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Alterations of gut bacteria Akkermansia muciniphila and 
Faecalibacterium prausnitzii in late post-transplant period after 
liver transplantation 

Alexander Kukov %"\, Milena Peruhova °, Atanas Syarov‘™, Iskra Altankova ~4, 
Nonka Yurukova Tio} Andrei Goncharov eD, Radoslava Vazharova c(), Anoaneta 
Mihova *4\, Tsvetelina Velikova *4\, Yordanka Uzunova °\ 

“University Hospital “Lozenets”, Laboratory of Clinical Immunology, Sofia, Bulgaria 
’ Faculty of Medicine, Sofia University “St. Kliment Ohridski”, University Hospital “Lozenets”, Clinic of Gastroenterology, Sofia, 


© Faculty of Medicine, Sofia University “St. Kliment Ohridski”, University Hospital “Lozenets”, Laboratory of Medical Genetics, Sofia, 


4 Faculty of Medicine, Sofia University “St. Kliment Ohridski”, Department of Clinical Immunology, Sofia, Bulgaria 
° Faculty of Medicine, Sofia University “St. Kliment Ohridski”, University Hospital “Lozenets”, Clinic of Pediatrics, Sofia, Bulgaria 



Article history: 

Received 18 November 2021 
Received in revised form 15 
December 2021 

Accepted 17 January 2022 


Akkermansia muciniphila 
Faecalibacterium prausnitzii 
Gut microbiota 

Liver transplantation 


Introduction: Recent studies have shown that the intestinal microbiota can modulate certain 
systemic metabolic and immune responses, including liver graft function and the development 
of complications in patients after liver transplantation (LT). Akkermansia muciniphila (AKM) and 
Faecalibacterium prausnitzii (FAEP) are two of the most abundant gut commensal bacteria, with 
mucosa-protective and anti-inflammatory effects that are important for maintaining normal 
intestinal homeostasis and gut barrier function. Our objective was to quantify levels of 
Akkermansia muciniphila and Faecalibacterium prausnitzii in immunosuppressed patients with 

Materials and methods; Fecal samples from 23 liver transplant patients (15 adults and 8 
children) and 9 non-LT controls were examined. Bacterial DNA was isolated from the samples 
using the stool DNA isolation kit and the obtained DNA was analyzed with commercially 
available qPCR kit for AKM and FAEP. 

Results: We found a statistically significant decrease in the amount of AKM and FAEP compared 
to the control group. The median values were: for AKM 8.75 for patients and 10.25 for the control 
group (p = 0.030), and for FAEP 9.72 and 10.47, p = 0.003, respectively. In children after LT, this 
difference was also statistically significant: AKM (p = 0.051) and FAEP (p = 0.014). In contrast 
no statistically significant differences were found between adult patients and controls, AKM (p 
= 0.283) and FAEP (p = 0.056), although the amount of both bacteria showed tendency for 

Conclusions: In this pilot study, we found a reduction in the total amount of the two studied 
bacteria in transplanted patients compared to the control healthy group. 

© 2022 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the 
CC BY license (http://creativecommons. org/licenses/by/4.0/). 

* Corresponding author. 

E-mail address: 

ISSN: 2695-5075 / © 2022 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the CC BY license 
(http://creativecommons. org/licenses/by/4.0/). 



Alteraciones de las bacterias intestinales Akkermansia muciniphila y 
Faecalibacterium prausnitzii en el postrasplante tardio tras trasplante hepatico 



Historia del articulo: 
Recibido 18 Noviembre 2021 
Recibido en forma revisada 
15 Diciembre 2021 
Aceptado 17 Enero 2022 

Palabras clave: 
Akkermansia muciniphila 
Faecalibacterium prausnitzii 
Microbiota intestinal 
Trasplante de higado 


