Liver cirrhosis and antibiotic therapy but not TIPS application leads to a shift of the intestinal bacterial communities: A controlled, prospective study

The gut–liver axis is discussed to play an important role in hepatic cirrhosis. Decompensated liver cirrhosis is associated with portal hypertension, which can lead to a variety of complications. Transjugular intrahepatic portosystemic shunt (TIPS) is an established treatment option for the complications of portal hypertension. In this study we focused on the effect of TIPS on intestinal microbial composition in cirrhotic patients.

7][18] In addition, changes in the intestinal bacterial composition are reported to play a specific role in the development and the clinical course of liver disease.[21][22] Bacterial infections are now regarded a major complication in advanced liver cirrhosis, leading to high morbidity and mortality. 23ontaneous bacterial peritonitis (SBP), one of the most common bacterial infections in patients with liver cirrhosis, is an example of how bacterial translocation into the portal system leads to hepatic inflammation and activation of the host immune system. 24One reason why the bacteria in the intestine seem to be a major factor in these disease entities is that a disruption in the intestinal barrier can lead to bacterial translocation, which is another hit for the already injured liver.
Another important aspect in liver cirrhosis is small intestinal bacterial overgrowth, implicating changes in the bacterial growth in the small intestine, caused by changes in the intestinal motility and a delayed transit time, also with a consequence of bacterial translocation. 25However, whether an altered intestinal microbial composition in patients with liver cirrhosis is the reason or a consequence of the hepatopathy resulting in portal hypertension is still a matter of debate.
The effects of TIPS on the intestinal microbial composition in a prospective, controlled setting has not been examined yet, to the best of our knowledge.We hypothesize that TIPS application with a consecutive reduction of portal hypertension has an effect on the intestinal bacterial communities, which-in consequence-can lead to certain clinical consequences such as hepatic encephalopathy (HE).
In this study we evaluated 17 well-characterized patients with liver cirrhosis, all of whom received TIPS for clinical indications and examined their bacterial compositions before and after TIPS application.
The patients who underwent TIPS were compared to 13 cirrhotic patients without TIPS application and 18 healthy volunteers, who served as control groups.Clinical characteristics of the participants were obtained and 16S rRNA gene amplicon sequencing was performed to specify the bacterial composition in the different groups.Since antibiotic therapy is also discussed to have an impact on gut microbiota, we also stratified the patients based on whether antibiotic therapy was administered or not.
We aimed to identify whether the intestinal bacterial communities differed between cirrhotic patients and healthy controls or between patients who received TIPS and the control group.Child-Pugh score (CPS), 26 the model for end-stage liver disease (MELD) score, 27 and the presence of ascites and comorbidities were recorded.Regarding BMI, the study participants were stratified into underweight (BMI <18.49kg/m 2 ), normal (BMI 18.5-24.99kg/m 2 ), overweight (BMI 25-29.99kg/m 2 ), obesity stage I (BMI 30-34.99kg/m 2 ), obesity stage II (BMI 35-39.99kg/m 2 ), and obesity stage III (BMI ≥40 kg/m 2 ), respectively. 28Use of antibiotic therapy during the sample collection was also documented.

| Study design
Stool samples were collected to assess the luminal intestinal bacterial composition at different time points (d-1 before TIPS installation, d5 after TIPS, and d30 after TIPS).The patients were on the ward for samples d-1 and d5 collection; while sample d30 was obtained in the outpatient clinic.In patients with liver cirrhosis and without TIPS placement, we also collected three stool samples at the same time intervals for comparison with those of the TIPS group.
While in the healthy control group, only one stool sample was collected.
To compare the effect of TIPS on intestinal bacterial communities, we compared the samples collected at different time points with each other (d-1 with d-1, d5 with d5, and d30 with d30).And to compare the differences in the intestinal microbial communities between patients with liver cirrhosis and the healthy controls, only one sample (d5) was used for calculation to avoid systematic error when comparing different samples.

