Cholecystitis leads to gallbladder inflammation or even perforation, tissue death, gangrene, fibrosis, and shrinking of the gallbladder, which is a hospitalized disease with increasing medical and financial burden (Wadhwa et al., 2017; Wang et al., 2021). The cardinal symptoms were fever, pain, and nausea with increasing levels of clinical indicators, such as serum transaminases, alkaline phosphatase, and total bilirubin (Chisholm et al., 2019). Laparoscopic cholecystectomy was commonly applied for treating this disease (Loozen et al., 2018).

Although the pathogenicity in cholecystitis still needs further exploration, bacteria, as one of the important risk factors, were closely correlated with its poor operative outcomes (Liu et al., 2015; Yun and Seo, 2018). Moreover, positive bile culture rates from laparoscopic cholecystectomy patients ranged from 25.1 to 60.3%, suggesting the presence of bacteria in the cholecystitis patients’ bile (Nitzan et al., 2017; Yun and Seo, 2018). Due to the limitation of culture conditions, most bile bacterial isolates from clinical samples are aerobic bacteria especially Gram stain-negative Enterobacteriaceae, such as Escherichia coli, Klebsiella spp., Enterobacter spp., and less frequently anaerobic bacteria, including Bacteroides and Clostridium spp., as well as microaerophilic Helicobacter pylori (Nitzan et al., 2017; Cen et al., 2018). For culture-independent methods, denaturing gradient gel electrophoresis (DGGE) was applied for the investigation of microbiota composition (Liu et al., 2015); however, low throughput and complex operation were its limitations. With the advent of high-throughput sequencing platforms and bioinformatic analysis pipelines, metagenome sequencing analysis including 16S rRNA amplicon sequencing and whole genomic shotgun sequencing have revealed the abundant diversity composition of the bile microbiome (Wu et al., 2013; Liu et al., 2015). Meanwhile, owing to nearly 80% of the human intestinal bacteria were unculturable (Tajeddin et al., 2016), with the advancement in bacterial culturomics technology (Tidjani Alou et al., 2020), it is reasonable to infer that abundant bile bacteria are waiting to be isolated.

Proper identification method of the isolated bacteria was a highly essential step in the culture-dependent workflow. The traditional polyphasic taxonomic strategy aims to generate phenotypic and phylogenetic information of a bacterium, such as its shape, color, size, staining properties, host range, pathogenicity, indole test, gelatin liquefaction test, motility test, and assimilation of carbon sources (Das et al., 2014). In addition to biochemical identification, flow cytometry and MALDI-TOF mass spectrometry can also be used for rapid identification for cultured isolates (Das et al., 2014; Jang and Kim, 2018). Despite each approach having its own advantages and limitations, such as higher cost or rough results of flow cytometer and equipment popularity of MALDI-TOF mass spectrum hindered their application. Considerable advancement in culturable bacterial identification and taxonomy methods using genotypic and phylogenetic information has taken place such as chemotaxonomy, numerical taxonomy, and DNA–DNA hybridization, housekeeping gene amplification, and sequencing (16S rRNA gene, gyrB, rpoABCD, etc.) and whole-genome sequencing (Das et al., 2014). As the primary gene target for bacterial identification and comprehensive consideration of cost performance, the 16S rRNA gene was often used for preliminary screening. Then whole-genome sequencing was used for in-depth exploration of functional genes. In recent years, cultivation-dependent microbiota studies of the human digestive tract (Browne et al., 2016; Lagier et al., 2016), respiratory tract (Fonkou et al., 2018), genitourinary tract (Thomas-White et al., 2018), oral cavity (Fonkou et al., 2018; Martellacci et al., 2020), and even in other model animals (Lagkouvardos et al., 2016; Liu et al., 2020) mostly adopted this strategy and had isolated hundreds of previously unknown bacteria inhabiting the human body. In addition, live bacteria could facilitate the follow-up mechanism researches regarding their impact on physiological and pathological functions, such as the bile salt (salt ion form of bile acid) metabolism test. Bile acids are cholesterol-derived natural surfactants and are regarded as important digestive hormones that are produced in the liver and secreted into the duodenum via the gallbladder to regulate numerous physiological processes in the host (Harris et al., 2018). They are known to affect lipid digestion, antibacterial defense, glucose metabolism, host metabolism, cancer progression, and innate and adaptive immunity (Shapiro et al., 2018; Hang et al., 2019; Song et al., 2020). The biotransformation of bile acids (BAs) by intestinal bacteria may have an important role in cholesterol gallstone formation and colon carcinogenesis via TGR and FXR (farnesoid-X receptor) receptors (Salvioli et al., 1982; Funabashi et al., 2020). So far, bacteria are mainly involved in two categories of bile acid metabolism. Bile salt hydrolase (BSH) with deconjugation activity hydrolyzes the amide bond that links the bile acid side chain to glycine or taurine and takes part in the transformation process from conjugated bile acid to unconjugated bile acid (Ridlon et al., 2006). Besides, the dehydroxylase activity participates in the transformation process from primary bile acid to secondary bile acid (White et al., 1980). These metabolic activities have mostly been found in intestinal bacteria (Kuipers et al., 2020). However, the ability of bile acid biotransformation by bile bacteria has not been reported to date.

