Le travail de thèse a eu pour but de contribuer à une meilleure connaissance de la dangerosité pour l’homme des souches STEC d’origine bovine, en explorant la corrélation pouvant exister entre la présence du gène stx chez de telles souches et la réalité de son expression. La quantification de l’expression du gène stx2 présent chez 46 souches STEC bovines a été réalisée à l’aide d’un test ELISA commercial détectant spécifiquement les shiga toxines, le test ProSpecT® Shiga toxin (OXOID). L’ensemble des résultats de validation préalable obtenus pour ce test a permis de considérer qu’il pouvait être valablement appliqué à l’étude du panel de souches d’E. coli O157 :H7 bovines collectées au laboratoire, tout en déterminant les limites méthodologiques, et donc d’interprétation. Utilisé comme outil de quantification de la production de Stx2 par les souches du panel choisi, et dans deux conditions expérimentales différentes (présence ou absence d’induction par l’enrofloxacine), ce test a permis de mettre en évidence que seulement 15,2% des souches d’E.coli O157:H7/H- étudiés produisent des quantités significatives de Stx2 détectables sans induction, et ce à des niveaux variables. En revanche, la majorité de ces isolats, bien que n'exprimant pas la protéine Stx2 de manière constitutive, produit des quantités significatives de Stx2 en présence de concentrations subinhibitrices d'enrofloxacine, antibiotique de la famille des fluoroquinolones et utilisé en médecine vétérinaire. Enfin, des mutants résistants à l'enrofloxacine sélectionnés à partir de certaines souches d’E. coli O157:H7, produisent, après induction par l'enrofloxacine, 3 fois plus de toxine Stx2 que les souches sauvages. Les mutants sont également inductibles en utilisant des doses d'enrofloxacine 100 fois supérieures à celles utilisables pour les souches sauvages. L’ensemble de ces résultats montre (i) la corrélation, ou non, qui peut exister entre la présence du gène stx2 et son expression, (ii) que la proportion inductible des souches STEC bovines est potentiellement importante, (iii) que l’enrofloxacine induit fortement l’expression du gène stx2 chez les souches STEC bovines et que (iv) l’induction par l’enrofloxacine conduit à des taux d’expression du gène stx2 supérieurs chez des souches résistantes aux fluoroquinolones que chez les souches sensibles. Au final, cette étude contribue à documenter la variabilité des niveaux d’expression des gènes stx et illustre le risque que des STEC issus de bovins puissent devenir plus fréquemment pathogènes pour l'homme suite à l'usage croissant des fluoroquinolones vétérinaires. / The present study contributed to a better knowledge of the pathogenicity of STEC for humans by quantifying the expression of the stx2 gene from a panel of 46 cattle STEC isolates by ELISA. Succesful validation experiments of the ProSpecT® Shiga toxin ELISA (OXOID) first concluded to its capability to be used for a valuable quantification of the Stx2 protein. Stx2 expression was tested in presence and absence of subtherapeutic concentrations of enrofloxacin, an antibiotic of the fluoroquinolones family used in veterinary medicine. Whereas only 15.2% of the strains displayed significant amounts of detectable Stx2 in absence of induction, most of them were shown to be inducible, and at various levels, in presence of subtherapeutic concentrations of enrofloxacin. Also, enrofloxacin-resistant mutants of Stx2-producing E. coli O157:H7 were selected and produced 3-fold higher Stx2 levels than native strains after induction with enrofloxacin. Mutants were also inducible using hundred-fold higher enrofloxacin concentrations than the useful ones for native strains. At the end, these results show (i) the inconstant and variable expression of the stx2 gene from cattle STEC isolates in native conditions, (ii) the potentially high number of inducible STEC isolates in cattle, (iii) that enrofloxacin is a strong inducer of the stx2 expression in cattle STEC isolates and (iv) that the stx2 gene is stronger induced in isolates resistant to fluoroquinolones compared to susceptible ones. Finally, this all study documents the variable expression of the stx2 gene and also suggests that E. coli O157:H7 from cattle may become more frequently pathogenic to humans as a side-effect of the increasing use of veterinary fluoroquinolones.