Introduccién: Estudios recientes han demostrado que la microbiota intestinal puede modular 
determinadas respuestas metabdlicas e inmunitarias sistémicas, entre ellas la funcion del injerto 
hepatico y el desarrollo de complicaciones en pacientes tras un trasplante hepatico (TH). 
Akkermansia muciniphila (AKM) y Faecalibacterium prausnitzii (FAEP) son dos de las bacterias 
comensales intestinales mas abundantes, con efectos protectores de la mucosa y 
antiinflamatorios que son importantes para mantener la homeostasis intestinal normal y la 
funcién de barrera intestinal. Nuestro objetivo fue cuantificar los niveles de Akkermansia 
muciniphila y Faecalibacterium prausnitzii en pacientes inmunodeprimidos con TH. 
Materiales y métodos: Se examinaron muestras fecales de 23 pacientes trasplantados de higado 
(15 adultos y 8 nifios) y 9 controles sin TH. El ADN bacteriano se aisl6 de las muestras utilizando 
el kit de aislamiento de ADN de heces y el ADN obtenido se analizo con el kit qPCR disponible 
comercialmente para AKM y FAEP. 
Resultados: Encontramos una disminuci6n estadisticamente significativa en la cantidad de AKM 
y FAEP en comparacion con el grupo control. Los valores medianos fueron: para AKM 8,75 para 
los pacientes y 10,25 para el grupo control (p = 0,030), y para FAEP 9,72 y 10,47, p = 0,003, 
respectivamente. En nifios tras TH, esta diferencia también fue estadisticamente significativa: 
AKM (p = 0,051) y FAEP (p = 0,014). Por el contrario, no se encontraron diferencias 
estadisticamente significativas entre pacientes adultos y controles, AKM (p = 0,283) y FAEP (p 
= 0,056), aunque la cantidad de ambas bacterias mostr6 tendencia a la reduccion. 
Conclusiones: En este estudio piloto, encontramos una reduccion en la cantidad total de las dos 
bacterias estudiadas en pacientes trasplantados en comparacion con el grupo control sano. 

© 2022 Los Autores. Publicado por Iberoamerican Journal of Medicine. Este es un articulo en acceso abierto 

bajo licencia CC BY (http://creativecommons. org/licenses/by/4.0/). 

HOWTO CITE THIS ARTICLE: Kukov A, Peruhova M, Syarov A, Altankova I, Yurukova N, Goncharov A, Varzharova R, Mihova A, 
Velikova T, Uzunova Y. Alterations of gut bacteria Akkermansia muciniphila and Faecalibacterium prausnitzii in late post- 
transplant period after liver transplantation. Iberoam J Med. 2022;4(1):45-51. doi: 10.53986/ibjm.2022.0010. 

some bacteria and their metabolites have a beneficial effect 


Patients with end-stage liver diseases (ESLD), such as 
cirrhosis of different etiologies, autoimmune liver diseases, 
genetically linked liver malformations and other have a poor 
prognosis and liver transplantation (LT) is a life-saving 
treatment for them. Scientific studies discuss the influence 
of gut bacteria on normal and pathological liver function. 
With the advent of advanced molecular biological 
techniques to facilitate culture-independent characterization 
of microbiota, it is possible to study in details different 
bacteria in the human microbiome [1]. This allows us to 
understand their role in the clinical setting during and after 
LT. The connection between intestinal microbiome, 
immunity and its dysregulation is well established, as well 
as the changes that occur in the post-transplant period under 
the influence of immunosuppressive therapy. A number of 
studies have shown that changes in the microbiome may be 
associated with acute graft rejection, as well as other post- 
transplant complications. Gut microbiota modulate some 
systemic and immune responses via multiple mechanisms 
that may affect allograft function [2]. It has been found that 

on innate and acquired immunity, reducing T-cell activation 
and promoting induction of regulatory T cells (Tregs) [3]. 
This is essential for building and maintaining graft tolerance 

The barrier functions of the intestinal mucosa are very 
sensitive to dysbiosis (condition when the gut bacteria 
become imbalanced) and are often disrupted which could 
lead the bacterial metabolites to enter the circulation [1]. Gut 
dysbiosis could alter the barrier functions of the mucosa; 
followed by reduced production of mucin, which coincides 
with the loss of the beneficial microorganism Akkermansia 
muciniphila (AKM) [5]. 
(FAEP), which is a bacterium with potent anti-inflammatory 
properties, via nuclear factor «B inhibition and induction of 
Treg [6], is also of great interest for research. It has been 
found that the decrease in the total amount of FAEP is 
accompanied by overgrowth of some opportunistic species, 
which increases the risk of infections in patients [7]. 

