Clostridium perfringens (C. perfringens) causes diseases such as necrotic enteritis in poultry. Current methods for early detection of C. perfringens, including culture, and molecular techniques (PCR and qPCR), have logistical limitations in detecting pathogens on-site because they require specialized equipment and long sample preparation time. This study describes developing a highly sensitive on-site LAMP assay combined with a simple DNA extraction method to detect C. perfringens. Results showed that the LAMP assay was highly specific and sensitive with the optimal reaction conditions: 63°C for 30 mins with primer concentration of 1.2 mM FIP/BIP and 0.2 mM F3/B3. The HotSHOT method was the most convenient DNA extraction method for the on-site LAMP assay with respect to the processing time, cost, and minimal equipment requirement. Therefore, the HotSHOT DNA extraction method followed by the LAMP assay could serve as a rapid on-site molecular test for C. perfringens in the field.

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Clostridium perfringens is a significant human pathogen, causing 250,000 cases of food poisoning in addition to several thousand potentially lethal cases of gas gangrene each year in the United States. Historically, work in this field has centered around toxin production, as C. perfringens can produce over 13 toxins. This work expands the knowledge of the starvation-response of C. perfringens, which includes several potential virulence factors, sporulation, motility and biofilm formation. Sporulation protects cells from a variety of stresses, including starvation. Efficient sporulation requires the transcriptional regulator CcpA, mediator of catabolite repression. Sporulation is repressed by glucose, but, surprisingly, in a CcpA-independent fashion. C. perfringens cells in a biofilm are resistant to a number of environmental stresses, including oxygen and antibiotics. Biofilm formation is repressed by glucose, and other carbohydrates, independently of CcpA. Gliding motility, a type four pili (TFP)-dependent phenomenon, affords C. perfringens with a mechanism for moving across a solid surface in response to carbohydrate starvation, while carbohydrates supplementation at high levels delay the initiation of the motility response. CcpA is required for the proper initiation of motility, a ccpA^-C. perfringens strain showed a considerable increase in the time to initiation of motility on lactose and galactose, and was unable to move at all in the presence of glucose. Gliding motility represents the most significant finding of this work. TFP were previously undescribed in any Gram-positive bacterial species, and this work produced genetic evidence suggesting their presence in all members of the clostridia, and physical evidence for TFP-dependent gliding motility in a second species, C. beijerinckii. / Ph. D.

Clostridium perfringens is a spore-forming anaerobic Gram-positive rod which has gliding motility through type IV Pili (TFP). Since the discovery of TFP in Gram-positive bacteria is relatively new, more studies are required to understand the mechanism and interaction of the proteins of this machinery. Moreover, the similarities between TFP and type 2 secretion system (T2SS) suggest that C. perfringens has also a T2SS. We studied the localization of TFP ATPases, PilB1, PilB2 and PilT in Bacillus subtilis to compare the localization in an organism other than C. perfringens and which lacks any known genes similar to TFP. Unlike the case in C. perfringens, PilB1 in B. subtilis localized to the poles in the absence of PilT, with some central foci at the future division sites. Colocalization of PilB1 was also studied with PilT and the results suggested that PilB1 needs PilT to migrate from the poles to the center. Localization of PilB2 in B. subtilis, was similar to the results in C. perfringens and to the localization of PilB1 in B. subtilis. We have not been able to co-express PilB2 with PilT yet. Succeeding in this study will help us better understand the interactions between PilB proteins and PilT. In another project, we studied a von Willebrand factor Type A-Domain Containing protein (vWA) which is secreted from C. perfringens strain 13. We overexpressed and purified this protein and tested the effects on mammalian cells. We found that the vWA is probably not a toxin but since it seems to bind to macrophage membranes, we propose that the vWA could be part of a toxin complex, probably the subunit of the complex that binds to the host cells. / Master of Science