| TIPS
TIPS was performed by experienced senior radiologists (T.H., J.C.K., and M.A.W.) of the Division of Radiology at the University Medical Center Rostock.The procedure is briefly described as follows.After local anesthesia using 1% lidocaine, ultrasound-guided puncture of the right internal jugular vein was performed and an 8-Fr introducer system (Radifocus Introducer II; Terumo, Tokyo, Japan) was inserted.
The central venous pressure was monitored throughout the procedure.After gaining venous access, piritramide (Piritramide, Hameln, Germany) and midazolam (Midazolam, Hameln, Germany) were intravenously administered in individual doses for analgesia and sedation, while vital parameters including respiration, pulse, and oxygen saturation were monitored.The inferior vena cava was probed using an atraumatic guidewire with a flexible core (polytetrafluoroethylene [PTFE] coated guidewire; Angiomed, Karlsruhe, Germany), through which the 8-Fr TIPS Guiding Catheter (OptiMed, Karlsruhe, Germany) and the modified Ross needle (TIPS puncture needle with Obturator; OptiMed) were advanced.This system was used to selectively probe the right hepatic vein.The transparenchymal puncture of the right branch of the portal vein was performed under ultrasound guidance, adjusting the angle of the needle tip manually beforehand.The guidewire and catheter were advanced into the main portal vein or into the portal venous system with this system.After removing the needle, a 5-Fr multi-hole ring catheter (PIG Tempo 5; Cordis, Miami Lakes, FL, USA) was inserted.
The portal venous outlet pressure was measured in the region of the confluence.Subsequently, contrast medium (Imeron 300; Bracco, Geneva, Switzerland) was injected into the portal venous system, depending on the anatomical conditions, indications, and collateral circulations, originating from the distal splenic vein in all cases.Existing varices toward the esophagus were probed and embolized with a mixture of Histoacryl and Lipiodol until flow cessation using a 4-Fr Cobra2 catheter without side holes (C2 super torque; Cordis).After inserting an Amplatz wire (InQwire; Merit Medical, South Jordan, UT, USA) and preparing the parenchymal tract, a percutaneous transluminal angioplasty (PTA) balloon (8 mm in diameter, 6 cm in length; Mustang; Boston Scientific, Marlborough, MA, USA) was used.
Implantation was either performed using a bare-metal stent (10 mm in diameter, 6 cm in length; E-Luminexx, BARD, Franklin Lakes, NJ, USA), or through a 10-Fr sheath (Flexor Check-Flow, Cook Medical, Bloomington, IN, USA) with an ePTFE-covered stent graft (VIATORR; GORE, Flagstaff, AZ, USA).The stent was dilated and molded to 8 mm using the PTA balloon until the portosystemic pressure gradient was reduced to less than 12 mmHg.During the procedure, heparin (Ratiopharm, Ulm, Germany) was administered based on the coagulation parameters.
Finally, central venous and portal venous pressures were determined and a control phlebography was performed.

| Preparation of 16S rRNA gene sequencing libraries, sequencing run, and data analysis
The isolated DNA was amplified with the bacterial 16S rRNA gene primers Bakt_341F (5'-CCTACGGGNGGCWGCAG-3') and Bakt_805R (5'-GACTACHVGGGTATCTAATCC-3'). 29 Samples were polymerase chain reaction (PCR)-amplified and processed according to the Illumina protocol using a 500 cycle V2 chemistry kit on an Illumina MiSeq machine (Illumina, San Diego, CA, USA).The raw sequence data from Illumina were processed with the QIIME 2 microbiome analysis package 30 using default settings.Quality filtering and chimera identification were carried out using the DADA2 plugin for QIIME. 31The sequences assigned to chloroplasts, mitochondria, eukaryotes, and Archaea were excluded because the primer set employed in the analysis has a very limited coverage of these groups.The amplicon PCR, the index PCR, a quantity and a quality control, and the sequencing of the individual libraries as a pool in one Illumina MiSeq run was performed, as described in a previous study. 16The raw sequences of the study were deposited at the Short Sequence Read Archive (SRA) under the accession number PRJEB21819.

