Dissertations / Theses on the topic 'Bacillus cereus group species'

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 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.

Bacillus cereus (Bc) et Bacillus thuringiensis (Bt) sont deux espèces génétiquement proches. Bc est une bactérie pathogène que peuvent causer des gastro-entérites d’origine aliméntaire. Bt est une bactérie entomopathogène, dont le cycle de vie dans la larve d’insecte est contrôlé par des systèmes de quorum sensing, comme le système Rap/Phr, que régule processus tels que la sporulation, la formation de biofilm et la conjugaison. La présence des ces genès a été identifiée dans les plasmides, et ces eleménts ont été associés à l’adaptation des spécies dans sont niche ecologique. Le but de cette étude est de comprendre le rôle des plasmides dans ces bactéries. Pour la première étude l’insecte larvae, le niche privilegie de Bt, ont été infectées par souches de Bc et Bt, avec un contenu plasmidique diffèrent. Le fitness a été evallué par le comptage de cellules végétatives et spores dans quatre temps. Les souches de Bt et Bc ont été classées dans cinq groups par rapport à sont fitness. Dans ces groups le plasmide a affecté le fitness de la bactérie positive ou négativement. Les résultats ont démontré que les souches du group du B. cereus que reçoivent a pathogène plasmid ne est pas suffisant pour une augmentation effectif de la population bactérienne, i.e., coloniser l’hôte. La deuxième étude a permis caractériser le système rap/phr porté par le plasmide cryptique pHT8_1. Les résultats démontrent que la protéine Rap8 inhibe la sporulation dans la l’insecte. L’activité de cette protéine est inhibée par le peptide de signalisation Phr8. Le système Rap/Phr8_1 a permis les bactéries exercer un strict contrôle sur la sporulation, un processus important pour assurer la survie et la dissémination des bactéries. L’ensemble des résultats de la deuxième étude montrent que les plasmides peuvent fournir avantages pour l’adaptation et evolution de B. thuringiensis dans son niche ecologique, alors que les résultats de la première étude indiqués que les souches de Bc group doivent avoir un contenu génétique approprié pour exhiber un fitness élévé en permettant une optimal multiplication and dissemination de populations bactérienne dans l’insect larvae
Bacillus cereus (Bc) and Bacillus thuringiensis (Bt) are two closely related species. Bc is a pathogenic species responsible for gastroenteritis by food-borne. Bt is an entomopathogenic bacterium, which the lifecycle in insect larvae is controlled by quorum sensing systems, such as Rap/Phr, which regulates processes such as sporulation, biofilm formation and conjugation. The presence of these genes in plasmids has been described, furthermore, plasmids have been involved in bacterial adaptation to their ecological niche. In order to understand the role of the plasmids to these species, two complementary works were carried out. First, insect larvae, a privileged ecological niche of Bt strains, were infected with Bc and Bt strains harboring different plasmid contents. Their fitness were evaluated by vegetative cells and spore counts at four time points. Bt and Bc strains were classified into five groups according to the bacterial fitness. In these groups, the plasmid affects positively or negatively the bacterial fitness. The results demonstrated that for B. cereus group strains, getting a pathogenicity plasmid is not enough to effectively increase bacterial population, colonizing insect hosts. The second study characterized the rap/phr system encoded by the cryptic plasmid pHT8_1. The Rap8 protein inhibited the sporulation process in insect larvae. This protein was directly inhibited by the active signaling peptide Phr8. The Rap8/Phr8 system may allow the bacteria to exert a tight control of the sporulation process in the host cadaver for optimizing the multiplication, the survival and the dissemination of the bacteria. Thus, the results of the second study showed that the plasmids can provide advantages for the adaptation and the evolution of B. thuringiensis in its ecological niche, while the results of the first study indicate that B. cereus group strains must have a suitable genetic background to display a high fitness allowing optimal multiplication and dissemination of the bacterial population within insect larvae

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.