Many factors can affect the gut microbiota after LT, such as 
antibiotics, pro/prebiotics, and immunosuppressive therapy 
that can additionally modify the baseline gut bacterial 

Faecalibacterium prausnitzii 

dysbiosis present in ESLD, emphasizing the importance of 


understanding the impact of gut microbiota post LT [1, 8]. 
However, it should be noted that there are age related 
changes in the gut microbiota with infant microbiota being 
relatively volatile [9]. Calcineurin (CND, 
including tacrolimus and cyclosporine A (CsA), are the 
main immunosuppressive drugs used in the treatment of 
patients after LT [10]. Optimal dosing is extremely 
important for a better allograft outcome. It has been shown 
that both high and low doses of CNI could suppress some 
beneficial bacteria, such as Faecalibacterium prausnitzii 
[11]. Mycophenolate (MMF) is __ potent 
immunosuppressive agent used as adjunctive therapy in 
prevention of allograft rejection. MMF has been shown to 
be associated with frequent diarrhea and dysbiosis, as well 
as a reduction in some beneficial bacteria including AKM 
[10]. The mammalian target of rapamycin (mTOR) 
inhibitors, such as sirolimus and everolimus, have a 
favorable adverse event profile and are effective in 
protecting kidney function in LT patients. Everolimus 
appear to have little impact on intestinal microbiota [12]. 
We studied the distribution and quantities of AKM and 
FAEP in Bulgarian patients after LT and subjected to 
immunosuppressive therapy. The aim of this pilot study was 
to investigate the prevalence and quantity of these two 
bacteria in the late post-transplant period. 




This study was performed between May 2021 and October 
2021 at the University Hospital "Lozenets", Sofia, Bulgaria. 
In total 32 individuals, children and adults, designed into 
two groups: control group (9 persons) and patients after LT 
(23 persons) were included in the study. Patients were 
enrolled according to 
transplantation at least 3 months after the procedure and on 
immunosuppressive therapy; without systemic or gut 
infections and antibiotic usage at least two months before 
enrolment; no data for acute rejection; no autoimmune 
diseases, diabetes and carcinoma. Exclusion criteria: active 
hepatitis B hepatitis C 
immunodeficiency virus infections or tuberculosis; alcohol 
and cigarette abuse, acute diarrhea. All patients with LT 
were in good general condition. Control groups (children 
and adults) were healthy individuals without infections and 
antibiotic treatment in the last 2 months. 

inclusion criteria: with liver 

virus, virus, human 

The study was conducted following the ethical guidelines of 
the Declaration of Helsinki and was approved by the local 
ethics committee (protocol No:2/2021). All participants 
have declared and signed their informed consent. Parents or 
guardians signed the informed consent for children (< 18 

years old). The liver transplantions were performed at the 
University Hospital (Sofia), being also 
performed the follow-up monitoring in the post-transplant 



Fecal samples were obtained from children and adult 
patients admitted to the hospital for routine examination. 
Sterile stool collection tubes (Prima, 20 ml) were used; the 
samples were immediately transported to the laboratory and 
stored at - 20°C until later analysis. 


Bacterial DNA was isolated from the fecal samples using the 
stool DNA isolation kit (QIAamp Fast DNA Stool Mini Kit, 
ref.51604, Qiagen, Germany), according to the 
manufacturer's guidelines, with a slight modification. 
Briefly, 1 ml InhibitEX buffer and sand particles were added 
to 200 mg feces; the samples were incubated at 95°C for 10 
minutes, to promote better lysis of Gram-positive bacteria. 
Additional homogenization and lysis of the samples was 
done on Precellys 24 (Peqlab,Gmbh) for 15 sec. at 4500 rpm. 
The resulting homogenate was centrifuged (3 min at 17000 
g) and samples were processed for DNA isolation. The 
concentration and purity of the DNA was determined 
spectrophotometrically using a  NanoDrop 2000 
spectrophotometer (Thermo Scientific, USA). The mean 
purity of the DNA was (mean + SD) 1.90 + 0.10, ratio 
Abs260/280 nm. The DNA samples were stored at -20°C 
until further testing was performed. 