| Statistical analysis
PAST software package version 3.22 was used for the statistical analysis. 32Samples for β-diversity analysis were normalized using Center Log Ratio (CLR) transformation.For Chao1 estimations Explicet was used, 33 which performs a rarefaction-based analysis through bootstrapping.A Kruskal-Wallis test and a post-hoc Dunn's pairwise test was used to calculate significant differences between the numbers of amplicon sequence variants (ASVs) in the samples.
Differences in the bacterial community composition were tested by permutational multivariate analysis of variance (PerMANOVA) combined with a pairwise PerMANOVA test between all pairs of groups as a post-hoc test corrected by the sequential Bonferroni method.The similarity between bacterial communities were visualized by principal coordinates analysis (PCA) based on CLR normalized data.P > 0.05 was regarded as statistically significant.

| Characteristics of the participants
Between January 2018 and January 2021, 17 patients with liver cirrhosis and portal hypertension-related complications received TIPS.Thirteen patients with liver cirrhosis who did not undergo TIPS and 18 healthy controls served as the control groups.The characteristics of the patients and the controls at d-1 are depicted in Table 1.The etiology of cirrhosis included alcoholic cirrhosis (n = 12 in the TIPS group, n = 11 in the non-TIPS group), NAFLD/NASH (n = 2 in the TIPS group, n = 1 in the non-TIPS group), hemochromatosis (n = 1 in the TIPS group, none in the non-TIPS group), and idiopathic (n = 2 in the TIPS group, n = 1 in the non-TIPS group), respectively.Refractory ascites was the most common indication for TIPS, which was presented in 13 (76.5%)patients, followed by recurrent variceal bleeding (n = 2) and hepatorenal syndrome (n = 2).TIPS led to a significant reduction of hepatic venous pressure gradient (HVPG) from 24.8 ± 6.7 mmHg before TIPS and 9.1 ± 3.4 mmHg after TIPS (P < 0.01, F = 3.81; Figure 1).

| Effect of TIPS on the intestinal microbial composition
When comparing the different groups at baseline (healthy controls as well as cirrhotic patients about to undergo TIPS placement or not), the liver cirrhotic groups showed a significant difference in the intestinal bacterial compositions compared to the healthy control group in the PCA (P < 0.01, F = 2.924; Figure 2A).However, when comparing the T A B L E 1 Characteristics of the patients with liver cirrhosis and healthy controls at baseline.α-diversity, the Chao1 index was not significantly different (Figure 2B).
We stratified patients with cirrhosis according to the etiology to alcohol-related and nonalcohol-related and compared their microbial compositions; the results showed that the etiology was not associated with a specific difference in intestinal microbiota (data not shown).
When comparing the luminal microbiota samples of patients undergoing TIPS and those without TIPS (d-1 vs d5 vs d30), no significant difference was found at the different time points (P > 0.01, F = 0.78; Figure 3A).Again, the diversity between the different groups measured by the Chao1 index was not different (Figure 3B).Stratification of patients according to the indications for TIPS (refractory ascites, recurrent variceal hemorrhage, and hepatorenal syndrome), BMI, liver function (CPS), and individual laboratory test results (international normalized ratio [INR], albumin, bilirubin, and creatinine) did not reveal specific associations of the luminal intestinal bacterial composition (data not shown).

| Effect of antibiotic therapy on the intestinal bacterial composition
To investigate whether antibiotic therapy has an effect on intestinal bacterial composition, we differentiated between liver cirrhotic patients who received antibiotic therapy (n = 14) and those without antibiotic therapy (n = 16); we used probe d-5 for this comparison.
Included in the antibiotic therapy group was the use of rifaximin in the prevention of hepatic encephalopathy.We found that the use of antibiotics led to a significant shift of the bacterial communities (Figure 4A) (P = 0.016, F = 1.491).In addition, the use of antibiotics led to a significant decrease in the bacterial diversity (Dunn's test, P = 0.03) (Figure 4B).Without antibiotic treatment, the α-diversity in patients with liver cirrhosis, measured by the Chao1 index, was comparable to that in the healthy control group (Dunn's test, P = 0.84).The effect of antibiotic therapy on the intestinal bacterial composition was further visualized for the 30 most abundant bacterial species in a heatmap (Figure 5).Antibiotic therapy led to a shift of the abundances in a variety of bacterial groups.

