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Theoretical outcomes and applications of taxonomic studies on the genera Enterococcus and Lactobacillus
No full textQuesto progetto di dottorato, dal titolo “Theoretical outcomes and applications of taxonomic
studies on the genera Enterococcus and Lactobacillus”, si colloca nel contesto della tassonomia
batterica. Tale disciplina scientifica si propone di descrivere la diversità degli organismi viventi con
lo scopo di caratterizzarli e di raggrupparli in un sistema gerarchico ordinato. È convinzione diffusa
che il sistema di classificazione più stabile e affidabile sia quello che si basa sulle reali relazioni
genealogiche degli organismi. Tuttavia, data la loro semplicità strutturale, lo studio dei batteri è
sempre stato fortemente influenzato dallo sviluppo scientifico e dalle tecnologie di indagine
disponibili.
I microrganismi ricoprono notevole interesse in diverse aree scientifiche, quali la medicina, la
genetica, l’ecologia e trovano ampia applicazione nell’industria alimentare, farmaceutica e nel
settore ambientale. Lo studio della biodiversità esistente è indispensabile per impiegare al meglio i
microrganismi, per scoprire nuove applicazioni biotecnologiche nonché per approfondire i rischi
derivanti dalla presenza di batteri indesiderati o pericolosi. La tassonomia, inoltre, ha il compito di
assegnare un nome univoco ad ogni organismo fino ad ora caratterizzato, in modo tale da consentire
lo scambio delle informazioni disponibili tramite l’utilizzo di una terminologia comune e di fornire
strumenti utili alla corretta identificazione di nuovi isolati.
Nel corso di questo progetto sono state approfondite alcune problematiche tassonomiche legate
a specie appartenenti ai generi Lactobacillus ed Enterococcus, che includono batteri di notevole
interesse ecologico e applicativo, in particolare, nella produzione di alimenti fermentati dove
vengono impiegati come commensali o starter. Alcune specie, inoltre, sono comuni abitanti del
tratto gastro-intestinale dell’uomo e degli animali e possono influire sulla composizione della
microflora intestinale, sull’assorbimento dei nutrienti e sullo stato di salute dell’ospite. Data la loro
diffusione in ambienti così differenti e il ruolo che ricoprono in diversi settori, l’approfondimento
tassonomico di questi generi risulta particolarmente rilevante. Le ricerche svolte nell’ambito di
questo progetto sono state affrontate con l’ausilio di approcci differenti a seconda dell’organismo
preso in esame, della problematica da risolvere e del livello tassonomico investigato (tra specie
distinte, strettamente correlate o fra individui di una stessa specie).
Nella prima parte della tesi (capitolo 2) sono state approfondite le relazioni filogenetiche tra
enterococchi tramite l’analisi di sequenza di sei diversi marcatori molecolari (16S rDNA, recA,
hsp60, pheS, atpA, rpoA) e il confronto dell’organizzazione genica delle regioni adiacenti al gene
recA. Questo approccio ha permesso di evidenziare la presenza di gruppi di specie maggiormente
correlate tra loro e di valutare un probabile ordine di speciazione. I dati di sequenza ottenuti sono
stati analizzati per individuare dei polimorfismi specie-specifici sui geni 16S rRNA, recA e hsp60,
in corrispondenza dei quali sono state disegnate delle sonde da impiegare a fini diagnostici. Queste
sonde, utilizzate per lo sviluppo di un prototipo di DNA-microarray tramite deposizione meccanica,
hanno consentito di differenziare, sulla base dei profili di ibridazione ottenuti, le specie di
enterococchi che si ritrovano più frequentemente in campioni clinici e in matrici alimentari, alcune
delle quali, come E. durans ed E. hirae, sono in genere difficilmente distinguibili con altri metodi
molecolari e fenotipici.
Nella seconda parte (capitoli 3 e 4) sono state affrontate due problematiche di classificazione tra
specie strettamente correlate (L. ingluviei e L. thermotolerans, L. vaccinostercus e L. durianis), che
mostrano una elevata conservazione dei marcatori molecolari e proprietà fenotipiche molto simili.
