Characterization of biliary microbiota dysbiosis in acute cholecystitis: A reduction in the biodiversity of the bile microbiome

doi:10.21203/rs.3.rs-2178148/v1

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Characterization of biliary microbiota dysbiosis in acute cholecystitis: A reduction in the biodiversity of the bile microbiome

https://doi.org/10.21203/rs.3.rs-2178148/v1

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Background

Previous studies have shown that bacterial infections are closely associated with most common biliary diseases, such as biliary tract infection and gallbladder stone formation. Acute cholecystitis usually occurs in patients with acute infections of the biliary system, and severe infections can easily lead to life-threatening sepsis. This study explored the structural differences in the bile microbiome in patients with acute and chronic cholecystitis and the relationship with acute and chronic cholecystitis.

Results

A total of 18 patients in the acute cholecystitis group and 8 patients in the control group were enrolled in the analysis. The composition of the biliary microbiota significantly differed between acute cholecystitis patients and chronic cholecystitis controls. Principal coordinate analysis (PCoA) and principal component analysis (PCA) revealed that the microbial communities obtained from the chronic cholecystitis controls clustered separately from those from the acute cholecystitis patients. We observed that many kinds of bacteria, such as Burkholderia, Bradyrhizobium, Phreatobacter and Comamonas, were significantly higher in chronic cholecystitis controls than in acute cholecystitis patients.

Conclusions

The diversity of the bile microbiome in patients with acute cholecystitis is lower than that in patients with chronic cholecystitis. Patients with acute cholecystitis may have a bile microbial imbalance that might related to acute infections.

Acute cholecystitis

Bile microbiome

Microbiota dysbiosis

16S rDNA sequencing

Biliary tract infection is one of the most common biliary tract diseases. Acute cholecystitis (AC) usually occurs in patients with acute infections of the biliary system, and in elderly patients with severe infections, life-threatening sepsis can easily develop. Most acute cholecystitis is also closely associated with gallbladder stones.

Gallstone disease is widespread worldwide, with factors such as sex, age, diet and lifestyle influencing its occurrence. Bile is synthesized in the liver and stored in the gallbladder. It is mainly composed of bile acids (BAs), cholesterol, phospholipids and proteins, and its main physiological function is to facilitate the absorption of fats in the intestine. Normal bile specimens are difficult to obtain, and microorganisms were previously thought to be absent from normal human bile. A recent study of bile samples from the gallbladder of liver transplant donors revealed the presence of a complex microbial ecosystem within the bile of normal subjects with hepatobiliary disease[1]. Moreover, several studies have shown that bacterial infections are closely associated with gallbladder stone formation. For instance, Escherichia coli, Salmonella and Pseudomonas aeruginosa play an important role in the formation of gallbladder stones[2–4]. However, the relationship between acute cholecystitis and gallbladder microbes has not been revealed.

Effective and timely antibiotic therapy plays a crucial role in acute attacks of biliary system infections. The Tokyo guidelines (TG18) also recommend the timely and effective use of antibiotics and state that bacterial cultures should be performed to guide antibiotic use[5]. Since bacterial cultures are incomplete in terms of bacterial species, they are of little clinical relevance for early treatment guidance. 16S rDNA sequencing technology can rapidly clarify microorganisms within bile samples, provide rapid diagnosis of microbial species, allow timely adjustment of antibiotic treatment regimens and is expected to become one of the most important diagnostic techniques for the early treatment of biliary tract infections.

In this paper, we used the 16S rDNA sequencing method to find bacteria in the bile of patients with acute and chronic cholecystitis and analysed the microbiome information. We found that when acute cholecystitis was induced, there were corresponding changes in the microbiome within the bile. Changes in the bile microbiome may be associated with acute episodes of biliary tract infections. The study of the imbalance of the microbiome in bile is helpful to reveal the pathogenesis of acute biliary tract infections and provide help for early and precise intervention.

Characteristics of patients

A total of 26 patients with cholecystitis were enrolled in the analysis. Diagnostic criteria for acute cholecystitis were implemented according to the Tokyo guidelines (TG18) base on clinical symptoms and auxiliary examination [6]. Patients with acute cholecystitis were divided into three groups according to cholecystolithiasis (Group A, n = 7), cholecystolithiasis and common bile duct stones (Group B, n = 7), and cholecystolithiasis, common bile duct stones and acute pancreatitis (Group C, n = 4). Patients with chronic cholecystitis and cholecystolithiasis (Group D, n = 8) were included as the control group. The general characteristics and clinical parameters of the four groups are shown in Table 1. There were no significant differences between the groups in terms of gender, age and previous hypertension. Compared with control group, patients in the acute cholecystitis group were more likely to have higher WBC, TBil, ALT, GGT and body temperature levels, which were associated with an acute inflammatory response (Group A) or acute cholangitis (Group B and C). Consequently, further analysis of bile samples from these four groups of patients was performed.

