Fei Liu et al. examined tissue samples of cancerous bladder mucosa from patients diagnosed with BCa, consisting of 22 carcinoma tissues and 12 adjacent normal tissues. This study confirmed the occurrence of bladder microbiota dysbiosis in BCa patients. The researchers observed that Proteobacteria was the predominant phylum in both cancerous and noncancerous tissues, with a significantly greater abundance in cancerous tissues. In cancerous tissues, the presence of Cupriavidus, Acinetobacter, and Escherichia‐Shigella increases markedly (Wang et al., 2007). Previous research has indicated that certain trivalent pesticide-related chemicals can induce protein carbonylation and oxidative DNA damage in human urothelial cells, potentially leading to BCa (Liu et al., 2019). Interestingly, the enrichment of harmful chemical products, which are subject to metabolic processes, might correlate with the significantly elevated abundance of the genus Cupriavidus. This genus was the most abundant bacteria found in both cancerous and noncancerous bladder tissues, despite notable differences in their levels. Cupriavidus, known from prior studies as an organophosphorus pesticide-degrading microorganism, is thought to break down harmful substances absorbed by the body, subsequently excreting them into the bladder through specific enzymes (Wang et al., 2007).

Naomi Nadler et al. collected tissue samples from ten patients undergoing TURBT and analyzed them for bacterial aggregates using Fluorescence in situ Hybridization (FISH). Dense biofilms were identified in the urothelial cancer tissue of two samples, and spherical bacteria were confirmed in one sample. Notably, both patients had negative preoperative urine cultures. These findings suggest a potential link between biofilm formation and BCa (Nadler et al., 2021).

Bacterial aggregates, commonly referred to as biofilms, often attach to the apical epithelial cells of the bladder and are known as umbrella cells. These cells typically have a protective layer of sulfated polysaccharide aminoglycans. Disruption of this layer may lead to pathological bladder changes and chronic inflammation in the genitourinary system (Garrett, 2015). During biofilm formation, bacteria such as Aeromonas hydrophila and Pseudomonas aeruginosa can augment the production of extracellular proteases, including serine proteases, metalloproteinases, and elastases (Swift et al., 1999). Some pathogens, such as Streptococcus pyogenes, may cause invasive diseases by degrading intercellular junctions in conjunction with the host cysteine protease calpain (Sumitomo et al., 2013).

Studies on bacterial biofilms in colorectal cancer have shown that certain bacteria, such as Escherichia coli, can drive cancer development. Escherichia coli produces the genotoxin colibactin, which causes mutagenic DNA damage through covalent DNA binding to a chemical moiety known to promote tumorigenesis (Garrett, 2015). Further research is required to understand this relationship, particularly whether bacterial dysbiosis and biofilm formation in the bladder exhibit oncogenic features similar to those in colorectal cancer. Establishing a causal link could make microbiome modulation a significant therapeutic area.

Wei Tse Li et al. identified a range of microbes, including E. coli, the butyrate-producing bacterium SM4/1, and a species of Oscillatoria, that were correlated with the expression of classic EMT-associated genes, such as E-cadherin, vimentin, SNAI2, SNAI3, and TWIST1. Notably, the Escherichia coli str. K-12 substr. showed the most significant correlations with EMT-related gene expression. They also observed significant correlations between microbial abundance and the expression of extracellular matrix (ECM) genes, particularly those related to collagens and elastin. The presence of the Escherichia coli O157:H7 strain exhibited a significant correlation with the expression of ECM proteins (Li et al., 2021).

EMT is a key process in several cancers that enhances metastatic potential by increasing cell mobility and decreasing cell–cell adhesion (Franzen et al., 2015; Ogawa et al., 2020). During EMT, epithelial cells transform into mesenchymal cells, enabling detachment from the basement membrane and invasion into adjacent tissues. These mesenchymal cells can migrate to distant sites and revert back to epithelial cells through mesenchymal–epithelial transition, initiating metastasis. In MIBC, the importance of EMT is highlighted by the upregulation of mesenchymal cell markers, such as N-cadherin and P-cadherin, and the downregulation of epithelial cell markers, such as E-cadherin (Yun and Kim, 2013; Franzen et al., 2015). ECM proteins play a vital role in cancer cell invasion and metastasis, with proteins such as collagens, laminins, and fibronectins associated with survival in urothelial bladder cancer (Brunner and Tzankov, 2007). During metastasis and invasion, ECM proteins degrade and integrins rearrange, facilitating EMT (Brunner and Tzankov, 2007; Jiang et al., 2017). Microbes, known for releasing proteases including collagenases, are thought to influence ECM protein turnover, although this has not yet been confirmed in BCa studies (Alfano et al., 2016).