Escherichia coli produtora de toxina Shiga (STEC) está entre os patógenos envolvidos em surtos de doenças transmitidas por alimentos devido ao consumo de vegetais. No entanto, até agora, os relatos sobre a presença de STEC em vegetais no Brasil são escassos. Esse microrganismo é veiculado por alimentos, contaminados direta ou indiretamente por fezes animais, sendo responsável por um amplo espectro de doenças que compreende desde diarréia leve que pode evoluir para colite hemorrágica (CH), até síndrome hemolítico-urêmica (SHU) e púrpura trombocitopênica trombótica (PTT). O presente estudo teve como objetivo investigar a presença de STEC em vegetais orgânicos cultivados na região metropolitana da cidade de São Paulo, Brasil, caracterizando os fatores de virulência stx1, stx2, eae e ehx, bem como o sorotipo. Um total de 200 amostras de vegetais orgânicos (folhas verdes), obtido a partir de três produtores foi analisado quanto à presença de cepas de STEC. Caldo triptona de soja (TSB) suplementado com vancomicina (8 mg / L), cefixima (50 µg / L) e telurito de potássio (2,5 mg / L) foi utilizado na etapa de pré-enriquecimento, com incubação a 37ºC / 24 h, seguido por semeadura em MacConkey Sorbitol (SMAC) e CHROMagar STEC (CHROM). Após incubação a 37ºC / 24 h, as colônias suspeitas foram confirmadas por testes bioquímicos e submetidas a PCR objetivando a detecção dos genes de virulência stx1, stx2, eae, ehx, e os genes fliCH7 e rfbO157. Entre as 200 amostras de vegetais orgânicos analisadas, 30 (15%) foram positivas para E. coli, mas nenhum isolado apresentou os genes de virulência pesquisados. Nossos resultados indicam baixo risco de infecção devido ao consumo destes produtos frescos em São Paulo, Brasil. No entanto, são necessárias mais pesquisas, abrangendo um maior número de amostras e área pesquisada, uma vez que este patógeno já foi encontrado no meio ambiente em estudos anteriores e poucas pesquisas investigaram a presença de STEC em vegetais no Brasil. / Shiga toxin producing Escherichia coli (STEC) strains are among the pathogens involved in foodborne disease outbreaks due to consumption of vegetables. However, reports on the presence of STEC in vegetables in Brazil are lacking. STEC is an important pathogen transmitted by food, directly or indirectly contaminated with animal feces, responsible for a broad spectrum of diseases varying from mild diarrhea to hemorrhagic colitis (HC), syndrome hemolytic uremic (HUS) and thrombotic thrombocytopenic purpura (TTP). This study aimed at investigating the presence of STEC in organic vegetables in the metropolitan region of São Paulo city, Brazil, characterizing the virulence factors stx1, stx2, eae and ehx as well as identifying the serotype. A total of 200 samples of organic vegetables (green leafy), obtained from three organic producers was analyzed for the presence of STEC strains. Tryptic Soy Broth (TSB) supplemented with vancomycin (8mg/L), cefixim (50µg/L) and potassium telurite (2.5mg/L) was used in the pre enrichment step with incubation at 37°C/24 h, followed by plating onto Sorbitol-MacConkey (SMAC) agar and CHROMagar STEC (CHROM). After incubation at 37°C/24 h, presumptive colonies were confirmed by biochemical tests and submitted to PCR targeting the detection of stx1, stx2, eae and ehx virulence genes, as well as fliCH7 and rfbO157. Among the 200 organic vegetable samples analyzed for STEC strains, 30 (15%) were positive for E. coli, but none of them showed the virulence genes studied. These findings indicate low risk of infection due to the consumption of these fresh produce in Sao Paulo, Brazil. However, more research is required, covering a larger number of samples and area, since this pathogen has already been found in the environment in previous studies, and few research investigating the presence of STEC in vegetables has been reported in Brazil.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Natalia Cernicchiaro / David G. Renter / Cattle shed Shiga toxin-producing Escherichia coli (STEC) in their feces. Therefore, cattle pose a risk to contaminate produce, water, and beef products intended for human consumption. The United States Department of Agriculture Food Safety and Inspection Service consider seven STEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, non-intact beef products. Contrary to O157, the frequency and distribution of non-O157 serogroups and virulence genes have not been well-established in cattle. Therefore, the objectives of my thesis research were: 1) to appraise and synthesize data from peer-reviewed literature on non-O157 serogroup and virulence gene prevalence, and 2) to determine the prevalence of seven STEC in feedlot cattle feces across seasons. A systematic review and meta-analysis of published literature were conducted to gather, summarize, and interpret the existent data regarding non-O157 serogroup and virulence gene prevalence in cattle. Random-effects meta-analyses were used to obtain pooled non-O157 fecal prevalence estimates for continents worldwide and meta-regression analyses were conducted to evaluate effects of specific factors on between-study heterogeneity. Results indicated that non-O157 serogroup and virulence gene fecal prevalence significantly differed (P < 0.05) by geographic region, with North America yielding the highest pooled prevalence estimate worldwide. While previous research has demonstrated a strong seasonal shedding pattern of STEC O157, data regarding the seasonality of non-O157 STEC shedding in cattle is very limited. A repeated cross-sectional study was conducted to obtain serogroup and virulence gene prevalence data for the seven STEC in pre-harvest cattle feces, in summer and winter. We found that non-O157 serogroups were recovered in fecal samples collected in both seasons but virulence genes, thus STEC, were rarely detected in summer and undetected in winter. In conclusion, non-O157 STEC are present in cattle feces at very low frequencies, but STEC O103 and O157 significantly differed (P < 0.05) between seasons. Overall, the research described in this thesis greatly contributes to the limited body of data regarding non-O157 serogroup and virulence gene distribution in cattle and provides a better understanding of two major risk factors, season and geographic distribution, associated with STEC fecal shedding in cattle.

