Littérature scientifique sur le sujet « Bacillus cereu »

Concentrations of Colony Forming Units (CFU) were determined for two entomopathogenic bacteria (Pantoea agglomerans and Bacillus cereus), at the egg, larval, pupal, and adult stages of agriculturally important Phyllophaga and Cyclocephala white grubs, which were collected in five agroecosystems in Costa Rica. L2 and L3 larvae of Phyllophaga elenans collected in all regions where the study was conducted were extensive carriers of Pantoea agglomerans and Bacillus cereu. L2 and L3 larvae of Phyllophaga obsoleta, Phyllophaga menetriesi, Cyclocephala sanguinicollis and Cyclocephala castaniella found in the Central Valley and Central Pacific regions were carriers of Pantoea agglomerans and Bacillus cereus bacteria. In 60% to 90% of larvae in all white grub varieties studied, Pantoea agglomerans showed greater concentrations of CFU than Bacillus cereu, which showed the lowest CFU concentration. Egg, pupal, and adult mortality in all Phyllophaga species was due to Pantoea agglomerans in 62%, 80% and 22.5% of the cases, respectively. A possible antagonistic interaction between Pantoea agglomerans and Bacillus cereus is also discussed. In general, it was noted that light and larval manipulation were the main stress factors affecting these scarabids.

This study aimed to trace the dynamics of biofilm formation by vegetative cells and endospores of Bacillus cereus DL5 and Bacillus subtilis 168. Counts of B. cereus DL5 and B. subtilis 168 vegetative cells and spores either attached to glass wool or, correspondingly, planktonic cells were determined by standard plate-counting methods. Results from this study highlighted the biofilm-forming potential of both spores and vegetative cells of two different Bacillus species. It was shown that once Bacillus spores had attached to a surface, the spores germinated under favorable (B. cereus DL5) and even unfavorable (B. subtilis 168) nutrient conditions, resulting in biofilms containing both spores and vegetative populations. Furthermore, it was suggested that vegetative B. cereus DL5 cells exhibited a low propensity for spore formation in attached and planktonic growth forms in nutrient-limited growth medium. By contrast, vegetative B. subtilis 168 cells readily formed spores in planktonic and attached microcosms when exposed to nutrient-limited growth conditions. Sporulation in attached Bacillus populations is an important practical consideration for many food industries, such as dairy processing, where bacilli are routinely isolated from populations attached to processing-equipment surfaces.

Bacillus cereus is aerobic, positive gram, and spore-forming bacilli bacteria. The enterotoxin of this bacteria can cause food poisonous that manifest as diarrhea and vomitus. Some previous study has been performed to explore the antibacterial effect of Indian borage leaf, but none of them were explore the antibacterial effect of Indian borage against Bacillus cereus. Hence this study was design to explore the antibacterial activity of ethanol extract of Indian borage against Bacillus cereus. This was an experimental study with post only control group design. Ethanol extract was extracted by maceration methods and antibacterial activity against Bacillus cereus was evaluated by disc diffusion methods. The obtained ethanol extract was dissolved into some concentraions (1 g/ml, 0.8 g/ml, 0.6 g/ml, 0.4 g/ml, 0.2 g/ml). Data was analysed by One Way ANOVA and followed by Post hoc Test Tukey HSD using SPSS 25. Ethanol extract of Coleus amboinicus showed significant difference at the two highest concentration group against the lowest concentration (P- Value < 0.05). The average of inhibition zone diameter from the lowest (0.2 gr/ml) and highest (1.0 gr/ml) concentration were 14.87 mm and 31.50 mm, respectively. Overall, ethanol extract of Indian borage leaves had potential antibacterial activity against Bacillus cereus. This antibacterial activity increase followed by the increase of the concentration.

Totally 75 raw milk samples were analysed with the methods employing the media compared &ndash; MYPA, PEMBA, Brilliance^TM Bacillus cereus agar, and HiCrome Bacillus agar. The reference method with MYPA seems to be the most suitable for dairy plants laboratories because there is only low risk of mistaken identity. However, the samples containing miscellaneous micro-flora should be heat-inactivated before plating. Both positive and negative strains (totally 132) were isolated. Twelve strains, which could cause problems in the evaluation of the plates, were selected and identified by phenotyping and by PCR methods for Bacillus sp., B. cereus, and B. licheniformis. The PCR methods differed in their selectivity within particular bacilli group, within genera Bacillus, and within raw milk microflora.

Astronauts remote from Earth, not least those who will inhabit the Moon or Mars, are vulnerable to disease due to their reduced immunity, isolation from clinical support, and the disconnect from any buffering capacity provided by the Earth. Here, we explore potential risks for astronaut health, focusing on key aspects of the biology of Bacillus anthracis and other anthrax-like bacilli. We examine aspects of Bacillus cereus group genetics in relation to their evolutionary biology and pathogenicity; a new clade of the Bacillus cereus group, close related to B. anthracis, has colonized the International Space Station (ISS), is still present, and could in theory at least acquire pathogenic plasmids from the other B. cereus group strains. The main finding is that the genomic sequence alignments of the B. cereus group ISS strains revealed a high sequence identity, indicating they originated from the same strain and that a close look to the genetic variations among the strains suggesting they lived, or they are living, in a vegetative form in the ISS enough time to accumulate genetic variations unique for each single strains.

