Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach

Christian Milani; Giulia Alessandri; Marta Mangifesta; Leonardo Mancabelli; Gabriele Andrea Lugli; Federico Fontana; Giulia Longhi; Rosaria Anzalone; Alice Viappiani; Sabrina Duranti; Francesca Turroni; Renato Costi; Alfredo Annicchiarico; Andrea Morini; Leopoldo Sarli; Maria Cristina Ossiprandi; Douwe van Sinderen; Marco Ventura

doi:10.1128/mSystems.00404-20

Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach

mSystems. 2020 Jul 28;5(4):e00404-20. doi: 10.1128/mSystems.00404-20.

Authors

Christian Milani^{1

2}, Giulia Alessandri³, Marta Mangifesta¹, Leonardo Mancabelli¹, Gabriele Andrea Lugli¹, Federico Fontana¹, Giulia Longhi^{1

4}, Rosaria Anzalone⁴, Alice Viappiani⁴, Sabrina Duranti¹, Francesca Turroni^{1

2}, Renato Costi⁵, Alfredo Annicchiarico⁵, Andrea Morini⁵, Leopoldo Sarli⁵, Maria Cristina Ossiprandi^{2

3}, Douwe van Sinderen⁶, Marco Ventura^{7

2}

Affiliations

¹ Laboratory of Probiogenomics, Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parma, Italy.
² Microbiome Research Hub, University of Parma, Parma, Italy.
³ Department of Veterinary Medical Science, University of Parma, Parma, Italy.
⁴ GenProbio srl, Parma, Italy.
⁵ Department of Medicine and Surgery, University of Parma, Parma, Italy.
⁶ APC Microbiome Institute and School of Microbiology, Bioscience Institute, National University of Ireland, Cork, Ireland.
⁷ Laboratory of Probiogenomics, Department of Chemistry, Life Sciences, and Environmental Sustainability, University of Parma, Parma, Italy marco.ventura@unipr.it.

Abstract

16S small-subunit (SSU) rRNA gene-based bacterial profiling is the gold standard for cost-effective taxonomic reconstruction of complex bacterial populations down to the genus level. However, it has been proven ineffective in clinical and research settings requiring higher taxonomic resolution. We therefore developed a bacterial profiling method based on the internal transcribed spacer (ITS) region employing optimized primers and a comprehensive ITS database for accurate cataloguing of bacterial communities at (sub)species resolution. Performance of the microbial ITS profiling pipeline was tested through analysis of host-associated, food, and environmental matrices, while its efficacy in clinical settings was assessed through analysis of mucosal biopsy specimens of colorectal cancer, leading to the identification of putative novel biomarkers. The data collected indicate that the proposed pipeline represents a major step forward in cost-effective identification and screening of microbial biomarkers at (sub)species level, with relevant impact in research, industrial, and clinical settings.IMPORTANCE We developed a novel method for accurate cataloguing of bacterial communities at (sub)species level involving amplification of the internal transcribed spacer (ITS) region through optimized primers, followed by next-generation sequencing and taxonomic classification of amplicons by means of a comprehensive database of bacterial ITS sequences. Host-associated, food, and environmental matrices were employed to test the performance of the microbial ITS profiling pipeline. Moreover, mucosal biopsy samples from colorectal cancer patients were analyzed to demonstrate the scientific relevance of this profiling approach in a clinical setting through identification of putative novel biomarkers. The results indicate that the ITS-based profiling pipeline proposed here represents a key metagenomic tool with major relevance for research, industrial, and clinical settings.

Keywords: ITS; metagenomics; profiling.