O presente trabalho foi planejado com o objetivo de estudar os atributos do teste tuberculínico e da abreugrafia em confronto com o diagnóstico bacteriológico da tuberculose. A população de estudo foi constituída por 15.056 pessoas com 15 e mais anos de idade, matriculadas no Centro de Saúde de Ribeirão Preto durante 6 meses consecutivos em 1973. Constatou-se que o valor predictivo do teste tuberculínico negativo foi de 99,98 por cento e o valor predictivo positivo da abreugrafia foi de 14,45 por cento ; a realização de abreugrafia apenas em reatores fortes elevaria o seu valor predictivo positivo para 18,96 por cento melhorando consideravelmente a eficácia desse instrumento sem prejuízo da sua sensibilidade. Esses resultados permitem recomendar o emprêgo do teste tuberculínico em Saúde Pública visando a exclusão de tuberculose doença bem corno para a triagem de adultos para exame abreugráfico do tórax. / This study was designed to evaluate the tuberculin test and roentgenphotography attributes in comparison with the bacteriological diagnosis of tuberculosis. The study population consisted of 15.056 persons aged 15 or above, registered at the Health Center of Ribeirão Preto, Brazil, during 6 consecutive months in 1973. It was observed that the predictive value of negative tuberculin test was of 99,98 per cent and the predictive value of abnormal roentgenphotography was of 14,45 per cent ; when the late was taken only of \"strong reactors\" its predictive positive value raised to 18,96 per cent , what increased its efficacy without impairing sensibility. These findings permit to recommend the use of the tuberculin test as a Public Health routine procedure to exclude tuberculosis disease in adults as well as a screening procedure for roentgenphotography examination.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Universiẗat, Diss., 2004--Bonn.

Submitted by LUCAS EDUARDO PILON null (lucaseduardopilon@yahoo.com.br) on 2016-03-07T12:00:11Z No. of bitstreams: 1 Tese Lucas Eduardo Pilon.pdf: 1670960 bytes, checksum: 4da72578d702faee29a2735a07314cd0 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-03-07T13:38:08Z (GMT) No. of bitstreams: 1 pilon_le_dr_jabo.pdf: 1670960 bytes, checksum: 4da72578d702faee29a2735a07314cd0 (MD5) / Made available in DSpace on 2016-03-07T13:38:08Z (GMT). No. of bitstreams: 1 pilon_le_dr_jabo.pdf: 1670960 bytes, checksum: 4da72578d702faee29a2735a07314cd0 (MD5) Previous issue date: 2016-02-05 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A mastite em ovelhas de dupla aptidão é reconhecida por afetar a qualidade do leite. Staphylococcus coagulase-negativos (SCN) são os principais micro-organismos responsáveis pela doença, e o tratamento ao final da lactação, pode contribuir para a cura e prevenção de casos subclínicos na lactação consecutiva. Entretanto, fatores de virulência e mecanismos de resistência apresentados por SCN podem reduzir as taxas de cura. Os objetivos deste trabalho foram identificar no leite de ovelhas tratadas e não tratadas à secagem com antimicrobianos, as espécies de SCN antes e após o tratamento e identificar nesses micro-organismos a presença dos genes mecA, icaA, icaC, icaD, bap, bhp, sea, seb, sec, sed e tsst-1, determinar o perfil clonal das principais espécies identificadas e relacionar os casos de cura após o tratamento com a presença/ausência dos respectivos genes. Sessenta ovelhas foram divididas em três grupos experimentais: G1, controle, metades mamárias que não receberam antimicrobiano; G2, metades mamárias em que foram administrados 10 mL de cloxacilina-benzatina 100 mg via intramamária / estrutura convencional; G3, metades mamárias em que foram administrados 86 mL de cloxacilina-benzatina 50 mg via intramamária / estrutura nanoencapsulada. As amostras de leite foram coletadas à secagem e aos 15 e 30 dias pós-parto da lactação seguinte. As análises para identificação das espécies de SCN foram realizadas por meio de testes bioquímicos e Internal Transcribe Spacer (ITS-PCR), e a pesquisa dos genes responsáveis pelos fatores de virulência e pela resistência à oxacilina foram realizados por meio da técnica de reação em cadeia de polimerase (PCR). Dentre