Cette thèse s’est intéressée à évaluer le niveau de risque représenté par les bactéries du groupe Bacillus cereus dans des aliments tunisiens et à tester l’efficacité de leur contrôle par le traitement des surfaces industrielles par des bactériophages. Une collection de 191 isolats a été créée à partir de 687 matrices alimentaires. Près de 40 % des isolats se sont avérés appartenir au groupe, avec une forte diversité génétique (143 profils PFGE et 99 profils ERIC-PCR) et un profil thermique intermédiaire (signatures 16S rDNA-1 m et-2 p). Près de 60 % des isolats du groupe appartiennent au groupe phylogénétique III, potentiellement pathogène. Les spores présentent majoritairement un taux d’adhésion plus fort que les cellules végétatives. Douze groupes toxinogènes ont été mis en évidence.Au moins un des gènes de chacun des complexes NHE et HBL sont présents, associés ou non à bceT, cytK 2 et ces. Après 18 h d’incubation à 30°C, près de 71% des isolats sont cytotoxiques. Différentes combinaisons de facteurs de virulence sont associées au potentiel cytotoxique et un lien apparait clairement entre cytotoxicité et type d’aliment. La collection s’est montrée sensible à de nombreux antibiotiques, alors qu’elle présente une résistance à l'ampicilline et à la novobiocine. Sur les 7 bactériophages sélectionnés, 5 possèdent un profil protéique unique alors qu’ils présentent tous une taille de génome et des profils de restriction similaires. Ils permettent de prévenir et de les traiter la formation de biofilms. Ce travail confirme le risque sanitaire lié à la présence du groupe B. cer
This thesis focused on evaluating the level of risk represented by Bacillus cereus group bacteria in Tunisian food and testing the effectiveness of their control by treating industrial surfaces with bacteriophages. A collection of 191 isolates was created from 687 food matrices. Nearly 40% of the isolates were found to belong to the group, with high genetic diversity (143 PFGE profiles and 99 ERIC-PCR profiles) and an intermediate thermal profile (signatures 16S rDNA-1 m and-2 p). Nearly 60% of the group's isolates belong to the phylogenetic group III, which is potentially pathogenic. Spores have a higher rate of adhesion than vegetative cells. Twelve toxigenic groups have been identified.At least one of the genes of each of the NHE and HBL complexes are present, whether or not associated with bceT, cytK 2 and these. After 18 hours of incubation at 30°C, nearly 71% of the isolates are cytotoxic. Different combinations of virulence factors are associated with cytotoxic potential and a clear link appears between cytotoxicity and food type. The collection has been shown to be sensitive to many antibiotics, while it is resistant to ampicillin and novobiocin. Of the 7 bacteriophages selected, 5 have a unique protein profile while all have similar genome size and restriction profiles. They are used to prevent the formation of biofilms and to treat them. This work confirms the health risk associated with the presence of the B. cereus group in Tunisian foods and the promising role of bacteriophages as biocontrol tools

Orientadores: Arnaldo Yoshiteru Kuaye, Dirce Yorika Kabuki
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Enterococcus faecium e Enterococcus faecalis são espécies de patógenos oportunistas que infectam principalmente imunocomprometidos. Estas espécies são encontradas em produtos lácteos e possuem capacidade de formar biofilme em superfícies que contatam com os alimentos. A sua remoção é muito dependente dos procedimentos de higienização. Os Enterococcus spp. utilizam o sistema de comunicação célula-célula (quorum sensing) para a formação de biofilmes. A formação de biofilme mono e multiespécie, a eficácia dos procedimentos de higienização no controle destes biofilmes e a produção de moléculas sinalizadoras de quorum sensing por cepas de E. faecalis, E. faecium, Bacillus cereus e Listeria monocytogenes foram avaliadas. Os ensaios foram realizados com cupons de aço inoxidável e variando-se a temperatura (7, 25 e 39 °C) e