- Email: [email protected]
Bacteria and pathogenesis of gallstones
2050
CORRESPONDENCE
the fixed neutrophil ELISA yields a level of sensitivity not achievable by assays based on microscopic observations. Our conclusion that the pANCA associated with type 1 AIH may be an independent and selective marker for that disease 1 is based on the following findings: (1) there is no correlation between serum ANCA titer and laboratory indices of inflammation or histological patterns of disease activity and (2) the titers of ANCA do not correlate with ANA titers. The relationship of this patient population pANCA with the pANCA described for PSC remains to be elucidated, but several distinctions can be discerned. On average, the ANCA titers of severe type 1 AIH were found to be very high (mean, 1:11,410).1 However, inspection of the data presented,1 not surprisingly, indicates that a percentage of the type 1 AIH population displays ANCA titers in the range of those observed for PSC. 4 Additionally, the studies of both Hardarson et al) and Mulder et al. 6 have shown pANCA titers of patients with AIH to be both high (up to 1:1280) and on average higher than those with PSC (without ulcerative colitis). In agreement with Drs. Bansi, Fleming, and Chapman, the fact that the type 1 AIH-associated pANCA was found to react with monocytes as well as neutrophils suggests that the antigen(s) involved may be different from that recognized by the pANCA of either PSC or ulcerative colitis. This point cannot be further addressed until the actual pANCA-reactive antigens are identified for the diseases in question. Although it is difficult to address the differences in IgG isotype distribution between our studies and those of Bansi et al., 2 it is likely that the already detailed patient population differences account for the disparate results. ALDA VIDRICH, M.D. STEPHAN R. TARGAN, M.D.
Cedars-Sinai Medical Center P.O. Box 48750 Los Angeles, California 90048-1865 1. Targan SR, Landers C, Vidrich A, Czaja AJ. High-titer antineutrophil cytoplasmic antibodies in type-1 autoimmune hepatitis. Gastroenterology 1995;108:1159-1166. 2. Bansi DS, Chapman RW, Fleming KA. Antineutrophil cytoplasmic antibody titer but not IgG subclass distinguishes between primary sclerosing cholangitis and autoimmune hepatitis (abstr). Gastroenterology 1995; 108:A1028. 3. Saxon A, Shanahan F, Landers CJ, Ganz T, Targan S. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. J Allergy Clin Immunol 1990;86: 202-210. 4. Duerr RH, Targan SR, Landers C, LaRusso NF, Lindsay K, Wiesner RH, Shanahan F, Neutrophil cytoplasmic antibodies: a link between primary sclerosing cholangitis and ulcerative colitis. Gastroenterology 1991; 100:1385-1391. 5. Hardarson S, LaBrecque DR, Mitros FA, Nell GA, Goeken JA, Antineutrophil cytoplasmic antibody in inflammatory bowel disease and hepatobiliary diseases. Am J Clin Pathol 1993;99:277-281. 6. Mulder AHL, Horst G, Haagsma EB, Limburg PC, Kleibeuker JH, Kallenberg CGN. Prevalence and characterization of neutrophil cytoplasmic antibodies in autoimmune liver diseases. Hepatology 1993;17:411-417.
Bacteria and Pathogenesis of Gallstones Dear Sir: The recent paper by Swidsinsky et al.t and the editorial by Soloway and Crowther in the same issue 2 raise once again the question whether bacteria play a role in the initial formation of cholesterol, mixed, or composite gallstones or whether their role must be confined to the pathogenesis of brown pigment gallstones) 6 Gallstones from 20
GASTROENTEROLOGY Vo1.109, No. 6
patients with negative bacterial cuiture were examined, and ribosomal RNA gene fragments were amplified from 16 of them. Sequences typical of enterobacteria, with 97% similarity to Escherichia coli, were isolated from 5 patients (25%). Propionibacteria-related DNA was found in the stones of 9 patients (45%). The authors conclude that "most cholesterol gallstones harbor bacterial DNA," despite negative bacterial culture. As outlined in the editorial, the authors acknowledge that "the mere presence of bacterial DNA in these cholesterol gallstones does not imply a cause and effect relationship." However, "although all gallstones are not formed by the same mechanism," Swidsinsky et al. "are convinced that the actual role of bacteria in the formation of composite stones ( 7 0 % - 9 0 % cholesterol) remains to be determined." To bolster a cause and effect relationship, Soloway and Crowther suggest repeating the current study and trying to show that "if bacterial DNA could not be reproducibly shown in the region of the stone nidus, this would tend to argue against bacterial initiation of stone formation." This statement supports the view that the opposite (i.e., the presence of bacteria in the central position of the stone) is unequivocal proof of the basic role of bacteria in the pathogenesis of a given stone. Both the article and the editorial outline that "bacteria are seen to preferentially colonize pigmented areas of mixed stones" and that "most cholesterol stones ate composite stones, i.e., have a pigment center or section." Many investigators share the opinion that bacteria may play a role in the initial formation of this "pigmented" center, v which usually appears "brown" at visual inspection. In the editorial, an exciting hypothesis based on the presence of Propionibacterium aches is also reported to explain the initial formation of some types of cholesterol gallstones. This hypothesis suggests the activation of an "extracellular lipase that catalyses the total hydrolysis of triacyiglycerols to free fatty acids and glycerol" and "the possible production of a phospholipase C, which has been recently identified as a promoter of cholesterol precipitation.'8 On the basis of the prospective survey of 1590 consecutive surgical patients with bile tract diseases 4-5 undergoing systematic follow-up, stone analysis (and stone culture in selected cases), bile analysis and bile culture, and comparison of the content (gallstone and bile) to the container (gallbladder wall; also cultured in selected cases), we have accumulated data that are against a basic role of bacteria in the pathogenesis