as espécies identificadas S. warneri prevaleceram nos três grupos experimentais. Nenhuma amostra foi positiva para o gene mecA. O único gene relacionado com a produção de enterotoxinas encontrado foi o sec. Dentre os genes relacionados com a produção de biofilme, icaD foi o único identificado nos três grupos experimentais. Staphylococcus warneri, S. simulans e S. epidermidis apresentaram clones na mesma metade mamária no pré e pós-parto das ovelhas. A cloxacilina benzatina nanoparticulada 50mg / 86 mL foi eficiente para reduzir a mastite subclínica no pós-parto de ovelhas (P= 0,0192). Staphylococcus warneri, S. simulans, S. epidermidis e S. xylosus foram as espécies de maior ocorrência. Os genes icaA, icaC, icaD e bap foram encontrados no momento da desmama e no pós-parto, os genes sec e icaD estão associados à ausência de cura da mastite subclínica no pós-parto. Ovelhas em que foram isolados SCN portadores de genes responsáveis pela formação de biofilme não apresentaram resultados satisfatórios quando submetidas a esquemas de controle e ao tratamento da mastite subclínica. Os genes sec e icaD, estão associadas à ausência de cura microbiológica da mastite subclínica no pós-parto. Staphylococcus epidermidis e S. xylosus portador do gene bap estão associados à reinfecção. / Mastitis in dual-purpose sheep is recognized to affect their milk quality. Coagulase-negative Staphylococcus (CNS) is the main microorganism responsible for this disease and treatment at the end of lactation may contribute towards curing it and preventing subclinical cases during the consecutive lactation. However, virulence factors and resistance mechanisms presented by CNS may reduce the cure rates. The aims of this study were to identify CNS species in sheep’s milk with and without treatment with antimicrobials at the time of drying off and, in these microorganisms, to identify presence of the genes mecA, icaA, icaC, icaD, bap, bhp, sea, seb, sec, sed and tsst-1, determine clonal profile of the main species identified and correlate the cases of cure after treatment with the presence or absence of the respective genes. Sixty sheep were divided into three experimental groups: G1 (control), mammary glands that did not receive antimicrobials; G2, mammary glands to which 10 mL of cloxacillin-benzathine (100 mg) was administered via the intramammary route in a conventional structure; and G3, mammary glands to which 86 mL of cloxacillin-benzathine (50 mg) was administered via the intramammary route in a nanoencapsulated structure. Milk samples were collected at the time of drying off and 15 and 30 days after lambing in the next lactation. The analyses to identify the CNS species were performed by means of biochemical tests and internal transcribe spacer polymerase chain reaction (ITS-PCR), and the genes responsible for the virulence factors and resistance to oxacillin were investigated by means of the PCR technique. Among the species identified, Staphylococcus warneri was most prevalent in the three experimental groups. None of the samples were positive for the gene mecA. The only gene relating to production of enterotoxins that was found was sec. Among the genes relating to biofilm production, icaD was the only one identified in the three experimental groups. Staphylococcus warneri, S. simulans and S. epidermidis presented clones in the same mammary gland before and after lambing. The dose of 86 mL of nanoparticulate cloxacillin-benzathine (50 mg) was efficient for reducing subclinical mastitis after lambing (P = 0.0192). Staphylococcus warneri, S. simulans, S. epidermidis and S. xylosus were the species that occurred most frequently. The genes icaA, icaC, icaD and bap were found at the time of weaning and in the postpartum period. The genes sec and icaD were correlated with absence of cure for subclinical mastitis after lambing. Sheep in which CNS carrying genes responsible for biofilm formation was isolated did not present satisfactory results when they were subjected to regimens for controlling and treating subclinical mastitis. The genes sec and icaD were correlated with absence of microbiological control over subclinical mastitis after lambing. Staphylococcus epidermidis and S. xylosus carrying the gene bap were correlated with reinfection. / FAPESP: 2012/23044-0

