Prevalence and Phylogenetic Analysis of Fusobacterium nucleatum and Virulent Factor FadA among Ulcerative Colitis Precancerous and Colorectal Carcinoma Patients in the Iraqi Kurdish Population

1. Introduction

Colon cancer is one of the significant health problems, with an annual incidence of 1.7 million ( 1 ); nearly about 6.3 million people in the world have been diagnosed with CRC, that caused about 860000 death in 20018 ( 1 , 2 ) all over the world, and this disease recorded the high morbidity in a year. In Iraq, colorectal cancer has increased in the last decades; its mortality rates increased by about 10% ( 3 ), and it has become the second and third most common cancer in women and men, respectively ( 4 ).

The microbiota that habituates the human intestinal tract form an ecosystem consisting of Archei, Bacteria, Fungi and Virus ( 5 ), including around 10¹⁴ bacteria. The highest amount is in the colon ( 6 , 7 ) over 1000 species referring mainly to 4 identified phyla in human, of which each individual have 160 species ( 8 ). The investigations and evidence demonstrated that microbiota has a crucial role in maintaining intestinal mucosal homeostasis and act as a protective epithelial barrier against other invasive pathogens, have an essential role in the promotion of wound repair ( 9 , 10 ). Symbiotic relationship confirmed between commensal bacteria and healthy host, altering gut microbiota population, and irritable bowel disease leads to dysbiosis which contributed the CRC ( 11 , 12 ).

Fusobacterium nucleatumis a gram-negative obligate anaerobic, non-motile, non-spore former bacteria spindle-shaped with pointed ends, and they are commensal microorganisms. Its natural habit is the oral cavity that employs the circulation system to reach the neoplastic cells in the intestine, where the Fap2 mediated in hemagglutination for transporting ( 13 - 17 ), although they act as opportunistic pathogens that can transmit from its natural habitat to other places through the bloodstream, e.g. from the oral cavity to gastrointestinal tract that leads to gastrointestinal carcinogenesis, or placenta and lead to preterm labour ( 18 , 19 ) or urogenital tract ( 20 , 21 ) then become pathogenic, also able to form biofilm as a structural supporting to other bacteria and associated with periodontal health and disease ( 22 - 25 ), from lung and pleural infection ( 26 ), bacteremia and liver abscesses, pancreatic cancer ( 27 ) appendicitis ( 28 ).

Fusobacterium nucleatum has several virulence factors that promote exploring the mechanism of CRC production. The most common basic biological characteristic associated with pathogenicity is; adhesin surface protein, which has a critical role in biofilm formation through the coaggregation of bacteria in the oral cavity and on the right side of a CRC patient's colon ( 29 ). It has invasive factors that influence the entrance of the bacteria to the host cells, like Fad A, which is selectively bound to E-cadherin and has a role in activating B- catenin signalling pathways, thus inducing oncogenic and inflammatory responses ( 30 ). Fusobacterium nucleatum possesses the most significant adhesion membrane protein, Fap2, which can inhibit natural killer cell cytotoxicity through binding to human immune inhibitory receptor T cell and ITIM domain. Hashemi Goradel, Heidarzadeh ( 31 ) recently showed that Fap 2 dependent invasions induce secretion of proinflammatory cytokines, ILK-8, and CXCL that promote CRC cell migration.

The main aim of this investigation is to determine the prevalence of detected subtypes of Fusobacterium nucleatum in ulcerative typical, colitis, Polyps and malignant CRC patients based on 16S rRNA sequencing. Then to detect the ratio of adhesive virulence factor FadA gene via PCR.

2. Materials and Methods

2.1. Subjects

The present study included four groups of subjects depending on their colonoscopy results and histopathological findings. The first group was regarded as control healthy people ( 25 ); the second group included patients who have ulcerative colitis ( 18 ); the third group were patients affected with precancerous polyps ( 19 ); and the last group were patients who have colorectal cancer ( 38 ). The studied subjects were collected in the OGD department from three hospitals within Erbil city from June 2021 until March 2023. The Ethical Committee approved this work of the College of Science at Salahaddin university. We obtained written informed consent from the patients who participated in this investigation before performing the study.

2.2. Colonoscopy Sample Collection

During this test, a flexible colonoscope and a viewing tube, a tool for removing tissue, were applied to check the colorectal region. Before starting the procedure, patients received sedative drugs. Then the observed abnormal noted growth was taken during a colonoscopy. The samples were collected during this process from the target region. All specimens were handled in phospho-buffer saline (PBS), directly transferred to the research center laboratory at Erbil polytechnic university, and stored at -80c.

