Microbial Pathogenesis 83-84 (2015) 57e63
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Cholesterol gallstones and bile host diverse bacterial communities with potential to promote the formation of gallstones Yuhong Peng a, Yang Yang a, Yongkang Liu b, Yuanyang Nie a, Peilun Xu a, Baixue Xia a, Fuzhou Tian c, Qun Sun a, * a Key Laboratory of Bio-resource and Bio-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, PR China b The 452nd Hospital of PLA, Chengdu, Sichuan 610000, PR China c Chengdu Military General Hospital, Chengdu, Sichuan 610083, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 December 2014 Received in revised form 4 May 2015 Accepted 6 May 2015 Available online 7 May 2015
The prevalence of cholesterol gallstones has increased in recent years. Bacterial infection correlates with the formation of gallstones. We studied the composition and function of bacterial communities in cholesterol gallstones and bile from 22 cholesterol gallstone patients using culture-dependent and culture-independent methods. Altogether fourteen and eight bacterial genera were detected in cholesterol gallstones and bile, respectively. Pseudomonas spp. were the dominant bacteria in both cholesterol gallstones and bile. As judged by diversity indices, hierarchical clustering and principal component analysis, the bacterial communities in gallstones were different from those in bile. The gallstone microbiome was considered more stable than that of bile. The different microbial communities may be partially explained by differences in their habitats. We found that 30% of the culturable strains from cholesterol gallstones secreted b-glucuronidase and phospholipase A2. Pseudomonas aeruginosa strains showed the highest b-glucuronidase activity and produced the highest concentration of phospholipase A2, indicating that Ps. aeruginosa may be a major agent in the formation of cholesterol gallstones. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cholesterol gallstones Bile Bacterial community PCR-DGGE b-Glucuronidase Phospholipase A2
1. Introduction The prevalence of cholesterol gallstones, a common disease including gallstones, common bile duct gallstones, and intrahepatic bile duct gallstones, has increased in recent years, especially in the Western world [1]. In China, along with the population aging, obesity, hyperlipidemia and diabetes, the incidence of the cholesterol gallstones has risen to 10% [2e4]. The secretion of cholesterol in bile and abnormal metabolism are considered as the primary pathophysiological causes in the formation of cholesterol gallstones. Factors that are thought to or known to promote the formation of cholesterol gallstones include estrogen and progestogens, cholesterol-lowering medications, obesity and rapid weight loss [5,6]. The consistency and relevance of symptomatic gallbladder stones in identical twins is significantly higher than in double egg twins, indicating that both genetic and environmental
* Corresponding author. E-mail address: [email protected] (Q. Sun). http://dx.doi.org/10.1016/j.micpath.2015.05.002 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
factors are involved [7]. The frequent Cyp7a1 polymorphism in A allele in Chinese patients with cholesterol gallstone implies that gallstone formation is related to the expression of genes [8]. Other potential factors associated with the formation of cholesterol gallstones include mucoprotein, aminopeptidase N and vesicular protein [9,10]. Bacterial colonization correlate with the formation of gallstones. Bacterial infection has been proposed as a key factor in the pathogenesis of pigment gallstones [11]. Some study confirmed that nanobacteria may use its self-propagation to cause the formation of black pigment gallstones in rabbits [12,13]. Meanwhile the role of bacteria in cholesterol gallstones formation has aroused increasingly more attention. Swidsinski et al. [14] used RT-PCR (reverse transcription e polymerase chain reaction) to release an 80% detection rate of bacterial DNA in cholesterol gallstones. Chen et al. [15] showed that the detection rate of bacterial DNA in bile, gallstone and biliary tract mucous membrane was 77%, 84% and 62%, respectively. Bacteria may play a critical role in the formation of cholesterol crystals [16]. Alterations in the gastrointestinal microbiome may change aspects of
58
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
cholesterol gallstone pathogenesis and then these changes may impact cholelithogenesis [17]. Pseudomonas aeruginosa and Enterococcus faecalis shortened the cholesterol crystallization time in the model bile indicating that these species may be crucial in the formation of the cholesterol gallstones [18]. These findings indicated that bacterial community composition might influence the formation of cholesterol gallstones. Thus, it is crucial to understand the structure of bacterial community and its potential connection in the pathogenesis of cholesterol gallstones. Bacteria speed up the gallstone formation by secrefing crystal formation factors. b-glucuronidase and phospholipase A2 have been identified as the major factors leading to the formation of gallstones. The role of b-glucuronidase in gallstone formation process has been fully recognized [19]. The hydrolysis of phospholipids by phospholipase A2 consequently caused the crystallization of cholesterol and finally led to the formation of gallstones [20]. Furthermore, multidrug-resistance efflux pump proteins expressed by bacteria may help bacteria to survive in bile [21e25]. Traditionally characterizing bacteria has relied on cultivation. However, most of the bacteria in are non-cultivable by conventional methods. Therefore, culture-independent methods like polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) have been widely used to study microbial communities present in biological samples [26e28]. To get a more wide view on the microbial communities in gallstones and bile, we used both cultivation and the culture-independent PCR-DGGE method. In addition, we tested the b-G activity and the PLA2 production of the isolated bacteria to better understand the bacterial community in the cholesterol gallstones and to assess which bacteria have potential to promote the formation of cholesterol gallstones. 2. Material and methods 2.1. Patients and sample collection The 22 cholesterol gallstone samples (S1eS22) and 12 bile samples (B1eB12) from 22 gallbladder-stone patients were provided by Chengdu Military General Hospital. The 12 male and 10 female provided a signed informed consent to partake in the study. The average age of the patients was 45.1 ± 14.2 (SD). The patients did not receive any antibiotics or probiotics before the study. Gallstones were removed aseptically from the gallbladder of each patient. At the same time, gallbladder bile was aspirated from indwelling catheters into 10 ml sterile tubes from 12 patients. All samples were stored in their original tubes at 40 C until further analyses. The gallstones were classified as cholesterol gallstones with above 70% cholesterol content by Fourier transform infrared spectroscopy as described [29,30]. 2.2. Total DNA extraction Cholesterol gallstones (180e200 mg) were homogenized by grinding with a sterile mortar and then by mechanical disruption with a bead beater. Total DNA from homogenized gallstones and bile samples was extracted by DNA Stool Kit (OMEGA) following the manufacturer's instructions. 2.3. PCR amplification of V3 region of 16S rDNA PCR amplification was performed in S1000™ Thermal Cycler (BIO-RAD) with reagents obtained from TaKaRa (Japan). In the first step of a nested PCR method, 16S rRNA gene fragments were amplified using 20 pmol of each primer 27F (50 -AGA GTT TGA TCM TGG CTC AG-30 ) and 1492R (50 -TAC GGC TAC CTT GTT ACG ACT T-30 ) in 50 ml of amplification buffer (250 mM of each dNTP, 10 mM
TriseHCl, 1.5 mM MgCl2, 50 mM KCl and 2.5 U Taq DNA polymerase) with 5 ml sample DNA as the template. The amplification was performed with the following temperature profile: an initial denaturation at 94 C for 5 min; 30 cycles of denaturation at 94 C for 1 min, annealing at 55 C for 1 min and extension at 72 C for 1.5 min; and a final extension at 72 C for 10 min. The amplification products were subjected to electrophoresis in 1% (w/v) agarose gel and extracted by Gel Extraction Kit (OMEGA) according the manufacturer's instructions. The extracted products were used as template for amplification of V3 region of 16S rDNA. The V3 region was amplified using primers 338F (50 -ACT CCT ACG GGA GGC AGC AG-30 ) with a GCclamp (50 -CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-30 ) at the 50 end and 518R (50 -GTA TTA CCG CGG CTG CTG GCA-30 ) in 50 ml of amplification buffer as described above with 5 ml extract as the template. The amplification was performed with the following temperature profile: 5 min at 95 C; 10 cycles of denaturation at 94 C for 30 s, annealing for 30 s with decreasing the temperature by 0.5 C per cycle from 60 to 55 C, extension at 72 C for 35 s; 25 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 30 s; and a 7 min final extension at 72 C. The amplification products were subjected to electrophoresis in 1% agarose gels (Invitrogen) using a D2000 DNA Marker (TIANGEN BIOTECH, Beijing, China) as a molecular weight standard. The gels were run at 120 V for 20 min in 1 TAE, stained with 0.1 ml ml1 (v/ v) Green View and evaluated as described [31,32].
2.4. DGGE analysis DGGE was performed with the D-Code™ universal mutation detection system (BioRad, Lab., USA). The amplification products from cholesterol gallstone samples were separated in 8% (w/v) polyacrylamide gels in a denaturing gradient of 40%e65% (100% denaturant defined as 7 M urea and 40% formamide in 1 TAE buffer, pH 7.4) at 110 V and 60 C for 6.5 h. The amplification products from bile samples were separated in a denaturing gradient of 45%e55% under conditions described above for separating preferably bands. Gels were stained with SYBR Green (Invitrogen) for 45 min, visualized with a UV transilluminator (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed with Quantity One software (Bio-Rad, USA).
2.5. Amplification and sequencing of excised DGGE bands Selected bands were excised from the DGGE gel, resuspended in sterile water and kept at 4 C for 24 h before amplification of the V3 region as described above. The amplification products were sequenced at Invitrogen (Shanghai, China). The sequences were compared with NCBI database to determine the closest match sequence.