Altogether, the objectives of the study are to (1) cultivate and isolate a large number of bile bacteria of cholecystitis patients and whole-genome sequence parts of isolates, (2) conduct comparative analyses between our isolates and gut isolates in public databases, which revealed different pathways in our isolates, and (3) evaluate the bile salt metabolic capacity of biliary bacteria in vitro. This work provided new insights into the bile bacterial communities based on genomes and the biotransformation potentiality of human bile bacteria.

Identification and characterization of antibiotic resistance genes (ARGs) and virulence factors of pathogens are crucial in understanding bacterial pathogenesis and their interactions with the host, and in the development of novel drugs, vaccines, and molecular diagnostic tools. Furthermore, detecting virulence or resistance markers improved outbreak monitoring and therapeutic management. Currently, the use of the next-generation sequencing platforms has allowed great progress in this field (Bakour et al., 2016). ARGs were identified based on ABRicate.¹ ABRicate searched on the databases, including NCBI Bacterial Antimicrobial Resistance Reference Gene Database, CARD (Jia et al., 2017), ARG-ANNOT (Gupta et al., 2014), and ResFinder (Zankari et al., 2012) for predicting ARGs. The virulence factors of the genome were identified based on the VFDB database (Chen et al., 2016).

The bile bacteria, including the Vibrio genus especially Vibrio cholerae (Asnis et al., 1996), Escherichia coli, Klebsiella spp., Enterococcus (Yun and Seo, 2018), Staphylococcus, Corynebacterium (Backert et al., 2018), and Lactobacillus salivarius, have been studied extensively (Woo et al., 2002). Tajeddin et al. (2016) found that enterobacteria were the dominant biliary bacteria based on cultivate-dependent methods. As an alternative animal model of humans, most of the bile and gall bladder bacteria of healthy pigs were Streptococcus alactolyticus, Staphylococcus epidermidis, and Corynebacterium testudinoris, which suggested that healthy host bile may also contain bacteria (Jimenez et al., 2014). In our previous research and the work of others, the bile pathogenic bacteria were considered a predisposing factor in gallbladder disorders (Liu et al., 2015; Shen et al., 2015; Tajeddin et al., 2016). Furthermore, metagenomic results indicated that many potential bile bacteria still cannot be isolated and cultivated (Liu et al., 2015). With more cultivated and detected conditions termed “culturomics” emerging (Lagier et al., 2016), many previously unavailable “dark matter bacteria” from bile would be isolated.

In this study, we characterized the isolated bacterial genome from the fresh bile specimens from four cholecystitis patients and evaluated its bile acid biotransformation ability in vitro. Our results showed that the number of cultivable bile bacteria exceeds expectations, spanning 138 bacterial isolates that were distributed in 14 genera. In particular, three bacterial species (i.e., Providencia sp. D135, Psychrobacter sp. D093, and Vibrio sp. D074) were not represented in existing reference genome databases. Enterobacteriaceae had the most isolated strains (28 strains) under aerobic conditions, in agreement with previous studies (Tajeddin et al., 2016). Although there was little research on bile anaerobes, we obtained 63 strains under anaerobic conditions, however, most of them were facultative anaerobes. The possible reasons were the small sample size (n = 4) and the nutrient composition of the medium, which was not specifically optimized or not rich enough for bile bacteria. For instance, Lagier et al. (2016) reported that liquid media precultivation operation can effectively promote the diversity of isolated bacteria. In addition, the centrifugal operation may lose some lighter bacteria; therefore, after centrifugation, the supernatant can be aseptically filtered (Spin-X Centrifuge Tube Filter, Corning Inc.) and concentrated, and the concentrated part will be used for cultivation to obtain more bacteria. The isolated bacterial genomes could extend the databases, which are the basis for metagenomics analysis (Lagier et al., 2016).

Whole-genome sequencing was performed on 35 bacterial species and compared with gut isolates. Our results showed that many pathways, such as biofilm formation, ABC transporters, and quorum sensing, were enriched in bile bacteria, which may contribute to communicate with each other and resist the antibacterial high concentration of bile salts (Davidson et al., 2008; Moons et al., 2009; Rutherford and Bassler, 2012). Bile salts promoted the biofilm formation of bile bacteria, and the biofilms of bacteria-laden gallstones influenced the illness severity (Stewart et al., 2007). Especially, the biofilm formation ability seems to be a necessary condition for bile resistance and polymicrobial infection in this organ (Tajeddin et al., 2016; Zheng et al., 2017). Therefore, this high abundance of functional pathways of bile bacteria, such as biofilm formation, was related to its living environment and physiological characteristics.

