Microbiota and Hepatic Encephalopathy: microbial dynamics and metabolism upon prebiotic, antibiotic and probiotic treatment

CENTRO RICERCA e INNOVAZIONE

Microbiota and Hepa/c Encephalopathy:

microbial dynamics and metabolism upon

prebio/c, an/bio/c and probio/c treatment

Andrea Mancini

_{, Francesca Campagna}

_{, Piero Amodio}

_{, Cecilia Pravadelli}

_{, Kieran Tuohy}

1 _{Nutri(on and Nutrigenomic Group, Department of Food Quality and Nutri(on, Research and Innova(on Centre, Fondazione Edmund Mach}

(FEM), 38010 San Michele all’Adige (TN), Italy; 2_{Department of Food Science, University of Parma, 43100 Parma, Italy;} 3_{Department of}

Medicine-DIMED, University of Padova, 35128 Padova, Italy; 4_{Gastroenterology Unit, Santa Chiara Hospital, 38123 Trento, Italy.}

We inves(gated how gut microbiota modula(on by prebio(cs, an(bio(cs and probio(c treatments affected microbial ammonia produc(on and the rela(ve abundance of important members of the gut bacteria, within the cirrho(c environment.

[1] Rose, C.F. Clin Pharmacol Ther. 2012 Sep;92(3):321-31. [2] Dhiman, R.K. Metab Brain Dis. 2013 Jun;28(2):321-6. [3] Bajaj, J.S., et al. J Hepatol. 2014 May;60(5):940-7.

[4] Sharma, P., Sharma, B.C. Metab Brain Dis. 2013 Jun;28(2):313-20. [5] Kavish, R., et al. Metab Brain Dis. 2013 Jun; 28(2): 307–312. [6] Bajaj, J.S. Aliment Pharmacol Ther. 2016

Jan;43 Suppl 1:11-26 [7] Sharma, B.C. and Singh, J. Metab Brain Dis. 2016 Apr 28.

Introduc/on

Objec/ves

1.  Gut microbiota modula(on is a poten(al target for relieving the symptoms of HE by regula(ng colonic ammonia produc(on;

2.  In general lack of a direct correla(on between microbial modifica(on and NH₃ reduc(on for the tested treatments, suggest a modula(on in ammonia produc(on rather than increased size of the “colonic ammonia sink” via microbial biomass alone, as a possible mode of ac(on.

The data pave the way to further inves(ga(ons on gut metabolic ac(vity and microbial cross-talk in the presence of Lactulose, Rifaximin and VSL#3.

Conclusions

1.  Stool samples collec(on from ten cirrho(c pa(ents (age 66±3.3; Child-Pugh A (n=9) and B (n=1); average MELD score 9±2.8) and three healthy subjects (age 63±2.5)

2.  Sample inoculum in a 24-hour batch culture fermenta(ons model at controlled pH (6.8) Condi(ons tested: no treatment (ctrl), Lactulose (1%), Rifaximin (616μg/ml), Lactulose/Rifaximin, VSL#3 (ini(al [ ] of 108_{cell/ml), Lactulose/VSL#3,} Rifaximin/VSL#3, Lactulose/Rifaximin/VSL#3 3.  Microbial enumera(on by Cytofluorimetric FISH (FCM-FISH) at 0, 4, 10 and 24 hours, using specific probes 4.  NH₃ and SCFA were quan(fied at 0, 4, 10 and 24 hours

Study design

andrea.mancini@fmach.it

NH₃ concentra,on assessed by colorimetric method on the batch culture fermenta,on supernatant (mean ± sd, N = 10, in triplicates). *p-value≤0.05, **p-value ≤0.01, paired t-test, treatment vs ctrl.

Results

Take home messages

1) Ammonia level assay

2) ∆T bacterial enumera/on

Average bacteria count differences, between ,mes 5, 10, 24 and 0 for the considered FISH probes (mean ± sd, N = 10). *p-value≤0.05, **p-value ≤0.01, ***p-value ≤0.001, paired t-test, treatment vs ctrl. ∆T5; ∆T10; ∆T24 Ctrl, Control; Lact, Lactulose; Rifax, Rifaximin.

Contact informa(on:

Gut ammonia produc(on by microbial ac(vity is one of the main factors implicated in Hepa(c Encephalopathy (HE) [1, 2, 3]. Ammonia reducing strategies target mainly gut microbiota, by administra(on of prebio(cs (Lactulose) [4], an(bio(cs (Rifaximin) [5, 6] and probio(cs (VSL#3) [7]. Although the role of ammonia in the pathophysiology of HE is well established, the factors governing its produc(ons by the gut microbiota are poorly understood.

Bifidobacterium sp. Bacteroides/Prevotella Clostriudium histolyticum/perfringes

Desulfovibrionales/Desulfomonales Enterobacteriaceae sp. Cell density

Average SFCA produc,on differences, between ,mes 5, 10, 24 and 0 for the considered FISH probes (mean ± sd, N = 10). *p-value≤0.05, **p-value ≤0.01, ***p-value ≤0.001, paired t-test, treatment vs ctrl. ∆T5; ∆T10; ∆T24 Ctrl, Control; Lact, Lactulose; Rifax, Rifaximin.

3) ∆T SFCA produc/on

Acetate Propionate Butyrate

•  NH₃ is reduced by all the treatments in a (me dependent and combina(on manner, except for VSL#3 alone

•  Over 24 hours NH₃ removal was retained although the efficacy of Lactulose was greater at T10

•  VSL#3 NH_{3 removal was augmented by the}

presence of prebio(c and an(bio(c

•  NH₃ reduc(on appears to be directly linked to increase in rela(ve abundance in Bifidobacteria, bever induced by prebio(c and an(bio(c

•  The Bifidogenic ac(vity of Lactulose impact posi(vely on SCFA produc(on, with poten(al cross-feeding process for Priopionate and Butyrate ** _** * * ** ** ** _** * * NH 3 μg /ml