Metagenomic Signatures of Colorectal Cancer in the Jordanian Population: A Regional Case-Control Analysis Using 16S rRNA Profiling
Abstract
1. Introduction
2. Materials and Methods
2.1. Study Design and Ethical Approval
2.2. Sample Collection
2.3. DNA Extraction and Quantification
2.4. 16S rRNA Amplicon Library Preparation
2.5. Library Quantification and Pooling
2.6. Sequencing on Illumina iSeq 100 Platform
2.7. Bioinformatics and Taxonomic Analysis
3. Results
3.1. Overview of Sequencing and Taxonomic Classification
3.2. Microbial Community Structure in Healthy Controls and CRC Patients
3.3. Microbial Diversity Metrics Reveal Dysbiosis in Colorectal Cancer
3.4. Potential Bacterial Biomarkers Identified via LDA
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CRC | Colorectal cancer |
rRNA | Ribosomal RNA |
GI | Gastrointestinal |
SCFAs | Short-chain fatty acids |
F/B | Firmicutes/Bacteroidetes |
HDACs | Histone deacetylases |
IRB | Institutional Review Board |
KHCC | King Hussein Cancer Center |
SBS | Sequencing-by-Synthesis |
QC | Quality control |
repseqs | Representative sequences |
LDA | Linear Discriminant Analysis |
SD | Standard deviation |
HFD | High-fat diet |
LPS | Lipopolysaccharides |
References
- Hillman, E.T.; Lu, H.; Yao, T.; Nakatsu, C.H. Microbial ecology along the gastrointestinal tract. Microbes Environ. 2017, 32, 300–313. [Google Scholar] [CrossRef] [PubMed]
- Ghosh, S.; Pramanik, S. Structural diversity, functional aspects and future therapeutic applications of human gut microbiome. Arch. Microbiol. 2021, 203, 5281–5308. [Google Scholar] [CrossRef]
- Sommer, F.; Bäckhed, F. The gut microbiota—Masters of host development and physiology. Nat. Rev. Microbiol. 2013, 11, 227–238. [Google Scholar] [CrossRef]
- Rinninella, E.; Raoul, P.; Cintoni, M.; Franceschi, F.; Miggiano, G.A.D.; Gasbarrini, A.; Mele, M.C. What is the Healthy Gut Microbiota Composition? A Changing Ecosystem across Age, Environment, Diet, and Diseases. Microorganisms 2019, 7, 14. [Google Scholar] [CrossRef]
- Curtis, H.; Blaser, M.J.; Dirk, G.; Kota, K.C.; Rob, K.; Liu, B.; Wang, L.; Sahar, A.; White, J.R.; Badger, J.H. Structure, function and diversity of the healthy human microbiome. Nature 2012, 486, 207–214. [Google Scholar] [CrossRef]
- Fan, Y.; Pedersen, O. Gut microbiota in human metabolic health and disease. Nat. Rev. Microbiol. 2021, 19, 55–71. [Google Scholar] [CrossRef] [PubMed]
- Wang, T.; Cai, G.; Qiu, Y.; Fei, N.; Zhang, M.; Pang, X.; Jia, W.; Cai, S.; Zhao, L. Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers. ISME J. 2012, 6, 320–329. [Google Scholar] [CrossRef] [PubMed]
- Kostic, A.D.; Chun, E.; Robertson, L.; Glickman, J.N.; Gallini, C.A.; Michaud, M.; Clancy, T.E.; Chung, D.C.; Lochhead, P.; Hold, G.L. Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe 2013, 14, 207–215. [Google Scholar] [CrossRef]
- Hu, L.; Liu, Y.; Kong, X.; Wu, R.; Peng, Q.; Zhang, Y.; Zhou, L.; Duan, L. Fusobacterium nucleatum Facilitates M2 Macrophage Polarization and Colorectal Carcinoma Progression by Activating TLR4/NF-κ B/S100A9 Cascade. Front. Immunol. 2021, 12, 658681. [Google Scholar] [CrossRef]