Table 1

Characteristics of patients with cholecystitis
Characteristic	A	B	C	D	p valve
Patients, n	7	7	4	8	-
Female, n (%)	3 (42.9%)	5 (71.4%)	2 (50%)	5 (62.5%)	0.476
Age (years)	80.57 ± 7.21	70.00 ± 10.31	72.25 ± 12.92	68.75 ± 3.69	0.058
WBC (10⁹/L)	10.50 ± 2.98	9.16 ± 3.85	11.03 ± 5.03	5.30 ± 1.28	0.014
TBil (µmol/L)	37.99 ± 22.65	44.91 ± 22.89	100.25 ± 53.60	17.11 ± 7.76	＜0.001
ALT (U/L)	61.24 ± 59.54	252.08 ± 271.64	413.25 ± 490.55	14.03 ± 4.12	0.035
GGT (U/L)	133.29 ± 161.67	387.86 ± 403.87	506.50 ± 398.60	25.95 ± 8.40	0.021
AST (U/L)	108.71 ± 157.89	495.14 ± 617.92	408.50 ± 281.01	20.39 ± 5.56	0.056
Body temperature (°C)	37.94 ± 1.20	37.97 ± 1.03	37.32 ± 1.45	36.56 ± 0.52	0.045
Previous hypertension n (%)	4 (57.1%)	2 (28.6%)	2 (50%)	3 (37.5%)	0.751
WBC: white blood cell; TBil: total bilirubin; ALT: alanine aminotransferase; GGT: gamma glutamyl transpeptidase; AST: aspartate aminotransferase; -: no relevant information.

The Distribution And Diversity Of The Bile Microbiome

16S rDNA gene sequencing was employed for bacterial identification. Raw reads averaged 82,552, 84,974, 84,361 and 84,854 per sample in Groups A, B, C, and D, respectively. After quality filtering, an average of 76,549, 79,125, 75,535 and 77,478 high-quality sequence tags were respectively obtained. We used SILVA (Release 138) and the NT-16S database to classify sequences.

As shown in Fig. 1a, Proteobacteria was the most abundant phylum in the whole population (Group A, B C and D). Compared with control Group D (mean relative abundance of 3.07%), Firmicutes was more abundant in acute cholecystitis Groups A, B and C (mean relative abundance of 18.70%, 7.72% and 24.70%, respectively). In contrast, Chloroflexi was more abundant in Group D (1.69%) than in patient Groups A, B and C (less than 0.01%). Significantly, there was an increasing trend in the abundance of Actinobacteriota, Fusobacteriota and Verrucomicrobiota in each A, B and C patient group compared to those in Group D.

At the genus level (Fig. 1b), Burkholderia was significantly abundant in the bile samples of the control group (mean relative abundance of 41.24%) compared with those of the acute cholecystitis Groups A, B and C (mean relative abundance of 13.19%, 11.08% and 1.09%, respectively). In contrast, Escherichia and Aeromonas were more abundant in acute cholecystitis Groups A, B and C (20.10%, 49.14% and 33.44% for Escherichia and 15.17%, 14.51% and 6.92% for Aeromonas, respectively) than in the control group (11.73% for Escherichia and 0.01% for Aeromonas). In addition, the abundance of Klebsiella and Enterococcus were highest in Group A (12.7% and 12.92%, respectively), and Clostridium was highest in Group C (22.77%). All data showed significant differences in the bile microbiome between patients with acute and chronic cholecystitis.

Comparison Of The Bile Microbiome In The Four Groups

Principal coordinate analysis (PCoA) (weighted UniFrac distance) and principal component analysis (PCA) were employed to study the differences among the four groups of bile specimens (Fig. 2a, b). We noticed that microbial communities obtained from the bile of patients with chronic cholecystitis controls (Group D) clustered separately from those obtained from the bile of acute cholecystitis patients (Group A, B and C). However, there were no significant differences among acute cholecystitis patients in Groups A, B and C.

The operational taxonomic unit (OTU)-based microbial diversity was also estimated, and we visualized the similarities among the samples. Venn diagrams showed that 37 OTUs overlapped in all groups, while 100 OTUs appeared in the control group and Group A, 90 OTUs appeared in the control group and Group B, and 101 OTUs overlapped in the control group and Group C (Fig. 2). 2c). In addition, the exclusive OTUs of the control, Groups A, B and C were 542, 314, 530 and 260, respectively. The total OTUs were 713, 592, 836 and 498, respectively. This result showed that the biliary microbiota significantly differed among the four groups at the genus level, indicating a correlation between the microbiome and biliary tract diseases.