Enterobacteriaceae had the largest isolated strains (28 strains) under aerobic conditions in this study which was consistent with the previous studies (Yun and Seo, 2018) and had been identified to be closely related to cholecystitis in our previous research (Liu et al., 2015). ARGs and virulence factors analyses of the bacterial isolates further revealed that important Gram-negative opportunistic pathogen of the Enterobacteriaceae, such as Escherichia coli, Citrobacter sp. CRE-46, Klebsiella aerogenes, and Klebsiella pneumoniae, encoded the higher number of ARGs and the virulence factors than other bile species. This result was inconsistent with those of previous studies that less than 5% resistance was observed in bile isolated bacteria against carbapenem, beta-lactam antibiotics, glycopeptide antibiotics, and linezolid via the antibiotic susceptibility tests (Yun and Seo, 2018). This difference may be caused by different test methods and their sensitivity. Therefore, the combination of the bioinformatics and the verification of conventional drug-susceptibility testing method on the pivotal isolates in the next step may have positive significance in the quicker selection of the most suitable antibiotics for clinical applications.

In humans, the major bile salts included taurocholic acid (TCA) and glycocholic acid (GCA) derived from cholic acid (CA), while taurochenodeoxycholic acid (TCDCA) and glycochenodeoxycholic acid (GCDCA) derived from chenodeoxycholic acid (CDCA). The salts of their 7-alpha-dehydroxylated derivatives, deoxycholic acid (DCA), and lithocholic acid (LCA), are also found, with derivatives of cholic, chenodeoxycholic, and deoxycholic acids accounting for over 90% of human biliary bile acids (Hofmann, 1999). Bile acids are closely related to human health and can be metabolized by gut bacteria, which generated free bile acid by deconjugation and generated secondary bile acids by 7α-and 7β-dehydroxylation, such as deoxycholic acid and lithocholic acid (Ridlon et al., 2016; Wang and Jia, 2016; Marion et al., 2019; Song et al., 2019; Funabashi et al., 2020). Secondary bile acids, especially DCA, have been listed as carcinogens and promoters of CRC (Yang and Yu, 2018). High-fat diet-induced high levels of DCA in the intestine are also considered an important risk factor for CRC. Herein, based on the phylogenetic tree of isolates, we selected 24 bile bacterial strains and detected their bile salt deconjugation and transformation abilities. Lysinibacillus genus had strong bile acid biotransformation ability (Figure 5), which was consistent with previous reports that Paenibacillus and Lysinibacillus widely carried BSH (Song et al., 2019). This ability of bile bacteria may allow them to overcome the bile salt damaging effect. Although this study provided direct evidence in vitro for the bile bacteria involved in the biotransformation of bile acid, however, we are not sure that the bacteria-mediated transformations occur in the gallbladder or not so far. The reason is that the presence of in vivo bile acid transformations performed by certain isolated microorganisms lacks rigor since the same strain may display completely different functional performance between its in vivo and in vitro activity (Marion et al., 2019). For instance, Clostridium hiranonis did not deconjugate tauro-conjugated bile acids in germ-free mice, although its ability to deconjugate taurocholic acid (TCA) was shown in vitro (Narushima et al., 1999). Importantly, our research results provided new insights for screening bile salt metabolizing bacteria, which many studies focused on gut bacteria (Lucas et al., 2021).

Our data helps in understanding the genetic basis of the physiology, biochemical pathways, and evolution of the isolates and provides a preliminary exploration in bile acid transformations by bile bacteria; however, the sample size and culture conditions were relatively small. Therefore, larger-scale bacterial isolation and identification, more in-depth screening isolates with bile acid transformation ability in vivo by metabolite detection method, and verifying the impact of metabolite on gallbladder and even whole-body health are to be investigated in the next step.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA726166, PRJNA726166.

The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the Hunan Provincial People’s Hospital (Ethical approval number: 2020-54). The patients/participants provided their written informed consent to participate in this study.

YM, QY, SL, YX, and XHM contributed to conception and design of the study. SL, SZ, YZ, QL, and AZ organized the formal analysis. GW, RL, MX, TJ, YS, XL, SC, XT, and XD performed methodology. YM, XCM, CW, and YX were responsible for project administration. QY, SL, and SZ wrote the first draft of the manuscript. All authors were involved in preparing the manuscript and contributed to manuscript revision, read, and approved the submitted version.

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.

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