Effects of probiotics in the treatment of alcoholic

hepatitis: randomized controlled multicenter study

Lactobacillus subtilis/Streptococcus faecium

Lactobacillus rhamnosus R0011/acidophilus R0052

Ki Tae Suk,1 Dong Joon Kim,1 Moon young Kim,2 Soon Koo Baik,2

Young Don Kim,3 Gab Jin Cheon,3 Dae Hee Choi,4 Dong Hoon Shin,1

Eun Ji Kim,1 Hak Cheol Kwon,5 Sung Hun Kim5

Human gut microbiota: complex ecosystem (>>1,000 species, >1kg)

Functions: barrier, digestion, and immune system…..

Important role in many diseases (IBD, IBS, DM, obesity…)

‘‘One of the hottest areas in medicine”

Nat Rev Gastroenterol Hepatol. 2011 Dec 20;9(2):72-4

J Hepatol. 2013 May;58(5):1020-7

Floch et al. Gastroenterology 1970:59;76

Microbiota & Liver Disease

Microbial factor Mediator Clinical implications

Small intestinal bacterial overgrowth (SIBO) Multiple NAFLD, ALD

Alteration in the composition of microbiota Multiple Obesity, ALD

Bacterial translocation LPS, endotoxin SBP, portal hypertension

Direct effects of bacterial metabolites Acetaldehyde NAFLD, ALD

Exacerbate activation of

hepatic innate immune

system

Fibrosis

Hepatitis

Alcohic liver disease

NAFLD

Cancer

Activation of pro-inflammatory pathway

anti-apoptotic pathway

anti-viral pathway Hepatology. 2014 Jan;59(1):328-39

Healthy condition Pathologic condition

Hepatology 2011;54:562-572

Fecal Microbial Communities

Healthy group Liver cirrhosis group

In patients with liver cirrhosis, the increase of pathogenic bacteria such as

Streptococcaceae and the reduction of beneficial bacteria may affect prognosis.

Alcoholic Liver Disease & Microbiota

Alcoholic hepatitis

Enterococcus ↓

E. coli ↑

Lactobacillus

reduce endotoxin

improve steatohepatitis

restore gut flora

However, most relevant evidences are from animal studies.

Few human studies are available.

Hepatology, 2004:39;1441–1449

J Clin Gatroenterol 2005;39:540-543

Alcohol Dis, 2000;8:1–4

Hepatology. 2014 Jan;59(1):328-39

Hypothesis & Aim

Pathophysiological importance of this association is not yet fully understood.

Few human data are available in regard to alcoholic liver disease.

We evaluated the therapeutic effects of probiotics in patients with alcoholic hepatitis

Design -Multicenter, randomized, clinical trial at 4 university hospitals-

Alcoholic

hepatitis

Admission

Randomization

Day 7 Day 0

Placebo + Legalon®

Probiotics + Legalon®

Day 1

• Liver enzymes

• TNF-a / IL-1b / IL-10

• LPS

• Stool culture (study 1)

Stool PCR-DGGE (study 2)

• Liver enzymes

• TNF-a / IL-1b / IL-10

• Lipopolysaccharide (LPS)

• Stool culture (study 1)

Stool PCR-DGGE (study 2)

Stool culture: E. coli & Enterococcus species

Colony forming unit (CFU) per gram stool were counted

and bacteria were identified by using Vitek II system

Stool PCR DGGE:16S rRNA V3

Alcohol. 2008 Dec;42(8):675-82

Hepatology, 2004;34:1441–1449

PCR-Denaturing Gradient Gel Electrophoresis

(DGGE)

Primer sets for bacterial identification/DGGE

Bacterial variability map. E. coil 16S rRNA gene sequence annotated with bacteria and “universal” priming sites

and variable regions V1-V9.

KEY

totally conserved conserved variable highly variable

>75% variable regions priming sites

• Alcoholic hepatitis

AST/ALT >2 & elevated AST (ALT) level

Alcohol >60 g/day (M), >40 g/day (F) during 7 days before screening

Last drinks: within 48 hours prior to admission

• All patients: hospitalization & no alcohol drinking during 7 days.