- Cuevas-Ramos, G.; Petit, C.R.; Marcq, I.; Boury, M.; Oswald, E.; Nougayrède, J.-P. Escherichia coli induces DNA damage in vivo and triggers genomic instability in mammalian cells. Proc. Natl. Acad. Sci. USA 2010, 107, 11537–11542. [Google Scholar] [CrossRef]
- Louis, P.; Hold, G.L.; Flint, H.J. The gut microbiota, bacterial metabolites and colorectal cancer. Nat. Rev. Microbiol. 2014, 12, 661–672. [Google Scholar] [CrossRef] [PubMed]
- Niekamp, P.; Kim, C.H. Microbial Metabolite Dysbiosis and Colorectal Cancer. Gut Liver 2023, 17, 190–203. [Google Scholar] [CrossRef] [PubMed]
- Zhang, H.; Tian, Y.; Xu, C.; Chen, M.; Xiang, Z.; Gu, L.; Xue, H.; Xu, Q. Crosstalk between gut microbiotas and fatty acid metabolism in colorectal cancer. Cell Death Discov. 2025, 11, 78. [Google Scholar] [CrossRef]
- Jones, J.; Shi, Q.; Nath, R.R.; Brito, I.L. Keystone pathobionts associated with colorectal cancer promote oncogenic reprograming. PLoS ONE 2024, 19, e0297897. [Google Scholar] [CrossRef]
- Schmitt, M.; Greten, F.R. The inflammatory pathogenesis of colorectal cancer. Nat. Rev. Immunol. 2021, 21, 653–667. [Google Scholar] [CrossRef]
- Schulz, M.D.; Atay, Ç.; Heringer, J.; Romrig, F.K.; Schwitalla, S.; Aydin, B.; Ziegler, P.K.; Varga, J.; Reindl, W.; Pommerenke, C. High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity. Nature 2014, 514, 508–512. [Google Scholar] [CrossRef]
- Wirbel, J.; Pyl, P.T.; Kartal, E.; Zych, K.; Kashani, A.; Milanese, A.; Fleck, J.S.; Voigt, A.Y.; Palleja, A.; Ponnudurai, R. Meta-analysis of fecal metagenomes reveals global microbial signatures that are specific for colorectal cancer. Nat. Med. 2019, 25, 679–689. [Google Scholar] [CrossRef]
- Minot, S.S.; Li, N.; Srinivasan, H.; Ayers, J.L.; Yu, M.; Koester, S.T.; Stangis, M.M.; Dominitz, J.A.; Halberg, R.B.; Grady, W.M. Colorectal cancer-associated bacteria are broadly distributed in global microbiomes and drivers of precancerous change. Sci. Rep. 2024, 14, 23646. [Google Scholar] [CrossRef]
- Zackular, J.P.; Baxter, N.T.; Iverson, K.D.; Sadler, W.D.; Petrosino, J.F.; Chen, G.Y.; Schloss, P.D. The gut microbiome modulates colon tumorigenesis. mBio 2013, 4, e00692-13. [Google Scholar] [CrossRef] [PubMed]
- Zeller, G.; Tap, J.; Voigt, A.Y.; Sunagawa, S.; Kultima, J.R.; Costea, P.I.; Amiot, A.; Böhm, J.; Brunetti, F.; Habermann, N. Potential of fecal microbiota for early-stage detection of colorectal cancer. Mol. Syst. Biol. 2014, 10, 766. [Google Scholar] [CrossRef]
- Franzosa, E.A.; McIver, L.J.; Rahnavard, G.; Thompson, L.R.; Schirmer, M.; Weingart, G.; Lipson, K.S.; Knight, R.; Caporaso, J.G.; Segata, N. Species-level functional profiling of metagenomes and metatranscriptomes. Nat. Methods 2018, 15, 962–968. [Google Scholar] [CrossRef]