Using a reversed passive latex agglutination assay, about 85% of psychrotrophic Bacillus spp. tested were shown to produce diarrhoegenic toxin during growth on brain heart infusion broth at 25°C. The majority of these strains were identified as Bacillus cereus or cereus-related strains. However, a number of other species was capable of synthesizing the toxin. Further investigation of four psychrotrophic Bacilli showed that the toxin was produced during growth in milk at temperatures ranging from 6 to 21°C. Toxin production increased with increasing temperatures and was not synthesized in appreciable quantities until the bacterial count exceeded 1 × 107 cfu/ml.

ABSTRACT The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.

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

This paper presents the results of studies of the antagonistic activity of bacteria of the genus Bacillus (Bacillus subtilis, Bacillus thuringiensis, Bacillus mycoides and Bacillus cereus) under prolonged exposure to ionizing radiation in relation to bacteria of the E. coli group. It was found that bacteria of the genus Bacillus exhibit antagonistic activity of varying degrees of severity. It was found that the bacterial strains Bacillus subtilis, Bacillus thuringiensis and Bacillus mycoides showed a high level of antagonistic activity. Low antagonistic activity was characteristic of Bacillus cereus bacteria.

The cultural-morphological and biochemical properties of 83 selected strains of organic substance destructor were studied. The analysis of nucleotide sequences of the genome of bacteria Bacillus cereus Fd 2 was performed.

"The objective of this study was to develop quantitative microbial exposure assessment models for Bacillus cereus in packaged rice cakes (PRC). Probability distribution for growth of B. cereus in PRC was estimated and effects of thermal processing and acidification on extending the shelf-life of PRC were quantitatively assessed. Heat penetration curves at cold point for retort process and pasteurization were successfully predicted using heat transfer simulation model (RMSE < 0.77 ºC). The retort process showed a better sterilization effect than the pasteurization process, but degraded the quality of rice cakes such as color, shape, and texture. The final contamination level in PRC of slab shape package (> 6.63 log CFU/g at 95% level) was lower than that in randomly packed sample (> 7.77 log CFU/g at 95% level) because the cold point in the slab shape package was closer to the surface. Acidification significantly inhibited the growth of B. cereus and also affected the inactivation of B. cereus. A combination of acidification and low temperature pasteurization extended the shelf-life of PRC, while minimizing quality degradation of products (< 0.43 log CFU/g at 95% level)."

Rapid detection of pathogens and hazardous elements in fresh fruits and vegetables after harvest requires the use of advanced sensor technology at each step in the farm-to-consumer or farm-to-processing sequence. Fourier-transform infrared (FTIR) spectroscopy and the complementary Raman spectroscopy, an advanced optical technique based on light scattering will be investigated for rapid and on-site assessment of produce safety. Paving the way toward the development of this innovative methodology, specific original objectives were to (1) identify and distinguish different serotypes of Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus cereus by FTIR and Raman spectroscopy, (2) develop spectroscopic fingerprint patterns and detection methodology for fungi such as Aspergillus, Rhizopus, Fusarium, and Penicillium (3) to validate a universal spectroscopic procedure to detect foodborne pathogens and non-pathogens in food systems. The original objectives proposed were very ambitious hence modifications were necessary to fit with the funding. Elaborate experiments were conducted for sensitivity, additionally, testing a wide range of pathogens (more than selected list proposed) was also necessary to demonstrate the robustness of the instruments, most crucially, algorithms for differentiating a specific organism of interest in mixed cultures was conceptualized and validated, and finally neural network and chemometric models were tested on a variety of applications. Food systems tested were apple juice and buffer systems. Pathogens tested include Enterococcus faecium, Salmonella enteritidis, Salmonella typhimurium, Bacillus cereus, Yersinia enterocolitis, Shigella boydii, Staphylococus aureus, Serratiamarcescens, Pseudomonas vulgaris, Vibrio cholerae, Hafniaalvei, Enterobacter cloacae, Enterobacter aerogenes, E. coli (O103, O55, O121, O30 and O26), Aspergillus niger (NRRL 326) and Fusarium verticilliodes (NRRL 13586), Saccharomyces cerevisiae (ATCC 24859), Lactobacillus casei (ATCC 11443), Erwinia carotovora pv. carotovora and Clavibacter michiganense. Sensitivity of the FTIR detection was 103CFU/ml and a clear differentiation was obtained between the different organisms both at the species as well as at the strain level for the tested pathogens. A very crucial step in the direction of analyzing mixed cultures was taken. The vector based algorithm was able to identify a target pathogen of interest in a mixture of up to three organisms. Efforts will be made to extend this to 10-12 key pathogens. The experience gained was very helpful in laying the foundations for extracting the true fingerprint of a specific pathogen irrespective of the background substrate. This is very crucial especially when experimenting with solid samples as well as complex food matrices. Spectroscopic techniques, especially FTIR and Raman methods are being pursued by agencies such as DARPA and Department of Defense to combat homeland security. Through the BARD US-3296-02 feasibility grant, the foundations for detection, sample handling, and the needed algorithms and models were developed. Successive efforts will be made in transferring the methodology to fruit surfaces and to other complex food matrices which can be accomplished with creative sampling methods and experimentation. Even a marginal success in this direction will result in a very significant breakthrough because FTIR and Raman methods, in spite of their limitations are still one of most rapid and nondestructive methods available. Continued interest and efforts in improving the components as well as the refinement of the procedures is bound to result in a significant breakthrough in sensor technology for food safety and biosecurity.