Chronic Salmonella Typhi Infection and Gallbladder Cancer

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Introduction

There is a correlation between chronic S. typhi (S. enterica serovar Typhimurium) infection and gallbladder cancer. This connection was first reported in India, where typhoid is endemic and the 1992 incidence of gallbladder cancer was 13.5 per 100,000 people in the population, and both tend to be endemic in many of the same regions.1 Furthermore, chronic typhoid carrier state has been demonstrated to be the single most important risk factor for development of gallbladder cancer, which is generally very rare, in patients with cholesterol-based gallstones.2 S. typhi causes typhoid fever, which is an acute bacterial disease. The bacteria are transmitted mainly through feces. However, a small percentage of individuals who suffer an acute infection become asymptomatic carriers whose infections persist for many years after the illness. S. typhi achieves this carrier state by creating biofilms on the cholesterol-based gallstones residing within the gallbladder. Persistent S. typhi infection—known as the chronic typhoid carrier state—can lead to chronic inflammation of the gallbladder, in which the bacteria produce potentially carcinogenic toxins and metabolites. The result is carcinoma of the gallbladder epithelium. One key feature of the gallbladder is that it concentrates not only bile salts but also toxins—an effect that has been shown to amplify their mutagenic effects. One such carcinogen is bacterial β-glucuronidase, which, in addition to the other secondary bile acids that are produced from bacterial enzyme processing and concentration in the gallbladder, is mutagenic.3

Role of biofilms in chronic S. typhi carriage

In a study conducted in mice, it was found that S. typhi is able to colonize the gallbladder and persist in an asymptomatic carrier state 4. Mice were fed a lithogenic diet, which included 1% cholesterol and 0.5% cholic acid supplementation, and it was found that significant amounts of S. typhi could be recovered from the gallstones of mice infected with S. typhi but not from those of the control mice. Significantly, it also took much longer for mice on lithogenic diets to recover from acute S. typhi infection than it took for the control mice, and these bacteria could be recovered in stool samples of the mice with cholesterol gallstones for over a year. Consistent results were obtained in vitro, affirming the specific role of cholesterol in this process. Thus, asymptomatic S. typhi carriage is facilitated by formation of biofilms on cholesterol gallstones.5 The mechanism through which S. typhi remains in the gallbladder for extended periods of time depends on the very specific binding affinity between the bacteria and cholesterol. This affinity, as well as the biofilm formation process, is related to the O-antigen capsule. Research of the genes involved in the process of creating this capsule found that they are upregulated when S. typhi is grown in a concentrated bile environment. This capsule plays a structural role in the EPS matrix of the biofilm and has accordingly been identified as is a potential therapeutic target.6 It is crucial to successful carriage, aids the bacteria in adapting to its host, and has been characterized in multiple studies.7

[FIGURE 1: I found a great image from a Nature review article (Gonzalez-Escobedo et. al. 2011, Fig. 2), but I am not sure how to go about obtaining the permission to use it. It has three parts and shows both illustrations and electron micrograph images of biofilm formation on gallstones.]

Mechanisms of S. typhi carcinogenicity in the gallbladder: how chronic carriage can lead to cancer

Bile as a mutagen of S. typhi
Bile salts, which act as detergents chemically and have been characterized as able to damage DNA, have a mutagenic effect on S. typhi that allows the bacteria to thrive in the gallbladder. Furthermore, by metabolizing these salts and also the cholesterol of the gallstones, S. typhi bacteria themselves are capable of producing compounds that are mutagenic to the gallbladder epithelium.8 One proposed mechanism of mutation of S. typhi is oxidative damage related to the conjugation that characterizes bile salt chemistry. Bile is exceptionally concentrated in the gallbladder, with levels exceeding 15% even in normal individuals. Thus, this factor could select for S. typhi survival within this niche environment through genome rearrangements and possible polymorphisms that result in increased fitness of S. typhi in chronic carriers.9 Analysis of the frequency of S. typhi genome rearrangements post exposure to bile salts shows that these mutations tend to occur in several common locations and have particular manifestations. One study found that bile, particularly in very high concentrations, can act as a mutagen in vivo for S. typhi for a variety of particular gene targets including DNA adenine methylase, which works to decrease bile sensitivity and reduce rate of mutation in normal bacteria. Although bile is not generally characterized as a strong mutagen, its high concentration and the long time of exposure are demonstrated to contribute to mutagenic potential and lead to cancerous changes of the gallbladder epithelium.10

