Дисертації з теми "Bacillus cereu"

Bacillus thuringiensis è un batterio formante spora, che appartiene al gruppo dei Bacillus cereus. Fu inizialmente caratterizzato per la sua capacità di produrre un cristallo parasporale attivo contro diverse specie di insetti appartenenti agli ordini Lepidotteri, Ditteri e Coleotteri. Grazie alla sua attività insetticida, viene utilizzato in tutto il mondo in silvicoltura e agricoltura per il controllo degli infestanti. Studi recenti riportano la presenza di determinanti genetiche per fattori di virulenza di B. cereus, come l’emolisina BL (HBL), l’enterotossina non emolitica (NHE), la citotossina K e l’enterotossinaT, in ceppi di B. thuringiensis. Poichè ceppi commerciali di B. thuringiensis sono stati rinvenuti sotto forma di spora in derrate alimentari trattate, è parso importante approfondire i potenziali rischi derivanti dalla presenza di questo batterio nella catena alimentare. Studi filogenetici basati sull’analisi dei geni cromosomici, hanno dato esiti contrastanti e, ad oggi, non è chiaro se B. cereus e B. thuringiensis possano essere considerati varietà della stessa specie o due specie differenti. Questo lavoro di tesi è stato condotto con l’obiettivo di approfondire le conoscenze sul comportamento di microrganismi appartenenti al genere Bacillus presenti in prodotti alimentari di origine vegetale, con l’ausilio delle nuove tecnologie molecolari basate sullo studio del genoma e riservando particolare attenzione ai ceppi di B. thuringiensis impiegati come base per bio-pesticidi. L’analisi del profilo patogenico di 10 ceppi di B. thuringiensis presenti in commercio, ha evidenziato un’elevata distribuzione dei geni nhe, hbl, bceT e cytK codificanti per 4 noti fattori di virulenza di B. cereus. I geni enterotossici sono stati individuati tramite tecnica di PCR in tutti i ceppi analizzati. L’analisi con RT-PCR ha evidenziato l’espressione di tutti i geni delle enterotossine. La produzione della tossina HBL è stata confermata con il test RPLA per un ceppo appartenente alla sub-specie kurstaki ampiamente utilizzato per prodotti bio-pesticidi. Queste caratteristiche, e le difficoltà di discriminazione tra B. cereus e B. thuringiensis, hanno suggerito la possibilità che il ruolo di B. thuringiensis nelle intossicazioni alimentari associate a B. cereus, sia stato sottostimato. Un punto importante di questo studio, ha riguardato lo sviluppo di un modello alimentare a base vegetale che consentisse di studiare il comportamento di spore di B. thuringiensis dopo la simulazione di un trattamento industriale. Le tecniche di microscopia a scansione e della microanalisi a raggi X, sono state applicate al modello alimentare, con lo scopo di analizzare la superficie delle spore di B. thuringiensis e la loro capacità di interazione con la matrice alimentare. Particolare attenzione è stata riservata allo studio dei cambiamenti morfologici e chimici delle spore di B. thuringiensis durante il processo di germinazione nel modello alimentare. E’ stata osservata una rapida evoluzione del ciclo biologico di B. thuringiensis rispetto ad altri microrganismi formanti spora come alcune subspecie appartenenti al genere Clostridium (Bassi et al. personal communication). L’esocrescita delle cellule vegetative e il livello massimo di duplicazione cellulare, sono stati raggiunti entro due ore dall’attivazione delle spore in alimento. La tecnica dell’RT-qPCR è stata utilizzata per quantificare l’espressione, in alimento, delle determinanti genetiche per i fattori di virulenza responsabili delle intossicazioni alimentari attribuite a B. cereus. I trascritti delle enterotossine sono stati individuati, in quantitativi variabili, durante tutti gli stadi di crescita considerati, evidenziando un forte incremento durante la fase di crescita logaritmica del microrganismo oggetto di studio. La produzione della componente L2 dell’enterotossina HBL, coinvolta nella sindrome diarroica, nel modello alimentare, è stata determinata, anche se a bassi quantitativi, già durante l’inizio della fase di crescita logaritmica. Queste osservazioni hanno dimostrato la capacità di B. thuringiensis di completare un intero ciclo biologico in una matrice alimentare, sottoposta alla simulazione di un processo industriale, dando luogo alla produzione di enterotossine, come già osservato in terreni di laboratorio. Al fine di gestire i rischi connessi alla possibile presenza di B. thuringiensis nel settore alimentare, si è deciso di procedere con l’identificazione e l’inattivazione di sistemi generali di regolazione della virulenza, attraverso la costruzione di mutanti nulli. Accanto al tradizionale sistema della ricombinazione omologa, è stato considerato un meccanismo innovativo che sfrutta la mobilità degli introni del gruppo II, per generare inattivazioni geniche altamente specifiche. Sebbene siano stati eseguiti numerosi esperimenti, nessuno ha condotto all'inattivazione cromosomica desiderata. Nel tentativo di porre le basi scientifiche per la gestione dei rischi associati alla germinazione delle spore di B. thuringiensis nei prodotti alimentari, e per ottenere più informazioni sul ciclo di vita di questo microrganismo, è stata effettuata un’analisi microarray del trascrittoma di B. thuringiensis in 4 diverse fasi del ciclo biologico nel modello alimentare: da spora dormiente a cellule vegetativa/sporulante. E’ stato possibile confermare che l'mRNA è un componente delle spore batteriche ed evidenziare come le spore siano equipaggiate con una grande quantità di trascritti, probabilmente utili a fronteggiare le prime fasi del processo di germinazione. Le spore dormienti contengono ribosomi; durante i primi 40 minuti dopo l'attivazione della spora, la sintesi di rRNA e proteine ribosomiali aumenta fortemente. Una significativa e determinante attivazione di geni polifunzionali è stata osservata nelle spore in germinazione: la maggior parte dei geni coinvolti nell’attività metabolica (house-keeping, fattore d’inizio della traduzione, proteine ribosomali e fattori di allungamento) sono risultati indotti in questa fase dell’analisi microarray. Nelle cellule vegetative di B. thuringiensis sono stati individuati un elevato numero di trascritti per proteine coinvolte nella regolazione di differenti processi biologici, compresi la resistenza a differenti composti anti-microbici e ad agenti di stress ossidativo. E’ stato ipotizzato che le cellule di B. thuringiensis attivino questi sistemi, in risposta a stimoli esterni, come meccanismo di difesa e adattamento alle condizioni ambientali rinvenute nel modello alimentare. In accordo con i dati riportati in letteratura, i geni associati alla virulenza di B. cereus, sono risultati indotti nell’analisi microarray, specialmente durante la tarda fase di crescita cellulare. L’analisi trascrittomica si è dimostrata non solo un metodo idoneo per lo studio del processo di germinazione ed esocrescita delle spore di B. thuringiensis, ma anche un sistema utile per valutare il comportamento di batteri patogeni in alimento. I dati ottenuti hanno permesso di approfondire le conoscenze sulla versatilità metabolica di B. thuringiensis facendo emergere le caratteristiche di virulenza di questo potenziale patogeno alimentare. Dal momento che B. thuringiensis è ampiamente utilizzato in agricoltura biologica, dovrebbe essere efettuato un attento monitoraggio dei ceppi impiegati. In letteratura sono diffusamente riportati i rischi associati al patogeno alimentare B.cereus, ma quelli relativi a B. thuringiensis sono spesso sottovalutati. Dai dati ottenuti in questo studio, si potrebbe insinuare che B. thuringiensis sia responsabile di parte dei focolai di tossinfezione alimentare attribuiti a B. cereus: i produttori alimentari e le autorità responsabili per la sicurezza alimentare, dovrebbero dunque considerare il potenziale rischio associato alla presenza di residui di biopesticidi a base di B. thuringiensis nella catena alimentare.
Bacillus thuringiensis is a spore forming bacterium that belongs to the Bacillus cereus family. It was first characterized for its ability to produce a parasporal crystal active against several insect species, especially Lepidoptera, Diptera, and Coleoptera. Due to its insect activities it is worldwide used in forestry and agriculture to control pests. Recent studies showed that most of the genetic determinants for B. cereus virulence, such as haemolysin BL (HBL), non haemolytic enterotoxin (NHE), cytotoxin K, and bc-D-ENT enterotoxin, are harboured by B. thuringiensis strains. Since B. thuringiensis can contaminate food, being residual in spore form after treatment in the fields, it is ever more urgent to deepen investigate the potential risks arising from the presence of B. thuringiensis in food industry. Phylogenetic studies based on the analysis of chromosomal genes bring controversial results, and it is unclear whether B. cereus and B. thuringiensis are varieties of the same species or different species (Ivanova et al. 2003). Hence, what may seem to be a minor problem of taxonomy may therefore have serious implications for virulence and pathogenicity. This work of thesis was aimed to achieve a deeper scientific information on the food-associated Bacilli, taking the advantage of new genome based molecular approaches, focusing the attention on B. thuringiensis strains used as commercial biopesticides. The in vitro pathogenic profile of ten commercial B. thuringiensis strains, was characterised by the high distribution of the nhe, hbl, bceT and cytK genes, coding for respectively four B. cereus associated virulence factors. Enterotoxin genes were detected by PCR in all the strains analyzed. RT-PCR analysis confirmed the enterotoxin genes expression. Toxin productions was detected by RPLA test in the strains belonging to the widely used subsp. kurstaki. These features and the difficult discrimination between B. thuringiensis and B. cereus, suggested that the role of B. thuringiensis in outbreaks of foodborne disease may have been underestimated. The development of a vegetable based food model, that would allow to asses the behaviour of B. thuringiensis spores, after the simulation of an industrial processing treatment, was an important point in this study. The analysis of Bacillus spore envelope, and its ability to interact with food environment, have been performed using SEM and SEM X-ray microanalysis applied to the food model proposed. In more detail, particular attention was devoted to morphological and chemical changes of B. thuringiensis spores during germination process in food. We observed a rapid evolution of the B. thuringiensis biological cycle compared to that of other spore forming bacteria like Clostridium spp. (Bassi et al. 2008, personal communication). Interesting was that only two hours after spore activation, cell outgrowth was completed and cell division was at the maximum level. RT-qPCR analysis were performed to quantify the expression, in food, of the major virulence genes involved in B. cereus-associated food borne disease. Toxin mRNAs were detected, in variable amounts, at all investigated growth stages of B. thuringiensis, with a strong increase during the log phase of microorganism growth. Although no information on the B. cereus toxin expression in food are available, previous in vitro studies on B. cereus enterotoxins production, reported that the highest toxin level is achieved during the late log/early stationary phase. The production of the L2 component of HBL enterotoxin, involved in the diarrhoeal syndrome was detected in food model, even in low amount, during the early log phase. We concluded that B. thuringiensis can complete an entire life cycle in food systems after an industrial processing simulation, producing enterotoxins as observed in broth cultures. Given this finding, the need to identify systems for manage the risks associated with B. thuringiensis in industrial fields has became clear. An experimental approach was described in this work of thesis. Identification and inactivation of general systems for regulating virulence, through null mutants construction, were considered to evaluate changes in growth performance, cellular metabolism and toxins expression, in the studied microorganism. Besides homologous recombination, the mobility mechanism of group II introns were assessed to generate highly specific chromosomal gene disruption in B. thuringiensis. A novel approach and several experiments were performed to achieve the desired chromosomal inactivation, however no attempts gave the expected results. In order to manage risks associated with B. thuringiensis outgrowth in foodstuffs, and to gain more information on its life cycle, a microarray transcriptome analysis of B. thuringiensis in four different stages of the biological cycle, was performed from dormant spore to vegetative/sporulating cells. We could emphasized that mRNA is a component of bacterial spores. We discovered that spores are equipped with a large amount of transcripts probably useful to front the next steps of outgrowth. Dormant spores contained populations of ribosomes; during the first 40 minutes after spore activation, rate of both rRNA and ribosomal proteins synthesis strongly increased. A basic and strong activation of polyfunctional genes seemed to begin in germinant spores: most of the genes involved in the metabolic activity (house-keeping genes, translation initiation factor, ribosomal proteins, and elongation factors) were overrepresented at this time in microarray analysis. A large number of transcripts for protein involved in the regulation of different biological process, including resistance to different antimicrobial compounds and oxidative stress agents, were found to be present in B. thuringiensis vegetative cells. We hypothesized that B. thuringiensis cells may activate these systems in response to external stimuli for cell defence and adaptation to changing environmental conditions in food model. The transcripts for germination proteins (ger type) found in spore, are an index of the expression of this genes in previous sporulation stage and suggested the importance during dormancy, to monitor the environment for proper outgrowth conditions. This finding could explain the ability of B. cereus-like microrganism to occupy and complete a full life cycle within several different environmental niches. According to literature data, all the associated virulence genes, represented in microarray analysis, were up-regulated especially during the late stage of cell growth. Transcriptomic has been demonstrated to be not only a powerful tool to study the germination and outgrowth of B. thuringiensis spores, but also a suitable method to assess the environmental response to bacterial pathogens in food. Data obtained, provide new basic knowledge on Bacillus cereus group. These data extends our knowledge on the metabolic versatility of B. thuringiensis and also added to our view of virulence traits of this potential food-pathogen. Since B. thuringiensis is widely used and popular in biological farming, a careful monitoring of the strains used should be justified. Literature reports widespread the risks associated with the food-pathogen B. cereus, but those related to B. thuringiensis are often underestimated. From data obtained in this study we could assume that B. thuringiensis could actually be responsible for many of the food borne outbreaks previously attributed to B. cereus; taking this enterotoxigenic potential into account, as well as the fact that B. thuringiensis cannot be separated from B. cereus at the chromosomal level, food producers and food authorities, responsible for food safety, should consider the risk of B. thuringiensis insecticide residue in the food chain.

