O objetivo deste estudo foi verificar a ocorrência de S. aureus e E. coli O157: H7 no leite de vacas com mastite subclínica e no leite de mistura de 42 propriedades leiteiras localizadas em duas regiões do Estado de São Paulo: São Carlos e Ribeirão Preto. Paralelamente, entre os isolados de S. aureus foi objetivo identificar os produtores de toxinas e determinar sua origem epidemiológica. O isolamento de S. aureus foi realizado em agar Baird-Parker (35ºC, 48h) e a confirmação bioquímica através da catalase, coagulase, termonuclease, produção de acetoína e fermentação aeróbia da maltose. O isolamento de E. coli O157: H7 foi realizado em agar Sorbitol MacConkey MUG (35ºC, 24h). Para confirmação utilizaram-se as provas do IMVC e sorologia através do kit Soro Anti E. coli O157. Para a detecção da TSST-1 e das enterotoxinas A, B, C e D utilizou-se aglutinação reversa passiva em látex (RPLA). A identificação epidemiológica dos isolados de S. aureus foi realizada por eletroforese em gel de campo pulsado (PFGE). A ocorrência de S. aureus no leite individual nas regiões 1 (São Carlos) e 2 (Ribeirão Preto) foram 3,9% e 6,7%, respectivamente. Animais pertencentes às propriedades leiteiras com produção entre 400 L e 1.000 L/dia apresentaram maior risco de veiculação de S. aureus através do leite quando comparadas com propriedades com produção 1.000 L/dia. Quanto ao leite de mistura, verificou-se que a ocorrência de S. aureus foi a mesma em ambas as regiões (19%). A produção simultânea das enterotoxinas B e C foi observada em 4,7% dos isolados de leite individual, enquanto que 4,7% produziram enterotoxina A e toxina TSST-1. A produção de TSST-1, isoladamente, foi constatada em 14,3% dos isolados de leite individual e em 25% do leite de mistura. Houve similaridade genética entre os isolados de S. aureus, evidenciando sua dispersão epidemiológica entre as propriedades avaliadas. A ocorrência de E. coli O157: H7 no leite individual foi 1% na região 1 e 1,6% na região 2. No leite de mistura não foi detectada E. coli O157: H7. Ressalte-se a importância de medidas preventivas para assegurar a qualidade do leite durante a ordenha, a fim de evitar a ocorrência de microrganismos patogênicos, principalmente S. aureus, e conseqüentemente prevenir riscos de veiculação de toxinfecções através deste alimento. / The aim of this study was to verify the occurrence of S. aureus and E. coli O157: H7 in the milk from dairy cows with subclinical mastitis and in the bulk milk from 42 dairy farms located in two regions of São Paulo State (Region 1: São Carlos, Region 2: Ribeirão Preto). Among the S. aureus strains isolated, the aim was to identify the toxin producers and their epidemiological origin. The isolation of S. aureus was conducted using Baird-Parker agar, and the strains were confirmed by catalase, coagulase, thermonuclease, maltose aerobic fermentation and acetoin production. The isolation of E. coli O157: H7 was conducted using Sorbitol MacConkey MUG agar. The strains were confirmed by IMVC and serology using anti E. coli O157 sera. Rapid passive latex agglutination was used for detection of TSST-1 and enterotoxigenic strains of S. aureus. The epidemiological identification was performed using pulsed field gel electrophoresis (PFGE). S. aureus was isolated from 3.9% and 6.7% of the individual milk samples from regions 1 and 2, respectively. Dairy cows belonging to farms with milk production ranging from 400 to 1.000 L/day showed higher risk of S. aureus carrying-over, when compared with dairy farms with milk production < 400 L/day and > 1.000 L/day. In bulk milk samples, the occurrence of S. aureus was the same in both regions evaluated (19%). The simultaneous production of enterotoxin B and C was observed in 4.7% of strains isolated from individual milk samples, while 4.7% produced both enterotoxin A and TSST-1. TSST-1 production alone was observed in 14.3% of S. aureus strains isolated from individual milk and 25% of bulk milk samples. S. aureus strains tested by PFGE demonstrated genetic similarity, showing the dispersion patterns of this microorganism among dairy farms. E. coli O157: H7 was isolated from 1% and 1.6% of individual milk samples from regions 1 and 2, respectively, although it was not detected in bulk milk samples. The importance of preventive measures to ensure milk quality during milking extraction is stressed, aiming to avoid pathogenic agents, mainly S. aureus, and therefore, to prevent the carry-over of food borne diseases to humans through milk.