Interactions between tumor cells and the extracellular microenvironment are critical in cancer development and progression. The interplay between ECM components and bacterial products regulates tissue homeostasis, dysregulation of these processes may create protumorigenic niches, potentially contributing to disease relapse. Understanding the relationship between Escherichia coli and the extracellular microenvironment offers valuable insights into the pathogenesis and progression of BCa.

Filippo Pederzoli and his team conducted a study on paired samples from 21 male and 8 female patients, including urine, neoplastic (Npl), and non-neoplastic (non-Npl) tissues. Their findings revealed a greater abundance of the genus Burkholderia in tumor tissues than in non-tumor tissues, regardless of the patient’s gender (Pederzoli et al., 2020). In a parallel study, Bassel Mansour’s research group corroborated these results. They investigated microbiome differences in urine and tissue samples from BCa patients and noted a greater abundance of Burkholderia in cancerous tissues (Mansour et al., 2020). This observation implies a potential role for this genus in the pathobiology of BCa, similar to recent findings in colorectal cancer versus healthy colon mucosa (Alomair et al., 2018). However, the role of this taxon appears to extend beyond merely acting as a trigger for neoplasia. Recent studies have suggested Burkholderiales as a potential “anticancer probiotic.” For instance, in an animal sarcoma model, it was demonstrated that the efficacy of immunotherapy with CTLA-4 antibody was influenced by the composition of the microbiota, particularly by Bacteroides fragilis, Bacteroides thetaiotaomicron, and Burkholderiales (Vétizou et al., 2015). Additionally, the transplantation of these bacteria into antibiotic-conditioned animals showed a protective effect against colitis induced by CTLA-4 blockade.

Wei Tse Li et al. discovered a strong negative correlation between the presence of Oscillatoria, a Cyanobacteria member, and the expression of EMT-promoting genes (Li et al., 2021). Although Cyanobacteria are less characterized in humans, their presence has been detected in the gut (Almeida et al., 2019). Importantly, Oscillatoria species produce butylated hydroxytoluene, a natural antioxidant, which might explain its association with reduced EMT (Babu and Wu, 2008). This connection is significant as oxidative stress and reactive oxygen species are known to regulate ECM proteins and the EMT process (Jiang et al., 2017).

Bassel Mansour et al. compared the microbiota composition in cancerous tissues and urine samples from the same patient set. They noted a surprisingly high presence of Cyanobacteria—7% and 8% in urine and tissue samples, respectively—a finding previously undocumented (Mansour et al., 2020).

Mansour, Ádám Monyók, and their team subsequently investigated the tissue microbiome in BCa patients compared to that in benign prostatic hyperplasia patients and healthy volunteers. The authors focused on the mRNA levels of hBDs in tissue and the levels of defensin in urine. Oxyphotobacteria, a Cyanobacterium genus linked to tumor formation (Miao et al., 2016), exhibited a significantly greater abundance in the BCa group, especially in patients with low urinary hBD-1 levels (Mansour et al., 2022).

Defensins, notably hBDs, are critical antimicrobial peptides that play a role in tumor cell dissolution, immune cell attraction, and interactions with complement factors (Adyns et al., 2023). Human β-Defensin-1 (hBD-1) can modify HER2 signal transduction and has been shown to suppress BCa growth (Sun et al., 2019). The ability of hBD-1 to attract cells expressing C-C chemokine receptor 6 (CCR6) suggests its potential for recruiting immune cells, such as Th cells, Tregs, imDCs, and neutrophils (Yang et al., 1999; Biragyn et al., 2001; Wu et al., 2003). The dual antitumor and antibacterial activities of hBD-1, along with the influence of bacterial presence on the production of hBD-2 and hBD-3 (Adyns et al., 2023), highlight the significance of understanding the role of hBDs in antitumoral immune responses and their potential impact on immunotherapy effectiveness.

LAB, a group of gram-positive bacteria within the phylum Firmicutes, are primarily categorized into the genera Lactobacillus, Streptococcus and Lactococcus (Sharma et al., 2020).