of cholesterol gallstones. We have documented by scanning electron microscopy that (1) bacteria are present not only on the surface but also in the center of brown pigment stones4'9; (2) calcium palmitate is a specific compound of infectious brown gallstones because it is almost never present in large amounts ( > 1 0 % 15% of the stone dry weight) in other types of gallstones<5; (3) the activity of phospholipase AI and other phospholipases, either of bacterial or pancreatic origin, is extremely high in the bile of patients with recurrent common duct brown gallstones after sphincterotomyff but not in the bile of patients with other types of stones. However, we founded our hypothesis that suggests the crucial role of E. coli in the pathogenesis of brown gallstones not on the mere presence of bacteria resembling E. coli in the center of brown stones 4'9 but on the following findings: (1) in a previous sterile common duct, harboring other types of gallstones, brown stones began to occur only after the onset of bile infection by E. coli4; and (2) calcium palmitate was found both in the center and in the periphery of brown gallstones in hundreds of alternate tan and light layers together with bilirubinate and cholesterol, suggesting that calcium palmitate precipitation was an early event, occurring from the beginning and remaining unchanged throughout the entire stone growth. 5 These findings were subsequently confirmed by others. 6'::-:3
December 1995
In our opinion, infection by E. toll, more than by other types of bacteria, 5 triggers a global hydrolysis responsible not only for the precipitation of calcium bilirubinate but also of calcium palmitate, as suggested by the extremely high levels of bacterial [~-glucuronidase3 and phospholipase4'9 together with a series of other physicochemical and biological changes that we have considered typical of the "infectious mechanisms. ''4'5 Bacteria can adhere to cholesterol, mixed, or even black stones. 14a5 Additionally, the shell of gallstones, even if partly calcified, is not impermeable to the surrounding bile. Bile and gallstones have continuous interchanges among them. During our follow-up study of patients with uncalcified gallstones, we sometimes observed late calcification not only in the periphery but also in the stone center. Similar findings also have been reported by Wolpers. :6 In addition, the gas-containing center of some mixed gallstones (Mercedes-Benz sign), after stone embedding in a given solution, tends to acquire the same composition as the solution and appears radiopaque to "in vitro" x-rays if the solution contains contrast medium for cholangiography (i.e., large proteic molecules). A similar event (i.e., the passage from outside inside) is possible if stones are embedded in infected bile (unpublished observations). These data suggest more caution before stating that what is found in the center is by definition antecedent to what is found in the stone periphery. In addition, we have shown that the apparently "brown" center of some cholesterol gallstones has not the same composition and structure as a brown microstone, iv Similar findings have also been reported by others. TM Furthermore, in our prospective study, we documented that while brown stones (or a brown periphery) can form because of secondary bacterial infection, in patients with previous cholesterol or black gallstones, 4 the opposite was never observed. In particular, in patients with multiple reoperations on the bile tract (up to four) for "infectious" brown gallstones, no stones other than brown ones are likely to form after the onset of the hydrolytic mechanism determined by bacterial infection) Therefore, it is unlikely that bacteria, at least those responsible for major hydrolytic changes, play a transient or intermittent "causative" role in the initial formation of cholesterol gallstones because the cascade of biological events triggered by bile tract infection is usually self-maintaining5 and not self-limiting, as suggested in the editorial. Obviously, bacteria transiently passing through the bile tract, as well as all other foreign bodies, can promote or facilitate stone nucleation. However, this is a nonspecific phenomenon that must be kept distinct from a specific pathogenetic role of a given bacterial species. In conclusion, while not criticizing the specific role of molecular genetics in the detection of bacterial colonization of cholesterol gallstones in the presence of a negative bacterial culture, we suggest caution before making inferences concerning a direct cause and effect relationship between what can be a minor finding or a secondary event and the initial phenomena that lead to cholesterol gallstone formation. As stated in the editorial, it is not correct "to dismiss the importance of bacteria in the pathogenesis of cholesterol gallstones simply because the bacterial titers were believed to be too low to be clinically significant," but it also could be incorrect or premature to support new pathogenetic hypotheses on the basis of what could be a casual finding or a late event, not influencing significantly the initial steps of stone formation. The increasing availability of more sophisticated methods of detection and identification of chemical compounds or biological agents such as bacteria has shown with increasing frequency the presence of (1) minor components in trace amounts in gallstones, likely due to coprecipitation:9'2°; (2) phospholipasts in the bile of patients with cholesterol gallstones, 8 but in a concentration that is hundreds to thousands folds inferior to that
CORRESPONDENCE 2051
found in the bile of patients with brown gallstones:2; and (3) bacteria or traces of their previous presence in the stones of patients with negative culture.: However, because in the future almost everything will be found everywhere, it is of paramount importance to state which is which and to distinguish minor factors, late events, or curiosities from basic components or fundamental factors that play a crucial role from the beginning in the pathogenesis of the various types of gallstones. FRANCESCO CETTA, M.D.