Orientador: Arnaldo Yoshiteru Kuaye / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-07T02:52:52Z (GMT). No. of bitstreams: 1 Borges_MariadeFatima_D.pdf: 904414 bytes, checksum: dab57575c28ef69977aa61268afabf0d (MD5) Previous issue date: 2006 / Resumo: O queijo é considerado um veículo freqüente de patógenos de origem alimentar e, em especial os queijos frescos artesanais. Dentre estes, destaca-se o queijo de coalho por ser comumente elaborado a partir de leite cru e sob condições insatisfatórias de higiene, em pequenas indústrias que não adotam de forma plena as Boas Práticas de Fabricação (BPF). Portanto, a contaminação microbiológica deste produto assume destacada relevância para a saúde pública, pelo risco de causar doenças transmitidas por alimentos. Neste trabalho, foi realizado um diagnóstico da contaminação por coliformes fecais, Escherichia coli, Listeria monocytogenes, Salmonella sp. e Staphylococcus spp. na linha de produção de queijo de coalho em uma indústria de laticínios, na região metropolitana de Fortaleza - CE. Um total de 100 amostras de alimentos, incluindo leite cru, leite pasteurizado, coalhada, queijo, e 165 amostras ambientais, incluindo ar ambiente, superfícies de equipamentos, móveis, utensílios, embalagem de polietileno, drenos, pisos, paredes e luvas utilizadas pelos manipuladores, foi coletado durante a fabricação de cinco lotes diferentes, a intervalos de 45-50 dias, no período de maio a outubro de 2004. As amostras ambientais foram analisadas quanto à presença de L. monocytogenes, Staphylococcus spp., enquanto as amostras de alimentos, também, foram analisadas quanto ao número mais provável (NMP) de coliformes totais e fecais, pesquisa de E. coli e de Salmonella sp. A contagem e identificação das bactérias analisadas foi realizada pelos métodos preconizados pelo Bacteriological Analytical Manual (FDA), exceto para L. monocytogenes que foi analisada segundo metodologia do Canadiam Health and Food Branch. Os isolados característicos do gênero Listeria foram identificados utilizando-se o kit API® Listeria e por meio da amplificação de fragmentos dos genes hly e actA, utilizando a técnica PCR. Cem isolados característicos de Staphylococcus sp. foram identificados pelo sistema API ¿ Staph (BioMérieux) e os 23 isolados identificados como S. aureus foram confirmados através da amplificação de um fragmento do gene femA pela técnica da PCR. A pesquisa dos genes sea, seb, sec, sed, see, sei e sej foi realizada em 32 cepas de Staphylococcus, utilizando PCR. A detecção de enterotoxinas foi realizada em 20 amostras compostas, através do método imunoenzimático ELFA empregando o sistema automatizado VIDAS® Staph enterotoxin II (BioMérieux). O leite cru apresentou elevada população de bactérias do grupo coliformes totais e fecais, com confirmação da presença de E. coli, evidenciando deficiências nas condições higiênico-sanitárias durante o processo de obtenção do leite. A presença de E. coli não foi constatada no leite pasteurizado, na coalhada e no queijo. Os níveis de coliformes totais e fecais detectados apresentaram-se superiores ao limite legal estabelecido para queijo de coalho (1.0 x 103NMP/g) em apenas um lote. Em 100% das amostras dos alimentos analisadas verificou-se ausência de Salmonella spp. A avaliação de 18 isolados característicos do gênero Listeria através do kit API® Listeria revelou três isolados de L. monocytogenes ¿atípicas¿, que não foram confirmadas, uma vez que não apresentaram amplificação dos fragmentos específicos para os genes hly e actA, indicando assim a possível ausência desse patógeno no ambiente de produção de queijo de coalho. A população de Staphylococcus sp. e de Staphylococcus coagulase positiva reduziram de 1,5 x 107UFC/mL e 5,0 x 106UFC/mL no leite cru para zero e no leite pasteurizado, respectivamente. Staphylococcus coagulase positiva foi detectado em 100% das amostras de leite cru (25/25) e em 8% das amostras de queijos (2/25). As contagens de Staphylococcus sp. em equipamentos e utensílios oscilaram entre <102 a 3,2 x 