Bacterial DNA in mixed cholesterol gallstones

THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 1999 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.

Vol. 94, No. 12, 1999 ISSN 0002-9270/99/$20.00 PII S0002-9270(99)00673-5

Bacterial DNA in Mixed Cholesterol Gallstones Dong Ki Lee, M.D., Phillip I. Tarr, M.D., W. Geoffrey Haigh, Ph.D., and Sum P. Lee, M.D., Ph.D. Department of Medicine, VA Medical Center; Department of Pediatrics, Children’s Hospital and Regional Medical Center; and the Departments of Medicine, Pediatrics, and Microbiology, University of Washington School of Medicine, Seattle, Washington

OBJECTIVE: Numerous investigators have proposed a role for bacteria in biliary lithogenesis. We hypothesized that bacterial DNA is present in gallstones, and that categorical differences exist between gallstone type and the frequency of bacterial sequences. METHODS: Polymerase chain reaction (PCR) was used to amplify bacterial 16S rRNA and uidA (encoding Escherichia coli [E. coli] ␤-glucuronidase) genes in different types of gallstones. PCR products were sequenced. RESULTS: Bacterial 16S rRNA and uidA DNA sequences in E. coli were detected in all brown pigment, common bile duct, and mixed cholesterol gallstones (n ⫽ 14). In contrast, only one (14%) of seven pure cholesterol gallstones yielded a PCR product. Most (88%) mixed cholesterol gallstones yielded PCR amplification products from their central, as well as their outer, portions. Sequenced products possessed 88 –98% identity to 16S rRNA genes of E. coli and Pseudomonas species. CONCLUSIONS: Bacterial DNA sequences are usually present in mixed cholesterol (to 95% cholesterol content), brown pigment, and common bile duct, but rarely in pure cholesterol gallstones. The presence of bacterial ␤-glucuronidase is also suggested. The role of bacteria and their products in the formation of mixed cholesterol gallstones, which comprise the majority of cholesterol gallstones, warrants further study. (Am J Gastroenterol 1999;94: 3502–3506. © 1999 by Am. Coll. of Gastroenterology)

INTRODUCTION Pigmented gallstones (PGS) and cholesterol gallstones (CGS) are believed to have different origins (1). Considerable data (2– 4) implicate bacteria in the formation of low (⬍40%) cholesterol content brown pigment gallstones (PGS) (5). Because bacterial ␤-glucuronidase, phospholipases, and bile acid hydrolases catalyze biliary lipid hydrolysis, yielding calcium-sensitive anions and calcium salt precipitates (6), such precipitates are niduses for, and constituents of, brown PGS. In contrast, the formation of pure CGS is believed to depend mainly on cholesterol saturation and solubility (7). Microbiological analyses of bile and electron microscopic

imaging of stones have implicated bacteria in CGS development (8 –11), but these studies did not distinguish pure from mixed CGS. Human bile is usually sterile (12–14), but transient, so-called “benign” and asymptomatic bactobilia occurs (15, 16), and CGS can be associated with bile infection (17–20). Also, the presence of rhamnose, a constituent of the lipopolysaccharide of Gram-negative bacteria, in 50% of CGS (21) suggests bacterial involvement in CGS development. Recently, Swidsinski et al. (22) documented DNA homologous to bacterial rRNA in CGS. These data indicate a possible association between bacteria and the formation of biliary stones with as much as 90% cholesterol content. We examined gallstones of varying cholesterol content to test the hypothesis that categorical differences exist between gallstone type and the presence of bacterial sequences. We also analyzed the resulting sequences to determine the species from which the DNA originated.