Shiga Toxin produzierende Escherichia coli (STEC) sind wichtige Erreger von Lebensmittelinfektionen und gelten als Hauptverursacher für die Ausbildung einer hämorrhagischen Colitis und dem lebensbedrohlichen hämolytisch-urämischen Syndrom. Als Hauptvirulenzfaktor und wichtigstes Charakteristikum der STEC wird die Fähigkeit angesehen, Shiga Toxine (Stx) zu produzieren. Die genetische Information für deren Produktion ist im Genom lambdoider Prophagen kodiert. Eine Antibiotikatherapie bei STEC-assoziierten Infektionen wird sehr kontrovers diskutiert, da sowohl die Produktion der Stx als auch die Freisetzung der Bakteriophagen in vitro durch antibiotisch wirkende Substanzen stimuliert werden kann. Der enterohämorrhagische E. coli O157:H7 Stamm EDL933 beherbergt insgesamt 18 lambdoide Prophagen und prophagenähnliche Elemente, darunter den Stx1-kodierenden Phagen CP-933V und den Stx2-kodierenden Phagen BP-933W. Ziel der vorliegenden Arbeit war es, den Einfluss des Gyrasehemmers Norfloxacin auf das Gesamttranskriptom von EDL933 mit Hilfe der DNA-Microarraytechnologie und unter Verwendung des MWG E. coli O157 Arrays umfassend zu untersuchen und insbesondere die Expression der Prophagengene zu analysieren. Hierfür wurde der E. coli O157:H7 Stamm EDL933 mit 200 ng/ml Norfloxacin inkubiert, Gesamt-RNA isoliert und diese mittels reverser Transkription in cDNA synthetisiert, wobei ein Einbau von fluoreszenzmarkierten Nukleotiden erfolgte. Diese cDNA wurde mit den auf dem E. coli O157 Array befindlichen Oligonukleotiden hybridisiert. Die Auswertung der Fluoreszenzintensitäten ermöglicht eine Analyse der Genexpression. Infolge der Induktion von EDL933 mit 200 ng/ml Norfloxacin zeigten 118 Spots eine Hochregulation und 122 eine Deregulation von Genen an. Bei genauerer Betrachtung resultierte auf Grund der Inkubation mit Norfloxacin eine erhöhte Expression von 52 Genen der Stx-kodierenden Phagen CP-933V und BP-933W. Insbesondere erfolgte eine erhöhte Regulation von Genen der späten Region des BP-933W. Das stxA2-Gen wurde dabei im Vergleich zur nicht-induzierten Kultur 158-fach stärker exprimiert. Im Falle des Stx1-Phagen wiesen nur einige Gene der frühen Region eine gesteigerte Genaktivität auf. Auffallend war die Hochregulation einzelner Gene der nicht Stx-kodierenden Phagen. Gene des Primärstoffwechsels, u. a. Gene der Aminosäurebiosynthese, des Energiehaushaltes und der Zellteilung zeigten eine verminderte Genaktivität nach Induktion. Diese Ergebnisse weisen darauf hin, dass die verwendete Konzentration von Norfloxacin große Auswirkungen auf das Gesamttranskriptom des untersuchten Stammes haben und insbesondere die Genexpression von zehn im Genom des EDL933 befindlichen Prophagen erhöht wurde. Die durch die Microarrayversuche erhaltenen Expressionsraten wurden durch Quantifizierung der cDNA mittels RT Real-Time PCR Untersuchungen überprüft. Zusammenfassend ist festzustellen, dass Norfloxacin neben der antibiotischen Wirkung in der hier verwendeten geringen Konzentration multiple Effekte auf E. coli O157:H7 ausübt und die Auswirkungen in Zukunft noch detaillierter untersucht werden müssen. Die DNA-Microarraytechnologie und der kommerziell erhältliche E. coli O157 Array ermöglichen diese umfassenden Analyse des Transkriptionsprofils. Im Rahmen dieser Arbeit konnte diese Technologie für Untersuchungen der