2.6. Isolation and identification of culturable bacteria Cholesterol gallstone and bile samples were inoculated onto modified tryptic soy broth (TSB). The plates were incubated at 37 C under aerobic conditions (CO2 concentration 5%). DNA was extracted from bacterial mass taken from single colonies by Bacterial DNA Extraction kit (TIANGEN, Beijing, China) while the rest of the colony was preserved for further testing. The 16S rDNA was amplified using primers 27F and 1492R as described above and sequenced at Invitrogen (Shanghai, China). The sequences were compared with NCBI database to determine the closest match sequence.
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
2.7. Determining the b-glucuronidase activity and phospholipase A2 production of the isolated bacteria The isolates were grown in liquid modified TSB media for 24 h. After cultivation, cells were dissolved by adding 180 ml lysozyme (TIANGEN, Beijing, China) in 5 ml culture solution and incubating for 35 min at 37 C. The cells were pelleted at 10,000 r min1 for 2 min. b-Glucuronidase activity was tested as described [33]. Briefly, 2 ml p-nitrophenyl- b-D-glucuronide was added to 2 ml supernatant, the solution was incubated for 1 h at 37 C, after which the reaction was terminated by adding 0.4 ml NaOH (0.1 mol l1). 2 ml PBS (0.05 mol l1, pH 7.0) served as a negative control. Standards for quantifying the amount of released p-nitrophenol were done using from 10 mmol l1 to 2 mmol l1 dilutions of p-nitrophenol in PBS (0.05 mol l1, pH 7.0). After adjusting zero using the negative control, OD at 405 nm of the standards and samples was measured. Enzyme activity (U l1) was defined as 1 mmol p-nitrophenol released from p-nitrophenylb-D-glucuronide in one minute. The concentration of phospholipase A2 was measured by PLA2 ELISA Kit according the manufacturer's instructions. 2.8. Data analysis Quantity One software was used to convert individual DGGE lanes to densitometric profiles. The indices of species richness, evenness and the Shannon Diversity were calculated based on the profiles. Cluster analysis of the band patterns was performed with the unweighted-pair group method using average linkages. Principal component analysis was performed by SPSS 17.0 based on the number and relative intensity of the band of each DGGE fingerprints as described [34]. Multiple samples comparisons were performed using one-way ANOVA. Statistical significance was set at P < 0.05. 3. Results 3.1. Profiling of bacterial communities in cholesterol gallstones and bile using PCR-DGGE
59
higher in the gallstones than in the bile. In the cluster analysis, the DGGE band patterns of the cholesterol gallstones samples differed from those of bile samples (Fig. 5). The six cholesterol gallstones samples and six bile samples formed three clusters. The gallstone samples clustered together with pairwise similarity coefficients from 0.33 to 0.73 (Cluster C), and the bile samples formed two clusters (Cluster A and B), which had very low similarity with the gallstone cluster. A principal component analysis on the bacterial communities in cholesterol gallstones and bile explained 67.39% (PC1) and 32.61% (PC2) of the total variance (Fig. 6). As in the cluster analysis, the bile samples and cholesterol gallstone samples clustered separately in PCA, suggesting that the bacterial communities in cholesterol gallstones differed significantly from those in bile, while the cholesterol gallstone communities had small differences with one another. In contrast, the difference within the bile samples was great. 3.3. Identification, b-glucuronidase activity and phospholipase A2 production of the isolated bacteria The diversity and function of culturable bacteria from cholesterol gallstones was assessed by isolating them and analyzing their b-glucuronidase activity and phospholipase A2 production. In total 93 strains was isolated from gallstone samples and identified based on 16S rDNA sequence. The isolates represented seven species, out of which all but Bacillus amyloliquefaciens had been identified with the culture-independent approach (Tables 1 and 3). Altogether 35 out of 93 stains showed b-glucuronidase activity and produced phospholipase A2. Ps. aeruginosa strains showed highest and Pseudomonas stutzeri strains the lowest b-glucuronidase activities (Table 3). The b-glucuronidase activities of Ps. stutzeri and B. amyloliquefaciens strains were significantly lower than those of the other species (P > 0.05). There were no statistically significant differences in phospholipase A2 production between the producing strains (Table 3). Both the b-glucuronidase activity and the phospholipase A2 production suggested that these strains could potentially induce the formation of cholesterol gallstones. 4. Discussion
The diversity of bacterial communities in cholesterol gallstones and bile was assessed using 16S-rDNA PCR-DGGE. The differences between samples were high and every sample had its specific bands (Figs. 1 and 2). A total of 19 bands were selected for amplification, sequencing, and identification by searching the National Center for Biotechnology Information (NCBI) database. Altogether 14 bacterial genera were identified (identity above than 97%) (Tables 1 and 2). Genera Brucella, Lachnospiraceae, Escherichia, Citrobacter, Shinella, Lactococcus and Lactobacillus were identified only from the gallstones, but not in bile, while the genus Acinetobacter only in bile. According to the statistical analysis of relative abundances, Pseudomonas was the dominant genus in both cholesterol gallstones and bile (Fig. 3). In addition, the relative abundance of the genera Pseudomonas, Bacillus, Klebsiella, Clostridium, Staphylococcus and Enterobacter was significantly higher in cholesterol gallstones than in bile (P > 0.05, n ¼ 3). 3.2. The comparison of bacterial communities in cholesterol gallstones and bile We used cholesterol gallstones and surrounding bile from 22 patients to compare the difference of bacterial communities between these two environments. The Shannon diversity index (H) was significantly higher (P > 0.05) in the gallstones than in the bile (Fig. 4). Likewise, the richness and evenness were significantly
Microbiome is important in many immunological and metabolic diseases [35], as its variation in the intestinal microbiome may change cholesterol gallstone pathogenesis. The gut microbiome of gallstone patients was shown to be significantly different from that of healthy people [36]. In this research, both culture-independent and culture-dependent analyses were performed to compare microbial communities in cholesterol gallstones and bile. To our knowledge, this is the first study to apply the PCR-DGGE to clarify the composition of bacterial communities within cholesterol gallstones. Dysfunction of the intestinal mucosa barrier may result in translocation of the bacteria into bile duct where the bacteria may promote the formation of gallstones [37]. Bacteria that inhabit the gastrointestinal tract (GIT) were also detected in the cholesterol gallstones [36]. In addition to genera previously reported, we detected Escherichia, Brucella, Citrobacter, Shinella, Aurantimonas, Lachnospiraceae and Lactobacillus in the gallstones, among which Citrobacter, Lactobacillus and Aurantimonas are common members of the GIT microbiome [38]. After Lactobacillus infected gallbladder, the outer membrane proteins of Lactobacillus could stimulate humoral immunity and cellular immunity in human body [39]. The presence of GIT bacteria in cholesterol gallstones indicated that once getting into the gallbladder, the GIT bacteria might trigger the immune system and accelerate the formation of cholesterol
60
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
Fig. 1. DGGE fingerprints of V3 region in bacterial 16S rDNA from bacterial communities in cholesterol gallstones. The bacteria identified on the sequence analyses of the selected bands are summarized in Table 1.
gallstones. Brucella is a pathogen that can invade the body through the digestive tract mucosa [40,41]. Our results indicated that Brucella could collaborate with other bacteria to accelerate the formation of cholesterol crystals. It has been proposed that Ps. aeruginosa plays a major role in the infection of biliary tract and more attention should be paid on its role in cholelithiasis [42,43]. Enterococcus faecium is both a member of the GIT microbiome and a common agent in biliary tract infection. Moreover, both Ps. aeruginosa and E. faecium shorten the time of the formation of cholesterol crystals [18]. The abundance of E. faecium, Klebsiella spp. and Enterobacter spp. in human bile has increased significantly from 1988 to 2010 [44]. Our DGGE results showed that Pseudomonas, Enterococcus, Klebsiella and Enterobacter were all relatively abundant in cholesterol gallstones, indicating that these four genera
Table 1 The bacteria identified based on the sequence analysis of selected bands in DGGE fingerprints from bacterial communities in cholesterol gallstones. Band no.
Closest NCBI database match
Similarity (%)
Accession no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Uncultured Bacillus sp. Brucella melitensis Lachnospiraceae Klebsiella oxytoca Escherichia sp. Citrobacter sp. Clostridium clostridioforme Staphylococcus sp. Shinella sp. Enterococcus durans Enterococcus faecium Enterobacter cloacae Enterobacter asburiae Enterobacter hormaechei Lactococcu spiscium Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas stutzeri Lactobacillus sp.
99 100 97 99 98 97 98 100 98 100 99 99 100 99 99 99 99 100 99
KF581441.1 KF771410.1 EF704617.1 KC593550.1 JQ828856.1 EU360138.1 KC143063.1 FJ937931.1 JQ684917.1 KF918537.1 HE646384.1 KC509581.1 CP007546.1 KF862929.1 KF983318.1 KJ184550.1 KF913776.1 KF850545.1 HQ420284.1
were potentially connected with cholesterol crystallization as dominant population. As judged by diversity indices, hierarchical clustering and Table 2 The bacteria identified based on the sequence analysis of selected bands in DGGE fingerprints from bacterial communities in bile.
Fig. 2. The DGGE fingerprint of V3 region in bacterial 16S rDNA from bacterial communities in bile. The bacteria identified based on the sequence analyses of the selected bands are summarized in Table 2.
Band no.
Closest NCBI database match
Similarity (%)
Accession no.