104UFC/cm2. Identificou-se 12 espécies de Staphylococcus através do kit API® Staph, sendo nove coagulase negativa e três coagulase positiva. No leite cru observou-se alta freqüência de espécies coagulase positiva, com prevalência de S. aureus. Nas demais amostras de produtos, luvas dos manipuladores, superfícies de equipamentos e utensílios a prevalência foi de espécies coagulase negativa. A presença de enterotoxinas estafilocócicas foi constatada em todas as amostras de um mesmo lote processado, desde a matéria prima até o produto final (queijo). De um total de 23 cepas de S. aureus identificados fenotípicamente (API® Staph - BioMérieux), 82,6% (19/23) foram positivas para o gene femA, demonstrando maior especificidade e poder discriminatório da analise molecular. A presença dos genes sea e sec foi detectada em 37,5% (12/32) das cepas analisadas, sendo estas pertencentes a cinco espécies de Staphylococcus / Abstract: Cheese is considered to be a frequent vehicle of food borne pathogens, especially fresh artisan cheeses. Amongst these, ¿coalho¿ cheeses stand out since they are frequently made from non-pasteurised milk, under unsatisfactory conditions of hygiene, in small-scale industries that fail to completely adopt the Good Manufacturing Practices (GMP). Thus the microbiological contamination of this product assumes prominent relevance with respect to public health, due to the risk of transmitting foodborne diseases. In this study a diagnosis for the contamination by faecal coliforms, E. coli, L. monocytogenes, Salmonella sp. and Staphylococcus spp. was carried out on the ¿coalho¿ cheese production line of a dairy in the metropolitan region of Fortaleza ¿ CE, Brazil. A total of 100 samples were taken from 5 different batches processed at intervals of 45-50 days from May to October, 2004, and comprised non-pasteurised and pasteurised milks, curd, cheese and 145 environmental samples, including the air, equipment and utensils surfaces, drains, floors, walls and gloves worn by cheese handlers. The environmental samples were analysed for the presence of L. monocytogenes and Staphylococcus spp., whilst the food samples were also analysed for the most probable number (MPN) of total and faecal coliforms and examined for the presence of E. coli and Salmonella spp. The isolation and counts of the microorganisms analysed were carried out using the methods presented in the Bacteriological Analytical Manual (FDA), except for L. monocytogenes, which was analysed according to the Canadian Health and Food Branch methodology. Isolates characteristic of the genus Listeria were identified using the API® Listeria kit and by amplifying fragments of the genes hly and actA using the PCR technique. A hundred isolates characteristic of Staphylococcus sp., were selected for identification by the API® - Staph system (BioMérieux). Twenty-three S. aureus strains were identified at the molecular level by amplifying a fragment of the gene femA. A search for the genes sea, seb, sec, sed, see, sei and sej was carried out by PCR in 32 strains of Staphylococcus. Enterotoxin detection was carried out for 20 compound samples using the ELFA immuno-enzymatic assay with the automatated VIDAS® Staph enterotoxin II (BioMérieux). The non-pasteurised milk presented a high count of the total and faecal coliform group with confirmation of the presence of E. coli, as evidence of deficiencies in the hygiene-sanitary conditions during milking. E. coli was not found in the pasteurised milk, curd or cheese, and the levels of total and faecal coliforms found were above the legal limits for ¿coalho¿ cheese (103MPN/mL) for one single batch. Salmonella spp. were absent in 100% of the samples analysed. Eighteen isolates characteristic of the genus Listeria were evaluated using the API® Listeria kit, and three ¿atypical¿ isolates of L. monocytogenes were found. These did not present amplification of the fragments specific for the genes hly and actA, raising the possibility of their absence in the ¿coalho¿ cheese production environment. The Staphylococcus sp. Population decreased from 1.5 x 107CFU/mL in the raw milk to zero in the pasteurised milk, whilst coagulase positive