Genexpression von E. coli O157 etabliert und validiert werden. / Infection with Shiga toxin producing Escherichia coli (STEC) is a serious cause of bloody diarrhea and sporadic cases and outbreaks of food-related diseases such hemorrhagic colitis and the hemolytic uremic syndrome (HUS) worldwide. The use of antibiotics in therapy of STEC-associated diseases has been discussed controversially. Release of phage-encoded Shiga toxins is the major virulence factor of enterhemorrhagic Escherichia coli (EHEC). The genome of the EHEC strain E. coli O157:H7 EDL933 contains 18 prophages or prophages elements, including the Stx1- and Stx2-encoding phages CP-933V and BP-933W. Stx-production and Stx-prophage induction can be stimulated by certain antibiotics, e.g. mitomycinC or UV light. The aim of this study was to investigate the influence of a low concentration of the gyrase inhibitor norfloxacin on the whole transcriptom of E. coli O157:H7 strain EDL933 and particularly on the gene expression of prophages using the DNA-microarraytechnology and the commercial available MWG E. coli O157 Array. To determine this, E. coli O157:H7 cultures were incubated with 200 ng/ml norfloxacin. Total RNA was isolated and labelled with fluorescence dyes during reverse transcription. Following this, the labelled cDNA was hybridized with the commercial E. coli O157 Arrays and the fluorescence intensities were measured, analysed and evaluated with appropriate software. Results of this study have indicated that a low concentration of norfloxacin have profound effects on the trancriptome of E. coli O157:H7. Under the conditions applied (200 ng/ml norfloxacin) and an incubation time of 120 minutes, 118 spots indicated a transcriptional upregulation and 122 spots a transcriptional downregulation of E. coli O157:H7 genes present on the array. In detail, 85 spots could be ascribed to EDL933 phage genes. Fifty-two of them could be assigned to the Shiga toxin encoding phages CP-933V and BP-933W, the others belonged to the non-Stx encoding phages or prophages elements existing in the EDL933 genome. Conspicuous, genes present in the late region of the BP-933W prophage were induced most strongly, up to 158-fold in the case of stxA2. Only some genes present in the early region of the Stx1 encoding phage CP-933V were induced upon induction with norfloxacin. Notably, only some genes of the non-Stx phages of EDL933 appeared to be induced after induction. The additional upregulated genes were related to recombination and stress functions and to E. coli O157:H7 RIMD0509952 genes. The majority of downregulated genes belonging to primary metabolism, such as amino acid biosynthesis, cell division and energy metabolism. In conclusion, an induction of E. coli O157:H7 strain EDL933 with 200 ng/ml norfloxacin has profound effects on the transcriptome of E. coli O157:H7, in particular on the global gene expression of more then ten prophages. The DNA-microarray technology and especially the E. coli O157 Array offer a modern tool for analysis of transcription profiles of the serious pathogen EHEC O157 in response to stress, e.g. antibiotics. In the context of this work, the DNA-microarraytechnology could be established and validated to provide the opportunity for further studies about the global effects on the whole transcriptome of E. coli O157.

