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

n2n3

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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

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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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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

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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

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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

50.00E+001.00E+016.60E+010.00E+000.00E+00NT0.00E+000.00E+000.00E+000.00E+000.00E+000.00E+00NT0.00E+000.00E+001.50E+048.00E+044.00E+020.00E+000.00E+000.00E+00

60.00E+001.00E+012.00E+010.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+002.00E+012.00E+016.00E+012.00E+010.00E+00

70.00E+000.00E+000.00E+000.00E+000.00E+000.00E+000.00E+002.00E+045.60E+054.60E+047.00E+034.80E+031.00E+010.00E+000.00E+004.00E+010.00E+003.00E+010.00E+000.00E+000.00E+00

80.00E+003.20E+014.00E+011.00E+010.00E+000.00E+000.00E+000.00E+000.00E+00NT0.00E+000.00E+000.00E+000.00E+00NT0.00E+000.00E+000.00E+000.00E+000.00E+00NT

9NT0.00E+000.00E+000.00E+000.00E+00NT0.00E+000.00E+004.40E+041.80E+042.00E+032.00E+020.00E+000.00E+000.00E+00NT1.00E+010.00E+000.00E+000.00E+000.00E+00

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