Background
Hepatitis B is caused by hepatitis B virus (HBV) infection and can lead to acute and chronic liver diseases [
1]. Patients with chronic Hepatitis B (CHB) often develop serious complications, such as cirrhosis and hepatocellular carcinoma (HCC). HCC is the third leading cause of cancer death worldwide [
2]. At present, safe and effective vaccines can prevent Hepatitis B [
3]. However, according to the WHO, there were as many as 1.1 million new cases of Hepatitis B in 2017 (
https://www.who.int/news-room/fact-sheets/detail/hepatitis-b). In China, 80% of all HCC diagnoses are attributed to Hepatitis B [
3]. Even in the USA, the incidence rate of HCC has increased rapidly [
4]. Therefore, Hepatitis B is a global public health problem threatening human health.
As of 2016, only 10.5% (27 million) of all estimated Hepatitis B patients knew that they were infected, and 4.5 million (16.7%) confirmed patients were receiving treatment (
https://www.who.int/news-room/fact-sheets/detail/hepatitis-b). HCC progresses rapidly; most patients are usually diagnosed at an advanced stage, and the prognosis of advanced liver disease is generally poor [
5]. Therefore, early prediction of Hepatitis B progression is of great value for the diagnosis and treatment of HCC.
Faecal microbiota transplantation (FMT) is considered to be a promising new treatment option for a variety of refractory diseases, including Hepatitis B [
6,
7]. Increasing evidence demonstrates that the gut microbiota is included in the pathogenesis of liver diseases [
8‐
10]. At present, studies on the intestinal flora in patients with Hepatitis B have mainly focused on patients with CHB or patients with advanced liver diseases caused by liver cirrhosis or HCC [
8‐
12]. As far as we know, there are no reports on the intestinal flora of asymptomatic HBV carriers using next-generation sequencing. Therefore, a cross-sectional study including Hepatitis B patients at different stages was conducted. We aim to have a comprehensive understanding of the intestinal flora of patients with hepatitis B by studying all non-cancerous hepatitis B patients.
Discussion
Early prediction of the progression of Hepatitis B is of great value for the diagnosis and treatment of HCC. Therefore, in this study, the features of the gut microbiota in Hepatitis B patients covering four non-cancerous stages, HBV carriers, CHB, liver cirrhosis, and acute-on-chronic liver failure, were investigated using 16S rRNA gene sequencing. We intended to identify the potential role of the gut microbiota in disease progression for early different stages of Hepatitis B patients.
Consistent with previous results, our PCoA results showed that age and sex had no significant effect on the composition of the intestinal flora [
9,
10,
17]. Hu and his colleagues showed that the intestinal microbial community was not affected by oral antiviral treatment [
9,
18], so antiviral therapy was not regarded as an exclusion criterion in this study. Patients who received antibiotics (including traditional Chinese medicine) within 3 months were excluded because it is reported that some members of the microbial community disappear from the community indefinitely [
19]. It is speculated that region and eating habits might affect the flora of Hepatitis B patients [
20,
21]. Hence, the participants enrolled in this study were all from the Pearl River Delta in Guangdong Province, South China.
Consistent with previous studies [
9,
10,
22], compared with the CK, there was no significant change in the α diversity of HBV carriers or patients with CHB. However, the cohort could be divided into five groups using β diversity, indicating that the intestinal flora has already changed in HBV carriers even though they have no specific symptoms.
The intestinal flora in all reported studies is not completely identical, and the participants come from different regions of China [
7,
9,
10]. Therefore, the composition of the intestinal flora may be influenced by diet [
20]. With the development of the disease,
g_Roseburia and
g_Fusobacterium decreased.
G_Roseburia produces short-chain fatty acids that could affect colon movement, immune maintenance and anti-inflammatory properties [
22]. Therefore, g_roseburia is associated with many metabolic pathways and serves as a beneficial bacteria [
23]. In our study, the potential pro-inflammatory strains, g_Escherichia-Shigella, g_Klebsiella
and g_Enterococcus, increased, similar to previous reports [
7,
10].
