A total of 20 colorectal cancer patients were included in this study ( Table 1). The age distribution showed a balanced proportion between the <60 years and ≥60 years groups, with 10 patients (50%) in each group. Based on gender, the number of male and female respondents was also balanced, with 10 patients (50%) in each group. Nutritional status was dominated by the normal category (14 patients; 70%), followed by underweight (3 patients; 15%), obese (2 patients; 10%), and overweight (1 patient; 5%).

The most common tumor location was the rectosigmoid, found in 16 patients (80%). Other locations were less common, namely the descending colon in 2 patients (10%), the ascending colon/cecum in 1 patient (5%), and the transverse colon in 1 patient (5%) ( Table 1).

A comparison of microbiota species in cancer tissue, normal mucosal tissue, and feces is shown in Table 2 (n = 20 each). Overall, Escherichia coli was the most frequently found species in all three types of samples, with the highest proportion in cancer tissue (8/20; 40%), compared to normal mucosal tissue (6/20; 30%) and feces (6/20; 30%).

In cancerous tissue, besides Escherichia coli, other bacterial species detected included Bacteroides fragilis (2/20; 10%), and each of the following at 1/20 (5%): Shigella sp., Shigella sonnei, Salmonella enterica, Pseudomonas sp., Bacteroides graminisolvens, and Aeromonas sp. No bacterial growth was observed in 4 of the cancer tissue samples (20%).

In normal mucosal tissue, aside from E. coli (30%), the species detected were B. fragilis (2/20; 10%), Salmonella enterica (2/20; 10%), and Enterobacter sp. (2/20; 10%). Species found in 1/20 (5%) of samples included Escherichia fergusonii, Klebsiella pneumoniae, Bacillus subtilis, Microbacterium paraoxydans, and Staphylococcus sp. There was no bacterial growth in 3 of the normal mucosa samples (15%).

In fecal samples, E. coli remained the most frequently detected organism (6/20; 30%), followed by Klebsiella pneumoniae (4/20; 20%), B. fragilis (3/20; 15%), and Faecalibacterium prausnitzii (2/20; 10%). Other species detected at 1/20 (5%) included Shigella sp., Shigella sonnei, Corynebacterium sp., and Oscillospiraceae bacterium. Additionally, one fecal sample showed no bacterial growth (5%).

A comparison of genera in cancer tissue, normal mucosal tissue, and feces is shown in Table 3. The most dominant genus in all sample types was Escherichia, with the highest proportion in cancer tissue (8/20; 40%), followed by normal mucosal tissue (7/20; 35%) and feces (6/20; 30%).

In cancer tissue, besides Escherichia, other genera identified were Bacteroides (3/20; 15%) and Shigella (2/20; 10%), as well as Salmonella, Pseudomonas, and Aeromonas, each detected in 1/20 samples (5%). No bacterial growth was observed in 4 cancer tissue samples (20%).

In normal mucosal tissue, after Escherichia (35%), the genera detected were Salmonella and Enterobacter (each 2/20; 10%), Staphylococcus (2/20; 10%), and Bacteroides, Klebsiella, and Microbacterium (each 1/20; 5%). No growth was found in 3 samples (15%).

In fecal samples, Escherichia (30%) remained the most prevalent genus, followed by Klebsiella (5/20; 25%), Bacteroides (3/20; 15%), and Faecalibacterium (2/20; 10%). Other genera, each detected in 1/20 samples (5%), included Shigella, Corynebacterium, and Oscillospira, and 1 sample showed no growth (5%).

Proteobacteria was the most dominant phylum across all sample types, accounting for 13/20 (65%) in cancer tissue, 12/20 (60%) in normal mucosal tissue, and 12/20 (60%) in fecal samples. The phylum Bacteroidetes was detected at the same proportion in cancer tissue and feces (3/20; 15% each), but at a lower proportion in normal mucosal tissue (1/20; 5%). Firmicutes was not detected in cancer tissue, but was found in normal mucosal tissue (2/20; 10%) and feces (3/20; 15%). Actinobacteria was not detected in cancer tissue and was only found in normal mucosal tissue (1/20; 5%) and feces (1/20; 5%). No bacterial growth was observed in 4 cancer tissue samples (20%), 3 normal mucosal samples (15%), and 1 fecal sample (5%).