Les spores bactériennes sont omniprésentes dans l'environnement et contaminent fréquemment les aliments. L'histoire des bactéries productrices des spores dans leurs habitats naturels est mal connu. Quelle peut-être l'influence de ces conditions de sporulation sur les propriétés des spores bactériennes ? Ont-elles une incidence sur la sécurité microbiologique des aliments? La germination est une étape clé dans la transformation de la spore dormante en cellule végétative métaboliquement active. L'objectif de cette thèse est d'étudier l'impact de la température de formation sur la germination des spores de Bacillus cereus, un agent d'intoxications alimentaires fréquentes chez l'homme. Nos résultats ont montré que les spores formées à basses températures (15°C pour deux souches psychrotrophes LM9 et D 15 et 20°C pour la souche-type ATCC 14579) avaient des capacités de germination en réponse aux agents nutritionnels inosine et L-alanine nettement plus élevées que celles des spores formées à 37°C. Les spores formées à 37°C présentaient aussi une bien meilleure résistance à un traitement thermique à 90°C, résistance pouvant être attribuée à des teneurs en acide dipicolinique et en cations divalents, plus élevées. Le caractère hydrophobe des spores, exprimant la capacité d'adhérence des spores, était plus prononcé quand les spores étaient formées à 15°C et les observations au microscope électronique ont aussi montré que la morphologie de l'exosporium, auquel le caractère hydrophobe des spores est principalement attribué, était également affectée. La température de sporulation affecte aussi bien la phase précoce de germination (marquée par la libération de l'acide dipicolinique), que la phase tardive correspondant à la dégradation du cortex. L'étude par microscopie électronique de l'ultra-structure des spores formées à différentes températures a montré des différences importantes dans la morphologie des tuniques, les enveloppes protéiques externes de la spore impliquées dans les phases précoces et tardive du processus de germination. De nettes différences dans la composition des extraits de tuniques des spores formées à 15°C et à 37°C en protéines étaient détectées par analyse électrophorétique. Nos résultats suggèrent que la température de sporulation affecte le processus d'assemblage des protéines des tuniques, et par conséquence la capacité des spores à germer
Bacterial spores are ubiquitous in the environment and frequently contaminate food The history of spore forming bacteria in their native habitats is largely unknown. Sporulation conditions have ther an influence on spore properties and an incidence on microbiological food safety? Germination is a key step in the transformation of the dormant spore into vegetative cell metabolicly active. The objective of this thesis is to study the impact ofsporulation temperature on the germination of spores of Bacillus cereus, an agent of frequent human food poisonings. Our results showed that the spores formed at low temperatures (15°C for two psychrotrophic strains LM9 and D15 and 20°C for the type-strain ATCC 14579) had an anhanced germination capacities in response to the nutrient germinant inosine and L-alanine than those of spores formed at 37°C. Spores formed at 37°C had also a much better resistance to a heat treatment at 90°C, resistance being attributed to higher contents afdipicolinic acid and of divalent cations. The spore hydrophobicity, expressing the capacity of spore adhesion, was more marked when the spores were formed at 15°C and the observations under the electron microscope have showed that the morphology ofexosporium, to which the hydrophobic character of the spores is mainly attributed, was also affected. The temperature ofsporulation affects both the early phase of germination (marked by the release ofdipicolinic acid), and the late phase corresponding to cortex lysis The study by electronic microscopy of the ultrastructure of spores formed at various temperatures showed important differences in the morphology of coats, external spore layers known to be implicated in early and late phases of the germination process. Clear differences in the composition in proteins of coats extracts of spores formed at 15°C and 37°C were detected by electrophoretic analysis. Our results suggest that the Sporulation temperature affects the process of coats proteins assembly, and consequentely the capacity of spores to germinate

O grupo do Bacillus cereus reúne microorganismos de grande importância econômica, médica e também em questões de biodefesa, o que se reflete no fato dele conter um grande número de genomas sequenciados intimamente relacionados, como Bacillus anthracis, B. cereus e B. thuringiensis. As ferramentas atuais de bioinformática aplicadas a tal conjunto de informações nos proporcionam grande oportunidade para análises genômicas comparativas completas. Os membros deste grupo tem muito de sua especificidade atribuída aos seus plasmídeos, os quais variam em tamanho e número. Seus cromossomos apresentam um alto nível de sintenia com diferenças limitadas no conteúdo genético, tornando questionáveis as interpretações sobre a especiação dos membros deste grupo. Este trabalho visa contribuir ao esclarecimento sobre a proximidade genética entre estes três constituintes do grupo do Bacillus cereus sensu lato anteriormente citados mediante identificação e caracterização de RNAs não codificadores, auxiliando na compreensão taxonômica, no entendimento dos fatores de virulência, na interação patógeno hospedeiro e em interações ecológicas como o comportamento de B. thuringiensis no controle de pragas da agricultura e vetores de doenças. Para a identificação dos ncRNAs, foi produzido um conjunto de dados composto por 35 genomas completos dos organismos de interesse, sendo 9 B. anthracis, 13 B. cereus, 12 B. thuringiensis e também 1 B. weihenstephanensis, obtidos à partir dos bancos de dados GOLD e NCBI. Tais genomas foram classificados considerando-se a metodologia de montagem após os sequenciamentos, tipo de anotação, manual ou automática e impacto das publicações resultando em 26 escolhidos. Em seguida o material foi processado no software Artemis V.16.0.0 para extração das regiões intergênicas, então submetidas a três métodos diferentes de inferência computacional para identificação de RNAs não codificadores. O primeiro método foi o processamento via Infernal V.1.1 / banco de dados Rfam V.11.0, o segundo foi o processamento via sRNAscanner V.1.9, e finalmente uma análise comparativa com o banco de dados da UTFPR que reúne ncRNAs da literatura, com base no Non-coding RNA Databases Resource (NRDR). Os dados foram então carregados para um banco de dados PostgreSQL V.9.1. Para a caracterização das sequências obtidas, foram criadas tabelas relacionais contendo os dados das 2208 famílias Rfam, agrupando os dados públicos dos sites FTP Rfam e instituto SANGER. Ainda como auxílio à busca e caracterização, os genomas completos foram carregados em banco de dados. Os resultados dos três métodos de descoberta foram pesquisados via consultas diretas no banco de dados (linguagem SQL) mediante agrupamento de regiões contíguas sobrepostas. Foram identificados 181 ncRNA candidatos, distribuidos em 12 famílias exclusivas para um ou outro grupo: B. anthracis (2), B. cereus (5) e B. thuringiensis (5). Posteriormente os candidatos foram caracterizados por espécie e cepa nas 23 famílias identificadas.
The Bacillus cereus group gathers microorganisms of great economic importance and also at medical and biodefense issues, this is reflected in the fact that it contains a large number of sequenced genomes closely related organisms as Bacillus anthracis, B. cereus and B. thuringiensis. Current bioinformatics tools applied to this set of information provides us great opportunity to complete comparative genomic analyzes. Members of this group has plenty of its specificity attributed to their plasmids, which vary in size and number. Their chromosomes have a high level of synteny with limited differences in genetic content, which makes questionable the interpretations on speciation to the members of this group. This work aims to contribute to the clarification of the genetic proximity between these three constituents of the cereus group by identification and characterization of non-coding RNAs, contributing in taxonomic understanding, the understanding of virulence factors in host pathogen interaction and ecological interactions like the behavior of B. thuringiensis in the control of agricultural pests and disease vectors. With the purpose of identifying non-coding RNAs, it was produced a data set consisting of entire genomes of interest organisms, 9 B. anthracis, 13 B. cereus, 12 B. thuringiensis and also 1 B. weihenstephanensis, a total of 35 GenBank format genomes obtained from GOLD and NCBI databases. These genomes were classified considering the assembling methodology, after sequencing process, annotation type, manual or automatic as well the publications impact, resulting in 26 finally selected genomes. Data were then processed using Artemis V.16.0.0 program, for extraction of intergenic regions and submitted to three different methods of computational inference for non-coding RNAs identification. The first method, processing with Infernal V.1.1 / Rfam database V.11.0, the second was processing through sRNAscanner V.1.9, and finally a comparative analysis with the UTFPR database which gathers ncRNA literature based on Non-coding RNA Databases Resource (NRDR). Data from these three analyzes were loaded into a PostgreSQL V.9.1 database. Relational tables were created to the characterization of the obtained sequences. The tables contained all 2208 Rfam families data, grouping public FTP and Rfam institute SANGER sites data. As supporting means to search and characterization, complete genomes to the related organisms were loaded into a data base. Then the results of those three methods of discovery were searched through direct queries on the created database (SQL language) by grouping contiguous overlap regions. Thus, 181 ncRNA candidates were identified and further characterized by species, strain and ncRNA family. 181 ncRNA candidates were identified, distributed in 12 unique families to either group: B. anthracis (2), B. cereus (5) and B. thuringiensis (5). Later, candidates were characterized by species and strain on 23 identified families.

Die vorliegende Arbeit befasst sich mit der Entwicklung und Charakterisierung monoklonaler Antikörper (mAk) gegen die C-Komponente des Non-haemolytic Enterotoxin (Nhe)-Komplexes von Bacillus cereus sowie deren Einsatz zur Analyse der Rolle des NheC im Hinblick auf die Wirkungsweise von Nhe. NheC wurde in Escherichia coli rekombinant exprimiert und mittels Immunaffinitätschromatographie aufgereinigt. Nach Überprüfung durch SDS-PAGE und Quantifizierung mittels SYPRO® Ruby Protein Gel Färbung wurde das gereinigte Toxin als Immunogen eingesetzt. Nachfolgend konnten drei mAk vom IgG-Subtyp (mAk 2G8, 1E12, 2F10) und ein mAk vom IgM-Subtyp (mAk 3D6) gegen NheC hergestellt werden. Die Charakterisierung der mAk erfolgte mittels Enzymimmuntest (EIA), Western Immunblot und Immunfluoreszenzmikroskopie. Bei Verwendung eines indirekten EIAs erlaubten die mAk den sensitiven Nachweis von rNheC im unteren Nanogrammbereich (Nachweisgrenze 10 – 15 ng/ml). Untersuchungen zur Spezifität der mAk innerhalb des Nhe-Komplexes mittels indirekten EIAs und Western Immunblots zeigten, dass die mAk 2G8 und 1E12 substantielle Kreuzreaktionen mit der strukturverwandten NheB-Komponente aufweisen. Dies konnte durch Epitopanalysen und kompetitive Bindungstests noch bestätigt werden. Während die mAk 2G8, 1E12 und 3D6 mit allen überprüften B. cereus-Stämmen reagierten, weist das von dem mAk 2F10 erkannte Epitop eine stammspezifische Variabilität auf. In Zellkulturtests war keiner der vier mAk in der Lage die zytotoxische Aktivität des Nhe-Komplexes zu neutralisieren. Demgegenüber gelang es mit allen mAk mittels Immunfluoreszenz erstmals die direkte Bindung von NheC an die Zielzelle darzustellen. Unter Verwendung eines polyklonalen Kaninchen-Antiserums gegen NheC als Fangantikörper und der vier mAk gegen NheC als Nachweisantikörper wurden hochempfindliche Sandwich-EIAs für den Nachweis von gereinigtem NheC entwickelt. In natürlichen B. cereus-Überständen konnten jedoch nur geringe Mengen (< 10 %) des theroretisch vorhandenen NheC nachgewiesen werden. Die auf diesen Ergebnissen basierende Hypothese, dass NheC in Lösung an NheB gebunden vorliegt, konnte zunächst in artifiziellen Systemen bestätigt werden. Mangels SDS-Stabilität der NheB-NheC-Komplexe, erfolgte der Nachweis mittels intermolekularem Cross-Linking und Dot Blot-Analysen. Durch Kombination des NheC-spezifischen mAk 3D6 als Fangantikörper und des HRP-gekoppelten mAk 1E11 gegen NheB als Nachweisantikörper konnte ein spezifischer NheC/B-Sandwich-EIA zum Nachweis von NheB-NheC-Komplexen in B. cereus-Kulturüberständen etabliert werden. Zur Analyse der Funktion der NheB-NheC-Komplexe wurde zum einen die Komplexbildung mit der Zytotoxizität verglichen, und zum anderen per Durchflusszytometrie deren Zellbindung mittels NheB-Quantifizierung (durch mAk 1E11) bestimmt. Fazit ist, dass scheinbar sowohl eine definierte Menge an NheB-NheC-Komplexen, als auch ausreichend freies NheB vorhanden sein müssen, damit effiziente Zellbindung und Zytotoxizität von Nhe gewährleistet sind.