MATERIALS AND METHODS Gallstones and Patients We examined 21 gallstones removed aseptically from seven men and 14 women (mean age, 55 yr; range, 39 –74 yr). Nineteen stones were obtained from the gall bladder during open cholecystectomy in patients with (one patient) or without (18 patients) cholecystitis. Common bile duct (CBD) stones were collected from two patients with cholangitis by endoscopic retrograde cholangiopancreatography (ERCP). All stones were stored in sterile bottles at ⫺70°C until analyzed, within 3–10 months of collection. Gallstones were cultured using standard aerobic bacterial culture techniques. One patient with cholecystitis and two patients with CBD stones received antibiotics in the 48 h before surgery, but the remaining patients took no antibiotics in the month before the operation. Sample Preparation and DNA Extraction From Gallstones One stone from each patient was quadrisected with a fresh, sterile disposable steel blade. Interior stone material was loosened with a fresh surgical blade. DNA was extracted from the interiors of nine large (⬎12 mm diameter) mixed CGS (inner and outer halves of four stones; inner, middle, and outermost thirds of five stones), with blades changed

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between scrapings. Inert materials (e.g., tubings and swabs) collected at the same time during the surgical operations were processed identically (23), and subjected to polymerase chain reaction (PCR) to detect possible contamination during sample storage and processing. DNA was extracted by a modification of the method of Swidsinski et al. (22). Approximately 100 mg of crushed stones from each patient were incubated overnight at room temperature on a rotating platform in 700 ␮l of 1% sodium dodecyl sulfate in a sterile 5 ml polypropylene tube. Lithium chloride (7 mol/L) was then added to a final concentration of 1.5 mol/L. DNA was then extracted twice with phenol/ chloroform, precipitated by adding ethanol to a final concentration of 80% (v/v), adsorbed to a silica matrix (BioRad Laboratories, Hercules, CA), and eluted into 20 ␮l of elution buffer. DNA content was estimated from ethidium bromide staining of electrophoresed DNA. Identical procedures were performed with inert materials. PCR Conditions and Sequencing of Products PCR using primers (prepared by GIBCO-BRL, Gaithersburg, MD) specific for bacterial 16S rRNA (5⬘CCGGATC CTAATAGAGTTTGAT(C/T)(C/A)TGGCTCAG3⬘ and 5⬘GGCTGCAGTAATACCGC(G/T)(A/G)CTGCTGG CAC3⬘ (incorporating Pst I and Bam HI sites, respectively) [24]) and E. coli uidA (encoding ␤-glucuronidase) (5⬘GCG AAAACTGTTGGAATTGAT3⬘ and 5⬘TGCTCCATA ACTTCCTG3⬘ [25]) genes was performed in 100 ␮l containing ␮mol/L of each deoxyribonucleotide, 50 pmol of each primer, 4.5 mmol/L MgCl2 (including MgCl2 introduced with the PCR buffer), 1 ⫻ PCR buffer (50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L Tris-HCl [pH 9.0 at 25°C], 0.1% TritonX-100), 2.5 U of AmpliTaq DNA polymerase (Promega, Madison, WI), and 0.5 ng/␮l of extracted DNA. For 16S rRNA sequences, primers and template were subjected to an initial cycle at 95°C (3 min), 55°C (1 min), and 74°C (1 min), followed by 35 cycles of 95°C (1 min), 55°C (1 min), and 74°C (1 min), and a final incubation at 72°C (5 min). The hot-start technique was used to seek uidA sequences (25). Samples were then amplified for 35 cycles, with each cycle consisting of 94°C (1.5 min), 64°C (1.5 min), and 72°C (1.5 min). Reaction mixture without DNA template was examined simultaneously in every PCR reaction to confirm lack of contamination with template DNA. Amplified products were examined by 1.2% agarose gel electrophoresis in Tris-borate/EDTA buffer (26). PCR products were cloned into plasmid SK⫹ (Stratagene, La Jolla, CA) or the pT7 Blue T-vector (Novagen, Madison, WI) and sequenced using plasmid-specific primers by double-strand techniques with the Taq DyeDeoxy cyclesequencing kit (Perkin Elmer, Foster, CA). One in five clones was randomly selected for bidirectional sequencing to check the sequencing error rate. Resulting sequences were compared to databases accessed through the National Center for Biotechnology Information Geneinfo BLAST network server (24) on January 4, 1999.

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Quantitation of Cholesterol in Gallstones Representative gallstone portions were weighed and extracted overnight in 5 ml of chloroform/methanol (2:1). Extracts were separated by high-performance liquid chromatography (HPLC), using a 250-⫻-4.6 mm Zorbax ODS column (Phenomenex, Torrance, CA) with 100% methanol as mobile phase. Cholesterol was quantified with a Varex ELSD III mass detector (Alltech, Deerfield, IL) to calculate the cholesterol percentage by weight in each stone. Statistics Fisher’s exact test was used to determine the significance of differences between proportions (27).