| DISCUSSION
Liver cirrhosis is the advanced stage of different liver diseases, which is pathologically characterized by a diffuse fibrosing and nodule-forming change.Altered intestinal bacterial composition, a so-called dysbiosis, has been reported to play an important role in the development of liver diseases.5][36] However, whether the changes in the bacterial composition are the reason or a consequence of hepatopathy is still a matter of debate and has been intensively discussed previously. 37,38ver cirrhosis is a disease with high morbidity and mortality and its decompensated form can manifest with a variable clinical spectrum including ascites, gastrointestinal bleeding mainly from esophageal varices, and hepatorenal syndrome.One possible treatment option for portal hypertension is TIPS, which reduces the blood flow via the portal vein.These changes in the blood pressure of the splanchnic system might possibly have an effect on the ileocolonic venous microcirculation as well.Our hypothesis was that the reduced HVPG after TIPS might also have an effect on the intestinal microbial composition.F I G U R E 1 Hepatic venous pressure gradient (HVPG) (1) before and (2) after transjugular intrahepatic portosystemic shunt (TIPS).Noncapital letters (a,b) above the boxplots indicate statistical significance (P < 0.05).When groups share the same noncapital letter, then the differences between the groups are not statistically significant.
The association between TIPS in patients with liver cirrhosis and intestinal microbiota has already been described and focused mainly on the development of HE.In a case report of two patients with HE, the application of fecal microbial transplantation (FMT) after TIPS resulted in a decreased number of HE episodes. 39In another study by Li et al on 106 cirrhotic patients receiving TIPS, restauration of the gut microbiota was inversely related to episodes of HE after TIPS application. 40In the current study we focused on the effect of TIPS application on the intestinal microbial composition in cirrhotic patients with TIPS compared to that in cirrhotic patients without TIPS and healthy controls.In addition, in our patient cohort HE was not a major clinical problem in patients with liver cirrhosis who received TIPS.
To study the effects of TIPS application on the luminal microbiome, stool samples were collected before, shortly, and long after the TIPS application.As a control group we included patients with liver cirrhosis who did not receive TIPS application and were in the hospital due to other reasons.Another group of 18 healthy adults served as a healthy control group.Our results did not show a significant difference between the microbial composition of the patients who received TIPS and those who did not receive TIPS.Since even 30 days after TIPS application no significant change in the microbial communities was found, it might be possible that some changes might even appear later post-TIPS; however, we think this is unlikely.However, there was a significant difference in the microbial composition between patients with liver cirrhosis and the healthy controls.This result might go in hand with other studies, which showed an overall reduced gene richness in fecal samples of patients with liver cirrhosis. 41,42However, the luminal bacterial composition remained relatively stable over the first 4 weeks after TIPS.From our data we cannot exclude that the intestinal bacterial composition changes after this time point, a longterm extension of our study to address the effects of TIPS placement on the luminal microbiota would be needed.
To our best knowledge, this is the first controlled study to address the question whether TIPS application has a direct effect on the intestinal luminal microbiome.Although we performed a controlled, prospective analysis, there were some limitations to our study.First, we did not interventionally assessed the HVPG in the liver cirrhosis control group.Second, as in all clinical studies with complex diseases, a high multicollinearity in the parameters measured cannot be avoided.In our study, in a high percentage of patients, anti- Antibiotic therapy is known to have a strong effect on gut microbiota. 44In our study we also found that, on the one hand a shift of F I G U R E 5 Heatmap of the 30 most abundant bacteria derived from liver cirrhotic patients after 5 days in the clinic with and without antibiotic therapy in comparison to bacterial communities derived from healthy controls.
the intestinal bacterial communities under antibiotic therapy compared to fecal samples without prior antibiotic treatment, and on the other hand also a reduction in the bacterial diversity under antibiotic therapy.From our point of view, inclusion of a larger number of patients would have been unlikely to change the abovementioned results.Interestingly, antibiotic therapy led to a significant reduction of certain bacterial genera such as Faecalicaterium, Blautia, Alistipes, Akkermansia, and Roseburia, which, at least in the field of inflammatory bowel diseases, seem to have a protective effect for the host 45 and certain probiotic properties.Further studies are needed to address the question whether antibiotic therapy has a negative long-term effect on the health via changing the intestinal bacterial communities toward a so-called "dysbiosis" and if this effect could possibly be reversed by an additional probiotic therapy.
This study was approved by the Ethic Board of the University of Rostock (no.A2016-0109).Written informed consent was obtained from each participant prior to their enrollment.Patients with a clinical or image-based diagnosis of liver cirrhosis were recruited from the Division of Gastroenterology at the University Medical Center Rostock (Rostock, Germany).Adult patients aged 18 years or elder who provided the written informed consent were included.Exclusion criteria were those with any neoplasia, pregnancy, and advanced HE. TIPS was performed for clinical indications including refractory ascites, hepatorenal syndrome, and gastrointestinal bleeding after the initial endoscopic therapy.Patients who received TIPS in an emergency setting were excluded from the study.Patients with liver cirrhosis who were inpatient due to other reasons who did not undergo TIPS served as controls.In addition, 18 healthy controls without any comorbidities served as the healthy control group.Demographic characteristics of the participants were collected, including age, gender and body mass index (BMI).For patients with liver cirrhosis, etiology of cirrhosis (alcoholic, viral hepatitis, nonalcoholic fatty liver disease [NAFLD], nonalcoholic steatohepatitis [NASH], hemochromatosis, etc), consumption of alcohol (if have), Abbreviations: BMI, body mass index; INR, international normalized ratio; MELD, model for end-stage liver disease; NA, not applicable; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; SD, standard deviation; TIPS, transjugular intrahepatic portosystemic shunt.