Les exopolysaccharides - des polymères de sucres exportés - sont impliqués dans des fonctions essentielles de la physiologie bactérienne. Ce sont en effet des composants majeurs, respectivement, de la paroi bactérienne, des polymères secondaires attachés à cette paroi, des capsules, et de la matrice du biofilm. Bacillus thuringiensis est une bactérie entomopathogène, appartenant au groupe Bacillus cereus, capable de former un biofilm à l’interface air-liquide. Ce biofilm comporte deux structures distinctes: une pellicule flottant sur le milieu de culture et, en périphérie, en continuité avec celle-ci, un anneau adhérent sur les surfaces solides. Pour identifier les exopolysaccharides constitutifs de la matrice du biofilm chez cette bactérie, j'ai recherché, dans le génome séquencé de la souche 407 de B. thuringiensis, les différents loci chromosomiques susceptibles d'être impliqués dans la production de ces exopolymères. Deux loci ont étés identifiés, que nous avons appelé eps1 et eps2. Le locus eps1 avait déjà été décrit comme n'ayant aucun rôle dans la formation des biofilms chez B. cereus et sa fonction était restée inconnue. Nous avons montré que l'exopolysaccharide Eps1, dépendant du locus eps1, forme une capsule en phase stationnaire et en condition d'hypoxie. Cette capsule, qui présente des propriétés adhésives importantes sur des surfaces biotiques et abiotiques, permet l'adhésion du biofilm sur les surfaces solides et est requise pour la formation de l'anneau du biofilm. En accord avec ces résultats, nous avons observé que Eps1 n'est présent que dans l'anneau du biofilm. En revanche, l'exopolysaccharide Eps2 dépendant du locus eps2 est un élément essentiel de la matrice du biofilm et est nécessaire pour la formation de la pellicule. Enfin, à l'aide de marqueurs fluorescents, nous avons montré que deux souches mutantes capables de ne produire, respectivement, que Eps1 ou Eps2, se distribuent de façon hétérogène dans le biofilm lorsqu'elles sont mises en co-culture. En effet, la souche ne produisant que Eps1 est localisée dans l'anneau tandis que la souche ne produisant que Eps2 est localisée dans la pellicule. L'étude de la régulation de la transcription des loci eps1 et eps2 montre que ces deux loci sont régulés de façon identique. Le répresseur SinR, qui contrôle la formation de la composante protéique de la matrice du biofilm chez B. thuringiensis, n'a aucun effet sur la transcription de eps1 et eps2 chez cette bactérie. En revanche, cette transcription est activée par Spo0A et réprimée par AbrB. Enfin, le régulateur CodY réprime l’expression de ces loci en phase exponentielle, mais stimule cette expression en phase stationnaire tardive. Nos résultats montrent également un rétrocontrôle négatif d’Eps2 sur la production d’Eps1, suggérant l'existence d'une bascule ne permettant la production, au niveau d'une cellule isolée, que d'un seul de ces exopolysaccharides
Exopolysaccharides - polymers of exported sugars - are involved in essential functions of bacterial physiology. Exopolysaccharides are in fact major components, of the bacterial wall, secondary polymers attached to this wall, the capsules, and finally the biofilm’s matrix. Bacillus thuringiensis is an entomopathogenic bacterium of the cereus group, capable of forming a biofilm at the air-liquid interface. This biofilm has two distinct structures: a floating pellicle on the culture medium and, at the periphery, in continuity with the pellicle, a ring adhering to the solid surfaces. To identify the exopolysaccharides, which constitute the biofilm matrix in B. thuringiensis, I investigated the various chromosomal loci in the sequenced genome of B. thuringiensis strain 407. Two loci have been identified and were called eps1 and eps2. To date, no role in the formation of biofilms in B. thuringiensis had been attributed to eps1 locus. Our data showed that the exopolysaccharide Eps1, depending on the eps1 locus, forms a capsule in the stationary phase and in hypoxic conditions. This capsule, which has significant adhesive properties on biotic and abiotic surfaces, allows adhesion of the biofilm to the solid surfaces, thus forming of the biofilm ring. Consistently with these results, we observed that Eps1 is present only in the biofilm ring. We found that Eps2 exopolysaccharide depending on the eps2 locus is an essential element of the biofilm matrix and is necessary for the formation of the pellicle. We have shown using fluorescent markers that two mutant strains capable of producing only type of exopolysaccharides Eps1 or Eps2 are distributed heterogeneously in the biofilm when they are cocultured. The mutant strain producing only Eps1 is localized in the ring while the mutant strain producing only Eps2 is located in the pellicle. Our data show that the transcription of eps1 and eps2 loci is regulated identically by the same set of regulators. The SinR repressor, which controls the formation of the protein component of the biofilm’s matrix in B. thuringiensis, has no effect on the transcription of eps1 and eps2 in this bacterium. The transcription is activated by Spo0A and repressed by AbrB. The CodY regulator represses the expression of these loci in exponential phase, but activates it in the late stationary phase. Our results also show a negative feedback from Eps2 on the production of Eps1, suggesting the existence of a switch, allowing only one of these exopolysaccharides to be produced in an isolated cell

Orientador: Mirna Lucia Gigante
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O objetivo geral deste trabalho foi avaliar o efeito de diferentes matrizes na adesão e formação de biofilme em aço inoxidável por Bacillus cereus, bem como avaliar a eficiência dos procedimentos de higienização no controle de biofilmes de esporos desse micro-organismo. Nas duas primeiras etapas, avaliou-se a capacidade de adesão e formação de biofilme por B. cereus em aço inoxidável, com e sem prévio condicionamento da superfície, utilizando-se água, leite UHT desnatado e integral como matrizes e quatro diferentes tipos de inóculos, pool de células vegetativas de B. cereus isolados da indústria láctea, pool de esporos de B. cereus isolados da indústria láctea, células vegetativas da cepa de B. cereus ATCC 14579 e esporos da cepa de B. cereus ATCC 14579. Na terceira etapa do trabalho avaliou-se a influência da matriz condicionante (água e leite UHT integral), do meio de inoculação do pool de esporos de B. cereus (água e leite UHT integral) e do tempo de exposição (5 min (0,08h), 10, 24, 48 e 72 horas) sobre a adesão e formação de biofilme por B. cereus em aço inoxidável. Na quarta etapa, avaliou-se a eficiência de nove procedimentos de higienização na remoção dos biofilmes formados pelo pool de esporos de B. cereus em aço inoxidável. Todos os experimentos foram repetidos três vezes e os dados estatisticamente avaliados. A hidrofobicidade e o potencial zeta das superfícies dos esporos também foram avaliados. Os resultados das duas primeiras etapas indicaram que o pool de esporos de B. cereus isolados de indústria láctea apresentou a maior capacidade de adesão e formação de biofilme em aço inoxidável quando comparado aos outros tipos de inóculos, em todas as condições avaliadas. O maior grau de adesão de esporos de B. cereus (4,93 log UFC/cm2) foi observado ao se utilizar leite integral como matriz condicionante do aço inoxidável. Entretanto, comparando-se todas as matrizes, a menor adesão (3,01 log UFC/cm2) foi observada quando o pool de esporos de B cereus foi veiculado no leite integral sem prévio condicionamento da superfície. Na terceira etapa do trabalho observou-se que a adesão e formação de biofilme pelo pool de esporos de B. cereus foi maior quando inoculados em água, independente das matrizes de condicionamento. A adesão de B. cereus aumentou 1,02 e 0,3 log UFC/cm2 ao longo do tempo de exposição, quando o pool de esporos de B. cereus foi inoculado em água e leite integral, respectivamente. O biofilme de esporos veiculados na água apresentou maior resistência aos procedimentos de higienização. A sanitização com hipoclorito de sódio foi mais eficiente na remoção dos biofilmes quando comparada ao ácido peracético. O pool de esporos de B. cereus isolados da indústria láctea foi altamente hidrofóbico e apresentou carga negativa em uma ampla faixa de pH, com ponto isoelétrico de aproximadamente 3,0. Os esporos de B. cereus isolados da indústria láctea apresentaram maior capacidade de adesão ao aço inoxidável quando comparados aos outros inóculos avaliados, o que pode estar relacionado à alta hidrofobicidade e a baixa carga de superfície dos esporos
Abstract: The aim of this study was to evaluate the effect of different matrices on the adhesion and biofilm formation by Bacillus cereus on stainless steel, and to evaluate the effectiveness of sanitation procedures for controlling biofilm from spores of this microorganism. The first two parts were carried out in order to evaluate the adhesion and biofilm formation by B. cereus on stainless steel, with and without previous conditioning of the surface, using water, skim and whole UHT milk as matrices and four different types of inocula: a pool of B. cereus vegetative cells isolated from dairy industry, a pool of B. cereus spores isolated from dairy industry, vegetative cells of B. cereus ATCC 14579, and spores of B. cereus ATCC 14579. The third part of the study evaluated the effect of the conditioning matrix (water and whole UHT milk), the inoculation medium of pool of B. cereus spores (water and whole UHT milk) and exposure time (5 min (0.08h), 10, 24, 48 and 72 hours) on the adhesion and biofilm formation by B. cereus on stainless steel. In the fourth part, the effect of nine sanitation procedures on the removal of B. cereus spores biofilm was evaluated. All experiments were repeated three times and data were statistically evaluated. Hydrophobicity and zeta potential from spore¿s surface were also evaluated. Regarding the results to the first and second parts, the pool of B. cereus spores isolated from dairy industry had the highest ability of adhesion on stainless steel when compared to the other inocula, for all tested conditions. After stainless steel surface conditioning with whole milk, B. cereus spores showed the highest adhesion (4.93 log CFU/cm2). However, lower adhesion (3.01 log CFU/cm2) was observed when B. cereus spores were delivered in whole milk as compared to the other matrices, without previous conditioning of the surface. The results of the third part indicated that the adhesion and biofilm formation by the pool of B. cereus spores was higher when they were inoculated in water, regardless of the conditioning matrix. B. cereus spores adhesion increased by 1.02 and 0.3 log CFU/cm2 over exposure time, when the pool of B. cereus spores was inoculated into water and whole milk, respectively. Biofilm of B. cereus spores inoculated in water showed the highest resistance against all tested sanitation procedures. Sodium hypochlorite was the most effective sanitizer for removing all biofilms when compared to the peracetic acid. The pool of B. cereus spores isolated from dairy industry was highly hydrophobic and showed a negative charge at a wide pH range, with an isoelectric point of about 3.0. B. cereus spores isolated from dairy industry showed the highest ability to adhere on stainless steel when compared to the other inocula, which is possibly related to its higher hydrophobicity and lower spore surface charge
Doutorado
Tecnologia de Alimentos
Doutora em Tecnologia de Alimentos

The Bacillus cereus group of bacteria comprises B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B. weihenstephanensis. Species status has been allocated to these taxa largely according to pathogenic properties. B. anthracis is the causative agent of anthrax in ungulates and humans. B. thuringiensis is primarily an insect pathogen and B. cereus is associated with food poisoning and occasionally soft tissue infections in humans. One hundred and forty-six strains of the B. cereus group were examined by multilocus sequence typing (MLST) in which partial sequences for seven housekeeping genes (glpF, gmk, ilvD, pta, pur, pycA and tpi) were generated to provide a definitive sequence type (ST) for each strain. Statistical analyses of the data using pairwise comparisons between groups for (i) Fst (gene flow), (ii) shared mutations and (iii) fixed differences confirmed that the present designation of separate species status for members of the B. cereus group was inappropriate. Comparison of neighbour joining (NJ) trees derived from the concatenated sequence data with trees constructed for each allele individually indicated limited recombination between strains and a largely clonal structure to the group. Three major clades were recovered: clade 1 was made up of B. anthracis, B. cereus and rare B. ringiensis strains; clade 2 comprised a heterogeneous mixture of B. thuringiensis and B. cereus strains while clade 3 was composed of strains of B. cereus, B. mycoides and B. weihenstephanensis. Two B. pseudomycoides strains were distant outliers from the main tree. Four lineages were recognised in both clades 1 and 2 based on shared mutations within the lineages and fixed differences between them. B. anthracis strains and the emetic toxin-producing strains of B. cereus formed two clones within clade 1. A clonal group of entomopathogenic B. thuringiensis strains was identified in clade 2 and named the ‘Sotto’ lineage (after the predicted founder group). Strains of B. cereus that had been isolated from human wound infections and septicaemia, on the other hand, were distributed over clades 1 and 2, and were not restricted to a particular clonal group. Similarly, some serotypes of B. thuringiensis were found to have a clonal structure while others were heterogeneous. Representative strains from several serotypes of B. thuringiensis were examined by the RAPD (random amplified polymorphic DNA) method. Serovars israelensis and thuringiensis were strongly clonal, morrisoni and tolworthi were partially clonal while darmstadiensis and canadensis were heterogeneous. Serotype, MLST profile and RAPD did not always correlate with delta-endotoxin cry gene content. This may be due to the cry genes being located on plasmids and subject to transfer between strains. MLST does not support the separate species status of B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B. weihenstephanensis and an alternative classification based on DNA sequence data is proposed based on three main clades with nine distinct lineages. The proposed lineages were named to be consistent with current nomenclature, as far as possible.

Bacillus cereus est une bactérie largement disséminée dans la nature, contaminant ainsi les aliments en contact avec le sol. En France, cette bactérie est considérée comme le quatrième agent de toxi infection alimentaire collective. Pour être pathogène, B. cereus doit être capable de se multiplier lors des différentes étapes de transformation et notamment au cours de la réfrigération. Le but de cette étude a été d'étudier les mécanismes moléculaires de la réponse adaptative au froid et en particulier le rôle des hélicases à ARN de B. cereus ATCC 14579. Le gène cshA, codant pour une hélicase à ARN putative, a été identifié par une approche de mutagénèse aléatoire, comme jouant un important dans l’adaptation au froid de B. cereus. La souche ATCC 14579 possède 5 gènes codant pour des hélicases à ARN, cshA à cshE qui sont tous fortement surexprimés à 10°C par rapport à 37°C et quel que soit le stade de croissance considéré. La délétion simple des gènes cshA, cshB et cshC conduit à l’apparition de phénotypes cryosensibles, se traduisant par une incapacité d'adaptation au froid par rapport à la souche sauvage, associée à une modification de la morphologie cellulaire. De plus, CshA, CshB et CshC possèdent chacune un domaine de température où leur action est prépondérante. Elles semblent également être impliquées dans l’adaptation au stress oxydant et au stress basique, alors que CshD et E n’ont pas de rôle dans l’adaptation aux stress testés. Nous avons montré que CshA est indispensable à basse température, pour permettre le maintien de la stabilité des ribosomes avec lesquels elle interagit directement, mais aussi pour réguler la dégradation des ARNr. L’identification des partenaires protéiques interagissant avec CshA suggérent qu'elle puisse être également impliquée dans un complexe de dégradation des ARN
Bacillus cereus is a widespread bacteria, thus contaminating all raw materials in contact with soil. In France, B. cereus is considered as the fourth causative agent of foodborne illness. To be pathogenic, B. cereus should multiply during the various stages of food processing and particularly during preservation at low temperature. The aim of this study was to study molecular mechanisms of the adaptive response at low temperature and more precisely the involvement of the B. cereus ATCC 14579 RNA helicases. The cshA gene encoding a putative RNA helicase was identified by a random mutagenesis approach, as playing a major role in cold adaptation of B. cereus. The ATCC 14579 strain possesses 5 genes encoding putative RNA helicases, cshA to cshE, which were all strongly overexpressed at 10°C versus 37°C, whatever the growth stage. The simple deletion of cshA, cshB, and cshC lead to a cold-sensitive phenotype, resulting in an inability to adapt at 10 °C compared to the wild type strain, associated to a huge modification of cell morphology. In addition, CshA, CshB and CshC have a temperature range where their action is decisive. The role of these three RNA helicases also appears to be important in adaptation to oxidative and basic stresses while CshD and E did not appear to be involved in the adaptation to the tested stresses. The RNA helicase CshA has the most important role in adaptation to cold. We demonstrated that CshA is essential at low temperature to allow the maintenance of ribosome stability. CshA interacts directly with ribosomes, and also regulate rRNA degradation. The identification of protein partners that interact with CshA suggests that it could be involve in a complex of RNA decay