Interuniversity Center for Research in Hepatobiliary Diseases Institute of Surgical Clinics University of Siena Nuovo Policlinico 53100 Siena, Italy 1. Swidsinsky A, Ludwig W, Pahlig H, Priem F. Molecular genetic evidence of bacterial colonization of cholesterol gallstones. Gastroenterology 1995; 108:860-864. 2. Soloway RD, Crowther RS. Bacteria and cholesterol gallstones: molecular biology comes to gallstone pathogenesis. Gastroenterology 1995; 108:934-936. 3. Maki T. Pathogenesis of calcium bilirubinate gallstones. Ann Surg 1966; 164:90-100. 4. Cetta F. Bile infection documented as initial event in the pathogenesis of brown pigment biliary stones. Hepatology 1986:482489. 5. Cetta F. The role of bacteria in pigment gallstone disease. Ann Surg 1991;213:315-326. 6. Kaufman HS, Magnuson TH, Lillemoe KD, Frasca HA, Pitt HA. The role of bacteria in gallbladder and common duct stone formation. Ann Surg 1989; 209:584-592. 7. Diliberto JP, Marks JW, Shoenfieid LJ. Classification and pathogenesis of gallstones. In: Braasch JW, Tompkins RK, eds. Surgical disease of the biliary tract and pancreas. St. Louis, MO: Mosby, 1994:50-67. 8. Pattinson NR, Willis KE. Effect of phospholipase C on cholesterol solubilization in model bile. Gastroenterology 1991; 101:13391344, 9. Cetta F, Fonzi L, Rossi S, Belli M, Doretti C, Macris S. Scanning electron microscopy analysis of recurrent common duct stones. Gastroenterology 1984;86:13-14. 10. Cetta F, Lombardo F. The possible role of phospholipase in gallstone pathogenesis. Gastroenterology 1991;101:592-593. 11, Malet PF, Doberzves MA, Guanghua H, et al. Quantitative infrared spectroscopy of common bile duct gallstones. Gastroenterology 1988; 94:1217 -1221. 12. Soloway RD, Trotman BW, Maddrey WC, Nakajama F. Pigment gallstone composition in patients with hemolysis or infection/ stasis. Dig Dis Sci 1986;31:454-60. 13. Nakano T, Yanagisawa, Nakayama F. Phospholipase activity in human bile. Hepatology 1988;8:1560-1564. 14. Stewart L, Smith AL, Petlegrini CA, Motson RW, Way LW. Pigment gallstones form as a composite of bacterial microcolonies and pigment solids. Ann Surg 1987;206:242-250. 15. Wetter LA, Hamedeh RM, Griffiss J McL, Oesterle A, Aagaard B, Way LW. Differences in outer membrane characteristics between gallstone-associated bacteria and normal bacterial flora. Lancet 1994; 343:444-448. 16. Wolpers C. Gallenblasenstein. Basel, Switzerland: Karger, 1987. 17. Cetta F, Lombardo F, Giubbolini M, Rossi S. The early phases of cholesterol stone formation and growth in humans. Morphologic, structural and ultrastructural aspects (abstr). Gastroenterology 1993; 104:A356. 18. Malet PF, Williamson CE, Trotman BW, Soloway RD. The composition of pigment centers of cholesterol gallstones. Hepatology 1986; 6:477-481.
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19. Kaufman HS, Magnuson TH, Pitt HA, Frasca P, Lillemoe KD. The distribution of calcium salt precipitates in the core, periphery and shell of cholesterol, black pigment and brown pigment gallstones. Hepatoiogy 1994; 19:1124-1132. 20. Cetta F, Lombardo F, Giubbolini M, Baldi C, Cariati A. Quantity and location of stone compounds, not the mere presence in small amounts within the stone fabric, are important factors for the correct classification of gallstones and a better knowledge of their pathogenesis. Hepatology (in press). Reply. Dr. Cetta correctly points to the necessity of a cautious interpretation of bacterial DNA detected in "sterile" cholesterol gallstones. We welcome any further contributions to this discussion. However, we disagree with the statement that "in the future almost everything will be found everywhere" regarding our findings. We do not find everything everywhere, but evidence of only four groups of microorganisms: propionibacteria, a group of unknown gram-positive bacteria with a low DNA guanine-cytosine content, E. toll, and, most recently, some Flavobacterium relative. The bacteria detected with our methods in sterile gallbladder gallstones and those cultured from bile in clinical laboratories differ strongly in their percent of occurrence. Our current investigation of gallstones from patients with positive bile cultures suggests that, except for E. toll, bacteria that usually grow in bile are of a secondary, transient nature. Finally, not every polymerase chain reaction is a marvel of sensitivity. We could not detect < 100,000 bacteria when known dilutions of E. coli and Streptococcusfaecalis were treated as if they were purified from a gallstone. Because gallstone components retard the polymerase chain reaction and a lot of DNA is lost during extraction and purification, the actual bacterial load of a stone must be much higher. ALEXANDER SWIDSINSKI, M.D.