• Exclusion: viral hepatitis, autoimmune hepatitis, pancreatitis, delirium

tremens, hemochromatosis, Wilson’s disease, DILI, cancer, infection

need for antibiotics, severe AH, or obesity (BMI >30 kg/m2)

Patients & Methods

September 2010 – September 2012

130 patients were recruited

Randomization

Probiotics group (n = 65) Placebo group (n = 65)

Completed full therapy (n = 60) Completed full therapy (n = 57)

Excluded because of

patients’ refusal (n = 3),

early discharge (n = 1),

other (n = 1)

Excluded because of

patients’ refusal (n = 3),

early discharge (n = 5)

Lactobacillus subtilis/Streptococcus faecium

Study 1

Lactowel® , 1,500 mg/day

Stool culture: CFU per gram stool

Lactobacillus rhamnosus R0011/acidophilus R0052

December 2012 – September 2014 (Ongoing)

100/150 (66%) patients were recruited

Randomization

Probiotics group (n = 55) Placebo group (n = 45)

Completed full therapy (n = 50) Completed full therapy (n = 40)

Excluded because of

patients’ refusal (n = 3),

early discharge (n = 2),

Excluded because of

patients’ refusal (n = 3),

early discharge (n = 2)

(Lacidophil® , 120 mg/day)

PCR DGGE, 16S rRNA V3

Study 2, Interim data

Characteristics of Patients Study 1

Variables All patients (n = 117)

Male (n [%]) 75 (64)

Age (years) 52.7 ± 11.3

Presence of L/C (n [%]) 62 (53)

Day 1 Day 7 p value

Total protein (g/dL) 6.5 ± 1.0 6.6 ± 0.8 0.221

Albumin (g/dL) 3.7 ± 0.8 3.8 ± 0.6 0.032

AST (IU/L) 159 ± 182 66 ± 51 < 0.001

ALT (IU/L) 87 ± 136 55 ± 95 < 0.001

ALP (IU/L) 129 ± 48 110 ± 38 < 0.001

rGT (IU/L) 526 ± 768 331 ± 422 < 0.001

TB (mg/dL) 2.6 ± 4.3 1.6 ± 2.7 < 0.001

Cholesterol (mg/dL) 147 ± 63 151 ± 52 0.447

PT (seconds) 12.3 ± 2.0 11.8 ± 1.7 0.243

CP score in patients with L/C 7.7 ± 2.0 7.2 ± 2.0 < 0.001

Variable

(mean ± SD)

Probiotics group (n = 60) Placebo group (n = 57)

Day 1 Day 7 p value Day 1 Day 7 p value

Total protein (g/dL) 6.5 ± 1.1 6.6 ± 0.8 0.505 6.6 ± 1.5 6.8 ± 0.7 0.344

Albumin (g/dL) 3.5 ± 0.7 3.7 ± 0.6 0.038 3.8 ± 0.8 3.9 ± 0.7 0.346

AST (IU/L) 166 ± 213 64 ± 42 0.001 148 ± 130 69 ± 62 <0.001

ALT (IU/L) 83 ± 126 48 ± 49 0.010 93 ± 152 66 ± 136 0.001

ALP (IU/L) 132 ± 54 115 ± 44 0.001 124 ± 39 103 ± 27 <0.001

rGT (IU/L) 510 ± 629 334 ± 430 <0.001 553 ± 953 328 ± 417 0.030

TB (mg/dL) 3.2 ± 5.5 2.1 ± 3.4 0.005 1.8 ± 1.4 1.0 ± 0.7 <0.001

PT (seconds) 12.6 ± 2.1 12.1 ± 1.9 0.039 12.0 ± 1.7 11.4 ± 1.4 0.019

Cholesterol (mg/dL) 151 ± 67 149 ± 51 0.822 142 ± 57 155 ± 56 0.114

Study 1

Characteristics of Patients

TNF-α

IL1-b

Study 1

Day 1

Day 7

LPS Study 1

Changes of microbiota after probiotics & placebo

Variable

(mean ± SD)

Number of CFU per gram stool

Probiotics group (n = 49) Placebo group (n = 35)

Day 1 Day 7 p-value Day 1 Day 7 p-value

Escherichia coli 404 ± 289 224 ± 236 0.001 363 ± 291 298 ± 253 0.138

Enterococci 341 ± 286 302 ± 289 0.130 284 ± 325 434 ± 293 0.454

Variable

(mean ± SD)

Probiotics group (n = 50) Placebo group (n = 40)