[Truncated abstract] The applications of biosensors are rapidly expanding with the increased emphasis placed on the use of technology in the evaluation of food safety and also in military use. The United States food industry carried out 144.3 million microbiological tests in 1999 (Alocilja and Radke, 2003). These numbers are expected to rise with the recently implemented regulatory measures for food safety in the United States. In fact, similar trends in food safety are occurring on a global scale. Furthermore, with the recognition and establishment of Microbial Forensics as a new field of forensics, the interest in biosensor development for the detection of microbes will thrive. Moreover, the recent spate of biocrimes, notably the anthrax scares, has called for newer and improved techniques for the sensitive, rapid and reproducible detection of microbes. Biosensors have the capability to fill this role as an efficient device for microbial detection. There is a wide range of biosensors available for different purposes. In addition, their versatility allows for their overlap in many fields. The quartz crystal microbalance (QCM) is a biosensor that is cost-efficient, sensitive, field-deployable with the ability to perform automated, real-time assays within minutes. The QCM is a mass sensitive device that works on the principle where a change in mass deposited on the crystal is inversely proportional to the change in the resonant frequency of the crystal. Therefore, frequency decreases with increasing mass deposited. The QCM has been used in several studies as a biosensor for the detection of a number of viral and bacterial species. ... High antibody incubation concentration required a shorter antibody incubation duration. Conversely, low antibody incubation concentration required a longer antibody incubation duration. Furthermore, regardless of antibody incubation concentration, a distinct pattern in the rate of antibody binding with time was observed. One hour antigen incubation at ambient room temperature (22.5oC) was sufficient for the efficient binding of the antigens to the immobilized antibody layer. Extension of antigen binding time to 15 hours produced inconsequential differences in readings. The binding efficiency of the quartz crystals after a storage period of 2 to 4 weeks at ambient room temperature (22.5oC) fared better than the crystals that were refrigerated at 4oC. Results showed that 0.2M glycine hydrochloride is a poor reagent for the removal of the antigen layer on the quartz crystals for repeated assay use. The 16-mercaptohexadecanoic acid (MHDA) layer and adsorbed proteins on the quartz crystals can be removed by a mixture of sulphuric acid and hydrogen peroxide, known as a piranha process. This allows the crystals to be repeatedly recoated and reused. Overall, this research provides new insights into the preparation process of the quartz crystals for the specific detection of E. coli O157:H7. Conclusive results have been obtained for several tested parameters and suggestions have been raised for further studies in the optimization of the QCM for the E. coli O157:H7 detection process. With improved knowledge and recognition in the capability of the QCM as a biosensor, the QCM may soon be used in conjunction with conventional techniques for the rapid detection of E. coli O157:H7.

O objetivo deste estudo foi verificar a ocorrência de S. aureus e E. coli O157: H7 no leite de vacas com mastite subclínica e no leite de mistura de 42 propriedades leiteiras localizadas em duas regiões do Estado de São Paulo: São Carlos e Ribeirão Preto. Paralelamente, entre os isolados de S. aureus foi objetivo identificar os produtores de toxinas e determinar sua origem epidemiológica. O isolamento de S. aureus foi realizado em agar Baird-Parker (35ºC, 48h) e a confirmação bioquímica através da catalase, coagulase, termonuclease, produção de acetoína e fermentação aeróbia da maltose. O isolamento de E. coli O157: H7 foi realizado em agar Sorbitol MacConkey MUG (35ºC, 24h). Para confirmação utilizaram-se as provas do IMVC e sorologia através do kit Soro Anti E. coli O157. Para a detecção da TSST-1 e das enterotoxinas A, B, C e D utilizou-se aglutinação reversa passiva em látex (RPLA). A identificação epidemiológica dos isolados de S. aureus foi realizada por eletroforese em gel de campo pulsado (PFGE). A ocorrência de S. aureus no leite individual nas regiões 1 (São Carlos) e 2 (Ribeirão Preto) foram 3,9% e 6,7%, respectivamente. Animais pertencentes às propriedades leiteiras com produção entre 400 L e 1.000 L/dia apresentaram maior risco de veiculação de S. aureus através do leite quando comparadas com propriedades com produção 1.000 L/dia. Quanto ao leite de mistura, verificou-se que a ocorrência de S. aureus foi a mesma em ambas as regiões (19%). A produção simultânea das