1,721,051 research outputs found
TaqMan RT-qPCR targeting the pta gene for Clostridium tyrobutyricum quantification in animal feed, faeces, milk and cheese.
No full textLate blowing defect affects hard cheese production and Clostridium tyrobutyricum appears as the primary
cause of this spoilage. Our aimwas to develop a quantitative TaqMan real-time PCR method to detect this
agent in different matrices along the whole agro-dairy chain, using specific primers targeting the
phosphotransacetylase gene (pta). The optimised assay appeared to be sensitive and specific to quantify
C. tyrobutyricum. Results demonstrated a 100% efficiency with low numbers of cells and spores (approx.
14 mL 1 or g 1) also detected in broth and in milk matrix artificially contaminated with C. tyrobutyricum.
Combined with a DNA extraction method optimised to lyse both cells and spores, our method is suitable
for silage and hay, faeces, milk and hard cheese samples without any enrichment. The molecular
quantification by qPCR was found to be precise and could be used for the rapid determination of the
presence of C. tyrobutyricum
ENVIRONMENTAL RESPONSE OF SPORE FORMING BACTERIA: MOLECULAR STUDIES OF FOOD ASSOCIATED CLOSTRIDIA
No full textPer varie ragioni, tra cui le loro specifiche condizioni di crescita, la diagnosi di infezione e di contaminazione alimentare da clostridi presenta ancora numerose difficoltà sia a livello clinico-batteriologico che a livello molecolare.
In questo lavoro di tesi si è cercato di ampliare lo spettro di conoscenze riguardo i clostridi e la loro diffusione; durante il primo anno di ricerca è stato studiato, applicando nuove tecniche di microscopia, il processo di germinazione di Clostridium tyrobutyricum, uno dei batteri maggiormente responsabili del gonfiore tardivo dei formaggi a pasta dura; l’applicazione di tecniche di Real-Time PCR ha nel contempo reso possibile una determinazione quantitativa dello stesso in latte.
Successivamente, è stata condotta un’analisi di tipizzazione molecolare di clostridi nell’ambito di una filiera agro-zoo-casearia finalizzata alle matrici di processo al fine di individuare le possibili vie di diffusione dei microrganismi.
La parte finale del lavoro è stata dedicata allo studio di espressione genica di un altro Clostridium responsabile di gonfiore ma scelto perché geneticamente indistinguibile da Clostridium botulinum, ovvero il Clostridium sporogenes; l’analisi trascrizionale dei suoi geni durante le fasi vegetativa, di sporulazione, germinazione ed esocrescita ha permesso di assegnare diverse funzioni a geni singoli o a gruppi di geni allo scopo di utilizzare queste informazioni per formulare ipotesi future anche su altre specie di clostridi patogeni.For several reasons, including their specific growth requirements, the diagnosis of infections and food contamination caused by clostridia still presents much difficulty at the clinical, bacteriological and molecular levels. The main purpose of this work is to learn more about clostridia and their interactions with environment. First, new microscopy techniques have been used to study the germination process in Clostridium tyrobutyricum, an anaerobic bacterium responsible for late blowing defects during cheese ripening; meanwhile, the application of real-time PCR methods have been employed to enumerate C. tyrobutyricum cells and spores in milk. Then, a molecular genotyping has been set in order to identify the most common clostridia in a agro-dairy production aimed to detect the possible ways of diffusion of these microbial species.