1 2 3 4 5 6 7 8 9 10 11 12
Bacillus oleronius Klebsiella oxytoca Clostridium sp Uncultured Staphylococcus sp. Enterococcus durans Enterobacter asburiae Staphylococcus epidermidis Enterobacter aerogenes Pseudomonas aeruginosa Pseudomonas stutzeri Acinetobacter johnsonii Acinetobacter lwoffii
100 99 100 100 100 100 100 100 100 100 100 100
KF835577.1 KC593550.1 KJ194597.1 FJ937931.1 KJ631376.1 CP007546.1 KF862930.1 KJ011873.1 KJ461700.1 KJ185383.1 HG810396.1 KF704079.1
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
61
Fig. 3. The estimated abundances of identified members of bacterial communities in cholesterol gallstones and bile. * Difference significant at 0.05 level (n ¼ 3).
principal component analysis, the bacterial communities in gallstones were different from those in bile. Based on the low similarities between the bile communities, the bile microbiome could be considered as more dynamic. The different microbial communities may be partially explained by differences in their habitats. The bile salts are toxic to the bacteria. In addition, bile changes frequently because of enterohepatic circulation, whereas cholesterol gallstones provide a stable and protected ecological niche. The similarity of the gallstone bacterial communities indicated that bacteria which cause the formation of cholesterol gallstones may be same in different patients, and therefore more attention should be paid on these bacteria in the future. Both b-glucuronidase and phospholipase A2 are known risk factors in the formation of cholesterol gallstones. b-glucuronidase is a hydrolase found in all kinds of cells in the human body, especially in the liver. b-glucuronidase is either endogenous, secreted by cells, or exogenous, secreted by bacteria. The gallbladder tissue secretes endogenous b-glucuronidase [45]. Both endogenous and exogenous
Fig. 4. The comparison of Shannon diversity index (SDI), evenness and richness of bacterial communities from cholesterol gallstones and bile. * Difference significant at 0.05 level (n ¼ 4).
b-glucuronidase may participate into the formation of cholesterol gallstones. The activity of exogenous b-glucuronidase rises rapidly in early-stage biliary infection when the infection is treated [46]. Bacteria may secrete enzymes to accelerate the formation of insoluble precipitates which could serve as sites of nucleation in the formation of cholesterol gallstones [47]. According to Ma et al. [48], exogenous b-glucuronidase could make cholesterol in bile to precipitate into crystals because of cholesterol saturation. Tao et al. also proposed that during a bacterial infection, the inflammatory factors secreted by bacteria would accelerate the secretion of endogenous b-glucuronidase from both hepatocytes and biliary epithelial cells, after which these two factors would participate in the formation of cholesterol gallstones. Bacterial infection of biliary tract causes an increase in the amount of phospholipase A2 [49]. Phospholipase A2 may hydrolyze lecithin to disturb the balance of bile acid, cholesterol
Fig. 5. Unweighted-pair group method with average linkages analysis of the DGGE band patterns of V3 region in bacterial 16S rDNA from cholesterol gallstones and bile. Cluster A and B: bile samples. Cluster C: cholesterol gallstone samples.
62
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
Fig. 6. Principal component analysis of the relative optical densities produced by bacterial activities from cholesterol gallstones and bile samples. B1eB12: bile samples. S1eS22: cholesterol gallstone samples.
Table 3 The identification of isolated bacteria based on 16S rDNA sequence analysis, their b-glucosidase activities and phospholipase A2 production. Closet relative n (%)
Similarity (%)
Accession no.
b-G (U/l)
Pseudomonas aeruginosa 10(10.6) Enterobacter hormaechei 3(3.2) Enterococcus faecium 8(8.6) Klebsiella oxytoca 4(4.3) Pseudomonas stutzeri 3(3.2) Enterobacter cloacae 4(4.3) Bacillus amyloliquefaciens 3(3.2)
100 99 99 100 100 100 99
KF956583.1 KF836497.1 KF358453.1 KF254665.1 KJ185383.1 CP006580.1 KJ149810.1
33.6 15.6 19.0 16.8 12.1 27.4 12.4
and phosphatidic acid, ultimately resulting in decreased cholesterol saturation index. The decrease of saturation index makes cholesterol more likely to crystallize. We found that 30% of the culturable isolates from cholesterol gallstones secreted b-glucuronidase and phospholipase A2. Ps. aeruginosa strains showed the highest b-glucuronidase activity and produced the highest concentration of phospholipase A2, indicating that Ps. aeruginosa may be a major agent in the formation of cholesterol gallstones. E. faecium has been considered as a gallstone-associated bacterium, but its role in cholelithiasis was not clear. We showed that E. faecium secreted both b-glucuronidase and phospholipase A2 thereby clarifying the potential of E. faecium in gallstone formation. We found that Enterobacter hormaechei, B. amyloliquefaciens and Enterobacter cloacae secrete both b-glucuronidase and phospholipase A2. These bacteria are also b-lactamase producers [50,51]. b-lactamase gives resistance to antibiotics making these bacteria in gallbladder less vulnerable to antibiotic treatments. Therefore, determining which bacteria have infected gallbladder is important in choosing the appropriate antibiotics for treatment. In summary, we confirmed the association between bacteria and cholesterol gallstones. Using PCR-DGGE we found that at least 14 bacterial genera reside in cholesterol gallstones. Pseudomonas was the dominant bacteria in both bile and cholesterol gallstones. The bacterial communities in cholesterol gallstones and bile were significantly different. Strains representing seven species were isolated from the gallstones. Altogether 30% of the strains secreted b-glucuronidase and phospholipase A2. In future, these seven species should be taken into consideration when
± ± ± ± ± ± ±
16.7 4.0 9.9 4.9 2.1 16.6 11.3
PLA2 (ng/ml) 0.589291 0.588593 0.587404 0.586954 0.587213 0.589073 0.588547
± ± ± ± ± ± ±
0.010188 0.009276 0.009659 0.011004 0.001680 0.007744 0.011167
designing clinical prevention and treatment. Our results could provide basis for designing a detailed clinical therapeutic guideline. The role of the b-glucuronidase and phospholipase A2 producers in gallstone formation should be verified by further follow-up studies. Acknowledgments This research was supported by grants from the Natural Science Foundation of J1103518. References [1] K.J. Van Erpecum, Pathogenesis of cholesterol and pigment gallstones: an update, Clin. Res. Hepatol. Gastroenterol. 35 (2011) 281e287. [2] Q. Zeng, Y. He, D.C. Qiang, L.X. Wu, Prevalence and epidemiological pattern of gallstones in urban residents in China, Eur. J. Gastroenterol. Hepatol. 24 (2012) 1459. [3] X.G. Chen, J.Q. Liu, M.H. Peng, et al., Clinical epidemiological study on intrahepatic cholelithiasis: analysis of 8585 cases, Hepatobiliary Pancreat. Dis. Int. 2 (2003) 281. [4] C.Y. Chen, S.C. Shiesh, X.Z. Lin, Biliary sludge and pigment stone formation in bile duct-ligated guinea pigs, Dig. Dis. Sci. 44 (1999) 203. [5] B. Liu, V. Beral, A. Balkwill, J. Green, S. Sweetland, G. Reeves, et al., Gallbladder disease and use of transdermal versus oral hormone replacement therapy in postmenopausal women: prospective cohort study, Bmj 337 (2008) a386. [6] H. Volzke, S.E. Baumeister, D. Alte, W. Hoffmann, C. Schwahn, P. Simon, et al., Independent risk factors for gallstone formation in a region with high cholelithiasis prevalence, Digestion 71 (2005) 97e105. [7] D. Katsika, A. Grjibovski, C. Einarsson, F. Lammert, P. Lichtenstein, H.U. Marschall, Genetic and environmental influences on symptomatic gallstone disease: a Swedish study of 43,141 twin pairs, Hepatology 41 (2005) 1138e1143. [8] Z.Y. Jiang, P. Parini, G. Eggertsen, M.A. Davis, H. Hu, G.J. Suo, et al., Increased
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23] [24] [25]
[26]
[27]
[28]
[29]
[30]
