Alma Mater Studiorum – Università di Bologna

DOTTORATO DI RICERCA IN BIOLOGIA CELLULARE, MOLECOLARE E INDUSTRIALE Ciclo XXII

PROGETTO 3: BIOCATALISI APPLICATA E MICROBIOLOGIA INDUSTRIALE

Settore scientifico-disciplinare di afferenza: CHIM11

TITOLO DELLA TESI:

MOLECULAR CHARACTERIZATION OF THE HUMAN GUT MICROBIOTA: THE EFFECT OF AGING

Presentata da: Dott.ssa Elena Biagi

Coordinatore del Dottorato: Relatore: Prof. A. Hockhoeppler Prof.ssa Patrizia Brigidi

Esame finale anno 2010

Fact are stubborn things, but statistics are more pliable. Mark Twain, (1835-1919)

First draw your curve, then plot your readings. A. Bloch, in “The Murphy’s Law”

ABSTRACT

Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota. The study presented here is focused on the application and comparison of two different microarray approaches for the characterization of the human gut microbiota, the HITChip and the HTF-Microb.Array, with particular attention to the effects of the aging process on the composition of this ecosystem. By using the Human Intestinal Tract Chip (HITChip), recently developed at the Wageningen University, The Netherland, we explored the age-related changes of gut microbiota during the whole adult lifespan, from young adults, through elderly to centenarians. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the population and an enrichment of facultative anaerobes. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammation status, also known as inflamm-aging, as determined by a range of peripheral blood inflammatory markers. In parallel, we overtook the development of our own phylogenetic microarray with a lower number of targets, aiming the description of the human gut microbiota structure at high taxonomic level. The resulting chip was called High Taxonomic level Fingerprinting Microbiota Array (HTF- Microb.Array), and was based on the Ligase Detection Reaction (LDR) technology, which allowed us to develop a fast and sensitive tool for the fingerprint of the human gut microbiota in terms of presence/absence of the principal groups. The validation on artificial DNA mixes, as well as the pilot study involving eight healthy young adults, demonstrated that the HTF-Microb.Array can be used to successfully characterize the human gut microbiota, allowing us to obtain results which are in approximate accordance with the most recent characterizations. Conversely, the evaluation of the relative abundance of the target groups on the bases of the relative fluorescence intensity probes response still has some hindrances, as demonstrated by comparing the HTF.Microb.Array and HITChip high taxonomic level fingerprints of the same centenarians.

TABLE OF CONTENTS

1. INTRODUCTION 1

1.1 The human gut microbiota 1 1.1.1 Overview 1 1.1.2 Gut microbiota and immune system 5 1.1.3 Probiotics and prebiotics 6 1.2 The characterization of the gut bacterial ecosystem 7 1.2.1 From cultivation to molecular techniques 7 1.2.2 The HITChip technology 9 1.2.3 Pyrosequencing: a massive sequencing approach 10 1.2.4 The „omics‟ era 11 1.3 The aging process 13 1.3.1 Theories of aging and perspectives 13 1.3.2 Longevity 15 1.3.3 Aging and immune system 17 1.3.4 Aging and gut microbiota 19 1.3.5 Probiotics and prebiotics in the elderly 23

2. PROJECT OUTLINE 25

3. MATERIALS AND METHODS 28

3.1 Subjects recruitment and study groups 28 3.1.1 Subjects involved in the “Centenarians project” 28 3.1.2 Subjects recruited for