Differential Bacteria In Biliary Microbiota (Acute Cholecystitis Patients Versus Chronic Cholecystitis Controls)

The composition of the biliary microbiota significantly differed between acute cholecystitis patients (Groups A, B and C) and chronic cholecystitis controls (Group D). At the genus level, we observed that Burkholderia, Bradyrhizobium, Phreatobacter and Comamonas levels were significantly higher in chronic cholecystitis controls than in acute cholecystitis patients (Fig. 3). Similarly, the family levels of Burkholderiaceae, Bradyrhizobiaceae, Rhizobiales and Lactobacillaceae, the order levels of Burkholderiales and Rhizobiales, and the class levels of Alphaproteobacteria were significantly higher in chronic cholecystitis controls versus acute cholecystitis patients (Fig. 3).

With high-throughput sequencing and bioinformatics-based analysis, we extracted genomic information from the biliary flora and characterized the diversity of microbial community. The human faecal microbiome has been extensively studied as noninvasive biological samples are easily available. However, other human microbial sites have been less studied. Recent studies have shown that the natural microbiome also exists in the body fluids of all kinds of organisms, such as human milk, blood and bile [7, 8].

Recently, several studies have used large-scale sequencing to analyse the biliary microbiota in people with bile duct stones. However, it is difficult to establish a link between the microbiome and acute/chronic cholecystitis, and to the best of our knowledge, differences in the microbiome between patients with acute and chronic cholecystitis have not been reported. Our results show that the microbial diversity of bile samples from patients with acute cholecystitis is reduced compared to bile samples from chronic cholecystitis patients. Ye et al. [9] reported similar results when they observed the reduction of microbial diversity in patients with recurrent common bile duct stones (CBDS) compared to controls without CBDS. Shen et al. [10] found that the microbial diversity of faecal samples decreased in patients with gallstones compared to healthy controls. The bile microbiome of patients with acute cholecystitis deviated from a normal stable state, suggesting that acute attacks are closely related to biliary microbial environment.

There is an association between biliary microbiota dysbiosis and human diseases [11, 12]. In this work, we reported a reduction in bile microbiome biodiversity in patients with acute cholecystitis. From an ecological perspective, a drop in diversity can reduce ecosystem resilience, increasing the likelihood of ecosystem degradation [13]. At the genus level, we observed a significant increase in Escherichia in the biliary tract of patients with acute cholecystitis. To our knowledge, E. coli is common in the bile of patients with cholelithiasis [14,15] and is also highly expressed in the bile of patients with recurrent CBDS [9]. The probable existence of virulence genes in Escherichia and their resistance to multiple antibiotics may be responsible for their persistence in the biliary microenvironment [16]. In addition, we detected a significant increase in Burkholderia–Caballeronia–Paraburkholderia, which is a member of the Betaproteobacteria family, in the bile sample of the control group. According to previous literature, it was abundant in the gut microbiota, which could improve the diagnostic effect in patients with cholangiocarcinoma [17] and may be involved in the metabolic pathways of oesophageal cancer [18]. Furthermore, the decreased abundance of Burkholderia–Caballeronia–Paraburkholderia is a potential diagnostic signal for dysbiosis and disease risk [17].

In this pilot study, patients with acute cholecystitis were categorized into three subgroups: those without associated diseases (Group A), as well as those with other associated diseases, such as common bile duct stones (Group B), cholecystolithiasis, common bile duct stones and acute pancreatitis (Group C). It is better for us to more deeply analyse the possible causes of acute episodes of biliary tract infection without the interference of other comorbidities. We found that different bacteria were aberrantly expressed in different subpopulations, such as Actinobacteriota, Fusobacteriota and Verrucomicrobiota, which were highly expressed in Groups A, B and C, respectively. However, in general, other associated diseases had little impact on the microbiota analysis of acute and chronic cholecystitis, as bile samples from patients with acute cholecystitis had less microbial diversity than those from chronic cholecystitis.

Our study has some limitations. First, the sample size is only 26 and more samples should be needed to assess the robustness of the observed changes in the bile microbiome in the future. Second, since bile samples cannot be obtained from healthy volunteers for ethical reasons, patients with chronic cholecystitis was selected as the control group. We cannot characterize the biliary microbiota dysbiosis between acute cholecystitis patients and healthy controls. Third, antibiotic resistance genes based on whole genome sequencing may provide important guidance for subsequent treatment. Our future study will focus on this topic.

We characterized the biliary microbiota in acute cholecystitis patients with cholecystolithiasis (Group A) and other comorbidities (Groups B and C) to avoid complicated factors of associated diseases and compared them with chronic cholecystitis (Group D). We report significant changes in the bile microbial community. We believe our work is important in revealing the relationship between the bile microbiome and human health. The role of the microbiome in the pathogenesis of acute cholecystitis with bile duct stones needs to be further studies, and more relevant factors should be considered to explore the microbial ecology of the biliary tract in the future.

