The advent of the Omics Sciences, based on Genomics, Transcriptomics, Proteomics, and Metabolomics, enabled the study of the human microbiota from another perspective, leading to the discovery of a connection between microbiota and the health of the host and identifying the characteristics of the microbiome that can contribute to diseases (Marchesi and Ravel, 2015).

The intestinal microbiota is a system composed of microorganisms that settle in the gastrointestinal tract and preserve physiological and metabolic well-being. Particular attention in recent years has been paid to the study of bacterial colonization of the gastrointestinal tract. It has been demonstrated that some bacteria are associated with the development of cancer disease (de Almeida et al., 2018; Maekawa et al., 2018; Mendez et al., 2020; Nokhandani et al., 2021; Saab et al., 2021). A multi-omics approach may help elucidate the gut microbiota activity and show that selected intestinal bacterial communities could play a role in developing chronic inflammatory disorders and neoplasms. In this regard, the composition of the biliary microbiota has attracted the interest of research aimed at clarifying the role of specific players in stimulating chronic inflammation and producing carcinogenic metabolites in the biliary tract, gallbladder, and pancreas (Molinero et al., 2019; Mendez et al., 2020; Visekruna and Luu, 2021).

The gallbladder may be affected by numerous disorders ranging from infectious processes to chronic inflammatory diseases and cancer (Apstein and Carey, 1996; Ebata et al., 2016). Since the gallbladder is a small pouch that collects bile produced by the liver, a change in bile composition, especially in cholesterol, is responsible for cholelithiasis. In 1967, Drs. Scott and Khan, two scientists of the Royal Free Hospital, London, United Kingdom (Scott and Khan, 1967), published a seminal work that showed the presence of bacteria in the bile of subjects undergoing uncomplicated cholecystectomy. Subsequently, about 20 years later, Wells and others highlighted the concept that bile is not genuinely sterile. Its colonization may represent a risk factor for postoperative sepsis concerning patients undergoing biliary tract surgery (Wells et al., 1989). The biliary system is in continuous contact with the gut bacteria. In fact, microorganisms ascend from the intestinal tract to the gallbladder, and gut microbial products influence bile salt metabolism (Small, 2003; Ebata et al., 2016). Currently, the term human bile microbiota refers to the microbial ecosystem of the gallbladder (Ebata et al., 2016). Studies suggest that the microbiota may substantially influence the outcome of gallbladder disorders (van Velkinburgh and Gunn, 1999; Prouty and Gunn, 2000; Bina and Mekalanos, 2001; Sleator et al., 2005; Torres et al., 2007; Crawford et al., 2008). Bile microbiota composition may show Salmonella spp. and other enteric commensals and pathogens, such as Vibrio cholera, Campylobacter jejuni, Escherichia coli, and Listeria monocytogenes. The latter have also demonstrated their ability to survive in the biliary environment, even though bile salts display antimicrobial properties (Begley et al., 2005; Hardy et al., 2006; Hung et al., 2006).

Variations within the human microbiota, such as an imbalance in bacterial composition, changes in bacterial metabolic activities, or changes in bacterial distribution, are defined as dysbiosis (Ebata et al., 2016). The term gallbladder dysbiosis has become the subject of revived attention in both human and experimental animals (Ebata et al., 2016; Wang et al., 2017; Gutierrez-Diaz et al., 2018; Xu et al., 2018). Changes in the microbial composition can cause a dire imbalance favoring pathogenic bacteria. They can lead to inflammation that contributes to different diseases, such as obesity, diabetes mellitus, virgola, multiple sclerosis, and cancer. Studies on bacterial bile composition have detected microorganisms with a possible molecular biologic role in chronic inflammation and carcinogenesis (Nath et al., 2010; Koshiol et al., 2016; Sharma et al., 2017). Investigations on the culture of bile samples collected by endoscopic retrograde cholangiopancreatography (ERCP) have identified a pattern of bacteria isolated in patients with diseases of the biliary tract, gallbladder, and pancreas impacting the survival of our patients affected with cancer (Di Carlo et al., 2018; Serra et al., 2018; Di Carlo et al., 2019; Sergi et al., 2019; Serra et al., 2021). On the other hand, other bile components, such as cholesterol and its derivates, have been investigated by biochemistry. This data involves microbial activities and cholesterol in the genesis of benign and malignant gallbladder disorders (Crawford et al., 2008; Nath et al., 2010; Phelan et al., 2017; Sergi et al., 2019; Kiss et al., 2020; Gruner and Mattner, 2021).