Pertanto, il loro grado di similarità è stato determinato tramite ibridazione del DNA totale, l’unico
metodo attualmente valido per la delimitazione di una specie batterica. Dai risultati ottenuti è stato
possibile accertare che le unità tassonomiche non differenziabili rappresentano in realtà una singola
specie, erroneamente annotata nel sistema di classificazione con nomi diversi. Come conseguenza
di queste evidenze lo schema di classificazione del genere Lactobacillus e il sistema di
nomenclatura di questi organismi è stato corretto, in modo tale da consentire in futuro una più
chiara e non ambigua identificazione di isolati ambientali.
Nella terza parte (capitoli 5 e 6) è stata approfondita la biodiversità microbica a livello intraspecifico
di due specie che ricoprono un ruolo rilevante in diverse produzioni alimentari, L.
plantarum e L. delbrueckii. Per la specie L. plantarum è stato confrontato il contenuto genico di
ceppi di diversa origine tramite l’utilizzo di DNA-microarray commerciali su cui è rappresentato il
genoma completo del ceppo WCFS1, interamente sequenziato nel 2003. Gli esperimenti di
ibridazione hanno evidenziato una notevole eterogeneità genetica che correla con i profili fenotipici
dei ceppi considerati. Un ulteriore approfondimento di queste osservazioni consentirà, tramite
l’utilizzo di supporti bioinformatici dedicati, di valutare più chiaramente il legame tra specifici
determinanti genici e proprietà fisiologiche in un’ottica di genomica funzionale. Nel capitolo 6 è
riportata per la prima volta la presenza di due distinte vie metaboliche, una mediata da permeasi e
l’altra da un trasportatore fosfoenol-piruvato dipendente, responsabili del trasporto e della
degradazione del lattosio nella specie L. delbrueckii. La presenza/assenza e l’espressione di una o
dell’altra via metabolica sono legate all’ecologia, alle proprietà tecnologiche e all’evoluzione delle
quattro subspecie attualmente descritte, L. delbrueckii subsp. lactis, bulgaricus, delbrueckii e
indicus. I risultati ottenuti costituiscono un originale contributo per valutare con più rigore la
flessibilità di queste specie batteriche e la funzionalità dei singoli isolati che li rendono
differentemente adatti a sopravvivere in determinati ambienti (latte, vegetali) e a trovare impiego in
diversi processi dell’industria alimentare (produzione di yogurt o formaggi).
In conclusione, gli studi tassonomici riportati hanno contribuito a migliorare lo schema di
classificazione dei batteri investigati, chiarendo le eventuali ambiguità del sistema di nomenclatura.
Inoltre, è evidente come la ricerca di base possa avere notevoli risvolti applicativi quali
l’approfondimento di tratti funzionali, utili per la selezione di organismi di rilevanza tecnologica.
Non da ultimo, l’eterogeneità osservata consente di comprendere più chiaramente la flessibilità
genetica che spinge questi organismi ad evolvere rapidamente e ad adattarsi all’ambiente che li
circonda.The present thesis, titled “Theoretical outcomes and applications of taxonomic studies on the
genera Enterococcus and Lactobacillus”, concerns taxonomy of bacteria. This scientific discipline
aims at studying all living organisms with the final goal of characterizing and arranging them in an
orderly manner on the basis of their similarity and relationships. The classification scheme based on
the real genealogical relationships of living organisms is considered the most stable. However, as a
consequence of their simple structure, bacteria investigation has been strongly influenced by
scientific and technical advances.
Microbes certainly play a crucial role in several scientific area, such as medicine, genetics,
ecology and they are largely employed in food production industry, pharmacology and
environmental science. Thus, it is necessary to study existing biodiversity in order to use
microorganisms for human activities, to discover new biotechnological applications and to deep the
risks linked to the presence of unwanted or even dangerous bacteria. Moreover, taxonomy assigns a
unique name to each described organism, in order to provide information exchange by using a
common terminology and to provide tools or methods to improve the correct identification of new
isolates.
During this project different taxonomic aspects have been faced concerning some species
belonging to the genera Lactobacillus ed Enterococcus, which include relevant bacteria for
ecological and applicative purpose, in particular, for fermented food production where they are
largely employed as natural component or as starter. Some species are marked as common
inhabitant of the gastro-intestinal tract of human and animals and they can modulate the microflora
composition, nutrient assimilation as well as the general health status of the host. Due to their
presence in so different environments and their application in several human area, their study
appear particularly interesting and necessary. The present project has been carried out by using
different approaches based on the aspect to be evaluated, the organism and the taxonomic rank
under investigation (i.e. the comparison of distinct or strictly correlated species and the analysis of
strains belonging to the same taxonomic unit).