Chemical mechanisms of S. typhi mutagenic action
Chemical mechanisms through which carcinogenic compounds are created through bile salt metabolism in long-term S. typhi carriers have been proposed. For example, the action of the β-glucuronidase enzyme, which leads to deconjugation of conjugated toxins and bile acids in this niche under high concentrations, could render them carcinogenic to the host.11 Another proposed mechanism of mutagenicity is related to interactions with the cholesterol that form the structural basis of the gallstones. One study found that bacteria that colonize the gut could not only alter bile salts to a secondary bile form but also convert cholesterol into carcinogenic compounds, including cholesterol 5alpha,6alpha-epoxide.12 Additional studies demonstrated that S. typhi bacteria are capable of metabolizing a variety of chemicals to mutagenic cholic acid derivative forms in the presence of bile and cholesterol. Another commonly described factor that leads to mutagenicity in the host cells is the presence of β-glucuronidase.13 One study expanded on this finding to show that β-glucuronidase action on bile salt substrates may lead to the production of a high-energy, active intermediate that, in turn, binds to DNA and has mutagenic potential in S. typhi itself.14 Other mechanisms including the S. typhi genotoxin Cytolethal Distending Toxin B (CDT) may also play a role in this mutagenic and pathogenic process. This toxin is encoded by S. typhi but is able to create DNA lesions in target cells—including, potentially, cells of human hosts.15,16

[FIGURE 2: I found an image (Crawford et. al. 2010, Fig. 4) that is a composite of four different patients with different clinical presentations but have not yet obtained the permission to use it. Two were asymptomatic typhoid carriers who had bacterial biofilms on gallstones. A third was S. typhi positive but had gallstones of a non-cholesterol composition (calcium bilirubinate) and no biofilm. The fourth was not positive for S. typhi and did not have a biofilm.]

Epidemiological links between chronic S. typhi carriage and gallbladder cancer

Literature and published studies
The relationship between chronic S. typhi carriage and gallbladder cancer has been researched and characterized in studies conducted in sites worldwide. One major case-control study was performed in India and published in 2000. Controls, gallbladder carcinoma patients, and cholelithiasis (gallstone) patients were studied with respect to their typhoid carriage state. It was found that gallbladder cancer patients had significantly higher incidences of S. typhi than controls and cholelithiasis patients did, at 29.4%. The study concluded that the risk of developing gallbladder carcinomas in typhoid carriers was 8.47 times higher than in non-carriers.17 A 1994 study followed a cohort of 507 patients in Scotland following a 1964 typhoid outbreak. 16% of these were found to be chronic carriers. The major finding, however, was that these individuals were reportedly 167 times more likely to develop gallbladder cancer than the patients with acute infections but not chronic carriage were, thus stressing the importance of bacterial niche and time frame in S. typhi mutagenicity. Risk of other cancers of digestive system organs was also elevated—but less intensely by 1-2 orders of magnitude on a logarithmic scale.18 A 1979 case-control study conducted in the US looked into the association of hepatobiliary cancer and typhoid carrier state in deceased typhoid carriers who had been registered by the NYC health department between 1922-1975. The 471 carriers and 942 controls in the study were matched for sex, age at death, year of death, borough in which they died, and where they were born. The key findings were that chronic typhoid carriers died of hepatobiliary cancer six times more often than the controls did—a statistically significant difference. The mechanism of carcinogenicity was suggested to be “altered” bile salts acting as a carcinogen within the gallbladder, bile duct, and small bowel rather than due to an explanation such as cancer preceding typhoid infection or carcinogenicity of the typhoid treatment itself.19 Another team investigated the correlation among typhoid carriage, biofilms, and cholesterol-based gallstones. Through analysis of data taken from surgically removed human gallstones in Mexico, typhoid carriage and biofilms were identified in 4.9%, but neither was present without the other in any patients studied.20

[FIGURE 3: I was able to locate a map (originally created by the World Health Organization; published by the Oxford Journal of Medicine [1]) showing the global distribution of typhoid fever, but I have not yet obtained the official permission to use it.]

Specificity of association cancer of the gallbladder as opposed to other pathologies
Chronic S. typhi infection in humans is linked clearly to gallbladder cancer but not to other gallbladder pathologies. One study considered the role that bacterial degradation of primary bile acids could play in gall bladder carcinogenesis by studying a patient population consisting of individuals presenting with gallbladder carcinoma and also patients presenting only with gallstones. S. typhi bacteria were identified in the bile of 40% of the gallbladder carcinoma patients and 30% of the cholelithiasis patients. However, cancer patients but not cholelithiasis patients had significantly elevated secondary bile acid levels—specifically lithocholate and deoxycholate, which have both been linked to carcinogenicity in humans in numerous studies. Although cancer patients consistently had higher secondary bile levels than cholelithiasis patients did regardless of S. typhi carriage state, the cancer patients with bacteria in the bile had significantly higher secondary bile acid levels than the non-carrier cancer patients did. The conclusion, thus, was that bacterial degradation of primary bile acids in the gallbladder could be a factor in carcinogenesis, one whose effect is amplified by S. typhi mechanisms and presence, definitively linking the typhoid carrier state to carcinogenesis.21