IV Innere Klinik, Charite Abteihmg Gastroenterologie 10098 Berlin Germany Reply. Our editorial 1 was written with the objective of stimulating inquiry and debate about the possible role of bacteria in the pathogenesis of cholesterol gallstones; in this spirit, we welcome Dr. Cetta's comments. The underlying theme of Dr. Cetta's letter is that there is much less evidence for bacterial involvement in the formation of cholesterol stones than there is for the formation of brown pigment stones. This fact is incontrovertible. However, in part, our views have been misunderstood. Dr. Cetta takes our statement that "if bacterial DNA could not be reproducibly shown in the region of the stone nidus, this would tend to argue against bacterial initiation of stone formation" and inverts it to imply that we also believe that the presence of bacteria in the center of a stone "is an unequivocal proof of the basic role of bacteria in the pathogenesis of a given stone." This latter statement does not follow logically from our original statement, and it is patently untrue. Mere presence at the scene of the crime is not indicative of guilt. Repeated instances, however, may at least raise suspicion. Dr. Cetta's inclination to dismiss a possible role for bacteria in the formation of cholesterol gallstones stems from two main objections. First, bacterial DNA may enter a mature gallstone by diffusion; therefore, the bacteria whence the DNA came are not necessarily involved in formation of the stone. Proteins have been shown to diffuse into and out of gallstones,2 and the same may also be true of DNA. However, a simple diffusive method of entry suggests that the DNA content of a series of gallstones would be derived from all of the bacterial species that are routinely isolated from gallstones, bile,
GASTROENTEROLOGY Vo1.109, No. 6
or gallbladder. In this context, the relatively frequent identification of Propionibacterium DNA from cholesterol gallstones (45 %) by Swidsinski et al) was surprising because this bacterium is not normally considered a frequent inhabitant of the biliary tract; even in studies in which it has been regularly isolated, other bacteria are also seen with great frequency.4'5 Thus P. aches DNA appears to be overrepresented in the series of stones in the Swidsinski study for a simple diffusive method of entry to be considered probable. Second, the level of phospholipase A activity is lower in the bile-bathing cholesterol stones than it is in bile-bathing brown pigment stones, and cholesterol stones contain less calcium palmitate than might be anticipated if these stones were formed under the influence of phospholipase-producing bacteria. There is conflicting evidence about the ability of the nonspecific lipase that is produced by P. acnes to cleave the acyl chains from phosphatidylcholine6'V; thus, high levels of calcium palmitate precipitation would not always be anticipated. Probably of greater interest as far as cholesterol precipitation is concerned is P. aches phospholipase C, 8 which does not, of course, result in the liberation of free fatty acids but which has been shown to promote cholesterol precipitation in model bile. 9 We acknowledge that even ifP. acnes does play a role in cholesterol gallstone pathogenesis, none of the mechanisms discussed in the editorial may be relevant. These mechanisms merely seemed to be the most likely that could be advanced on purely theoretical grounds. The British surgeon Lord Moynihan (1865-1936) believed that "a gallstone is a tombstone erected to the memory of the organism within it." His Lordship was correct about the bacterial origins of brown pigment stones, as Maki, l° Cetta, 11 and others have shown, but it remains to be proven whether the same is true for cholesterol gallstones. P. acnes DNA was isolated from cholesterol gallstones with surprising frequency by Swidsinski et ai., 3 and this bacterium possesses a number of biochemical properties that may permit it to promote cholesterol precipitation. Whether it does so will only be uncovered by further investigation. ROGER D. SOLOWAY ROGER S. CROWTHER
Division of Gastroenterology Department of Internal Medicine University of Texas Medical Branch Galveston, Texas 77555-0764 1. Soloway RD, Crowther RS. Bacteria and cholesterol gallstones: molecular biology comes to gallstone pathogenesis. Gastroenterology 1995; 108:934-936. 2. Sanabria JR, Upadhya GA, Harvey RP, Strasberg SM. Diffusion of substances into human cholesterol gallstones. Gastroenterology 1994;106:749-754. 3. Swidsinsky A, Ludwig W, Pahlig H, Priem F. Molecular genetic evidence of bacterial colonization of cholesterol gallstones. Gastroenterology 1995; 108:860-864. 4. Goodhart GL, Levison ME, Trotman BW, Soloway RD. Pigment vs cholesterol lithiasis, bacteriology of gallbladder stone, bile and tissue correlated with biliary lipid analysis. Am J Dig Dis 1978;23:877-882. 5. Flinn WR, Olson DF, Oyasu R, Beal JM. Biliary bacteria and hepatic histopathologic changes in gallstone disease. Ann Surg 1977;185:593-597. 6. Kabongo Muamba ML. Exoenzymes of Propionibacterium acnes. Can J Microbiol 1982;28:758-761. 7. Ingham E, Holland KT, Gowland G, Cunliffe WJ. Partial purification and characterization of lipase (EC 3.1.1.3) from Propionibacterium acnes. J Gen Microbiol 1981; 124:393-401. 8. Nakamura T, Taniguchi H, Takeuchi K, Fujimura S, Pulverer G.