Patient selection and sample collection

The study was performed at The First People’s Hospital of Taicang, Taicang Affiliated Hospital of Soochow University. Between 2021 and 2022, biliary fluid was collected from 26 patients during percutaneous transhepatic cholangial drainage (PTCD) or percutaneous transhepatic gallbladder drainage (PTGD). All samples were immediately stored at -80°C before subsequent processing. The diagnosis was established based on preoperative imaging evidence by using colour Doppler ultrasonography, computed tomography (CT) and magnetic resonance cholangiopancreatography (MRCP). Patients were divided into four groups according to their clinical characteristics. Group A, acute cholecystitis and cholecystolithiasis; Group B, choledocholithiasis, acute cholecystitis and cholecystolithiasis; Group C: acute pancreatitis, choledocholithiasis, acute cholecystitis and cholecystolithiasis; Group D: chronic cholecystitis and cholecystolithiasis. This research work was approved by the Research Ethics Committee of Taicang The First People’s Hospital. All patients gave written informed consent for participation in this study.

Specimen Processing And Sequencing

DNA was extracted from bile samples using CTAB according to the manufacturer's protocol. The hypervariable V3-V4 region of the 16S rRNA gene was amplified in a total volume of 25 µL reaction mixture containing 25 ng of template DNA, 2.5 µL of each primer, 12.5 µL PCR premixture and PCR-grade water to adjust the volume with modified versions of primers 341F and 805R. PCR was conducted under the following conditions: denaturation at 98°C for 30 seconds; 32 cycles of denaturation at 98°C for 10 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 45 seconds; and then a final extension at 72°C for 10 minutes. The PCR products were purified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA). The amplicon pools were prepared for sequencing, and the Agilent 2100 Bioanalyzer (Agilent, USA) and the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA) were used to assess the size and quantity of the amplicon library, respectively. The libraries were sequenced on a NovaSeq PE250 platform by LC-Bio Technology Co., Ltd, Hang Zhou, Zhejiang Province, China.

Bioinformatic Analysis

Under the specific filtering conditions, quality filtering of the raw reads was performed to obtain high-quality clean tags according to fqtrim (v0.94). After dereplication using DADA2 software, we obtained a feature table and sequence. Alpha diversity and beta diversity were calculated using random normalization to the same sequences. Then, feature abundance was normalized using the relative abundance of each sample according to the SILVA (release 138) classifier. Beta diversity was calculated by QIIME2, and the graphs were drawn using the R package. Sequence alignment was Blast, and the feature sequences were annotated with the SILVA database (Release 138.1) for each representative sequence. Other diagrams were also implemented by the R package (v3.5.2).

Statistical analysis

The discrimination analysis was performed using online packages (LC-Bio Technology Co., Ltd., Hangzhou, China). The beta diversity analysis of four groups was performed using PCA and PCoA based on weighted UniFrac distance. Venn Diagram. 2.8 was used to obtained Venn diagrams. Heatmap analysis was implemented for bacteria with significant differences between acute cholecystitis groups (Group A, B, C) and the control group (Group D).

Statistical analysis was performed using SPSS 23.0. The clinical data were verbalized as mean ± standard deviation. The Fisher exact test was exploited for the comparison of clinical data about gender and previous hypertension, and one-way analysis variance (ANOVA) test was exploited for the comparison of all other clinical data between four groups in Table 1, P < 0.05 was considered significant.

Ethics approval and consent to participate

All methods were carried out in accordance with relevant guidelines and regulations. Ethical approval was obtained from the Ethical Review Committee, and written informed consent was provided by all patients before sampling.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests

Not applicable

Funding

Science and Technology Project of Taicang City, TC2020JCYL27

Authors' contributions

Danfeng Shen, Jingjing Xu, Peng Chang, Haibin Xu and Ye Shen wrote the main manuscript text and Zhiqiang Zhu prepared table 1 and figures 1-3. All authors reviewed the manuscript.

Acknowledgements

Not applicable

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No competing interests reported.

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Characterization of biliary microbiota dysbiosis in acute cholecystitis: A reduction in the biodiversity of the bile microbiome

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Characteristics of patients

The Distribution And Diversity Of The Bile Microbiome

Comparison Of The Bile Microbiome In The Four Groups

Differential Bacteria In Biliary Microbiota (Acute Cholecystitis Patients Versus Chronic Cholecystitis Controls)

Discussion

Conclusion

Methods

Patient selection and sample collection

Specimen Processing And Sequencing

Bioinformatic Analysis

Statistical analysis

Declarations

References

Additional Declarations

Status:

Version 1