Trillions of microbes have evolved with and continue to live on and within human beings. In 2001, Nobel Prize winner Joshua Lederberg introduced the new scientific term “microbiome”, which refers to the set of genes of all microorganisms that live in and within the human body (skin, mouth, intestine, etc.), that is, in their genome and thus an microecosystem of commensal, symbiotic and pathogenic microbes. A population of these “good” germs gradually colonizes the human body from birth, externally and internally, and forms the classic so-called “normal microbial flora”, currently reported as microbiota. A variety of environmental factors can affect human microbial imbalance, generally called dysbiosis, which has a close relationship with health and disease. Dysbiosis has been associated with numerous diseases, including cardiovascular diseases, cancer, respiratory diseases, diabetes (types 1 and 2), brain disorders, chronic kidney and liver diseases and inflammatory bowel diseases. Moreover, the microbiota seems to be specific to everyone and this specificity, maintained consistently over time in each host, confirms the hypothesis that the microbial profile is modelled on the genetic, metabolic, structural, and functional characteristics of everyone. In this context, attention in recent years has been turned to genomic and functional characterization of the microbiota to understand the extent to which microbial species provide a functional benefit to the host. Therefore in 2008, an international program was established by the United States National Institutes of Health (NIH), the “Human Microbiome Project”, or the scientific program for the microbiome. Current research on the human microbiota has become much more sophisticated and more comprehensive and along with the development of research on the microbiota, research on its habitat is also emerging: the mucus. However, most of the microbial diversity in any given environment, including the human body, has not yet been isolated and cultured and therefore hamper in-depth studies of effects on human health and disease. In nature, the way bacteria grow and develop is by forming biofilms. Nevertheless, most of the experiments carried in vitro are realized by using the planktonic culturing method, failing to recreate how bacteria grow and develop in real life. In this study, the current clinical microbiology knowledge about the microbiota and the various diseases relationships is investigated. By copying key properties of the human mucus, Bac3Gel® is a newly generation of advanced 3D substrates for culturing microorganisms, exhibiting micro-gradients that recreate several types of human habitat. In this way it is possible to sustain different microbiota in the laboratory to generate and evaluate novel solutions for healthcare and wellness. For this purpose, a set of seven different bacterial strains has been used: E. coli, E. faecalis, L. monocytogenes, A. baumannii, K. pneumoniae, S. aureus and P. aeruginosa. Each of these has a specific human environment in which it is preferentially expressed, or like the so-called ESKAPEs, these can colonize anywhere in the human organism. Once the culture conditions and appropriate media had been established, the bacteria were seeded onto these 3D substrates, which are gels reproduced in the laboratory according to a meticulous protocol. After carrying out the infection for 24 and 48 hours, gels and planktonic cultures have been analyzed with a newly technique, called spot plating, that can help to optimize conventional culture-based methods. Furthermore, qualitative analysis, using a confocal laser scanning microscopy and a fluorescent bacterial viability kit, were conducted in order to quantify the amounts of live and dead bacteria on the gels.
Trilioni di microbi si sono evoluti con gli esseri umani e continuano a vivere al loro interno. Nel 2001, il premio Nobel Joshua Lederberg ha introdotto il nuovo termine scientifico "microbioma", che si riferisce all'insieme dei geni di tutti i microrganismi che vivono all'interno del corpo umano (pelle, bocca, intestino, ecc.), cioè nel loro genoma e quindi un microecosistema di microbi commensali, simbiotici e patogeni. Una popolazione di questi germi "buoni" colonizza gradualmente il corpo umano fin dalla nascita, sia esternamente che internamente, e forma la classica cosiddetta "flora microbica normale", attualmente indicata come microbiota. Una varietà di fattori ambientali può influenzare lo squilibrio microbico umano, generalmente chiamato disbiosi, che ha una stretta relazione con la salute e la malattia. La disbiosi è stata associata a numerose patologie, tra cui malattie cardiovascolari, cancro, malattie respiratorie, diabete (tipo 1 e 2), disturbi cerebrali, malattie croniche dei reni e del fegato e malattie infiammatorie intestinali. Inoltre, il microbiota sembra essere specifico per ognuno e questa specificità, mantenuta costantemente nel tempo in ogni ospite, conferma l'ipotesi che il profilo microbico sia modellato sulle caratteristiche genetiche, metaboliche, strutturali e funzionali di ognuno. In questo contesto, negli ultimi anni l'attenzione è stata rivolta alla caratterizzazione genomica e funzionale del microbiota per capire in che misura le specie microbiche forniscono un beneficio funzionale all'ospite. Per questo motivo, nel 2008, è stato istituito un programma internazionale dai National Institutes of Health (NIH) degli Stati Uniti, il "Progetto Microbioma Umano", o programma scientifico per il microbioma. La ricerca attuale sul microbiota umano è diventata molto più sofisticata e completa e, insieme allo sviluppo della ricerca sul microbiota, sta emergendo anche la ricerca sul suo habitat: il muco. Tuttavia, la maggior parte della diversità microbica presente in un determinato ambiente, compreso il corpo umano, non è ancora stata isolata e coltivata e quindi ostacola studi approfonditi sugli effetti sulla salute e sulle malattie umane. In natura, i batteri crescono e si sviluppano formando biofilm. Tuttavia, la maggior parte degli esperimenti condotti in vitro sono realizzati con il metodo della coltura planctonica, non riuscendo a ricreare il modo in cui i batteri crescono e si sviluppano nella vita reale. In questo studio vengono analizzate le attuali conoscenze di microbiologia clinica sul microbiota e le relazioni con le varie malattie. Copiando le proprietà chiave del muco umano, Bac3Gel® è una nuova generazione di substrati 3D avanzati per la coltura di microrganismi, che presentano microgradienti che ricreano diversi tipi di habitat umano. In questo modo è possibile sostenere diversi microbioti in laboratorio per generare e valutare nuove soluzioni per la salute e il benessere. A questo scopo, è stato utilizzato un set di sette diversi ceppi batterici: E. coli, E. faecalis, L. monocytogenes, A. baumannii, K. pneumoniae, S. aureus e P. aeruginosa. Ognuno di questi ha un ambiente umano specifico in cui si esprime in modo preferenziale, oppure, come le cosiddette ESKAPE, possono colonizzare qualsiasi punto dell'organismo umano. Una volta stabilite le condizioni di coltura e i terreni appropriati, i batteri sono stati seminati su questi substrati 3D, che sono gel riprodotti in laboratorio secondo un protocollo meticoloso. Dopo aver effettuato l'infezione per 24 e 48 ore, i gel e le colture planctoniche sono stati analizzati con una nuova tecnica, chiamata spot plating, che può aiutare a ottimizzare i metodi convenzionali basati sulle colture. Inoltre, sono state condotte analisi qualitative, utilizzando un microscopio a scansione laser confocale e un kit di vitalità batterica fluorescente, per quantificare le quantità di batteri vivi e morti sui gel.
Studio in vitro di vari ceppi batterici del microbiota umano coltivati in diversi tipi di substrati 3D avanzati.
PELUSO, EMANUELA
2021/2022
Abstract
Trillions of microbes have evolved with and continue to live on and within human beings. In 2001, Nobel Prize winner Joshua Lederberg introduced the new scientific term “microbiome”, which refers to the set of genes of all microorganisms that live in and within the human body (skin, mouth, intestine, etc.), that is, in their genome and thus an microecosystem of commensal, symbiotic and pathogenic microbes. A population of these “good” germs gradually colonizes the human body from birth, externally and internally, and forms the classic so-called “normal microbial flora”, currently reported as microbiota. A variety of environmental factors can affect human microbial imbalance, generally called dysbiosis, which has a close relationship with health and disease. Dysbiosis has been associated with numerous diseases, including cardiovascular diseases, cancer, respiratory diseases, diabetes (types 1 and 2), brain disorders, chronic kidney and liver diseases and inflammatory bowel diseases. Moreover, the microbiota seems to be specific to everyone and this specificity, maintained consistently over time in each host, confirms the hypothesis that the microbial profile is modelled on the genetic, metabolic, structural, and functional characteristics of everyone. In this context, attention in recent years has been turned to genomic and functional characterization of the microbiota to understand the extent to which microbial species provide a functional benefit to the host. Therefore in 2008, an international program was established by the United States National Institutes of Health (NIH), the “Human Microbiome Project”, or the scientific program for the microbiome. Current research on the human microbiota has become much more sophisticated and more comprehensive and along with the development of research on the microbiota, research on its habitat is also emerging: the mucus. However, most of the microbial diversity in any given environment, including the human body, has not yet been isolated and cultured and therefore hamper in-depth studies of effects on human health and disease. In nature, the way bacteria grow and develop is by forming biofilms. Nevertheless, most of the experiments carried in vitro are realized by using the planktonic culturing method, failing to recreate how bacteria grow and develop in real life. In this study, the current clinical microbiology knowledge about the microbiota and the various diseases relationships is investigated. By copying key properties of the human mucus, Bac3Gel® is a newly generation of advanced 3D substrates for culturing microorganisms, exhibiting micro-gradients that recreate several types of human habitat. In this way it is possible to sustain different microbiota in the laboratory to generate and evaluate novel solutions for healthcare and wellness. For this purpose, a set of seven different bacterial strains has been used: E. coli, E. faecalis, L. monocytogenes, A. baumannii, K. pneumoniae, S. aureus and P. aeruginosa. Each of these has a specific human environment in which it is preferentially expressed, or like the so-called ESKAPEs, these can colonize anywhere in the human organism. Once the culture conditions and appropriate media had been established, the bacteria were seeded onto these 3D substrates, which are gels reproduced in the laboratory according to a meticulous protocol. After carrying out the infection for 24 and 48 hours, gels and planktonic cultures have been analyzed with a newly technique, called spot plating, that can help to optimize conventional culture-based methods. Furthermore, qualitative analysis, using a confocal laser scanning microscopy and a fluorescent bacterial viability kit, were conducted in order to quantify the amounts of live and dead bacteria on the gels.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15280