CORRESPONDENCE
the fixed neutrophil ELISA yields a level of sensitivity not achievable by assays based on microscopic observations. Our conclusion that the pANCA associated with type 1 AIH may be an independent and selective marker for that disease 1 is based on the following findings: (1) there is no correlation between serum ANCA titer and laboratory indices of inflammation or histological patterns of disease activity and (2) the titers of ANCA do not correlate with ANA titers. The relationship of this patient population pANCA with the pANCA described for PSC remains to be elucidated, but several distinctions can be discerned. On average, the ANCA titers of severe type 1 AIH were found to be very high (mean, 1:11,410).1 However, inspection of the data presented,1 not surprisingly, indicates that a percentage of the type 1 AIH population displays ANCA titers in the range of those observed for PSC. 4 Additionally, the studies of both Hardarson et al) and Mulder et al. 6 have shown pANCA titers of patients with AIH to be both high (up to 1:1280) and on average higher than those with PSC (without ulcerative colitis). In agreement with Drs. Bansi, Fleming, and Chapman, the fact that the type 1 AIH-associated pANCA was found to react with monocytes as well as neutrophils suggests that the antigen(s) involved may be different from that recognized by the pANCA of either PSC or ulcerative colitis. This point cannot be further addressed until the actual pANCA-reactive antigens are identified for the diseases in question. Although it is difficult to address the differences in IgG isotype distribution between our studies and those of Bansi et al., 2 it is likely that the already detailed patient population differences account for the disparate results. ALDA VIDRICH, M.D. STEPHAN R. TARGAN, M.D.
Cedars-Sinai Medical Center P.O. Box 48750 Los Angeles, California 90048-1865 1. Targan SR, Landers C, Vidrich A, Czaja AJ. High-titer antineutrophil cytoplasmic antibodies in type-1 autoimmune hepatitis. Gastroenterology 1995;108:1159-1166. 2. Bansi DS, Chapman RW, Fleming KA. Antineutrophil cytoplasmic antibody titer but not IgG subclass distinguishes between primary sclerosing cholangitis and autoimmune hepatitis (abstr). Gastroenterology 1995; 108:A1028. 3. Saxon A, Shanahan F, Landers CJ, Ganz T, Targan S. A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease. J Allergy Clin Immunol 1990;86: 202-210. 4. Duerr RH, Targan SR, Landers C, LaRusso NF, Lindsay K, Wiesner RH, Shanahan F, Neutrophil cytoplasmic antibodies: a link between primary sclerosing cholangitis and ulcerative colitis. Gastroenterology 1991; 100:1385-1391. 5. Hardarson S, LaBrecque DR, Mitros FA, Nell GA, Goeken JA, Antineutrophil cytoplasmic antibody in inflammatory bowel disease and hepatobiliary diseases. Am J Clin Pathol 1993;99:277-281. 6. Mulder AHL, Horst G, Haagsma EB, Limburg PC, Kleibeuker JH, Kallenberg CGN. Prevalence and characterization of neutrophil cytoplasmic antibodies in autoimmune liver diseases. Hepatology 1993;17:411-417.
Bacteria and Pathogenesis of Gallstones Dear Sir: The recent paper by Swidsinsky et al.t and the editorial by Soloway and Crowther in the same issue 2 raise once again the question whether bacteria play a role in the initial formation of cholesterol, mixed, or composite gallstones or whether their role must be confined to the pathogenesis of brown pigment gallstones) 6 Gallstones from 20
GASTROENTEROLOGY Vo1.109, No. 6
patients with negative bacterial cuiture were examined, and ribosomal RNA gene fragments were amplified from 16 of them. Sequences typical of enterobacteria, with 97% similarity to Escherichia coli, were isolated from 5 patients (25%). Propionibacteria-related DNA was found in the stones of 9 patients (45%). The authors conclude that "most cholesterol gallstones harbor bacterial DNA," despite negative bacterial culture. As outlined in the editorial, the authors acknowledge that "the mere presence of bacterial DNA in these cholesterol gallstones does not imply a cause and effect relationship." However, "although all gallstones are not formed by the same mechanism," Swidsinsky et al. "are convinced that the actual role of bacteria in the formation of composite stones ( 7 0 % - 9 0 % cholesterol) remains to be determined." To bolster a cause and effect relationship, Soloway and Crowther suggest repeating the current study and trying to show that "if bacterial DNA could not be reproducibly shown in the region of the stone nidus, this would tend to argue against bacterial initiation of stone formation." This statement supports the view that the opposite (i.e., the presence of bacteria in the central position of the stone) is unequivocal proof of the basic role of bacteria in the pathogenesis of a given stone. Both the article and the editorial outline that "bacteria are seen to preferentially colonize pigmented areas of mixed stones" and that "most cholesterol stones ate composite stones, i.e., have a pigment center or section." Many investigators share the opinion that bacteria may play a role in the initial formation of this "pigmented" center, v which usually appears "brown" at visual inspection. In the editorial, an exciting hypothesis based on the presence of Propionibacterium aches is also reported to explain the initial formation of some types of cholesterol gallstones. This hypothesis suggests the activation of an "extracellular lipase that catalyses the total hydrolysis of triacyiglycerols to free fatty acids and glycerol" and "the possible production of a phospholipase C, which has been recently identified as a promoter of cholesterol precipitation.'8 On the basis of the prospective survey of 1590 consecutive surgical patients with bile tract diseases 4-5 undergoing systematic follow-up, stone analysis (and stone culture in selected cases), bile analysis and bile culture, and comparison of the content (gallstone and bile) to the container (gallbladder wall; also cultured in selected cases), we have accumulated data that are against a basic role of bacteria in the pathogenesis of cholesterol gallstones. We have documented