The classification of microbiota into proteolytic and saccharolytic groups identified by corespondent. Proteolytic microbiota were the most frequently identified across all sample types, detected in 13/20 (65%) cancer tissue samples, 14/20 (70%) normal mucosal tissue samples, and 13/20 (65%) fecal samples. Saccharolytic microbiota were less common and showed similar proportions in cancer and normal mucosal tissues (3/20; 15% each), while in fecal samples they were detected in 6/20 (15%). No growth was recorded in 4/20 (20%) cancer tissue samples, 3/20 (15%) normal mucosal samples, and 1/20 (5%) fecal samples. Overall, these findings indicate differences in microbiota patterns among cancer tissue, normal mucosa, and feces, with Escherichia predominating at the species and genus levels and Proteobacteria being the dominant phylum across all sample types ( Tables 2 and 3).

The distribution of respondents by age in this study ( Table 1) shows an equal split between individuals aged ≥60 years and <60 years, suggesting that colorectal cancer (CRC) affects both older and younger adults. This is consistent with global epidemiological trends showing a rise in CRC incidence among younger populations, particularly in high-income countries, even as rates in older groups remain high or decline due to screening efforts and lifestyle changes.

The balanced gender distribution (50% male and 50% female) aligns with existing literature indicating that while CRC risk is slightly higher in males, the difference is often not as pronounced depending on the population studied, and both sexes experience similar disease burdens in clinical cohorts. This finding supports the notion that gender alone is not a definitive risk determinant in CRC, but interacts with lifestyle, genetic, and microbiota factors.

In terms of nutritional status, most patients (70%) were classified as normal, with smaller proportions being underweight, overweight, or obese ( Table 1). Nutritional status and diet are known to influence gut microbiota composition and metabolism, which in turn can affect CRC risk. For example, a diet high in processed red meats and low in fiber has been linked to an increased abundance of pro-carcinogenic gut bacteria and secondary bile acids that promote DNA damage and inflammation, whereas fiber intake increases protective short-chain fatty acids (SCFAs) that maintain mucosal integrity. ¹⁴

Taken together, the characteristics of the respondents reflect a clinically relevant CRC population and provide a basis for interpreting microbiota differences in subsequent analyses.

a.

Species Distribution

The microbial species profiling revealed that Escherichia coli was the most abundant species in cancer tissues (40%), normal mucosa (30%), and feces (30%) ( Table 2). The high relative abundance of E. coli across sample types supports extensive literature demonstrating the role of specific strains of E. coli, particularly those harboring the polyketide synthase (pks) genomic island, in CRC pathogenesis. These pks-positive strains produce colibactin, a genotoxin that induces DNA damage and mutational signatures commonly observed in human CRC genomes, suggesting a causal role in tumor initiation and progression. ¹⁵

Moreover, chronic interactions between colibactin-producing E. coli and epithelial cells stimulate continuous DNA damage responses and pro-inflammatory cycles that further promote carcinogenesis. The presence of E. coli in normal mucosa and feces in this study may reflect a broader state of dysbiosis that precedes or accompanies tumor development, consistent with findings that CRC-associated microbiota differ in both tumor and non-tumor tissues compared to healthy controls. ^{14, 16}

Other detected species such as Bacteroides fragilis, Shigella sp., and Pseudomonas sp. in cancer tissues emphasize the complexity of microbial communities in CRC. Enterotoxigenic B. fragilis (ETBF) strains, for example, are shown to induce chronic Th17-linked inflammation and epithelial disruption through toxins like BFT, which directly activate oncogenic pathways and inflammatory cascades that promote tumor growth in animal models and human cohorts. ^{15, 17} b.