This thesis concentrates on the Bacillus cereus group of organisms and interactions that they may encounter in their natural environment. Inorganic polyphosphate has been identified as an important factor of stress and survival in B. cereus. One of the aims of this project was to create knock out mutants of certain enzymes involved in polyphosphate metabolism in B. anthracis, the etiological agent of anthrax. Unfortunately, even though B. anthracis is very closely related to B. cereus and despite the application of published methods it was not possible to create these B. anthracis knockout mutants. In order to address the importance of inorganic polyphosphate in B. anthracis, a real time RT‐PCR assay was developed to monitor the mRNA levels of these enzymes when the bacterium is faced with harsh nutrient environments Real time RT‐PCR analysis showed that mRNA levels of the metabolizing enzymes were upregulated in low nutrient conditions but that the profiles of gene expression were varied when grown in a chemically defined media. In addition to abiotic stresses such as low nutrients, B. anthracis is also likely to face biotic stress such as predation by amoeba in the soil. Investigations were performed into the outcome of the interaction of B. cereus group bacteria with a model amoeba, Acanthamoeba polyphaga. Amoebae are bacterial predators but can also be utilised as hosts by bacterial symbionts and pathogens, such as Legionella pneumophila. It was theorised that amoebae may provide a host environment similar to that of the professional macrophages, which B. anthracis encounters in mammalian infection. These investigations confirmed that the B. cereus group bacteria demonstrate a range of interactions with amoeba cells, from surface attachment through to intracellular persistence. These studies went on to show that B. cereus, B. thuringiensis and B. anthracis can all be engulfed by amoebae when challenged in their vegetative form and that spores were able to survive, and apparently germinate. Finally these studies have identified a new developmental stage of the B. cereus group bacteria. When grown in static conditions, especially in the presence of amoeba, the bacterial cells cease to septate and large (often motile) continuous hyphae like filaments form. These filaments can be seen to “weave” together to form large “rope” like macrofibre structures which can even become visible by eye. Previously this macrofibre growth has also been seen in B. subtilis, suggesting it may be common to the whole genus. In the light of these findings we speculate that this group of pathogens have evolved complex behaviours to interact with soil amoeba in order to facilitate survival in harsh environmental conditions.

Orientador: Telma Teixeira Franco
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Este trabalho investigou a produção, extração e purificação da enzlma quitosanase. Planejamento fatorial fracionado 25-1 foi utilizado para obtenção de maiores níveis de quitosanase, avaliando o efeito das seguintes variáveis: concentração de quitosana, concentração de sulfato de amônio, pH, aeração e tempo de fermentação. As variáveis significativas para a produção da enzima quitosanase foram a concentração de sulfato de amônio, aeração, pH e as interações entre concentração de sulfato de amônio e aeração. A atividade máxima (l,5U/mL) foi alcançada em meio contendo 2,0% de quito sana, 4,0% de sulfato de amônio, aeração 10 (relação entre o volume do Erlenmeyer e o volume de meio de cultura), pH 5,8, 30°C em 16 horas de fermentação. Purificação primária da enzima quitosanase foi desenvolvida por partição em Sistemas de Duas Fases Aquosas (SDFA). Cinco composições de SDFA foram investigadas, sendo o melhor sistema para separação constituído de 22% PEG 1500 e 10% fosfato. Neste SDFA a enzima quitosanase foi recuperada na fase inferior (91%), obtendo-se um fator de purificação de 5,2. Purificação secundária da enzima quitosanase foi desenvolvida por cromatografia preparativa de troca iônica (CTI) em resina S-Sepharose (catiônica). A extração e purificação nas duas etapas (SDFA e CTI) resultaram em fator de purificação de 20 vezes e 66% de recuperação de quitosanase. Este processo de purificação desenvolvido é potencialmente interessante para a ampliação de escala, alcançando elevada recuperação em apenas duas etapas. A massa molecular da enzima quitosanase purificada (47 kDa) foi determinada por eletroforese em gel de poliacrilamida (SDS) e o ponto isoelétrico (8,8) foi determinado por focalização isoelétrica.
Abstract: A culture media for a wild strain of Baci/lus cereus was studied with regard to chitosanase production by means of experimental fractional fatorial design 25-1. The factors which were investigated were the chitosan concentration, the pH, the ammonium sulfate concentration, the aeration and the fennentation time. The factors having the strongest influence on chitosanase production were the ammonium sulfate concentration, aeration, pH and the interaction of the first two parameters. Optimal conditions for chitosan production (1.5 U/rnL) were in culture media containing 2.0% of chitosan, 4.0% of ammonium sulfate and with aeration 10 (Erlenmeyer flask volume and culture media volume ratio) at pH 5.8 at 30° for 16 hours. The enzyme was partially purified by partitioning in aqueous two-phase system (ATPS). Five different ATPS compositions were investigated for enzyme recovery and purity. The best system was made with 22% PEG 1,500, 10% phosphate, where the chitosanase was mainly collected in the botton phase (91% recovery and 5.2 fold the purification factor). A second-step purification was achieved by cation-exchange chromatography with S-Sepharose and salt gradient. The complete two-steps purification process developed achieved 66% chitosanase recovery and a 20 fold increase of the purification factor The apparent molecular weight of the chitosanase detennined by SDS-P AGE electrophoresis was 47 kDa and the observed isoelectric point was 8.8.
Mestrado
Mestre em Tecnologia de Alimentos

Bacillus cereus est une bactérie connue pour être le deuxième agent responsable de Toxi-Infections Alimentaires Collectives (TIAC) en France depuis 2012. Plusieurs cas d'infections locales et systémiques à B. cereus ont également été signalés. Par sa capacité à former des biofilms et à sporuler, B. cereus pose de vrais problèmes en agroalimentaire et en santé publique en résistant aux procédures de nettoyage et désinfection. Il reste néanmoins beaucoup d’interrogations sur les différences de toxicité observées chez les souches de B. cereus, des souches étant inoffensives pour l’Homme alors que d’autres sont mortelles. Or, les industries agroalimentaires et les hôpitaux ont besoin de savoir si une souche retrouvée dans leur environnement présente un danger et nécessite une intervention qui pourra avoir des répercussions économiques et humaines. Pour répondre à ces enjeux, mon travail a consisté à collecter et caractériser 564 souches isolées dans le cadre de TIAC et 56 souches isolées suite à des cas d’infections non-gastro-intestinales dans le but de les comparer avec des souches environnementales de B. cereus non reliées à des infections et identifier ce qui les différencie.A la suite de l’analyse complète des données épidémiologiques et cliniques des souches de B. cereus ainsi que leur typage et leur caractérisation sur un modèle de caractérisation génétique basé sur la détection de dix gènes présumés impliqués dans la virulence, des souches d’intérêts ont été sélectionnées pour faire l’objet d’une étude approfondie de toxicité et de transcriptomique. Cette première partie du travail a également permis d’approfondir les connaissances portant sur les foyers de TIAC causés par B. cereus et aussi de mettre en évidence des cas de contaminations croisées survenues au sein de plusieurs hôpitaux français pouvant conduire au décès du patient.L’étude portant sur la toxicité in vitro des souches sur trois modèles cellulaire eucaryotes a montré des différences significatives entre des B. cereus qui ont causé des infections et ceux non reliés à des infections. L’étude de trancriptomique différentielle a permis d’identifier une liste de marqueurs qui pourraient être utilisés, après validation, pour différencier les souches pathogènes et environnementales considérées comme non pathogènes. À la suite d’un transfert de connaissance et de méthode, ces marqueurs pourront être utilisés par les laboratoires de terrain en agro-alimentaire et les laboratoires d’analyses médicales pour aider à la prise de décision en cas de contamination à B. cereus
Bacillus cereus is the second cause of foodborne outbreaks (FBO) in France since 2012. Several cases of local and systemic infections caused by B. cereus were also reported. By its ability to form biofilms and spores, B. cereus arises real problems in the food industry and public health by resisting to cleaning and disinfection procedures. It remains many questions about the toxicity differences observed among B. cereus strains, several are harmless to humans while others can cause death. But the food industry and hospitals need to know if a strain found in their environment is unsafe and requires intervention that can have an economic and human impact. Face to these challenges, my work was to collect and characterize 564 strains isolated from FBO and 56 strains isolated from patients following cases of non-gastrointestinal infections in order to compare them with environmental strains of B. cereus and identify what differentiates them to others.By a full analysis of epidemiological and clinical data of B. cereus strains and their molecular typing and characterization by a genetic model based on the detection of ten genes potentially involved in virulence, interest strains have been selected to deal with toxicity and transcriptomic in depth. This first part has also allowed increasing knowledge about FBO caused by B. cereus and also to highlight cross-contaminations occurred in several French hospitals leading to death.The in vitro toxicity studies performed on three eukaryotic cell models showed significant differences between toxicity levels of B. cereus strains that have caused infections and those no linked to infections. The differential trancriptomic study has allowed identifying a list of markers that could be used, after validation, for differentiating pathogenic strains from those considering as non-pathogenic. Following the transfer of knowledge and methods, these markers could be used by food safety laboratories and medical laboratories to be a decision aid in case of B. cereus contamination

Le niveau initial de contamination des deux types de produits de Vème gamme à base de légumes par Bacillus cereus est inférieur a 100 ufc. G 1 et b. Cereus n'est pas détecté dans les denrées stockées a 4°C, même au delà de leur dlc. Les tests de confirmation et d'identification des isolats montrent que le groupe b. Cereus est un groupe hétérogène. Nos isolats se répartissent en 7 profils biochimiques. Les isolats poussant des 5°C (9,7% des isolats testés), sont peu thermorésistants à 90°C et faibles producteurs d'entérotoxines contrairement aux souches ne pouvant pas se multiplier au-dessous de 10°C (63%). Les isolats d'un profil biochimique, incapables de pousser des 10°C et d'hydrolyser l'amidon, sont très thermorésistants et seraient sans doute producteurs de toxine émétique. Quatre-vingt dix-sept virgule cinq pour cents des souches de b. Cereus isolées produisent au moins une entérotoxine et ont une activité cytotoxique d'un niveau variable. Les études de croissance de b. Cereus, dans un milieu courgette, montrent que cette dernière s'y multiplie dès 10°C mais avec une latence environ 20 fois plus longue que dans le milieu riche (jb). L'étude de l'effet de la température sur la croissance de b. Cereus, par le modèle d'Arrhenius, met en évidence deux domaines de températures séparés par une température critique aux environs de 13°C alors qu'il n'en est rien avec les deux autres modèles prédictifs, modèles de Ratkowsky et de Rosso

L’effet de la température et de la composition du milieu en nutriments sur les propriétés des spores (résistance et germination) de B. cereus a été largement étudié contrairement à l'effet de l'anaérobiose. Or, les cellules végétatives de B. cereus peuvent se retrouver dans une grande variété de milieux naturels avec un faible niveau d'oxygène (intestin, sol, lignes de traitement des aliments…) où la sporulation peut avoir lieu. Les spores produites dans ces conditions anaérobies pourraient donc avoir des propriétés particulières. Dans ce travail, un panel de 18 souches de B. cereus appartenant aux groupes phylogénétiques de II à VII a été étudié pour sa capacité à sporuler en anaérobiose dans un milieu de sporulation approprié que nous avons développé (MODS). En anaérobiose, la capacité de sporulation a été plus faible et plus hétérogène qu’en aérobiose. La souche AH187 a produit le niveau de spores le plus important en anaérobiose, elle a donc été choisie pour étudier les propriétés de ces spores. Les spores produites en anaérobiose étaient plus résistantes à la chaleur humide entre 90°C et 100°C, à 1M de NaOH, 1M d'acide nitreux et à la lumière pulsée. Aucune différence dans la résistance à 5 % de peroxyde d'hydrogène ou à 0.25 mM de formaldéhyde, ni aux UV-C, n'a été observée entre les deux conditions. En présence de L- alanine, les spores produites en anaérobiose germaient plus efficacement que celles produites en aérobiose tandis qu’aucune différence dans la germination n’a été observée en présence d'inosine. Aucune différence dans la taille des spores produites dans les deux conditions n’a été observée par microscopie électronique à transmission. Toutefois, les spores obtenues dans des conditions anaérobies avaient un exosporium endommagé ou dans certains cas un exosporium complètement détaché, contrairement aux spores produites dans des conditions aérobies. Afin de comprendre les différences dans la capacité de sporulation de B.cereus entre les 2 conditions, des PCR en temps réel (RT-PCR) ont été utilisées pour étudier l'expression des gènes de l'initiation de la sporulation spo0A, spo0B, spo0F, KinA et kinB. Les cinétiques d'expressions des gènes spo0A, spo0B, spo0F et KinA avaient la même tendance. Ils étaient caractérisés par une expression plus élevée en anaérobiose par rapport à l’aérobiose au début et à la fin de la phase exponentielle de croissance. En outre, l'expression du gène kinB était caractérisée par une augmentation en anaérobiose par rapport à l’aérobiose pour atteindre un pic entre 4 h (milieu de phase exponentielle) et 6 h (début de phase stationnaire) de croissance. Les gènes spo0A, spo0B, spo0F, KinA et kinB sont exprimés de manière différentielle entre l’aérobiose et l’anaérobiose. Ces données pourraient aider à comprendre la différence de capacité de sporulation de B. cereus entre la condition aérobie et anaérobie
The effect of temperature and nutrient composition of the medium on B. cereus spore properties (resistance and germination) has been extensively studied unlike to the effect of anaerobiosis. Nevertheless, B. cereus vegetative cells can be found in a large variety of natural environments with low oxygen level (intestine, soil, food processing line) where sporulation take place. Spores produced in these anaerobic environments could have particular properties. In this work, a panel of B. cereus strains belonging to phylogenetic groups II to VII was studied for their capacity to sporulate in anaerobiosis in an appropriate sporulation medium we developed (MODS). In anaerobiosis, sporulation ability was lower and more heterogeneous than in aerobiosis. The B. cereus AH187 strain produced the highest level of spores in anaerobiosis, it was therefore chosen to study spore properties. Spores produced in anaerobiosis were more resistant to wet heat from 90°C to 100 °C, 1M NaOH, 1M nitrous acid and pulsed light. No difference in resistance to 5 % hydrogen peroxide or 0.25 mM formaldehyde or UV-C was observed between these two conditions. In the presence of L-alanine, spores produced in anaerobiosis germinated more efficiently than spore produced in aerobiosis. No difference in germination was observed with inosine. No difference in the spores size produced in the two conditions was observed by transmission electron microscopy. However, spores obtained under anaerobic conditions had a damaged exosporium, or in some cases a completely detached exosporium, unlike spores produced under aerobic conditions. To understand differences in sporulation ability between both conditions, Real-time reverse transcription-PCR was used to study the expression the expression of sporulation initiation genes spo0A, spo0B, spo0F, kinA and kinB. The kinetics of gene expression spo0A, spo0B, spo0F and kinA had the same trend. They were characterized by a higher expression in anaerobiosis compared to aerobiosis at the beginning and the end of exponential growth phase. Furthermore, kinB gene expression was characterized by an increase in anaerobiosis compared to aerobiosis to achieve a peak between 4 (middle exponential phase) and 6 (early stationary phase) hours of growth. The spo0A, spo0B, spo0F, kinA and kinB genes are differentially expressed between aerobiosis and anaerobiosis. These data may help to understand the difference in B. cereus sporulation capacity between aerobic and anaerobic condition