o tempo (0, 1, 2, 4, 6 e 8 dias). Após 1 e 8 dias de contato nas temperaturas de 25 e 39 °C, os cupons foram submetidos a diferentes processos de higienização. Os sanitizantes testados foram: hipoclorito de sódio (0,2%), ácido peracético (0,2%), quaternário de amônio (3,0%) e biguanida (1,0%). A detecção das moléculas sinalizadoras de quorum sensing AI-2 foi realizada através da avaliação do gene luxS e de ensaio biológico de bioluminescência. Nenhum dos micro-organismos avaliados foi capaz de formar biofilmes a 7 ?C. Enterococcus sp. foram capazes de formar biofilmes, com contagens acima de 8 log ufc/cm2 para as temperaturas de 25 e 39 °C após 8 dias de contato. Em cultivo multiespécie, a temperatura 25 °C favoreceu o desenvolvimento do biofilme de L. monocytogenes (contagens acima de 6 log ufc/cm2). Por sua vez, a 39 °C observou-se o efeito negativo no desenvolvimento do biofilme de L. monocytogenes em cultivo misto, com redução significativa nas contagens ao longo do tempo (valores abaixo de 0,4 log ufc/cm2). As contagens de B. cereus, para ambas as temperaturas em diferentes tempos de exposição situaram-se abaixo de 4,1 log ufc/cm2. Em contrapartida, a contagem de esporos de B. cereus evoluiu ao longo do tempo, atingindo contagens em torno de 4,6 log ufc/cm2. A limpeza com tensoativo aniônico complementada por outra etapa (limpeza ácida, limpeza ácida + sanitização ou sanitização) foi capaz de remover os biofilmes mono e multiespécie em todas as condições testadas. O ácido peracético foi o sanitizante mais eficiente e a biguanida o menos eficiente. Todas as cepas de Enterococcus spp. e B. cereus apresentaram o gene luxS e induziram o fenômeno de bioluminescência em Vibrio harveyi BB170, indicando a presença de autoindutores AI-2
Abstract: Enterococcus faecium and Enteroccus faecalis are opportunistic pathogens species that infect mainly immunocompromised individuals. These species are found in dairy products and are capable of forming biofilms on surfaces that contact with food. Their removal is highly dependent on the cleaning procedures. It is known that enterococci use the cell-cell communication (quorum sensing) to biofilm formation. The formation of mono- and multi-species biofilm, the effectiveness of sanitization procedures to control these biofilms and the production of signaling molecules of quorum sensing (AI-2) by strains of E. faecalis, E. faecium, Bacillus cereus and Listeria monocytogenes were evaluated in this work. The biofilms were grown on stainless steel coupons at various incubation temperatures (7, 25 and 39 °C) and times (0, 1, 2, 4, 6 and 8 days). After 1 and 8 days of contact at 25 and 39 °C, the coupons were subjected to different sanitation procedures: anionic tensioactive cleaning, acid-anionic tensioactive cleaning, sanitization, anionic tensioactive cleaning + sanitization, acidic- anionic tensioactive cleaning + sanitization and chlorinated alkaline cleaning. The sanitizers tested were: sodium hypochlorite (0.2%), peracetic acid (0.2%), quaternary ammonium (3%), and biguanide (1%). The detection of AI-2 molecules was performed by evaluating the luxS gene and biological bioluminescence assay. None of the microorganisms evaluated was able to form biofilms at 7 °C. Enterococcus sp. were able to form biofilms, with counts above 8 log CFU/cm2 for the temperatures of 25 and 39 °C after 8 days of contact. In multi-species culture, the temperature of 25 °C favored the development of L. monocytogenes biofilms (counts above 6 log CFU/cm2). On the other hand, at 39 °C it was observed a negative effect in the development of L. monocytogenes biofilms in mixed culture, with a significant reduction in counts over time (values below 0.4 log CFU/cm2). The counts of B. cereus, for both temperatures at different exposure times were below 4.1 log CFU/cm2. In contrast, the spore counts of B. cereus evolved over time, reaching scores of around 4.6 log CFU/cm2. The anionic tensioactive cleaning complemented by an aditional step (acid cleaning, acid cleaning + sanitization or sanitization) was able to remove mono- and multi-species biofilms in all tested conditions. The peracetic acid was the most effective sanitizer and the less efficient was biguanide. All strains of Enterococcus spp. and B. cereus showed the luxS gene and induced the phenomenon of bioluminescence in Vibrio harveyi BB170, indicating the presence of AI-2 autoinducers