Verschiedene ELISA zur Erkennung von Stx1 und Stx2 in Rinderkotproben wurden auf ihrer Anwendbarkeit hin verglichen. Der ELISA nach RICHTER et al. (1997) unter Anwendung von Hydatidenflüssigkeiten von Echinococcus granulosus als Beschichtungssubstanz und monoklonalen Antikörper als Bindungsmolekül stellte sich als die praktikabelste Lösung im Vergleich mit dem ELISA mit monoklonalen Antikörpern (RANDALL et al., 1997) und einem ELISA mit kommerziellem Gb3 heraus. Der ELISA nach RICHTER et al. (1997) wurde gegen den klassischen Vero-Zell- Neutralisationstest validiert. Zu diesem Zweck wurden 100 E. coli-Feldisolate mit beiden Tests ausgewertet. Die zur Quantifizierung von Ergebnissen angewendete Coulter-Methode (Ermittlung der Überlebensraten der Vero-Zellen mit Hilfe des Z2-Partikelzählers) erwies sich als geeignet, während zwei Färbungsmethoden (KV- und MTT-Färbungsmethode) aufgrund von Einstellungsproblemen bei der spektralfotometrischen Messungen nicht zu auswertbaren Ergebnissen geführt hatten. Die Ergebnisse des ELISA und des Vero-Zell-Neutralisationstest wurden in einer „Two- Graphic Receiver-Operating Characteristic“ (TG-ROC)-Analyse verglichen. Die Indexwerte 0,020 und 0,040 wurden als „cut-off“ für den Stx1- bzw. Stx2-ELISA anhand dieser Analyse 79 festgelegt. Die geschätze Sensitivität und Spezifität für den Stx1-ELISA betrugen 100% (pu = 100%; po = 100%), für den Stx2-ELISA betrug die Sensitivität 100% (pu = 100%; po = 100%) und die Spezifität 94% (pu = 88,4%; po = 99,7%). Nach diesen Ergebnissen ist der ELISA ein geeigneter Test und kann den Vero-Zell-Neutralisationstest ersetzen. Der validierte ELISA wurde in einem Feldversuch parallel mit der MK1/MK2-PCR (KARCH und MEYER, 1989) eingesetzt. In einem Zeitraum vom 15.01.97 bis 31.12.99 wurden 1030 Rinderkotproben mit beiden Tests auf das Vorkommen von STEC untersucht. Die PCR wies stx-Gene bei 500 (49%) der Proben nach. Der ELISA wies nur 307 (30%) Stx-positive nach. Bei 264 positiven und 487 negativen Ergebnissen stimmten die Resultate zu 751 Proben (73%) überein. Der berechnete Kappa-Wert war 0,452. Der Kappa-Wert spricht für eine mäßige Übereinstimmung beider Tests. Diesen Ergebnissen nach sollte die PCR als „pre-screening“-Test favorisiert werden, obwohl eine Kombination beider Tests zum Stx-Nachweis am besten geeignet wäre.

Shiga toxin-producing E. coli (STEC) have important public health and food safety implications. Cattle are the primary reservoir for STEC, which are transmitted to humans via contact with cattle or related food products. Dairy farms in particular have been incriminated as an important source of STEC. The broad aim of this study was to examine in depth the epidemiology of STEC on the dairy farm. The presence of STEC on three Australian dairy farms was surveyed. This aimed to provide data on the prevalence and nature of STEC on Australian dairy farms, as well as to examine in more detail the pre-harvest/slaughter ecology of STEC. STEC, E. coli O157:H7 and E. coli O26:H11 prevalences were similar to those from dairy farms in other countries. Replacement heifers were the most important source of STEC on the farms. Calves excreted STEC from an early age, with faecal prevalence peaking at weaning. Higher STEC prevalence was also associated with group housing of calves during weaning. Calf isolates were potential human pathogens based on serotype and virulence markers. Clonal relationships between isolates were analysed. Calf isolates were diverse and had a high clonal turnover. STEC isolated from within the same farm had a higher genetic similarity than those from different farms. Vertical and horizontal transmission were both identified among cattle. The farm environment was also identified as an important source of STEC. Reasons for increased levels of STEC excretion by calves were investigated. Two broad hypotheses for higher faecal shedding were proposed and examined individually. The first was that an animal is more likely to excrete STEC when its exposure to STEC is greater, thus promoting inoculation of the gastrointestinal tract. Calves were experimentally inoculated with a traceable STEC strain to examine the infection dynamics of STEC within cattle groups, and explore the effect of calf management procedures. Calves which were housed in groups and co-jointly fed and managed had a higher prevalence of the inoculation strain than animals housed individually. The test strain was readily isolated from the hides and saliva of inoculated calves, as well as their immediate environments. Calves become infected with STEC via the faecal-oral route, iv either by direct contact with other calves, or indirectly through contact with faecally contaminated materials. The second hypothesis was that individual animals are variably susceptible to intestinal colonisation by STEC, which leads to differing magnitudes and durations of STEC carriage. Factors influencing colonisation susceptibility to STEC and the mechanisms behind these factors were also examined. In order to compare enteric colonisation under a range of different conditions, a suitable experimental system was developed. In vitro organ culture of explanted ruminant colonic tissues provided a laboratory model that was representative of in vivo bacterial-mucosal attachment. The degree of STEC colonisation was enumerated using an immunofluorescent filtration technique. The quantitative colonisation assay was applied to determine the effects of host-dependant variables on STEC colonisation. Colonic tissues from weaning calves and adult cattle did not differ significantly in their susceptibility to colonisation; nor did tissues from cattle fed either high forage or high grain diets. Colonic explants from sheep, however, demonstrated significantly higher numbers of adherent STEC than bovine explants. It was therefore concluded that while species-specific differences in host tissues may mediate STEC carriage differences, this did not explain in vivo variability in age and diet related excretion. Factors that indirectly affect the susceptibility of host tissues to colonisation were examined. E. coli O157:H7 cultured in media designed to represent the enteric contents of a well-fed ruminant colonised the colonic mucosa in reduced numbers, indicating that age and diet may be correlated with differences in STEC carriage and excretion because of differing physiological augmentation of the intra-enteric environment. In conclusion, while group dynamics and management practices may increase STEC shedding prevalences for cattle via increased STEC exposure, factors that modulate an individual ruminant’s gastrointestinal carriage of STEC have a significant role in mediating STEC excretion. Either directly or indirectly, species, age and diet can affect the numbers of STEC that colonise the bowel wall, thereby influencing the magnitude and duration of STEC excretion. Both of these features of ruminant STEC ecology should be addressed in order to reduce the pre-slaughter/harvest presence of STEC.