The last part concerns the study of expression patterns of Clostridium sporogenes, an apathogenic gram positive clostridium usually involved in food damage and frequently isolated from late bowled cheese; Clostridium sporogenes is genetically indistinguishable from Clostridium botulinum and is often used as a model for the toxic subtypes. The objective of this study is to use an array-based large-scale transcriptional analysis in order to study gene expression in four different steps of Clostridium sporogenes life cycle: vegetative cells, sporulating cells, dormant spores and germinating ones. Our aims includes being able to relate gene-expression patterns to specific phenotypes and to discover gene expression divergences between the different phases of living, germination and outgrowth of spore-forming bacteria. An important aim is to assign functions to groups of or individual C. sporogenes genes and use this information to formulate specific hypotheses for further testing also on pathogenic clostridia types
Genetic alterations in poorly differentiated endocrine carcinomas of the gastrointestinal tract
No full textThe molecular pathogenesis of poorly differentiated endocrine carcinomas of the gastrointestinal tract (GI PDECs) remains unclear. It has been suggested that these lesions either originate from multipotent stem cells that also can serve as the origin of nonendocrine adenocarcinomas or arise due to the dedifferentiation of well-differentiated endocrine carcinomas (WDECs)
Array-based transcriptional analysis of Clostridium sporogenes UC9000 during germination, cell outgrowth and vegetative life
No full textThe members of the genus Clostridium, including the spore-forming anaerobic bacteria, have a complex and strictly regulated life cycle, but very little is known about the genetic pathways involved in the different stages of their life cycle. Clostridium sporogenes, a Gram-positive bacterium usually involved in food spoilage and frequently isolated from late blowing cheese, is genetically indistinguishable from the proteolytic Clostridium botulinum. As the non-neurotoxic counterpart, it is often used as an exemplar for the toxic subtypes. In this work, we performed a microscopic study combined with a custom array-based analysis of the C. sporogenes cycle, from dormant spores to the early stationary phase.We identified a total of 211 transcripts in spores, validating the hypothesis that mRNAs are abundant in spores and the pattern of mRNA expression is strikingly different from that present in growing cells. The spore transcripts included genes responsible for different life-sustaining functions, suggesting there was transcript entrapment or basic poly-functional gene activation for future steps. In addition, 3 h after the beginning of the germination process, 20% of the total up-regulated genes were temporally expressed in germinating spores. The vegetative condition appeared to be more active in terms of gene transcription and protein synthesis than the spore, and genes coding for germination and sporulation factors seemed to be expressed at this point. These results suggest that spores are not silent entities, and a broader knowledge of the genetic pathways involved in the Clostridium life cycle could provide a better understanding of pathogenic clostridia type
Array-based transcriptional analysis of Clostridium sporogenes/botulinum during its vegetative cycle, germination process and outgrowth
No full textComparing natural and selected starter cultures in meat and cheese fermentations.
No full textIn the last decades, the inoculum of high counts of suitable microorganisms, defined as starter cultures, has become a widely adopted approach in the production of fermented foods. Here, we reviewed the recent works about the use of selected cultures or the exploitation of indigenous microbiota in the production of dry fermented sausages and dairy products. We found that the scientific literature is well consistent in indicating a significant advantage in the use of selected and natural starter cultures (SSC and NSC) as compared to adventitious microbiota (AM) in terms of acidification, sensory traits and acceptability of final ripened products, as well as in the control of undesired microorganisms. Anyway, a thorough understanding of the interactions at ecological level between the introduced strains and the autochthonous microbial communities is a challenge that needs to be addressed in the near future
A combination of SEM technique and X-ray microanalysis to study spore germination process of Clostridium tyrobutyricum
No full textUnderstanding the bacterial communities of hard cheese with blowing defect
Full text linkThe environment of hard cheese encourages bacterial synergies and competitions along the ripening
process, which might lead in defects such as clostridial blowing. In this study, Denaturing Gradient Gel
Electrophoresis (DGGE), a quantitative Clostridium tyrobutyricum PCR and next-generation Illuminabased
sequencing of 16S rRNA gene were applied to study 83 Grana Padano spoiled samples. The aimwas
to investigate the community of clostridia involved in spoilage, the ecological relationships with the
other members of the cheese microbiota, and the effect of lysozyme. Three main genera were dominant
in the analysed cheeses, Lactobacillus, Streptococcus and Clostridium, and the assignment at the species
level was of 94.3% of 4,477,326 high quality sequences. C. tyrobutyricum and C. butyricum were the most
prevalent clostridia. Hierarchical clustering based on the abundance of bacterial genera, revealed three
main clusters: one characterized by the highest proportion of Clostridium, a second where Lactobacillus
was predominant and the last, dominated by Streptococcus thermophilus. Ecological relationships among
species were found: cheeses characterized by an high abundance of S. thermophilus and L. rhamnosus
were spoiled by C. tyrobutyricum while, when L. delbrueckii was the most abundant Lactobacillus, C.
butyricum was the dominant spoiling species. Lysozyme also shaped the bacterial community, reducing
C. tyrobutyricum in favour of C. butyricum. Moreover, this preservative increased the proportion of
L. delbrueckii and obligate heterofermentative lactobacilli and lowered L. helveticus and non-starter
species, such as L. rhamnosus and L. casei
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