In the first part of this dissertation (chapter 2) is presented an evaluation of the phylogenetic
relationships among enterococci by using a comparative sequence analysis of six molecular markers
(16S rDNA, recA, hsp60, pheS, atpA, rpoA) and synteny of the region flanking recA gene. These
approaches allowed to underline the presence of distinct groups of closely related species and to
hypothesize their speciation order. The obtained gene sequence data of three target genes, 16S
rRNA, recA e hsp60, were analysed in order to design diagnostic probes in correspondence of
species-specific polymorphisms. The designed probes, employed to develop a prototype of spotted
DNA-microarray, permitted to distinguish, on the basis of the obtained hybridization profiles, the
Enterococcus species which are more frequently detected in clinical samples and in several food
production chains, e.g. E. durans and E. hirae, usually hardly differentiated by the use of other
molecular and phenotypic methods.
In the second part (chapters 3 and 4) other two aspects, involving closely related species (L.
ingluviei e L. thermotolerans, L. vaccinostercus e L. durianis) have been faced. These species
showed highly conserved molecular markers and similar phenotypic properties. Thus, their degree
of similarity has been determined by DNA-DNA hybridization, that is the unique valid method to
circumscribe a bacterial species. Obtained results permitted to evidence that these closely related
taxa belong to a single species, and they were wrongly classified with different names. As a
consequence of these evidences, the classification scheme of Lactobacillus genus and the
nomenclature of these organisms were corrected, in order to allow in the future a more clear and
unambiguous identification of natural isolates.
In the third part of the thesis (chapters 5 and 6) it is reported an exploration of microbial
biodiversity at inter-specific level, in particular of two relevant species largely employed in several
food production processes, i.e. L. plantarum and L. delbrueckii. Concerning the L. plantarum
species, the gene-content of strains of different isolation origin was evaluated by using commercial
DNA-microarray representing the complete genome of WCFS1 strain, sequenced in the 2003.
Hybridization experiments highlighted an consistent genetic heterogeneity which seems correlatine
with the phenotypic profiles of the considered strains. A further evaluation of these observations, by
the application of powerful bioinformatic tools, will permit to match specific genetic determinants
and physiological properties in a functional genomic perspective. In chapter 6 the presence of two
distinct metabolic pathways, responsible for lactose transport and degradation in L. delbrueckii
species, is reported for the first time: one involves a permease and the other a phosphoenol-pyruvate
dependent system. The presence/absence and transcription regulation of the first or of the second
metabolic pathway seemed linked to ecology, technological properties and evolution of the four
subspecies, L. delbrueckii subsp. lactis, bulgaricus, delbrueckii and indicus. The presented
observations represent an original contribution to appraise the flexibility of this bacterial species
and functionality of single isolates made them able to survive in different habits (milk, vegetable)
and to be employed in several food industry processes (e.g. yogurt or cheese production).
In conclusion, the reported taxonomic studies have contributed to improve the classification
scheme of the investigated bacteria and removing some ambiguous aspects of the nomenclature
system. Moreover, it is highlight the applications of basic research studies such as a deep evaluation
of functional traits and criteria, helpful to the selection of organism of technological relevance.
Finally, the observed heterogeneity furnish a more clear view of genetic flexibility which force
these organisms to rapidly evolve and to easily adapt to the several environments
Development of novel procedures to obtain innovative supplements based on phytocompounds and microorganisms.
No full textL’attività di ricerca è stata svolta al fine di studiare l’effetto di microrganismi fermentanti sui componenti metabolici di matrici vegetali, con l’obiettivo finale di ottenere un integratore alimentare con migliorate proprietà metaboliche di una selezionata varietà di agrumi. E' stata creata una collezione di microrganismi (lieviti e batteri) che potranno essere ulteriormente studiati per possibili applicazioni future. Alcuni di questi microrganismi sono stati impiegati nelle prove di fermentazione per valutare l'effetto sul profilo metabolico complessivo. E' stata valutata anche la possibilità di aggiungere dei microrganismi probiotici con proprietà benefiche documentate. Le prove di fermentazione hanno individuato diversi aspetti innovati da approfondire a livello aziendale con prove di fattibilità e stabilità dei prossibili integratore alimentare realizzabili.The research activity aimed at studying the effect of microrganisms on the metabolic profile of vegetable matrices. A collection of yeasts and bacteria were created. Some of them were chosen to ferment a selected variety of citrus fruits. Two selected probiotics strains with documented properties were added to the product. The analyses performed arose several innovative aspects to be evaluate in pre-indestrial setting with feasibility and stability tests in order to define which food supplement can be obtained
Evoluzione della tecnologia DNA-microarray per applicazioni di diagnostica molecolare.
