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Andreani, Nadia Andrea (2016) INTO THE BLUE: Spoilage phenotypes of Pseudomonas fluorescens in food matrices. [Tesi di dottorato]

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Abstract (inglese)

Spoilage induced by Pseudomonas strains is commonly found in a wide range of food products as a result of the ubiquitous presence of these strains and their ability to induce alteration through different mechanisms. Particular attention has been recently paid on those P. fluorescens strains able to induce a blue discolouration on several food matrices (e.g. dairy or meat products). Actually, poor data are available about this curious event that draw the attention of European consumer from 2010.
In the present manuscript a step-by-step investigation of the spoilage potential of Pseudomonas fluorescens species complex strains is reported, focusing in particular on the ability to produce an unpleasant blue pigment in food.
Firstly, some general information is given to the reader to understand the P. fluorescens group as food spoiler. Then, the application of a polyphasic approach is described with the aim to investigate 136 Pseudomonas fluorescens group strains. Additionally, the achievement and the analyses of draft genomes and transcriptomes for 4 P. fluorescens strains are described to investigate the biosynthetic pathways involved in the blue pigment production. The attempt to chemically characterise the blue molecule using MALDI-TOF mass spectrometry is also reported. Finally, the execution of a transposon-mediated mutagenesis is described to confirm previously obtained genomic data and to highlight further genes involved in the blue-pigment production.
The phenotypic and genotypic characterisation, based on the combination of classical microbiological tests and a MLST scheme, allowed the reconstruction of phylogenetic relationships among the isolates and the identification of a monophyletic group (named “the blue branch”) grouping all the blue-pigmenting and few uncoloured strains. The real involvement of these strains in the blue mozzarella event was confirmed by their ability to induce a blue discolouration on mozzarella cheese during a challenge test.
The genomic investigation confirmed the strict phylogenetic relationship between the strains belonging to the “blue branch”. Additionally, comparative genomic tools revealed the presence of a genetic cluster unique to the blue pigmenting strains containing a second copy of five trp genes, clearly involved in the blue pigment production. The biochemical characterisation of the pigment, hampered by strong issues of solubility, led to the conclusion that the molecule is an indigo-derivative.
Transposon-induced mutants confirmed the involvement of the previously identified unique cluster and the association of several genes affecting directly or indirectly the blue molecule production. Furthermore, the phenotypic characterisation of the mutants revealed a key role of iron in the production of the pigment, such as absence of any advantage of the wild-type strain in co-culture with a non-pigmenting mutant.
To conclude, the present work represents an exhaustive investigation of the spoilage potential of the blue-pigmenting P. fluorescens strains, giving to food industry reliable approaches to identify, track and prevent spoilage related to the growth of these interesting bacteria.

Abstract (italiano)

Le alterazioni causate da ceppi di Pseudomonas sono solitamente riscontrate in una grande varietà di alimenti a causa del loro essere ubiquitari e dalla loro capacità di indurre modificazioni organolettiche negli alimenti mediante diversi meccanismi. Particolare attenzione è stata posta su alcuni ceppi di P. fluorescens in grado di indurre una colorazione blu in diverse matrici alimentari (quali prodotti lattiero-caseari o carne). In realtà, poche informazioni sono ad oggi disponibili riguardo al curioso caso che ha attirato l’attenzione pubblica a partire dal 2010.
In questo lavoro è riportata un’analisi a più livelli del potenziale alternate dei ceppi appartenenti allo Pseudomonas fluorescens species complex, ponendo particolare attenzione alla capacità di produrre un indesiderato pigmento blu negli alimenti.
In primo luogo, ai lettori sono date delle informazioni generali per una migliore comprensione di P. fluorescens come alterante alimentare. In seguito, è descritta la messa a punto e applicazione di un approccio polifasico con l’obbiettivo di indagare 136 ceppi appartenenti al gruppo P. fluorescens. Inoltre, sono descritti l’ottenimento e le analisi dei genomi draft e dei trascrittomi di 4 ceppi di P. fluorescens con la finalità di comprendere il pathway biosintetico coinvolto nella produzione del pigmento blu. In aggiunta, è riportato il tentativo di caratterizzare chimicamente il pigmento mediante la metodica della spettrometria di massa MALDI-TOF. Infine, è riportata l’esecuzione della mutagenesi random con la finalità di confermare i risultati genomici precedentemente ottenuti e di individuare ulteriori geni coinvolti nella produzione del pigmento blu.
La caratterizzazione fenotipica e genotipica, basata sulla combinazione di metodiche di microbiologia classica e di uno schema MLST, ha permesso la ricostruzione delle relazioni filogenetiche tra gli isolati e l’identificazione di un gruppo monofiletico (chiamato “ramo blu”) che raggruppa tutti i ceppi pigmentanti e pochi ceppi non-pigmentanti. Il reale coinvolgimento dei ceppi blu nei casi di mozzarella blu è stato confermato dalla possibilità degli stessi di indurre un’anomala colorazione blu su mozzarella durante un challenge test.
Le analisi genomiche hanno confermato la stretta vicinanza filogenetica tra i ceppi del “ramo blu”. Inoltre, analisi di genomica comparativa hanno rivelato la presenza di un cluster genico unicamente presente nei ceppi blu, contenente una seconda copia di cinque dei sette geni per la biosintesi del triptofano, chiaramente coinvolto nella produzione del pigmento blu. La caratterizzazione biochimica del pigmento, resa difficoltosa da problemi di solubilità, ha portato alla conclusione che la molecola blu sia un derivato dell’indigo. I mutanti ottenuti mediante l’applicazione di trasposoni hanno confermato il coinvolgimento del cluster genico precedentemente identificato nella produzione del pigmento e l’associazione di ulteriori geni che influenzano direttamente o indirettamente la produzione della molecola blu. Inoltre, la caratterizzazione dei mutanti ha rivelato il ruolo importante del ferro nella produzione del pigmento e l’assenza di un effettivo vantaggio del ceppo wild-type posto in co-cultura con un mutante non pigmentante.
In conclusione, questo studio rappresenta un’indagine esaustiva del potenziale alterante dei ceppi blu, dando inoltre all’industria alimentare sistemi efficaci per identificare, tracciare e prevenire l’alterazione indotta da questi interessanti ceppi.

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Tipo di EPrint:Tesi di dottorato
Relatore:Cardazzo, Barbara
Dottorato (corsi e scuole):Ciclo 28 > Scuole 28 > SCIENZE VETERINARIE
Data di deposito della tesi:25 Gennaio 2016
Anno di Pubblicazione:25 Gennaio 2016
Parole chiave (italiano / inglese):Pseudomonas, Xanthomonas, Shewanella, pigment production, enzyme production, volatile compound production, SSO, Pseudomonas fluorescens group, Multilocus Sequence Typing, Food spoilage, Blue mozzarella, Comparative genomics and transcriptomics, transposon-induced mutants, phenotypic characterisation, blue-pigment biosynthesis.
Settori scientifico-disciplinari MIUR:Area 07 - Scienze agrarie e veterinarie > VET/04 Ispezione degli alimenti di origine animale
Struttura di riferimento:Dipartimenti > Dipartimento di Biomedicina Comparata ed Alimentazione
Codice ID:9109
Depositato il:21 Ott 2016 10:23
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