Bacillus cereus est une bactérie à coloration de Gram positive capable de s'adapter à un grand nombre de stress tels que les bas pH ou les bas potentiels d'oxydo-réduction (Eh). Si certains des mécanismes d'adaptation de B. cereus à chacun de ces stress sont connus, les intéractions entre ces deux facteurs sur la physiologie bactérienne n'ont jamais été étudiés. Nous nous sommes donc intéressés à l'effet des variations de Eh sur la résistance au stress acide de B. cereus, aux adaptations métaboliques de B. cereus à pH acide et à l'intéraction de cette bactérie avec son environnement redox. Les résultats obtenus ont démontré, dans un premier temps, que la survie de B. cereus au choc acide était légèrement augmentée sous atmosphères à Eh réducteurs par rapport aux atmosphères à Eh oxydants. Nous avons également observé une réorientation majeure du métabolisme de B. cereus exposé à pH acide depuis la fermentation des acides mixtes vers la fermentation butanediolique. Cette réorientation joue vraisemblablement un rôle important dans la résistance au stress acide de B. cereus. Dans un second temps, nous avons étudié les intéractions de B. cereus avec son environnement redox et nous avons montré l'importance des groupements thiols exofaciaux dans l'activité réductrice de cette bactérie. L'ensemble de ces résultats permettent de mieux comprendre la physiologie de B. cereus confronté à un stress acide et aux variations de Eh et ouvre la voie à de nombreuses pistes de recherches novatrices
Bacillus cereus is a Gram positive bacterium able to adapt and survive to numerous stress, including acid stress or oxydo-reduction potential (Eh) variations. Some adaptations are documeted for each of these two stress. However, the interaction between Eh and pH on B. cereus physiology was never studied. Here, we focus on the impact of Eh variation on the acid resistance of B. cereus, on the metabolic adaptation of these bacteria under low pH and on the interaction of bacterial cells with their redox environement. Results obtained demonstrate that the acid survival of B. cereus was slighlty higher under reductive Eh than under oxdative Eh. Concerning acid adaptations, we observed a major metabolic adjustement for cells cultivated at low pH with an important shift from mixed acid fermentation to butanediolic fermentation. Finally, we demonstrate the importance of exofacials thiols groups in the reductive abilities of B. cereus. All these conclusions will help to better understand the response of B. cereus exposed to acid stress and Eh

English-style crumpets (pH 6--8, aw 0.97--0.99) are a popular baked product enjoyed by consumers worldwide. However, over the past few years, outbreaks of food poisoning have been caused by the growth of Bacillus cereus in crumpets. This spore forming microorganism, which originates in flour, can easily survive the baking process and grow to >106 CFU/g within 3--5 days at ambient storage temperature. Therefore, control of this pathogen is essential to ensure the safety and marketability of English-style crumpets.
Initial studies were done to determine the effect of water activity ( aw), pH, modified atmosphere packaging (MAP), UV-light, bacteriocins, organic acids and esters, alone and in conjunction with each other, on the growth of B. cereus in model broth/agar systems.
B. cereus is a difficult microorganism to control in food using conventional preservation methods. Further studies are now under way to investigate novel methods to control the growth of this pathogen, particularly in high pH crumpets. (Abstract shortened by UMI.)

Enterotoxinproduction of Bacillus cereus under simulated intestinal conditions Bacillus cereus is an important food-borne pathogen. Enteropathogenic B. cereus produce enterotoxins in the humane intestine after ingestion of contaminated food and cause diarrhea. The most important enterotoxins are the nonhemolytic enterotoxin (Nhe) and the hemolysin BL (HBL) each consisting of three components. The toxic potential of B. cereus depends on the amount of produced enterotoxins and cannot be determined by simple detection of the toxin genes. We analyzed enterotoxin production and the cytotoxic potential of 19 B. cereus strains associated with food and food-borne diseases with different toxin gene profiles and different toxin expression levels both under simulated intestinal and laboratory conditions. The growth rates of all analyzed strains were reduced under simulated intestinal conditions. The highest enterotoxin titers, determined by EIA with specific monoclonal antibodies, were detected after 6 h under laboratory conditions and were strain-dependent. In contrast, the Toxin production efficiency (enterotoxintiter in relation to growth) was highest after 4 h under simulated intestinal conditions. Enhanced toxin production of all strains was observed after 2 h under simulated intestinal conditions in comparison to laboratory conditions and was caused by so far unknown factors secreted by the Caco-2-cells. The cytotoxic activity after 6 h was determined in WST-1-bioassays using Vero- and Caco-2-cells. The cytotoxicity titers were strain-dependent, dependent on enterotoxin titers and reduced under simulated intestinal conditions. In the latter, discrimination between high and low toxic strains was less distinct. PI influx in Caco-2-cells was mainly dependent on HBL, not Nhe. It was also shown that the enterotoxins were secreted completely by all B. cereus strains and that a possible defect in the secretion mechanism is not the reason for the highly variable amounts of enterotoxins. The results of this work show that cultivation under laboratory and simulated intestinal conditions cause differences in growth rate, enterotoxin production and cytotoxicity. So far unknown factors secreted by Caco-2-cells enhance enterotoxin production of B. cereus.

Bacillus cereus est un pathogène opportuniste à l'origine de deux types de toxi-infections alimentaires classées en syndrome émétique ou diarrhéique. Le syndrome diarrhéique résulte de la production d'entérotoxines (Hbl, Nhe et CytK) au niveau de l'intestin grêle de l'hôte, caractérisé par une atmosphère anaérobie et un faible potentiel d'oxydo-réduction (POR). La capacité de B. cereus à se développer et à produire des entérotoxines dans ces conditions est sous le contrôle de deux systèmes qui agissent, en partie, indépendamment du régulateur pléiotrope connu, PlcR (Phospholipase C Regulator). Il s'agit du système à deux composants ResDE et de la protéine Fnr (Fumarate Nitrate Reductase). Le but de cette étude a été de caractériser d'un point de vue fonctionnel l'implication du régulateur Fnr et du système ResDE dans la toxinogenèse de B. cereus. Les résultats ont montré que la régulation de la transcription de hbl et nhe était sous le contrôle direct et indirect de Fnr et de ResD. En aérobiose, la fixation de Fnr (forme Apo) sur les régions promotrices des gènes de structure des entérotoxines (pnhe et phbl) et des gènes de régulation (presDE, pfnr et pplcR) dépend des conditions redox. L'affinité de ResD pour pnhe, phbl, presDE, pfnr et pplcR dépend des séquences de ces régions promotrices et son affinité pour les régions promotrices presDE et pfnr dépend de son état de phosphorylation. ResD et ApoFnr sont capables de se fixer simultanément sur les régions promotrices étudiées et sont également capables d'interagir physiquement en l'absence d'ADN. Nous avons proposé un modèle de régulation de la toxinogenèse dans lequel ResDE et Fnr pourraient agir en synergie. Enfin des expériences de double hybride ont permis de mettre en évidence que la protéine PlcR pourrait interagir in vivo avec les régulateurs ResD et Fnr. La régulation de la toxinogenèse impliquerait donc la formation d'un complexe multi-moléculaire

Bacillus cereus est une bactérie pathogène opportuniste, responsable de deux types d'intoxications alimentaires. L'une causée par une toxine émétique (céreulide) est à l'origine du syndrome émétique, et l'autre causée par trois entérotoxines (Hbl, Nhe et CytK) est à l'origine du syndrome diarrhéique. Ce dernier est du à la production d'entérotoxines au niveau de l'intestin grêle de l'homme, caractérisé par une atmosphère anaérobie et un bas potentiel d'oxydoréduction (POR). L'objectif de cette thèse est de caractériser l'adaptation de B. Cereus et d'évaluer sa toxinogenèse face à un environnement anaérobie et réducteur comme souvent rencontré et impliqué dans la virulence d'autres pathogènes bactériens. Nos résultats ont montrés que la souche diarrhéique F4430/73 de B. Cereus dispose d'un métabolisme fennentaire très efficace qui lui permet de se développer dans des conditions de bas POR. D'autre part, nous avons montré que la production des entérotoxines est dépendante du métabolisme énergétique. Elle est favorisée par des conditions de croissance fennentaire et est d'autant plus importante que la fermentation est conduite à bas POR (POR=-148 mV). La régulation de l'expression des entérotoxines, en réponse à l'anaérobiose, s'effectue principalement au niveau transcriptionnel. Nous avons démontré l'existence de deux régulateurs contrôlant simultanément les voies fermentaires et la toxinogenèse. Le système à deux composants « ResDE » agit comme un senseur redox et la protéine à centre Fe-S « Fnr » agit comme un senseur de la fermentation. Nos résultats suggèrent que le système ResDE et la protéine Fnr appartiennent à une voie de régulation fonctionnant en partie indépendamment du régulateur pléiotrope de virulence PIcR, pour réguler l'expression des gènes des entérotoxines
Bacillus cereus is an opportunistic pathogenic bacteria responsible of two types of food-home diseases one caused by an emetic toxin (cereulide) responsible of the emetic syndrome, and the other caused by three enterotoxins (Hbl, Nhe and CytK) associated with the diarrhoeal syndrome. The diarrhoeal syndrome results from toxin production by B. Cereus in the host small intestine characterized by an anaerobic atmosphere and low oxidoreduction potential (ORP). The objective of this thesis is to characterize the B. Cereus adaptation and to evaluate its toxinogenesis when it encounter anaerobic and reduced environment often met and implied in the virulence of other pathogenic bacteria. Our results showed mat the diarrhoeal strain F4430/73 of B. Cereus has a very efficient fermentative metabolism which enables it to grow under low ORP conditions. Furthermore, we showed that the production of the enterotoxins is energy metabolism dependent. It is supported by fermentative growth conditions and is more important as fermentation is conducted at tow ORP (ORP=-148 mV). The regulation of enterotoxins expression in response to anaerobiosis is carried at the transcriptionnel level. We showed the involvement of two regulators controlling simultaneously the fermentative pathways and toxinogenesis. The two components system "ResDE" acts as redox sensor and the "Fnr" protein carrying the Fe-S cluster acts as fermentation sensor. Our results suggest that both ResDE and Fnr regulators belong to a redox regulatory pathway that at least partially functions independently of the pleiotropic virulence regulator PIcR to regulate enterotoxin gene expression