Doutorado
Tecnologia de Alimentos
Doutora em Tecnologia de Alimentos

Chez les bactéries sporulantes du genre Bacillus, des mécanismes importants tels que la sporulation et la virulence sont régulés par des systèmes de communication cellulaire qui impliquent des peptides de signalisation et des régulateurs de la famille RNPP (Rap, NprR, PlcR, PrgX). L'objectif de mon travail de thèse a été de déterminer le rôle du régulateur NprR chez les bactéries du groupe B. cereus. Ce travail se divise en trois parties complémentaires. La première partie a consisté à montrer que NprR est impliqué dans un système de communication cellulaire. Nous avons montré que NprR est un régulateur transcriptionnel de début de phase stationnaire qui est dépendant du peptide de signalisation NprX. Associé à NprX, NprR active la transcription du gène nprA qui code pour une protéase extracellulaire. Nous avons démontré que le peptide NprX est sécrété, maturé puis réimporté dans la cellule bactérienne par deux systèmes d'oligopeptide perméase (Opp et Npp). Une fois dans la cellule, la forme mature de NprX (vraisemblablement l'heptapeptide SKPDIVG) se lie à NprR et permet la transcription du gène nprA. Nous avons ensuite cherché à déterminer la fonction de ce régulateur au cours du cycle infectieux de B. thuringiensis (Bt) chez l'insecte. Nous avons montré que NprR est actif après la mort de l'insecte et permet aux bactéries de survivre, sous forme de cellules végétatives, dans les cadavres. Une analyse transcriptomique indique que NprR régule l'expression d'au moins 41 gènes qui codent notamment pour des enzymes dégradatives et un locus de gènes impliqués dans la production d'un peptide synthétisé de façon non ribosomique (la kurstakine). Nous avons démontré que les gènes codant pour les enzymes dégradatives s'expriment spécifiquement après la mort de l'hôte et que les produits de ces gènes sont essentiels pour hydrolyser différents substrats (protéines, lipides, chitine), ce qui suggère que Bt a un mode de vie nécrotrophe dans le cadavre. La kurstakine est essentielle pour la survie de Bt pendant son développement nécrotrophe et nous avons montré que cette molécule est nécessaire pour le swarming et la formation de biofilm. Par ailleurs, un mutant du gène nprR ne se développe pas et ne sporule pas efficacement dans le cadavre. L'ensemble de nos résultats indiquent que le necrotrophisme est un mode de vie hautement régulé, qui est essentiel dans le cycle infectieux de Bt car il contribue à la transmission horizontale de ce micro-organisme. Enfin, nous avons étudié la régulation de l'expression des gènes nprR et nprX. Nous avons montré que les gènes nprR-nprX sont co-transcrits à partir d'un promoteur dépendant de sigma-A (PA) situé en amont du gène nprR. La transcription à partir de ce promoteur débute lors de l'entrée en phase stationnaire et est contrôlée par deux régulateurs transcriptionnels: CodY et PlcR. Le répresseur CodY pourrait se lier à l'ADN en amont du promoteur PA et réprimer la transcription des gènes nprR-nprX pendant la phase exponentielle de croissance. Au début de la phase stationnaire, le contrôle négatif de CodY est levé et PlcR active la transcription de nprR-nprX en se liant à une boîte PlcR située en amont de PA. Nos résultats indiquent que nprX est également transcrit indépendamment de nprR à partir de deux promoteurs, PH et PE, respectivement dépendant de sigma-H et sigma-E. Les deux promoteurs permettent d'assurer la transcription de nprX en phase stationnaire tardive alors que la transcription à partir du promoteur PA est achevée. Cette étude met en évidence le role clé des régulateurs CodY, PlcR and Spo0A dans la régulation de l'expression des gènes nprR-nprX.