Staphylococcus decreased from 5.0 x 106CFU/mL in the raw milk to zero in the pasteurised milk. Coagulase positive Staphylococcus was detected in 100% of the raw milk samples (25/25) and in 8% of the cheese samples (2/25). Staphylococcus sp. counts oscillated between <102 and 3.2 x 104CFU/cm2 on the equipment and utensils. Both coagulase positive and negative Staphylococcus species were identified on the gloves of the cheese handlers. Twelve species of Staphylococcus were identified, 9 being coagulase negative and 3 being coagulase positive. A high frequency of coagulase positive species was observed in the raw milk, with prevalence for S. aureus. The prevalence of coagulase negative species was verified in the other samples of product, handlers¿ gloves, equipment and utensils surfaces. Staphylococcal enterotoxins were found in all the samples from one particular batch, from the raw material to the final product (cheese). Out of a total of 23 strains of S. aureus phenotypically identified (API® Staph ¿ BioMérieux), 82.6% (19/23) were positive for the gene femA, demonstrating the greater specificity and discriminatory power of the molecular analysis. The presence of the genes sea and sec was detected at a level of 37.5% (12/32) of the strains analysed of 5 Staphylococcus species / Doutorado / Doutor em Tecnologia de Alimentos

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Bacillus cereus is a ubiquitous organism and a potential foodborne pathogen that can cause two types of gastrointestinal diseases: emesis and diarrhea. The emetic syndrome is caused by a heat and acid stable peptide toxin that is pre-formed in food, while the diarrheal syndrome is associated to two 3-protein, heat labile enterotoxin complexes that are formed in the intestine after ingestion of the organism. There are many reports on the isolation and characterization of Bacillus cereus from various foods, however there are no studies on the levels, toxigenicity and physical characteristics of B. cereus isolated from U.S. retail spices. A huge part of spices sold in the U.S. are imported from developing nations. Developing nations lack hygienic practices during processing and packaging of spices, due to which there is a high chance of imported spices being contaminated with B. cereus. Therefore, the main objective of this thesis work was to characterize B. cereus spores from U.S. retail spices. Levels of aerobic spores and B. cereus spores were determined. B. cereus spores were further analyzed for their enterotoxigenic ability, growth characteristics and physical spore characteristics. In the 247 spice samples analyzed 77 were found to contain B. cereus, while 11 were positive for B. thuringiensis. Eighty four of the 88 spices tested possessed either one of the enterotoxin genes. None of the isolates tested positive for the emetic toxin (ces) gene. Seventy five of the B. cereus isolates grew at 12 °C, although only two isolates grew well at 9 °C. Seven selected diarrheal B. cereus spore strains had D95-values ranging from 0.64-3.53 min while the two emetic strains had D95-values of 7.04 min and 6.64 min. B. cereus grew well in pre-cooked rice. After 48 h, counts of 1.26 X 107 and 3.8 X 107 B. cereus/ 10 g were obtained in pre-cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 104 CFU/ 10g even after 48 h of incubation. The aerobic mesophilic bacterial population and B. cereus population of 0.1% crushed pepper in pre-cooked rice over a period of 48h at temperature 20 °C and 17 °C were also analyzed. Counts of B. cereus in pepper rice samples reached a maximum of 1600 MPN/ 10 g and 1100 MPN/ 10 g at 20 °C and 17 °C respectively while the aerobic mesophilic counts per 10 g were 2.4 X 108 and 4.4 X 106 at these temperatures. The low B. cereus counts and high aerobic mesophilic population indicates competition of nutrients in cooked rice by background flora other than B. cereus. The physical spore characteristics of five B. cereus and 3 B. thuringiensis strains were studied using transmission electron microscopy (TEM). Tubular, whip-like appendages were present in four B. cereus and two B. thuringiensis, while all seven isolates possessed exosporia.

