Background: Mastitis in dairy cattle is a common ailment worldwide. A cause of mastitis can be bacteria such as Escherichia coli. Mastitis is not a deadly ailment and sometimes the dairy cows show no symptoms but if certain virulence genes are present in the bacteria that cause the mastitis, the bacteria can be transmitted to humans and cause severe diseases. The potential presence of enterohemorrhagic Escherichia coli (EHEC) in particular would be a major concern for human health. Aim: The aim for this study was to analyze the presence of virulence genes known to be present in E.coli strains isolated from dairy cows with mastitis in Sweden. Method: A Qiagen BIO ROBOT EZ1 was used to purify DNA from 90 bacterial cultures. A panel of virulence genes were amplified and biotinylated from the purified DNA by PCR and an E.coli based DNA microarray was used to detect presumed virulence genes in E.coli. Result: There were no samples that had all the genes traditionally used to classify E.coli as EHEC or potential EHEC. 63 samples were analyzed without any problems but 27 samples were not fully analyzed. Conclusion: The DNA based microarray proved to be a reliable method to detect genes from pathogenic bacteria but it needed high concentration of purified DNA which was not always easy to obtain. There were some samples in this study that contained virulence genes.

Les Escherichia coli entérohémorragiques (EHEC) sont considérés comme l'un des plus importants groupes de pathogènes émergents responsables de toxi-infections alimentaires. Depuis ces dix dernières années, l'environnement est de plus en plus incriminé dans les épidémies à EHEC. Dans cette étude, nous nous sommes intéressés à la prévalence des E. coli producteurs de Shiga-toxines (STEC) dans les exploitations laitières ainsi qu'à leur survie dans les effluents d'élevages et le sol.<br />Au sein des exploitations laitières, une grande diversité de souches STEC, capables de persister sur de nombreux supports (abreuvoirs, murs et sol des enclos, etc), a été observée. Dans les effluents d'élevages (fumier et lisier bovins non traités), la survie des STEC non-O157 a été évaluée à plus de 90 jours. Lorsque les tas de fumier sont retournés (pratique d'assainissement menée par les exploitants) la survie est de seulement 45 jours ; la température élevée au cœur des andains (≥ 65°C) est associée au déclin important des STEC. <br />En ce qui concerne la survie dans les sols, nous avons montré qu'in vitro les souches STEC O26:H11 persistaient pendant plus d'une année dans différents types de sols mélangés à du fumier, même en présence de taux d'humidité faibles (< 0,08 g H2O g-1 sol sec). La température ambiante (i.e. 20°C versus 4°C) est significativement associée (P<0,001) à une inhibition marquée des STEC. In situ, la survie et le transfert des STEC dans le sol à partir de bouses naturellement contaminées ont également été évalués dans différentes stations d'un bassin versant d'altitude situé dans les Alpes du Nord. Les STEC sont capables de persister dans la matière fécale et sont détectés dans le sol rhizosphérique sous-jacent (jusqu'à 20 cm de profondeur) pendant environ 2 mois, jusqu'à la disparition complète des bouses.<br />Dans la rhizosphère, la survie des STEC peut être affectée par des populations microbiennes productrices de métabolites antibactériens. En prenant comme modèle de populations telluriques antagonistes, une souche de Pseudomonas productrice d'un antibiotique, le 2,4-diacétylphloroglucinol (Phl), aucun effet négatif de la production de cet antibiotique sur la survie d'E. coli O157:H7 dans la rhizosphère du blé n'a pu être cependant mis en évidence. <br />L'ensemble de ces résultats suggère que l'environnement constituerait un réservoir important en STEC dont le contrôle passe notamment par le respect de mesures d'hygiène rigoureuses en exploitations.

A l'heure actuelle, les Escherichia coli producteurs de Shiga-toxines (STEC) sont considérés comme des pathogènes émergents importants en santé publique. Cependant, il n'existe aujourd'hui aucune réglementation officielle stipulant les procédures à suivre pour l'échantillonnage et la recherche des STEC dans les denrées alimentaires. <br />Ce travail a pour objectif d'étudier les différents protocoles utilisés pour la recherche des STEC, de manière à pouvoir proposer aux industriels des protocoles optimisés leur permettant une réelle maîtrise du « danger STEC » dans leur filière. Dans ce but, la cinétique de croissance de diverses souches de STEC a, dans différentes conditions d'enrichissement, été suivie simultanément à celle de la flore annexe de la matrice, puis modélisée.<br />Notre étude souligne qu'un enrichissement trop court, comme les 6 heures d'incubation dans le cas de l'IMS, peut conduire à l'obtention de résultats faussement négatifs. Il s'avère néanmoins inutile, dans certaines conditions, de prolonger l'étape d'enrichissement car une interaction de type compétition simple avec la flore annexe arrête la croissance des STEC. Cet arrêt est plus ou moins rapide selon la matrice analysée et sa densité en flore annexe naturelle (de 4 à 7 h pour les fèces et de 10 à 12 h pour le steak haché dans nos expérimentations). Dans le lait, des interactions plus complexes entraînent un arrêt de la croissance des STEC avant celui de la flore naturelle (8,5 à 11 h dans nos expérimentations).<br />L'utilisation d'agents sélectifs a pour but de freiner la croissance de la flore annexe, ce qui peut avoir pour impact de prolonger la croissance des STEC. L'ajout de sels biliaires dans le milieu d'enrichissement a un effet positif dans le cas de l'enrichissement d'échantillons de fèces de bovins et de lait cru mais n'a pas d'effet significatif pour la matrice « steak haché ». En revanche, l'addition de novobiocine dans le milieu peut inhiber certaines souches de STEC non-O157:H7 et ralentir la croissance de E. coli O157:H7. L'usage de cet antibiotique, potentiellement responsable de résultats faussement négatifs, devrait être abandonné.<br />Par ailleurs, cette étude a permis d'optimiser le protocole de recherche de E. coli O157:H7 dans le steak haché (ISO 16140) en validant, d'une part, l'analyse d'une plus grosse masse d'échantillon dans un même volume de milieu (ratio plus élevé) et en réduisant, d'autre part, le temps d'analyse grâce à l'utilisation d'une température d'incubation plus élevée de 41,5°C.

Shiga toxin-producing E. coli (STEC) cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). All STEC produce one or both of two Shiga toxins, Stx1 and Stx2. STEC strains that produce Stx2 are more strongly associated with HUS than strains that produce Stx1 or both Stx1 and Stx2. Epidemiologic evidence indicates a recent increase in the rate of HUS among STEC outbreaks. The increasing rate of HUS could be explained by a shift in the toxin profiles of STEC strains. The purpose of this study was to examine trends in toxin profiles of human STEC O157 isolates from 1996 to 2008 and to assess whether an increase in the number of Stx2-only-producing strains could be correlated with a recent increase in HUS cases. Data from three independent datasets, collected from PulseNet, eFORS and NARMS, were used. Additionally, trends such as seasonal variations, geographical variations, gender differences, and age differences were examined for each toxin profile. Results from this study show a shift in the toxin profile of human STEC O157 strains in the United States, in that the proportion of Stx2-only producing strains has increased dramatically since 1996.

Master of Science / Food Science Institute - Animal Sciences and Industry / Randall K. Phebus / A variety of antimicrobial processes are used to reduce pathogen risks on commercially processed raw beef. Little research has evaluated chlorinated water on beef tissues, especially in a processing water dip scenario. Interest in nanobubble technology has increased due to its proposed surfactant properties, but it is undetermined whether this improves antimicrobial effectiveness of chlorine-based solutions in food applications. Benchtop studies were conducted to evaluate chlorinated nanobubble waters (0 to 11.94 ppm) against Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O121, O145, and O157:H7 (STEC-7), Salmonella spp., and USDA-approved non-pathogenic STEC surrogates 1) in pure culture with the goal of characterizing the lethality contributions of pH (5 or 7), temperature, free available chlorine level (FAC), inclusion of nanobubbles, or a combination thereof; 2) in select chlorinated nanobubble “red water” (water containing 0.1% beef purge) solutions; and 3) on the surface of lean and fat beef tissue. In pure culture solutions, surrogates demonstrated greater resistance (P ≤ 0.05) to chlorinated solutions (3.4-5.5 log CFU/mL reductions) with increased reductions at the higher (11.94 ppm) FAC levels. STEC-7 and Salmonella population reductions were also notably reduced (3.3-7.1 log CFU/mL) by the higher FAC concentrations. No definitive impacts of temperature, nanobubble inclusion, or acidic pH were observed. At an average 5.23 ppm FAC in red water, all microbial populations were reduced by > 6 log CFU/mL after 60 minutes. Reductions of target organisms on inoculated lean and fat tissues were ≤ 1 log CFU/g in red water; likely due to the inability to maintain FAC levels above 0.7 ppm in the presence of organic loading. An in-plant antimicrobial validation study of a proprietary raw beef manufacturing process was conducted to determine the effectiveness of a recirculating acidic nanobubble water system, chlorinated to 5 ppm FAC using EO water generated concentrate, against the USDA-approved STEC surrogates. Preliminarily, inoculated beef trim was introduced into the system targeting 5 ppm FAC; chlorine concentrate reinfusion rates were determined to establish applicable operational parameters and sampling strategies for the system. An optimized in-plant study was conducted. Meat inoculated at ~ 7 log CFU/g was introduced into the recirculating chlorinated nanobubble system every other day over 6 days, achieving an average 1.6 log CFU/g surrogate reduction on inoculated meat throughout the manufacturing process. Approximately 2.7 log CFU/g of residual surrogates were recovered on non-inoculated meat ~35 minutes after inoculated meat entered the system, indicating that harborage of microbial contamination on processing equipment can lead to subsequent contamination carry-over that must be controlled during processing. Surrogate organisms were recovered by enrichment only from non-inoculated meat 24 h after inoculated meat processing on alternate days, likely stemming from inadequately sanitized processing equipment after inoculated batch processing. Control of the residual surrogate population in the system following inoculation was accomplished through daily equipment sanitation and boosting recirculated processing water to 50 ppm during a 4-h sanitation period (no beef entering system). The optimized study will be used as an antimicrobial process validation against STEC and Salmonella spp. in beef manufacturing.

Orientador: Roberto de Oliveira Roça / Resumo: Um total de 180 bovinos Bos indicus (Nelore), terminados em confinamento, foi dividido em grupos com 6, 12 ou 24 horas de jejum pré-abate para verificar se a redução deste tempo resulta em melhor condição higiênica e sanitária durante o processo de abate. Amostras de fezes pré e pós-jejum, de pele e de carcaça foram colhidas e analisadas para ocorrências e contagens de bactérias aeróbias mesófilas, Escherichia coli genérica, coliformes totais, assim como, E. coli produtora de toxinas Shiga (STEC) e enteropatogênica (EPEC). As amostras de fezes também foram analisadas quanto ao pH e os teores percentuais de matéria seca (MS%) e de fibras em detergente neutro (FDN%) e ácido (FDA%). Os dados foram analisados com modelos logísticos, análises de variância e regressões, ao nível de 5% de significância. A extensão do tempo de jejum pré-abate reduziu a MS%, mas elevou o pH, FDN% e FDA% das fezes. O gradual aumento do jejum pré-abate resultou em maior excreção fecal de STEC (stx1 e stx2), entretanto, com decréscimo de aeróbios mesófilos, mas sem interferência na E. coli genérica. A extensão do tempo de jejum pré-abate também resultou na maior contaminação da pele por STEC (stx1) e EPEC, assim como, das carcaças por aeróbios mesófilos, E. coli genérica e coliformes totais. Dessa forma, conclui-se que a condição higiênica e sanitária durante o processo de abate de bovinos pode ser melhorada com a redução do tempo de jejum pré-abate. Para as indústrias da carne isto pode representar a re... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-09-21Bitstream added on 2014-06-13T19:35:22Z : No. of bitstreams: 1 paula_cjs_me_jabo.pdf: 334942 bytes, checksum: 17d2a17239376692690b930753f05b71 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Cachorros têm sido propostos como possíveis reservatórios de cepas de Escherichia coli patogênicas que causam infecções em cachorros e em humanos. Entre Janeiro e Dezembro de 2006, 92 cepas de E. coli foram analisadas para a detecção da presença de genes produtores de toxina Shigalike (stx 1 e stx 2) e o gene da intimina (eae). Doze cepas de E. coli Shigatoxigênica (STEC) foram detectadas por PCR como portadoras dos genes de Shiga toxina, 7 cepas do gene stx 1 (7,6%), 5 do stx 2 (5,4%) e nenhuma delas apresentando ambos os genes. Nove (9,8%) das cepas de E. coli apresentaram o gene eae e não eram produtoras de Shiga toxina. Os isolados de E. coli foram também examinados para a detecção dos genes codificadores de adesinas (pap, sfa e afa), hemolisina e aerobactina. A freqüência dos genes de virulência detectados nestes isolados foi de 12,0% pap, 1,0% sfa, 10,0% hemolisina e 6,5% aerobactina. Seis destes isolados foram caracterizados como cepas de E.coli patogênica extraintestinal (ExPEC). Entre as cepas de STEC e ExPEC foi encontrado um alto nível de resistência a agentes antimicrobianos (estreptomicina com 81% e 100% e cefalotina com 85,7% e 50% respectivamente) e algumas delas foram caracterizadas como E. coli resistentes a múltiplas drogas (MDREC), o que representa um motivo de preocupação devido ao risco de disseminação dos genes de resistência para a microbiota dos seres humanos. / Dogs have been proposed as a possible reservoir of the pathogenic Escherichia coli that causes infection in dogs and human beings. From January to December 2006, 92 E. coli isolates from 25 diarrheic dogs were analyzed by screening for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twelve Shiga toxin-producing E. coli (STEC) isolates were detected by PCR to harbor the Shiga toxin genes, 7 isolates the stx 1 (7.6%); 5 the stx 2(5.4%) and none both of them. Nine (9.8%) of the E. coli isolates studied were eae positive non Shiga toxin-producing. The E. coli isolates also were screened for the presence of adhesion-encoding genes (pap, sfa, afa), hemolysin and aerobactin genes. Virulence gene frequencies detected in those isolates were: 12.0% pap, 1.0% sfa, 10.0% hemolysin and 6.5% aerobactin. Ten isolates were characterized as extraintestinal pathogenic E. coli (ExPEC) strains. Among STEC and ExPEC isolates was found high level of resistance to antimicrobial agents and some of them as characterized as multidrug resistant E. coli (MDREC), what represent a reason for concern due the risk of dissemination of antimicrobial resistant genes to the microbiota of human beings.

Submitted by GUILHERME SICCA LOPES SAMPAIO null (guilhermeslsampaio@yahoo.com.br) on 2017-01-30T16:59:48Z No. of bitstreams: 1 Tese de Doutorado - Guilherme S. L. Sampaio (versão final).pdf: 1435860 bytes, checksum: d3afaf1a274ca2aaa24f6ebb66f4ae63 (MD5) / Rejected by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: Incluir o número do processo de financiamento nos agradecimentos da dissertação/tese. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-02-01T12:47:13Z (GMT) / Submitted by GUILHERME SICCA LOPES SAMPAIO null (guilhermeslsampaio@yahoo.com.br) on 2017-02-01T12:59:33Z No. of bitstreams: 1 Tese de Doutorado - Guilherme S. L. Sampaio (versão final).pdf: 1436609 bytes, checksum: a731559ab98fc75f6ee45c3b1a2e8208 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-02-01T13:09:02Z (GMT) No. of bitstreams: 1 sampaio_gsl_dr_bot.pdf: 1436609 bytes, checksum: a731559ab98fc75f6ee45c3b1a2e8208 (MD5) / Made available in DSpace on 2017-02-01T13:09:02Z (GMT). No. of bitstreams: 1 sampaio_gsl_dr_bot.pdf: 1436609 bytes, checksum: a731559ab98fc75f6ee45c3b1a2e8208 (MD5) Previous issue date: 2017-01-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Um total de 180 bovinos Bos indicus (Nelore), terminados em confinamento, foi dividido em grupos com 6, 12 ou 24 horas de jejum pré-abate para verificar se a redução deste tempo resulta em melhor condição higiênica e sanitária durante o processo de abate. Amostras de fezes pré e pós-jejum, de pele e de carcaça foram colhidas e analisadas para ocorrências e contagens de bactérias aeróbias mesófilas, Escherichia coli genérica, coliformes totais, assim como, E. coli produtora de toxinas Shiga (STEC) e enteropatogênica (EPEC). As amostras de fezes também foram analisadas quanto ao pH e os teores percentuais de matéria seca (MS%) e de fibras em detergente neutro (FDN%) e ácido (FDA%). Os dados foram analisados com modelos logísticos, análises de variância e regressões, ao nível de 5% de significância. A extensão do tempo de jejum pré-abate reduziu a MS%, mas elevou o pH, FDN% e FDA% das fezes. O gradual aumento do jejum pré-abate resultou em maior excreção fecal de STEC (stx1 e stx2), entretanto, com decréscimo de aeróbios mesófilos, mas sem interferência na E. coli genérica. A extensão do tempo de jejum pré-abate também resultou na maior contaminação da pele por STEC (stx1) e EPEC, assim como, das carcaças por aeróbios mesófilos, E. coli genérica e coliformes totais. Dessa forma, conclui-se que a condição higiênica e sanitária durante o processo de abate de bovinos pode ser melhorada com a redução do tempo de jejum pré-abate. Para as indústrias da carne isto pode representar a redução de custos operacionais e com a infraestrutura dos currais de abate, menos condenações e falhas no controle de produção, maior rendimento produtivo e incremento na validade comercial. A redução de STEC, além de ser um fomento à saúde publica, constitui-se em um estimulo à economia brasileira pela redução de barreiras comerciais e menor devolução de mercadorias. / A total of 180 Bos indicus (Nellore) cattle finished in feedlot were divided into groups of 6, 12 or 24 hours of pre-harvest fasting to verify if time reduction results in better hygiene and sanitary conditions during the slaughter process. Pre-fasting and post-fasting samples of feces, and post-fasting samples of hide, and carcass were collected and analyzed for the occurrence and counting of mesophilic aerobic bacteria, generic Escherichia coli, total coliforms, as well as Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC). The feces samples were also analyzed for pH and content percentage of dry matter (DM%), neuter detergent fiber (NDF%) and acid detergent fiber (ADF%). Data were analyzed with logistic models, analyses of variance and regressions at 5% significance. The extension of pre-harvest fasting time reduced DM% but increased pH, NDF% and ADF% of feces. The gradual increase of pre-harvest fasting time resulted in greater fecal excretion of STEC (stx1 and stx2) with reduction of mesophilic aerobic bacteria, but without interference in generic E. coli. The extension of this time also resulted in greater contamination of hide by STEC (stx1) and EPEC as well as of carcass by mesophilic aerobic bacteria, generic E. coli and total coliforms. Thus, it was concluded that the hygiene and sanitary condition during the slaughter process of bovines can be improved with the reduction of pre-harvest fasting time. For the meat industry, can represent a reduction of costs with operations and facilities of lairage pens, less with condemnations and failures in the production control besides a greater productive yield and increment in the commercial validity. STEC reduction, besides being a development in public health, becomes a stimulus to the Brazilian economy by decreasing commercial barriers and return of goods. / FAPESP: 2015/02208-3 / FAPESP: 2014/01109-9

Escherichia coli are bacteria found in bowels of warm blooded animals. Most subspecies are harmless and part of the normal gut flora. However, E. coli have the ability to exchange genetic material with other bacteria, and some E. coli have acquired genes coding for virulence factors. VTEC, E. coli with the ability to produce verotoxin are commonly found in cattle, but certain types can cause severe disease in humans, known as enterohaemorrhagic E. coli, EHEC.     In this study, isolates of E. coli and other bacteria in the family Enterobacteriaceae from calves were subtyped and clustered using MALDI-TOF. Ten strains were selected for experimental competition analysis against E. coli MG1655.     The aim of the study was to identify strains of bacteria with the potential to outcompete VTEC in the cattle host and decrease the risk of human infections.     Three of the bacterial strains were able to outcompete the laboratory strain, and in future studies these strains can be analysed when competing against VTEC. The rest of the strains were outcompeted. Four known strains of VTEC were analysed competing the laboratory strain, showing weak ability to compete. Finally, a highly pathogenic strain of VTEC was analysed against Escherichia coli Nissle 1917, known for its ability to outcompete many strains of bacteria. Nissle could not outcompete the tested VTEC strain under the tested conditions.     In conclusion the majority of the bacterial strains isolated from calves were identified as E. coli and three of the isolates showed good ability to compete against the laboratory strain.

BACKGROUND: Over the past 5 years, 634 cases of Shigatoxin E. coli (STEC) infection were reported to Tennessee Health Department 1. At our local children’s hospital, 4-5 children are hospitalized with STEC infection each year. Some of these children had no history of ingesting food items that could have placed them at risk to develop STEC infection; however, there are other ways that humans could get infected, such as exposure to contaminated water from cattle farms 2. GOALS: To determine if bodies of water in the city are contaminated with STEC. METHODS: Fifty (50) ml of water samples were collected from selected areas of Johnson City, TN. Samples were inoculated to Sorbitol McConkey Agar (SMA) plates under sterile techniques & incubated at 36C for 18 hours under aerobic conditions. RESULTS: Table 1 E. coli Strains Isolated from Water Samples Colony Types Founders Park Sinking Creek Carroll Creek Cherokee Creek Colorless (STEC) 14 (3.5) 24 (6) 32 (8) 35 (8.75) Pink (Non-STEC ) 8 (2) 3 (0.75) 7 (1.75) 4(1) DISCUSSION/ CONCLUSION: All sampled sites were positive for STEC. STEC is a normal flora of the gastrointestinal tract of cattle. Around city neighborhoods are pastures, as cattle farming is a major livelihood in Northeastern, TN. It is highly possible that water runoff from these pastures contaminates the waters around the city. Public health measures should be undertaken to inform the community that these waters are contaminated with STEC to prevent STEC infection. References: Reportable Conditions. TN Epi-news, TN Health Dept Issue 3, Volume 9, 2016 Escherichia coli O157:H7 Infections in Children Associated with Raw Milk & Raw Colostrum From Cows—California, 2006. MMWR Weekly, 57(23); 625-628, June 23, 2008.