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Application of next-generation sequencing to study ascitic microbiome in cirrhotic patients with or without
Ye Feng1, Chyi-Liang Chen2, Tsung-Hsing Chen3
1Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China; Institute for Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China.
Background:
Spontaneous bacterial peritonitis is an important cause of morbidity and mortality in cirrhotic patients with ascites. The key step in the pathogenesis of spontaneous bacterial peritonitis is bacterial translocation from intestinal lumen to mesenteric lymph nodes, and from there to the systemic circulation and ascitic fluid. We aimed to study the ascitic microbiota of cirrhotic patients with or without spontaneous bacterial peritonitis.
Methods:
Both the 16S polymerase chain reaction approach and the whole genome shotgun approach were adopted for the next-generation sequencing technology. We compared the results derived from the two methods.
Results:
The bacterial culture-negative ascites in cirrhotic patients, which even failed for amplification of 16S ribosomal DNA, were found to contain much less bacterial DNA than the culture-positive ones, indicating that the paucity of bacteria, instead of the difficulty of bacterial culture, was possibly the main reason for negative culture result of the ascites. Escherichia coli was the predominant species in all samples, and the bacteria of low abundance were also identified by the next-generation sequencing technology.
Conclusion:
Whole genome shotgun-based next-generation sequencing is an appropriate method for depicting the microbiome of ascites or of other specimens with a low abundance of bacterial DNA.
Insights
Spontaneous bacterial peritonitis in cirrhosis patients is linked to gut bacteria. Whole genome shotgun sequencing effectively identifies low-abundance bacteria in ascites fluid, improving diagnosis.
Area of Science:
- Gastroenterology
- Microbiology
- Genomics
Background:
- Spontaneous bacterial peritonitis (SBP) significantly increases morbidity and mortality in cirrhotic patients with ascites.
- Bacterial translocation from the gut lumen is a critical factor in SBP pathogenesis.
- Understanding ascites microbiota is crucial for managing SBP in cirrhosis.
Purpose of the Study:
- To investigate the microbial composition of ascites fluid in cirrhotic patients.
- To compare the effectiveness of different next-generation sequencing methods for analyzing ascites microbiota.
- To determine the role of bacterial load in culture-negative ascites.
Main Methods:
- Utilized 16S polymerase chain reaction (PCR) and whole genome shotgun (WGS) approaches for next-generation sequencing.
- Compared the diagnostic yield of 16S PCR and WGS for identifying bacteria in ascites.
- Analyzed bacterial DNA content in both culture-positive and culture-negative ascites samples.
Main Results:
- Culture-negative ascites, even those negative for 16S ribosomal DNA amplification, showed significantly lower bacterial DNA levels.
- Paucity of bacteria, not culture difficulty, is likely the primary reason for negative culture results in ascites.
- Next-generation sequencing identified low-abundance bacteria, with Escherichia coli being the predominant species across samples.
Conclusions:
- Whole genome shotgun sequencing is a suitable method for characterizing the microbiome of ascites.
- WGS-based next-generation sequencing is effective for analyzing specimens with low bacterial DNA abundance.
- This approach enhances the understanding of ascites microbiome in cirrhotic patients, aiding SBP diagnosis.
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