Clostridium perfringens is the causative agent of gas gangrene. A lethal infection in mice requires a large inoculum suggesting that the immune system is involved in inhibiting disease. Human monocytic cells and neutrophils killed C. perfringens in vitro when complement was present. Macrophages and neutrophils co-localized with C. perfringens in vivo when bacterial numbers were low. Depletion of neutrophils and monocytes in mice revealed that monocytic cells play a role in inhibiting C. perfringens gas gangrene in mice infected with an intermediate dose. C. perfringens can persist in the tissues and this could be mediated by persistence within macrophages. To examine if the toxin perfringolysin O (PFO) could mediate this, less active variants of PFO were used to examine what occurs between phagosomal escape and cell lysis. The mutant forms of PFO did mediate phagosomal escape in macrophages and were found within macrophages at higher numbers than wild-type C. perfringens. Our data were preliminary but may indicate that less active PFO mediates intracellular persistence. To investigate the role of sialidase in C. perfringens gas gangrene we made nanI-, nanJ-, and nanI-/nanJ- mutants. We observed that NanI is responsible for the majority of sialidase activity of C. perfringens strain 13, that NanJ is an extracellular sialidase, and that these genes are transcriptionally regulated by sialic acid. Murine infection trials revealed that these sialidases may be protective for mice during infection. In conclusion, murine monocytes inhibit disease onset and C. perfringens sialidase enhances mouse survival. However, the toxin PFO if less active promotes the survival of C. perfringens with macrophages. / Master of Science

Clostridium perfringens is a versatile pathogen capable of causing a wide array of diseases, ranging from clostridial food poisoning to tissue infections such as gas gangrene. An important factor in virulence as well as in the distribution of C. perfringens is its ability to form an endospore. The symptoms of C. perfringens food poisoning are directly correlated to the release of an enterotoxin at the end of the sporulation process. The sporulation process in C. perfringens is subject to carbon catabolite repression (CCR) by sugars, especially glucose. CCR is a regulatory pathway that alters transcription based on carbon source availability. In Gram-positive bacteria, the HPr kinase/phosphatase is responsible for this nutritional sensing by phosphorylating or dephosphorylating the serine-46 residue of HPr. HPr-Ser-P then forms a complex with the transcriptional regulator CcpA to regulate transcription. We were able to show here that purified recombinant C. perfringens HPr kinase/phosphatase was able to phosphorylate the serine-46 residue of HPr. When the codon for this serine residue is mutated through PCR mutagenesis to encode alanine, phosphorylation could not take place. We have also shown that in gel retardation assays, CcpA and HPr-Ser-P were able to bind to two DNA fragments containing putative C. perfringens CRE-sites, sequences where CcpA binds to regulate transcription. The genome sequence of a food poisoning strain of C. perfringens was searched for potential CRE-sites using degenerate sequences designed to match those CRE-sites CcpA was shown to bind. DNA fragments containing these newly identified CRE-sites were then used in gel retardation assays to determine whether CcpA binds to these CRE-sites, making them candidates for CCR regulation. These results, combined with comparisons of metabolic characteristics of a ccpA- strain versus wild-type C. perfringens, provide evidence that CcpA participates in the regulation of carbon catabolite repression in the pathogenic bacterium C. perfringens / Master of Science

