Gallstone disease (GSD) has been prevalent worldwide, especially in Western countries and China in the last decades (Fan et al., 2017; Scherber et al., 2017). Most of the gallstones in the gallbladder are the cholesterol type (approximately two-thirds), and the remaining are mainly pigment stones (Portincasa and Wang, 2012). It was previously suggested that a healthy human biliary system is sterile; however, several years ago, it was recognized that the gallbladder has a complex microbiota in non-pathological conditions (Verdier et al., 2015; Warburton et al., 2017). Despite the high worldwide prevalence of GSD, the role of the biliary microbiota in gallstone pathogenesis remains unclear. To date, knowledge about the composition of the biliary microbiota and its influence on the development of biliary disease is limited. Previous studies have linked biliary infection with gallstone development and indicated that bacteria may act as the nucleating factor initiating the formation of both pigment and cholesterol gallstones (Maki, 1966; Swidsinski and Lee, 2001; Stewart et al., 2002; Begley et al., 2005; Stewart et al., 2006). The presence of living bacteria in gallstones has been demonstrated using multiple methods (Swidsinski et al., 1995; Lee et al., 1999; Hazrah et al., 2004; Ramana Ramya et al., 2017). Moreover, several studies have shown that the alterations of bacteria in bile were linked to biliary diseases, such as cholelithiasis (Petrov et al., 2020), cholangiocarcinoma (Chen B. et al., 2019), common bile duct stones (Choe et al., 2021; Kim et al., 2021), biliary injury (Ying Liu et al., 2018), and primary sclerosing cholangitis (Pereira et al., 2017; Timur Liwinski et al., 2019). While works have reported that bacteria could be detected in gallstones, the bacterial spectrum remains unclear in cholelithiasis.

Fungi, as eukaryotes, are ancestrally and ecologically intrinsic to terrestrial life with multiple roles, extending to the regulobiotic. Notably, fungal species have been reported to colonize as commensals in many niches in healthy humans (Cui et al., 2013). Alterations within the fungi are associated with different diseases. The association between fungi and gastrointestinal disease has been well documented, with a special focus on candidiasis (Mukherjee et al., 2015). Studies performed in the past decade have demonstrated that fungi have a complex, multifaceted role in the gastrointestinal tract and actively and directly influence health and disease. A recent retrospective study found that Candida presented in bile at a low level (1.3%) by culture (Serra et al., 2021). However, the fungal organisms in bile and gallstone remain largely unclear. Fungi may have the potential to manipulate neighboring bacterial communities or vice versa. Therefore, interactions between the mycobiome and the bacteriome may also play a role in GSD.

In light of the above, we investigated both the fungal and bacterial profiles in paired bile and gallstone samples from patients with cholelithiasis using ITS and 16S rRNA gene high-throughput sequencing. To the best of our knowledge, this is the first study to uncover the fungal spectrum and its interaction with bacteria in both bile and gallstone. Thus, the current study provides insights into mycobiome in GSD. Importantly, the bile origin of the gallstone microbiota and fungal–bacterial interactions might contribute to the stone formation of cholelithiasis.

Increasing evidence has demonstrated the importance of microbiota in the development of many diseases. GSD is a major type of gallbladder disease, which affects 10–20% of the global adult population (Grigor'eva and Romanova, 2020). Multikingdom gallbladder microbiome analysis by metagenomic sequencing provides a new opportunity to explore the gallbladder mycobiome landscape in GSD. Accordingly, we used metagenomic sequencing to show for the first time that the gallbladder gallstone mycobiome in GSD subjects is similar to paired bile mycobiome, characterized by the same diversity and richness and a few differential gallstone-related fungal species. However, the interkingdom correlations between the mycobiome and bacteriome and the intrakingdom correlations within them were marked as more gallstone-related correlations. In addition, we first found that archaea (such as DSEG) were significantly thriving in bile, indicating the association thereof with gallbladder microbiome dysbiosis in GSD. These data fill an important gap in our current understanding of the potential role of gallbladder microbiome profile in GSD.

Research on the bile and gallstone microbiota and their role in biliary disease is quite rare. To our knowledge, no previous studies have described changes in the diversity of the human gallbladder mycobiome in GSD, although the fungus Candida in bile was recently reported by Serra et al. based on the culture method (Serra et al., 2021). To identify consistent gallbladder fungal signatures for bile and gallstone, we first combined both sex and types of gallstones to generally observe the changes. Our data showed a significant difference in evenness between bile and gallstone but not in the Shannon diversity and richness. One explanation could be that the species distribution was more uniform in gallstone than in bile (Supplementary Figure S9), which may have led to a more significant difference in the evenness of the samples. In addition, our PCoA results also suggest different trends of mycobiome between bile and gallstone samples. However, the pseudo-F values obtained in the PCoA remained low (1.81), indicating that larger unknown factors may contribute to variations between the subjects.