Parmi les desserts à base d’ovoproduits, l’île flottante est reconnue comme particulièrement sensible d’un point de vue microbiologique car sa commercialisation souffre de la survenue intempestive d’altérations que les industriels souhaitent maîtriser. Les travaux réalisés au cours de cette thèse avaient pour objectif de mieux appréhender ces phénomènes afin de mieux les contrôler. Nous avons montré que l’altération de l’île flottante concernait principalement la crème anglaise et qu’elle s’accompagnait d’un développement bactérien conséquent, d’une fréquente diminution du pH et de modifications sensorielles liées à l’aspect et à l’odeur. Les principales bactéries détectées ont été identifiées comme appartenant au groupe Bacillus cereus et aux genres Staphylococcus et Enterococcus. Le blanc d’œuf pasteurisé, utilisé pour la fabrication des œufs en neige, s’est avéré être une source de contamination possible.Cependant, l’implication de bactéries installées sous forme de biofilms dans l’environnement de production ou véhiculées par d’autres ingrédients a aussi été fortement envisagée. La ré-inoculation, en culture pure, d’une collection bactérienne représentative dans de la crème anglaise stérile a montré que différents types de modifications sensorielles et physico-chimiques s’exprimaient d’un genre bactérien à l’autre et qu’ils étaient notamment corrélés à la capacité des bactéries à consommer les sucres et les protéines de la crème anglaise et à produire des métabolites dont des composés volatils odorants. Ces résultats à l’appui, différents tests ont pu être proposés, p
Among the chilled egg products-based desserts, the French dessert “île flottante” is recognized as particularly sensitive from a microbiological point of view, because marketing is suffering from untimely spoilage occurrence that manufacturers wish to control. The work done in this thesis aimed to better understand these phenomena in order to better control them. We have shown that the dessert spoilage mainly concerned the custard cream and it was characterized by high bacterial count, frequent pH decreasing and sensory changes of appearance and smell. The main bacteria detected were identified as belonging to the Bacillus cereus group and Staphylococcus and Enterococcus genera. The possible involvement of bacteria from the pasteurized egg white, used for the egg white foaming, in the dessert spoilage issue was established.However, the involvement of bacteria from biofilms installed in the production environment or provided by other ingredients was also strongly suspected. The spoilage potential assessment of pure culture of a representative bacterial collection in sterile custard cream has shown that different types of sensory and physicochemical changes were expressed according to bacterial genus and that these changes were particularly correlated with the ability of bacteria to consume sugars and proteins of custard and to produce various volatile compounds with specific odorous. With these results, various tests have been proposed for a better control of the white egg batches orientation according to their microbiological quality and so to guarantee their safety with

Rozdziały (3 załączniki) w postaci publikacji naukowych w czasopismach FEMS Microbiology Ecology (Appendix 1) oraz PLOS ONE (Appendix 2 oraz Appendix 3).
Przedstawiciele B. cereus s.l. występują powszechnie w środowisku naturalnym i wywierają ogromny wpływ na zdrowie człowieka, przemysł spożywczy oraz rolnictwo. Te tlenowe laseczki z jednej strony produkują toksyny szkodliwe dla ludzi i zwierząt, ale znane są również z syntezy wtórnych metabolitów degradujących niebezpieczne związki chemiczne lub wspomagających wzrost roślin. Powyższe właściwości były intensywnie badane w odniesieniu do szczepów o szczególnym znaczeniu gospodarczym i medycznym. Tymczasem pokrewieństwo filogenetyczne i podłoże ekologicznej dywersyfikacji szczepów izolowanych z gleby nie jest dostatecznie poznane. Analizowałam strukturę genetyczną 297 szczepów B. cereus s.l. wyizolowanych z gleby (i) Narwiańskiego PN, (ii) Białowieskiego PN, (iii) Biebrzańskiego PN oraz (iv) gospodarstwa rolnego w Jasienówce. Zidentyfikowałam homogeniczną grupę bakterii (i) zdolną do wzrostu w niskiej temperaturze (ekotyp psychrotolerancyjny) oraz (ii) syntetyzującą ciemno-brązowy barwnik (ekotyp melaninowy). Analizy MLST wskazują na istnienie specyficznych genotypów wśród naturalnych populacji B. cereus s.l. Ponadto wykazałam istnienie trzech grup filogenetycznych, obejmujących zmienną liczbę B. cereus, B. thuringiensis i B. mycoides. Jednakże analizy typów sekwencyjnych i kompleksów klonalnych wskazują, iż środowiskowe izolaty B. cereus s.l. nie reprezentują jednego gatunku. Szczegółowe badania genetyczne, fenotypowe oraz biochemiczne środowiskowych szczepów B. cereus s.l., rzuciły nowe światło na ewolucję oraz ekologiczną adaptację tych bakterii.