To overcome the drawbacks encountered in a culture-dependent approach, the microbiota study may exploit the progress of the most recent omics technologies, computational analytics, and deep neural network applications (Milani et al., 2021). Currently, the microbial composition of an organ or a niche can be explored by detecting the genetic material of microbes through the next-generation sequencing (NGS) of conserved microbial genes (microbiome analysis) using the metagenomic DNA extracted from the whole sample. Metagenomic DNA can also be used for Whole Genome Sequencing (WGS) to obtain data on the genetic background of the microorganisms present in the sample (Cho and Blaser, 2012; Liu et al., 2012; Armour et al., 2019). Furthermore, metagenomics allows us to investigate the metataxonomic and functional profiles through the shotgun approach (Milani et al., 2021). Metagenomics can quickly provide data on different microbial communities in healthy individuals and subjects suffering from various diseases (Armour et al., 2019; Xing et al., 2019; Little et al., 2020; Galluzzo et al., 2021; Saab et al., 2021). As for other disorders, the advances in sequencing technologies and computational methods are facilitating the investigation of the gallbladder microbiotas and serving in the diagnostic and therapeutical approaches to benign and malignant tumors (Hsing et al., 2007).

The method of metatranscriptomic provides a snapshot of gene expression in a given sample at a given time under specific conditions by capturing the total mRNA. However, it is technically an arduous study if it has to be thorough because RNA samples are more challenging to handle than DNA samples.

Environmental conditions can also influence the composition of microorganisms. For example, in the case of the gallbladder, variations in bile composition, especially bile salt and fats (i.e., cholesterol, fatty acids, and lecithin), may affect the microbiome (Small, 2003; Boyer and Soroka, 2021). Metatranscriptomics could help understand the interactions between the biliary components, especially the bile acids of the lipid component, and the microbial species that are part of the microbial community of the gallbladder (Botero et al., 2005; Carvalhais et al., 2012; Aguiar-Pulido et al., 2016; de Vos et al., 2022). Metatranscriptomics, metaproteomics, and metabolomics are powerful integrative approaches to metagenomics, shaping precision medicine, i.e., the medical model that recommends the customization of healthcare with medical decisions and therapies being tailored to a subgroup of patients. In addition, omics technologies expose microbial activity and interaction, enabling a better understanding of the interplay between the microbial community and the environment (Aw and Fukuda, 2015; Ye et al., 2016; Armour et al., 2019; Milani et al., 2021). However, these studies may struggle to recruit a large number of patients. Therefore, multicenter studies and the establishment of biorepositories are critical for these investigations (Sergi, 2022a).

This review focuses on describing the current state of gallbladder disorders with multiple approaches, including metagenomics, metatranscriptomics, and metabolomics. This review aims to summarize several studies to increase available knowledge of the connection between biliary microbiota and gallbladder disease.