expression of LXR alpha, ABCG5, ABCG8, and SR-BI in the liver from normolipidemic, nonobese Chinese gallstone patients, J. Lipid Res. 49 (2008) 464e472. D.C. Zhang, J.B. Xiang, L.Y. Wang, Z.B. Xu, L.D. Sun, F. Zhou, et al., Comparative proteomic analysis of gallbladder bile proteins related to cholesterol gallstones, PloS One 8 (2013) 350e352. Y.T. Li, X.G. Zhu, X.L. Zhao, G.S. An, H.T. Jia, Expression of aminopeptidase N and its correlation with the gallstones formation, Chin. J. Biochem. Mol. Biol. 17 (2001) 425e429. T. Maki, Pathogenesis of calcium bilirubinate gallstone: role of E. coli, bglucuronidase and coagulation by inorganic ions, polyelectrolytes and agitation, Ann. Surg. 164 (1996) 90e100. L.M. Wang, W.L. Shen, J. Wen, X. An, L.Y. Cao, B.Q. Wang, An animal model of black pigment gallstones caused by nanobacteria, Dig. Dis. Sci. 51 (6) (2006) 1126e1132. E. Chabriere, D. Gonzalez, S. Azza, P. Durand, F.A. Shiekh, V. Moal, et al., Fetuin is the key for nanon self-propagation, Microb. Pathog. 73 (2014) 25e30. A. Swidsinski, M. Khilkin, H. Pahlig, S. Swidsinski, F. Priem, Time dependent changes in the concentration and type of bacterial sequences found in cholesterol gallstones, Hepatology 27 (1998) 662e665. G.P. Chen, L.M. Zhu, D. Cai, Research on bacterial infection in gallbladder in cholesterol gallstone patients, J. Hepatopancreatobiliary Surg. 19 (2007) 158e160, http://dx.doi.org/10.3969/j.issn.1007-1954.2007.03.007. L.M. Stewart, A.L. Smith, C.A. Pellegrini, R.W. Motson, L.W. Way, Pigment gallstones form as a composite of bacterial microcolonies and pigment solids, Ann. Surg. 206 (1987) 242e249. J.J. Fremont-Rahl, Z. Ge, C. Umana, M.T. Whary, N.S. Taylor, S. Muthupalani, et al., An analysis of the role of the indigenous microbiota in cholesterol gallstone pathogenesis, PloS One 8 (2013) e70657. L.M. Zhu, H. Zhang, J. Li, X.Z. Wang, D. Cai, Y.L. Zhang, Study on the mechanism of pronucleation of cholesterol crystal formation by bacteria, J. Hepatopancreatobiliary Surg. 21 (2009) 122e125, http://dx.doi.org/10.3969/ j.issn.1007-1954.2009.02.012. K. Ho, Rapid purification of beta-glucuronidase from human liver by immunoaffinity chromatography employing specific murine monoclonal igg binding to tresyl-activated agarose, Protein Expr. Purif. 2 (1991) 63e65. M. Kano, J. Shoda, T. Irimura, T. Ueda, R. Iwasaki, T. Urasaki, et al., Effects of long-term ursodeoxycholate administration on expression levels of secretory low-molecular-weight phospholipases A2 and mucin genes in gallbladders and biliary composition in patients with multiple cholesterol stones, Hepatology 28 (1998) 302e313. H. Nikaido, J.M. Pages, Broad-specificity efflux pumps and their role in multidrug resistance of gram-negative bacteria, FEMS Microbiol. Rev. 36 (2012) 340e363. J. Lin, O. Sahin, L.O. Michel, Q. Zhang, Critical role of multidrug efflux pump CmeABC in bile resistance and in vivo colonization of Campylobacter jejuni, Infect. Immun. 71 (2003) 4250e4259. W.A. Alrefai, R.K. Gill, Bile acid transporters: structure, function, regulation and pathophysiological implications, Pharm. Res. 24 (2007) 1803e1823. M. Begley, C.G.M. Gahan, C. Hill, The interaction between bacteria and bile, Fm Microbiol. Rev. 29 (2005) 625e651. G. Szakacs, A. Varadi, C. Ozvegy-Laczka, B. Sarkadi, The role of ABC transporters in drug absorption, distribution, metabolism, excretion and toxicity (ADME-Tox), Drug Discov. Today 13 (2008) 379e393. A. Endo, S. Okada, Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis, J. Biosci. Bioeng. 99 (2005) 216e221. D. Mamlouk, C. Hidalgo, M.J. Torija, M. Gullo, Evaluation and optimisation of bacterial genomic DNA extraction for no-culture techniques applied to vinegars, Food Microbiol. 28 (2011) 1374e1379. T. Garcia-Armisen, Z. Papalexandratou, H. Hendryckx, N. Camu, G. Vrancken, L. Vuyst, et al., Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library, Appl. Microbiol. Biotechnol. 87 (2010) 2281e2292. A. Swidsinski, P. Schlien, A. Pernthaler, U. Gottschalk, E. Barlehner, G. Decker, et al., Bacterial biofilm within diseased pancreatic and biliary tracts, Gut 54 (2005) 388e395. P. Portincasa, A. Moschetta, G. Palasciano, Cholesterol gallstone disease, Lancet
63
368 (2006) 230e239. [31] M. Azian, M.N. Hapizah, B. Khalid, Y. Khalid, A. Rosli, R. Jamal, et al., Use of the denaturing gradient gel electrophoresis (DGGE) method for mutational screening of patients with familial hypercholesterolaemia (FH) and familial defective apolipoprotein B100 (FDB), Malays. J. Pathol. 28 (2006) 7e15. [32] Y. Ying, Z.M. Lv, H. Min, J. Cheng, Dynamic changes of microbial community diversity in a photohydrogen producing reactor monitored by PCR-DGGE, J. Environ. Sci 9 (2008) 1118e1125. [33] X.H. Cao, X.L. Bai, Determination of b-Glucuronidase activity produced by E.coli, J. Chin. Inst. Food Sci. Technol. 3 (2003) 82e85, http://dx.doi.org/ 10.3969/j.issn.1009-7848.2003.04.019. [34] F.A. Oguntoyinbo, P. Tourlomousis, M.J. Gasson, A. Narbad, Analysis of bacterial communities of traditional fermented West African cereal foods using culture independent methods, Int. J. Food Microbiol. 145 (2011) 205e210. [35] K. Honda, D.R. Littman, The microbiome in infectious disease and inflammation, Annu. Rev. Immunol. 30 (2012) 759e795. [36] T. Wu, Z. Zhang, B. Liu, D. Hou, Y. Liang, J. Zhang, et al., Gut microbiota dysbiosis and bacterial community assembly associated with cholesterol gallstones in largescale study, BMC Genomics 14 (2013) 669, http://dx.doi.org/ 10.1186/1471-2164-14-669. [37] Y. Su, S.D. Wu, J.Z. Jin, Z.H. Zhang, Y. Fan, Role of intestinal barrier in pathogenesis of pigment gallstone in a guinea pig modelm, Hepatobiliary Pancreat. Dis. Int. 5 (2006) 443e448. [38] M. Rajilic-Stojanovic, W.M.D. Vos, The first 1000 cultured species of the human gastrointestinal microbiota, FEMS Microbiol. Rev. 38 (2014) 996e1047. [39] Y.L. Lightfoot, M. Mohamadzadeh, Tailoring gut immune responses with lipoteichoic acid-deficient Lactobacillus acidophilus, Front. Immunol. 4 (2013) 25. [40] T.A. Paixao, C.M. Roux, A.B. Hartigh, S. Sankaran-Walters, S. Dandekar, R.L. Santos, et al., Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O antigen, Infect. Immun. 77 (2009) 4197e4208. [41] M.C. Ferrero, C.A. Fossati, M. Rumbo, P.C. Baldi, Brucella invasion of human intestinal epithelial cells elicits a weak proinflammatory response but a significant CCL20 secretion, FEMS Immunol. Med. Microbiol. 66 (2012) 45e57. [42] A. Swidsinski, W. Ludwig, H. Pahlig, F. Priem, Molecular genetic evidence of bacterial colonization of cholesterol gallgallstones, Gastroenterology 108 (1995) 860e864. [43] D.K. Lee, P.I. Tarr, W.G. Haigh, S.P. Lee, Bacterial DNA in mixed cholesterol gallstones, Am. J. Gastroenterol. 94 (1999) 3502e3506. [44] W. Kwon, J.Y. Jang, E.C. Kim, J.W. Park, I.W. Han, M.J. Kang, et al., Changing trend in bile microbiology and antibiotic susceptibilities: over 12 years of experience, Infection 41 (2013) 93e102. [45] B. Yang, S.D. Zhu, Y.G. Zhao, H. Yue, X.J. Du, Y.S. Li, et al., Immunological electron microscopic study on relationship between hepatocytic endogenous b-glucuronidase and pigment stone formation, Chin. J. Hepatobiliary Surg. 6 (2000) 123e125. [46] N. Li, L.J. Xiao, S.W. Cheng, B.L. Xiao, W.B. Cheng, Q.Y. Li, The changes of bGlucuronidase in rabbit model with calcium bilirubinate stone, J. WCUMS 21 (1990) 185e187. [47] L. Vitetta, S.P. Best, A. Sali, Single and multiple cholesterol gallstones and the influence of bacteria, Med. Hyp. 55 (2000) 502e506. [48] W.J. Ma, Y. Zhou, A. Shrestha, H. Mao, F.Y. Li, N.S. Cheng, et al., Applying chemical bile duct embolization to achieve chemical hepatectomy in hepatolithiasis: a further experimental study, J. Surg. Res. 187 (2014) 113e121. [49] J. Shoda, M. Kano, T. Asano, T. Irimura, T. Ueda, R. Iwasaki, et al., Secretory low-molecular-weight phospholipases A2 and their specific receptor in bile ducts of patients with intrahepatic calculi: factors of chronic proliferative cholangitis, Hepatology 29 (1999) 1026e1036. [50] J.L.M. Sampaio, V.B. Ribeiro, J.C. Campos, F.P. Rozales, C.M. Magagnin, D.R. Falci, et al., Detection of OXA-370, an OXA-48-related class D b-lactamase, in Enterobacter hormaechei from Brazil, Antimicrob. Agents Chemother. 58 (2014). [51] C.M. Dierikx, E.V. Duijkeren, A.H.W. Schoormans, A.V. Essen-Zandbergen, K. Veldman, A. Kant, et al., Occurrence and characteristics of extended-spectrum-b-lactamase- and AmpC-producing clinical isolates derived from companion animals and horses, J. Antimicrob. Chemother. 67 (2012) 1368e1374.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Cholesterol gallstones and bile host diverse bacterial communities with potential to promote the formation of gallstones Yuhong Peng a, Yang Yang a, Yongkang Liu b, Yuanyang Nie a, Peilun Xu a, Baixue Xia a, Fuzhou Tian c, Qun Sun a, * a Key Laboratory of Bio-resource and Bio-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, PR China b The 452nd Hospital of PLA, Chengdu, Sichuan 610000, PR China c Chengdu Military General Hospital, Chengdu, Sichuan 610083, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 December 2014 Received in revised form 4 May 2015 Accepted 6 May 2015 Available online 7 May 2015
The prevalence of cholesterol gallstones has increased in recent years. Bacterial infection correlates with the formation of gallstones. We studied the composition and function of bacterial communities in cholesterol gallstones and bile from 22 cholesterol gallstone patients using culture-dependent and culture-independent methods. Altogether fourteen and eight bacterial genera were detected in cholesterol gallstones and bile, respectively. Pseudomonas spp. were the dominant bacteria in both cholesterol gallstones and bile. As judged by diversity indices, hierarchical clustering and principal component analysis, the bacterial communities in gallstones were different from those in bile. The gallstone microbiome was considered more stable than that of bile. The different microbial communities may be partially explained by differences in their habitats. We found that 30% of the culturable strains from cholesterol gallstones secreted b-glucuronidase and phospholipase A2. Pseudomonas aeruginosa strains showed the highest b-glucuronidase activity and produced the highest concentration of phospholipase A2, indicating that Ps. aeruginosa may be a major agent in the formation of cholesterol gallstones. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Cholesterol gallstones Bile Bacterial community PCR-DGGE b-Glucuronidase Phospholipase A2