Genus Distribution

Analysis of the genus level ( Table 3) reinforces the patterns observed at species and phylum levels, showing that Escherichia was consistently dominant across all sample types tumor tissue (40%), normal mucosa (35%), and feces (30%). The persistence of Escherichia suggests that its expansion is not limited to tumor sites but also affects surrounding tissues and fecal microbiota, indicating systemic dysbiosis rather than a local phenomenon alone. This aligns with evidence that facultative anaerobes, such as Escherichia, thrive in inflamed and oxygen-altered colonic microenvironments typical of CRC, where they can exacerbate inflammation and disrupt epithelial homeostasis. ^{14, 16}

In addition to Escherichia, genera like Bacteroides and Shigella were relatively more prevalent in cancerous tissues compared to normal mucosa and feces. Bacteroides includes strains capable of producing inflammatory molecules that modulate host signaling, contributing to mucosal barrier disruption and immune activation factors associated with CRC progression. ¹⁴ Although Shigella and other genera appeared at lower frequencies, their presence supports a shift toward opportunistic pathogenic bacteria in CRC patients.

Notably, Klebsiella showed higher representation in fecal samples than in tissues. Some Klebsiella species are known for eliciting pro-inflammatory responses, suggesting that fecal microbiota could reflect broader dysbiotic trends in CRC patients and might serve as a non-invasive indicator of disease-associated shifts.

Taken together, the genus distribution in Table 3 reinforces the notion that colorectal cancer is associated with a shift toward pro-inflammatory and potentially pathogenic genera, which coexist across tissue and fecal microbiomes. This pattern strengthens the hypothesis that microbial dysbiosis in CRC is systemic rather than isolated, and highlights the potential utility of genus-level profiling for diagnostic or prognostic purposes. c.

Phylum Distribution

Proteobacteria dominance at the phylum level (65% in cancer, 60% in normal mucosa, and 60% in feces) reflects a signature pattern of microbial dysbiosis in CRC. Proteobacteria enrichment, particularly of gamma-proteobacteria such as E. coli, has been associated with inflammation, epithelial barrier disruption, and oncogenic signaling in CRC. This phylum is often considered a marker of microbial imbalance, as it tends to increase when beneficial microbes like Firmicutes decline, resulting in pro-inflammatory and pro-carcinogenic interactions within the gut ecosystem. ^{14, 18}

In contrast, phyla such as Firmicutes and Bacteroidetes, which include many SCFA-producing and anti-inflammatory bacteria, are often reduced or altered in CRC. Loss of these protective bacteria can compromise mucosal integrity and immune modulation, further exacerbating tumorigenic processes.

The finding that proteolytic microbiota dominate across cancer, normal mucosa, and fecal samples suggests a shift in metabolic processes in the CRC gut environment. Proteolytic bacteria ferment amino acids and proteins into metabolites such as ammonia, hydrogen sulfide (H ₂S), and phenolic compounds that are associated with DNA damage, oxidative stress, and inflammation all pro-carcinogenic conditions. Conversely, saccharolytic bacteria that produce SCFAs like butyrate which has anti-inflammatory and anti-tumor effects by promoting colonocyte apoptosis and maintaining epithelial barrier function were relatively scarce in cancer tissues. Reduced SCFA production is a known feature of CRC dysbiosis and correlates with increased vulnerability to inflammation and tumor initiation (such as butyrate has been shown to have protective effects in CRC models). ^{14, 16}

The consistent patterns of microbiota alteration observed in this study highlight several translational implications: •

Microbiota as Biomarkers

The similarity of microbiota in fecal samples to CRC tissues, especially enrichment of E. coli and Proteobacteria, supports the use of fecal microbial profiling as a non-invasive diagnostic tool for CRC risk assessment and early detection, as proposed by recent microbial screening strategies.