Our review focuses on the role of omics’ technologies in identifying the changes in community biliary microbial composition or biliary dysbiosis (DeGruttola et al., 2016; Xing et al., 2019; Little et al., 2020; Saab et al., 2021) and biliary metabolites detected in the most common gallbladder disorders such as cholelithiasis, CC, GBC, and in patients with diabetes who underwent cholecystectomy (Ferslew et al., 2015; Shen et al., 2015; Stepien et al., 2016; Kujiraoka et al., 2017; Sharma et al., 2017; Kose et al., 2018; Näsström et al., 2018; Mohajeri et al., 2019; Kose et al., 2020). Animal-based studies showed a native biliary microbiota that changes in the course of gallbladder disease (Jimenez et al., 2014; Xing et al., 2019). It is not easy to study bile microbiota in healthy subjects for ethical reasons. Still, Molinero et al. collected bile samples from liver donors with no record of biliary or hepatic disorders and, via shotgun metagenomic analyses, corroborated the 16S rRNA gene data, detecting the existence of three main phyla - Actinobacteria, Bacteroidetes, Firmicutes-associated with some genera of the alpha division of Proteobacteria (Molinero et al., 2019). Moreover, these authors found in patients with cholelithiasis that Bacteroides, Escherichia, and Shigella were more abundant than in healthy subjects where the genera Sphingomonas (Proteobacteria) was abundant. Even though some enteric pathogens have a unique ability to resist the bactericidal effects of bile (Sistrunk et al., 2016), other bacteria can survive in bile thanks to bacterial gene encoding mechanisms of resistance to the biliary environment such as bile salt deconjugation and multidrug efflux pump proteins (Ruiz et al., 2013; Shen et al., 2015). Bile resistance-related genes, which could be crucial for bacterial survival, were identified by Shen et al. in patients with choledocholithiasis (Shen et al., 2015). These authors showed that in addition to the bacteria that make up the gut microbiota, other bacteria that colonize the oral cavity or upper respiratory tract could also become inhabitants of the biliary microbiota (Shen et al., 2015). This suggests that there is, indeed, a continuous change in the biliary microbiota.

The role of bacteria in gallstone formation is an old question (Cetta, 1991). Via omics technologies, bacterial genes associated with gallstones have been confirmed. Therefore, bacterial slime (i.e., glycocalyx), bacteria resistance in bile, and biofilm formation should play an essential role in gallstone formation (Shen et al., 2015; Kose et al., 2018; Molinero et al., 2019). It is well-known that extended periods of exposure to bile salts lead to biofilm formation among the enteric pathogens within the Enterobacteriaceae family. This concerns well-researched bacteria such as Salmonella and the Shigella species and other emerging pathogens including E. coli, K. pneumoniae, Enterococcus spp., and Clostridium spp. (Begley et al., 2005; Hardy et al., 2006; Hung et al., 2006; Ye et al., 2016; Nickerson et al., 2017; Kose et al., 2018; Nickerson and Faherty, 2018; Mullish and Allegretti, 2021).

Biofilm formation and anaerobic energy metabolism are the potential microbial mechanisms of bacteria involved in gallstone formation (Kose et al., 2018). Kose et al. have studied the bacterial composition of stones and found enterobacteria such as Klebsiellaspp., Escherichiaspp., Enterococcusspp., Salmonellaspp., and Enterobacter spp. played a role in the formation of gallstones (Kose et al., 2018).

Carcinogenesis due to chronic inflammation caused by pathogen infections has been recognized as one of the carcinogenesis patterns in humans (Nokhandani et al., 2021). Song et al. performed metagenomic shotgun sequencing on mucosal biopsy samples collected in patients with CC and CL (i.e., cholecystitis accompanied by biliary stones) and GBC (Song et al., 2020). Song et al. showed that Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria were detected in both groups. The authors analyzed the alpha diversity of the richness and uniformity of the species of the two groups during the development of GBC. They found differences in the composition of the biliary microbial community. Peptostreptococcus stomatis, Fusobacterium mortiferum, and Enterococcus faecium were present in more significant numbers in tissue samples of patients with GBC than those without neoplastic progression.

Moreover, to demonstrate a potential carcinogenic role in the different microbial compositions, these authors focused on the carbohydrate composition during the development of GBC. Bacterial bile detection via metagenomics in patients with GBC was studied in South America (Tsuchiya et al., 2018). Tsuchiya et al. reported a predominance of Fusobacterium nucleatum, E. coli, and Enterobacter spp. in Bolivian and Chilean patients with GBC (Tsuchiya et al., 2018). Fusobacterium nucleatum, a common component of the oral bacterial community, has recently emerged as a compelling candidate for causing human diseases given its prevalence in gut inflammation and cancer (Engevik et al., 2021).