by scanning electron microscopy that (1) bacteria are present not only on the surface but also in the center of brown pigment stones4'9; (2) calcium palmitate is a specific compound of infectious brown gallstones because it is almost never present in large amounts ( > 1 0 % 15% of the stone dry weight) in other types of gallstones<5; (3) the activity of phospholipase AI and other phospholipases, either of bacterial or pancreatic origin, is extremely high in the bile of patients with recurrent common duct brown gallstones after sphincterotomyff but not in the bile of patients with other types of stones. However, we founded our hypothesis that suggests the crucial role of E. coli in the pathogenesis of brown gallstones not on the mere presence of bacteria resembling E. coli in the center of brown stones 4'9 but on the following findings: (1) in a previous sterile common duct, harboring other types of gallstones, brown stones began to occur only after the onset of bile infection by E. coli4; and (2) calcium palmitate was found both in the center and in the periphery of brown gallstones in hundreds of alternate tan and light layers together with bilirubinate and cholesterol, suggesting that calcium palmitate precipitation was an early event, occurring from the beginning and remaining unchanged throughout the entire stone growth. 5 These findings were subsequently confirmed by others. 6'::-:3
December 1995
In our opinion, infection by E. toll, more than by other types of bacteria, 5 triggers a global hydrolysis responsible not only for the precipitation of calcium bilirubinate but also of calcium palmitate, as suggested by the extremely high levels of bacterial [~-glucuronidase3 and phospholipase4'9 together with a series of other physicochemical and biological changes that we have considered typical of the "infectious mechanisms. ''4'5 Bacteria can adhere to cholesterol, mixed, or even black stones. 14a5 Additionally, the shell of gallstones, even if partly calcified, is not impermeable to the surrounding bile. Bile and gallstones have continuous interchanges among them. During our follow-up study of patients with uncalcified gallstones, we sometimes observed late calcification not only in the periphery but also in the stone center. Similar findings also have been reported by Wolpers. :6 In addition, the gas-containing center of some mixed gallstones (Mercedes-Benz sign), after stone embedding in a given solution, tends to acquire the same composition as the solution and appears radiopaque to "in vitro" x-rays if the solution contains contrast medium for cholangiography (i.e., large proteic molecules). A similar event (i.e., the passage from outside inside) is possible if stones are embedded in infected bile (unpublished observations). These data suggest more caution before stating that what is found in the center is by definition antecedent to what is found in the stone periphery. In addition, we have shown that the apparently "brown" center of some cholesterol gallstones has not the same composition and structure as a brown microstone, iv Similar findings have also been reported by others. TM Furthermore, in our prospective study, we documented that while brown stones (or a brown periphery) can form because of secondary bacterial infection, in patients with previous cholesterol or black gallstones, 4 the opposite was never observed. In particular, in patients with multiple reoperations on the bile tract (up to four) for "infectious" brown gallstones, no stones other than brown ones are likely to form after the onset of the hydrolytic mechanism determined by bacterial infection) Therefore, it is unlikely that bacteria, at least those responsible for major hydrolytic changes, play a transient or intermittent "causative" role in the initial formation of cholesterol gallstones because the cascade of biological events triggered by bile tract infection is usually self-maintaining5 and not self-limiting, as suggested in the editorial. Obviously, bacteria transiently passing through the bile tract, as well as all other foreign bodies, can promote or facilitate stone nucleation. However, this is a nonspecific phenomenon that must be kept distinct from a specific pathogenetic role of a given bacterial species. In conclusion, while not criticizing the specific role of molecular genetics in the detection of bacterial colonization of cholesterol gallstones in the presence of a negative bacterial culture, we suggest caution before making inferences concerning a direct cause and effect relationship between what can be a minor finding or a secondary event and the initial phenomena that lead to cholesterol gallstone formation. As stated in the editorial, it is not correct "to dismiss the importance of bacteria in the pathogenesis of cholesterol gallstones simply because the bacterial titers were believed to be too low to be clinically significant," but it also could be incorrect or premature to support new pathogenetic hypotheses on the basis of what could be a casual finding or a late event, not influencing significantly the initial steps of stone formation. The increasing availability of more sophisticated methods of detection and identification of chemical compounds or biological agents such as bacteria has shown with increasing frequency the presence of (1) minor components in trace amounts in gallstones, likely due to coprecipitation:9'2°; (2) phospholipasts in the bile of patients with cholesterol gallstones, 8 but in a concentration that is hundreds to thousands folds inferior to that
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found in the bile of patients with brown gallstones:2; and (3) bacteria or traces of their previous presence in the stones of patients with negative culture.: However, because in the future almost everything will be found everywhere, it is of paramount importance to state which is which and to distinguish minor factors, late events, or curiosities from basic components or fundamental factors that play a crucial role from the beginning in the pathogenesis of the various types of gallstones. FRANCESCO CETTA, M.D.