Conclusion

Individuals who are long-term S. typhi carriers tend to be asymptomatic—though highly contagious—and have a significantly elevated risk of developing gallbladder carcinoma. S. typhi bacteria survive in the gallbladder niche by forming biofilms on cholesterol gallstones. The mutagenic effects of bile salts on S. typhi further facilitate this survival, which initially favored by structural mechanisms such as the O-antigen capsule, over long periods of time. Furthermore, S. typhi itself has a mutagenic effect on the host through metabolism of bile salts into carcinogenic secondary bile compounds and other genotoxic effects. This connection has been characterized pathologically and epidemiologically by studies performed on a global scale, in which the chronic S. typhi carrier state constitutes a key risk factor for gallbladder carcinoma.

References


1 Delhi Cancer Registry, Institute of Rotary Cancer Hospital, All India Institute of Medical Sciences, 1993;29:1992

2 Dutta U, Garg PK, Kumar R, Tandon RK (2000). Typhoid carriers among patients with gallstones are at increased risk for carcinoma of the gallbladder. Am. J. Gastroenterol. 95:784-787.

3 Nath G, Gulati AK, Shukla VK (2010). Role of bacteria in carcinogenesis, with special reference to carcinoma of the gallbladder. World J. Gastroenterol. 16:5395-5404.

4 Gonzalez-Escobedo G, Marshall JM, Gunn JS (2011). Chronic and acute infection of the gall bladder by Salmonella Typhi: understanding the carrier state. Nature Rev. Microbiol. 9:9-14.

5 Crawford RW, et al. (2010). Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage. Proc. Natl. Acad. Sci. U.S.A. 107:4353-4358.

6 Crawford RW, Gibson DL, Kay WW, Gunn JS (2008). Identification of a bile-induced exopolysaccharide required for Salmonella biofilm formation on gallstone surfaces. Infect. Immun. 76:5341-5349.

7 Gibson DL, et al. 2006. Salmonella produces an O-antigen capsule regulated by AgfD and important for environmental persistence. J. Bacteriol. 188:7722–7730.

8 Prieto AI, Ramos-Morales F, Casadesús J (2006). Repair of DNA damage induced by bile salts in Salmonella enterica. Genetics. 174:575-584.

9 Prieto AI, Ramos-Morales F, Casadesús J (2006). Repair of DNA damage induced by bile salts in Salmonella enterica. Genetics. 174:575-584.

10 Prieto AI, Ramos-Morales F, Casadesús J (2004). Bile-induced DNA damage in Salmonella enterica. Genetics 168:1787-1794.

11 Dutta U, Garg PK, Kumar R, Tandon RK (2000). Typhoid carriers among patients with gallstones are at increased risk for carcinoma of the gallbladder. Am. J. Gastroenterol. 95:784-787.

12 Chipman JK (1982). Bile as a source of potential reactive metabolites. Toxicology 25:99-111.

13 Connor TH, Forti GC, Sitra P, Legator MS (1979). Bile as a source of mutagenic metabolites produced in vivo and detected by Salmonella typhimurium. Environ. Mutagen., Vol. 1, ISS 3, 269-276.

14 Kinoshita N, Gelboin HV (1978). Beta-glucuronidase catalyzed hydrolysis of benzoapyrene-3-glucuronide and binding of DNA. Science 199:307-9.

15 Nath G, Gulati AK, Shukla VK (2010). Role of bacteria in carcinogenesis, with special reference to carcinoma of the gallbladder. World J. Gastroenterol. 16:5395-5404.

16 Haghjoo E, Galán JE. Salmonella typhi encodes a functional cytolethal distending toxin that is delivered into host cells by a bacterial-internalization pathway. Proc. Natl. Acad. Sci. USA 2004; 101: 4614-4619.

17 Shukla VK, Singh H, Pandey M, et al (2000). Carcinoma of the gall 
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18 Caygill C, Hill M, Braddick M, Sharp J (1994). Cancer mortality in chronic typhoid and paratyphoid carriers. Lancet 343:83-84.

19 Welton JC, Marr JS, Friedman SM (1979). Association between hepatobiliary cancer and typhoid carrier state. Lancet 313(8120):791-794.

20 Crawford RW, et al. (2010). Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage. Proc. Natl. Acad. Sci. U.S.A. 107:4353-4358.

21 Pandey M, Vishwakarma RA, Khatri AK, et al (1995). Bile bacteria and gall bladder carcinogenesis, J Surg. Oncol. 58:282-283.

Edited by Hannah Moore, a student of Nora Sullivan in BIOL187S (Microbial Life) in The Keck Science Department of the Claremont Colleges Spring 2013.