RESULTS Physical and Microbiological Characteristics of Gallstone Samples Pure and mixed CGS contained 95–98%, and 65–94% cholesterol, respectively. One CBD stone (cholesterol content, 62%) was extracted from a patient with another gall bladder stone and presumably originated in the gall bladder. The other CBD stone analyzed had a cholesterol content of 7%, was extracted from a patient without intrahepatic or gall bladder stones, and was presumably a primary CBD stone. Except for mixed CGS patient 1 (Table 1) whose culture yielded an E. coli, cultures of the extracted stones failed to produce bacteria. PCR of Bacterial DNA in Gallstones We detected 548 and 252 base-pair amplicons representing bacterial 16S rRNA and uidA DNA, respectively, in each of 14 mixed CGS, brown PGS, and CBD stones, but in only one of seven pure CGS (14%) (p ⫽ 0.00057, pure CGS compared to all other stones; p ⫽ 0.0013, pure CGS compared to mixed CGS). To exclude the possibility that the negative PCR reactions DNA extracted from pure CGS were caused by an inhibitor, we performed PCR with serially diluted extracts of pure CGS added to a fixed amount of lysate of E. coli HB101 (26). Inhibition of the reactions was not observed. Inert samples did not yield PCR products. DNA from the interiors from eight of nine mixed CGS (89%) yielded PCR amplicons. Sequencing of Bacterial Genes Eight to fourteen clones were sequenced from each stone yielding a PCR product. E. coli sequences were detected in all, and Pseudomonas sequences in all but one, brown PGS and CBD stones analyzed. Clostridium species and Stomatococcus mucilagenosus sequences were detected in one brown PGS (Table 1). E. coli and Pseudomonas were also the major DNA species identified in mixed CGS. In two stones, sequences with extensive homology to Staphylococcus, Clostridium sp. and Flavobacterium also were identified. These 16S rRNA sequences had 88 to 98% identity with known corresponding 16S rRNA sequences. uidA sequence was 98 –100% identical to the corresponding gene

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Table 1. Bacterial Sequences Detected in Gallstones

Mixed cholesterol gallstones 1 2 3 4 5 6 7 8 9 10 Brown-pigment gallstones 1 2 Common bile duct stones 1 2 Pure cholesterol gallstone 1

Cholesterol (%)

No. of Sequences

65 66 80 84 86 88 91 93 93 94

8 10 10 12 10 14 10 10 10 10

E. E. E. E. E. E. E. E. E. E.

Pseudomonas Pseudomonas Pseudomonas

91–98 88–97 88–95 90–98 88–94 91–98 91–97 93–98 89–96 91–98

16 25

10 10

E. coli, Pseudomonas, others‡ E. coli, Pseudomonas

92–98 90–94

7 62

11 6

E. coli, Pseudomonas E. coli, Pseudomonas

90–98 88–94

97

Bacteria Identified coli, coli, coli, coli, coli, coli, coli coli, coli, coli,

Pseudomonas, other* Pseudomonas Pseudomonas, other† Pseudomonas Pseudomonas Pseudomonas

Identity (%)

E. coli

* Staphylococcus, † Clostridium, Flavobacterium, ‡ Clostridium, Stomatococcus mucilaginosus. E. coli ⫽ Escherichia coli.

sequence in E. coli. Bidirectional sequencing yielded 100% sequence concordance to single-strand sequencing.