Interuniversity Center for Research in Hepatobiliary Diseases Institute of Surgical Clinics University of Siena Nuovo Policlinico 53100 Siena, Italy 1. Swidsinsky A, Ludwig W, Pahlig H, Priem F. Molecular genetic evidence of bacterial colonization of cholesterol gallstones. Gastroenterology 1995; 108:860-864. 2. Soloway RD, Crowther RS. Bacteria and cholesterol gallstones: molecular biology comes to gallstone pathogenesis. Gastroenterology 1995; 108:934-936. 3. Maki T. Pathogenesis of calcium bilirubinate gallstones. Ann Surg 1966; 164:90-100. 4. Cetta F. Bile infection documented as initial event in the pathogenesis of brown pigment biliary stones. Hepatology 1986:482489. 5. Cetta F. The role of bacteria in pigment gallstone disease. Ann Surg 1991;213:315-326. 6. Kaufman HS, Magnuson TH, Lillemoe KD, Frasca HA, Pitt HA. The role of bacteria in gallbladder and common duct stone formation. Ann Surg 1989; 209:584-592. 7. Diliberto JP, Marks JW, Shoenfieid LJ. Classification and pathogenesis of gallstones. In: Braasch JW, Tompkins RK, eds. Surgical disease of the biliary tract and pancreas. St. Louis, MO: Mosby, 1994:50-67. 8. Pattinson NR, Willis KE. Effect of phospholipase C on cholesterol solubilization in model bile. Gastroenterology 1991; 101:13391344, 9. Cetta F, Fonzi L, Rossi S, Belli M, Doretti C, Macris S. Scanning electron microscopy analysis of recurrent common duct stones. Gastroenterology 1984;86:13-14. 10. Cetta F, Lombardo F. The possible role of phospholipase in gallstone pathogenesis. Gastroenterology 1991;101:592-593. 11, Malet PF, Doberzves MA, Guanghua H, et al. Quantitative infrared spectroscopy of common bile duct gallstones. Gastroenterology 1988; 94:1217 -1221. 12. Soloway RD, Trotman BW, Maddrey WC, Nakajama F. Pigment gallstone composition in patients with hemolysis or infection/ stasis. Dig Dis Sci 1986;31:454-60. 13. Nakano T, Yanagisawa, Nakayama F. Phospholipase activity in human bile. Hepatology 1988;8:1560-1564. 14. Stewart L, Smith AL, Petlegrini CA, Motson RW, Way LW. Pigment gallstones form as a composite of bacterial microcolonies and pigment solids. Ann Surg 1987;206:242-250. 15. Wetter LA, Hamedeh RM, Griffiss J McL, Oesterle A, Aagaard B, Way LW. Differences in outer membrane characteristics between gallstone-associated bacteria and normal bacterial flora. Lancet 1994; 343:444-448. 16. Wolpers C. Gallenblasenstein. Basel, Switzerland: Karger, 1987. 17. Cetta F, Lombardo F, Giubbolini M, Rossi S. The early phases of cholesterol stone formation and growth in humans. Morphologic, structural and ultrastructural aspects (abstr). Gastroenterology 1993; 104:A356. 18. Malet PF, Williamson CE, Trotman BW, Soloway RD. The composition of pigment centers of cholesterol gallstones. Hepatology 1986; 6:477-481.
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19. Kaufman HS, Magnuson TH, Pitt HA, Frasca P, Lillemoe KD. The distribution of calcium salt precipitates in the core, periphery and shell of cholesterol, black pigment and brown pigment gallstones. Hepatoiogy 1994; 19:1124-1132. 20. Cetta F, Lombardo F, Giubbolini M, Baldi C, Cariati A. Quantity and location of stone compounds, not the mere presence in small amounts within the stone fabric, are important factors for the correct classification of gallstones and a better knowledge of their pathogenesis. Hepatology (in press). Reply. Dr. Cetta correctly points to the necessity of a cautious interpretation of bacterial DNA detected in "sterile" cholesterol gallstones. We welcome any further contributions to this discussion. However, we disagree with the statement that "in the future almost everything will be found everywhere" regarding our findings. We do not find everything everywhere, but evidence of only four groups of microorganisms: propionibacteria, a group of unknown gram-positive bacteria with a low DNA guanine-cytosine content, E. toll, and, most recently, some Flavobacterium relative. The bacteria detected with our methods in sterile gallbladder gallstones and those cultured from bile in clinical laboratories differ strongly in their percent of occurrence. Our current investigation of gallstones from patients with positive bile cultures suggests that, except for E. toll, bacteria that usually grow in bile are of a secondary, transient nature. Finally, not every polymerase chain reaction is a marvel of sensitivity. We could not detect < 100,000 bacteria when known dilutions of E. coli and Streptococcusfaecalis were treated as if they were purified from a gallstone. Because gallstone components retard the polymerase chain reaction and a lot of DNA is lost during extraction and purification, the actual bacterial load of a stone must be much higher. ALEXANDER SWIDSINSKI, M.D.