After extraction, fecal bacterial DNA was quantified and 
adjusted in order to obtain DNA 12.5 ng/ul. qPCRs included 
4 ul template (50 ng of DNA per reaction) and 16 ul of 
Reaction/Master mix. To quantify the amount of AKM and 
FAEP DNA we used MutaPLEX® AKM/FAEP real time 
PCR kit (Immundiagnostik AG, Germany), following the 
manufacturer's instructions. Samples were analyzed using 
LightCycler 480 II thermocycler (Roche). For quantification 
of Akkermansia muciniphila and  Faecalibacterium 
prausnitzii positive DNA in samples, a standard curve using 
standards was applied. The data obtained in copies per 
reaction were then converted to cells/g, according to the 
protocol and the results are presented in log io of bacteria 

cells per g feces. 



The Mann-Whitney Exact U test was used to compare the 
Control and Patients groups. In terms of statistical 
significance, p < 0.05 was significantly different. 


The average post-transplant period was 7 years (min-max: 2 
— 17y). The indications for transplantation in adults were 
decompensated _ liver etiologies 
(ethylism - 4, viral - 4, autoimmune - 2 and 6 with other 

cirrhosis of various 
etiology). In children, the cause for transplantation was: 
autoimmune hepatitis - 1, biliary atresia - 3, liver cirrhosis 
of unknown etiology - 4. Control groups included healthy 
individuals without LT. Gender and age of the subjects 
groups are presented in Table 1. 

measured amounts in the transplanted group were 
significantly lower than healthy controls (p = 0.014, Table 
3). The analysis of AKM in children found that 3 out of 8 
children (38%) had AKM in the fecal samples, eg. 62% of 
children with LT do not have AKM expression in their feces. 
The statistical comparison between the control and LT 
patient groups revealed reduced amounts of AKM in 
patients (p = 0.051, Table 3). 

The results of the research in adults are presented in Table 
4. The frequency of positive FAEP samples is 100% in the 
control group, while in the transplanted patients it is 73%. In 
27% of patients with LT, this bacterium was absent. AKM 
expression was also reduced in patients with LT - 67% 
versus 80% in the control group. Notably, in 33% of 
transplanted patients AKM were not detected. The median 
amounts for both bacteria in patients were reduced 
compared to healthy controls, but the differences were not 

Table 1: Demographic features of patients and control groups 
Control group 

Tested individuals 

(n= 9) 

Liver transplantation group 

Children 4 
Adults 5 

(n = 23) 
6 (4 - 12) 8 5/3 8 (2 - 17) 
40 (26 - 49) 15 12/3 50 (19 - 70) 

M: Male; F: Female. Age expressed as mean as well as minimum and maximum values. 

We studied the presence and amounts of AKM and FAEP in 
fecal samples of all enrolled persons. The results are shown 
on Table 2. We found that in the control group, all 
individuals had FAEP and AKM and only one lacked AKM. 
In patients with LT group, however, we found that FAEP 
was observed in 83% and AKM in only 52% of transplant 
recipients. These differences were statistically significant 
when the amounts of bacterial expression was compared for 
FAEP and AKM, respectively p = 0.003 and p = 0.03. AKM 
was not detected in 48% of the patients and FAEP in 17% of 
the patients. 

statistically significant (Table 4). 

One possible reason for the decreased bacterial expression 
in the gut of LT patients might be the immunosuppressive 
therapy of these patients. Seven adult patients were treated 
with tacrolimus and MMF, four - received tacrolimus alone, 
one patient was on everolimus and cyclosporine, and one on 
cyclosporine and MMF. Seven of the children with LT were 
treated with tacrolimus and one child (autoimmune 
hepatitis) was on cyclosporine, methylprednisolone and 
MMF. Our results concerning the reduced prevalence of 
both bacteria in LT patients, subjected to different 

Table 2: Prevalence and amounts of FAEP and AKM in fecal samples of control and liver transplanted patients 

Control group 
(n= 9) 

Bacteria te Median value 
Positive (%) . 
(min — max) 
FAEP a COUR): ~~ oe 1th 
AKM 8 (89%) (0.00 - 11.54) 

Liver transplantation group 

e273) = P value* 
We Median value 
Positive (%) F 
(min — max) 
19 (83%) (0.00 - 11.15) 0.003 
122%) (000-1157) ae 

*Mann-Whitney Exact U Test was used to compare the amounts of the two bacteria. p<0.05. FAEP: Faecalibacterium prausnitzii; 
AKM: Akkermansia muciniphila. 