Bacillus cereus is a Gram-positive, spore-forming bacterium that is frequently identified as the causative agent of food-borne diseases and is also implicated in food spoilage of especially dairy products. The capacity of B. cereus to form biofilms on different substrata is of great concern in the food industry. Not only does biofilm formation cause economic loss by equipment failure, but contamination of food products via biofilm cells also raises safety concerns. Bacterial biofilms have been defined as structmed multicellular communities that form through a complex developmental process. In contrast to Gram-negative bacteria, biofilm formation by Gram-positive bacteria has only recently been examined. Relatively few genes have been identified that are required for these bacteria to form biofilms and little is known about how they coordinate biofilm formation. In order to contribute to the advancement of knowledge regarding the process of biofilm formation in Gram-positive bacteria, the aim of this investigation was essentially to identify and characterize genes involved in B. cereus biofilm formation. To investigate. B. cereus ATCC 14579 was subjected to transposon mutagenesis with the Tn917-LTVI transposon. Screening of a collection of3 500 insertional mutants for the ability to form biofilms at the solid-liquid-air interface of glass surfaces led to the identification of eight biofilm-impaired mutants. Each of the mutants contained a single transposon insertion, and no significant differences were observed in the planktonic growth rate between the B. cereus wild-type and biofilm-impaired mutant strains. The chromosomal transposon insertion in three of the mutants mapped to genes involved in purine biosynthesis (pur A, purC and purL), while the transposon insertion in two other mutants mapped to the ftsE gene and to the promoter region of the motA gene, respectively. In one of the mutants the transposon was located in the intergenic region between two divergently transcribed genes, which encodes a murein hydrolase exporter and nucleoside hydrolase, respectively. In the final two biofilmimpaired mutants the transposon was respectively mapped to genes encoding a putative membrane spanning protein and a putative protein of unknown function. Results obtained by quantitative real-time PCR assays indicated that expression of each of the identified B. cereus ATCC 14579 genes, with the exception of the motA gene, was up-regulated in the biofilm population. In the case of motA, expression of the gene was down-regulated 3.2-fold in the biofilm population and results obtained during the course of this investigation indicated that motility, rather than the presence of flagella, is required for B. cereus biofilm formation. Although this result is in agreement with that reported previously for B. subtilis, none of the other genes identified in this investigation have previously been implicated in biofilm formation by Gram-positive bacteria.
Dissertation (MSc)--University of Pretoria, 2013.
Microbiology and Plant Pathology
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O leite e os produtos lácteos podem se contaminar com Bacillus cereus, um micro-organismo que dependendo do número, da estirpe e das condições de processamento e comercialização, além de produzir lipases e proteases causadoras de off-flavor nos produtos, está associado a duas síndromes distintas que atingem os humanos: uma de natureza diarréica causada pela toxinas não hemolítica nhe, hemolítica hbl e citotoxina cytk e outra emética, causada pela toxina denominada de cereulide ces. Avaliou-se a presença de B. cereus no leite pasteurizado, leite em pó, leite UHT, queijo ricota e sobremesas lácteas, bebidas lácteas e leite achocolatado comercializados supermercados da microrregião de Viçosa, Minas Gerais. nos Também, determinou-se a presença de fatores de virulência de B. cereus nos isolados e avaliou-se a produção de enterotoxina diarreica. A ocorrência de B. cereus em diferentes produtos lácteos foi avaliada utilizando métodos quantitavivo e qualitativo. Das 129 amostras analisadas, foi observada a presença de B. cereus em 69 (53,4%) considerando ambos os métodos. As contagens do micro-organismo variaram de 1,30 log UFC/mL no leite pasteurizado até 5,32 log UFC/g na ricota. A presença do B. cereus foi detectada no leite UHT apenas no teste qualitativo. Os 69 isolados que apresentaram caraterísticas fenotípicas de B. cereus foram confirmados como sendo dessa espécie pela técnica de PCR para o gene 16S, e foram classificados como pertencentes a três estirpes diferentes (B. cereus KAVK4, B. cereus SVK1 e B. cereus ATCC 4342), sendo predominante em todos os produtos analisados a estirpe B. cereus KAVK4. Dos 69 isolados analisados para a presença de genes produtores de enterotoxinas, 68 (98 %) apresentaram o gene nhe. Este gene foi expresso nos 68 isolados com a produção de pelo menos 6 ng/mL da nhe, limite mínimo de detecção desta toxina na metodologia utilizada. Dos 69 isolados confirmados como B. cereus, 40 (57 %) apresentam a amplificação do gene hbl, em 30 (75 %) destes constatou-se a produção de pelo menos 20 ng/mL da toxina hbl, limite de detecção do kit utilizado. A presença do gene cytk foi verificada nos 69 isolados analisados. A ocorrência de pelo menos um gene produtor de enterotoxina foi constatada em todos isolados, o que indicaria um alto potencial patogênico das estirpes presentes nos produtos lácteos.
Milk and dairy products may be contaminated with Bacillus cereus, a micro- organism that depending on the number, of the strain and of the processing and marketing conditions can produce lipases and proteases that cause off-flavor in dairy product and it is associated with two distinct syndromes that affect humans: one diarrhea caused by toxins, not hemolytic nhe, hemolytic hbl and cytotoxin k cytk and another emetic caused by toxin, called cereulide. The aim of this study was to evaluate the presence of B. cereus in milk products marketed in the supermarkets of Viçosa city, Minas Gerais, Brazil. Also, it was determined the presence of virulence factors of B. cereus isolates and it was evaluated the diarrheal enterotoxin production. The occurrence of B. cereus in different dairy products was evaluated using quantitavive and qualitative methods. Of the 129 samples analyzed, it was observed the presence of B. cereus in 69 (53.4%) considering both methods. The microorganism counts vary from 1.30 log UFC/ml in milk pasteurized up to 5.32 log UFC/g in the ricotta. The presence of B. cereus was detected in the UHT milk only in qualitative testing. The 69 isolates with phenotypic characteristics of B. cereus were confirmed as this species by PCR for gene 16S, and they were classified as belonging to three different strains (B. cereus KAVK4, B. cereus SVK1 and B. cereus ATCC 4342), being predominant in all the products analyzed strain B. cereus KAVK4. From the 69 isolates analyzed for the presence of enterotoxin producing genes, 68 (98%) had the nhe gene. This gene was expressed in the 68 isolates with the production of at least 6 ng / ml of nhe, minimum detection limit of the methodology of this toxin. From the 69 isolates confirmed as B. cereus, 40 (57%) showed the amplification of the gene hbl, 30 (75%) of them it was found containing at least 20 ng / mL of the hbl toxin, according kit detection limit .The presence of cytk gene was found in 69 isolates analyzed. The occurrence of at least one enterotoxin producing gene was found in all isolates, which would indicate a high potential pathogenic strains present in dairy products.

Bacillus cereus is the most prevalent pathogenic Bacillus species found in foods, causing food spoilage and two types of toxin-mediated food poisoning known as the diarrhoeal and emetic syndromes. Other Bacillus species, particularly B. subtilis, B. licheniformis, B. brevis, B. pumilus and B. thuringensis, have also been recognised as food poisoning bacteria of increasing concern, with reports of outbreaks of diarrhoeal or emetic food poisoning. This study involved a systematic ecological investigation of Bacillus species isolated from rice products, commonly associated with Bacillus emetic food poisoning, using cultural and molecular methods. A centrifugation-plating method, more sensitive than the conventional spread plating method, was developed and used to determine the occurrence and biodiversity of Bacillus species in rice, a well known source of B. cereus. Eight different Bacillus species, B. cereus/B. thuringiensis, B. mycoides, B. subtilis/B. mojavensis, B. licheniformis, B. pumilus, B. sphaericus/B. fusiformis and B. megaterium, as well as Paenibacillus species, identified by partial rDNA sequencing, were isolated from raw (uncooked) and cooked rice products. The diversity of the isolates at the subspecies (strain) level was investigated using the RAPD-PCR typing technique, which proved to be useful for differentiating strains of bacilli, revealing broad diversity among the strains. Generally, different genotypes were found in raw and cooked rice, with some isolates of the same RAPD pattern found in both raw and cooked rice. The toxigenic potential of Bacillus isolates were also determined by molecular and immunological analysis as well as an MEKC method, developed in this study for quantitative analysis of the emetic toxin, cereulide. The results revealed that most isolates from the B. cereus group were potentially or actually toxigenic and some isolates were able to produce both diarrhoeal and emetic toxins. Other Bacillus species outside the B. cereus group were also shown to produce cereulide.

Lorsqu'un biofilm se développe dans des conditions statiques, deux populations, sessiles et planctoniques, coexistent et peuvent échanger des cellules. Cependant, l'immigration de cellules planctoniques dans un biofilm n'a, jusqu'à présent, fait l'objet que de peu d'études dans le cas d'un biofilm monoespèce. Chez B. cereus, un pathogène alimentaire, notre équipe a récemment montré que des bactéries planctoniques mobiles peuvent pénétrer en profondeur à l'intérieur d'un biofilm formé en immersion. L'objectif du présent travail était, en partant de ces données, de déterminer le rôle du recrutement dans le développement du biofilm formé en interface air-liquide, et de caractériser ce processus d’un point de vue physiologique et moléculaire. Nous avons montré que la population planctonique est massivement recrutée par le biofilm, mais que, dans nos conditions expérimentales, ce recrutement ne contribue que de façon marginale à la croissance du biofilm. Nous avons mis au point deux dispositifs expérimentaux (en interface air-liquide et en immersion) permettant de quantifier le recrutement, ce qui nous a permis de cribler une banque de mutants, obtenue par mutagenèse aléatoire, pour sélectionner des clones inaptes à être recrutés. Le criblage de 1700 clones a abouti à la sélection d'un gène: Bthur002_62720. La délétion de ce gène par échange allélique affecte fortement la capacité du mutant à être recruté, et la complémentation rétablit le phénotype sauvage. Ce gène code pour une protéine probablement localisée dans l'enveloppe bactérienne. Il est porté par un plasmide, pCT8513, et pourrait être un élément mobile dont l'acquisition augmenterait fortement la capacité de la bactérie réceptrice à être recrutée par un biofilm. Enfin, nous avons mis en évidence le rôle du locus eps dans le recrutement des cellules planctoniques par un biofilm formé en interface air-liquide. Ce locus est homologue du locus epsA-O de Bacillus subtilis, requis chez cette espèce pour la production des exopolysaccharides de la matrice du biofilm. Chez B. cereus, nous avons montré que le locus eps est impliqué dans la formation d’une gaine d’exopolysaccharides faiblement liée à la paroi bactérienne. Cette couche d'exopolysaccharides contribue, avec d'autres exopolysaccharides d'origine inconnue, à la formation de la matrice du biofilm, et joue un rôle important dans l'adhésion de la bactérie sur des surfaces inertes et vivantes. En favorisant l'adhésion de la bactérie sur des surfaces vivantes, le locus eps pourrait faciliter son intégration dans le biofilm. Il pourrait également être impliqué dans le pouvoir pathogène de la bactérie
When biofilm is developing in static conditions, cell exchanges between sessile and planktonic coexisting population can emerge. Up to now, very few are known about the implication of planktonic cells integration in monospecies biofilm development. in B. cereus, a foodborne pathogen, our team have shown that motility is a key factor for biofilm development and for the deep penetration of motile planktonic bacteria inside a biofilm formed in immersed condition. Based on these data, the purpose of the present work was to determine the role of recruitment in the development of biofilm in air-liquid interface and to characterize this phenomenon physiologically and in a molecular aspect. We showed that a massive planktonic population is integrated in the developing biofilm, however, in our experimental conditions this recruitment contributes only marginally to the biofilm growth. We have developed two recruitment systems (in air-liquid interface and in immersion condition), to quantify recruitment, which has allowed us to screen a library of mutants obtained by random mutagenesis in order to select clones unable to be recruited by a preformed biofilm. Screening of 1700 clones resulted in the selection of a gene: Bthur002_62720. The deletion of this gene by allelic exchange strongly affects the ability of the mutant to be recruited, and complementation restored the wild type phenotype. This gene encodes a protein probably localized in the bacterial envelope. It is carried by a plasmid, pCT8513, and could be a mobile element whose acquisition would greatly increase the ability of the recipient bacterium to be recruited by a biofilm. Finally, we have highlighted the role of the eps locus in the recruitment of planktonic cells in a biofilm formed at air-liquid interface. This locus is homologous to Bacillus subtilis epsA-O locus, required in this species for the production of exopolysaccharides of the biofilm matrix. In B. cereus, we have shown that the eps locus is involved in the formation of an exopolysaccharides sheath weakly bound to the bacterial cell wall. This exopolysaccharides layer contributes with other exopolysaccharides of unknown origin, to the formation of the biofilm matrix, and plays an important role in the adhesion of bacteria on inanimate and living surfaces.By promoting bacterial adhesion on living surfaces; the eps locus could help bacteria integration into the biofilm. It could also be involved in the pathogenicity of bacteria

Bacillus cereus causes emesis and/or diarrhea following ingestion of contaminated food due to the production of emetic toxins and enterotoxins. SYBR Green I is used as an intercalating dye and its florescence increases as a result of DNA amplification during real-time PCR. A second-derivative plot is obtained at the end of the PCR run, where amplicons are differentiated based on their DNA melting temperature (Tm). DNA was extracted from Tryptic Soy Broth (TSB) and 2.5% Nonfat Dry Milk (NFDM)-grown B. cereus at cell densities of 10',106,105,104,0,102, and 101 cfu/ml. In order to detect the multiple virulence determinants in pathogenic B. cereus, specific primers were used to target three enterotoxigenic genes (hblC, nheA, and hblA), followed by melt-curve analysis to confirm identity. Conditions used for this experiment allowed for the reproducible distinction of melt curves (characteristic Tm) for each amplicon (hblC = 74.5°C in TSB and 75°C in NFDM; nheA = 78°C; and hblA = 85.5°C in TSB and 84°C in NFDM) with an assay sensitivity of 106 CFU/ml in TSB and 10' CFU/ml in NFDM. B. cereus, nheA expression was examined in cells grown in TSB using transcript-specific, real-time nucleic acid sequence-based amplification (NASBA) with SYBR Green II. NASBA was applied to ascertain relative levels of nheA expression, when cells were subjected to subinhibitory levels of chloramphenicol as a stressor. B. cereus demonstrated consistently high levels of nheA expression at 15 hours when grown in TSB containing subinhibitory concentration (SIC) chloramphenicol (15.625 mg/ml). Relative levels of nheA expression differed in stressed B. cereus cells grown during the 30 hours incubation.
Department of Biology

Dissertação de Mestrado Integrado em Medicina Veterinária
O Bacillus cereus sensu stricto é um patogénio alimentar que tem adquirido uma importância crescente nos últimos anos. Sendo uma bactéria com distribuição ubiquitária na natureza pode contaminar várias matérias-primas e alimentos, o que associado à sua capacidade de formação de esporos resistentes a condições severas faz com que possa permanecer viável após a confecção dos alimentos. Este microrganismo é reconhecido desde o início do século XX como agente de toxinfecção alimentar devido à sua capacidade de causar um síndrome diarreico, mas actualmente sabe-se que este agente possui também a capacidade de causar um síndrome emético, associado a uma toxina termostável pré-formada nos alimentos: a cereulida. Pensa-se que a incidência de toxinfecção alimentar por B. cereus sensu stricto seja subestimada, uma vez que não é uma doença de declaração obrigatória e mimetiza outros agentes, contudo nos últimos anos têm sido descritos casos graves de toxinfecção alimentar causados por esta bactéria, com hospitalizações e mortes. O objectivo deste estudo foi pesquisar a presença do gene que codifica a cereulida sintetase numa amostragem de bactérias do grupo B. cereus isoladas de alimentos e de vários pontos do país. Foram encontradas por PCR em tempo real 7 estirpes positivas num total de 106: 3 estirpes alimentares e 4 ambientais, tendo as estirpes positivas sido confirmadas presuntivamente como sendo B. cereus sensu stricto. A detecção de estirpes portadoras do gene da cereulida sintetase vem demonstrar a possibilidade de toxinfecção alimentar por estirpes eméticas B. cereus sensu stricto em Portugal. Os dados deste estudo permitem propor a adopção do PCR em tempo real como metodologia de rotina para pesquisa de estirpes eméticas de B. cereus sensu stricto, já que esta é uma técnica fiável que permite uma rápida obtenção de resultados. Uma vez que esta técnica permite também a fácil distinção em relação ao Staphylococcus aureus pode contribuir para a elucidação sobre qual a verdadeira incidência de toxinfecção por estirpes eméticas de B. cereus sensu stricto.
ABSTRACT - Bacillus cereus sensu stricto is a food pathogen which has grown in importance over the last few years. As it is a bacterium with ubiquitary distribution in nature a it can contaminate a variety of processed and raw foods. Since it has the ability to produce spores resistant to extreme conditions, it remains viable through food processing. This microorganism has been recognized as a food pathogen since early in the XXth century for its ability to cause diarrheic syndrome, but it’s now know that it can also cause emetic syndrome, due to the production of a heat stable toxin in food: cereulide. The incidence of B. cereus sensu stricto food-poisoning is thought to be underestimated since it is not a reportable disease and mimetizes other food pathogens, but even so over the last few years severe cases have occasionally been reported involving hospitalization or even deaths. The objective of this study was to investigate the presence of the cereulide synthetase gene in a sample of 106 strains of bacteria of the B. cereus group isolated from different foods and from several spots in Portugal. Using real time PCR 7 positive strains were found: 3 strains from food and 4 environmental, which were presumptively confirmed as B. cereus sensu stricto. The detection of strains carrying the cereulide synthetase gene shows that there is a possibility of food poisoning by emetic strains of B. cereus sensu stricto in Portugal. The data from this study allows to propose real time PCR as a routine methodology for identification of emetic strains of B. cereus sensu stricto, since this is a reliable technique with quick results. As this technique allows easy distinction from Staphylococcus aureus it may contribute to determining the true incidence of the emetic type of food poisoning caused by B. cereus sensu stricto.