The fungus Ophiocordyceps camponoti-floridani (Ophcf) infects Camponotus floridanus carpenter ants and manipulates them to climb to a high tree branch, bite down on foliage and die. Post ant death, the fungus grows out of the ant and spreads spores for reproduction. I investigated the function of an Ophcf gene product highly activated during the behavioral manipulation of these “zombie ants”; an enterotoxin. I have created an expression vector and heterologously expressed this enterotoxin in Cordyceps bassiana (Cbass), a related fungus that does not normally manipulate behavior. This process includes gene amplification, Golden Gate vector cloning in E. coli, A. tumefaciens-mediated transformation to Cbass, and RT-qPCR to verify heterologous gene expression. This was followed by carpenter ant infections with transgenic Cbass (EntB), wildtype Cbass (infected control), and sham (non-infected control) infected ants. Subsequent behavioral observations using tracking system MARGO (Werkhoven et al., 2019) have detected changes in activity levels of ants infected with both transgenic and WT Cbass compared to sham infected ants. This supports previously qualitative descriptions of increased activity caused during infection with WT Cbass (Trinh, 2020). There is a slight but insignificantly higher activity response from EntB compared to WT infected ants over the course of the trial that may be indicative of Ophcf induced changes that are different from general sickness behavior. Additional replicates are necessary to discern if these findings are statistically robust. Future studies should administer this enterotoxin expressing Cbass to observe inter-social behaviors of Carpenter ants. If the enterotoxin is sufficient to elicit one of the side effects of typical Ophcf infection, this would justify further characterization of the proteins and their functions in altering C. floridanus behavior. This characterization could yield information applicable to other parasite-host relationships as well.

Rotaviruses, members of the family Reoviridae, are the major etiologic agents of severe, acute dehydrating diarrhea in the young of many mammalian species, including humans, calves and foals. Recent estimates indicate an annual death toll of approximately 600,000 infants due to rotavirus, besides inflicting staggering economic burden worldwide. Most of these deaths occur in the developing countries and India is estimated to account for about a quarter of these deaths. Extensive molecular epidemiology studies carried out by our laboratory have revealed many interesting aspects about rotavirus diversity in this country. Molecular epidemiology of rotaviruses causing severe diarrhea in foals in two organized farms in northern India was carried out. These foal rotaviruses exhibited 5 different electropherotypes (E), E1-E5. Strains belonging to E1, E2 and E5 exhibited G10, P6[1]; G3 and G1 type specificities. Though the E1 strains possessed genes encoding G10 and P6[1] type outer capsid proteins, unlike the G10, P8[11] type strain I321, they exhibited high reactivity with the G6-specific MAb suggesting that the uncommon combination altered the specificity of the conformation-dependent antigenic epitopes on the surface proteins. Strains belonging to electropherotypes E3 and E4 were untypeable. Sequence analysis of the VP7 gene from E4 strains (Erv92 and Erv99), revealed that they represent a new VP7 genotype, G16. Nonstructural protein 4 (NSP4) of rotavirus is a multidomainal, multifunctional protein and is the first viral enterotoxin identified. We have recently reported that the diarrhea-inducing and double-layered particle (DLP)–binding properties of NSP4 are dependent on a structurally and functionally overlapping conformational domain that is conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail (Jagannath et al., J. Virol, pp 412-425, 2006). Further, a stretch of 40 amino acids (aa) from the C-terminus is predicted to be unstructured and highly susceptible to trypsin cleavage. We examined the role of this unstructured C-terminus of Hg18 NSP4 and SA11 NSP4 on the biological properties of NSP4 using a series of deletion and substitution mutants of the conserved proline and tyrosine residues in this region. Gel filtration, CD spectroscopy and Thioflavin T binding studies showed that these mutants have altered secondary structural contents and either failed to multimerize