Cholecystitis is an inflammatory disease of the digestive system, and exosomes are commonly studied as inflammatory mediators to investigate the pathogenesis of allergic diseases [33, 34]. Currently, most studies have focused on the bile and gut microbiota in healthy individuals and cholecystitis patients, with limited research on the microbial composition within exosomes of patients with acute and chronic cholecystitis. In this study, we analyzed the microbial communities in serum exosomes of patients with acute and chronic cholecystitis. Our findings revealed distinct microbial community characteristics in patients with acute cholecystitis as compared to those with chronic cholecystitis. Specifically, we observed reduced microbial diversity and specific changes in microbial abundance within the serum exosomes of patients with acute cholecystitis. Furthermore, diverse microbial metabolic pathways were also identified between the acute and chronic groups.

This study initially conducted α-diversity and β-diversity analyses on two groups of patients. Alpha-diversity serves as an indicator to measure species diversity within an ecosystem or community, while β-diversity metrics reveal relative differences in microbial communities between patients with acute and chronic cholecystitis [35]. Mora-Guzmán I et al. and Mintz D et al. found that there are differences in microbial profiles in the bile of cholecystitis patients compared to healthy individuals, with lower richness and diversity in patient microbiota [36, 37]. Similarly, in our study, we observed lower bacterial diversity and richness in the Acute group compared to the Chronic group, accompanied by significant alterations in microbial composition between the two groups. These findings indicated distinct microbial community compositions within exosomes in the blood of patients with acute and chronic cholecystitis.

Studies have shown the presence of diverse microbial communities in the biliary tract, where microbial dysbiosis might contribute to the formation of gallstones [17]. Previous research indicated that the Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria dominate the bile bacterial composition [38,39,40]. In our study, we observed similar results, particularly noting a significant increase in Proteobacteria in the exosomes of patients with acute cholecystitis. Proteobacteria are highly abundant in the biliary tract [17], constituting approximately 1% of the healthy human gut microbiota [41]. However, in our study, Proteobacteria accounted for over 50% of both acute and chronic cholecystitis exosomes, suggesting an alteration in the microbial composition of cholecystitis patients. An investigation into bile microbiota identified Gram-negative bacteria such as Escherichia coli, Enterobacteriaceae, Pseudomonas aeruginosa, and Klebsiella as predominant pathogenic taxa in cholecystitis patients [42]. Notably, Escherichia coli, Enterobacteriaceae, Pseudomonas aeruginosa, and Klebsiella are members of the Proteobacteria phylum [43]. Among these, Enterobacteriaceae is a major harmful member of the human microbiota [44], associated with various diseases including acute pelvic inflammatory disease, necrotizing enterocolitis, and urinary tract infections [45,46,47]. Liu et al. [48] demonstrated that isolates of Enterobacteriaceae from patients with acute cholecystitis could induce inflammation and morphological changes in animal gallbladders. Additionally, Enterobacteriaceae can impair the intestinal barrier, leading to the translocation of intestinal microbiota to the host’s bloodstream and biliary system [45]. These findings indicate that bacteria from the Enterobacteriaceae family might be the primary pathogenic microorganisms in acute cholecystitis. Sarah J Powers et al. [49] detected Pseudomonas aeruginosa in gallbladder tissue samples from common marmosets (Callithrix jacchus) with cholecystitis. In a study on the microbiota of gallstones and bile, Pseudomonas aeruginosa exhibited the highest glucuronic acid enzyme activity and produced higher concentrations of phospholipase A2, promoting gallstone formation and, consequently, acute cholecystitis [50]. These findings suggest that Pseudomonas aeruginosa might serve as a diagnostic biomarker for acute cholecystitis. In our study, we observed significant differences in the microbial composition within exosome in the blood of patients with acute and chronic cholecystitis. Further analysis revealed significant disparities in the Proteobacteria phylum between the two patient groups. Combined with previous research results, we speculate that varying levels of Proteobacteria could potentially serve as novel biomarkers for diagnosing acute and chronic cholecystitis. Additionally, in our study, we observed a significant decrease in the abundance of Firmicutes, Bacteroidetes, and Actinobacteria phyla in the exosome of patients with acute cholecystitis, indicating potential alterations in these microbial communities in both acute and chronic cholecystitis patients. However, the specific mechanisms underlying these changes require further in-depth investigation.