O presente trabalho teve como principal objetivo avaliar a produção de enzimas proteolíticas por Bacillus cereus. As fermentações foram conduzidas em Biorreator Biodesign, com aeração de 1vvm, temperatura de 37°C e agitação de 400 r.p.m. como parâmetros fixos. Dois meios diferentes foram utilizados, Meio Referência (MR) e Meio de Proteína de Soja PS60® (MPS1), em experimentos realizados com 24 horas. Para avaliação da produção de complexos enzimáticos, retirou-se amostras a intervalos de 3 horas para análise de valores de pH, densidade ótica e massa seca. A atividade proteolítica, concentração de proteína solúvel e açúcares redutores, contagem de células viáveis totais e esporos também foram investigadas. Os resultados demonstraram que ambos os meios propiciaram condições para o ótimo desenvolvimento do B. cereus. Os resultados também indicam que, embora os dois meios tenham apresentado um crescimento celular semelhante, o meio composto por proteína de soja propiciou a obtenção de um extrato bruto com atividade proteolítica mais elevada. Nas condições de fermentação empregadas, o meio MPS1 apresentou em 15 horas uma atividade enzimática de 18,7UmL-1.h-1.enquanto que o meio MR apresentou uma atividade proteolítica de 11,8 UmL-1.h-1.

Bacillus cereus est une bactérie très largement disséminée dans l'environnement. Certaines souches sont à l'origine de toxi-infections alimentaires de type émétique ou diarrhéique. Afin de coloniser l'intestin et induire un syndrome diarrhéique, B. cereus doit s'adapter aux variations d'oxygénation et de potentiel d'oxydoréduction rencontrées, se multiplier et sécréter des toxines. En comparant les capacités métaboliques et les capacités de sécrétion de l'entérotoxine Nhe de quatre souches de B. cereus, nous avons mis en évidence que la L-lactate déshydrogénase A (LdhA) était quelles que soient les conditions de croissance impliquée à la fois dans le métabolisme fermentaire et dans la toxinogénèse de B. cereus. LdhA contribue au maintien du ratio NAD+/NADH intracellulaire et son absence affecte les capacités d'expression de plusieurs gènes d'entérotoxines en anaérobiose et en aérobiose chez la souche F4430/73. Afin de déterminer si l'effet observé sur les toxines était indirect et dépendant du régulateur rédox Rex, un mutant ne synthétisant plus Rex a été construit. La caractérisation de ce mutant a mis en évidence le rôle de Rex dans le métabolisme de B. cereus, dans la réponse au stress oxydant et dans la toxinogénèse. De part sa structure et sa capacité a lié l'ADN, Rex pourrait être un facteur de transcription contrôlant l'expression des gènes codant les entérotoxines. Sa capacité de fixation sur l'ADN est dépendante du ratio NAD+/NADH. Afin de déterminer si LdhA pouvait réguler directement l'expression des gènes des toxines, ldhA a été exprimé chez E. coli et purifiée sous la forme d'une protéine fusion. Les premières expériences de retard sur gel n'ont pas permis de mettre en évidence de fixation sur les régions promotrices de toxines
Bacillus cereus is a widespread bacteria. Some strains are responsible of food-borne classified as emetic and diarrhoel syndromes. To colonize such environment and induce diarrhea, B. cereus must be able to grow in the various oxidoreduction potential and oxygenation conditions encountered in the gastrointestinal tract and to secrete toxins. By comparing the catabolic capacities of four strains of the B. cereus group grown under anoxic and oxic conditions in relation to their capacities for expressing Non hemolytoc enterotoxin (Nhe), we identified Lactate dehydrogenase A (LdhA) as both involved in the fermentative metabolism of B. cereus and toxinogenesis. We showed that disruption of ldhA affected the fermentative capacity of anaerobically grown F4430/73 cells and the expression of several enterotoxin genes under both anaerobiosis and aerobiosis. To determine whether the observed effect in the toxins expression was indirect and dependant of Rex, a rex mutant was built. The characterization of this mutant revealed the role of Rex in the metabolism of B. cereus, oxidative stress and toxins production. Rex could be a transcription factor controlling the expression of genes encoding enterotoxins. Ability of DNA binding is dependant on the ratio of NAD+/NADH. To determine whether LdhA could directly regulate the expression of toxins genes, LdhA was expressed in E. coli and purified as a fusion protein. The first gel retardation experiments have failed to demonstrate fixation of LdhA in the promoter regions of toxins

Bacillus cereus is primarily a soil-dwelling bacterium, however it is also pathogenic bacterium, which is implicated in many food poisoning outbreaks throughout the world. It is responsible for both non-gastrointestinal and gastrointestinal infection with the former leading to many working days lost. Two types of infection exist, the emetic Infection and the diarrhoeal infection. The emetic infection is caused by a heat stable protein, which causes vomiting and stomach cramps. The diarrhoeal infection is caused by several different toxins including haemolysin BL (HBL), non-haemolytic enterotoxin (NHE), B. cereus enterotoxin T (bceT), cytotoxin K (cytK), the recently discovered Hlyll toxin and enterotoxin Fm (entFM). The purpose of this project was to develop a reporter system that could be used to investigate the environmental conditions necessary for the production of Bacillus cereus enterotoxin T (bceT) after human ingestion. A plasmid was constructed that carried the bceT gene, which would ultimately be integrated Into the B. cereus chromosome after manipulation.

Dissertação de Mestrado Integrado em Medicina Veterinária
RESUMO - Assiste-se na atualidade a uma necessidade crescente de desenvolver novos métodos de descontaminação, uma vez que os métodos convencionais são insuficientes para eliminar agentes patogénicos resistentes. O projeto “Descontaminação por Aerossol Gasoso de Partículas Oxidantes” (DRACO), desenvolvido pela Unidade Militar Laboratorial de Defesa Biológica e Química, em parceria com o Centro de Investigação da Academia Militar, tem por objetivos testar e desenvolver abordagens inovadoras para a descontaminação operacional. Neste âmbito pretende testar a eficácia de um novo descontaminante, baseado em nano partículas oxidantes. A tecnologia em teste, designada por dryVHP (formulação sólida de peróxido de hidrogénio vaporizado) foi desenvolvida pela Delox, uma startup inovadora da Faculdade de Ciências da Universidade de Lisboa. Esta tecnologia tem como objetivo permitir a criação de uma nova geração de sistemas de descontaminação com maior compatibilidade para materiais e eletrónica, permitindo também a redução do impacto ambiental e redução dos recursos humanos em cenários de contaminação biológica. Pretende-se que esta tecnologia seja empregue em áreas civis na descontaminação de infraestruturas, mas também na descontaminação de interiores, edifícios e viaturas militares. Deste modo, propomo-nos neste trabalho testar a eficácia deste novo descontaminante, comparando o seu potencial esporicida ao de outros descontaminantes líquidos (peróxido de hidrogénio líquido a 10% e ácido peracético a 0.1%), recorrendo ao uso de esporos de Bacillus cereus, como substituto de Bacillus anthracis. A estirpe de B. cereus NCTC 11143 estudada neste trabalho produz duas frações de esporos, do topo e do fundo, que se diferenciam entre si essencialmente pelas suas propriedades físico-químicas, nomeadamente a nível da hidrofobicidade. As experiências de descontaminação foram efetuadas com estas subpopulações de esporos de B. cereus em separado e testaram-se duas superfícies de plástico diferentes, uma limpa e outra suja com óleo alimentar. O peróxido de hidrogénio líquido a 10% e o ácido peracético a 0.1% demonstraram ação esporicida para as duas frações de esporos de B. cereus tanto quando aplicado em superfícies limpas, como em superfícies sujas. Por outro lado, dryVHP, disperso por vaporização pela Delox demonstrou-se apenas eficaz na descontaminação dos esporos do fundo (hidrofílicos), não exercendo ação esporicida nos esporos do topo (hidrofóbicos). Pensa-se que a diferente hidrofobicidade das duas frações de esporos esteja relacionada com as suas resistências diferenciais aos descontaminantes testados. O presente estudo permitiu destacar que o modo de aplicação do descontaminante é um fator condicionante do mecanismo de ação das substâncias ativas oxidantes, e contribuiu para aumentar o conhecimento no âmbito da descontaminação de esporos de B. cereus.
ABSTRACT - Nowadays, there is a growing need to develop new decontamination methods since conventional ones are insufficient to eliminate resistant pathogens. The "Decontamination by Aerosol Gas Decontamination of Oxidizing Particles" (DRACO) project, developed by the Biological Defense and Chemical Laboratory Unit in partnership with the Military Academy Research Center, aims to test and develop innovative approaches for operational decontamination. In this context, the main goal is to test the effectiveness of a new decontaminant, based on oxidizing nanoparticles. The decontaminant technology under test is called dryVHP (a solid formulation of vaporized hydrogen peroxide) and was developed by Delox, an innovative start-up from the Faculty of Science of the University of Lisbon. This technology aims to provide the creation of a new generation of decontamination systems with greater compatibility for materials and electronics, also allowing the reduction of environmental impact and reduction of human resources in biological contamination scenarios. This technology is intended to be used in civilian areas in the decontamination of infrastructures, but also in the decontamination of interiors, buildings, and military vehicles. Thus, the purpose of this work is to test the effectiveness of this new decontaminant, comparing its sporicidal potential to other liquid decontaminants (liquid hydrogen peroxide at 10% and peracetic acid at 0.1%), using Bacillus cereus spores as a substitute for Bacillus anthracis. The strain of B. cereus NCTC 11143 studied in this work produces two fractions of endospores, the top and bottom fraction, which differ essentially by their physicochemical properties, namely hydrophobicity. The decontamination experiments were carried out with these separate B. cereus spore subpopulations and two different plastic surfaces were tested, a clean one and a soiled one with cooking oil. Liquid hydrogen peroxide at 10% and peracetic acid at 0.1% were shown to possess sporicidal action for both subpopulations of B. cereus spores when applied to clean and dirty surfaces. On the other hand, gaseous hydrogen peroxide (dryVHP) dispersed by vaporization by Delox was shown to be effective only in decontaminating bottom (hydrophilic) spores and did not exert sporicidal action on the spores of the top (hydrophobic). The different surface hydrophobicity these two spore fractions carry is thought to be responsible for their differential resistances with the decontaminants tested. The present study highlighted that the application method of the decontaminant is a conditioning factor on the mechanism of action of the oxidizing active substances and contributed to increase knowledge in the field of decontamination of B. cereus spores.
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Bacillus cereus 8A, isolado de solo da região sul do Brasil, produz uma bacteriocina, cereína 8A, que apresenta potencial para utilização como bioconservante no controle de microrganismos patogênicos e deteriorantes de alimentos. Neste trabalho foram realizados estudos de caracterização e otimização no processo de obtenção da cereína semi-purificada. Na primeira etapa do trabalho foi estudada a cinética de inativação térmica para a cereína 8A. Amostras da bacteriocina foram tratadas em diferentes combinações de tempo/temperatura no intervalo de 0-30min. e 70-82°C e os parâmetros termodinâmicos e cinéticos para a inativação da cereína 8A foram calculados. Os resultados revelaram que a inativação segue uma reação de primeira ordem com valores de k entre 0.059 e 0.235 min-1. A diminuição e o aumento dos valores de D e k, respectivamente, com o aumento da temperatura, indicaram uma rápida inativação de bacteriocina a temperaturas mais elevadas. Os resultados sugerem que a cereína 8A é uma bacteriocina relativamente termoestável com um valor de z de 21.98ºC e energia de ativação de 105.7 kJ mol-1. Em um segundo momento, a capacidade dos quelantes EDTA e lactato de sódio em diferentes concentrações e da cereína 8A (3200 UA/ml-1) em inibir e inativar a Salmonella Enteritidis foram analisadas. Os resultados indicaram que todos os tratamentos testados, EDTA, lactato de sódio e cereína 8A, sozinhos e em combinação, causaram uma redução significativa nos valores de OD600 em culturas de S. Enteritidis. A combinação de cerein 8A acrescido EDTA 100 mol l-1 resultou em um tratamento mais eficaz para reduzir o número de células viáveis de S. enteritidis. A microscopia de eletrônica de transmissão revelou paredes de células danificadas e perda de materiais protoplasmáticos nas células tratadas. As células de S. enteritidis tratadas com 8A cereína sozinha mostraram pequenos poros e o tratamento não afetou a maioria das células. Quando o agente quelante EDTA foi adicionado, as células mostraram-se mais danificadas. As lesões na parede celular tornaram-se mais intensas com a combinação de cereína 8A acrescida de EDTA, inclusive demonstrando uma notável perda do material intracelular, como observado nos resultados dos tratamentos com EDTA 50 e 100 mmol l-1 mais cereína 8A. A última etapa deste estudo investigou o particionamento da cereína 8A em dois sistemas de extração líquido-líquido, um com Triton X-114 e outro PEG com sais inorgânicos, que são considerados promissores para fins de bioseparação e depuração. Os resultados indicaram que cereína 8A particiona preferencialmente na fase rica em micela no sistema com Triton X-114 na concentração de 4 % e sua atividade antimicrobiana foi preservado. Em ATPS, os melhores resultados foram relativos ao coeficiente de partição obtidos com PEG e sulfato de amônio, quando o cloreto de sódio foi adicionado, o valor do Kb aumentou significativamente e mostrou o melhor rendimento de recuperação quando comparado com o sistema micelar. A purificação convencional resulta em um fator de purificação superior, mas em uma recuperação menor em comparação com métodos de particionamento. Estes resultados representam um importante passo no sentido de desenvolver um método de separação para cereína 8A e de um modo mais geral, para outras biomoléculas de interesse.
The bacteriocin cerein 8A, produced by a strain of B. cereus 8A, isolated from soil of south of Brazil with potential application against food spoilage and pathogenic bacteria.At the first phase of the study, the kinetics of thermal inactivation was studied for the bacteriocin cerein 8A. Samples of cerein 8A were treated at different time-temperature combinations in the range of 0-30 min and 70-82°C and the thermodynamic and kinetic parameters for bacteriocin inactivation were calculated. Results showed that inactivation followed a first-order reaction with k-values between 0.059 and 0.235 min-1. D- and k-values decreased and increased, respectively, with increasing temperature, indicating a faster bacteriocin inactivation at higher temperatures. Results suggest that cerein 8A is a relatively thermostable bacteriocin with a z-value of 21.98 °C and Ea of 105.7 kJ mol-1. In a second time, the ability of chelators EDTA and sodium lactate on different concentrations and cerein 8A (3200 UA/ml-1) to inhibit and inactivate Salmonella Enteritidis was investigated. The results indicated that all treatments tested, namely EDTA, sodium lactate and cerein 8A, alone and in combination, caused a significant reduction in the OD600 values of S. Enteritidis cultures. The addition of bacteriocin plus EDTA resulted in higher inhibition in comparison with the bacteriocin alone; the greater the concentration of EDTA, the greater the inhibitory effect. Transmission electron microscopy showed damaged cell walls and loss of protoplasmic material in treated cells. The cells of S. Enteritidis treated with cerein 8A alone showed small pores and the treatment does not affect the majority of cells. When the chelating agent EDTA was added the cells appeared more damaged. The injuries in cell wall become more marked with the combination of EDTA plus cerein 8A, including noticeable discharge of intracellular material, as shown for treatments with 50 and 100 mmol l- 1EDTA plus cerein 8A. The last step of this study investigated the partitioning of cerein 8A in two liquid–liquid extraction systems that are considered promising for bioseparation and purification purposes. In aqueous two phase micellar systems Triton X-114 was chosen as the as phase-forming surfactant. Aqueous two-phase systems were prepared of PEG and inorganic salts and the addition of sodium chloride was investigated in this system. Results indicated that cerein 8A partitions preferentially to the micelle rich-phase in the system with 4% Triton X-114 concentration and its antimicrobial activity was preserved. In ATPS, the best results concerning to the partition coefficients (Kb) were obtained with PEG + ammonium sulphate, when sodium chloride was added the value of Kb increase significantly and showed the best recovery yield when compared with micellar systems. The conventional purification results a higher purification fold, but a minor recovery in comparison with partitioning methods. The successful implementation of this peptide partitioning, from a suspension containing other compounds, represents an important step towards developing a separation method for cerein 8A, and more generally, for other biomolecules of interest.