The separate analysis in Children and Adults groups shows 
that this trend is more pronounced in children than in adult 
transplant subjects. In all studied children (controls and 
patients with LT) we found the presence of FAEP, but the 

immunosuppressive regimens, are shown on Table 5. 
The following conclusions can be drawn from Table 5: 
1. FAEP was detected in all children fecal samples. In 
contrast, the prevalence of AKM in both patient 


groups was lower in comparison with controls. 
Furthermore, two adult patients were found to be 
double negative. 

2. It should be noted that all regimens of used 
immunosuppressive therapy most likely influence 
AKM growth, as it was not detected in 10 out of 23 
patients with LT (43%). We can assume that the 
calcineurine inhibitor tacrolimus might suppress 
both studied bacteria, but apparently its 
combination with MMF enhances this effect. 

3. The two double-negative patients (lacking both 
FAEP and AKM) are on therapy with MMF and 
tacrolimus or everolimus. 

in several disease conditions [13]. Moreover, both species 
are now shown to have a role in a well-functioning gut and 
thus are considered as promising next generation probiotics 
[14, 15]. 

In our study, we found a reduction in the prevalence and 
amount of AKM and FAEP in patients in the late post- 
transplant period compared to the control healthy group. 
This was more markedly seen in children with LT, where 
AKM was not detected in 62% of the studied samples. The 
limitations of the current study include small number of 
patients and control individuals, single testing of all 
participants, single-center design, the COVID 19 pandemic, 
which complicates the 

further enrolment of 

Table 3: Distribution and quantity of FAEP and AKM in children control and liver transplantation groups 

Control group 

Bacteria a4) 5 
re, Median value 
Positive (%) 
(min — max) 
FAEP 4 (100%) 10.47 (10.16 - 10.68) 
AKM 4 (100%) 10.85 (9.77 - 11.54) 

Liver transplantation group 
(n= 8) 

oe Median value EEG 
Positive (%) 4 
(min — max) 
8 (100%) 9.63 (8.41 - 10.58) 0.014 
3 (38%) 0.00 (0.00 - 11.20) 0.051 

*Mann-Whitney Exact U Test was used to compare the amounts of the two bacteria. p<0.05. FAEP: Faecalibacterium prausnitzii; 
AKM: Akkermansia muciniphila. 

Table 4: Distribution and quantity of FAEP and AKM in adult control and liver transplantation groups 

Control group 

Bacteria ine) = 
ye Median value 
Positive (%) i 
(min — max) 
FAEP 5 (100%) 10.47 (10.31 - 11.12) 
AKM 4 (80%) 9.71 (0.00 - 11.22) 

Liver transplantation group 
(n= 8) 

Are Median value Evalue 
Positive (%) 3 
(min — max) 
11 (73%) 9.88 (0.00 - 11.15) 0.056 
10 (67%) 9.00 (0.00 - 11.57) 0.283 

*Mann-Whitney Exact U Test was used to compare the amounts of the two bacteria. p<0.05. FAEP: Faecalibacterium prausnitzii; 
AKM: Akkermansia muciniphila. 


Akkermansia muciniphila and Faecalibacterium prausnitzii 
are two commensal bacteria, symbiotic and numerically 
abundant members of the gut microbiota. Recent studies 
have demonstrated their possible association with dysbiosis 

immunosuppressed patients. 

Recently, it has been shown that after LT there is a decrease 
in gut bacterial diversity and dysbiosis [1, 8]. It has been 
found that changes in gut microbial composition can result 
in disruption of the mucosal barrier, facilitating the 
translocation of bacteria and microbial products pathogen 
associated molecular patterns in the portal circulation 
affecting the inflammatory cytokine milieu in the liver [16]. 

Table 5: Patients with liver transplantation with no detection of AKM or/and FAEP in context of immunosuppressive 

FAEP negative AKM negative FAEP and AKM 
Treatment n=4 n=10 double negative 
(adults) (5 children and 5 adults) ri) 

Tacrolimus 0 6" 0 
Tacrolimus + MMF 3 2 1 
Everolimus + CsA 0 1 0 
Everolimus + MMF 1 1 1 

*5 children + I adult patients. FAEP: Faecalibacterium prausnitzii; AKM: Akkermansia muciniphila; MMF: Mycophenolate 
mofetil; CsA: Cyclosporine A. 


