthy Humans: Safety test of T4 phage in Switzerland and BangladeshSlide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26