Day 1 Day 7 p value Day 1 Day 7 p value

Total protein (g/dL) 6.8 ± 0.8 6.9 ± 0.7 0.243 6.5 ± 1.0 6.7 ± 0.7 0.325

Albumin (g/dL) 3.8 ± 0.7 3.8 ± 0.7 0.285 3.7 ± 0.6 3.8 ± 0.6 0.412

PT (seconds) 12.5 ± 3.6 12.1 ± 3.2 0.482 12.6 ± 3.9 12.4 ± 3.2 0.524

AST (IU/L) 188 ± 320 68 ± 52 0.012 116 ± 133 68 ± 72 0.029

ALT (IU/L) 113 ± 192 42 ± 37 0.004 90 ± 167 39 ± 44 0.023

rGT (IU/L) 432 ± 501 335 ± 403 0.018 442 ± 405 345 ± 353 0.009

TB (mg/dL) 1.9 ± 2.3 1.3 ± 1.9 <0.001 2.8 ± 3.6 2.0 ± 3.4 0.001

TNF-α 79 ± 67 84 ± 222 0.243 163 ± 513 131 ± 32 0.084

IL 10 41 ± 144 48 ± 144 0.345 49 ± 147 28 ± 90 0.117

LPS 1.13±1.04 0.47± 0.62 <0.001 0.92 ± 1.23 0.48 ± 0.50 0.056

Characteristics of Patients

Study 2, Interim data

Study 2, Interim data

Stool DGGE

Line : type of microbiota

Density: amount of microbiota No cirrhosis

Probiotics group

Bacteroides

coprocola

unidentified

Enterobacter sp.

Enterobacter sp.

Day1 Day 7 Day1 Day 7

Case 1 Case 2

Cirrhosis

Escherichia coli

Enterobacter sp.

Day1 Day 7 Day1 Day 7

Case 5 Case 6

• At day 1, dominant type of bacteria was evident.

• The most dominant type of bacteria was G (-) bacteria.

• The density of G (-) bacteria (E. coli , Enterobacter) was decreased at day 7.

• Cirrhosis: dominant type of bacteria → various different types of bacteria (diversity)

Study 2, Interim data Placebo group

Escherichia coli

Enterobacter sp.

Enterobacter sp.

No cirrhosis Cirrhosis Day1 Day 7 Day1 Day 7 Day1 Day 7 Day1 Day 7

Case 9 Case 10 Case 13 Case 14

• At day 1, dominant type of bacteria was evident.

• The most dominant type of bacteria was G (-) bacteria.

• The density of G (-) bacteria (Enterobacter, Bacteroides) was increased at day 7.

• Microbial distribution: dominant bacteria → other dominant bacteria (simplification)

Study 2, Interim data

Stool DGGE

Study 2, Interim data

One patient was treated by placebo and probiotics

Probiotics treatment is associated with change in gut microbiota.

Case 16

Study 2, Interim data

Summary of stool DGGE analysis (n=16) Dominant type of bacteria

Change in distribution after Tx Day 1 Day 7

Probiotics No LC

(n=4)

Unidentified No

• Disappeared

• Different types of bacteria

LC

(n=4)

E. coli

Enterobacter

No

• Disappeared

• Various different types of bacteria

Placebo No LC

(n=4)

E. coli Enterobacter

• Dominant type of bacteria

→ other dominant type of bacteria

LC

(n=4)

E. coli

Bacteroides

Enterobacter

• Dominant type of bacteria

→ other dominant type of bacteria

B

B

Summary

• Immediate abstinence is a most important treatment for

patients with AH.

• Serum LPS was decreased by probiotics, especially, in patients

with LC.

• In the stool culture, the number of CFU in E. coli was

significantly decreased by probiotics.

• In the stool analysis, most dominant type of bacteria was G (-)

bacteria in AH.

• In the stool analysis, probiotics treatment reduced the density of

G (-) bacteria and placebo treatment increased the density of G

(-) bacteria.

• Probiotics treatment changed microbial distribution from

dominant type of G (-) bacteria to different types of bacteria.

• Placebo treatment simplified microbial distribution from

dominant type of G (-) bacteria to other dominant type of G (-)

bacteria.

Summary

Conclusion

7 days of oral supplementation with probiotics is

associated with improvement of LPS and

restoration of the bowel microbiota in AH.

Thank You For Attention!