Master of Public Health / Department of Diagnostic Medicine and Pathobiology / Robert Larson / Among animal products consumed by humans, ground beef has been reported as one of the most common vehicles for STEC outbreaks in humans. In the United States, cull dairy cattle contribute as one of the primary sources for ground beef. The objective of this study was to determine the prevalence and concentration of 7 Shiga toxin-producing Escherichia coli serogroups (STEC-7; O26, O103, O111, O121, O45, O145, and O157) and associated virulence genes (Shiga toxin 1 and 2 (stx1, stx2), intimin (eae), and enterohomolysin (ehxA)) in the feces of cull dairy cattle processed in commercial slaughter plants during summer months. Fecal swab samples (n=183) were collected from three processing plants, one in California and two in Pennsylvania. At each plant at least 60 to 65 cattle were selected, and the samples were obtained by swabbing the mucosal surface of the recto-anal junction using a sterile cotton-tipped applicator. To determine prevalence, all samples were subjected to culture-based detection methods that included enrichment, serogroup-specific immunomagnetic separation and plating on selective media, followed by polymerase chain reaction for serogroup confirmation and virulence gene detection. Pre-enriched fecal samples were subjected to spiral plating to determine the concentration of STEC-7. A sample was considered STEC positive if a recovered isolate harbored one of the 7 target O genes, stx1, and/or stx2. Of the 183 fecal swab samples collected, 23 (12.6%) harbored at least one O157, O26, O103, or O111 serogroup, with their associated virulence genes. However, none of the fecal samples from this cattle population carried STEC at high-levels (>10⁴ CFU/g). This study has provided important information on STEC-7 prevalence from dairy cattle that enter the ground beef processing system. However, there is still a need to determine prevalence and concentration of STEC in cull dairy cattle during winter months as well as in other sources of ground beef production (e.g., imported lean beef, cull beef).

Submitted by Letícia Fernanda Lavezzo (leticialavezzo.unesp@hotmail.com) on 2016-03-23T20:23:19Z No. of bitstreams: 1 Dissertação_Letícia_Fernanda_Lavezzo.pdf: 1419993 bytes, checksum: 8e337d69094b988dba50c9a0bee85085 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-24T18:21:25Z (GMT) No. of bitstreams: 1 lavezzo_lf_me_jabo.pdf: 1419993 bytes, checksum: 8e337d69094b988dba50c9a0bee85085 (MD5) / Made available in DSpace on 2016-03-24T18:21:25Z (GMT). No. of bitstreams: 1 lavezzo_lf_me_jabo.pdf: 1419993 bytes, checksum: 8e337d69094b988dba50c9a0bee85085 (MD5) Previous issue date: 2016-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O lodo de esgoto é uma alternativa como fertilizante orgânico na agricultura, porém em sua composição pode apresentar patógenos que oferecem risco ao homem e ao ambiente. Objetivou-se, com o presente estudo, avaliar a fertilidade do solo, e a presença de ovos viáveis de helmintos, coliformes termotolerantes, Escherichia coli para os patótipos EHEC, EPEC e STEC e a atividade enzimática das enzimas protease, redutase do nitrato e urease no solo após dezoito anos de aplicações anuais de lodo de esgoto em um Latossolo Vermelho eutroférrico (LVef) e Latossolo Vermelho distrófico (LVd). O lodo utilizado foi obtido na SABESP de Franca, São Paulo e o experimento foi instalado em delineamento de blocos cazualiados, sendo 4 tratamentos e 5 repetições. Os tratamentos foram T1: controle, apenas com aplicação de adubação mineral, T2: 5, T3: 10 e T4: 20 Mg ha-1 de LE. Antes de ser incorporado ao solo, realizou-se análise do lodo para ovos viáveis de helmintos e coliformes termotolerantes. Aos 40 dias, coletou-se amostras de solo na profundidade de 0-10 cm para avalição de ovos viáveis de helmintos no solo. Aos 70 dias, coletou-se amostras de solo na profundidade de 0-20cm para a análise da fertilidade. Para a análise de coliformes termotolerantes, seguindo a técnica de tubos múltiplos, as amostras foram coletadas no dia 0, 26, 40 e aos 78 dias. Para a realização da reação em cadeia da polimerase (PCR) para identificar a presença de Escherichia coli, coletou-se amostras de solo antes do início do experimento, no dia 0, aos 26 dias, 40, 58, 78, 110 e 146 dias. Para a avaliação da atividade enzimática, as amostras foram coletadas em profundidade de 0-10cm, nos dias 0, 40, 78 e aos 146 dias. Os atributos químicos do solo apresentaram efeito significativo entre os tratamentos utilizados. A análise do solo incorporado com o resíduo apresentou ausência total de ovos viáveis de helmintos no solo após 40 dias da aplicação do LE. Os valores de termotolerantes nos solos variaram entre zero a 1,1x106 Número Mais Provável de Sólidos Totais durante o período de 0 a 26 dias. Para análise de Escherichia coli do lodo e do solo, mostrou ausência dos patótipos de EHEC, EPEC e STEC por meio dos primers para os genes stx1, stx2 e aea. A atividade enzimática das enzimas proteases, redutase do nitrato e urease, ao londo do experimento, não apresentaram diferença estatística entre tratamentos. A aplicação do lodo de esgoto por 18 anos consecutivos influenciou nos atributos químicos do solo, não apresentou risco potencial de contaminação do solo por ovos de helmintos e Escherichia coli e não diferiu na atividade enzimática do solo. / The sewage sludge is an alternative as organic fertilizer to use in agriculture, but in its composition may have pathogens that offer to humans and the