enterotoxinas B e C foi observada em 4,7% dos isolados de leite individual, enquanto que 4,7% produziram enterotoxina A e toxina TSST-1. A produção de TSST-1, isoladamente, foi constatada em 14,3% dos isolados de leite individual e em 25% do leite de mistura. Houve similaridade genética entre os isolados de S. aureus, evidenciando sua dispersão epidemiológica entre as propriedades avaliadas. A ocorrência de E. coli O157: H7 no leite individual foi 1% na região 1 e 1,6% na região 2. No leite de mistura não foi detectada E. coli O157: H7. Ressalte-se a importância de medidas preventivas para assegurar a qualidade do leite durante a ordenha, a fim de evitar a ocorrência de microrganismos patogênicos, principalmente S. aureus, e conseqüentemente prevenir riscos de veiculação de toxinfecções através deste alimento. / The aim of this study was to verify the occurrence of S. aureus and E. coli O157: H7 in the milk from dairy cows with subclinical mastitis and in the bulk milk from 42 dairy farms located in two regions of São Paulo State (Region 1: São Carlos, Region 2: Ribeirão Preto). Among the S. aureus strains isolated, the aim was to identify the toxin producers and their epidemiological origin. The isolation of S. aureus was conducted using Baird-Parker agar, and the strains were confirmed by catalase, coagulase, thermonuclease, maltose aerobic fermentation and acetoin production. The isolation of E. coli O157: H7 was conducted using Sorbitol MacConkey MUG agar. The strains were confirmed by IMVC and serology using anti E. coli O157 sera. Rapid passive latex agglutination was used for detection of TSST-1 and enterotoxigenic strains of S. aureus. The epidemiological identification was performed using pulsed field gel electrophoresis (PFGE). S. aureus was isolated from 3.9% and 6.7% of the individual milk samples from regions 1 and 2, respectively. Dairy cows belonging to farms with milk production ranging from 400 to 1.000 L/day showed higher risk of S. aureus carrying-over, when compared with dairy farms with milk production < 400 L/day and > 1.000 L/day. In bulk milk samples, the occurrence of S. aureus was the same in both regions evaluated (19%). The simultaneous production of enterotoxin B and C was observed in 4.7% of strains isolated from individual milk samples, while 4.7% produced both enterotoxin A and TSST-1. TSST-1 production alone was observed in 14.3% of S. aureus strains isolated from individual milk and 25% of bulk milk samples. S. aureus strains tested by PFGE demonstrated genetic similarity, showing the dispersion patterns of this microorganism among dairy farms. E. coli O157: H7 was isolated from 1% and 1.6% of individual milk samples from regions 1 and 2, respectively, although it was not detected in bulk milk samples. The importance of preventive measures to ensure milk quality during milking extraction is stressed, aiming to avoid pathogenic agents, mainly S. aureus, and therefore, to prevent the carry-over of food borne diseases to humans through milk.

Shiga Toxin produzierende Escherichia coli (STEC) sind wichtige Erreger von Lebensmittelinfektionen und gelten als Hauptverursacher für die Ausbildung einer hämorrhagischen Colitis und dem lebensbedrohlichen hämolytisch-urämischen Syndrom. Als Hauptvirulenzfaktor und wichtigstes Charakteristikum der STEC wird die Fähigkeit angesehen, Shiga Toxine (Stx) zu produzieren. Die genetische Information für deren Produktion ist im Genom lambdoider Prophagen kodiert. Eine Antibiotikatherapie bei STEC-assoziierten Infektionen wird sehr kontrovers diskutiert, da sowohl die Produktion der Stx als auch die Freisetzung der Bakteriophagen in vitro durch antibiotisch wirkende Substanzen stimuliert werden kann. Der enterohämorrhagische E. coli O157:H7 Stamm EDL933 beherbergt insgesamt 18 lambdoide Prophagen und prophagenähnliche Elemente, darunter den Stx1-kodierenden Phagen CP-933V und den Stx2-kodierenden Phagen BP-933W. Ziel der vorliegenden Arbeit war es, den Einfluss des Gyrasehemmers Norfloxacin auf das Gesamttranskriptom von EDL933 mit Hilfe der DNA-Microarraytechnologie und unter Verwendung des MWG E. coli O157 Arrays umfassend zu untersuchen und insbesondere die Expression der Prophagengene zu analysieren. Hierfür wurde der E. coli O157:H7 Stamm EDL933 mit 200 ng/ml Norfloxacin inkubiert, Gesamt-RNA isoliert und diese mittels reverser Transkription in cDNA synthetisiert, wobei ein Einbau von fluoreszenzmarkierten Nukleotiden erfolgte. Diese cDNA wurde mit den auf dem E. coli O157 Array befindlichen Oligonukleotiden hybridisiert. Die Auswertung der Fluoreszenzintensitäten ermöglicht eine Analyse der Genexpression. Infolge der Induktion von EDL933 mit 200 ng/ml Norfloxacin zeigten 118 Spots eine Hochregulation und 122 eine Deregulation von Genen an. Bei genauerer Betrachtung resultierte auf Grund der Inkubation mit