2.3. Sample Collection from Surgical Operation

For malignant cases, surgical samples were obtained from CRC patients. Specimens were treated with the same procedure of colonoscopy sample collection. For this study, biopsies were also taken for histopathological assessment.

2.4. Histopathology

Colon tissues were obtained immediately following surgery and directly fixed within formaldehyde. The process of histopathological slide preparation was made via embedding in paraffin. The staining process was performed using Hematoxylin and eosin stains added to the paraffin section (3–5 µm thickness) to identify the grade and the other histological findings for each sample.

2.5. DNA Extraction

Genomic DNA was extracted from tissue biopsies using the Favor Prep^TM Tissue Genomic DNA Extraction Mini Kit (Favorgen Biotech crop, Taiwan). Briefly, the tissue samples were cut up to 25 mg and ground into a microcentrifuge tube. The samples were processed according to the manufacturer’s protocol. Each of provided buffers, Proteinase K and absolute ethanol, were added to the samples before applying the spin column. The samples underwent centrifugation and ran through column filters using a specific eluting buffer. Finally, the DNA quality and quantity of the extracted samples were measured using a NanoDrop spectrophotometer (Thermo Scientific, MA, USA).

2.6. The F. Nucleatum 16S rRNA-Based PCR and FadA-Specific PCR

The extracted DNA samples were used for running conventional PCR and gel electrophoresis protocols. A specific sequence from 16S rRNA of F. nucleatum was used for a blast of primer design in the NCBI primer blast and Gene Runner program. Then the primer specificity was rechecked by nucleotide blast in the NCBI database. The primer 16S rRNA gene for F. nucleatum Forward was (F: 5′-AGA GTT TGA TCC TGG CTC AG -3′), and 16S rRNA Reverse was (R: 5′-GTC ATC GTG CAC ACA GAA TTG CTG-3′) with a product size of (380-bp) region, whereas FadA primers Forward were (F:5′-CAC AAG CTG ACG CTG CTA GA -3′) and FadA Reverse (R: 5′-TTA CCA GCT CTT AAA GCT TG -3′) with PCR band size of (232-bp) region.

The conventional PCR was performed on the extracted DNA samples using amplification reaction in a Thermal Cycler with 25 µl solution prepared as follows: (12.5 µl Master mix +0.5 µl F primer +0.5 µl R primer +3 µl DNA extracted from colon tissue samples +8.5 µl PCR-Garde DW).

The PCR protocol for F. nucleatum was performed with an initial 5 min at 94 °C, then 30 cycles repeated; each cycle consists of a denaturation process at 94 °C for 30 sec, annealing at 58 °C for 30 sec and extension at 72 °C for 1 min, with 10 min at 72 °C as a final extension. FadA PCR protocol was performed with an initial 4 min at 94 °C, then 30 cycles repeated; each cycle consists of denaturing process at 94 °C for 30 sec, an annealing process at 55.8 °C for 30 sec and an extension at 72 °C for 40 sec, with 6 min at 72 °C as a final extension.

The PCR products of both protocols underwent the electrophoresis process by using 1.5% agarose gel in Tris-borate buffer (pH=8.0). The bands were visualized with safe stain via a gel documentation system.

2.7. 16S rRNA Sequencing and Phylogenetic Analysis

The F. nucleatum-positive PCR products were sent to the Molecular genetics Laboratory in Zheen international hospital, Erbil, for 16S rRNA sequencing using the same primer. The obtained data were compared to the reference 16S rRNA sequences at the NCBI database via applying nucleotide BLAST. Later, sequence alignment and Phylogenetic analysis were conducted using the bootstrap method. Then the phylogenetic tree was constructed based on the 16S rRNA nucleotide sequences using Molecular Evolutionary Genetic Analysis (MEGA11) software.

2.8. Statistical Analysis

Chi-squared test was applied to find the difference regarding the detection of F. nucleatum and FadA among all groups. All statistical analyses were performed using GraphPad Prism version 9. The values of P<0.05 were considered to be statistically significant.

3. Results

Out of one hundred studied patients, 54% were male, and 46% were female. The colonoscopy results were obtained, and the tissue biopsies were classified according to them. Molecular screening for F. nucleatum using conventional PCR technique reported highly significant differences among the studied groups (P<0.001). Approximately half of the patients (49%) suffering from CRC were positive for F. nucleatum, while the other groups, including normal, Ulcerative colitis and Precancerous polyps, recorded 16%, 23% and 40%, respectively (Figure 1).