environment risks. The present study objective was to evaluate soil fertility, and the presence of viable helminth eggs, fecal coliforms, Escherichia coli for pathotypes EHEC, EPEC and STEC and the enzymatic activity of protease enzymes, nitrate reductase and urease in the soil after eighteen years of annual applications of sewage sludge in an Oxisol (LVef) and Oxisol (LVd). The sludge used was obtained in SABESP Franca, São Paulo and the experiment was installed in designing cazualiados blocks, 4 treatments and 5 repetitions. Treatments were T1: control, only with application of mineral fertilizer, T2: 5, T3: T4 10 and 20 Mg ha-1 LE. Before being incorporated into the soil, there was sludge analysis for viable helminth eggs and fecal coliforms. At 40 days, it is collected soil samples at a depth of 0-10 cm for viable helminth eggs evaluation in the soil. After 70 days it is collected soil samples at a depth of 0-20cm for fertility analysis. For fecal coliforms analysis, following the technique of multiple pipes, the samples were collected at day 0, 26, 40 and 78 days. To carry out the polymerase chain reaction (PCR) for the presence of Escherichia coli was collected from soil samples before the beginning of the experiment at day 0, after 26 days 40, 58, 78, 110 and 146 days . For the evaluation of enzyme activity, samples were collected at a depth of 0-10cm, on days 0, 40, 78 and 146 days. The soil chemical properties showed significant effects between treatments. Soil testing embedded with the residue showed complete absence of viable helminth eggs in the soil 40 days after the application of the LE. The values in thermotolerant soil ranged from zero to 1,1x106 Most Probable Number of total solids during the period from 0 to 26 days. For Escherichia coli analysis sludge and soil, showed absence of pathotypes EHEC, EPEC and STEC through primers for stx1, stx2 and aea. The enzymatic activity of protease enzymes, nitrate reductase and urease to the experiment, showed no statistical difference between treatments. The application for 18 consecutive years sewage sludge influenced the soil chemical properties, showed no potential risk of soil contamination by helminth eggs and Escherichia coli and did not differ in the enzymatic activity of the soil.

High-throughput sequencing (HTS) has revolutionized bacterial genomics. Its unparalleled sensitivity has opened the door to analyzing bacterial evolution and population genomics, dispersion of mobile genetic elements (MGEs), and within-host adaptation of pathogens, such as Escherichia coli. One of the defining characteristics of intestinal pathogenic E. coli (IPEC) pathotypes is a specific repertoire of virulence factors (VFs). Many of these IPEC VFs are used as typing markers in public health laboratories to monitor outbreaks and guide treatment options. Instead, extraintestinal pathogenic E. coli (ExPEC) isolates are genotypically diverse and harbor a varied set of VFs -- the majority of which also function as fitness factors (FFs) for gastrointestinal colonization. The aim of this thesis was the genomic characterization of pathogenic and commensal E. coli with respect to their virulence- and antibiotic resistance-associated gene content as well as phylogenetic background. In order to conduct the comparative analyses, I created a database of E. coli VFs, ecoli_VF_collection, with a focus on ExPEC virulence-associated proteins (Leimbach, 2016b). Furthermore, I wrote a suite of scripts and pipelines, bac-genomics-scripts, that are useful for bacterial genomics (Leimbach, 2016a). This compilation includes tools for assembly and annotation as well as comparative genomics analyses, like multi-locus sequence typing (MLST), assignment of Clusters of Orthologous Groups (COG) categories, searching for protein homologs, detection of genomic regions of difference (RODs), and calculating pan-genome-wide association statistics. Using these tools we were able to determine the prevalence of 18 autotransporters (ATs) in a large, phylogenetically heterogeneous strain panel and demonstrate that many AT proteins are not associated with E. coli pathotypes. According to multivariate analyses and statistics the distribution of AT variants is instead significantly dependent on phylogenetic lineages. As a consequence, ATs are not suitable to serve as pathotype markers (Zude et al., 2014). During the German Shiga toxin-producing E. coli (STEC) outbreak in 2011, the largest to date, we were one of the teams capable of analyzing the genomic features of two isolates. Based on MLST and detection of orthologous proteins to known E. coli reference genomes the close phylogenetic relationship and overall genome similarity to enteroaggregative E. coli (EAEC) 55989 was revealed. In particular, we identified VFs of both STEC and EAEC pathotypes, most importantly the prophage-encoded Shiga toxin (Stx) and the pAA-type plasmid harboring aggregative adherence fimbriae. As a result, we could show that the epidemic was caused by an unusual hybrid pathotype of the O104:H4 serotype. Moreover, we detected the basis of the antibiotic multi-resistant phenotype on an extended-spectrum beta-lactamase (ESBL) plasmid through comparisons to reference plasmids. With this information we proposed an evolutionary horizontal gene transfer (HGT) model for the possible emergence of the pathogen (Brzuszkiewicz et al., 2011). Similarly to ExPEC, E. coli isolates of bovine mastitis are genotypically and phenotypically highly diverse and many studies struggled to determine a positive association