- Bars-Cortina, D.; Ramon, E.; Rius-Sansalvador, B.; Guinó, E.; Garcia-Serrano, A.; Mach, N.; Khannous-Lleiffe, O.; Saus, E.; Gabaldón, T.; Ibáñez-Sanz, G. Comparison between 16S rRNA and shotgun sequencing in colorectal cancer, advanced colorectal lesions, and healthy human gut microbiota. BMC Genom. 2024, 25, 730. [Google Scholar] [CrossRef]
- Wu, N.; Yang, X.; Zhang, R.; Li, J.; Xiao, X.; Hu, Y.; Chen, Y.; Yang, F.; Lu, N.; Wang, Z.; et al. Dysbiosis signature of fecal microbiota in colorectal cancer patients. Microb. Ecol. 2013, 66, 462–470. [Google Scholar] [CrossRef]
- Mira-Pascual, L.; Cabrera-Rubio, R.; Ocon, S.; Costales, P.; Parra, A.; Suarez, A.; Moris, F.; Rodrigo, L.; Mira, A.; Collado, M. Microbial mucosal colonic shifts associated with the development of colorectal cancer reveal the presence of different bacterial and archaeal biomarkers. J. Gastroenterol. 2015, 50, 167–179. [Google Scholar] [CrossRef] [PubMed]
- Zhang, H.; Wu, J.; Ji, D.; Liu, Y.; Lu, S.; Lin, Z.; Chen, T.; Ao, L. Microbiome analysis reveals universal diagnostic biomarkers for colorectal cancer across populations and technologies. Front. Microbiol. 2022, 13, 1005201. [Google Scholar] [CrossRef]
- Tjalsma, H.; Boleij, A.; Marchesi, J.R.; Dutilh, B.E. A bacterial driver–passenger model for colorectal cancer: Beyond the usual suspects. Nat. Rev. Microbiol. 2012, 10, 575–582. [Google Scholar] [CrossRef]
- Kaindi, D.W.M.; Kogi-Makau, W.; Lule, G.N.; Kreikemeyer, B.; Renault, P.; Bonfoh, B.; Otaru, N.; Schmid, T.; Meile, L.; Hattendorf, J.; et al. Colorectal cancer-associated Streptococcus infantarius subsp. infantarius differ from a major dairy lineage providing evidence for pathogenic, pathobiont and food-grade lineages. Sci. Rep. 2018, 8, 9181. [Google Scholar] [CrossRef] [PubMed]
- Jans, C.; Meile, L.; Lacroix, C.; Stevens, M.J. Genomics, evolution, and molecular epidemiology of the Streptococcus bovis/Streptococcus equinus complex (SBSEC). Infect. Genet. Evol. 2015, 33, 419–436. [Google Scholar] [CrossRef]
- de Almeida, C.V.; Taddei, A.; Amedei, A. The controversial role of Enterococcus faecalis in colorectal cancer. Ther. Adv. Gastroenterol. 2018, 11, 1756284818783606. [Google Scholar] [CrossRef]
- Shi, F.; Liu, G.; Lin, Y.; Guo, C.L.; Han, J.; Chu, E.S.; Shi, C.; Li, Y.; Zhang, H.; Hu, C. Altered gut microbiome composition by appendectomy contributes to colorectal cancer. Oncogene 2023, 42, 530–540. [Google Scholar] [CrossRef] [PubMed]
- Li, G.; Zhao, D.; Ouyang, B.; Chen, Y.; Zhao, Y. Intestinal microbiota as biomarkers for different colorectal lesions based on colorectal cancer screening participants in community. Front. Microbiol. 2025, 16, 1529858. [Google Scholar] [CrossRef] [PubMed]
- Jahani-Sherafat, S.; Alebouyeh, M.; Moghim, S.; Amoli, H.A.; Ghasemian-Safaei, H. Role of gut microbiota in the pathogenesis of colorectal cancer; a review article. Gastroenterol. Hepatol. Bed Bench 2018, 11, 101. [Google Scholar] [PubMed]
- Singh, R.P.; Halaka, D.A.; Hayouka, Z.; Tirosh, O. High-fat diet induced alteration of mice microbiota and the functional ability to utilize fructooligosaccharide for ethanol production. Front. Cell. Infect. Microbiol. 2020, 10, 376. [Google Scholar] [CrossRef]