No full textI DNA-microarray sono strumenti innovativi costituiti da un supporto rigido di piccole dimensioni su cui sono immobilizzate molecole di DNA a singolo filamento, definite sonde. Essi sono stati sviluppati originariamente per eseguire analisi dei profili trascrizionali, ma il potenziale di questa tecnologia si esprime pienamente anche in altri tipi di applicazioni. Infatti, essi si
stanno dimostrando degli ottimi strumenti di diagnostica
molecolare per il rilevamento, tramite ibridazione, di
specifici marcatori di sequenza presenti nei campioni da analizzare. Le finalità diagnostiche per le quali possono essere impiegati i DNA-microarray sono molteplici e coinvolgono i più disparati ambiti del mondo scientifico.
In questa rassegna si intendono presentare alcune delle principali tipologie di DNA-microarray che hanno già trovato applicazione in ambito diagnostico e alcune piattaforme che hanno le potenzialità per farlo
Relevance of probiotic strain naming: scientific and commercial aspects of nomenclature
No full textThe reliable identification of probiotic strains is a fundamental prerequisite for correct labelling of probiotic products. Taxonomic analysis of bacterial strains consists in the attribution of genus and species names to microrganisms, and the lowest taxonomic rank recognized in bacterial taxonomy is the subspecies. However, development of investigation techniques results is a deepen comprehension of microbial biodiversity, thus evidencing the existence of intra- and sub-specific heterogeneity. Technical and taxonomic improvements are particularly useful to highlight and to detect strain-specific peculiarities, characterising the properties enable to distinguish distinct probiotic strains.
Therefore, taxonomy and nomenclature appear to be relevant scientific disciplines in order to undoubtedly link each bacterial species and strains with all data available related to each organism, from their isolation until the most recent investigations. From the scientific point of view, nomenclature undergoes changes in time, which are the results of improvement in taxonomic characterization of bacteria and it may result in classification and/or name adjustments. However, changes of names might produce confusion in the consumer's perceptions of product reliability in the tracking of literature data for probiotic strains and product patenting. Issues related to the scientific and commercial aspects of bacterial taxonomy and nomenclature are discussed
Technological and genetic aspects of Lactobacillus delbrueckii subsp. indicus involved in the production of novel fermented milks.
No full textFour Lactobacillus strains isolated from an Indian fermented milk have recently been grouped in the new subspecies L. delbrueckii subsp. indicus (1). Previous studies have evidenced that these strains ferment lactose constitutively, like L. delbrueckii subsp. bulgaricus (1). However, lactose degradation is probably related to the presence of a peculiar genetic equipment since any amplification of the lac operon genes was achieved. No additional information is currently available on technological and genetic properties of these strains. In the present study, the ability of the four Indian strains to induce pleasant organoleptic traits when grown in milk-based media of different composition was investigated. Physical and chemical parameters were monitored during milk fermentation in order to establish the performances of the examined strains. In addition, sequences of genes encoding some lactose-PTS system components were obtained suggesting that lactose degradation in the Indian strains is probably due to the presence of these genetic determinants. Finally, a comparison of technological and genetic characteristics displayed by the different L. delbrueckii subspecies was conducted. The data obtained indicate that L. delbrueckii subsp. indicus strains show interesting properties and may be applied in association with S. thermophilus for the production of novel fermented milks
The role of genotype and phenotype in the infraspecific taxonomy of Lactobacillus species.
No full textData recently obtained for two infraspecific groups within the species Lactobacillus delbrueckii and Lactobacillus plantarum led to the description of two novel subspecies: Lactobacillus delbrueckii subsp. indicus (Dellaglio et al., 2005) and Lactobacillus plantarum subsp. argentoratensis (Bringel et al., in press), respectively.