efficiently or multimerized with altered conformation. The C-terminal ten residues appear to play a regulatory role on multimerization. Proline 168, tyrosine 166 and methionine 175 appear to be critical determinants of DLP binding activity whereas, proline 165 and tyrosine 85 and 131 appears to determine the affinity of binding to DLP in the context of NSP4 ∆N72. Deletion and substitution mutants exhibited severely reduced diarrhea inducing ability and DLP binding property. Of great biological significance is the drastic decrease in the diarrhea inducing ability of the N- and C- terminal deletion mutant ∆N94 ∆C29 that exhibited about 11,000-fold increase in DD50 than the wild type (WT) ∆N72. These studies revealed that the predicted unstructured C-terminus is an important determinant of biological properties of NSP4. Extensive efforts to crystallize the complete cytoplasmic tail (CT) of NSP4 were unsuccessful and to date, the structure of only a synthetic peptide corresponding to aa 95-135 has been reported. Our recent studies indicate that the interspecies variable regions from aa 135-141 as well as the extreme C-terminus are critical determinants of virus virulence and diarrhea-inducing ability of the protein. Here, we examined the crystallization properties of several deletion mutants and report the structure of a mutant recombinant NSP4 from symptomatic (SA11) and asymptomatic (I321) strains that lacked the N-terminal 94 and C-terminal 29 aa (NSP4: 95-146) at 1.67 Å and 2.7Å, respectively. In spite of the high-resolution data, electron density for the stretch of 9 residues from the C-terminus could not be seen suggesting its highly flexible nature. The crystal packing showed a clear empty space for this region. Extension of the unstructured C-terminus beyond aa 146 hindered crystallization under the experimental conditions. The present structure revealed significant differences from that of the synthetic peptide in the conformation of amino acids at the end of the helix as well as crystal packing owing to the additional space required to accommodate the unstructured virulence-determining region. Conformational differences in this critical region effected by the presence or absence of proline or glycine at specific positions in the unstructured C-terminus, could form the basis for the wide range of variation seen in the diarrhea-inducing ability of NSP4 from different strains in newborn mouse pups. Although symptomatic and asymptomatic strains do not generally differ in the presence or absence of the conserved prolines or glycines, they contain a few additional changes that could alter the unique conformation required for optimal biological activity. In conclusion, we demonstrate that the predicted unstructured C-terminal region is indeed highly flexible and is an important determinant of biological functions of the NSP4, mutations in which probably correlates with the virulence properties of the virus.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-05-12T20:45:00Z No. of bitstreams: 2 Dissertação - Mariana de Andrade Ferreira - 2015.pdf: 2863875 bytes, checksum: 62c67ecc0abf8022b7bf54c6de62a20c (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-05-13T12:05:05Z (GMT) No. of bitstreams: 2 Dissertação - Mariana de Andrade Ferreira - 2015.pdf: 2863875 bytes, checksum: 62c67ecc0abf8022b7bf54c6de62a20c (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Made available in DSpace on 2016-05-13T12:05:05Z (GMT). No. of bitstreams: 2 Dissertação - Mariana de Andrade Ferreira - 2015.pdf: 2863875 bytes, checksum: 62c67ecc0abf8022b7bf54c6de62a20c (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) Previous issue date: 2015-08-28 / Staphylococcus aureus is an important foodborne pathogen, able to produce extracellular toxins and to express antimicrobial resistance. Among the foods involved in staphylococcal food poisoning, stands out the cheese, especially when manufactured under improper hygienic and sanitary conditions. The objectives of this study were to characterize Staphylococcus aureus isolated from artisanal and industrialized Minas frescal cheeses, to determine their antimicrobial susceptibility profile as well as the genetic similarity among the isolates. The isolates were also tested for staphylococcal enterotoxins (SE) genes and other virulence factors. Fifty-six artisanal raw milk cheeses sold at street fairs and 10 industrialized