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Pathogenic Escherichia coli strains cause a large number of diseases in humans, including diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, urinary tract infections, and neonatal meningitis, while in animals they cause diseases such as calf scours and mastitis in cattle, post-weaning diarrhea and edema disease in pigs, and peritonitis and airsacculitis in chickens. The different E. coli pathotypes are characterized by the presence of specific sets of virulence-related genes. Therefore, it is not surprising that pathogenic E. coli constitutes a genetically heterogeneous family of bacteria, and they are continuing to evolve. Rapid and accurate molecular methods are critically needed to detect and trace pathogenic E. coli in food and animals. They are also needed for epidemiological investigations to enhance food safety, as well as animal and human health and to minimize the size and geographical extent of outbreaks. The serotype of E. coli strains has traditionally been determined using antisera raised against the >180 different O- (somatic) and 53 H- (flagellar) antigens. However, there are many problems associated with serotyping, including: it is labor-intensive and time consuming; cross reactivity of the antisera with different serogroups occurs; antisera are available only in specialized laboratories; and many strains are non-typeable. Molecular serotyping targeting O-group-specific genes within the E. coli O-antigen gene clusters and genes that are involved in encoding for the different flagellar types offers an improved approach for determining the E. coli O- and H-groups. Furthermore, molecular serotyping can be coupled with determination of specific sets of virulence genes carried by the strain offering the possibility to determine O-group, pathotype, and the pathogenic potential simultaneously. Sequencing of the O-antigen gene clusters of all of the known O-groups of E. coli is now complete, and the sequences have been deposited in the GenBank database. The sequence information has revealed that some E. coli serogroups have identical sequences while others have point mutations or insertion sequences and type as different serogroups in serological reactions. There are also a number of other ambiguities in serotyping that need to be resolved. Furthermore, new E. coli O-groups are being identified. Therefore, there is an essential need to resolve these issues and to revise the E. coli serotype nomenclature based on these findings. There are emerging technologies that can potentially be applied for molecular serotyping and detection and characterization of E. coli. On a related topic, the genome sequence of thousands of E. coli strains have been deposited in GenBank, and this information is revealing unique markers such as CRISPR (clustered regularly interspaced short palindromic repeats) and virulence gene markers that could be used to identify E. coli pathotypes. Whole genome sequencing now provides the opportunity to study the role of horizontal gene transfer in the evolution and emergence of pathogenic E. coli strains. Whole genome sequencing approaches are being investigated for genotyping and outbreak investigation for regulatory and public health needs; however, there is a need for establishing bioinformatics pipelines able to handle large amounts of data as we move toward the use of genetic approaches for non-culture-based detection and characterization of E. coli and for outbreak investigations.

whole genome sequencing --- detection --- pathogenic E. coli --- E. coli characterization --- Molecular serotyping --- Genetic Markers --- subtyping --- outbreak investigation --- Shiga toxin-producing E. coli --- virulence genes