Figure 1. Percent values for detection of F. nucleatum among typical, ulcerative colitis, precancerous polyps and colorectal carcinoma samples using specific primer. Chi-square analysis revealed a significant difference between the studied group regarding the percent positive values for F. nucleatum at P<0.001

The molecular identification process started with amplifying a specific region using a specific primer. Then, the electrogram bands with 380 PB were confirmed according to the DNA ladder measure (Figure 2a). Among the 34 positive samples, only 18 samples were sent for DNA sanger sequencing. The 16S rRNA partial sequencing revealed the various subtypes of F. nucleatum (Figure 2c).

Figure 2. Molecular identification of F. nucleatum in clinically studied biopsies. a: Gel electrogram showing positive lanes and opposing lanes. The positive lane (1-7, 10 and 12) recorded 380 pb band size. b: Chromatogram sample showing the sequencing result. c: NCBI nucleotide blast result for partial sequencing of 16S rRNA gene

The results of phylogenetic construction via the MEGA 11 software program reported 10 strains of F. nucleatum according to the NCBI blast for 16S r RNA partial sequencing. The most prevalent subtype was F. nucleatum subtype animalis strain KCOM 3229 with 7 taxa. Then 2 taxa belonged to the uncultured Fusobacterium clone MS055A1 (Figure 3).

Figure 3. Phylogenetic tree analysis in human colon cancer tissues detected various F. nucleatum sub-sp. strains were obtained during 16S rRNA alignment. The most prevalent subtype was F. nucleatum subsp. animalis with 7 taxa

The screening for FadA positivity among F. nucleatum-positive patients showed that only 7 cases (20.59%) possessed the FadA gene (Figure 4a). Among these 7 cases, 3 were CRC patients, while 2 were suffering from precancerous polyps (Figure 4b). The positive results were confirmed through gel electrophoresis, which resulted in 232 bp bands (Figure 5).

Figure 4. Detection of FadA positive gene among studied clinical biopsies. a: Percent values for detecting F. nucleatum with positive and negative FadA using specific primer. b: FadA-positive and negative bacteria are frequent among typical ulcerative colitis, precancerous polyps and colorectal carcinoma samples

Figure 5. Molecular identification of FadA gene among clinical samples which recorded positive F. nucleatum. Gel electrogram showing positive lanes and negative lanes. The positive lane (1 and 5) recorded 232 pb band size

4. Discussion

The present study determined the prevalence of various subtypes of F. nucleatum in healthy individuals and patients with ulcerative colitis, precancerous polyps and colorectal cancer in the Iraqi-Kurdish population. In addition to detecting the ratio of F. nucleatum, which carries the FadA gene.

Several pieces of evidence indicated the association between the alteration of the gastrointestinal microbiome and with carcinogenesis mechanisms of colorectal carcinoma ( 32 ). It becomes one of the critical risk factors for tumorigenesis, tumour metastasis ( 33 ) and immune modulation against cancer ( 34 ). Our findings regarding our samples supported the relationship between the rate of F. nucleatum infection and inflammation and cancer progression status.

Our findings suggested a strong relationship between CRC and F. nucleatum subtype animalis (about half of the phylogenetic tree based on 16S rRNA sequencing results belonged to F. nucleatum subtype animalis). This result was supported by Borozan, Zaidi ( 35 ), who concluded that F. nucleatum, especially subtype animalis, strongly correlated with a higher colorectal cancer-specific mortality and specific somatic mutated genes. The possible reason might be due to the immunomodulatory action of F. nucleatum subtype animalis ( 36 ) or the induction of inflammatory cytokines that promote colorectal carcinogenesis ( 37 ).

Molecular detection of FadA results showed that only 20% of F. nucleatum cases were positive for the FadA gene. A similar study observed that FadA gene positive was prevalent among CRC F. nucleatum positive cases ( 38 ). It has been demonstrated that the FadA gene may play a key role in colorectal carcinogenesis via modulating E-cadherin/β-catenin signalling ( 39 ).

This finding suggested that F. nucleatum subtype animalis strain KCOM 3229 is the most prevalent strain in colorectal cancer patients based on specific primer for 16S rRNA partial sequence; the FadA gene was found in lesser than a quarter of the F. nucleatum positive cases.

Authors' Contribution

Study concept and design: H. J. T.

Taher, H. J1 *, Kamel, F. H2

Acquisition of data: H. J. T.

Analysis and interpretation of data: H. J. T.

Drafting of the manuscript: F. H. K.

Critical revision of the manuscript for important intellectual content: F. H. K.

Statistical analysis: F. H. K.

Administrative, technical, and material support: F. H. K.

Ethics

The Ethical Committee approved this work of the College of Science at Salahaddin University. We obtained written informed consent from the patients who participated in this investigation before performing the study.

Conflict of Interest

The authors declare that they have no conflict of interest.

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