In this exploratory study to identify more potential taxa that might be associated with bile and gallstone, we included fungal species with a relative abundance of >0.005%. As a result, we found markedly differential fungal species, including taxa with lower abundance in gallstone samples. To date, limited information is available about the role of these differential fungal species in GSD, especially for the taxa with very low abundance (such as C. sublineola). Further functional research on the fungal markers might help understand the role of the mycobiome in gallbladder diseases.

Ascomycota and Basidiomycota constitute the major phyla of the kingdom fungi in the gallbladder. It was previously revealed that the Ascomycota:Basidiomycota ratio in the gut could represent a fungal dysbiosis index to differentiate diseases (Sokol et al., 2017; Cao et al., 2021). We found that it was higher in gallstone than in bile, implying a dysbiosis in bile, thus leading to stone formation in the gallbladder. Genera Aspergillus and Candida are two of the predominant members in the gallbladder mycobiome, some of which are widely reported to be pathogenic to humans and animals. One reason for the phenomena might be that members of Aspergillus can survive in both highly acidic and mildly basic conditions and at a wide range of temperatures in addition to producing secondary metabolites mycotoxins that are capable of causing disease and death in humans (Bennett and Klich, 2003). Importantly, Candida spp. are commonly associated with humans. The genus Candida comprises over 10 species that are known to induce opportunistic infections in humans, especially C. albicans (Yapar, 2014; Kapitan et al., 2019). In line with that, C. albicans was the most abundant species in both bile and gallstone.

Numerous studies have identified bile bacterial alterations in biliary diseases, but there is limited evidence to compare gallbladder microbiome differences between bile and gallstone. Our results are consistent with those of previous studies, demonstrating that phyla, Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, predominate in the bile bacteriome (Shen et al., 2015; Molinero et al., 2019). We found that GSD subjects were associated with significant alterations in bacterial composition between bile and gallstone samples. Notably, kingdom bacteria was significantly enriched in gallstone, demonstrating that bacteria could be a key factor contributing to stone formation. Proteobacteria which include many pathogenic species was enriched in gallstone, implying its potential role contributing to the formation of stone (Timur Liwinski et al., 2019). A recent study demonstrated that bile acid and cholesterol metabolism dysfunction contributed to cholesterol gallstone formation (Hu et al., 2022). We observed a significant increase in the cholesterol-reducing anaerobic genus E. coprostanoligenes in gallstone of patients with GSD (Ren et al., 1996; Le et al., 2022). One possible explanation could be that the gallstones obtained in this study were mostly cholesterol stones (85.3%). In addition, B. plebeius, which was found in the human gut and could become an opportunistic pathogen if it escapes the gut, was thriving in gallstone. ³ Clostridium sensu stricto 1, referring to the Clostridium cluster I, which has a potential causative capacity, was associated with gallstone (Lopetuso et al., 2013). In addition, Clostridiales which can produce trimethylamine (TMA), the precursor of trimethylamine N-oxide (TMAO), was a biomarker in gallstone (Wang et al., 2015). Consistent with this, Chen Y. et al. (2019) found that high TMAO levels were positively associated with the presence of gallstone disease in humans. These results indicate that Clostridiales may contribute to GSD by a mechanism based on its metabolite TMA/TMAO signaling.

Archaea are widespread microorganisms that live in a variety of natural and host-associated ecosystems (Baker et al., 2020). Several aquatic microbiota types such as DSEG and Rhodobacteraceae were dominant in bile, which might be because of bile’s liquid environment, indicating that the archaea are a major group that thrives in extreme environments. However, it should be noted that we assigned the Archaea according to data of 16S rRNA genes for bacteria and not the archaea-specific genes in this study. Further research on the archaeal markers and functions might help uncover the role of the archaeome in gallbladder diseases.

It should be noted that understanding the microbial interactions are of particular significance in human health and disease. Indeed, both positive and negative connections between bacteria and fungi in the gut have been reported (Richard and Sokol, 2019). Thus, we analyzed the bacterial and fungal interkingdom and intrakingdom interactions in gallbladder. In general, our data indicate a complex microbial ecologic community in gallstones and further demonstrate that the unique environment of gallstones promotes microbial interactions; in turn, the complex interactions may boost the formation and growth of gallstones in the gallbladder.

In addition, we found a reconstruction of microbial co-occurrence network in gallstone. First, the number of nodes increased in the network of gallstones. Second, the key modules changed within a network of gallstones. Third, the relationships altered between the taxa, and even in the same two taxa, such as the Muribaculum and Rothia (positive in bile, but negative in gallstone; Figures 5A,B; Supplementary Tables S3, S4). Interestingly, the ratio of positive and negative correlations seems to maintain a “relative balance” (positive/negative = 1.3) in the bacteria–bacteria network in both bile and gallstone. The ratio is close to 2 (1.8) in the networks of fungi–fungi and bacteria–fungi in bile, whereas it is close to 1 in these networks in gallstone. This suggests that (1) microbial interactions tend to affect homeostasis in the gallbladder, and that (2) fungal disorder in bile might play a vital role in GSD.