G_Lachnospira [
10],
g_Prevotella_9 [
24],
g_Clostridium [
7],
g_Eubacterium]_eligens_group, and
g_Ruminococcus_1 [
9,
11] showed a downward trend, while
Veillonella [
9,
10] and g_ysipelatoclostridium were enriched with disease progression. Of note, the first cluster increased and the last two strains decreased in Group A compared with the CK. This phenomenon indicates that the floral composition of HBV carriers has its own particularity compared with those of patients who have clinical symptoms. Similar results were also found according to Gram staining and the microbial function of our cohort. HBV carriers can keep a balance with the virus during their entire life without clinical symptoms. According to our research results, we speculate that the change of the flora of HBV carriers may play an active role in the struggle between the human body and the virus. FMT has been proven to play a potential role in the treatment of refractory diseases including liver disease [
5,
6]. Therefore, HBV carriers may be more suitable for FMT than healthy people. Of course, more studies are needed to verify our hypothesis.
In our cohort, 40 strains with an LDA greater than 4 were identified by LEfSe, and these 40 strains belonged to the Top 3 most abundant phylum.
G_Roseburia and
g_Faecalibacterium were the most dominant in the CK, consistent with the previous reports [
9,
11].
G_Prevotella_9,
g_Clostridium,
g_Lachnospira, and
g_Lachnospiraceae_unclassified were the main bacteria in Group A. With the aggravation of disease,
G_Prevotella_9,
g_Clostridium, and
g_Lachnospira first increased in Group A and then decreased in Groups B-D, which further explained the specificity of Group A. The bacteria
g_Bacteroides was enriched in patients with CHB, live cirrhosis, and HCC [
9], but in our study, it only increased in CHB patients at the order level. The dominant bacteria in Group D were
g_Enterococcus, g_Klebsiella, g_Lactobacillus, g_Veillonella, and
g_Escherichia-
Shigella, which were also in the Top 30 most dominant bacteria in this study. These bacteria have been proved to play potential roles in the development of Hepatitis B [
7‐
10]. This result indicates that there is usually a higher abundance of the flora that may become molecular markers.
Correlation analysis showed that Lactobacillus was positively correlated with disease development, while Clostridium had a negative correlation with disease progression. These findings were confirmed by RDA with the TOP 10 most abundant flora. The serum sodium concentration is considered to be an important predictor of liver transplantation survival [
25].
G_Hungatella increases in patients with chronic kidney disease and primary IgA nephropathy [
26,
27]. In our study,
g_Hungatella was negatively correlated with the serum sodium concentration. The abundance of
g_Sutterella decreases in patients with bacteremia or bloodstream infection, indicating that
g_Sutterella is related to erythrocytopoiesis [
28]. In our study,
g_Sutterella was positively correlated with the red blood cell count. In accordance with this result, the identification of
g_Hungatella and
g_Sutterella was not a simple calculation result, but it had practical clinical significance. The Child-Pugh score is widely used for liver function evaluation [
29]. We analysed the correlation between the intestinal microflora and the Child-Pugh score and identified 15 highly correlated flora, all of which were discovered by LDA or the AUC. RDA showed that some floras were highly correlated with the staging of Hepatitis B. According to the above results, we can directly judge the role of a bacterium through the relationship between it and the progression of Hepatitis B.
Zeng et al. showed that, compared with the healthy control group, lipid metabolism of patients with Hepatitis B was significantly improved [
9]; however, this difference was not found in our study via a PICRUst prediction. The highest metabolic functions were the same as those reported by Zeng and Liu et al
. [9,
10]. Four of the six pathways had no significant differences, which indicated that these metabolic pathways play an important role in maintaining basic functions. Short-chain fatty acids are the main products of dietary fibre fermentation and reduce mucosal injury and intestinal permeability [
30,
31]. In our study, with the development of the disease, Acetyl-CoA fermentation to butanoate II and glycerol degradation to butanol decreased; correspondingly, the immune system and energy metabolism decreased, while infectious diseases increased. Through the detection of microbial community functions, we found that a correlation with the environmental adaptation function could directly identify the role played by a certain bacterium. Bacteria that are positively related to environmental adaptation are beneficial, while those that are negatively related to environmental adaptation are harmful.