of putative VFs. Instead the general E. coli pathogen-associated molecular pattern (PAMP), lipopolysaccharide (LPS), is implicated as a deciding factor for intramammary inflammation. Nevertheless, a mammary pathogenic E. coli (MPEC) pathotype was proposed presumably encompassing strains more adapted to elicit bovine mastitis with virulence traits differentiating them from commensals. We sequenced eight E. coli isolates from udder serous exudate and six fecal commensals (Leimbach et al., 2016). Two mastitis isolate genomes were closed to a finished-grade quality (Leimbach et al., 2015). The genomic sequence of mastitis-associated E. coli (MAEC) strain 1303 was used to elucidate the biosynthesis gene cluster of its O70 LPS O-antigen. We analyzed the phylogenetic genealogy of our strain panel plus eleven bovine-associated E. coli reference strains and found that commensal or MAEC could not be unambiguously allocated to specific phylogroups within a core genome tree of reference E. coli. A thorough gene content analysis could not identify functional convergence of either commensal or MAEC, instead both have only very few gene families enriched in either pathotype. Most importantly, gene content and ecoli_VF_collection analyses showed that no virulence determinants are significantly associated with MAEC in comparison to bovine fecal commensals, disproving the MPEC hypothesis. The genetic repertoire of bovine-associated E. coli, again, is dominated by phylogenetic background. This is also mostly the case for large virulence-associated E. coli gene cluster previously associated with mastitis. Correspondingly, MAEC are facultative and opportunistic pathogens recruited from the bovine commensal gastrointestinal microbiota (Leimbach et al., 2017). Thus, E. coli mastitis should be prevented rather than treated, as antibiotics and vaccines have not proven effective. Although traditional E. coli pathotypes serve a purpose for diagnostics and treatment, it is clear that the current typing system is an oversimplification of E. coli's genomic plasticity. Whole genome sequencing (WGS) revealed many nuances of pathogenic E. coli, including emerging hybrid or heteropathogenic pathotypes. Diagnostic and public health microbiology need to embrace the future by implementing HTS techniques to target patient care and infection control more efficiently. / Eines der definierenden Charakteristika intestinal pathogener E. coli (IPEC) Pathotypen ist ein spezifisches Repertoire an Virulenzfaktoren (VFs). Viele dieser IPEC VFs werden als Typisierungsmarker benutzt. Stattdessen sind Isolate extraintestinal pathogener E. coli (ExPEC) genotypisch vielfältig und beherbergen verschiedenartige VF Sets, welche in der Mehrheit auch als Fitnessfaktoren (FFs) für die gastrointestinale Kolonialisierung fungieren. Das Ziel dieser Dissertation war die genomische Charakterisierung pathogener und kommensaler E. coli in Bezug auf ihren Virulenz- und Antibiotikaresistenz-assoziierten Gengehalt sowie ihre phylogenetische Abstammung. Als Voraussetzung für die vergleichenden Analysen erstellte ich eine E. coli VF-Datenbank, ecoli_VF_collection, mit Fokus auf Virulenz-assoziierte Proteine von ExPEC (Leimbach, 2016b). Darüber hinaus programmierte ich mehrere Skripte und Pipelines zur Anwendung in der bakteriellen Genomik, bac-genomics-scripts (Leimbach, 2016a). Diese Sammlung beinhaltet Tools zur Unterstützung von Assemblierung und Annotation sowie komparativer Genomanalysen, wie Multilokus-Sequenztypisierung (MLST), Zuweisung von Clusters of Orthologous Groups (COG) Kategorien, Suche nach homologen Proteinen, Identifizierung von genomisch unterschiedlichen Regionen (RODs) und Berechnung Pan-genomweiter Assoziationsstatistiken. Mithilfe dieser Tools konnten wir die Prävalenz von 18 Autotransportern (ATs) in einer großen, phylogenetisch heterogenen Stammsammlung bestimmen und nachweisen, dass viele AT-Proteine nicht mit E. coli Pathotypen assoziiert sind. Multivariate Analysen und Statistik legten offen, dass die Verteilung von AT-Varianten vielmehr signifikant von phylogenetischen Abstammungslinien abhängt. Deshalb sind ATs nicht als Marker für Pathotypen geeignet (Zude et al., 2014). Während des bislang größten Ausbruchs von Shiga-Toxin-produzierenden E. coli (STEC) im Jahre 2011 in Deutschland waren wir eines der Teams, welches die genomischen Eigenschaften zweier Isolate analysieren konnte. Basierend auf MLST und Detektion orthologer Proteine zu bekannten E. coli Referenzgenomen konnte ihre enge phylogenetische Verwandschaft und Ähnlichkeit des gesamten Genoms zum enteroaggregativen E. coli (EAEC) 55989 aufgedeckt werden. Im Detail identifizierten wir VFs von STEC und EAEC Pathotypen, vor allem das Prophagen-kodierte Shiga-Toxin (Stx) und ein Plasmid des pAA-Typs kodierend für aggregative Adhärenz-Fimbrien. Die Epidemie wurde demnach durch einen ungewöhnlichen Hybrid-Pathotyp vom O104:H4 Serotyp verursacht. Zusätzlich identifizierten wir die Grundlage für den multiresistenten Phänotyp dieser Ausbruchsstämme auf einem Extended-Spektrum-beta-Laktamase (ESBL) Plasmid über Vergleiche mit Referenzplasmiden. Mit diesen Informationen konnten wir ein horizontales Gentransfer-Modell (HGT) zum Auftreten dieses Pathogenen vorschlagen (Brzuszkiewicz et al., 2011). Ähnlich zu ExPEC sind E. coli Isolate boviner Mastitiden genotypisch und phänotypisch sehr divers, und viele Studien scheiterten am Versuch eine positive Assoziation vermeintlicher VFs nachzuweisen. Stattdessen gilt Lipopolysaccharid (LPS) als entscheidender Faktor zur