Fei Liu’s research indicated a notable difference in the abundance of Lactobacillus between cancerous and noncancerous tissues, with a greater prevalence in noncancerous tissues (Wang et al., 2007). Lactobacillus, a well-studied probiotic, is known for its health-promoting mechanisms, such as colonizing resistance, acid production, and pathogen exclusion. It has also been linked to cancer, as cell-free supernatants from Lactobacillus can reduce the invasion ability of metastatic tumor cells in vitro (Siddiqui et al., 2012; Pearce et al., 2014). The predominant strain Lactobacillus in women’s bladders and its potential protective role against urothelial bladder cancer have not been determined, although there is evidence suggesting its role in reducing chronic inflammation and enhancing immune responses (Kato et al., 1984; Kato et al., 1988; Takagi et al., 2001; Matsumoto et al., 2009; Shida and Nomoto, 2013).

Wei Li’s study sequenced and analyzed bacterial genomes from cancer and adjacent tissues of 18 BCa patients with no urinary tract infection and negative urine culture. The research has revealed the presence of bacteria in various bladder layers, predominantly in the outer layer of the mucosa. Linear discriminant analysis Effect Size (LEfSe) revealed significant differences in microbial communities, highlighting Staphylococcus at the genus level as significantly more abundant in BCa tissues. This finding aligns with multiple cases of Staphylococcus in the urine cultures of BCa patients (Li, 2021). Similarly, Bassel Mansour’s team noted a greater abundance of Staphylococcus in tumor tissues, correlating with elevated levels of hBD-2 and hBD-3 in urine (Mansour et al., 2022). The inducible production of antibacterial HBD2 and HBD3 is affected by bacteria. Elevated levels of HBD2 were shown to cause treatment failure in anticancer immunotherapy (Adyns et al., 2023). Staphylococcus, particularly saprophytic Staphylococcus, is commonly associated with urinary tract infections. Its role in chronic infections and potential link to the development of BCa warrant further investigation. The impact of saprophytic Staphylococcus on bladder epithelial and cancer cells could be a valuable direction for future research.

Since 1976, BCG (Mycobacterium bovis, Bacillus Calmette–Guérin) has been used as one of the most successful antitumor immunotherapies, particularly for the treatment of BCa (Morales et al., 2017). This specific strain from the Actinobacteria phylum and Mycobacterium genus not only activates the immune system against BCa but also directly induces tumor cell apoptosis (Yu et al., 2015), necrosis (See et al., 2009), or oxidative stress (Shah et al., 2014). The lower incidence of BCa in women might be partially explained by the significantly greater presence of Actinomycetes, including the Mycobacterium genus, in the female urinary microbiome (Lewis et al., 2013).

Mónica Parra-Grande et al. observed a significantly greater abundance of Actinobacteria in nonneoplastic bladder mucosa than in tumor tissues (P = 0.014). Although not statistically significant at the genus level, Propionibacterium was more abundant in the non-tumor mucosa (P = 0.08) (Parra-Grande et al., 2022). Propionibacterium freudenreichii, notable for its probiotic potential and commercial relevance, has been suggested to be a protective agent against colorectal cancer. Research by Casanova et al. indicated that this bacterium could be useful as a probiotic for early-stage colorectal cancer prevention (Casanova et al., 2018). This finding supports the hypothesis that a microbiota rich in Actinomycetes might be linked to the lower incidence of BCa in women (Raoult, 2017), suggesting that a preventive effect similar to that of the BCG vaccine (composed of Actinomycetes), known for its protective role in BCa treatment and relapse prevention (Whiteside et al., 2015b).

Conversely, the research team led by Bassel Mansour and Ádám Monyók reported a greater abundance of another Actinobacteria phylum member, the Corynebacterium genus, in tumor specimens than in non-tumor specimens. This increased presence correlated with elevated levels of hBD-2 and hBD-3 in the urine (Mansour et al., 2022), suggesting a possible role of Corynebacterium in BCa pathogenesis.

The Bifidobacterium genus is a strictly anaerobic, gram-positive, pleomorphic rod-shaped bacterium that belongs to the Actinobacteria phylum. Owing to their morphological and physiological similarities with Lactobacillus, they were historically classified as members of the Lactobacillus genus for a significant portion of the 20th century. Only recently have they been acknowledged as a distinct genus separate from Lactobacillus (Turroni et al., 2011). Many bifidobacteria are used as active ingredients in a variety of so-called functional foods due to their perceived health-promoting or probiotic properties, such as protection against pathogens mediated through the process of competitive exclusion, bile salt hydrolase activity, immune modulation, and the ability to adhere to mucus or the intestinal epithelium (Liévin et al., 2000; Ouwehand et al., 2002; Stanton et al., 2005).