Norfloxacin eine erhöhte Expression von 52 Genen der Stx-kodierenden Phagen CP-933V und BP-933W. Insbesondere erfolgte eine erhöhte Regulation von Genen der späten Region des BP-933W. Das stxA2-Gen wurde dabei im Vergleich zur nicht-induzierten Kultur 158-fach stärker exprimiert. Im Falle des Stx1-Phagen wiesen nur einige Gene der frühen Region eine gesteigerte Genaktivität auf. Auffallend war die Hochregulation einzelner Gene der nicht Stx-kodierenden Phagen. Gene des Primärstoffwechsels, u. a. Gene der Aminosäurebiosynthese, des Energiehaushaltes und der Zellteilung zeigten eine verminderte Genaktivität nach Induktion. Diese Ergebnisse weisen darauf hin, dass die verwendete Konzentration von Norfloxacin große Auswirkungen auf das Gesamttranskriptom des untersuchten Stammes haben und insbesondere die Genexpression von zehn im Genom des EDL933 befindlichen Prophagen erhöht wurde. Die durch die Microarrayversuche erhaltenen Expressionsraten wurden durch Quantifizierung der cDNA mittels RT Real-Time PCR Untersuchungen überprüft. Zusammenfassend ist festzustellen, dass Norfloxacin neben der antibiotischen Wirkung in der hier verwendeten geringen Konzentration multiple Effekte auf E. coli O157:H7 ausübt und die Auswirkungen in Zukunft noch detaillierter untersucht werden müssen. Die DNA-Microarraytechnologie und der kommerziell erhältliche E. coli O157 Array ermöglichen diese umfassenden Analyse des Transkriptionsprofils. Im Rahmen dieser Arbeit konnte diese Technologie für Untersuchungen der Genexpression von E. coli O157 etabliert und validiert werden. / Infection with Shiga toxin producing Escherichia coli (STEC) is a serious cause of bloody diarrhea and sporadic cases and outbreaks of food-related diseases such hemorrhagic colitis and the hemolytic uremic syndrome (HUS) worldwide. The use of antibiotics in therapy of STEC-associated diseases has been discussed controversially. Release of phage-encoded Shiga toxins is the major virulence factor of enterhemorrhagic Escherichia coli (EHEC). The genome of the EHEC strain E. coli O157:H7 EDL933 contains 18 prophages or prophages elements, including the Stx1- and Stx2-encoding phages CP-933V and BP-933W. Stx-production and Stx-prophage induction can be stimulated by certain antibiotics, e.g. mitomycinC or UV light. The aim of this study was to investigate the influence of a low concentration of the gyrase inhibitor norfloxacin on the whole transcriptom of E. coli O157:H7 strain EDL933 and particularly on the gene expression of prophages using the DNA-microarraytechnology and the commercial available MWG E. coli O157 Array. To determine this, E. coli O157:H7 cultures were incubated with 200 ng/ml norfloxacin. Total RNA was isolated and labelled with fluorescence dyes during reverse transcription. Following this, the labelled cDNA was hybridized with the commercial E. coli O157 Arrays and the fluorescence intensities were measured, analysed and evaluated with appropriate software. Results of this study have indicated that a low concentration of norfloxacin have profound effects on the trancriptome of E. coli O157:H7. Under the conditions applied (200 ng/ml norfloxacin) and an incubation time of 120 minutes, 118 spots indicated a transcriptional upregulation and 122 spots a transcriptional downregulation of E. coli O157:H7 genes present on the array. In detail, 85 spots could be ascribed to EDL933 phage genes. Fifty-two of them could be assigned to the Shiga toxin encoding phages CP-933V and BP-933W, the others belonged to the non-Stx encoding phages or prophages elements existing in the EDL933 genome. Conspicuous, genes present in the late region of the BP-933W prophage were induced most strongly, up to 158-fold in the case of stxA2. Only some genes present in the early region of the Stx1 encoding phage CP-933V were induced upon induction with norfloxacin. Notably, only some genes of the non-Stx phages of EDL933 appeared to be induced after induction. The additional upregulated genes were related to recombination and stress functions and to E. coli O157:H7 RIMD0509952 genes. The majority of downregulated genes belonging to primary metabolism, such as amino acid biosynthesis, cell division and energy metabolism. In conclusion, an induction of E. coli O157:H7 strain EDL933 with 200 ng/ml norfloxacin has profound effects on the transcriptome of E. coli O157:H7, in particular on the global gene expression of more then ten prophages. The DNA-microarray technology and especially the E. coli O157 Array offer a modern tool for analysis of transcription profiles of the serious pathogen EHEC O157 in response to stress, e.g. antibiotics. In the context of this work, the DNA-microarraytechnology could be established and validated to provide the opportunity for further studies about the global effects on the whole transcriptome of E. coli O157.