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

A proposta apresentada se baseia na introdução de um estágio intermediário de desinfecção previamento ao tratamento biológico visando intensificar os efeitos do estágio seguinte destinado à desinfecção convencional. Para estudo de caso foram aplicadas as técnicas de ozonização e radiação UV combinadas em instalações piloto que simulam duas condições seqüenciais de desinfecção. O desempenho do método proposto foi avaliado através de exames microbiológicos de amostras do efluente anaeróbio previa e posteriormente à desinfecção, utilizando indicadores de contaminação por bactérias (Escherichia coli e coliformes totais), vírus (colifagos) e protozoário (Clostridium perfringens). Os resultados obtidos no sistema combinado pré-desinfecção/desinfecção revelaram eficiência de inativação superior quando comparada ao procedimento convencional. Nas análises de E.coli, por exemplo, a aplicação de apenas 1 mg de ozônio/L ou 51 mW de radiação/'CM POT.2', na primeira etapa de desinfecção, foi suficiente para se alcançar 1 log acima do valor correspondente ao método convencional. Mesmo indicadores mais resistentes como C. perfringens apresentaram redução da fração N/No da ordem de 1 log em relação ao método proposto. Além disso, estes níveis de inativação foram alcançados mesmo sob a influência de elevada concentração de SST, SSV e DQO na entrada na unidade piloto destinada à pré-desinfecção. Entre as seqüências de experimentação investigadas ('O IND.3'/'O IND.3', 'O IND.3'/UV, UV/'O IND.3' e UV/UV) não foram observadas grandes variações. De modo semelhante, os resultados revelaram que a relação N/No, para os indivíduos submetidos ao sistema combinado, não foi afetada pelo aumento no tempo de exposição ao agente inativante ('O IND.3': 5, 7, 10 min; UV: 30, 60, 120s). Considerando as baixas dosagens de 'O IND.3' e UV aplicadas na primeira etapa, somada às condições limitadas de desempenho do sistema real examinado, os níveis de inativação alcançados sugerem grande potencialidade de utilização do método alternativo proposto, comprovando sua viabilidade técnica / The present proposition is based on the introduction of an intermediate disinfection stage before the biological treatment, in order to intensify the effects of the next stage employed in conventional disinfection. Studies were performed using combined ozonization and UV radiation techniques, in a model installation that simulates two sequential disinfection conditions. The performance of the method was evaluated using microbiological exams of samples taken from the anaerobic effluent before and after the disinfection. Bacterial (Escherichia coli and total coliforms), viral (coliphages) and protozoan (Clostridium perfringens contamination tracers were used in such exams. Results obtained by combining pre-disinfection and disinfection reveal superior inactivation efficiency as compared to the conventional procedure. For example, in the E. coli analysis the application of only 1 mg of ozone/L or 51 mW/'CM POT.2' of radiation in the first disinfection stage was enough for achieving 1 log above the convention method. Even more resistant tracers, such as C. perfringens, showed aproximatelly 1 log of reduction in the N/No fraction in the proposed method. Besides, these inactivation levels were achieved even for high concentrations of SST, SSV and DQO in the entrance of the pre-disinfection unit. No significant variations were observed among the disinfection sequences ('O IND.3'/'O IND.3','O IND.3'/UV,UV/'O IND.3', and UV/UV). Similarly, the results showed that the N/No relation, for individuals submitted to the combined system, was not affected by the increase of the exposition time to the inactivation agent ('O IND.3': 5, 7, 10 min; UV: 30, 60, 120 s). Taking into account the low dosages of 'O IND.3' and UV applied in the first stage and the limited performance conditions of the real system, the achieved inactivation levels suggest a great potential for the alternative method proposed, demonstrating its technical viability