Mónica Parra-Grande et al. reported an increased abundance of Bifidobacterium in NMIBC tissues (Whiteside et al., 2015b). Bifidobacterium plays a crucial role in microbial homeostasis and anti-inflammatory responses in the mucosa (Di Giacinto et al., 2005; Turroni et al., 2011), and can inhibit IL-6 secretion (Trikha et al., 2003), indicating its possible influence on tumorigenesis. Vyara Matson’s study revealed a link between enriched Bifidobacterium and improved immunotherapy response in melanoma patients (Morales et al., 2017). Parra-Grande’s findings suggest that a lower abundance of Bifidobacterium in MIBC than in NMIBC could indicate bladder mucosal damage and a protective factor against BCa (Parra-Grande et al., 2022).

In their study, Fei Liu et al. reported that Prevotella_9 was more prevalent in non-cancerous tissues (Wang et al., 2007). Prevotella, an integral part of the gut microbiome, plays a crucial role in the digestion of fiber and complex carbohydrates. Its abundance is closely linked to dietary habits, particularly when it is high in fiber. Members of the Prevotella genus have been implicated in various cancers, including colorectal, oral, and stomach cancers (Feng et al., 2023). For instance, an increase in Prevotella has been noted in the gut microbiome of colorectal cancer patients (Bonnet et al., 2014). Some Prevotella species may contribute to cancer development by affecting the tumor microenvironment, for example, through modulating local immune responses or producing metabolic byproducts. There is also evidence suggesting that Prevotella may impact the host immune system, potentially playing a role in tumor immune evasion (Chen et al., 2022). While the general research on Prevotella highlights its potential significance in cancer development, additional studies are needed to elucidate the specific role and mechanisms of Prevotella_9 in cancer. The greater abundance of Prevotella_9 in non-cancerous tissues, as found by Fei Liu, might indicate distinct characteristics and roles of this subtype within the Prevotella genus.

Several studies included in this summary have conducted comparative analyses of BCa tissues, adjacent normal tissues, or non-tumor tissue samples. Bassel Mansour’s team highlighted significant differences in microbial composition between tumor and non-tumor tissue samples (Mansour et al., 2022). Fei Liu’s team found that, compared with noncancerous tissues, cancerous tissues had lower species richness and diversity, with notable differences in beta-diversity (Wang et al., 2007). Similarly, Mónica Parra-Grande’s team observed greater microbial diversity in non-tumor bladder mucosa than in tumor tissues, aligning with global indicators of microbiome diversity and richness (Parra-Grande et al., 2022). However, Rebecca M. Rodriguez’s team reported no significant statistical differences in total reads, relative abundance, or positivity ratio between paired tumor and adjacent normal samples (Rodriguez et al., 2020), a finding echoed by Wei Li et al (Li, 2021).

Regarding microbiota variations in BCa tissues of different stages and grades, Mónica Parra-Grande’s team noted significant differences in microbial composition among different tumor grades (Parra-Grande et al., 2022). Jian-Xuan Sun’s team reported similar microbial compositions in MIBC and NMIBC tumor tissues, but with notably greater microbial diversity in NMIBC tissues (Sun et al., 2023). These findings suggest a potential link between the microbiome and tumor biology. Conversely, Bassel Mansour et al. observed no differences in beta-diversity across different tumor grades and stages or in relation to diabetes and hypertension (Mansour et al., 2022). Similarly, Rebecca M. Rodriguez et al. reported no significant differences when paired tumor and normal tissues were stratified according to sex, race, anatomical site, or tumor stage (Rodriguez et al., 2020).

In the context of sex differences, Bassel Mansour et al.’s studies indicated significant diversity variations between male and female tissue samples (Mansour et al., 2020; Mansour et al., 2022), whereas Filippo Pederzoli et al.’s research showed no differences in alpha- and beta-diversity based on sex (Pederzoli et al., 2020). The study of the BCa tumor microbiota is a burgeoning field, with discrepancies among studies highlighting the need for larger sample sizes and further research.