- Larsen, J.M. The immune response to Prevotella bacteria in chronic inflammatory disease. Immunology 2017, 151, 363–374. [Google Scholar] [CrossRef]
- Arthur, J.C.; Perez-Chanona, E.; Mühlbauer, M.; Tomkovich, S.; Uronis, J.M.; Fan, T.-J.; Campbell, B.J.; Abujamel, T.; Dogan, B.; Rogers, A.B. Intestinal inflammation targets cancer-inducing activity of the microbiota. Science 2012, 338, 120–123. [Google Scholar] [CrossRef]
- Lamaudière, M.T.; Arasaradnam, R.; Weedall, G.D.; Morozov, I.Y. The colorectal cancer gut environment regulates activity of the microbiome and promotes the multidrug resistant phenotype of ESKAPE and other pathogens. Msphere 2023, 8, e00626-22. [Google Scholar] [CrossRef] [PubMed]
- Pleguezuelos-Manzano, C.; Puschhof, J.; Rosendahl Huber, A.; van Hoeck, A.; Wood, H.M.; Nomburg, J.; Gurjao, C.; Manders, F.; Dalmasso, G.; Stege, P.B. Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli. Nature 2020, 580, 269–273. [Google Scholar] [CrossRef]
- Nowrouzian, F.L.; Oswald, E. Escherichia coli strains with the capacity for long-term persistence in the bowel microbiota carry the potentially genotoxic pks island. Microb. Pathog. 2012, 53, 180–182. [Google Scholar] [CrossRef]
- Zou, S.; Fang, L.; Lee, M.H. Dysbiosis of gut microbiota in promoting the development of colorectal cancer. Gastroenterol. Rep. 2018, 6, 1–12. [Google Scholar] [CrossRef]
- Cai, P.; Yang, Q.; Lu, J.; Dai, X.; Xiong, J. Fecal bacterial biomarkers and blood biochemical indicators as potential key factors in the development of colorectal cancer. mSystems 2025, 10, e00043-25. [Google Scholar] [CrossRef]
- Qin, Y.; Tong, X.; Mei, W.-J.; Cheng, Y.; Zou, Y.; Han, K.; Yu, J.; Jie, Z.; Zhang, T.; Zhu, S. Consistent signatures in the human gut microbiome of old-and young-onset colorectal cancer. Nat. Commun. 2024, 15, 3396. [Google Scholar] [CrossRef] [PubMed]
- Fung, K.Y.; Cosgrove, L.; Lockett, T.; Head, R.; Topping, D.L. A review of the potential mechanisms for the lowering of colorectal oncogenesis by butyrate. Br. J. Nutr. 2012, 108, 820–831. [Google Scholar] [CrossRef] [PubMed]
- Ferreira-Halder, C.V.; de Sousa Faria, A.V.; Andrade, S.S. Action and function of Faecalibacterium prausnitzii in health and disease. Best Pract. Res. Clin. Gastroenterol. 2017, 31, 643–648. [Google Scholar] [CrossRef] [PubMed]
- Fang, Y.; Yan, C.; Zhao, Q.; Xu, J.; Liu, Z.; Gao, J.; Zhu, H.; Dai, Z.; Wang, D.; Tang, D. The roles of microbial products in the development of colorectal cancer: A review. Bioengineered 2021, 12, 720–735. [Google Scholar] [CrossRef]
- Ma, X.; Yu, X.; Guo, H.; He, Y.; Wen, S.; Yu, T.; Wang, W. The alleviating effect of Akkermansia muciniphila PROBIO on AOM/DSS-induced colorectal cancer in mice and its regulatory effect on gut microbiota. J. Funct. Foods 2024, 114, 106091. [Google Scholar] [CrossRef]