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Pathogenic Escherichia coli strains cause a large number of diseases in humans, including diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, urinary tract infections, and neonatal meningitis, while in animals they cause diseases such as calf scours and mastitis in cattle, post-weaning diarrhea and edema disease in pigs, and peritonitis and airsacculitis in chickens. The different E. coli pathotypes are characterized by the presence of specific sets of virulence-related genes. Therefore, it is not surprising that pathogenic E. coli constitutes a genetically heterogeneous family of bacteria, and they are continuing to evolve. Rapid and accurate molecular methods are critically needed to detect and trace pathogenic E. coli in food and animals. They are also needed for epidemiological investigations to enhance food safety, as well as animal and human health and to minimize the size and geographical extent of outbreaks. The serotype of E. coli strains has traditionally been determined using antisera raised against the >180 different O- (somatic) and 53 H- (flagellar) antigens. However, there are many problems associated with serotyping, including: it is labor-intensive and time consuming; cross reactivity of the antisera with different serogroups occurs; antisera are available only in specialized laboratories; and many strains are non-typeable. Molecular serotyping targeting O-group-specific genes within the E. coli O-antigen gene clusters and genes that are involved in encoding for the different flagellar types offers an improved approach for determining the E. coliO- and H-groups. Furthermore, molecular serotyping can be coupled with determination of specific sets of virulence genes carried by the strain offering the possibility to determine O-group, pathotype, and the pathogenic potential simultaneously. Sequencing of the O-antigen gene clusters of all of the known O-groups of E. coli is now complete, and the sequences have been deposited in the GenBank database. The sequence information has revealed that some E. coli serogroups have identical sequences while others have point mutations or insertion sequences and type as different serogroups in serological reactions. There are also a number of other ambiguities in serotyping that need to be resolved. Furthermore, new E. coli O-groups are being identified. Therefore, there is an essential need to resolve these issues and to revise the E. coli serotype nomenclature based on these findings. There are emerging technologies that can potentially be applied for molecular serotyping and detection and characterization of E. coli. On a related topic, the genome sequence of thousands of E. coli strains have been deposited in GenBank, and this information is revealing unique markers such as CRISPR (clustered regularly interspaced short palindromic repeats) and virulence gene markers that could be used to identify E. coli pathotypes. Whole genome sequencing now provides the opportunity to study the role of horizontal gene transfer in the evolution and emergence of pathogenic E. coli strains. Whole genome sequencing approaches are being investigated for genotyping and outbreak investigation for regulatory and public health needs; however, there is a need for establishing bioinformatics pipelines able to handle large amounts of data as we move toward the use of genetic approaches for non-culture-based detection and characterization of E. coli and for outbreak investigations.

whole genome sequencing --- detection --- pathogenic E. coli --- E. coli characterization --- Molecular serotyping --- Genetic Markers --- subtyping --- outbreak investigation --- Shiga toxin-producing E. coli --- virulence genes

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Pathogenic Escherichia coli strains cause a large number of diseases in humans, including diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, urinary tract infections, and neonatal meningitis, while in animals they cause diseases such as calf scours and mastitis in cattle, post-weaning diarrhea and edema disease in pigs, and peritonitis and airsacculitis in chickens. The different E. coli pathotypes are characterized by the presence of specific sets of virulence-related genes. Therefore, it is not surprising that pathogenic E. coli constitutes a genetically heterogeneous family of bacteria, and they are continuing to evolve. Rapid and accurate molecular methods are critically needed to detect and trace pathogenic E. coli in food and animals. They are also needed for epidemiological investigations to enhance food safety, as well as animal and human health and to minimize the size and geographical extent of outbreaks. The serotype of E. coli strains has traditionally been determined using antisera raised against the >180 different O- (somatic) and 53 H- (flagellar) antigens. However, there are many problems associated with serotyping, including: it is labor-intensive and time consuming; cross reactivity of the antisera with different serogroups occurs; antisera are available only in specialized laboratories; and many strains are non-typeable. Molecular serotyping targeting O-group-specific genes within the E. coli O-antigen gene clusters and genes that are involved in encoding for the different flagellar types offers an improved approach for determining the E. coliO- and H-groups. Furthermore, molecular serotyping can be coupled with determination of specific sets of virulence genes carried by the strain offering the possibility to determine O-group, pathotype, and the pathogenic potential simultaneously. Sequencing of the O-antigen gene clusters of all of the known O-groups of E. coli is now complete, and the sequences have been deposited in the GenBank database. The sequence information has revealed that some E. coli serogroups have identical sequences while others have point mutations or insertion sequences and type as different serogroups in serological reactions. There are also a number of other ambiguities in serotyping that need to be resolved. Furthermore, new E. coli O-groups are being identified. Therefore, there is an essential need to resolve these issues and to revise the E. coli serotype nomenclature based on these findings. There are emerging technologies that can potentially be applied for molecular serotyping and detection and characterization of E. coli. On a related topic, the genome sequence of thousands of E. coli strains have been deposited in GenBank, and this information is revealing unique markers such as CRISPR (clustered regularly interspaced short palindromic repeats) and virulence gene markers that could be used to identify E. coli pathotypes. Whole genome sequencing now provides the opportunity to study the role of horizontal gene transfer in the evolution and emergence of pathogenic E. coli strains. Whole genome sequencing approaches are being investigated for genotyping and outbreak investigation for regulatory and public health needs; however, there is a need for establishing bioinformatics pipelines able to handle large amounts of data as we move toward the use of genetic approaches for non-culture-based detection and characterization of E. coli and for outbreak investigations.