DISCUSSION Our data suggest that the pathogenesis of mixed CGS (with noncholesterol content as low as 6%) is more likely to resemble that of brown PGS and CBD stones than that of pure CGS. These data also suggest that the bacteria that play a role in gallstone formation are intestinal in origin; all bacterial species we identified have close homology to normal human gut flora (28). A precedent exists for ascending biliary infection, caused by sphincter of Oddi incompetence, duodenal diverticulum, or both (29, 30), being associated with the formation of brown PGS. We considered the possibility that the sequences identified represent postformation colonization of gallstones from ascending duodenal organisms, bacteria removed by Kupffer cells from the portal vein (31–33), or contamination during or after surgical extraction. However, two lines of evidence suggest that simple bacterial colonization of stones is not the source of the DNA we detected. First, inner as well as outer sections of the mixed CGS possessed amplifiable bacterial DNA. Had secondary bacterial colonization of a stone’s surface been the source of the DNA detected, we would have expected to detect such sequences on the periphery, but not in the core, of such stones. Nonetheless, we cannot exclude the possibility that bacteria or bacterial DNA diffused into the core portion of the stone after its formation (34), or were introduced during the cutting. Second, pure CGS only rarely possessed bacterial DNA sequences. If secondary bacterial colonization of biliary stones was common, we would have expected that pure CGS, which were

handled identically to the mixed CGS, would have contained amplifiable bacterial DNA. Products from the species implicated could plausibly contribute to gallstone initiation or growth. E. coli ␤-glucuronidase can produce calcium bilirubinate from conjugated bilirubin (35), and Pseudomonas phospholipases A1 and C (36) produce calcium palmitate and calcium stearate from biliary lecithin (6). The cleavage of biliary lecithin might decrease cholesterol solubilization and accelerate nucleation (37). Our demonstration of E. coli uidA sequences in all gallstones other than pure CGS supports the contention that bacteria capable of producing lithogenic enzymes had been in the biliary systems of the patients with these stones. We found evidence of a higher proportion of aerobic Gram-negative organisms (i.e., E. coli and Pseudomonas), and a lower proportion of anaerobes (i.e., Propionobacterium acnes), associated with biliary stones than did Swidsinski et al. (22). We also demonstrated a higher degree of homology of the bacterial sequences obtained to known bacterial sequences. The 2–12% discordance between the sequences we detected and known E. coli and Pseudomonas 16S rRNA sequences is unlikely to have been caused by a sequencing error, as the opposite strand was identical in the clones in which opposite-strand sequencing was performed. Also, PCR fidelity is approximately 99.9% (38). Therefore, it is possible that the variant sequences represent biliary bacteria with minor phylogenetic differences from E. coli or Pseudomonas. Indeed, recently described nanobacteria (39) are novel and possibly lithogenic bacteria that might play a role in gallstone formation. Swidsinski et al. (22) propose that a cut-off cholesterol content of 90% discriminates between the presence or absence of bacterial genes in CGS, whereas we detected bac-

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terial sequences in stones with as little as 6% noncholesterol content. These data strongly suggest that factors other than cholesterol solubility should be considered when pondering the mechanisms underlying the formation of mixed CGS. Bacteria may contribute to stone formation and alter, and perhaps even dictate, the cholesterol content of the concretion. Bacterial associations with gallstones may have additional relevant clinical implications. Biliary obstruction from stones is much more often complicated by inflammation and infection than is malignant obstruction. Is cholecystitis, cholangitis, or pancreatitis related to bacteria “boarding” in the stones? Most gallstones in Western countries are mixed CGS, and mixed CGS stones are being increasingly identified in Asia (40, 41). We propose that CGS may be a more heterogeneous group than has been recognized and that bacteria may contribute to the formation of mixed CGS, although probably not to the formation of most gallstones with ⬎ 95% cholesterol content. A better understanding of the role of bacteria in biliary lithogenesis may illuminate ways to prevent gallstones.

Bacterial DNA in Gallstones

8. 9. 10. 11.

12.

13. 14.

15. 16.

ACKNOWLEDGMENTS We thank Mr. Kan Lam for microbiological analysis, Dr. Harry Yim for scientific advice, Ms. Yoo-Lee Yea for technical support, and Ms. Christine A. Merrikin for secretarial assistance. Dr. Sum P. Lee is supported in part by the Medical Research Service of the Department of Veterans Affairs. Reprint requests and correspondence: Sum P. Lee, M.D., Ph.D., Gastroenterology Section (GI-111), Department of Medicine, VA Medical Center, 1660 South Columbian Way, Seattle, WA 98108 – 1597. Received Feb. 12, 1999; accepted Aug. 9, 1999.

17. 18. 19. 20.

21.

22.

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