1. Introduction The prevalence of cholesterol gallstones, a common disease including gallstones, common bile duct gallstones, and intrahepatic bile duct gallstones, has increased in recent years, especially in the Western world [1]. In China, along with the population aging, obesity, hyperlipidemia and diabetes, the incidence of the cholesterol gallstones has risen to 10% [2e4]. The secretion of cholesterol in bile and abnormal metabolism are considered as the primary pathophysiological causes in the formation of cholesterol gallstones. Factors that are thought to or known to promote the formation of cholesterol gallstones include estrogen and progestogens, cholesterol-lowering medications, obesity and rapid weight loss [5,6]. The consistency and relevance of symptomatic gallbladder stones in identical twins is significantly higher than in double egg twins, indicating that both genetic and environmental
* Corresponding author. E-mail address: [email protected] (Q. Sun). http://dx.doi.org/10.1016/j.micpath.2015.05.002 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
factors are involved [7]. The frequent Cyp7a1 polymorphism in A allele in Chinese patients with cholesterol gallstone implies that gallstone formation is related to the expression of genes [8]. Other potential factors associated with the formation of cholesterol gallstones include mucoprotein, aminopeptidase N and vesicular protein [9,10]. Bacterial colonization correlate with the formation of gallstones. Bacterial infection has been proposed as a key factor in the pathogenesis of pigment gallstones [11]. Some study confirmed that nanobacteria may use its self-propagation to cause the formation of black pigment gallstones in rabbits [12,13]. Meanwhile the role of bacteria in cholesterol gallstones formation has aroused increasingly more attention. Swidsinski et al. [14] used RT-PCR (reverse transcription e polymerase chain reaction) to release an 80% detection rate of bacterial DNA in cholesterol gallstones. Chen et al. [15] showed that the detection rate of bacterial DNA in bile, gallstone and biliary tract mucous membrane was 77%, 84% and 62%, respectively. Bacteria may play a critical role in the formation of cholesterol crystals [16]. Alterations in the gastrointestinal microbiome may change aspects of
58
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
cholesterol gallstone pathogenesis and then these changes may impact cholelithogenesis [17]. Pseudomonas aeruginosa and Enterococcus faecalis shortened the cholesterol crystallization time in the model bile indicating that these species may be crucial in the formation of the cholesterol gallstones [18]. These findings indicated that bacterial community composition might influence the formation of cholesterol gallstones. Thus, it is crucial to understand the structure of bacterial community and its potential connection in the pathogenesis of cholesterol gallstones. Bacteria speed up the gallstone formation by secrefing crystal formation factors. b-glucuronidase and phospholipase A2 have been identified as the major factors leading to the formation of gallstones. The role of b-glucuronidase in gallstone formation process has been fully recognized [19]. The hydrolysis of phospholipids by phospholipase A2 consequently caused the crystallization of cholesterol and finally led to the formation of gallstones [20]. Furthermore, multidrug-resistance efflux pump proteins expressed by bacteria may help bacteria to survive in bile [21e25]. Traditionally characterizing bacteria has relied on cultivation. However, most of the bacteria in are non-cultivable by conventional methods. Therefore, culture-independent methods like polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) have been widely used to study microbial communities present in biological samples [26e28]. To get a more wide view on the microbial communities in gallstones and bile, we used both cultivation and the culture-independent PCR-DGGE method. In addition, we tested the b-G activity and the PLA2 production of the isolated bacteria to better understand the bacterial community in the cholesterol gallstones and to assess which bacteria have potential to promote the formation of cholesterol gallstones. 2. Material and methods 2.1. Patients and sample collection The 22 cholesterol gallstone samples (S1eS22) and 12 bile samples (B1eB12) from 22 gallbladder-stone patients were provided by Chengdu Military General Hospital. The 12 male and 10 female provided a signed informed consent to partake in the study. The average age of the patients was 45.1 ± 14.2 (SD). The patients did not receive any antibiotics or probiotics before the study. Gallstones were removed aseptically from the gallbladder of each patient. At the same time, gallbladder bile was aspirated from indwelling catheters into 10 ml sterile tubes from 12 patients. All samples were stored in their original tubes at 40 C until further analyses. The gallstones were classified as cholesterol gallstones with above 70% cholesterol content by Fourier transform infrared spectroscopy as described [29,30]. 2.2. Total DNA extraction Cholesterol gallstones (180e200 mg) were homogenized by grinding with a sterile mortar and then by mechanical disruption with a bead beater. Total DNA from homogenized gallstones and bile samples was extracted by DNA Stool Kit (OMEGA) following the manufacturer's instructions. 2.3. PCR amplification of V3 region of 16S rDNA PCR amplification was performed in S1000™ Thermal Cycler (BIO-RAD) with reagents obtained from TaKaRa (Japan). In the first step of a nested PCR method, 16S rRNA gene fragments were amplified using 20 pmol of each primer 27F (50 -AGA GTT TGA TCM TGG CTC AG-30 ) and 1492R (50 -TAC GGC TAC CTT GTT ACG ACT T-30 ) in 50 ml of amplification buffer (250 mM of each dNTP, 10 mM
TriseHCl, 1.5 mM MgCl2, 50 mM KCl and 2.5 U Taq DNA polymerase) with 5 ml sample DNA as the template. The amplification was performed with the following temperature profile: an initial denaturation at 94 C for 5 min; 30 cycles of denaturation at 94 C for 1 min, annealing at 55 C for 1 min and extension at 72 C for 1.5 min; and a final extension at 72 C for 10 min. The amplification products were subjected to electrophoresis in 1% (w/v) agarose gel and extracted by Gel Extraction Kit (OMEGA) according the manufacturer's instructions. The extracted products were used as template for amplification of V3 region of 16S rDNA. The V3 region was amplified using primers 338F (50 -ACT CCT ACG GGA GGC AGC AG-30 ) with a GCclamp (50 -CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG T-30 ) at the 50 end and 518R (50 -GTA TTA CCG CGG CTG CTG GCA-30 ) in 50 ml of amplification buffer as described above with 5 ml extract as the template. The amplification was performed with the following temperature profile: 5 min at 95 C; 10 cycles of denaturation at 94 C for 30 s, annealing for 30 s with decreasing the temperature by 0.5 C per cycle from 60 to 55 C, extension at 72 C for 35 s; 25 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 30 s; and a 7 min final extension at 72 C. The amplification products were subjected to electrophoresis in 1% agarose gels (Invitrogen) using a D2000 DNA Marker (TIANGEN BIOTECH, Beijing, China) as a molecular weight standard. The gels were run at 120 V for 20 min in 1 TAE, stained with 0.1 ml ml1 (v/ v) Green View and evaluated as described [31,32].
2.4. DGGE analysis DGGE was performed with the D-Code™ universal mutation detection system (BioRad, Lab., USA). The amplification products from cholesterol gallstone samples were separated in 8% (w/v) polyacrylamide gels in a denaturing gradient of 40%e65% (100% denaturant defined as 7 M urea and 40% formamide in 1 TAE buffer, pH 7.4) at 110 V and 60 C for 6.5 h. The amplification products from bile samples were separated in a denaturing gradient of 45%e55% under conditions described above for separating preferably bands. Gels were stained with SYBR Green (Invitrogen) for 45 min, visualized with a UV transilluminator (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed with Quantity One software (Bio-Rad, USA).
2.5. Amplification and sequencing of excised DGGE bands Selected bands were excised from the DGGE gel, resuspended in sterile water and kept at 4 C for 24 h before amplification of the V3 region as described above. The amplification products were sequenced at Invitrogen (Shanghai, China). The sequences were compared with NCBI database to determine the closest match sequence.