The pre- and post-LT comparative analysis observed a 
decrease in gut microbial diversity in the early post- 
transplant period (1 month), with improvement in diversity 
after at least 6 months [17]. Furthermore, in a qPCR-based 
study of 111 LT recipients it was found that the amount of 
FAEP was significantly reduced in recipients, which 
corresponds results [7]. 
prausnitzii 1s an anaerobe with a fecal-mucosal distribution, 
one of the major producers of SCFAs (short-chain fatty 
acids) of which butyrate is the main energy source for 

with our Faecalibacterium 

colonic epithelium and possesses potent anti-inflammatory 
properties [18]. This bacterium is considered as an anti- 
inflammatory with an essential role for the maintenance of 
the colonic mucosa, the induction of regulatory T cells [18] 
and the regulation of Treg/Th17 balance [19]. 

Our data shows a significant reduction of AKM load in LT 
patients. In the literature, there are discrepant data. 
Satapathy SK et al. found loss of AKM in patients after LT, 
especially in patients with recurrent NAFLD. It has been 
suggested that AKM may play a protective role in the 
development of complications (such as de novo NAFLD) 
after LT [20]. However, Sun et al., found an increase in 
potentially beneficial bacteria, such as AKM, Blautia and 
Clostridiales cluster XIVa, 3 months after LT. The authors 
suggested that the transplanted liver could significantly 
improve gut function, hence leading to an increase in 
[21]. A. 
muciniphila is an anaerobic, mucin-degrading bacterium 
[15], which is considered to have a protective role in the 
barrier function of the intestinal mucosa by strengthening 

beneficial bacteria in the fecal microbiota 

the contacts between intestinal epithelium cells (tight 
junctions). Furthermore, it has been suggested that a 
decrease in the total amount of AKM can be associated with 
thinning of the mucin layer, which may contribute to liver 
inflammation [5]. 

In addition, we try to analyze our data in the context of the 
applied immunosuppressive therapy. The results showed 
that AKM is mostly affected by tacrolimus and the 
combination of tacrolimus with MMF. The available in 
literature data on this topic is limited and there is 
unequivocal opinion on how tacrolimus affects AKM. Most 
studies have been performed on mouse models of LT and 
immunosuppressive treatment. Same as us, some authors 
indicate a reduction in the load of this bacterium after 
tacrolimus treatment [22], in contrast others report that 
tacrolimus increases the amount of AKM [10]. It has been 
found in mice that the medium dose of tacrolimus increased 
the amount of FAEP, while both low and high dose of 
immunosuppressant reduced the bacteria [11]. Concerning 
FAEP, we found a reduction in the bacterial load after 

combined immunosuppression of tacrolimus/everolimus 
and MMF, whereas patients in tacrolimus only do not 
showed lack of F. prausnitzii. Intriguingly, it has been 
observed that in patients who require higher drug doses to 
reach optimal tacrolimus plasma concentrations, the amount 
of FAEP is increased [23]. The authors found that some 
Clostridiales including FAEP could transform tacrolimus 
into a less potent metabolite. The aforementioned study 
provides evidence for new pathways of tacrolimus 
metabolism and the role of the gut microbiota in it. 


Our pilot study shows that in patients with LT on 
immunosuppressive therapy in the late post-transplant 
period, the frequency and amount of beneficial bacteria 
AKM and FAEP in gut microbiota are significantly reduced 
in comparison with healthy individuals. This may be due to 
a variety of possible factors, one of which is 
immunosuppressive therapy. Studies in a larger number of 
patients are needed for confirmation of our results and 

further analysis. 


This work was funded by the scientific project grant Ne80- 
10-127/26.03.2021 of the Sofia University “St. Kliment 
Ohridski’, Sofia, Bulgaria and was executed in University 
Hospital "Lozenets", Sofia, Bulgaria. We appreciate the 
generosity of the patients and the staff of all clinics for their 
dedication and careful sample collection. We are thankful to 
Dr. Georgi Vasilev, MD, PhD for statistical help. 


The authors declare no conflict of interest. 


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