IV Innere Klinik, Charite Abteihmg Gastroenterologie 10098 Berlin Germany Reply. Our editorial 1 was written with the objective of stimulating inquiry and debate about the possible role of bacteria in the pathogenesis of cholesterol gallstones; in this spirit, we welcome Dr. Cetta's comments. The underlying theme of Dr. Cetta's letter is that there is much less evidence for bacterial involvement in the formation of cholesterol stones than there is for the formation of brown pigment stones. This fact is incontrovertible. However, in part, our views have been misunderstood. Dr. Cetta takes our statement that "if bacterial DNA could not be reproducibly shown in the region of the stone nidus, this would tend to argue against bacterial initiation of stone formation" and inverts it to imply that we also believe that the presence of bacteria in the center of a stone "is an unequivocal proof of the basic role of bacteria in the pathogenesis of a given stone." This latter statement does not follow logically from our original statement, and it is patently untrue. Mere presence at the scene of the crime is not indicative of guilt. Repeated instances, however, may at least raise suspicion. Dr. Cetta's inclination to dismiss a possible role for bacteria in the formation of cholesterol gallstones stems from two main objections. First, bacterial DNA may enter a mature gallstone by diffusion; therefore, the bacteria whence the DNA came are not necessarily involved in formation of the stone. Proteins have been shown to diffuse into and out of gallstones,2 and the same may also be true of DNA. However, a simple diffusive method of entry suggests that the DNA content of a series of gallstones would be derived from all of the bacterial species that are routinely isolated from gallstones, bile,
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or gallbladder. In this context, the relatively frequent identification of Propionibacterium DNA from cholesterol gallstones (45 %) by Swidsinski et al) was surprising because this bacterium is not normally considered a frequent inhabitant of the biliary tract; even in studies in which it has been regularly isolated, other bacteria are also seen with great frequency.4'5 Thus P. aches DNA appears to be overrepresented in the series of stones in the Swidsinski study for a simple diffusive method of entry to be considered probable. Second, the level of phospholipase A activity is lower in the bile-bathing cholesterol stones than it is in bile-bathing brown pigment stones, and cholesterol stones contain less calcium palmitate than might be anticipated if these stones were formed under the influence of phospholipase-producing bacteria. There is conflicting evidence about the ability of the nonspecific lipase that is produced by P. acnes to cleave the acyl chains from phosphatidylcholine6'V; thus, high levels of calcium palmitate precipitation would not always be anticipated. Probably of greater interest as far as cholesterol precipitation is concerned is P. aches phospholipase C, 8 which does not, of course, result in the liberation of free fatty acids but which has been shown to promote cholesterol precipitation in model bile. 9 We acknowledge that even ifP. acnes does play a role in cholesterol gallstone pathogenesis, none of the mechanisms discussed in the editorial may be relevant. These mechanisms merely seemed to be the most likely that could be advanced on purely theoretical grounds. The British surgeon Lord Moynihan (1865-1936) believed that "a gallstone is a tombstone erected to the memory of the organism within it." His Lordship was correct about the bacterial origins of brown pigment stones, as Maki, l° Cetta, 11 and others have shown, but it remains to be proven whether the same is true for cholesterol gallstones. P. acnes DNA was isolated from cholesterol gallstones with surprising frequency by Swidsinski et ai., 3 and this bacterium possesses a number of biochemical properties that may permit it to promote cholesterol precipitation. Whether it does so will only be uncovered by further investigation. ROGER D. SOLOWAY ROGER S. CROWTHER
Division of Gastroenterology Department of Internal Medicine University of Texas Medical Branch Galveston, Texas 77555-0764 1. Soloway RD, Crowther RS. Bacteria and cholesterol gallstones: molecular biology comes to gallstone pathogenesis. Gastroenterology 1995; 108:934-936. 2. Sanabria JR, Upadhya GA, Harvey RP, Strasberg SM. Diffusion of substances into human cholesterol gallstones. Gastroenterology 1994;106:749-754. 3. Swidsinsky A, Ludwig W, Pahlig H, Priem F. Molecular genetic evidence of bacterial colonization of cholesterol gallstones. Gastroenterology 1995; 108:860-864. 4. Goodhart GL, Levison ME, Trotman BW, Soloway RD. Pigment vs cholesterol lithiasis, bacteriology of gallbladder stone, bile and tissue correlated with biliary lipid analysis. Am J Dig Dis 1978;23:877-882. 5. Flinn WR, Olson DF, Oyasu R, Beal JM. Biliary bacteria and hepatic histopathologic changes in gallstone disease. Ann Surg 1977;185:593-597. 6. Kabongo Muamba ML. Exoenzymes of Propionibacterium acnes. Can J Microbiol 1982;28:758-761. 7. Ingham E, Holland KT, Gowland G, Cunliffe WJ. Partial purification and characterization of lipase (EC 3.1.1.3) from Propionibacterium acnes. J Gen Microbiol 1981; 124:393-401. 8. Nakamura T, Taniguchi H, Takeuchi K, Fujimura S, Pulverer G.