Prodeta commercialise le Paciflor®, un probiotique utilisé dans l'alimentation animale. La société souhaitait à la fois mieux connaître les caractéristiques de la souche Bacillus cereus (B. C. ) CIP 5832 et optimiser la production industrielle des spores de cette même bactérie, principal composant du produit. Les cultures ont été réalisées en micro-plaques, en fioles, et dans des fermenteurs automatisés de 1,5 et 15 L. Le contrôle des paramètres des fermentations en bioréacteur rend possible une extrapolation ultérieure. Dans un souci d'efficacité, l'utilisation de la méthodologie des plans d'expériences nous a permis de minimiser le nombre de nos essais. Les différents résultats mettent en exergue plusieurs caractéristiques de la souche B. C. CIP 5832. En particulier, conformément à la littérature, nous avons confirmé l'importance de cations tels que le calcium et le magnésium dans la sporulation et le rôle de la L-alanine dans la germination. In fine, nous proposons un procédé de fermentation qui assure la germination, la croissance et la sporulation du B. C. CIP 5832 en milieu industriel concentré. Les conditions de culture définies permettent d'augmenter de plus d'un tiers la thermo-résistance des spores. Ces résultats, très encourageants, demandent à être confortés : il faut s'assurer que les nouvelles conditions de culture n'altèrent pas les effets zootechniques du probiotique lors de la fabrication d'aliments pour le bétail sous forme granulée et au stockage

Le groupe Bacillus cereus regroupe des bactéries taxonomiquement proches et diverses par leurs phénotypes. Les génomes de ces bactéries sont assez plastiques et le flux des gènes par des éléments mobiles entre les souches proches pourrait jouer un rôle important dans leur évolution. Parmi ces éléments mobiles, les prophages peuvent avoir un impact sur l’évolution de ces bactéries qui sont des pathogènes émergents. Dans cette étude, nous avons séquencé, analysé et étudié la fonctionnalité du prophage phIS3501 qui est intégré dans le gène hlyII de la souche B. Thuringiensis ATCC35646. L’excision de phIS3501 aboutit à la formation d’un gène hlyII intact codant une toxine potentiellement active. La similarité de ce mode de régulation avec celui mis en évidence chez Staphylococcus aureus indique une sélection pour ce type de système chez les bactéries pathogènes. Une étude in silico a été réalisée sur 14 génomes du groupe B. Cereus pour leurs contenus en prophages. 37 prophages et résidus de prophages ont été détectés. Les résultats issus de l’analyse et de la génomique comparative suggèrent que les prophages sont très répandus dans ce groupe de bactéries. L’échange génétique entre ces bactéries est fréquent et l’évolution se fait par le biais de recombinaisons par l'intermédiaire de ces prophages. Plusieurs gènes qui n’ont pas une relation directe avec le développement des phages ont été trouvés dans les génomes de ces prophages. Ces gènes peuvent avoir un rôle dans la conversion lysogénique de ces bactéries et pourraient participer à l’émergence de la pathogénicité par une expansion clonale ou un transfert de gènes horizontal
The Bacillus cereus group comprises bacteria taxonomically close with various phenotypes. The genomes of these bacteria are quite plastic and gene flow between closely strains could play an important role in their evolution. Among mobile elements, prophages may have an impact on the evolution of these emerging bacterial pathogens. In this study, we sequenced and analyzed the functionality of phIS3501, a prophage, which is integrated into the hlyII gene of B. Thuringiensis ATCC35646. Excision of phIS3501 resulted in the formation of an intact hlyII gene coding for a potentially active toxin. The similarity of this mode of regulation with that demonstrated in Staphylococcus aureus suggests a selection for this type of system in pathogenic bacteria. An in silico study was conducted on 14 genomes of the B. Cereus group for their prophages content. 36 prophages and prophage remnants were detected. The results of this analysis and comparative genomics suggest that prophages are widespread in this group of bacteria. The genetic exchange between these bacteria is common facilitating evolution through prophage-mediated recombination. Several genes that have no direct relationship with the development of phages were found in the genomes of these prophages. These genes may have a role in lysogenic conversion and could participate in the pathogenicity emergence by a clonal expansion or horizontal gene transfer

Le groupe Bacillus cereus est composé de huit espèces de bactéries à Gram positif sporulantes qui peuvent coloniser plusieurs niches écologiques. Les espèces les plus importantes sont B. cereus, une bactérie ubiquitaire du sol et un pathogène opportuniste; B. thuringiensis, un entomopathogène très utilisé comme biopesticide; et B. anthracis l’agent de la maladie du charbon. Bien que ces espèces présentent différents phénotypes, elles sont étroitement liées génétiquement et leurs facteurs de virulences principaux sont portés par des plasmides. Le cycle infectieux de B. thuringiensis dans la larve d’insecte est régulé par l’activation séquentielle de systèmes de quorum sensing de la famille RNPP. Parmi eux, les systèmes Rap-Phr, caractérisés chez B. subtilis, ont très peu été étudiés dans le groupe B. cereus. Ces systèmes régulent divers processus bactériens importants dont la sporulation. L’objectif de cette étude est d’analyser les systèmes Rap-Phr dans le groupe B. cereus, pour connaitre leur distribution, leur localisation et leur diversité afin d’obtenir une vue globale de ces systèmes chez ces bactéries. De plus, leur possible implication dans la régulation du processus de sporulation a été prédite sur la base de données structurales décrites chez RapH de B. subtilis. Les gènes rap, toujours associés à un gène phr, sont présents dans toutes les souches étudiées avec une moyenne de six gènes rap-phr par souche et avec 30% de ces systèmes qui sont portés par des plasmides. Les souches de B. thuringiensis portent six fois plus de systèmes Rap-Phr plasmidiques que les souches de B. cereus. Par ailleurs, les souches phylogénétiquement proches possèdent un profil de gènes rap-phr similaire. Un tiers des protéines Rap sont prédites pour inhiber la sporulation et ces protéines sont préférentiellement localisées sur les plasmides et donc plus fréquemment présentes chez B. thuringiensis que chez B. cereus. Cette prédiction a été partiellement validée par des tests de sporulation suggérant que les résidus impliqués dans cette activité chez B. subtilis sont conservés mais insuffisants pour prédire cette fonction. Le système Rap63-Phr63 porté par le plasmide pAW63 de la souche B. thuringiensis HD73 a ensuite été caractérisé. La protéine Rap63 a un effet modéré sur la sporulation et retarde l’expression des gènes régulés par Spo0A. La Rap63 est inhibée par son peptide Phr63, dont la forme mature correspond à l’extrémité C-terminale du pro-peptide. Les résultats de sporulation dans l’insecte suggèrent une activité synergique des systèmes Rap63-Phr63 et Rap8-Phr8 (porté par le pHT8_1) dans la régulation de la sporulation. Malgré la similarité entre les Phr63 et Phr8 aucun cross-talk n’a pu être mis en évidence, ce qui confirme la spécificité de ces systèmes de communication cellulaire. L’ensemble de ces résultats démontre la grande diversité des systèmes Rap-Phr dans le groupe B. cereus et souligne l’impact des systèmes plasmidiques dans le développement de ces bactéries. Par conséquent, les plasmides sont des éléments importants pour l’adaptation et la survie de ces bactéries et particulièrement pour B. thuringiensis
The Bacillus cereus group of Gram positive spore forming bacteria is comprised by eight species that are able to colonize several ecological niches. The most important species are B. cereus, a ubiquitous soil bacterium and an opportunistic pathogen; B. thuringiensis, an entomopathogen widely used as biopesticide; and B. anthracis, the causative agent of anthrax. Even if they present different phenotypes, they are genetic closely related and their main virulence factors are encoded on plasmids. The infectious cycle of B. thuringiensis in the insect larvae is regulated by the sequential activation of quorum sensing systems from the RNPP family. Among them, the Rap-Phr was extensively studied in B. subtilis but just punctually in B. cereus group species. The Rap-Phr systems were shown to regulate various bacterial processes, including the sporulation. The objective of this study was to analyze the Rap-Phr systems in the B. cereus group, regarding their distribution, location and diversity to achieve an overview of these systems in these bacteria. Moreover, their possible involvement in the control of the sporulation process was predicted based on structural data described for RapH in B. subtilis. The rap genes, always associated with a phr gene, were present in all 49 studied strains with an average of six rap-phr genes per strain and 30% were located on plasmids. Comparison among B. cereus and B. thuringiensis strains revealed that the last one harbors six-fold more plasmid rap-phr system then the former. Moreover, phylogenetic closer strains possess a similar profile of rap-phr genes. Interestingly, 32% of the Rap proteins were predicted to inhibit sporulation and these proteins were preferentially located on plasmids and therefore in B. thuringiensis strains. This prediction was partially validated by sporulation efficiency assays suggesting that residues identified in B. subtilis as involved in the phosphatase activity are conserved but not sufficient to predict the sporulation function. Then, the plasmid-borne Rap63-Phr63 system from pAW63 plasmid of B. thuringiensis HD73 strain was further studied. The Rap63 protein moderately inhibits the sporulation and delays the expression of Spo0A-regulated genes. Rap63 is counteracted by its cognate Phr63 peptide, which mature form corresponds to the C-terminal end of the pro-peptide. Sporulation assays in insect larvae suggest a synergistic activity of Rap63-Phr63 and Rap8-Phr8 (from pHT8_1 of B. thuringiensis HD73 strain) systems on sporulation efficiency. Despite the similarities of Phr63 and Phr8 no cross-talk was found between these two systems, confirming their specificity. Altogether, these results reveal the high diversity of the Rap-Phr systems in the B. cereus group and highlight the relevance of the plasmid-borne systems to cell development. Therefore, the results demonstrated the importance of the plasmids in the adaptation and the survival of these bacteria, especially for B. thuringiensis