intramammären Entzündung. Gleichwohl wurde ein mammärer pathogener E. coli (MPEC) Pathotyp vorgeschlagen, der mutmaßlich Stämme umfasst, welche eher geeignet sind eine bovine Mastitis auszulösen und über Virulenz-Merkmale von Kommensalen abgegrenzt werden können. Wir sequenzierten acht E. coli Isolate aus serösem Eutersekret und sechs fäkale Kommensale (Leimbach et al., 2016). Bei zwei Mastitisisolaten wurden die Genome vollständig geschlossen (Leimbach et al., 2015). Anhand der genomischen Sequenz des Mastitis-assoziierten E. coli (MAEC) Stamms 1303 wurde das Gencluster zur Biosynthese seines O70 LPS O-Antigens aufgeklärt. Wir analysierten die phylogenetische Abstammung unserer Stammsammlung plus elf bovin-assoziierter E. coli Referenzstämme, aber konnten weder MAEC noch Kommensale bestimmten Phylogruppen innerhalb eines Core-Genom Stammbaums aus Referenz-E. coli eindeutig zuordnen. Eine ausführliche Gengehalt-Analyse konnte keine funktionelle Konvergenz innerhalb von Kommensalen oder MAEC identifizieren. Stattdessen besitzen beide nur sehr wenige Genfamilien, die bevorzugt in einer der beiden Pathotypen vorkommen. Weder eine Gengehalt- noch eine ecoli_VF_collection-Analyse konnte zeigen, dass eine signifikante Assoziation von bestimmten Virulenzfaktoren mit MAEC, im Vergleich zu bovinen fäkalen Kommensalen, besteht. Damit wurde die MPEC Hypothese widerlegt. Auch das genetische Repertoire von Rinder-assoziierten E. coli wird durch die phylogenetische Abstammung bestimmt. Dies ist überwiegend auch bei großen Virulenz-assoziierten Genclustern der Fall, die bisher mit Mastitis in Verbindung gebracht wurden. Dementsprechend sind MAEC fakultative und opportunistische Pathogene, die ihren Ursprung als Kommensale in der bovinen gastrointestinalen Mikrobiota haben (Leimbach et al., 2017). Obwohl traditionelle E. coli Pathotypen in der Diagnostik und Behandlung einen Zweck erfüllen, ist es offensichtlich, dass das derzeitige Typisierungs-System die genomische Plastizität von E. coli zu sehr vereinfacht. Die Gesamtgenom-Sequenzierung (WGS) deckte viele Nuancen pathogener E. coli auf, einschließlich entstehender hybrider oder heteropathogener Pathotypen. Diagnostische und medizinische Mikrobiologie müssen einen Schritt in Richtung Zukunft gehen und HTS-Technologien anwenden, um Patientenversorgung und Infektionskontrolle effizienter zu unterstützen.

Les Escherichia coli producteur de Shiga-toxines (STEC) sont des bactéries pathogènes majeures en santé publique. Elles sont à l'origine d'épidémies de colites hémorragiques et d'une centaine de cas de syndrome hémolytique et urémique par an, en France. Une classification des STEC (plus de 200 sérotypes) a été établie sur la base d'évaluations du risque par l'étude de marqueurs moléculaires directement impliqués dans la virulence, et l'analyse de données épidémiologiques. La virulence n'est cependant pas toujours clairement associée à des facteurs connus ou suffisamment décrits. L'hypothèse des travaux de thèse a été que le niveau de risque associé aux souches pourrait non seulement s'expliquer par le profil de virulence mais également par des spécificités dans les propriétés métaboliques des sérotypes dont les capacités de résistance aux anti-microbiens. Peu de données sont disponibles sur la physiologie des STEC, hormis les propriétés métaboliques exploitées dans le développement de milieux de culture spécifiques. La grande majorité des études se sont concentrées sur le sérogroupe O157 et très peu sur les non-O157.L'objectif de ce travail a été d'approfondir les connaissances sur les capacités métaboliques des souches de STEC afin d'étudier les relations avec le niveau de risque associé aux souches. Ce travail a été divisé en trois parties : (i) l'étude de la résistance au tellurite de potassium (K2TeO3), un oxyanion fortement dommageable pour les membranes, (ii) l'étude des profils de métabolisation de substrats carbonés et (iii) l'étude des capacités de résistance aux antibiotiques et autres antimicrobiens. Une grande variabilité dans les profils de résistance au K2TeO3 a été observée, ainsi qu'un phénomène d'émergence de mutants spontanés. L'utilisation du tellurite pour la détection des STEC peut induire une sous-estimation de leur prévalence. Les classifications des souches en fonction de leur niveau de risque ont pu cependant être reliées à des profils métaboliques particuliers dont la capacité de résistance à certains antimicrobiens. Ces données ont permis d'observer des évolutions verticales spécifiques de certains sérogroupes mais également certaines convergences évolutives inter-sérogroupes. Ces travaux devraient permettre de faire évoluer la spécificité des méthodologies d'identification et de classification des STEC. Ces données pourront être employées par les gestionnaires du risque pour une identification plus fine des STEC pathogènes / Shiga-toxin producing Escherichia coli (STEC) are major human pathogenic bacteria. These bacteria can cause hemorrhagic diarrheal diseases and Haemolytic and Uremic Syndromes. A risk assessment of more than 200 STEC serotypes has been performed using molecular markers of virulence and epidemiological datasets. However, virulence is not always associated with known or sufficiently described factors, questioning the reliability of such classifications. The hypothesis of this PhD work was that risk levels associated with STEC strains should be related not only to virulence specificities but also other metabolic properties such as a specialization for particular C-sources or resistances towards