B. cereus s.l. are widespread in natural environments and have a significant impact on human health, food industry, and agriculture. On one hand, members of this group synthetize various toxins harmful to humans, herbivores and invertebrates. On the other hand, they are also known as producers of various secondary metabolites whereby they degrade pollutants and promote the growth of plants and animals. These aspects have been intensively studied especially with regard to the B. cereus group members with the highest impact on human health and economy. Meanwhile, the phylogenetic relationships and the basis of ecological diversification of soil B. cereus s.l. remains largely undescribed. I investigated the genetic structure of 297 B. cereus s. l. soil isolates from diverse habitats in Northeastern Poland, such as (i) the Narew NP, (ii) the Białowieża NP, (iii) Biebrza NP, and (iv) agricultural land in Jasienowka. I identified homogenous groups of bacteria able to (i) growth in low temperature (psychrotolerant ecotype) and (ii) synthesis a dark pigment (melanotype). The MLST analysis indicates the existence of specific genotypes within the natural B. cereus s.l. populations. Phylogenetic studies revealed three major clades, in which B. cereus, B. thuringiensis and B. mycoides were intermixed. However, analysis of sequence types and clonal complexes indicate that environmental B. cereus s.l. do not represent one species. Detailed genetic, phenotypic and biochemical analyses of the environmental B. cereus s.l. strains shed new light on the evolution and ecological adaptation of these bacteria to specific soil habitats differing in scope of human activity.
Wydział Biologiczno-Chemiczny. Instytut Biologii.

博士
臺灣大學
微生物與生化學研究所
96
Bacillus cereus foodborne diseases are a major concern worldwide. In Taiwan for the period 1991 to 2006, outbreaks due to B. cereus were exceeded only by Vibrio parahaemolyticus and Staphylococcus aureus. Five different enterotoxins and one emetic toxin of B. cereus have been characterized. To amplify all of the enterotoxin and emetic-specific sequences of the species in the B. cereus group, a multiplex polymerase chain reaction (multiplex PCR) with 12 primer pairs was established. The assay was successfully applied to analyze the toxigenic potential of 162 isolated B. cereus group strains. Results showed that there were 10 toxigenic patterns for all the test strains. All of the B. cereus strains carried at least one toxin gene. More than 70% of B. mycoides strains carried no known toxin genes. The toxin profiles and toxin genes of B. mycoides strains were significantly different from B. cereus strains although the two species were closely related. The results suggested that many B. mycoides strains might be less prone to cause food poisoning. It also indicated the importance of detecting the toxin genes together with the detection of the species in the B. cereus group. Conventional bacteriological methods for the detection and identification of species of the B. cereus group require individual biochemical confirmation and are laborious and time-consuming. PCR is a choice of rapid detection but can not quantify the contamination level. In practice, it is necessary to quantify the contamination level of B. cereus group cells. We selected nhe coding for Nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated using 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2>0.993), wide dynamic quantification range (102-107 CFU/g for cooked rice and chicken, 103-107 CFU/mL for milk, and 104-107 CFU/g for feces), and adequate relative accuracy (85.5-101.1%). For the low level contaminations, a most probable number (MPN) real-time PCR assay was developed that could detect as low as 100 CFU/mL. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification. The cytotoxicity titers of Nhe components varied considerably and the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates. In order to examine the regulatory mechanism of different Nhe-producing strains, the expression level of their nhe mRNA was determined by real-time reverse transcription PCR. Five candidates of internal controls were evaluated by three softwares. Meanwhile, the growth curves of different Nhe-producing strains were also compared. All studied candidate of internal control genes reached high expression stability. The growth curves showed no significant difference but the nhe mRNA expression level of high Nhe-producing strains was significantly higher than low Nhe-producing strains. The results indicated that the different Nhe expression levels between B. cereus strains may not be due to their growth difference but may be controlled at the transcribed level of nhe gene expression.