2.6. Isolation and identification of culturable bacteria Cholesterol gallstone and bile samples were inoculated onto modified tryptic soy broth (TSB). The plates were incubated at 37 C under aerobic conditions (CO2 concentration 5%). DNA was extracted from bacterial mass taken from single colonies by Bacterial DNA Extraction kit (TIANGEN, Beijing, China) while the rest of the colony was preserved for further testing. The 16S rDNA was amplified using primers 27F and 1492R as described above and sequenced at Invitrogen (Shanghai, China). The sequences were compared with NCBI database to determine the closest match sequence.
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
2.7. Determining the b-glucuronidase activity and phospholipase A2 production of the isolated bacteria The isolates were grown in liquid modified TSB media for 24 h. After cultivation, cells were dissolved by adding 180 ml lysozyme (TIANGEN, Beijing, China) in 5 ml culture solution and incubating for 35 min at 37 C. The cells were pelleted at 10,000 r min1 for 2 min. b-Glucuronidase activity was tested as described [33]. Briefly, 2 ml p-nitrophenyl- b-D-glucuronide was added to 2 ml supernatant, the solution was incubated for 1 h at 37 C, after which the reaction was terminated by adding 0.4 ml NaOH (0.1 mol l1). 2 ml PBS (0.05 mol l1, pH 7.0) served as a negative control. Standards for quantifying the amount of released p-nitrophenol were done using from 10 mmol l1 to 2 mmol l1 dilutions of p-nitrophenol in PBS (0.05 mol l1, pH 7.0). After adjusting zero using the negative control, OD at 405 nm of the standards and samples was measured. Enzyme activity (U l1) was defined as 1 mmol p-nitrophenol released from p-nitrophenylb-D-glucuronide in one minute. The concentration of phospholipase A2 was measured by PLA2 ELISA Kit according the manufacturer's instructions. 2.8. Data analysis Quantity One software was used to convert individual DGGE lanes to densitometric profiles. The indices of species richness, evenness and the Shannon Diversity were calculated based on the profiles. Cluster analysis of the band patterns was performed with the unweighted-pair group method using average linkages. Principal component analysis was performed by SPSS 17.0 based on the number and relative intensity of the band of each DGGE fingerprints as described [34]. Multiple samples comparisons were performed using one-way ANOVA. Statistical significance was set at P < 0.05. 3. Results 3.1. Profiling of bacterial communities in cholesterol gallstones and bile using PCR-DGGE
59
higher in the gallstones than in the bile. In the cluster analysis, the DGGE band patterns of the cholesterol gallstones samples differed from those of bile samples (Fig. 5). The six cholesterol gallstones samples and six bile samples formed three clusters. The gallstone samples clustered together with pairwise similarity coefficients from 0.33 to 0.73 (Cluster C), and the bile samples formed two clusters (Cluster A and B), which had very low similarity with the gallstone cluster. A principal component analysis on the bacterial communities in cholesterol gallstones and bile explained 67.39% (PC1) and 32.61% (PC2) of the total variance (Fig. 6). As in the cluster analysis, the bile samples and cholesterol gallstone samples clustered separately in PCA, suggesting that the bacterial communities in cholesterol gallstones differed significantly from those in bile, while the cholesterol gallstone communities had small differences with one another. In contrast, the difference within the bile samples was great. 3.3. Identification, b-glucuronidase activity and phospholipase A2 production of the isolated bacteria The diversity and function of culturable bacteria from cholesterol gallstones was assessed by isolating them and analyzing their b-glucuronidase activity and phospholipase A2 production. In total 93 strains was isolated from gallstone samples and identified based on 16S rDNA sequence. The isolates represented seven species, out of which all but Bacillus amyloliquefaciens had been identified with the culture-independent approach (Tables 1 and 3). Altogether 35 out of 93 stains showed b-glucuronidase activity and produced phospholipase A2. Ps. aeruginosa strains showed highest and Pseudomonas stutzeri strains the lowest b-glucuronidase activities (Table 3). The b-glucuronidase activities of Ps. stutzeri and B. amyloliquefaciens strains were significantly lower than those of the other species (P > 0.05). There were no statistically significant differences in phospholipase A2 production between the producing strains (Table 3). Both the b-glucuronidase activity and the phospholipase A2 production suggested that these strains could potentially induce the formation of cholesterol gallstones. 4. Discussion
The diversity of bacterial communities in cholesterol gallstones and bile was assessed using 16S-rDNA PCR-DGGE. The differences between samples were high and every sample had its specific bands (Figs. 1 and 2). A total of 19 bands were selected for amplification, sequencing, and identification by searching the National Center for Biotechnology Information (NCBI) database. Altogether 14 bacterial genera were identified (identity above than 97%) (Tables 1 and 2). Genera Brucella, Lachnospiraceae, Escherichia, Citrobacter, Shinella, Lactococcus and Lactobacillus were identified only from the gallstones, but not in bile, while the genus Acinetobacter only in bile. According to the statistical analysis of relative abundances, Pseudomonas was the dominant genus in both cholesterol gallstones and bile (Fig. 3). In addition, the relative abundance of the genera Pseudomonas, Bacillus, Klebsiella, Clostridium, Staphylococcus and Enterobacter was significantly higher in cholesterol gallstones than in bile (P > 0.05, n ¼ 3). 3.2. The comparison of bacterial communities in cholesterol gallstones and bile We used cholesterol gallstones and surrounding bile from 22 patients to compare the difference of bacterial communities between these two environments. The Shannon diversity index (H) was significantly higher (P > 0.05) in the gallstones than in the bile (Fig. 4). Likewise, the richness and evenness were significantly
Microbiome is important in many immunological and metabolic diseases [35], as its variation in the intestinal microbiome may change cholesterol gallstone pathogenesis. The gut microbiome of gallstone patients was shown to be significantly different from that of healthy people [36]. In this research, both culture-independent and culture-dependent analyses were performed to compare microbial communities in cholesterol gallstones and bile. To our knowledge, this is the first study to apply the PCR-DGGE to clarify the composition of bacterial communities within cholesterol gallstones. Dysfunction of the intestinal mucosa barrier may result in translocation of the bacteria into bile duct where the bacteria may promote the formation of gallstones [37]. Bacteria that inhabit the gastrointestinal tract (GIT) were also detected in the cholesterol gallstones [36]. In addition to genera previously reported, we detected Escherichia, Brucella, Citrobacter, Shinella, Aurantimonas, Lachnospiraceae and Lactobacillus in the gallstones, among which Citrobacter, Lactobacillus and Aurantimonas are common members of the GIT microbiome [38]. After Lactobacillus infected gallbladder, the outer membrane proteins of Lactobacillus could stimulate humoral immunity and cellular immunity in human body [39]. The presence of GIT bacteria in cholesterol gallstones indicated that once getting into the gallbladder, the GIT bacteria might trigger the immune system and accelerate the formation of cholesterol
60
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
Fig. 1. DGGE fingerprints of V3 region in bacterial 16S rDNA from bacterial communities in cholesterol gallstones. The bacteria identified on the sequence analyses of the selected bands are summarized in Table 1.
gallstones. Brucella is a pathogen that can invade the body through the digestive tract mucosa [40,41]. Our results indicated that Brucella could collaborate with other bacteria to accelerate the formation of cholesterol crystals. It has been proposed that Ps. aeruginosa plays a major role in the infection of biliary tract and more attention should be paid on its role in cholelithiasis [42,43]. Enterococcus faecium is both a member of the GIT microbiome and a common agent in biliary tract infection. Moreover, both Ps. aeruginosa and E. faecium shorten the time of the formation of cholesterol crystals [18]. The abundance of E. faecium, Klebsiella spp. and Enterobacter spp. in human bile has increased significantly from 1988 to 2010 [44]. Our DGGE results showed that Pseudomonas, Enterococcus, Klebsiella and Enterobacter were all relatively abundant in cholesterol gallstones, indicating that these four genera
Table 1 The bacteria identified based on the sequence analysis of selected bands in DGGE fingerprints from bacterial communities in cholesterol gallstones. Band no.
Closest NCBI database match
Similarity (%)
Accession no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Uncultured Bacillus sp. Brucella melitensis Lachnospiraceae Klebsiella oxytoca Escherichia sp. Citrobacter sp. Clostridium clostridioforme Staphylococcus sp. Shinella sp. Enterococcus durans Enterococcus faecium Enterobacter cloacae Enterobacter asburiae Enterobacter hormaechei Lactococcu spiscium Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas stutzeri Lactobacillus sp.
99 100 97 99 98 97 98 100 98 100 99 99 100 99 99 99 99 100 99
KF581441.1 KF771410.1 EF704617.1 KC593550.1 JQ828856.1 EU360138.1 KC143063.1 FJ937931.1 JQ684917.1 KF918537.1 HE646384.1 KC509581.1 CP007546.1 KF862929.1 KF983318.1 KJ184550.1 KF913776.1 KF850545.1 HQ420284.1
were potentially connected with cholesterol crystallization as dominant population. As judged by diversity indices, hierarchical clustering and Table 2 The bacteria identified based on the sequence analysis of selected bands in DGGE fingerprints from bacterial communities in bile.
Fig. 2. The DGGE fingerprint of V3 region in bacterial 16S rDNA from bacterial communities in bile. The bacteria identified based on the sequence analyses of the selected bands are summarized in Table 2.
Band no.
Closest NCBI database match
Similarity (%)
Accession no.