cheeses commercialized in supermarkets of Goiânia, Goiás were analyzed between June and August 2014. S. aureus was confirmed in 19 samples (33.9%) of artisanal cheese by detection of femA gene, in which 29 isolates were obtained. These isolates were submitted to the antimicrobial susceptibility test and classified into nine different profiles (A - I). Thirteen isolates (44.8%) were resistant to penicillin and three (10.3%) to tetracycline, with two (7.4%) resistant to both. The Multiplex PCR technique was performed to detect virulence genes that code for the production of hemolysins (Hla and Hlb), toxic shock syndrome toxin (TSST-1), exfoliative toxins (ETa and ETb) and enterotoxins (SEA - SEE, SEG - SEJ, SEM - SEO). Genes encoding TSST-1 and exfoliative toxins were not detected. All the isolates amplified for the hla gene and 14 (48.3%) for the hbl gene. The seh gene was the most frequently detected (n=11, 37.9%) followed by seo gene (n = 3; 10.3%), seg, sem and sen genes (n = 2, 6.9%) and sec and sei genes (n = 1, 3.4%). In one isolate (3.4%), four enterotoxins genes were detected, and in another, six (3.4%). The comparison performed by Pulsed Field Gel Electrophoresis technique revealed 18 different DNA banding patterns which were grouped into five clusters. The genotyping found high genetic similarity among the isolates. Identical isolates were obtained from different samples and one sample showed more than one genetically different isolate. It was identified up to four different isolates from the same sample. The high prevalence of S. aureus in a widely consumed product like Minas fresh cheese, as well as the detection of toxin encoding genes identified in this study, warns of the necessity to reduce the contamination levels in this type of cheese through monitoring and controling the production and trade of the product. / S. aureus é um importante patógeno de origem alimentar com capacidade de produção de toxinas extracelulares e resistência antimicrobiana. Entre os alimentos envolvidos em intoxicação alimentar estafilocócica, destaca-se o queijo, principalmente quando fabricado em condições higienicossanitárias impróprias. Os objetivos deste estudo foram caracterizar S. aureus isolados de queijo Minas frescal artesanal e industrializado, determinar o perfil de suscetibilidade a antimicrobianos, bem como determinar a similaridade genética entre os isolados. Os isolados foram também testados para genes de enterotoxinas estafilocócicas (SE) e outros fatores de virulência. Foram analisadas 56 amostras de queijo Minas frescal de fabricação artesanal e dez de fabricação industrial comercializados em feiras livres e supermercados de Goiânia-GO, coletadas entre junho e agosto de 2014. S. aureus foi confirmado em 19 amostras (33,9) de queijo artesanal através da detecção do gene femA, onde 29 isolados foram obtidos. Estes isolados foram submetidos ao teste de susceptibilidade aos antimicrobianos e classificados em nove diferentes perfis (A - I). Treze isolados (44,8%) foram resistentes à penicilina e três (10,3%) à tetracilina, sendo dois (7,4%) resistentes a ambos. A técnica de multiplex PCR foi realizada para a detecção de genes de virulência que codificam a produção de hemolisinas (Hla e Hlb), TSST-1, toxinas esfoliativas (ETa e ETb) e enterotoxinas (EEA - EEE, EEG - EEJ, EEM - EEO). Genes que codificam as toxinas TSST-1 e esfoliativa não foram detectados. Todos os isolados amplificaram para o gene hla e 14 (48,3%) para o gene hlb. O gene seh foi o mais frequentemente detectado (n=11; 37,9%), seguido do gene seo (n=3; 10,3%), genes seg, sem, sen (n=2; 6,9%) e os genes sec e sei (n=1; 3,4%). Um isolado (3,4%) amplificou genes para quatro enterotoxinas e outro (3,4%) para seis. A comparação dos 29 isolados de S. aureus feita por PFGE revelou 18 padrões de bandas diferentes de DNA agrupados em cinco clusters. A genotipagem demonstrou alta similaridade genética entre os isolados. Isolados idênticos foram obtidos de amostras diferentes e uma mesma amostra apresentou mais de um isolado geneticamente diferente. Identificou-se até quatro diferentes isolados da mesma amostra. A alta prevalência de S. aureus nas amostras de queijo Minas Frescal, bem como a detecção de genes para produção de toxinas, alertam para a necessidade de reduzir os níveis de contaminação neste tipo de queijo através de monitoramento e controle da produção e comércio do produto.