Subspecies are the lowest taxonomic ranks with standing in nomenclature and are based on the recognition of phenotypic and/or genotypic heterogeneities (Rosselló-Mora & Amann, 2001). The descriptions of the aforementioned subspecies were the result of a polyphasic approach. It allowed to highlight that, in both species, the genotypic/genomic diversity was more marked than the phenotypic one. Moreover, in the case of L. delbrueckii, the phenotypic similarity between two subspecies was due to a phenomenon of convergent evolution: two different genetic equipments were responsible of the same phenotypic manifestation (Germond et al., 2003).
On the basis of those results, we present some considerations on the contribution that phenotypic and genotypic/genomic data may give to the delineation of subspecies. The experimental results presented make us suggest that prominent role in infraspecific taxonomy should be given to genotype since laboratory conditions could be misleading in the evaluation of “in vivo” expressed traits (phenotype) and, most of all, genotypic/genomic analysis is fundamental to understand which potentialities an organism could express
Growth in milk: a challenging task for strains of the species Lactobacillus delbrueckii.
No full textThe ability of lactic acid bacteria (LAB) to grow in milk is a consequence of the presence and expression of genes responsible for the degradation of milk sugars and casein. The genes coding for the utilization of lactose and galactose (the main sugars of milk) are organized into the lac and gal operons, respectively. The genetic equipment involved in the proteolytic activity of LAB in milk is complex and still not well characterized. The aim of this research was to investigate some genetic determinants implicated in the catabolism of lactose, galactose and casein in the four known subspecies of L. delbrueckii, i.e. the subspecies delbrueckii, bulgaricus, lactis, and indicus. In particular some aspects were examined: the expression of the lacZ gene, encoding the β-galactosidase enzyme; the sequence of lacR gene and the presence of galT gene, encoding the galactosyl-1-phosphate uridyl transferase. In addition, the presence of the gene prtB, encoding a cell-wall anchored protease, was evaluated and associated with the ability to grow in milk. Differences in the distribution and/or regulation of these genes among the four subspecies of L. delbrueckii, were underlined and related to their adaptation to specific ecological niches
Applicazione della tecnologia DNA-microarray per la caratterizzazione tassonomica e funzionale dei batteri lattici.
No full textExploring gut microbiota composition of the two caves coleoptera species Neobathyscia pasai and Neobathyscia mancinii
No full textNeobathyscia mancinii (Jeannel, 1924) and Neobathyscia pasai (Ruffo, 1950) are two cave-dwelling Coleoptera (Leiodidae, Cholevinae,), which inhabit two caves located in the Prealps of the Veneto Region (Damati Cave and Tana delle Sponde Cave, respectively). In detail, N. mancinii is confined in the internal part of Damati Cave characterized by a constant temperature during the year, while N. pasai mostly colonizes the cave entrance where the temperature is more variable and the energy sources might be more abundant (Latella et al. 2009; Bernabò et al., 2011).
Little is known about the behaviour and the physiological adaptation of these troglobite insects to cave environments, which are characterized by specific conditions as perpetual darkness, high humidity and sporadic food sources.
It has been observed that the complex microbial community associated to insects plays substantial biological roles for the host organisms and contributes to host fitness and adaptation (Colman et al. 2012; Shi et al. 2013).
The present project aimed at characterizing the gut microbiota of N. pasai and N. mancinii as a unique opportunity to gain insights about the behaviour of these insects and their eating habits in the cave environment.
A total of 90 insects (60 N. pasai and 30 N. mancinii) were collected from December 2013 to April 2014 both in Damati and Tana delle Sponde caves and they were pooled in samples of ten insects each. Total DNA was extracted from each pool and subjected to PCR amplification using universal primer designed on ribosomal gene sequences. Denaturing gradient gel electrophoresis (DGGE) was applied in order to investigate bacterial and fungal communities present in the gut of these insects. Selected DGGE bands were subjected to sequencing to identify the most relevant members of these complex communities at genus and species level. Results provide novel information on adaptation to cave environment of N. pasai and N. mancinii. Moreover, insects could be seen as natural reservoir of bacteria, therefore contributing to the microbial community involved in natural processes in the underground
Differentiation of Enterococcus species based on a triple-target genes diagnostic DNA-microarray.
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