- Gubernatorova, E.O.; Gorshkova, E.A.; Bondareva, M.A.; Podosokorskaya, O.A.; Sheynova, A.D.; Yakovleva, A.S.; Bonch-Osmolovskaya, E.A.; Nedospasov, S.A.; Kruglov, A.A.; Drutskaya, M.S. Akkermansia muciniphila-friend or foe in colorectal cancer? Front. Immunol. 2023, 14, 1303795. [Google Scholar] [CrossRef]
- Wang, N.; Fang, J.-Y. Fusobacterium nucleatum, a key pathogenic factor and microbial biomarker for colorectal cancer. Trends Microbiol. 2023, 31, 159–172. [Google Scholar] [CrossRef]
- O’keefe, S.J. Diet, microorganisms and their metabolites, and colon cancer. Nat. Rev. Gastroenterol. Hepatol. 2016, 13, 691–706. [Google Scholar] [CrossRef]
- Tamburini, S.; Shen, N.; Wu, H.C.; Clemente, J.C. The microbiome in early life: Implications for health outcomes. Nat. Med. 2016, 22, 713–722. [Google Scholar] [CrossRef]
- El-Sayed, A.; Kapila, D.; Taha, R.S.I.; El-Sayed, S.; Mahen, M.R.A.; Taha, R.a.; Alrubaiy, L. The role of the gut microbiome in inflammatory bowel disease: The middle east perspective. J. Pers. Med. 2024, 14, 652. [Google Scholar] [CrossRef]
- King, C.H.; Desai, H.; Sylvetsky, A.C.; LoTempio, J.; Ayanyan, S.; Carrie, J.; Crandall, K.A.; Fochtman, B.C.; Gasparyan, L.; Gulzar, N.; et al. Baseline human gut microbiota profile in healthy people and standard reporting template. PLoS ONE 2019, 14, e0206484. [Google Scholar] [CrossRef] [PubMed]
- Markowiak-Kopeć, P.; Śliżewska, K. The effect of probiotics on the production of short-chain fatty acids by human intestinal microbiome. Nutrients 2020, 12, 1107. [Google Scholar] [CrossRef] [PubMed]
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Alanagreh, L.; Mustafa, M.A.; Al-Zghoul, M.B.; Massadeh, M.I.; Batiha, O.; Sughayer, M.; Almashakbeh, R.T.; Abu Suilike, H.B.; Tout, F.S.; Alzoughool, F. Metagenomic Signatures of Colorectal Cancer in the Jordanian Population: A Regional Case-Control Analysis Using 16S rRNA Profiling. Microorganisms 2025, 13, 1963. https://doi.org/10.3390/microorganisms13081963
Alanagreh L, Mustafa MA, Al-Zghoul MB, Massadeh MI, Batiha O, Sughayer M, Almashakbeh RT, Abu Suilike HB, Tout FS, Alzoughool F. Metagenomic Signatures of Colorectal Cancer in the Jordanian Population: A Regional Case-Control Analysis Using 16S rRNA Profiling. Microorganisms. 2025; 13(8):1963. https://doi.org/10.3390/microorganisms13081963
Chicago/Turabian StyleAlanagreh, Lo’ai, Minas A. Mustafa, Mohammad Borhan Al-Zghoul, Muhannad I. Massadeh, Osamah Batiha, Maher Sughayer, Rashed Taiseer Almashakbeh, Haya Bader Abu Suilike, Faten S. Tout, and Foad Alzoughool. 2025. "Metagenomic Signatures of Colorectal Cancer in the Jordanian Population: A Regional Case-Control Analysis Using 16S rRNA Profiling" Microorganisms 13, no. 8: 1963. https://doi.org/10.3390/microorganisms13081963
APA StyleAlanagreh, L., Mustafa, M. A., Al-Zghoul, M. B., Massadeh, M. I., Batiha, O., Sughayer, M., Almashakbeh, R. T., Abu Suilike, H. B., Tout, F. S., & Alzoughool, F. (2025). Metagenomic Signatures of Colorectal Cancer in the Jordanian Population: A Regional Case-Control Analysis Using 16S rRNA Profiling. Microorganisms, 13(8), 1963. https://doi.org/10.3390/microorganisms13081963