whole genome sequencing --- detection --- pathogenic E. coli --- E. coli characterization --- Molecular serotyping --- Genetic Markers --- subtyping --- outbreak investigation --- Shiga toxin-producing E. coli --- virulence genes --- whole genome sequencing --- detection --- pathogenic E. coli --- E. coli characterization --- Molecular serotyping --- Genetic Markers --- subtyping --- outbreak investigation --- Shiga toxin-producing E. coli --- virulence genes

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Suite à la demande de certains producteurs wallons de beurre au lait cru confrontés à un problème récurrent de contamination de leur produit en E. coli, DiversiFerm a souhaité étudier cette problématique pour mieux la comprendre et pouvoir apporter aux producteurs des conseils afin d’y remédier. L’étude de la contamination en E coli du beurre au lait cru fait l’objet de ce travail. 
E. coli est une bactérie dont la présence ou non est un indicateur d’hygiène du procédé de fabrication. Les normes énoncées dans le Règlement (CE) n°2073/2005 encadrent le taux de contamination dans les denrées alimentaires afin d’offrir une garantie d’hygiène aux consommateurs. 
Le processus de fabrication du beurre compte plusieurs étapes : la traite, qui n’entre pas dans le cadre de ce travail, l’écrémage, la maturation, le barattage, le lavage, le malaxage et le conditionnement. L’étude repose sur le suivi de trois cycles de production de beurre au lait cru dans six fermes distinctes confrontées à la présence répétée d’E. coli dans le beurre produit. La méthode de travail pour cette étude a consisté à prélever des échantillons de surfaces (cinq points de prélèvement et trois répétitions) et des produits présents au cours du processus de fabrication du beurre : lait entier, crème, lait écrémé, crème maturée et beurre (cinq répétitions par produit). Ces prélèvements ont eu lieu à différentes étapes du processus de fabrication, à savoir avant et après l’écrémage, après la maturation et après le barattage. La présence et les dénombrements de E. coli ont été réalisés à l’aide de Petrifilms. La contamination mesurée traduit la qualité du nettoyage de l’équipement en ce qui concerne les 
échantillons de surfaces, et de la qualité microbiologique pour cette bactérie dans les produits laitiers. 
Selon la classification du règlement européen, sur les 18 lots de beurres échantillonnés et testés dans cette étude, un seul lot était de qualité satisfaisante, trois étaient acceptables et les 14 autres insatisfaisants, soit 78%. 
Les résultats des prélèvements de surfaces montrent qu’un point critique de contamination est le 
tuyau d’arrivée du lait entier. Un deuxième point a pu être mis en évidence : l’orifice de passage du lait entier vers l’écrémeuse. La propreté de ces deux points est donc primordiale. Pour ce qui est de la contamination en E. coli dans les produits laitiers, l’étude a essayé d’identifier les facteurs pouvant l’influencer. Une corrélation pour le taux d’E. coli ou de coliformes a pu être établie par régression linéaire entre plusieurs couples de produits. Des tests statistiques ANOVA ont, eux, permis de révéler l’importance de la qualité microbiologique en E. coli du lait entier utilisé, la qualité étant liée dans cette étude au type d’équipement de traite utilisé (salle ou robot de traite) et à la propreté de cet équipement évaluée à la sortie du lactoduc. En effet, dans les exploitations de l’étude utilisant des robots, le lait entier était mis en attente d’être écrémé dans un réservoir tampon pendant plusieurs heures sans refroidissement, permettant ainsi le développement d’E. coli. Des tests GLM ont montré l’impact favorable de l’utilisation de ferments pour la maturation de la crème. Enfin, l’effet du mélange de différents lots de crème maturée pour un même barattage a également pu être mis en évidence dans les résultats. 
Des pistes de solution ont été émises pour remédier à la contamination en E. coli chez les producteurs de l’étude. Celles-ci pourraient être appliquées à d’autres producteurs confrontés à une problématique semblable. Ces pistes consistent en l’amélioration du nettoyage de l’équipement, la diminution du temps d’attente du lait entier non refroidi avant l’écrémage, et l’ensemencement de la crème avec des ferments lactiques pour la maturation. 