1 2 3 4 5 6 7 8 9 10 11 12
Bacillus oleronius Klebsiella oxytoca Clostridium sp Uncultured Staphylococcus sp. Enterococcus durans Enterobacter asburiae Staphylococcus epidermidis Enterobacter aerogenes Pseudomonas aeruginosa Pseudomonas stutzeri Acinetobacter johnsonii Acinetobacter lwoffii
100 99 100 100 100 100 100 100 100 100 100 100
KF835577.1 KC593550.1 KJ194597.1 FJ937931.1 KJ631376.1 CP007546.1 KF862930.1 KJ011873.1 KJ461700.1 KJ185383.1 HG810396.1 KF704079.1
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
61
Fig. 3. The estimated abundances of identified members of bacterial communities in cholesterol gallstones and bile. * Difference significant at 0.05 level (n ¼ 3).
principal component analysis, the bacterial communities in gallstones were different from those in bile. Based on the low similarities between the bile communities, the bile microbiome could be considered as more dynamic. The different microbial communities may be partially explained by differences in their habitats. The bile salts are toxic to the bacteria. In addition, bile changes frequently because of enterohepatic circulation, whereas cholesterol gallstones provide a stable and protected ecological niche. The similarity of the gallstone bacterial communities indicated that bacteria which cause the formation of cholesterol gallstones may be same in different patients, and therefore more attention should be paid on these bacteria in the future. Both b-glucuronidase and phospholipase A2 are known risk factors in the formation of cholesterol gallstones. b-glucuronidase is a hydrolase found in all kinds of cells in the human body, especially in the liver. b-glucuronidase is either endogenous, secreted by cells, or exogenous, secreted by bacteria. The gallbladder tissue secretes endogenous b-glucuronidase [45]. Both endogenous and exogenous
Fig. 4. The comparison of Shannon diversity index (SDI), evenness and richness of bacterial communities from cholesterol gallstones and bile. * Difference significant at 0.05 level (n ¼ 4).
b-glucuronidase may participate into the formation of cholesterol gallstones. The activity of exogenous b-glucuronidase rises rapidly in early-stage biliary infection when the infection is treated [46]. Bacteria may secrete enzymes to accelerate the formation of insoluble precipitates which could serve as sites of nucleation in the formation of cholesterol gallstones [47]. According to Ma et al. [48], exogenous b-glucuronidase could make cholesterol in bile to precipitate into crystals because of cholesterol saturation. Tao et al. also proposed that during a bacterial infection, the inflammatory factors secreted by bacteria would accelerate the secretion of endogenous b-glucuronidase from both hepatocytes and biliary epithelial cells, after which these two factors would participate in the formation of cholesterol gallstones. Bacterial infection of biliary tract causes an increase in the amount of phospholipase A2 [49]. Phospholipase A2 may hydrolyze lecithin to disturb the balance of bile acid, cholesterol
Fig. 5. Unweighted-pair group method with average linkages analysis of the DGGE band patterns of V3 region in bacterial 16S rDNA from cholesterol gallstones and bile. Cluster A and B: bile samples. Cluster C: cholesterol gallstone samples.
62
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
Fig. 6. Principal component analysis of the relative optical densities produced by bacterial activities from cholesterol gallstones and bile samples. B1eB12: bile samples. S1eS22: cholesterol gallstone samples.
Table 3 The identification of isolated bacteria based on 16S rDNA sequence analysis, their b-glucosidase activities and phospholipase A2 production. Closet relative n (%)
Similarity (%)
Accession no.
b-G (U/l)
Pseudomonas aeruginosa 10(10.6) Enterobacter hormaechei 3(3.2) Enterococcus faecium 8(8.6) Klebsiella oxytoca 4(4.3) Pseudomonas stutzeri 3(3.2) Enterobacter cloacae 4(4.3) Bacillus amyloliquefaciens 3(3.2)
100 99 99 100 100 100 99
KF956583.1 KF836497.1 KF358453.1 KF254665.1 KJ185383.1 CP006580.1 KJ149810.1
33.6 15.6 19.0 16.8 12.1 27.4 12.4
and phosphatidic acid, ultimately resulting in decreased cholesterol saturation index. The decrease of saturation index makes cholesterol more likely to crystallize. We found that 30% of the culturable isolates from cholesterol gallstones secreted b-glucuronidase and phospholipase A2. Ps. aeruginosa strains showed the highest b-glucuronidase activity and produced the highest concentration of phospholipase A2, indicating that Ps. aeruginosa may be a major agent in the formation of cholesterol gallstones. E. faecium has been considered as a gallstone-associated bacterium, but its role in cholelithiasis was not clear. We showed that E. faecium secreted both b-glucuronidase and phospholipase A2 thereby clarifying the potential of E. faecium in gallstone formation. We found that Enterobacter hormaechei, B. amyloliquefaciens and Enterobacter cloacae secrete both b-glucuronidase and phospholipase A2. These bacteria are also b-lactamase producers [50,51]. b-lactamase gives resistance to antibiotics making these bacteria in gallbladder less vulnerable to antibiotic treatments. Therefore, determining which bacteria have infected gallbladder is important in choosing the appropriate antibiotics for treatment. In summary, we confirmed the association between bacteria and cholesterol gallstones. Using PCR-DGGE we found that at least 14 bacterial genera reside in cholesterol gallstones. Pseudomonas was the dominant bacteria in both bile and cholesterol gallstones. The bacterial communities in cholesterol gallstones and bile were significantly different. Strains representing seven species were isolated from the gallstones. Altogether 30% of the strains secreted b-glucuronidase and phospholipase A2. In future, these seven species should be taken into consideration when
± ± ± ± ± ± ±
16.7 4.0 9.9 4.9 2.1 16.6 11.3
PLA2 (ng/ml) 0.589291 0.588593 0.587404 0.586954 0.587213 0.589073 0.588547
± ± ± ± ± ± ±
0.010188 0.009276 0.009659 0.011004 0.001680 0.007744 0.011167
designing clinical prevention and treatment. Our results could provide basis for designing a detailed clinical therapeutic guideline. The role of the b-glucuronidase and phospholipase A2 producers in gallstone formation should be verified by further follow-up studies. Acknowledgments This research was supported by grants from the Natural Science Foundation of J1103518. References [1] K.J. Van Erpecum, Pathogenesis of cholesterol and pigment gallstones: an update, Clin. Res. Hepatol. Gastroenterol. 35 (2011) 281e287. [2] Q. Zeng, Y. He, D.C. Qiang, L.X. Wu, Prevalence and epidemiological pattern of gallstones in urban residents in China, Eur. J. Gastroenterol. Hepatol. 24 (2012) 1459. [3] X.G. Chen, J.Q. Liu, M.H. Peng, et al., Clinical epidemiological study on intrahepatic cholelithiasis: analysis of 8585 cases, Hepatobiliary Pancreat. Dis. Int. 2 (2003) 281. [4] C.Y. Chen, S.C. Shiesh, X.Z. Lin, Biliary sludge and pigment stone formation in bile duct-ligated guinea pigs, Dig. Dis. Sci. 44 (1999) 203. [5] B. Liu, V. Beral, A. Balkwill, J. Green, S. Sweetland, G. Reeves, et al., Gallbladder disease and use of transdermal versus oral hormone replacement therapy in postmenopausal women: prospective cohort study, Bmj 337 (2008) a386. [6] H. Volzke, S.E. Baumeister, D. Alte, W. Hoffmann, C. Schwahn, P. Simon, et al., Independent risk factors for gallstone formation in a region with high cholelithiasis prevalence, Digestion 71 (2005) 97e105. [7] D. Katsika, A. Grjibovski, C. Einarsson, F. Lammert, P. Lichtenstein, H.U. Marschall, Genetic and environmental influences on symptomatic gallstone disease: a Swedish study of 43,141 twin pairs, Hepatology 41 (2005) 1138e1143. [8] Z.Y. Jiang, P. Parini, G. Eggertsen, M.A. Davis, H. Hu, G.J. Suo, et al., Increased
Y. Peng et al. / Microbial Pathogenesis 83-84 (2015) 57e63
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23] [24] [25]
[26]
[27]
[28]
[29]
[30]