We found that some probiotics may play a negative role in disease development. For example, lactic acid bacteria are protobacteria in the gastrointestinal tract and vagina, which are generally considered to be probiotics [
32]. In our study, Lactobacillus was negatively associated with environmental adaptation, energy metabolism and the immune system, indicating that it might have negative effects. Although further functional studies are needed to investigate the influence of the increased abundance of g_Lactobacillus on the progression of Hepatitis B, our results indicated that the intake of lactic acid bacteria should be cautious [
10]. In summary, through comprehensive analysis of 16S rRNA gene sequencing data, we can basically distinguish the role of bacteria in the progression of Hepatitis B, thus providing data support for FMT.
Although our study provided novel information for the potential role of the intestinal microflora in predicting the progression of Hepatitis B patients in different non-cancerous stages, there are limitations in this study. First, this cross-sectional study only showed the correlation between the gut microflora and the progression of Hepatitis B but did not provide direct causal evidence. Second, the 16S rRNA gene sequencing strategy may not reflect the actual information of the microbial community. Therefore, further studies using metagenomics next-generation sequencing are needed to investigate the potential causal mechanisms that link the gut microbiota and Hepatitis B. Third, although we outlined the significance of intestinal flora based data from asymptomatic HBV carriers, the case number of 24 in this group leads to certain statistical limitations, and further studies with large samples are needed. Additionally, the specificity of the intestinal microflora differences in cases of other liver diseases with different underlying aetiologies in the same population was not under the purview of the present study, which limited the specificity of the data with respect to HBV.
Materials and methods
Participants
Healthy volunteers were regarded as the healthy controls group. Patients treated in the Department of Infectious Diseases of the Third Affiliated Hospital of Sun Yat-Sen University from January 2019 to March 2020 were regarded as candidates. Hepatitis B virus infection was confirmed by being positive for the hepatitis B surface antigen (HBsAg). Hepatitis B was staged according to the Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2015 Edition) issued by the Chinese Medical Association. All the participants in this study had lived in the Pearl River Delta in Guangdong Province for more than 3 years. Patients who were infected by human immunodeficiency virus (HIV) or hepatitis C virus (HCV) or had other diseases, such as alcoholic hepatitis, fatty liver disease, acute or chronic infectious diseases, autoimmune diseases, or non-Hepatitis B liver diseases, were excluded. Patients with a body mass index (BMI) (kg/m2) of less than 18.5 or more than 23.9 were excluded. Patients who with any gastrointestinal disease, which may be linked to a leaky gut and bacterial translocation to the liver, were ruled out. In addition, patients who took antibiotics or traditional Chinese medicine orally within the previous 3 months were excluded. This study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-Sen University. Written informed consent to participate in this study was provided by the participants.
Clinical testing
General information such as medical history and residence was collected during the inquiry. Routine bloodwork, reticulocyte counts, liver and kidney function, blood lipid profile, and coagulation function were determined. In the healthy controls group, only routine blood tests, reticulocyte count and liver and kidney function were determined. The patients were tested for virus-related indicators. Liver biopsy or imaging examinations including trans-abdominal ultrasound, computed X-ray tomography (CT), or nuclear magnetic resonance (NMR) were performed when necessary.
Faecal samples collection and 16S rRNA gene sequencing
Every participant involved in 16S rRNA gene sequencing provided a fresh tail stool sample in the morning. The samples were collected in sterile plastic tubes and stored in a − 80 °C refrigerator as soon as possible. The 16S rRNA sequencing was performed by LC-Bio Technology Co., Ltd, Hang Zhou, Zhejiang Province, China. In brief, DNA was extracted from stool samples using an E.Z.N.A. ®Stool DNA Kit (D4015, Omega, Inc., USA) following the manufacturer’s instructions. After determination by 1.2% agarose gel electrophoresis, total DNA was eluted in 50 μL Elution buffer, and stored at − 80 °C until further study.
Regarding sequencing, the 16S rRNA V4 region was amplified using primers 515F(5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The 5′ ends of the primers were tagged with specific barcodes per sample and sequencing universal primers. Polymerase chain reaction products were identified by 2% agarose gel electrophoresis, purified with pure XT beads (Beckman Coulter Genomics Company, Danvers, Massachusetts, USA), and quantified with quantum bits (Nitrogen, USA). After being assessed on an Agilent 2100 Bioanalyser (Agilent, USA) and Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), the libraries were sequenced on NovaSeq PE250 platform according to the manufacturer’s recommendations.
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