Des études ultérieures pourront approfondir le sujet et vérifier les hypothèses émises. Following the request of some Walloon raw milk butter producers faced with a recurrent problem of E. coli contamination of their product, DiversiFerm wished to study the issue to understand it better and to be able to provide producers with recommendations to remedy it. The study of Escherichia coli contamination in raw milk butter is the subject of this paper. 
E. coli is a bacterium whose presence or absence is an indicator of hygiene for the manufacturing process. The standards set out in Regulation (EC) No 2073/2005 govern the level of contamination in food products in order to provide consumers with a guarantee of hygiene. 
The butter manufacturing process includes several stages: milking, which is outside the scope of this work, skimming, maturation, churning, washing, kneading and packaging. 
The study relies on the monitoring of three production cycles of raw milk butter on six different farms faced with the repeated presence of E. coli in the resulting butter. The method of work for this study consisted of taking samples of surfaces (five sampling points and three repetitions) and samples of the products present during the butter production process: whole milk, cream, skimmed milk, ripened cream and butter (five repetitions per product). These samples were taken at different stages of the manufacturing process, i.e. before and after skimming, after maturation and after churning. The E. coli in the samples were cultured on Petrifilm on the same day so that they could be enumerated. The level of E. coli measured on the Petrifilms reflects the quality of the cleaning of the equipment for the surface samples and the microbiological quality for this bacterium in the dairy products. Based on the classification of the European Regulation, out of 18 batches of butter sampled and tested in this study, only one batch was of satisfactory quality, three were acceptable and the other 14 unsatisfactory, i.e. 78%. 
The results of the surface sampling show that a critical point of contamination is the whole milk supply pipe. A second point was found to be the whole milk inlet to the skimmer. The cleanliness of these two points is therefore of paramount importance. As for E. coli contamination in dairy products, the study tried to identify possible contributing factors. A correlation for E. coli or coliform levels could be established by linear regression between several product pairs. Statistical ANOVA tests revealed the importance of the microbiological quality in E. coli of the whole milk used, the quality being linked in this study to the type of milking equipment used (parlour or milking robot) and to the cleanliness of this equipment rated at the outlet of the milk duct. 
Indeed, on those farms participating in the study which use robots, the whole milk was left to stand in the buffer tank for several hours without cooling before skimming, thus allowing the development of E. coli. GLM tests showed the favorable impact of using ferments for cream maturation. Finally, the effect of mixing different batches of ripened cream for the same churning could also be seen in the results. 
Suggestions for solutions to the E. coli contamination of the producers in the study were made. 
These solutions may be applicable to other producers facing a similar problem. The solutions include improving equipment cleaning, reducing the waiting time of uncooled whole milk before skimming, and inoculating the cream with lactic ferments for maturation. 
Additional studies may further investigate the subject and verify the hypotheses.

Beurre --- Lait cru --- Escherichia coli --- E. coli --- Petrifilm --- Hygiène alimentaire --- Normes microbiologiques --- Butter --- Raw milk --- Escherichia coli --- E. coli --- Petrifilm --- Food safety --- Microbiological standards --- Sciences du vivant > Microbiologie

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Fed-batch Fermentation is primarily a practical guide for recombinant protein production in E. coli using a Fed-batch Fermentation process. Ideal users of this guide are teaching labs and R&D labs that need a quick and reproducible process for recombinant protein production. It may also be used as a template for the production of recombinant protein product for use in clinical trials. The guide highlights a method whereby a medium cell density - final Ods = 30-40 (A600) - Fed-batch Fermentation process can be accomplished within a single day with minimal supervision. This process can also be d

Biology --- Health & Biological Sciences --- Microbiology & Immunology --- Escherichia coli. --- E. coli (Bacterium) --- Escherichia