In this review, we included the gut dysbiosis analysis in T2DM patients. They did not show a difference in Fusobacterium levels between the new-onset T2DM and long-termT2DM subjects, indicating that the duration of diabetes may not have a significant effect on Fusobacterium. On the other hand, the cholecystectomy significantly increased the amount of the Fusobacterium in long-term T2DM patients, which was reported to cause opportunistic infections or aggravate high-fat diet-induced metabolic disorders (Wei et al., 2021). Nevertheless, further studies, including a large sample and multicenter studies, may be needed to discriminate between physiology and pathology (Brennan and Garrett, 2019; Brennan et al., 2021).

Several scientific contributions mentioned the role of Enterococcus spp. in gallbladder disease (Shen et al., 2015; Kose et al., 2018; Tsuchiya et al., 2018; Molinero et al., 2019; Song et al., 2020). The role of Enterococcus spp. is under discussion because it has been detected in benign and malignant disorders of the gallbladder (Maekawa et al., 2018; Song et al., 2020). The disadvantage of E. faecium in bile and gut microbe composition is debated. The dangerous role of this bacterium has been proposed due to its ability to cause genomic DNA instability. There is also evidence that this microorganism predisposes the host to mutations toward carcinogenesis (de Almeida et al., 2018; Maekawa et al., 2018). In other studies, the multidrug-resistant Gram-negative pathogens and Enterococcus spp. were prevalent in biliary and pancreatic disorders (Di Carlo et al., 2018; Serra et al., 2018; Di Carlo et al., 2019; Sergi et al., 2019; Serra et al., 2021).

Metabolomics has focused on metabolites in serum or other body fluids, allowing the early diagnosis of gallbladder diseases. Consequently, it can help clinicians manage the gallbladder’s acute and chronic inflammatory processes. Näsström et al. identified metabolites that distinguish between infection and colonization due to Salmonella typhi or S. paratyphi A (Näsström et al., 2018). Sharma et al. analyzed eleven selected metabolites in patients with CC versus controls and showed that the metabolite dosage could identify the inflammation process and help to reduce its progress (Sharma et al., 2017). Similarly, Ranjan and Sinha stress that the serum dosage of specific metabolites in gallbladder inflammatory processes could help predict the risk of tumor evolution (Ranjan and Sinha, 2019a). Metabolomics might help us better understand the role of bile microbiota in cancer pathogenesis. In particular, Stepien et al. analyzed nutrition and specific AA in subjects with hepatobiliary cancer. Despite the authors’ efforts, it was challenging to affirm a correlation between the studied metabolites and cancer patients’ food habits. In conclusion, they used the term “perturbation” about the circulating AA metabolites dosage levels in HCC (Stepien et al., 2016). As indicated above, Zhang et al. underlined the difficulties of making an early diagnosis of biliary and gallbladder cancer compared to liver cancer because this organ is a particular metabolically active organ with constant exposure to circulating metabolites (Zhang et al., 2021). Associations between diversity, the taxonomic profile of bile microbiota, and bile BA levels were evidenced in patients with cholelithiasis (Petrov et al., 2020). Petrov et al. showed a correlation between primary biliary acids and bile microbiota composition (Petrov et al., 2020). At the same time, fecal microbiota dysbiosis was primarily associated with secondary BAs. All studies analyzing BAs focus on the harmful role of secondary BAs and their metabolites. The most significant interaction with the gut microbial community is probably converting primary BAs to secondary BAs (Ferslew et al., 2015; Mohajeri et al., 2019; Petrov et al., 2020; Sinha et al., 2020). Microorganisms in the bile are relevant only if we show that they activate the metabolic enzymatic process, modulating the primary and secondary BAs circuit.