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

Orientador: Manoel Victor Franco Lemos / Banca: Fernando Antonio de Ávila / Banca: Felipe Perecin / Resumo: Escherichia coli O157:H7 é uma cepa de importância crescente por estar associada a vários surtos graves de doença em humanos, a maioria derivada do consumo de carne bovina crua ou mal cozida. Os bovinos constituem seu reservatório mais importante, aventando-se a hipótese de que mudanças do regime alimentar em confinamentos atuariam favoravelmente ao aparecimento de cepas shigatoxigênicas. Neste estudo objetivou-se verificar, comparativamente durante o abate, a prevalência desse sorotipo e o comportamento de métodos indicadores como a contagem total de microrganismos viáveis (CTMV) e de contaminação fecal - coliformes totais e E. coli, em amostras de fezes e em carcaças de bovinos terminados a pasto e em confinamento, possibilitando a disponibilização de subsídios necessários aos programas de Análise de Perigos e Pontos Críticos de Controle (APPCC) e de Análise de Risco (RA), empregados na redução do risco de doenças transmitidas por alimentos. Identificados os lotes de acordo com a terminação (dez de cada tipo), desses foram aleatoriamente colhidas e analisadas 100 amostras de suabe retal, 100 amostras de carcaças e 67 amostras de "recortes" da desossa (carne industrial) utilizando-se, para a E. coli O157- H7, técnica automatizada de PCR. À exceção de uma única amostra de recortes (0,37%), as demais, tanto de fezes quanto de carcaças, foram negativas para a cepa pesquisada. Além de contatar-se uma prevalência muito baixa, não se evidenciou diferenças entre os tipos de terminação dos animais. Os resultados dos indicadores - CTMV, de coliformes totais e E. coli, foram considerados aceitáveis em 91%, 85% e 93% das amostras, respectivamente, oferecendo suporte e concordância com a baixa prevalência encontrada. / Abstract: Escherichia coli O157:H7 is an important strain that has been associated with outbreaks of serious disease in humans, most being derived from consumption of raw or poorly cooked beef. It is likely that cattle are an important reservoir, suggesting the possibility that changes in feedlot diet favor the emergence of shigatoxigenic strains of E. coli. This study is intended to verify, comparatively during bovine slaughter, the occurrence of E. coli O157:H7 associated with the sampling results obtained by means of general indicator methods (total viable count) and fecal contamination indicators (coliforms and E. coli). Samples will be taken from both excreta and carcasses of cattle finished either on pasture or feedlot, allowing the provision of subsidies necessary for Hazard Analysis and Critical Control Points (HACCP) and Risk Analysis (RA) programs and applied in the reduction of the risk of foodborne diseases. After identification of batches according to the type of finishing (feedlot or pasture), samples were randomly collected and analyzed. 100 rectal swabs, 100 samples from carcasses sponging, and 67 samples of "sliced meat" from the boning room (industrial meat). An automatic PCR technique for detection of E. coli O157:H7 was used. Except for one sample of sliced meat (0.37%), all others, both for excreta and carcasses, were negative for the O157:H7 E. coli strain. There were no significant differences in prevalence between the types of cattle finishing of the animals. The results of the indicators methods (TVC, coliforms and E. coli); were considered acceptable in 91%, 85% and 93% of tested samples, respectively, supporting and in agreement with low prevalence of O157:H7 found. / Mestre