碩士
國立中興大學
食品科學系
86
B. cereus is one of the food pathogens which may cause foodbornedisease. It is also one of the important items for food inspection. Thus, tounderstand the relationship between its enterotoxins and cytotoxicity andto develop the multiplex PCR system for its detection is important. Based on the hblA gene for B component of hemolysin BL, bceTand entFM genes for enterotoxins, PCR primers aimed for the detectionof B. cereus enterotoxins have been developed. When the PCR resultsfor these enterotoxins specific primers were compared with the results ofcytotoxicity study using CHO cells, it was found that strains are positivewith any of these PCR primer pairs would show the positive cytotoxicityresult. The results for CHO cells cytotoxicity study were the same asthose obtained from immunoassay studies using BDE-VIA kit. As forthe multiplex PCR system, it was found that for hemolysin BL or bceTgene detection, when only one target strain was present in skim milk andcooked rice, the detection sensitivity reached to N*100 CFU per ml andper gram if a preculture step was performed prior to PCR. When twotarget strains were mixed to the food sample, however, it was found thatthe ratio of original cell numbers should be within 102 so that the targetstrains with lower cell numbers could be detectable. On the work forspecific PCR detection of B. cereus (not the B. cereus group cells), afterseries designing of PCR primers from 16S rRNA genes, we found PCRprimers which could allow the specific detection of B. cereus butexempted the interference from B. anthracis, B. mycoides and a standardstrain of B. thuringiensis. Four B. thuringiensis strains which interferethe PCR detection of B. cereus strains, however, were confirmed as B.thuringiensis strains since they generate cry gene specific product andthey were shown to produce parasporal crystals as examined by themicroscope. Sequence assay for the 16S rRNA genes showed that thesefour B. thuringiensis strains have the same DNA sequcnce as B. cereusstrains. Furthermore, on the position of base No. 165, two bases, ie. Cand T were observed. Whether two or more 16S rRNA operons would befound for B. thuringiensis strains need to be further investigated.

碩士
國立中興大學
食品科學系
85
Abstract Bacillus cereus is one of the important food pathogenes which may cause food poisoning. To investigate the relationship between B. cereus group cells and their toxigenicities and to develop rapid methods for the detection of B. cereus group cells and their hemolysin BL and/or B. cereus BceT enterotoxin is important. Based on the gene sequences coding for phospholipase C and sphingomyelinase, 16S rRNA, hblA of hemolysin BL B component and bceT, we have developed some novel PCR primers for the specific detection of B. cereus group cells and their genes coding for hemolysin BL and/or BceT toxins. The molecular weights generated from these PCR primers are 558 and 433 bp for B. cereus cells and 691 as well as 297 bp for hemolysin BL and BceT toxin, respectively. All the B. cereus group cells, such as B. cereus, B. thuringiensis, B. mycoides and B. anthracis would generate the expected PCR products with molecular weights equal to 558 or 433 bp, depending on the primers used. Of the 54 B. cereus strains tested, 18 were found to be hemolysin BL producing strains and 22 were bceT gene containing strains. In addition, for the 18 B. cereus strains containing hblA gene of hemolysin BL B component, further confirmation of their toxigenicity using blood agar hemolysis method and BCET-RPLA kit were performed. Results indicate that the PCR method might be used for the reliable detection of hemolysin BL productivity. Also, none of the bacterial strains other than B. cereus group cells would generate the false positive reaction. Thus, specificities of these PCR primers were assured. For food samples, such as whole milk, cooked pork, egg and cooked rice, which were inoculated with B. cereus cells, if a 8 hr preculture step was performed for target cells prior to the PCR, as low as N×100 cells per gram of the food sample could be detected. Also, the hemolysin BL and BceT toxin genes were detectable too.

碩士
國立中興大學
食品科學系
90
Abstract Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus group, Salmonella spp., pathogenic Escherichia coli are common pathogens which may cause food poisoning cases. Conventional methods for the detection of these bacteria spp. need the use of selection and differentiation medium followed by biochemical and serological identification steps. Such process is time consuming and laborious. Thus, development of rapid methods for the detection of these bacteria species is important. Biochip may be one of the choices for such purpose. Owing to the fact that 16S rRNA gene sequences have been used for bacteria identification and classification, in this study we thus tried to develop a 16S rRNA gene based biochip for the simultaneous detection of several different species of food pathogens. We found that 10 oligonucleotides arrayed on a biochip could be used for the differentiation of 5 genus of pathogenic bacteria. These bacteria genus are Salmonella spp., E coli, Bacillus cereus group strains, Staphylococcus spp. and Vibrio spp. etc. Five distinct chip hybridization patterns could be obtained for these 5 genus of bacteria spp. In additions, the hybridization pattern allowed us to find three specific oligonucleotide probes which in combination with a universal primer, could be used for the PCR primers specific for the detection of Salmonella spp. and E. coli. For pure culture, after 8 hrs preculture step, the detection sensitivity for theses bacteria was 100 CFU/ assay. For target cells in food samples, however, detection was not sensitive enough even after 8 hrs preculture step. In conclusion, the oligonucleotide array could be used for the identification of pure cultured bacteria but in use of it for food inspection, the process need some improvements.