Overall, the examination of the literature to write this review was not straightforward because the various methodologies were often heterogeneous. However, the articles in this review provide an array of research and a platform concerning the kind of human sample (serum, tissue, urine, e.g.), the heterogeneity of gallbladder disorders (cholelithiasis, cholecystitis, cancer, and patients with or without cholecystectomy), and the heterogeneity of analyzed elements (e.g., the composition of microbiota, BAs, and biliary metabolites), which may be useful for more congruent studies in the future. The human samples analyzed ranged from bile blood to non-blood tissue or other sites sampled using oral cavity or rectal swabs (Shen et al., 2015; Kujiraoka et al., 2017; Kose et al., 2018; Molinero et al., 2019; Kose et al., 2020; Lee et al., 2020; Song et al., 2020). It is noteworthy that metatranscriptomic studies in patients with gallbladder disease are missing. The lack of such studies is probably due to technical drawbacks in handling RNA molecules. In fact, mRNA is notoriously unstable, and sample integrity can be compromised before sequencing. In addition, distinguishing between host and microbial RNA can be challenging, although enrichment kits are now readily available on the market (Leimena et al., 2013; Mukherjee et al., 2017). Finally, much of the collected RNA comes from ribosomal RNA. Its dominant abundance can drastically reduce mRNA coverage (Mukherjee et al., 2017). Most metatranscriptomic studies focused on gut dysbiosis and the interplay of food intake and gut microbiota (Islam et al., 2011; Martinez et al., 2013; Rath et al., 2018; Franzosa et al., 2019). On this issue, metatranscriptomic studies conducted on laboratory animals such as mice or human fecal samples have recently shown that the harmful action of secondary BAs derives from primary BAs in the genesis of intestinal inflammatory processes and pancreatic cancer (Hildebrandt et al., 2009; Islam et al., 2011; Martinez et al., 2013; Zhang et al., 2016; Just et al., 2018). In addition, metatranscriptomic studies focused on RNA sequencing (RNA seq) also allow functional changes to be discovered to decipher what can affect antibiotic resistance gene expression after exposure to antibiotics. They can also study changes in the virulence factors of specific pathogens and epigenomic studies of some neoplastic conditions (Sharma et al., 2010; Korry et al., 2020; Auld et al., 2022).

In our systematic review, no article found Candida spp. in biliary dysbiosis. However, other authors have reported Candida spp. in biliary tract disorders, and Candida albicans is documented in the gut microbiome and gut dysbiosis (Rodolico et al., 2017; Gutierrez et al., 2020).

Current knowledge on the composition of the microbiota, its modifications concerning various physiological and pathological conditions, and the numerous mechanisms by which it can interact with the host have progressed rapidly in recent years. This aspect is due to the biotechnology pace over the last couple of years. The contribution of “omics” sciences, opening new perspectives on the role of the microbiota in the development of various systems and on the pathogenesis of many morbid conditions, has been remarkable in all fields of medicine. However, in many cases, the causal link between alterations of the bile microbiota and pathology remains to be consolidated, and the mechanisms that underlie it as it remains to strengthen the research field on the role of bile dysbiosis in rare conditions, such as gallbladder diseases.

Translating a newly developed methodology from the research laboratory to the clinical laboratory must consider analytical validity, clinical advantageousness, and financial responsibility. Therefore, using this approach in the routine clinical context and the implementation in laboratory information systems are not yet recommended (Sergi, 2022b). However, we need to stay tuned because it could be beneficial for identifying genera and species in the nearest future after validation and certification according to regulatory agencies, such as the College of American Pathologists.

Overall, the recent development of omics approaches can globally quantify cellular changes at different molecular levels by combining data from multiple omics methodologies. These include the study of the genome, transcriptome, proteome, interactome, epigenome, metabolome, lipidome, and microbiome (Muller et al., 2013; Segata et al., 2013; Heintz-Buschart et al., 2016a; Heintz-Buschart et al., 2016b). This approach is called multi-omics and, in comparison to single omics, gathers information from multiple “omes” to better understand complex diseases (Heintz-Buschart et al., 2016a; Heintz-Buschart et al., 2016b; Kaysen et al., 2017; Lloyd-Price et al., 2019; Mendez et al., 2020). Future efforts are needed to make these new high-cost technologies available in treatment centers. The authors hope that this review will stimulate research and create a network of collaboration between research centers focusing on animal models, implementing biorepositories, and treatment centers for gallbladder diseases.