expression of LXR alpha, ABCG5, ABCG8, and SR-BI in the liver from normolipidemic, nonobese Chinese gallstone patients, J. Lipid Res. 49 (2008) 464e472. D.C. Zhang, J.B. Xiang, L.Y. Wang, Z.B. Xu, L.D. Sun, F. Zhou, et al., Comparative proteomic analysis of gallbladder bile proteins related to cholesterol gallstones, PloS One 8 (2013) 350e352. Y.T. Li, X.G. Zhu, X.L. Zhao, G.S. An, H.T. Jia, Expression of aminopeptidase N and its correlation with the gallstones formation, Chin. J. Biochem. Mol. Biol. 17 (2001) 425e429. T. Maki, Pathogenesis of calcium bilirubinate gallstone: role of E. coli, bglucuronidase and coagulation by inorganic ions, polyelectrolytes and agitation, Ann. Surg. 164 (1996) 90e100. L.M. Wang, W.L. Shen, J. Wen, X. An, L.Y. Cao, B.Q. Wang, An animal model of black pigment gallstones caused by nanobacteria, Dig. Dis. Sci. 51 (6) (2006) 1126e1132. E. Chabriere, D. Gonzalez, S. Azza, P. Durand, F.A. Shiekh, V. Moal, et al., Fetuin is the key for nanon self-propagation, Microb. Pathog. 73 (2014) 25e30. A. Swidsinski, M. Khilkin, H. Pahlig, S. Swidsinski, F. Priem, Time dependent changes in the concentration and type of bacterial sequences found in cholesterol gallstones, Hepatology 27 (1998) 662e665. G.P. Chen, L.M. Zhu, D. Cai, Research on bacterial infection in gallbladder in cholesterol gallstone patients, J. Hepatopancreatobiliary Surg. 19 (2007) 158e160, http://dx.doi.org/10.3969/j.issn.1007-1954.2007.03.007. L.M. Stewart, A.L. Smith, C.A. Pellegrini, R.W. Motson, L.W. Way, Pigment gallstones form as a composite of bacterial microcolonies and pigment solids, Ann. Surg. 206 (1987) 242e249. J.J. Fremont-Rahl, Z. Ge, C. Umana, M.T. Whary, N.S. Taylor, S. Muthupalani, et al., An analysis of the role of the indigenous microbiota in cholesterol gallstone pathogenesis, PloS One 8 (2013) e70657. L.M. Zhu, H. Zhang, J. Li, X.Z. Wang, D. Cai, Y.L. Zhang, Study on the mechanism of pronucleation of cholesterol crystal formation by bacteria, J. Hepatopancreatobiliary Surg. 21 (2009) 122e125, http://dx.doi.org/10.3969/ j.issn.1007-1954.2009.02.012. K. Ho, Rapid purification of beta-glucuronidase from human liver by immunoaffinity chromatography employing specific murine monoclonal igg binding to tresyl-activated agarose, Protein Expr. Purif. 2 (1991) 63e65. M. Kano, J. Shoda, T. Irimura, T. Ueda, R. Iwasaki, T. Urasaki, et al., Effects of long-term ursodeoxycholate administration on expression levels of secretory low-molecular-weight phospholipases A2 and mucin genes in gallbladders and biliary composition in patients with multiple cholesterol stones, Hepatology 28 (1998) 302e313. H. Nikaido, J.M. Pages, Broad-specificity efflux pumps and their role in multidrug resistance of gram-negative bacteria, FEMS Microbiol. Rev. 36 (2012) 340e363. J. Lin, O. Sahin, L.O. Michel, Q. Zhang, Critical role of multidrug efflux pump CmeABC in bile resistance and in vivo colonization of Campylobacter jejuni, Infect. Immun. 71 (2003) 4250e4259. W.A. Alrefai, R.K. Gill, Bile acid transporters: structure, function, regulation and pathophysiological implications, Pharm. Res. 24 (2007) 1803e1823. M. Begley, C.G.M. Gahan, C. Hill, The interaction between bacteria and bile, Fm Microbiol. Rev. 29 (2005) 625e651. G. Szakacs, A. Varadi, C. Ozvegy-Laczka, B. Sarkadi, The role of ABC transporters in drug absorption, distribution, metabolism, excretion and toxicity (ADME-Tox), Drug Discov. Today 13 (2008) 379e393. A. Endo, S. Okada, Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis, J. Biosci. Bioeng. 99 (2005) 216e221. D. Mamlouk, C. Hidalgo, M.J. Torija, M. Gullo, Evaluation and optimisation of bacterial genomic DNA extraction for no-culture techniques applied to vinegars, Food Microbiol. 28 (2011) 1374e1379. T. Garcia-Armisen, Z. Papalexandratou, H. Hendryckx, N. Camu, G. Vrancken, L. Vuyst, et al., Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library, Appl. Microbiol. Biotechnol. 87 (2010) 2281e2292. A. Swidsinski, P. Schlien, A. Pernthaler, U. Gottschalk, E. Barlehner, G. Decker, et al., Bacterial biofilm within diseased pancreatic and biliary tracts, Gut 54 (2005) 388e395. P. Portincasa, A. Moschetta, G. Palasciano, Cholesterol gallstone disease, Lancet
63
368 (2006) 230e239. [31] M. Azian, M.N. Hapizah, B. Khalid, Y. Khalid, A. Rosli, R. Jamal, et al., Use of the denaturing gradient gel electrophoresis (DGGE) method for mutational screening of patients with familial hypercholesterolaemia (FH) and familial defective apolipoprotein B100 (FDB), Malays. J. Pathol. 28 (2006) 7e15. [32] Y. Ying, Z.M. Lv, H. Min, J. Cheng, Dynamic changes of microbial community diversity in a photohydrogen producing reactor monitored by PCR-DGGE, J. Environ. Sci 9 (2008) 1118e1125. [33] X.H. Cao, X.L. Bai, Determination of b-Glucuronidase activity produced by E.coli, J. Chin. Inst. Food Sci. Technol. 3 (2003) 82e85, http://dx.doi.org/ 10.3969/j.issn.1009-7848.2003.04.019. [34] F.A. Oguntoyinbo, P. Tourlomousis, M.J. Gasson, A. Narbad, Analysis of bacterial communities of traditional fermented West African cereal foods using culture independent methods, Int. J. Food Microbiol. 145 (2011) 205e210. [35] K. Honda, D.R. Littman, The microbiome in infectious disease and inflammation, Annu. Rev. Immunol. 30 (2012) 759e795. [36] T. Wu, Z. Zhang, B. Liu, D. Hou, Y. Liang, J. Zhang, et al., Gut microbiota dysbiosis and bacterial community assembly associated with cholesterol gallstones in largescale study, BMC Genomics 14 (2013) 669, http://dx.doi.org/ 10.1186/1471-2164-14-669. [37] Y. Su, S.D. Wu, J.Z. Jin, Z.H. Zhang, Y. Fan, Role of intestinal barrier in pathogenesis of pigment gallstone in a guinea pig modelm, Hepatobiliary Pancreat. Dis. Int. 5 (2006) 443e448. [38] M. Rajilic-Stojanovic, W.M.D. Vos, The first 1000 cultured species of the human gastrointestinal microbiota, FEMS Microbiol. Rev. 38 (2014) 996e1047. [39] Y.L. Lightfoot, M. Mohamadzadeh, Tailoring gut immune responses with lipoteichoic acid-deficient Lactobacillus acidophilus, Front. Immunol. 4 (2013) 25. [40] T.A. Paixao, C.M. Roux, A.B. Hartigh, S. Sankaran-Walters, S. Dandekar, R.L. Santos, et al., Establishment of systemic Brucella melitensis infection through the digestive tract requires urease, the type IV secretion system, and lipopolysaccharide O antigen, Infect. Immun. 77 (2009) 4197e4208. [41] M.C. Ferrero, C.A. Fossati, M. Rumbo, P.C. Baldi, Brucella invasion of human intestinal epithelial cells elicits a weak proinflammatory response but a significant CCL20 secretion, FEMS Immunol. Med. Microbiol. 66 (2012) 45e57. [42] A. Swidsinski, W. Ludwig, H. Pahlig, F. Priem, Molecular genetic evidence of bacterial colonization of cholesterol gallgallstones, Gastroenterology 108 (1995) 860e864. [43] D.K. Lee, P.I. Tarr, W.G. Haigh, S.P. Lee, Bacterial DNA in mixed cholesterol gallstones, Am. J. Gastroenterol. 94 (1999) 3502e3506. [44] W. Kwon, J.Y. Jang, E.C. Kim, J.W. Park, I.W. Han, M.J. Kang, et al., Changing trend in bile microbiology and antibiotic susceptibilities: over 12 years of experience, Infection 41 (2013) 93e102. [45] B. Yang, S.D. Zhu, Y.G. Zhao, H. Yue, X.J. Du, Y.S. Li, et al., Immunological electron microscopic study on relationship between hepatocytic endogenous b-glucuronidase and pigment stone formation, Chin. J. Hepatobiliary Surg. 6 (2000) 123e125. [46] N. Li, L.J. Xiao, S.W. Cheng, B.L. Xiao, W.B. Cheng, Q.Y. Li, The changes of bGlucuronidase in rabbit model with calcium bilirubinate stone, J. WCUMS 21 (1990) 185e187. [47] L. Vitetta, S.P. Best, A. Sali, Single and multiple cholesterol gallstones and the influence of bacteria, Med. Hyp. 55 (2000) 502e506. [48] W.J. Ma, Y. Zhou, A. Shrestha, H. Mao, F.Y. Li, N.S. Cheng, et al., Applying chemical bile duct embolization to achieve chemical hepatectomy in hepatolithiasis: a further experimental study, J. Surg. Res. 187 (2014) 113e121. [49] J. Shoda, M. Kano, T. Asano, T. Irimura, T. Ueda, R. Iwasaki, et al., Secretory low-molecular-weight phospholipases A2 and their specific receptor in bile ducts of patients with intrahepatic calculi: factors of chronic proliferative cholangitis, Hepatology 29 (1999) 1026e1036. [50] J.L.M. Sampaio, V.B. Ribeiro, J.C. Campos, F.P. Rozales, C.M. Magagnin, D.R. Falci, et al., Detection of OXA-370, an OXA-48-related class D b-lactamase, in Enterobacter hormaechei from Brazil, Antimicrob. Agents Chemother. 58 (2014). [51] C.M. Dierikx, E.V. Duijkeren, A.H.W. Schoormans, A.V. Essen-Zandbergen, K. Veldman, A. Kant, et al., Occurrence and characteristics of extended-spectrum-b-lactamase- and AmpC-producing clinical isolates derived from companion animals and horses, J. Antimicrob. Chemother. 67 (2012) 1368e1374.