Escherichia coli O157:H7 can contaminate dropped apples used for juicing via contact with manure or fecally tainted irrigation water and attach to the flesh of the apple through bruises and wounds where surface sanitizers are not effective. The goal of this project was to determine the efficacy of gamma irradiation at the maximum allowed dose of 1000 Gy to inactivate Escherichia coli O157: H7 in whole apples used for juicing. Whole apples were punctured to simulate wounds which were then inoculated with an outbreak strain of E.coli O157:H7 and subjected to gamma irradiation at doses upto 1000 Gy. The D-value of the E.coli O157:H7 strain was 334 Gy indicating that irradiation at 1000 Gy would result in a 3-log reduction of this pathogen. Contaminated apples were also stored for 3 weeks at refrigerated temperature during which time E.coli O157:H7 survived but did not grow. The inoculated apples were juiced, and the juice was stored up to 72 h. There was no change in counts of E.coli O157:H7 in the juice from the control apples, but irradiation at >600 Gy reduced counts by >3 logs, and survivors were not detected after 72 h storage. Sensory testing of juice treated at 652 Gy indicated consumers could tell the difference from control juice, due mostly to greater sweetness of the juice from irradiated apples. These results show that E.coli O157:H7 can easily survive in bruised apples and the juice made from them. Irradiation at 1000 Gy can provide significant lethality of E.coli O157:H7 in apples and juice conferring a greater level of safety without negative effects on sensory quality.

Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (Feb. 23, 2007). Includes bibliographical references.

The secondary metabolites of citrus such as limonoids and flavonoids constitute an important part of human diet. The present work was undertaken to elucidate the effect of citrus limonoids and flavonoids on the bacterial cell-cell signaling in Vibrio harveyi, Escherichia coli O157:H7 and Salmonella Typhimurium LT2. The first experiment was focused on purification of limonoids from grapefruit and sour orange seeds. The limonoids were extracted using organic solvents and purified by chromatographic techniques. A total of ten limonoids (7 aglycones and 3 glucosides) were purified. Currently, simultaneous measurement of aglycones and glucosides of limonoids is not available. To address this limitation, an analytical method using high performance liquid chromatography was developed with the capability of measuring both aglycones and glucosides in a single run. Furthermore, its applicability in the fruit and juice samples was demonstrated. The third study investigated the V. harveyi cell-cell signaling inhibitory potential of purified limonoids. Isolimonic acid, ichangin, obacunone and nomilin were showed potent inhibitory activity. Furthermore, isolimonic acid and ichangin inhibit the signal transduction pathway by up-regulating the response regulator luxO. Isolimonic acid was also found to be a potent inhibitor of Escherichia coli O157:H7 cell-cell signaling in the fourth study. The results demonstrated that isolimonic acid inhibits the autoinducer/epinephrine mediated cell-cell signaling, biofilm and virulence in QseBC and QseA dependent fashion. Further investigations using limonin analogues, in the fifth study, demonstrated that the analogue limonin-7-methoxime inhibited the E. coli biofilm in type 1 pili and antigen 43 dependent-fashion, by preventing the binding of the adhesins to plastic surfaces. Another limonoid, obacunone was demonstrated to attenuate the Salmonella virulence by repressing Salmonella Pathogenicity Island 1 (SPI-1) in EnvZ/OmpR dependent mecahnism. The seventh study showed that naringenin, among the flavonoids, was the most potent inhibitor of V. harveyi and E. coli O157:H7 cell-cell signaling. Furthermore, naringenin was found to repress the (SPI-1) in PstS-HilD dependent fashion in the eighth study. In conclusion, the current project identified several limonoids and flavonoids with cell-cell signaling inhibitory property in three bacterial species.