The modern Next Generation Sequencing technologies represent a crucial component in the study of microorganisms and microbial communities thanks to the huge quantity of data they can provide in a short period of time. These technologies allow the identification and characterization of microorganisms exploiting a vast number of bioinformatic tools that can replace the standard in vitro typing techniques, resulting in savings of time and resources. Genomics and metagenomics can be applied in different fields and they can provide information on single microorganism or on entire microbial communities. We focused our studies on two ecological niches: food matrices and human gut microbiome, due to their relevance to human health. The first work of this thesis is a comparative genomic study of 12 Leuconostoc carnosum strains isolated from meat-based products. This bacterium is a known colonizer of meat-based food matrices, it plays a role in spoilage, but preservative effects have also been proposed for some strains. In our study we performed whole genome sequencing for all the strains, and after genome assembly we identified their genomic features, the presence of plasmids, and genes related to antibiotic resistance, bacteriocins production, biogenic amines synthesis. We also reconstructed their metabolic pathways. The comparison revealed that the strains are closely related and share most of the metabolic features, confirming the adaptation to the meat environment due to the presence of 23 peptidase genes in their core genome. With this study we provided a deeper insight into the genomic and metabolic features of this bacterium ubiquitous in meat products. The second project of the thesis aimed to investigate through an inedited metagenomic strategy the presence of beta-glucuronidases (GUS) in the human gut microbiome. Beta-glucuronidases (GUS) produced by gut microbiome bacteria can remove glucuronic acid moieties from a vast range of compounds and metabolites, like drugs and xenobiotics. These molecules are conjugated with glucuronic acid in the liver to be excreted in the gastrointestinal tract, so the action of GUS may reactivate them allowing the reabsorption, with unpredictable and different efficacy of drugs and negative effect on health. GUS are classified in classes by the differences in the catalytic site. 60 shotgun sequenced metagenomic samples from healthy subjects, ascribed to five geographically distinct cohorts, have been retrieved. From this dataset, bacterial composition has been defined and a novel pipeline to investigate distribution the different GUS has been developed and utilized. Beta-diversity calculated on Bray-Curtis dissimilarity index has been used to determine the distances among samples and determine the differences among samples in terms of GUS type distribution and abundance of bacteria containing GUS. Since the structural differences in the enzyme involve a different substrate specificity, and taking into account the ratio of bacterial community harbouring GUS genes, we can assess that the microbiota composition can alter the excretion of certain drugs or xenobiotics, and determine a wide interindividual variability in terms of response to drugs. In the third part of the thesis, I present metagenomic analysis carried out in two other different studies, the former aimed to investigate the microbial composition in enriched human faecal samples to identify gut mucin degraders, and the latter focused on the description of the microbiota of Hermetia illucens larvae reared for food or feed consumption.

Le moderne tecnologie di Next Generation Sequencing sono un componente cruciale nello studio di microrganismi e comunità microbiche grazie all’enorme quantità di dati fornita in breve tempo. Con queste tecnologie è possibile identificare e caratterizzare i microrganismi utilizzando un ampio numero di tool bioinformatici che possono sostituire le classiche tecniche di tipizzazione in vitro, portando un risparmio di tempo e risorse. La genomica e la metagenomica possono essere applicate a vari campi e forniscono informazioni sia riguardo un singolo organismo, sia su intere comunità microbiche. I nostri studi sono stati concentrati su due nicchie ecologiche: matrici alimentari e microbiota intestinale umano, entrambi importanti per la salute umana. Un prima parte della tesi descrive la comparazione genomica di 12 ceppi di Leuconostoc carnosum isolati da prodotti a base di carne. Questo batterio è un noto colonizzatore di queste matrici, ricopre un ruolo nel loro deterioramento, ma alcuni ceppi presentano effetti utili alla preservazione. Abbiamo eseguito un sequenziamento whole genome per tutti i ceppi, e dopo l’assembly sono state identificate le caratteristiche genomiche, la presenza di plasmidi, di geni responsabili di antibiotico-resistenza, produzione di batteriocine, sintesi di ammine biogene e abbiamo ricostruito i loro pathway metabolici. La comparazione ha rivelato che i ceppi sono strettamente correlati e condividono la maggior parte delle caratteristiche metaboliche, evidenziando l’adattamento all’ambiente di isolamento grazie alle 23 peptidasi presenti nel genoma core. Questo studio fornisce un approfondimento genomico e metabolico su questo batterio ubiquitario nei prodotti a base di carne. Un secondo progetto della tesi è stato indirizzato a indagare la presenza delle beta-glucuronidasi (GUS) nel microbiota intestinale umano attraverso una strategia metagenomica inedita. Le GUS prodotte dai batteri del microbiota sono capaci di rimuovere le porzioni di acido glucuronico da molti composti e metaboliti, come farmaci e xenobiotici. Queste molecole vengono coniugate con l’acido glucuronico nel fegato per l’escrezione attraverso il tratto gastrointestinale, quindi l’azione enzimatica potrebbe riattivarle permettendone il riassorbimento comportando un’alterazione dell’efficacia del farmaco ed effetti negativi sulla salute. Le GUS sono organizzate in classi in base alle differenze nel loro sito catalitico. Sono stati utilizzati 60 metagenomi ottenuti con shotgun sequencing provenienti da soggetti sani divisi in coorti in base alla provenienza geografica. Da questo set di dati, è stata definita la composizione batterica ed è stata sviluppata ed impiegata una nuova strategia per indagare la distribuzione delle diverse GUS. La beta diversità calcolata con gli indici di Jaccard e Bray-Curtis è stata impiegata per determinare le distanze tra i campioni e determinare le differenze tra essi in termini di distribuzione del tipo di GUS e abbondanza dei batteri che le contengono. Poiché alle differenze strutturali dell’enzima corrisponde una diversa specificità di substrato, e considerando la proporzione delle comunità batteriche contenenti GUS, possiamo valutare che la composizione del microbiota può alterare l’escrezione di alcuni farmaci o xenobioti e determinare un’ampia variabilità interindividuale in termini di risposta al farmaco. Un’ultima parte del progetto di tesi descrive l’applicazione dell’approccio metagenomico in due diversi studi: il primo incentrato a indagare la composizione microbica di campioni fecali arricchiti per identificare specie intestinali capaci di degradare la mucina, e il secondo focalizzato sulla descrizione del microbiota di larve di Hermetia illucens allevate per consumo umano o animale.