Importantly, in this study, we found that the bacterial communities are largely different, while the fungal colonies are similar between bile and gallstone in GSD. This interesting phenomenon could be explained by three points. First, the bacteria were indeed the most major members of microorganisms in bile while fungi were a smaller group than bacteria. Moreover, we detected a group of archaea accounting for a large proportion in bile which hardly were detected in gallstone in bacterial analysis (such as DSEG, Figure 2). Second, the number of negative interactions (e.g., resource competition) of bacteria–bacteria network was more than those of fungi–fungi in bile and gallstone (Table 2). Therefore, this might cause a large change of bacteria not fungi. Last, there are distinct types of environments between bile and gallstone—liquid for bile whereas solid for gallstone, as well as disparate environmental factors, mainly water, bile acids/salts, and inorganic ions for bile but cholesterol, calcium salts, and bilirubin for gallstone. Thus, there are more room and resource of bile than gallstone to survive for microbes so that bacteria altered largely for surviving. Altogether, it is implied that most fungi and a small part bacteria detected in current study might contribute to the formation of gallstone.

Hu et al. identified the gut bacterial Desulfovibrionales as a key taxon contributing to cholesterol gallstone formation (Hu et al., 2022). Furthermore, Desulfovibrionales are responsible for metabolizing dietary and host-derived sulfur-containing compounds (Peck et al., 2019). In line with this, bacterial sulfur metabolism was significantly active in gallstone according to function prediction analysis in this study. Lipopolysaccharide (LPS) is an endotoxin derived from the outer membrane of Gram-negative bacteria. LPS can cause many health problems, particularly in people who have thyroid and autoimmune thyroid disorders. Indeed, we found high thyroid hormone synthesis activity of bacteria in gallstones. In addition, LPS can cause an acute inflammatory response by triggering the release of a great number of inflammatory cytokines (Lopes, 2016). One possible interpretation is that the subjects recruited in this study have chronic cholecystitis complications.

Notably, this study had some limitations. First, because of ethical and social issues, we could not obtain healthy bile samples as the control group. Accordingly, collecting bile samples from the gallbladders of subjects without hepatobiliary disease from liver transplant donors for the control or reference bile microbiota group may be an alternative method (Molinero et al., 2019).

Second, we used the marker gene (16S/ITS) amplicon sequencing technology for microbial analysis, which did not provide the functional information of microbiota in the gallbladder. Meanwhile, we used the reference database approach for bacterial and fungal reads classification, which may allow the loss of a considerable part of novel microbiota owing to the absence of a reference data. As such, shotgun metagenomic sequencing is an alternative solution. In addition, an archaea-specific analysis method is further required.

Third, the gut microbiome community is also likely to be linked with the formation of gallstones (Hu et al., 2022; Zhao et al., 2022). Our study did not excavate the spectrum of the gut microbiome. Thus, the location difference of gut not only the feces need to be further explored. Moreover, the microbial metabolites (such as secondary bile acids) are now suggested to be very important in health and disease (de Vos et al., 2022; Hu et al., 2022). Metabonomics may be a useful approach to uncover the GSD-related microbial metabolites in the future.

Fourth, because we did not include patients with longitudinal follow-up and prognosis, we were unable to study the possibility of preventing GSD by evaluating the risk of GSD before its progression. Future studies should recruit individuals with prognoses to validate the value of predicting full-blown GSD by gallbladder microbial profiles.

Lastly, other external factors, including diet and lifestyle changes—which are known to affect the microbiome (David et al., 2014; Strasser et al., 2021)—were not assessed in this study. Nonetheless, these findings provided new insights into our understanding of the role of bacteriome and mycobiome alterations in bile and gallstone. Further larger-scale studies are needed to explore the confounding factors and cause–effect between microbiome and GSD.

This study demonstrated that large and complex bacteria and fungi inhabited in the gallbladder of patients with GSD, and gallstone was characterized by significantly altered bacterial taxonomic composition. In addition, the bacterial–bacterial, fungal–fungal and bacterial–fungal correlations were strengthened compared to bile in the gallbladder of patients with GSD. As such, mycobiome, bacteriome, and even archaeome and their interactions might contribute to the formation of gallstone. We believe that our first paired bile–gallstone microbiome study has taken an important step in this field.

The data presented in the study are deposited in the Genome Sequence Archive (https://ngdc.cncb.ac.cn/gsa) repository in the National Genomics Data Center (NGDC), accession numbers CRA008937 and CRA008920.

The studies involving human participants were reviewed and approved by the Institutional Ethics Review Board of the Third People’s Hospital of Chengdu. The patients/participants provided their written informed consent to participate in this study.

YL, TZ, and JH designed the study. YJL and QY obtained the grants. JH analyzed the data and obtained plots. JT, XZ, KY, AZ, and YL enrolled the patients and participated in the clinical part of the study. JH and TZ wrote the draft of the manuscript. All the authors participated in the discussion and reviewed the manuscript.

This work was supported by the National Natural Science Foundation of China (82170887), the Chengdu High-level Key Clinical Specialty Construction Project, and the Science and Technology Project of The Health Planning Committee of Sichuan Municipality (20PJ211).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.