**1. Introduction**

Gallbladder cancer (GBC) is the most common malignancy of the biliary tract with an aggressive clinical course and short median survival [1]. While being a rarity in the western world, GBC is one of the major causes of cancer-related morbidity and mortality in South Asian and Southeast Asian countries [2]. Females are more commonly affected than males. According to the cancer statistics of 2020, GBC accounts for 0.6% of the total cancer cases and is associated with 0.9% of total cancer-related deaths [3]. Around 10% of the global GBC burden is contributed by India, with the Northern, Central, and North-eastern parts as the highest contributors [4]. Only 10% of cases present at an early stage which can be owed to the aggressive tumor biology of this cancer and the lack of effective screening techniques for its early detection [5]. Chronic inflammation of the gallbladder remains a major factor in the pathogenesis of GBC, although the causes are multifactorial. Gall stones, heavy metals, environmental toxins, and carcinogens have

all been implicated in chronic irritation of the gallbladder mucosa, thereby leading to dysplasia and subsequent development of neoplasia.

The landscape of the microbiome populating our digestive tract has received a lot of scientific attention in recent years [5]. There is ample evidence linking the human microbiome and its metabolites to carcinogenesis. It is proven that balanced flora or microbial eubiosis is related to health while dysbiosis or unbalanced flora can lead to various diseases, including cancers [6, 7]. There can be multiple triggers causing dysbiosis, including fluctuations in the environment, inflammation, infection, medications, dietary changes, or genetic predisposition. The International Agency for Research on Cancer labeled ten microbial species as carcinogens [8]. Around 15–20% of cancers are linked to microbial pathogens, with *Helicobacter pylori* (*H. pylori*), human papillomavirus, Hepatitis B virus, and hepatitis C virus being the four predominant species, driving 90% of infection-associated cancers [6, 9, 10]. However, there is very limited information available on the microbial species inhabiting the human gall bladder, except for a few cultivable species of bacteria associated with cholelithiasis [11, 12]. It was seen that the biliary tract has an abundance of Enterobacteriaceae [13]. Microorganisms in the common bile duct of patients having gallstones were more commonly those that inhabited the human respiratory tract and oral cavity rather than intestinal microbes [14]. Very recently, culture-negative bile samples acquired from normal gallbladders were evaluated using 6S ribosome gene analysis. A very simple and less diverse bacterial flora was found comprising the Firmicutes, Proteobacteria, and Actinobacteria phyla [15].

Detection of some bacteria does not indicate its causality in inflammation or cancer. However, recent amassing evidence indicates that microbiota dysbiosis and chronic inflammation contribute to carcinogenesis [16]. Several reports point towards strains of Salmonella and Helicobacter colonizing the gall bladder and are linked to an escalated risk of developing GBC [17, 18]. Premalignant lesions were found to be coexisting with chronic Salmonella infestation, despite the absence of gallstones [19]. Various experimental studies and epidemiologic data support the induction of carcinogenesis due to dysbiosis of the gallbladder microbiome. However, results indicating only cultivable species limit these claims. Also, despite the proximity of a large diverse microflora reservoir in the gut, little is known about its impact on the human bile microbiome. In this chapter, we aim to provide a comprehensive review of all the available literature on the gut and biliary microbiome and their association with GBC.