The microbial community can influence various host metabolic reactions, disease progression, and signaling pathways, thereby regulating growth processes and the onset of chronic diseases [24, 51]. In this study, functional metabolism and physiological pathways affected by acute and chronic cholecystitis were predicted through Tax4Fun analysis. Our findings revealed differential enrichment in 36 pathways between the two patient groups, including amino acid metabolism, carbohydrate metabolism, membrane transport, and signal transduction. Amino acid metabolism encompasses several amino acids. For instance, histidine, as an anti-inflammatory amino acid, can reduce the levels of reactive oxygen species in the body. Studies have shown that histidine expression is decreased in patients with chronic cholecystitis, thereby promoting inflammatory responses in cholecystitis [52]. Moreover, decreased histidine concentrations have been observed in inflammatory chronic kidney diseases [53]. Our study further suggested that the microbial composition in patients with acute and chronic cholecystitis might impact these metabolic pathways.

Currently, the diagnosis of cholecystitis primarily relies on imaging examinations such as ultrasound, CT, and HIDA scans, supplemented by comprehensive evaluation of detailed medical history, thorough clinical examinations, and laboratory test results [54]. These diagnostic modalities have been demonstrated to possess good accuracy in clinical practice. This study revealed significant differences in the microbial communities within serum exosomes of patients with acute and chronic cholecystitis. However, due to the time-consuming nature of microbial analysis and the imperative for immediate surgical intervention once acute cholecystitis is diagnosed, the clinical application of microbial analysis remains supplementary. Gallstones are considered one of the predisposing factors for biliary tract infections. Meanwhile, there have been significant changes in the distribution and resistance of pathogenic microorganisms causing biliary tract infections [55]. In the early stages of acute cholecystitis, bile is typically sterile and becomes infected as a secondary event [56]. Previous studies have indicated that approximately 9-42% of patients undergoing elective laparoscopic cholecystectomy develop bile infections, with the incidence of positive bile cultures in patients with acute cholecystitis rising to 35–65% [56], and to 44% in patients with chronic cholecystitis [55]. For patients with moderate to severe acute cholecystitis, early pathogen eradication based on antibiotics is crucial in limiting systemic sepsis and local inflammation following cholecystectomy [57]. Appropriate initial antibiotic therapy should not be delayed while waiting for culture test results, as this delay may increase the mortality rate of patients with biliary tract infections [43]. Hence, appropriate antibiotic therapy should commence immediately following diagnosis [38]. Gram-negative bacterial strains constitute the major pathogenic microbial population in cholecystitis, with typical gram-negative bacteria (such as Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) belonging to the Proteobacteria family [42]. In this study, we found that Proteobacteria accounted for over 50% of the exosomal microbiota in patients with both acute and chronic cholecystitis. It has been shown that Gram-negative bacterial strains have low susceptibility to cephalosporins, quinolones, and ampicillin [42]. Therefore, alternative antibiotics may need to be considered when selecting antibiotic therapy. These findings suggest that microbial analysis could provide better guidance for treatment [42].

However, this study has certain limitations. Firstly, we only observed changes in the microbial composition of serum exosomes in patients with acute and chronic cholecystitis in this study. However, existing research suggests that the gut microbiota might also be associated with gallbladder diseases. Secondly, the composition of microbiota is influenced by various factors, but this study did not assess the impact of other factors on microbial changes, such as age, gender, diet, and lifestyle. Additionally, once clinically diagnosed with acute cholecystitis, patients require early surgical treatment, while microbial composition analysis may require more time. Therefore, there is a need for further optimization of the workflow for microbiota analysis in the future. Despite these limitations, the findings of this study offer a novel insight into the role of microbial composition in acute and chronic cholecystitis, providing a new approach to the diagnosis of these conditions. However, the conclusions of this study are currently applicable only at the laboratory level. Future research should be conducted on a larger scale to explore the role of microbiota in acute and chronic cholecystitis and to optimize the technology for wider clinical application in the future.