Tecniche genomiche e metagenomiche per la caratterizzazione di batteri e comunità microbiche in nicchie ecologiche rilevanti per la salute umana / Francesco Candeliere , 2021 Mar 19. 33. ciclo, Anno Accademico 2019/2020.

Tecniche genomiche e metagenomiche per la caratterizzazione di batteri e comunità microbiche in nicchie ecologiche rilevanti per la salute umana

CANDELIERE, FRANCESCO
2021

Abstract

The modern Next Generation Sequencing technologies represent a crucial component in the study of microorganisms and microbial communities thanks to the huge quantity of data they can provide in a short period of time. These technologies allow the identification and characterization of microorganisms exploiting a vast number of bioinformatic tools that can replace the standard in vitro typing techniques, resulting in savings of time and resources. Genomics and metagenomics can be applied in different fields and they can provide information on single microorganism or on entire microbial communities. We focused our studies on two ecological niches: food matrices and human gut microbiome, due to their relevance to human health. The first work of this thesis is a comparative genomic study of 12 Leuconostoc carnosum strains isolated from meat-based products. This bacterium is a known colonizer of meat-based food matrices, it plays a role in spoilage, but preservative effects have also been proposed for some strains. In our study we performed whole genome sequencing for all the strains, and after genome assembly we identified their genomic features, the presence of plasmids, and genes related to antibiotic resistance, bacteriocins production, biogenic amines synthesis. We also reconstructed their metabolic pathways. The comparison revealed that the strains are closely related and share most of the metabolic features, confirming the adaptation to the meat environment due to the presence of 23 peptidase genes in their core genome. With this study we provided a deeper insight into the genomic and metabolic features of this bacterium ubiquitous in meat products. The second project of the thesis aimed to investigate through an inedited metagenomic strategy the presence of beta-glucuronidases (GUS) in the human gut microbiome. Beta-glucuronidases (GUS) produced by gut microbiome bacteria can remove glucuronic acid moieties from a vast range of compounds and metabolites, like drugs and xenobiotics. These molecules are conjugated with glucuronic acid in the liver to be excreted in the gastrointestinal tract, so the action of GUS may reactivate them allowing the reabsorption, with unpredictable and different efficacy of drugs and negative effect on health. GUS are classified in classes by the differences in the catalytic site. 60 shotgun sequenced metagenomic samples from healthy subjects, ascribed to five geographically distinct cohorts, have been retrieved. From this dataset, bacterial composition has been defined and a novel pipeline to investigate distribution the different GUS has been developed and utilized. Beta-diversity calculated on Bray-Curtis dissimilarity index has been used to determine the distances among samples and determine the differences among samples in terms of GUS type distribution and abundance of bacteria containing GUS. Since the structural differences in the enzyme involve a different substrate specificity, and taking into account the ratio of bacterial community harbouring GUS genes, we can assess that the microbiota composition can alter the excretion of certain drugs or xenobiotics, and determine a wide interindividual variability in terms of response to drugs. In the third part of the thesis, I present metagenomic analysis carried out in two other different studies, the former aimed to investigate the microbial composition in enriched human faecal samples to identify gut mucin degraders, and the latter focused on the description of the microbiota of Hermetia illucens larvae reared for food or feed consumption.
Genomic and metagenomic approaches for the characterization of bacteria and microbial communities of ecological niches relevant for the human health
19-mar-2021
ROSSI, Maddalena
File in questo prodotto:
File Dimensione Formato  
Tesi definitiva Candeliere Francesco.pdf

Open Access dal 19/03/2024

Descrizione: Tesi definitiva Candeliere Francesco
Tipologia: Tesi di dottorato
Dimensione 15.78 MB
Formato Adobe PDF
15.78 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

Licenza Creative Commons
I metadati presenti in IRIS UNIMORE sono rilasciati con licenza Creative Commons CC0 1.0 Universal, mentre i file delle pubblicazioni sono rilasciati con licenza Attribuzione 4.0 Internazionale (CC BY 4.0), salvo diversa indicazione.
In caso di violazione di copyright, contattare Supporto Iris

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1238973
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact