No description available.

Summary : Enterococcus faecium possesses a low affinity PBP5 that, like in other enterococci, is an important factor contributing to the intrinsic resistance to penicillin. The structure of PBP5 soluble form in complex with benzylpenicillin has been solved at 2.1 Å resolution. On the basis of this 3D structure, it was proposed that the I623KEKQDEKG631 turn connecting β3 and β4 strands and constituting one wall of the active site, as well as, R464 involved in a salin-bridge near the active site could be responsible for the low β-lactam affinity of PBP5fm The k2/K of sPBP5fm acylation by various b-lactams have been determined and, as expected for a low affinity PBP, they are very low. Mutants were constructed to determinate the role of the residues pointed in the 3D structure as related to the observed low affinity. For the sPBP5fm∆I7G mutant, the acylation rate constants are similar to those measured for sPBP5fm. However, when compared to the wild type, the mutants without salin-bridge show a higher affinity that could be explained as the consequence of an easier accessibility to the active site for the inactivator. This study allows mapping active site cavity and determines the importance of structural elements to understand the low affinity of PBP5. Furthermore, three news β-lactams (ceftaroline, ceftobiprole and ME1036) were tested against PBP5fm and they all exhibit inhibitory activity against the protein. Ceftaroline appears as the best inhibitor of sPBP5fm as well as the best inhibitor while tested on Enterococcus faecium cultures. Résumé : Enterococcus faecium possède une protéine PBP5 de faible affinité, qui est un facteur important de la résistance intrinsèque à la pénicilline. La structure de la forme soluble dans PBP5 complexés avec la benzylpénicilline a été résolue à 2,1 Å. Sur la base de cette structure 3D, il a été proposé que la boucle I623KEKQDEKG631 reliant brins β3 et β4 et pouvant constituer une barrière pour atteindre le site actif, ainsi que, R464 impliqué dans un pont-salin près du site actif qui pourrait être responsable de la faible affinité β-lactamines de PBP5fm. Dans un 1er temps, les k2/K de sPBP5fm sauvage par diverses β-lactamines ont été déterminées, et comme prévu, elles sont très faibles. Dans un 2nd temps, les mutants ont été produits, purifiés et caractérisés de manière à déterminer le rôle des résidus choisis dans la faible affinité. Pour le mutant sPBP5fmΔI7G, les constantes de vitesse acylation sont similaires à ceux mesurée pour sPBP5fm sauvage. Tandis que les mutants sans le pont salin R464-D481 montrent une relative plus grande affinité par rapport au type sauvage, qui pourrait être expliqué par un accès plus facile de linhibiteur au site actif. Cette étude permet une première cartographie de la cavité du site actif de PBP5fm et qui détermine limportance ou non déléments de structure pour comprendre la faible affinité. Trois nouvelles β-lactamines (ceftaroline, ceftobiprole et ME1036) ont été testés contre PBP5fm et elles présentent une activité inhibitrice de PBP5fm. La ceftaroline apparaît comme le meilleur inhibiteur de PBP5fm ainsi que le meilleur inhibiteur lors des essais sur les cultures Enterococcus faecium.

No description available.

Enterococcus faecium is a leading cause of hospital-acquired infection, disproportionally affecting immunocompromised and critically ill patients. Despite infection control measures, rates of vancomycin-resistant E. faecium (VREfm) bacteraemias have failed to decline in the United Kingdom, and Cambridge University Hospitals (CUH) report the highest numbers nationally. The aims of my PhD were to use epidemiological and genomic surveillance data to establish risk factors for acquisition and infection with E. faecium in patients at CUH, and to use a One Health approach to consider possible sources for hospital patients by relating bloodstream-associated isolates with those cultured from livestock and the environment in the same geographic region. A retrospective matched nested case control study was performed to determine risk factors for VRE bacteraemia relating to antibiotic exposure. 235 cases were matched to 220 controls for length of admission, year, specialty and ward type. Multivariable analysis demonstrated that duration of exposure to parenteral vancomycin, fluoroquinolones and meropenem were independently associated with VRE bacteraemia. This provides evidence for the importance of antimicrobial stewardship targeting high-risk antibiotics in patients at risk of VRE bacteraemia. VREfm bacteraemia may be complicated by disease recurrence. Whole genome sequencing was used to distinguish between relapse and reinfection in 14 episodes of recurrent VREfm bacteraemia. This demonstrated that 10 (71%) episodes were due to reinfection with a new strain, with reinfection being more likely with increasing time between two positive cultures. This study also evaluated 9 patients with blood cultures positive for both VREfm and vancomycin-susceptible E. faecium (VSEfm), the majority (78%) of which were found to be unrelated strains. More than half of all study isolates from these two patient groups were closely related to another isolate causing bacteraemia at CUH, suggesting that hospital acquisition of VREfm is a driver for infection and recurrence. A cross-sectional study of E. faecium in raw and treated wastewater from 20 municipal water treatment plants across the East of England revealed widespread dissemination of healthcare-associated lineages of VREfm in all sampled locations including rural areas, and environmental release in treated wastewater in 17/20 locations. Wastewater isolates were genetically intermixed with isolates causing bacteraemia at CUH, including highly related isolates indicating recent transmission between the two reservoirs. These findings are consistent with widespread distribution of healthcare-associated VREfm in community populations. A One Health approach incorporating sampling from livestock (10 pork, 10 cattle, 9 poultry farms) detected no VREfm in animals whilst 2 independent meat surveys demonstrated VREfm in 1-2% of uncooked products. Genomic comparison of >1400 E. faecium isolates from livestock, meat, wastewater and almost 800 people with bloodstream infection demonstrated that livestock and human isolates were genetically distinct. Analysis of the accessory genome added further evidence for distinct gene content associated with niche adaptation. An analysis of mobile genes encoding antibiotic resistance revealed limited evidence of sharing between human and animal populations. A prospective longitudinal study in haematology patients at CUH over 6 months revealed high rates of VREfm carriage (63% of cases) and environmental contamination (49% of samples). Genomic analysis elucidated complex colonisation dynamics with frequent loss and acquisition of subtypes, including unsuspected acquisition of new VREfm subtypes in patients already colonised with VREfm, and multiple transmission chains involving patients and the environment, including some leading to bacteraemia. These findings highlight the shortcomings of infection control and environmental cleaning and provide the basis for revised interventions.

Summary Objectives and hypothesis: This thesis concerns the study of plasmids of vancomycin- resistant enterococci isolated from feces of American crows in the years 2012 - 2013 period. The hypothesis is that, in various environments, there is one or more types of epidemiologically significant vanA gene-carrying plasmids that are capable of horizontally spread. Methods: Based on PFGE method the number and size of plasmids were detected in selected isolates of vancomycin-resistant E. faecium. Using PCR method the isolates were subjected to detection of genes of replicases, relaxases and toxin-antitoxin system of plasmid-bound resistance genes. Using 19 primers were characterized types of Tn1546. Results: Of the 12 tested vancomycin-resistant isolates of E. faecium the following number and size of plasmids was proven using PFGE method: 2 isolates contained two plasmids (17%), 3 isolates contained three plasmids (25 %), 5 isolates contained four plasmids (42 %) and 2 isolates contained five plasmids (17 %). All isolates (n = 12) were then subjected to the detection of genes of replicases, relaxases and toxin-antitoxin system for typing of plasmids from each plasmid families. RepA_N family of plasmids: genes characterizing plasmids related to pRUM: rep17 in 11 isolates (92 %), gene Axe-Txe was detected in 5 isolates (42 %) genes characterizing plasmids related to pLG1: rep20 in 7 isolates (58 %) genes characterizing plasmids related to pAD1: relpAD1 gene was detected in one isolate (8 %) Inc18 family of plasmids: genes characterizing plasmids related to pIL501: rep1 gene detected in one case (8 %) genes characterizing plasmids related to pRES25: rep2 gene in 2 isolates (17 %) genes characterizing plasmids related to pEF1: relpEF1 detected in 11 isolates (92 %) pHTB family of plasmids: genes characterizing plasmids related to pHTB: rep22 gene was detected in 4 isolates (33%) and in 2 isolates gene relpHTB was detected (17%) RCR family of plasmids: genes characterizing plasmids related to pRI: positive detection of Rep14 gene in 8 isolates (67%) and in 4 isolates relpRI gene was detected Small theta-replicating plasmids: genes characterizing plasmids related to pEF418 plasmids: rep18a gene in 2 isolates (17%) genes characterizing plasmids related to pB82: rep18b gene was detected in one isolate (8%) genes characterizing plasmids related to pCIZ2: relpCIZ2 gene was detected in 9 isolates tested (75%) Types of transposon Tn1546 Using the PCR method types of Tn1546 were characterized. In 4 isolates (n = 12; 33 %) Tn1546 was characterized as a F3 type. In one isolate (8 %) Tn1546 was characterized as a type F5, in one isolate (8 %) as a type PP-16. In 6 isolates Tn1546 was untypeable. Most likely these are new, yet unknown types. Conclusion: This is the first study of plasmids of vancomycin-resistant isolates E. faecium isolated from feces of American crows. These results emphasize not only a high proportion of plasmids in individual isolates, but also a high proportion of genes with horizontal transfer.

Submitted by Juliana Jabur Damiao Polete null (36492619870) on 2016-05-19T02:12:33Z No. of bitstreams: 1 DISSERTAÇÃO - JULIANA WITZLER 28-03-16.pdf: 908490 bytes, checksum: 813f3b334ef44efd2e6b6abb35e2702e (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-05-23T14:22:27Z (GMT) No. of bitstreams: 1 witzler_jjp_me_arafcf.pdf: 908490 bytes, checksum: 813f3b334ef44efd2e6b6abb35e2702e (MD5) / Made available in DSpace on 2016-05-23T14:22:27Z (GMT). No. of bitstreams: 1 witzler_jjp_me_arafcf.pdf: 908490 bytes, checksum: 813f3b334ef44efd2e6b6abb35e2702e (MD5) Previous issue date: 2016-03-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A utilização de microrganismos probióticos para a manutenção da saúde bucal tem despertado o interesse da comunidade científica, tendo em vista que estudos indicam que tais microrganismos podem conferir benefícios como: atividade anticariogênica, tratamento de doenças periodentais e halitose, e redução na população de microrganismos patogênicos relacionados a patologias bucais. Nesse sentido, o objetivo do presente trabalho foi o desenvolvimento e a caracterização de uma pastilha alimentícia diet contendo o microrganismo Enterococcus faecium CRL 183 e a avaliação do potencial da referida cepa probiótica em sobreviver em saliva humana e inibir a multiplicação da cepa patogênica Streptococcus mutans ATCC 25175. O microrganismo E. faecium CRL 183 foi microencapsulado por extrusão e coacervação em matriz complexa. A técnica de coacervação complexa apresentou o melhor desempenho em relação à manutenção da viabilidade durante o período de armazenamento à temperatura ambiente (23ºC), sendo selecionada para a produção das pastilhas potencialmente probióticas. As pastilhas foram processadas em três tratamentos: PC – pastilha controle, sem adição do probiótico; PPP1 – com adição do microrganismo probiótico e PPP2 – pastilha com adição do microrganismo probiótico e de inulina (prebiótico). Análises microbiológicas (viabilidade e segurança), físico-químicas (umidade, atividade de água, pH e cor) e sensoriais (testes de aceitação e intenção de compra) foram conduzidas no tempo inicial e ao longo do tempo de estocagem. A sobrevivência do probiótico à saliva e a inibição da multiplicação da cepa patogênica, foram avaliadas através da inoculação da cepa pura e das pastilhas em saliva e da técnica de difusão em poços, respectivamente. A cepa de E. faecium teve sua viabilidade reduzida (p<0,05) imediatamente após a produção das pastilhas e durante o tempo de armazenamento à temperatura ambiente, em ambos os tratamentos PPP1 e PPP2. Na etapa de análise sensorial, as pastilhas apresentaram médias de aceitação superiores a seis para os atributos aroma, textura e impressão global, sem diferirem significativamente entre si (p<0,05). A adição da cepa probiótica e da substância prebiótica reduziu a aceitação do produto em relação à aparência e cor e melhorou a impressão dos consumidores em relação ao sabor. As análises físico-químicas revelaram que as formulações PC, PPP1 e PPP2 não diferiram entre si em relação aos parâmetros físico-químicos avaliados, com exceção do parâmetro cor. Todos os tratamentos apresentaram características microbiológicas adequadas durante os 28 dias de estudo. A cepa E. faecium CRL 183 inoculada em saliva humana na forma de pastilhas e de células livres teve sua população de células viáveis aumentada após 24 horas de incubação. O teste de difusão em poços evidenciou que a cepa probiótica foi capaz de inibir a multiplicação de S. mutans ATCC 25175 nas condições do estudo. Os resultados obtidos indicam que a associação do probiótico com inulina melhorou a aceitação do produto em relação ao atributo sabor e que a cepa probiótica sobrevive à saliva humana e apresenta potencial para inibir a multiplicação do microrganismo causador de cárie dental – S. mutans ATCC 25175. No entanto, a queda brusca da viabilidade da cepa probiótica durante o armazenamento das pastilhas, indica a necessidade de aprimoramento do processo de obtenção das mesmas, além de adequação da embalagem utilizada à matriz alimentícia. / The interest in the local effect of probiotic microorganisms is increasing among the scientific community, since studies have indicated that such microorganisms can present anticariogenic activity, help on the treatment of periodontal and halitosis diseases, and reduction in the population of pathogenic microorganisms associated to oral pathologies. The main purpose of this study was the development and characterization of a diet lozenge containing the probiotic strain Enterococcus faecium CRL 183. Its potential of surviving in the human saliva environment and the inhibition of the pathogenetic strain Streptococcus mutans ATCC 25175, were also evaluated. The probiotic strain E. faecium CRL 183 was microencapsulated through the extrusion and complex coacervation techniques. The last one was selected to the next step of the study (lozenges production), as it showed the best performance in comparison to the extrusion technique, regarding viability maintenance during the storage period at room temperature (23ºC). The lozenges were produced through three different treatments: PC – control formulation, without the probiotic; PPP1 – probiotic formulation; PPP2 – probiotic formulation with inulin addition. Microbiologic (viability and security), physicochemical (moisture, water activity, pH and color) and sensorial (acceptance and purchase intention) analyses were conducted during the storage period. The probiotic survival to human saliva was evaluated through inoculation of the pure probiotic strain and the probiotic lozenges in saliva. The agar diffusion technique was used to evaluate the E. faecium CRL 183 inhibition potential against the pathogenic strain, S. mutans ATCC 25175. The probiotic strain had the viability decreased after the lozenges production and also during the storage period at room temperature for the PPP1 and PPP2 treatments. At sensorial analysis, lozenges showed acceptance averages higher than 6 to flavor, texture and global impression attributes, with no significant difference among then (p<0,05). The addition of the probiotic strain and the prebiotic ingredient reduced the product acceptance regarding appearance and color and improved the flavor impression. The physicochemical analysis revealed that the formulations PC, PPP1 and PPP2 did not differ among themselves concerning the parameters evaluated, except for color. The microbiologic results obtained during the storage were appropriated to the confectionery category. The E. faecium strain inoculated in saliva as lozenges and as free cells had the cell viability increased after 24 h of incubation. The diffusion test showed that the probiotic strain was able to inhibit the growth of S. mutans ATCC 25175 in the study conditions. The results suggested that the association of probiotic microorganism and inulin improved the acceptance of the product, in relation to the flavor attribute, and that the probiotic strain survived in the human saliva and has potential to inhibit the multiplication of dental caries-causing organism – S. mutans ATCC 25175. However, the drastic viability reduction during the storage period indicates the necessity of a process refinement and also the adjustment of the packaging to food matrix.

Orientador: Arnaldo Yoshiteru Kuaye, Laura Maria Bruno / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T22:18:13Z (GMT). No. of bitstreams: 1 Carvalho_JulianeDoeringGasparin_D.pdf: 1954120 bytes, checksum: 4ba23fd784559801ff2b5568f4ba02c0 (MD5) Previous issue date: 2007 / Resumo: O conhecimento da microbiota lática dos queijos de Coalho produzidos de forma artesanal a partir de leite cru, e suas propriedades tecnológicas são fundamentais para preservar as características originais do produto tradicional em queijos de Coalho industrializados, elaborados com leite pasteurizado. Para alcançar este conhecimento, foi realizado um trabalho de pesquisa dividido em três etapas: I) caracterização físico-química de queijos de Coalho artesanais produzidos no Ceará e de sua microbiota lática; II) estudo do comportamento das bactérias ácido láticas (BAL) durante o processamento do queijo; III) caracterização de propriedades tecnológicas das culturas láticas isoladas a partir deles. As análises físico-químicas caracterizaram as amostras avaliadas como sendo queijo de médio conteúdo de umidade (42%), baixa acidez (0,24%), com pH de 6,30; elevada atividade de água (0,959) e teor de NaCl de 2,88%. Dentre as BAL (643) isoladas destas amostras, foram encontrados os gêneros Enterococcus (59,6%), Lactobacillus (22%), Streptococcus (12,8%), Lactococcus (1,7%) e Leuconostoc (0,6%). A identificação de gênero não foi conclusiva para 3,3% de isolados. As espécies prevalecentes foram Enterococcus faecium, Lactobacillus paracasei subsp. paracasei, Streptococcus thermophilus e Lactococcus lactis subsp. lactis. O acompanhamento da evolução da microbiota lática em amostras de leite cru, massa de queijo e do produto final, coletadas em duas unidades produtoras, revelou a presença de Lactococcus no leite e sua ausência no queijo. A presença de Enterococcus aumentou das amostras de matéria-prima para o queijo, indicando a transferência e multiplicação destes microrganismos ao longo do processamento. Estes resultados evidenciaram uma seleção de microrganismos resistentes às temperaturas elevadas no processamento do queijo, durante o cozimento da massa. Quanto às propriedades tecnológicas avaliadas, 15 isolados foram considerados produtores rápidos de ácido, com predominância dos Lactococcus e Streptococcus (40% cada). Os Lactobacillus exibiram maior variabilidade e extensão proteolítica, além de maior produção de aroma. As culturas analisadas mostraram boa tolerância a 3 e 4% de sal. As cepas de Enterococcus faecium apresentaram a maior produção de bacteriocinas ativas contra Listeria spp., com potencial de emprego na produção de queijo de Coalho, como cultura protetora / Abstract: Understanding the lactic microbiota of the artisanal Coalho cheeses produced from raw milk, and its technological properties, is important to preserve the characteristics of the traditional product in the industrialized Coalho cheeses, elaborated with pasteurized milk. In order to achieve such knowledge, a research work was carried out in three stages: I) the physical-chemical characterization of the artisanal Coalho cheeses from Ceara-Brazil and its lactic microbiota, II) the study of the behaviour of the lactic acid bacteria (LAB) along the processing of cheese, III) characterization of technological properties of the lactic cultures isolated from the cheese. The physical-chemical analyses characterized the evaluated cheese samples with medium moisture content (42%), low acidity (0.24%), pH of 6.30, high water activity (0.959) and 2.88% NaCl content. Amongst the 643 LAB isolated from these samples, Enterococcus (59.6%), Lactobacillus (22%), Lactococcus (1.7%), Leuconostoc (0.6%) and Streptococcus (12.8%) genera were found. The identification was not conclusive for 3.3% of the isolates. The main species were Lactococcus latis subsp. latis, Lactobacillus paracasei subsp. paracasei, Streptococcus thermophilus and Enterococcus faecium. Following the evolution of lactic microbiota in raw milk, curd and cheese samples collected in two dairies, Lactococcus was found to be present in the milk, but absent in the cheese. The presence of Enterococcus increased from the raw material to the cheese samples, indicating the transference and multiplication of these microorganisms throughout the cheesemaking. Such results evidenced a selection of high temperature resistant microorganisms at the curd cooking stage of cheesemaking. According to the technological properties evaluated, 15 isolates were considered fast producers of acid, with predominance of the Lactococcus and Streptococcus (40% each). The Lactobacillus showed high variability and provided the widest range of proteolytic activity and production of flavour. The lactic cultures also showed good tolerance to 3 and 4 % of NaCl. Strains of Enterococcus faecium produced active bacteriocins against Listeria spp., with potential use in the production of Coalho cheese like protective culture / Doutorado / Doutor em Tecnologia de Alimentos

Master of Science / Food Science / Sajid Alavi / Validation studies on process equipment are an important step for effective pathogenic control during dry expanded pet food manufacturing. The preconditioner is used to hydrate, mix and pre-cook raw materials before extrusion of pet food. The High-Intensity-Preconditioner (HIP) was designed with two independently driven shafts, thus offering control of both shaft speed and rotational direction with potential for improving residence time and thus pathogen inactivation. Residence time distribution (RTD) of raw dog food mix was impacted by the HIP process parameters (average residence time varying between 104-178 s for dry experiment and 65-177 s with steam addition) depending on shaft speed and direction. In general, increase in shaft speed resulted in shorter residence time with the larger shaft having a greater impact than the smaller shaft. Rotational direction of shafts also had an effect on average residence time (a maximum difference of 37 s was noticed between treatments with different shaft directions and the same speed). The uniformity of residence time distribution (difference of 97-132 s between 15 and 85 percentiles of the cumulative RTD) also varied considerably with process conditions, with uniformity increasing with shaft speed.  Enterococcus faecium (ATCC® 8459™) was chosen as a surrogate for Salmonella for microbial inactivation studies on the HIP. Both HIP shaft speed (200 and 300 rpm) and process temperature (67-70°C and 89-91°C) impacted E.faecium survival. Lower shaft speed (corresponding to longer residence time) or higher temperature led to greater E.faecium inactivation. A 5 log CFU/g of E.faecium was reduced using selective agar (m-Enterococcus or mE agar) after treatment with high temperature, but approximately 3.5 log CFU/g of E.faecium reduced on non-selective agar (Brain Heart Infusion or BHI agar). Uneven heat distribution, inadequate residence time and system instability might have negatively affected the inactivation. Microbial inactivation, with E.faecium as surrogate, was also studied for the complete dry expanded pet food process using a pilot-scale single-screw extruder with a regular double shaft preconditioner. Meal was inoculated with E.faecium at 6 log CFU/g and processed. Preconditioner downspout temperature ranged from 89-94°C and extrusion die temperature was between 120-140°C. Complete inactivation was observed after extrusion.

Orientador: Elizeu Antonio Rossi / Banca: Alice Yoshiko Tanaka / Banca: Iracilda Zeppone Carlos / Resumo: Tendo em vista verificar algumas características essenciais aos microrganismos para serem considerados como probióticos, o objetivo deste trabalho foi avaliar *in vitro* de características probióticas do *Enterococcus faecium *CRL183 e do* Lactobacillus helveticus *ssp* jugurti* 416. Primeiramente foi testada a capacidade destes microrganismos em resistir aos pH 1.5, 2.0, 3,0 e 4,0 em meio de cultivo específico para cada espécie. Em seguida foi realizado o teste de sobrevivência ao trânsito gastrintestinal, onde, foram simuladas as condições do estômago e do intestino delgado, determinando-se a viabilidade dessas bactérias frente à pepsina em pH 2.0 e a pancreatina em pH 8.0. A resistência aos sais biliares foi avaliada inoculando o *Enterococcus faecium *CRL183 e do* Lactobacillus helveticus * ssp* jugurti* 416 em meio de cultivo suplementado com 0,1; 0,2; 0,3 e 0,5% de Oxgall. Já para teste de produção da hidrolase de sais biliares, foi vericada a ocorrência da mudança de cor das colônias ou precipitação dos sais biliares taurodeoxicolico e glicodeoxicolico (TDCA e GDCA). No estudo de auto-agregação e coagregação essas cepas foram colocadas separadamente (auto-agregação) e associados (coagregação) e verificada a densidade óptica (*DO*560). Foi testada a produção de substâncias antagônicas pelos dois microrganismos estudado frente a *Escherichia coli* 0157: H7, *Listeria monocytogenes* V2 e *Salmonella Enteritidis *por meio do teste *spot-on-the lawn*.* *Os resultados demonstraram que os microrganismos resistiram a todos os pH. Essas bactérias mostraram também ser tolerantes ao teste de sobrevivência ao trânsito gastrintestinal e também no teste de sobrevivência a sais biliares, porém neste teste, o *Enterococcus faecium *CRL 183 obteve um tempo de retardo menor em relação ao *Lactobacillus helveticus ssp. jugurti* 416. Já os resultados obtidos...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this work was to assess in vitro the probiotic characteristics of Enterococcus faecium CRL183 and Lactobacillus helveticus ssp jugurti 416, having as an aim to verify some characteristics which are essential to the microorganisms, in order to be considered as probiotic. Firstly, the capacity of these microorganisms to resist to pH 1.5, 2.0, 3.0and 4.0 was tested in a specific culture medium for each species. Then the survival test to the gastrointestinal passage was done, and the conditions of the stomach and small intestine were simulated, so determining the viability of these bacteria comparing with pepsin at pH 2.0 and pancreatin at pH 8.0. The resistance to bile salts was assessed by inoculating Enterococcus faecium CRL183 and Lactobacillus helveticus ssp jugurti 416 in a culture medium supplemented with 0,1; 0,2; 0,3 and 0,5 % of Oxgall. However, for the production test of the hydrolase of bile salts, it was verified the occurrence of change in color of the colonies, or the precipitation of the bile salts taurodeoxycholic and glycodeoxycholic (TDCA and GDCA). In the auto-aggregation and co-aggregation studies, these strains were placed separately (auto-aggregation) and associated (co-aggregation), and then the optical density was verified (DO560), for completion and adhesion intestinal epithelium was tested Enterococcus faecium CRL183 and Lactobacillus helveticus ssp jugurti 416 with the Escherishia coli 0157: H7. The production of antagonist substances by both studied microorganisms was tested in comparison with Escherishia coli 0157: H7, Listeria monocytogenes V2 and Salmonella enteriotidis, by using the spot-on-the-lawn test. The results demonstrated that the microorganisms resisted to all pH. These bacteria also showed to be tolerant to the survival test to the gastrointestinal passage and also to the survival test to bile salts, though in this test,...(Complete abstract click electronic access below) / Mestre

Orientador: Arnaldo Yoshiteru Kuaye / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T12:29:08Z (GMT). No. of bitstreams: 1 Fernandes_MegdaSilva_M.pdf: 3935080 bytes, checksum: 25bc1e5988870cf168c5403b4c8d2aba (MD5) Previous issue date: 2010 / Resumo: A ricota é um tipo de queijo fresco de origem italiana, obtido pela precipitação das proteínas do soro do queijo por acidificação associada ao calor. Por suas características nutricionais, físico-químicas e bioquímicas apresenta-se propícia ao desenvolvimento microbiano. No processamento deste produto destacam-se o Bacillus cereus, pela sua capacidade de esporular e ser um contaminante potencial do leite e do ambiente e as bactérias do gênero Enterococcus, pela característica ubíqua, habilidade de sobrevivência à condições diversas de pH, temperatura e salinidade e ocorrência em casos de infecções hospitalares. Os objetivos deste trabalho foram: a) verificar as possíveis fontes de contaminação de ricota por B. cereus e Enterococcus spp. ao longo do processamento; b) identificar as espécies de enterococos, avaliar o potencial de patogenicidade e o perfil de resistência destas espécies a antibióticos de uso clínico; e, c) avaliar a conformidade das amostras de ricota aos padrões microbiológicos legais. Amostras de leite cru e pasteurizado, soro de queijo, ricotas antes e após embalagem, superfícies diversas do ambiente e do ar obtidas em três coletas de laticínio da região Sul de Minas Gerais foram submetidas à determinação de B. cereus e Enterococcus spp. As contagens de B. cereus em leite cru, pasteurizado e soro de queijo, foram de 1,4 x104, 1,2 x103 e 1,0 x103 UFC/ml, respectivamente, e, apenas uma amostra de ricota final apresentou valor maior que 102 UFC/g. Dentre as 60 amostras ambientais, destaca-se a forma de moldagem da ricota, que apresentou contaminação persistente e contagem de até 1,7x107 UFC/unidade de B. cereus. Enterococcus spp. foram encontradas em todas as amostras de ricota, com contagens entre 103 e 107 UFC/g, e em todas de leite cru, com contagens de até 1,9 x106 UFC/ml. Nas superfícies de forma e tela, vassoura, parede e ralo foram encontrados valores superiores a 105 UFC/unidade; já para tanques, bancada da área de embalagem e caixa de recolhimento do soro os números foram superiores a 102 UFC/unidade. De um total de 136 isolados, confirmados para o gênero Enterococcus, 71,3% (97/136) foram confirmados para a espécie E. faecium e 20,6% (28/136) para E. faecalis, pela técnica de PCR. Os isolados (66) de E. faecium e E. faecalis das amostras de produto final submetidas aos testes fenotípicos resultaram em 89,4% (59/66) positivos para hemólise, nenhum para gelatinase (0/66) e 98,5% (65/66) positivos para termonuclease. A maioria dos isolados de E. faecium e E. faecalis mostrou resistência a pelo menos três dos 5 antimicrobianos testados, destacando-se que 100% deles apresentaram resistência à vancomicina. De 15 amostras de ricota avaliadas após 21 dias de armazenamento sob refrigeração, 13,3% (2/15) estavam em desacordo com o padrão legal para estafilococos coagulase positiva e em nenhuma delas foi detectada a presença de Salmonella, Listeria monocytogenes e coliformes termotolerantes. A natural e inevitável contaminação da matéria-prima e do ambiente de processamento de ricota por B. cereus e Enterococcus spp., bactérias estas potencialmente patogênicas, tem na eficiência dos programas de higienização um fator indispensável para o seu controle / Abstract: The ricotta is a type of fresh cheese of Italian origin, obtained by precipitation of proteins from cheese whey by acidification associated with the heat. Because of its nutritional, physicochemical and biochemical characteristics it is conducive to microbial growth. On the processing of this product it can be emphasized the Bacillus cereus, due to its ability to sporulate and be a potential contaminant of milk and the environment, and the bacteria of the genus Enterococcus, due its ubiquitous characteristic, ability to survive the various conditions of pH, temperature and salinity and appearance in cases of hospital infections. The objectives of the present work were: (a) to verify the possible sources of ricotta contamination by B. cereus and Enterococcus spp. during processing; (b) to identify the species of enterococci, evaluate the pathogenic potential and the resistance profile of these species to antibiotics of clinical use; and (c) to assess the conformity of samples of ricotta under legal microbiological standards. Samples of raw and pasteurized milk, cheese whey, ricotta before and after packaging, various surfaces of the environment and of air obtained from three collections on a dairy industry located in southern Minas Gerais were subjected to the determination of B. cereus and Enterococcus spp. The counts of B. cereus in raw and pasteurized milk and in cheese whey were 1,4 x104, 1,2 x103 and 1,0 x103 CFU/ml, respectively, and only one sample of final ricotta had levels of B. cereus higher than 1,7x107 CFU/unity. Among the 60 environmental samples, it can be highlighted the mold where the ricotta is shaped, which showed persistent contamination and count up to 1,7x107 CFU/unity for B. cereus. All the samples of ricotta and raw milk showed the presence of Enterococcus spp. with counts between 103 and 107 CFU/g and up to 1,9 x106 CFU/ml, respectively. On the surfaces of the mold, mesh, broom, wall and drain it were found counts higher than 105 CFU/unity; for the tank, stand in the area of packaging and box for the collection of serum the counts were higher than 102 CFU/unity. Over 136 isolated of the genus Enterococcus, 71,3% (97/136) were confirmed for the species E. faecium and 20,6% (28/136) for E. faecalis by PCR technique. The isolates (66) of E. faecium and E. faecalis taken from the samples of the final product submitted to phenotypic tests resulted in 89,4% (59/66) positive for hemolysis, none for gelatinase (0 / 66) and 98,5% (65/66) positive for thermonuclease. Most of the isolates of E. faecium and E. faecalis showed resistance to at least three of the 5 antimicrobials, highlighting that 100% of them were resistant to vancomycin. From 15 samples of ricotta evaluated after 21 days of refrigerated storage, 13,3% (2/15) were in disagreement with the legal standard for coagulase-positive staphylococci and none were detected the presence of Salmonella, Listeria monocytogenes and thermotolerant coliforms. The natural and inevitable contamination of raw-materials and of the processing environment of ricotta by B. cereus and Enterococcus spp., which are potentially pathogenic bacteria, have in the efficiency of the hygiene programs a essential factor for its control / Mestrado / Mestre em Tecnologia de Alimentos