various antimicrobials. Few data were available on STEC physiology, with the exception of datasets regarding the metabolic properties exploited for the development of specific culture media. The aim of this work was to improve knowledge on STEC metabolic capacities and investigate relationships with their respective risk level. This work was divided into three parts: (i) a study of potassium tellurite (K2TeO3) resistance, an oxyanion highly toxic for cell membranes, (ii) a study of carbon metabolic profiles, and (iii) an exhaustive study of antibiotic and other antimicrobial resistances. A great variability in K2TeO3 resistance profiles was observed, as well as a phenomenon leading to the emergence of significant numbers of spontaneous mutants. The use of tellurite for STEC detection was found to lead to an underestimation of their prevalence in food products. Specific metabolic profiles including resistance to certain antimicrobial substances were found related to STEC classifications into risk levels. These data allowed us to observe specific vertical evolutions of these phenotypes per serogroup but some intergroup evolutionary convergences were also observed. These datasets led to the proposal of novel STEC detection and identification schemes. These schemes can be used by risk assessment managers for a better appreciation of STEC risk hazards among food and environmental samples

O consumo crescente de carne de aves requer uma contínua atenção na provisão de alimentos seguros para o consumidor. Entre os agentes emergentes de relevância para a Saúde Pública destaca-se a Escherichia coli produtora de Shiga toxina (STEC), causadora de severa colite hemorrágica e síndrome urêmica hemolítica. Apesar da importância deste microrganismo na carne de ruminantes, sua presença em carne de aves tem sido pouco investigada. O presente estudo teve como objetivo avaliar a ocorrência de STEC em carcaças de aves congeladas comercializadas na cidade de Xanxerê/SC. Um total de 246 carcaças adquiridas em oito supermercados da cidade, de acordo com a disponibilidade na gôndola, foi submetido à rinsagem individual com água peptonada tamponada 1% e submetida à enumeração de coliformes totais e Escherichia coli e pesquisa de STEC pelo protocolo descrito na ISO/TS 13136:2012(E). Após 24 horas de incubação de uma alíquota da rinsagem a 36°C, foi realizada a pesquisa de genes de virulência (stx1, stx2 e eae) por PCR multiplex. Das carcaças amostradas, a maioria era frango “in natura” (57%), seguidos de galinha (16%), galeto (15%), frango temperado (11%) e frango caipira (2%). A enumeração de coliformes totais no líquido de rinsagem resultou em mediana de 5,2 UFC.g-1 (variando de 1 a 2.836,1 UFC.g-1) e de Escherichia coli foi 0,6 UFC.g-1 (variando de 1 242 UFC.g-1), demonstrando uma qualidade higiênico-sanitária satisfatória do produto. Todos os líquidos de rinsagem originados das 246 carcaças avaliadas foram negativos para os genes stx1 e stx2, indicando ausência de STEC nas amostras. Em 12 carcaças (4,88%) foi confirmada a presença de E.coli eae+, indicando a presença do patotipo enteropatogênico (EPEC). Conclui-se que as carcaças de aves comercializadas em Xanxerê apresentam um padrão higiênico sanitário adequado e ausência de STEC considerando uma prevalência detectável de 1,5% na amostra analisada. Por outro lado, detectou-se a presença de EPEC, a qual deve ser investigada para caracterizar as cepas encontradas em termos de potencial importância para a saúde do consumidor. / The increasing consumption of poultry meat requires continued attention in the provision of safe food for the consumer. Emerging agents of relevance to Public Health include Shiga toxin-producing Escherichia coli (STEC), which causes severe hemorrhagic colitis and hemolytic uremic syndrome. Despite the importance of this microorganism in beef, its presence in poultry meat has been few investigated. The present study aimed to evaluate the occurrence of STEC in frozen poultry carcasses marketed in the city of Xanxerê/SC. A total of 246 carcasses purchased from eight city supermarkets, according to on-shelf availability, was submitted to individual rinsing with 1% buffered peptone water and submitted to enumeration of total coliforms and Escherichia coli and STEC detection by the protocol described in ISO / TS 13136: 2012 (E). After 24 hours of incubation at 36 ° C an aliquot of the rinsing, virulence gene (stx1, stx2 and eae) were investigated by multiplex PCR. Of the carcasses sampled, the majority were fresh poultry meat (57%), followed by chicken (16%), young chicken (15%), seasoned poultry meat (11%) and “caipira”type chicken (2%). The enumeration of total coliforms in the rinsing liquid resulted in a median of 5.2 CFU.g-1 (ranging from 1 to 2836.1 CFU.g-1). The median of Escherichia coli was 0.6 CFU.g-1 (ranging from 1 to 242 CFU.g-1), demonstrating a good sanitary and hygienic quality of the product. All rinse liquids from the 246 carcasses tested were negative for the stx1 and stx2 genes, indicating absence of STEC in the samples. In 12 carcasses (4.88%) the presence of E.coli eae + was confirmed, indicating the presence of the enteropathogenic pathotype (EPEC). It is concluded that the poultry carcasses marketed in Xanxerê have an good hygienic sanitary standard and absence of STEC considering a detectable prevalence of 1.5% in the sample analyzed. On the other hand, we detected the presence of EPEC, which should be further investigated to characterize the strains in terms of potential importance for consumer health.