F I G U R E 2
Comparison of intestinal bacterial communities between healthy controls, and liver cirrhotic patients without and with transjugular intrahepatic portosystemic shunt (TIPS).(A) Principal component analysis (PCA) and (B) Chao1 diversity of intestinal bacterial communities derived liver cirrhotic patients after 5 days in the clinic without TIPS (open circles), with TIPS (filled circles), and healthy controls (green).The liver cirrhotic group showed a significant difference in the intestinal bacterial compositions compared to the healthy control group (P < 0.01, F = 2.924).Noncapital letters (a) above the boxplots indicate statistical significance (P < 0.05).When groups share the same noncapital letter, then the differences between the groups are not statistically significant.F I G U R E 3 Effect of transjugular intrahepatic portosystemic shunt (TIPS) on the intestinal bacterial communities.(A) Principal component analysis (PCA) and (B) Chao1 diversity of bacterial communities derived TIPS patients before (green), after 5 days (orange) and after 30 days (blue) of TIPS application.Noncapital letters (a) above the boxplots indicate statistical significance (P < 0.05).When groups share the same noncapital letter, then the differences between the groups are not statistically significant.
biotic therapy was needed, also leading to an altered intestinal microbial composition.In a recent study published by Gitto et al addressing the effect of TIPS placement on the intestinal microbial composition, patients with antibiotic therapy were excluded.43While Gitto et al identified in 8 out of 13 patients a considerable modification in the gut bacterial community composition after TIPS, the TIPS samples in our study did not show any specific pattern, which might possibly be associated with a different study protocol.

F I G U R E 4
Effect of antibiotic therapy on the intestinal bacterial composition in patients with cirrhosis.(A) Principal component analysis (PCA) and (B) α-diversity of bacterial composition derived from liver cirrhotic patients after 5 days in the clinic with (red) and without antibiotic therapy (blue) in comparison to intestinal bacterial communities derived from the healthy controls.Different noncapital letters (a,b) above the boxplots indicate statistically significant difference (P < 0.05).