Die im Biogasprozess anfallenden Gärreste werden oftmals als Wirtschaftsdünger verwendet. Krankheitserreger, die sich in den Gärresten befinden können über die Düngung in die Lebensmittelkette gelangen. Die Möglichkeit einer Vermehrung von Bakterien in den Biogasanlagen sowie deren Ausbreitung schürt die Bedenken der Öffentlichkeit. Das Ziel dieser Arbeit war es, Nachweismethoden für die Untersuchung von Proben aus Biogasanlagen zu etablieren, die Praxistauglichkeit dieser anhand von Proben aus Biogasanlagen zu überprüfen und die mikrobielle Belastung dieser Proben hinsichtlich ausgewählter Indikatorkeime zu erfassen. Bei den Indikatorkeimen handelte es sich um Clostridium perfringens, Clostridium botulinum, Enterokokken, Escherichia coli, ESBL-bildende Enterobaceriaceae und Salmonellen. Für die Etablierung der bakteriologischen Nachweismethoden wurde autoklavierter Gärrest mit einer definierten Keimmenge beimpft und auf verschiedene Nährmedien aufgebracht. Diese wurden bebrütet, ausgezählt und die KbE/ml berechnet. Mittels Probitanalyse wurde für jedes Medium die untere Grenze für den Nachweis aus beimpftem Gärrest bestimmt. Bei den Nährmedien handelte es sich um Brilliance™ Salmonella Agar, XLT4 Agar und XLD Agar für den Nachweis von Salmonella spp. Für E. coli wurden Tergitol 7 Lactose TCC Agar und Brilliance™ E. coli/Coliform Selektiv Agar verwendet. Der Nachweis von Enterokokken erfolgte mittels Slanetz Bartley Agar und Enterococcus Selektivagar. Für die ESBL-bildenden Enterobacteriaceae wurde der Brilliance™ ESBL Agar eingesetzt. Die getesteten Nährmedien zum Nachweis von C. perfringens waren Membran Clostridium Perfringens (mCP) Selektivnährboden sowie Tryptose Sulphite Cycloserine (TSC) Agar überschichtet mit TSC Agarbasis. Für C. botulinum erfolgte der Nachweis auf Eigelb Laktose Agar. Darüber hinaus wurde eine PCR zur C. perfringens Toxintyp-Bestimmung nach dem Protokoll von VAN ASTEN et al. (2009) etabliert. Zum Nachweis von C. botulinum wurde die PCR nach dem Protokoll von HILL et al. (2010) eingesetzt. Bei der Untersuchung der Praxistauglichkeit wurden Proben aus zehn Biogasanlagen des Freistaates Sachsen entnommen und untersucht. Hierbei handelte es sich um Proben aus Abschnitten vor, während und nach der Fermentation. Anhand der ermittelten Nachweisgrenze sowie der Handhabung wurden die folgenden Nährmedien für die Untersuchung der Biogasanlagen-Proben ausgewählt: Brilliance™ Salmonella Agar, XLT4 Agar, Brilliance™ E. coli/Coliform Selektiv Agar, Slanetz Bartley Agar, Brilliance™ ESBL Agar, TSC Agar überschichtet mit TSC Agarbasis und Eigelb Laktose Agar. Für die Anzucht anaerober Bakterien wurden die Proben vor der Beimpfung der Agarplatten erhitzt. Zudem erfolgte eine Anreicherung des zuvor erhitzten Probenmaterials in TPYG Bouillon. Diese wurde genutzt, um daraus aufgereinigte DNA mittels PCR auf C. botulinum und C. perfringens zu untersuchen. Die verwendeten Nährmedien wurden im Praxistest positiv evaluiert. Die Ergebnisse für die Proben aus den Biogasanlagen zeigten, dass, mit Ausnahme von C. perfringens, alle Indikatororganismen während des Biogasprozesses einer Reduktion unterlagen. Die durchschnittliche anaerobe Lebendkeimzahl belief sich auf 107 bis 108 KbE/g Probe. E. coli erfuhr eine Reduktion um bis zu vier Zehnerpotenzen. Enterokokken wurden um 1 bis 2 log10 Stufen reduziert. ESBL-bildende Enterobacteriaceae konnten in sechs der zehn Biogasanlagen nachgewiesen werden. Hierbei handelte es sich überwiegend um E. coli und Klebsiella spp. In keiner der Proben konnten Salmonellen oder C. botulinum nachgewiesen werden. Typ A war der am häufigsten nachgewiesene C. perfringens-Toxintyp. Das β2-Toxin-Gen wurde in 20 Fällen nachgewiesen. Einmal konnte C. perfringens Typ C, β2-Toxin-Gen-positiv detektiert werden. Der hygienische Status der Gärreste entsprach in etwa dem hygienischen Status von Gülle. In Abhängigkeit vom Indikatorkeim war eine Verbesserung des Status durch eine Reduktion der Keimzahl festzustellen.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal