Occurrence of potentially highly pathogenic free-living amoebae in ready made salads from restaurants in Jatai, Goias, Brazil

Free-living amoebae (FLA) are part of a group of protozoa found worldwide and in the most diverse environments. They resist various temperatures and disinfection methods, and are a risk to human health. Pathogenic strains grow at high temperatures and under hyperosmolarity conditions. Some FLA genera are mainly related to primary amoebic meningoencephalitis (PAM), skin ulcerations, corneal lesions, kidney and lung infections and keratitis. Therefore, studies that assess the pathogenic potential of FLA are public health issues of great concern. We aimed to evaluate the pathogenic potential of FLA isolated in salads from restaurants in vitro, using osmotolerance and thermotolerance tests. Forty-five isolates were used from ready-made salads purchased in restaurants in Jatai, Goias. Twelve isolates subjected to the osmotolerance test (26.6%) showed growth in 0.5 M mannitol, 18 (40.0%) in 1.0 M mannitol and 16 (35.5%) in 1.5 M mannitol, 13 (28.8%) isolates did not show growth. Four isolates that underwent the thermotolerance test (8.9%) showed growth at 25°C, 8 (17.8%) showed growth at 30°C, 3 (6.7%) showed growth at 37°C and 30 (66.7%) did not show growth. With the indices obtained in the present study, we concluded that 15.6% of the isolates were osmotolerant and thermotolerant. Our findings highlight a public health problem once these FLA are associated with harboring or being harbored by microorganisms responsible for diseases such as diarrhea and meningitis. Measures are required to improve food hygiene and so avoid FLA-related health problems.

Caracterização do gene do choque térmico (HSP-70. 1) e sua relação com características de produção em bovinos leiteiros criados no semiárido brasileiro / Characterization of the thermal shock gene (HSP-70. 1) and its relationship with production characteristics in dairy cattle reared in the Brazilian semiarid

Objetivou-se com este trabalho avaliar a diversidade genética do gene HSP-70.1 e associar os polimorfismos encontrados com a performance de vacas leiteiras das raças Holandesa, Girolando (5/8H-G) e Sindi criadas em região do semiárido brasileiro. Os polimorfismos foram identificados e avaliados pela técnica de PCR-RFLP, usando-se a enzima de restrição EcoRII. Avaliou-se a variabilidade genética por meio do índice de diversidade padrão e da análise de variância molecular (AMOVA). Os polimorfismos identificados foram avaliados sobre as características de produção de leite. Foram identificados sete alelos, os quais demonstraram que houve polimorfismo para a região gênica analisada, e alguns alelos foram compartilhados entre os rebanhos. As raças bovinas Holandesa e Sindi foram similares geneticamente para o gene analisado. A AMOVA demonstrou que há variação genética entre os rebanhos e dentro deles, com a maior parte da variação ocorrendo dentro dos rebanhos para todos os grupos avaliados. Houve efeito dos alelos identificados sobre a produção de leite dos rebanhos das raças Holandesa (P<0,0001) e Girolando (P<0,0117). O gene HSP-70.1 foi polimórfico na população de bovinos leiteiros estudada, sendo, portanto, um marcador molecular promissor para avaliar a produção de leite de raças criadas em região semiárida.(AU)

The objective of this work was to evaluate the genetic diversity of the HSP-70.1 gene and to associate the polymorphisms found with the performance of Holstein, Girolando (5/8H-G) and Sindi dairy cows raised in region of the Brazilian semiarid. Polymorphisms were identified and evaluated using the PCR-RFLP technique using the EcoRII restriction enzyme. Genetic variability was evaluated using the standard diversity index and molecular variance analysis (AMOVA). The identified polymorphisms were evaluated on the characteristics of milk production. They were identified from the seven alleles, demonstrating that there was polymorphism for the analyzed gene region and some alleles were shared among the herds. The Holstein and Sindi bovine breeds were genetically like the analyzed gene. AMOVA demonstrated that there is genetic variation between and within the herds, with most of the variation occurring within the herds for all groups evaluated. There was effect of the alleles identified on the production of milk herds of Holstein and (P<0.0001) Girolando (P<0.0117) breeds. The HSP-70.1 gene was polymorphic in the population of dairy cattle studied, and therefore a promising molecular marker to evaluate milk production of breeds created in semiarid regions.(AU)

Thermotolerance of Histoplasma capsulatum at 40 °C predominates among clinical isolates from different Latin American regions

ABSTRACT The yeast phase of 22 Histoplasma capsulatum clinical isolates from Mexico, Argentina, Colombia, and Guatemala and three reference strains, one from Panama and two from the United States of America (USA), were screened for thermosensitivity characteristics using different analyses. Growth curves at 0, 3, 6, 12, 24, and 30 h of incubation at 37 and 40 °C, the growth inhibition percentage at 40 °C, and the doubling time at 37 and 40 °C were determined for all yeasts studied. Most of the isolates examined exhibited thermotolerant phenotypes at 40 °C, whereas a thermosensitive phenotype at 40 °C was only detected in the Downs reference strain from the USA. Growth inhibition values lower than 33.8% supported the predominance of the thermotolerant phenotype at 40 °C. The doubling time means found for the different isolates were 5.14 h ± 1.47 h at 37 °C and 5.55 h ± 1.87 h at 40 °C. This is the first report to underscore the predominance of thermotolerant and delayed doubling time phenotypes in H. capsulatum clinical isolates from different regions of Latin America.

Germination temperatures affect the physiological quality of seeds of lettuce cultivars / Qualidade fisiológica de sementes de alface sob diferentes temperaturas na germinação

The objective of this study was to evaluate the physiological quality allied to biochemical quality of lettuce seeds by germination and enzymes expression at 20, 25, 30, 35, 40 and 42ºC. Germination speed index and percentage of germination were estimated. Isoenzyme expressions were assessed by alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), catalase (CAT), esterase (EST), pyruvate decarboxylase (PDC) and glutamate oxaloacetate transferase (GOT). The experiment consisted of a completely randomized design in a factorial scheme 4x6, with four cultivars and six different temperatures, with four replications. The highest germination and vigor were observed for cv. 'Everglades' at 35°C, which proved that this cultivar is thermotolerant. Catalase can be considered a genetic marker for the identification ofthermotolerant lettuce cultivars. Cultivar 'Everglades' has potential to be used in lettuce breeding programs aimed at cultivars tolerant to high temperatures during germination.

O objetivo deste estudo foi avaliar a qualidade fisiológica e bioquímica de sementes de alface por meio da germinação e expressão de enzimas a 20, 25, 30, 35, 40 e 42ºC. As variáveis velocidade de germinação e o índice de velocidade de germinação foram estimadas. As expressões das enzimas alcool desidrogenase (ADH), malato desidrogenase (MDH), catalase (CAT), esterase (EST), piruvate descarboxilase (PDC) e glutamato oxaloacetato transferase (GOT) foram avaliadas. Para análise dos genótipos foi empregado o delineamento inteiramente casualizado em esquema fatorial 4x6, testando quatro cultivares e seis diferentes temperaturas, com quatro repetições. A maior germinação e vigor foram observadas para a cv. 'Everglades' a 35°C, o que prova que esta cultivar é termotolerante. A catalase pode ser considerada um marcador para a identificação de cultivares de alface termotolerantes. A cultivar 'Everglades' tem potential para uso em programas de melhoramento visando tolerância à alta temperatura durante a germinação.

Perfil térmico de rutina proveniente da fava d’anta (Dimorphandra gardneriana Tulasne) / Thermal profile of rutina from fava d'anta (Dimorphandra gardneriana Tulasne)

Dimorphandra gardneriana Tulasne é conhecida como fava d’anta, é uma árvore brasileira, naturalmente do Cerrado e Caatinga, têm uma importância ecológica e funcional, seus frutos são ricos em flavonoides (rutina, quercetina, isoquercitrina). O objetivo deste trabalho foi estudar a estabilidade térmica da fava d’anta e da rutina purifica através da termogravimetria. As amostras foram analisadas no Termogravimétrico Shimadzu, TGA-51, no Laboratório de Materiais do IFPI, onde foram acondicionadas em porta-amostras de platina, com atmosfera de nitrogênio e com fluxo de 50 mL/min. A temperatura foi elevada a 600°C, com taxa de aquecimento de 2 °C/min. Obteve-se sucesso com a purificação da fava d’anta, pois a rutina obtida teve maior uniformidade nos eventos térmicos e maior estabilidade térmica em relação a fava.

Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Evaluation of alkali and thermotolerant lipase from an indigenous isolated Bacillus strain for detergent formulation

Background: Lipases are used in detergent industries to minimise the use of phosphate-based chemicals in detergent formulations. The use of lipase in household laundry reduces environmental pollution and enhances the ability of detergent to remove tough oil or grease stains. Results: A lipase-producing indigenous Bacillus subtilis strain [accession no. KT985358] was isolated from the foothills of Trikuta mountain in Jammu and Kashmir, India. The lipase (BSK-L) produced by this strain expressed alkali and thermotolerance. Lipase has an optimal activity at pH 8.0 and temperature 37°C, whereas it is stable at pH 6.0­9.0 and showed active lipolytic activity at temperatures 30 to 60°C. Furthermore, lipase activity was found to be stimulated in the presence of the metal ions Mn2+, K+, Zn2+, Fe2+ and Ca2+. This lipase was resistant to surfactants, oxidising agents and commercial detergents, suggesting it as a potential candidate for detergent formulation. BSK-L displayed noticeable capability to remove oil stains when used in different washing solutions containing buffer, lipase and commercial detergent. The maximum olive oil removal percentage obtained was 68% when the optimum detergent concentration (Fena) was 0.3%. The oil removal percentage from olive oil-soiled cotton fabric increased with 40 U/mL of lipase. Conclusions: This BSK-L enzyme has the potential for removing oil stains by developing a pre-soaked solution for detergent formulation and was compatible with surfactants, oxidising agents and commercial detergents.

Microbiology as an indicator of environmental health of Ilha Comprida lagoons- SP / Microbiologia como indicador da saúde ambiental das lagoas de Ilha Comprida - SP

With an area of 150 Km², the municipality of Ilha Comprida has limited sources of freshwater, sediments and nutrients. However, the sanitary aspects, aiming at Public Health, should be evaluated by determining the densities and the distribution of fecal pollution indicators, mainly bacteria from the group of thermotolerant coliforms. Thus, the objective of this work was to analyze the seasonal variations in the densities of thermotolerant coliforms in the lagoons of the municipality of Ilha Comprida (SP). For that, water samples were collected, in the period between 2014 and 2016, in lagoons of 13 localities (Juruvaúva, City Mar Sul, City Mar Norte, São Januário, Jardim Portugal, Atlântico, Icaraí, Adriana Sul, Adriana Norte, Porto Velho 2, Xandú, Jardim da Barra, Laguna Ponta da Praia) located in the municipality of Ilha Comprida (SP). The results showed that 21.90% of the samples were above the limit established by CONAMA. High densities of bacteria were found in the Icaraí lagoon. The highest averages of thermotolerant coliform densities were recorded in summer and spring. It is concluded that the seasonal variation interferes with the density of thermotolerant coliforms

Com uma área de 150 km², o município de Ilha Comprida tem fontes limitadas de água doce, sedimentos e nutrientes. No entanto, os aspectos sanitários, que visam a Saúde Pública, devem ser avaliados determinando as densidades e a distribuição de indicadores de poluição fecal, principalmente bactérias do grupo de coliformes termotolerantes. Assim, o objetivo deste trabalho foi analisar as variações sazonais nas densidades de coliformes termotolerantes nas lagoas do município de Ilha Comprida (SP). Para isso, foram coletadas amostras de água, no período entre 2014 e 2016, em lagoas de 13 localidades (Juruvaúva, Cidade do Mar do Sul, Cidade do Mar do Norte, São Januário, Jardim Portugal, Atlântico, Icaraí, Adriana Sul, Adriana Norte, Porto Velho 2, Xandú, Jardim da Barra, Laguna Ponta da Praia), localizada no município de Ilha Comprida (SP). Os resultados mostraram que 21,90% das amostras estavam acima do limite estabelecido pelo CONAMA. Foram encontradas altas densidades de bactérias na lagoa de Icaraí. As médias mais elevadas das densidades de coliformes termotolerantes foram registradas no verão e na primavera. Conclui-se que a variação sazonal interfere com a densidade de coliformes termotolerantes

Breed comparison for heat adaptation in rams using multivariate analysis / Comparação entre raças para adaptação ao calor em carneiros usando análise multivariada

In this study, a multivariate analysis of morphological and physiological characteristics was performed on clinically healthy rams from six breeds (Santa Ines, Bergamasca, Dorper, Texel, Ile de France and Hampshire Down) to determine if these characteristics were able to separate and determine the most important variables in the differentiation of breeds for heat adaptation. To characterize the thermal environment, mean temperature was 23°C and relative humidity ranged between 30.6-55.6%. Morphological and physiological data were subjected to multivariate statistical tests including principal components (PRINCOMP), clustering (CLUSTER), discriminant (DISCRIM), step-bystep (STEPDISC) and canonical (CANDISC) analyses, using the Statistical Analysis System (SAS®). A multiple analysis of variance (MANOVA) was carried out with the variables defined as important by the discriminant analysis. The principal components analysis for biometric characteristics and scrotum-testicle, for physiological characteristics and body temperature as well as the characteristics of the skin and hair explained 60, 70 and 67 % of the total variation, respectively. The dendrogram showed a clear separation between the breeds studied and the existence of two distinct groups, one formed by the Texel and the other by the other breeds, considering all the characteristics used in the study. The most useful morphological parameters to explain heat tolerance were diameter of hair, layer thickness of hair at withers, 12th thoracic vertebra and rump, withers height, thoracic and scrotal circumferences, body weight, anterior and posterior shin perimeters, hair and epidermis brightness as well as the content of red and yellow in the epidermis. Among physiological characteristics, respiratory rate was better than rectal temperature and heart rate to explain changes caused by thermal stress. From the multivariate and variance analyzes it can be concluded that the Santa Ines breed was the most tolerant to heat stress as it presented a highly pigmented epidermis, a shorter hair of larger diameter, the lower layer thickness of hair at withers, 12th thoracic vertebra and rump, the lower temperatures in the testicle and at the 12th thoracic vertebra as well as the lower respiratory rate and rectal temperature value.

Neste estudo, foram realizadas análises multivariadas das características fisiológicas e morfológicas em carneiros clinicamente saudáveis de seis raças (Santa Inês, Bergamácia, Dorper, Texel, Ile de France e Hampshire Down) para determinar se essas características foram capazes de separar as raças e determinar as variáveis mais importantes na diferenciação das raças na adaptação ao calor. Os dados foram submetidos a testes estatísticos multivariados, incluindo as análises de componentes principais (PINCOMP), agrupamento (CLUSTER), discriminante (DISCRIM), step-by-step (STEPDISC) e canônica (CANDISC), utilizando o pacote estatístico SAS®. A análise variância de múltipla (MANOVA) foi realizada com as variáveis definidas como importante pela análise discriminante. A análise dos componentes principais para características biométricas e escroto-testiculares, para as características fisiológicas e de temperatura corporal e para as características da pele e pelo explicaram 60, 70 e 67% da variação total, respectivamente. O dendrograma mostrou uma clara separação entre as raças estudadas e a existência de dois grupos distintos, um formado pela raça Texel e o outro pelas raças Dorper, Hampshire Down, Ile de France, Santa Inês e Bergamácia, considerando todas as características avaliadas. As características morfológicas mais importantes para explicar a tolerância ao calor foram o diâmetro do pelo, as espessuras das camadas de pelo na cernelha, na décima segunda vértebra torácica e na garupa, a altura da cernelha, as circunferências torácica e escrotal, o peso corporal, os perímetros das canelas anterior e posterior, as luminosidades da pele e do pelo, bem como os teores de pigmentação vermelho e amarelo na epiderme. Entre as características fisiológicas a frequência respiratória foi melhor que a temperatura retal e a frequência cardíaca para explicar as mudanças causadas pelo estresse térmico. A partir das análises multivariada e de variância pode-se concluir que a raça Santa Inês foi a mais tolerante ao estresse térmico, uma vez que apresentou epiderme altamente pigmentada, pelo mais curto e de diâmetro maior, menores espessuras das camadas de pelo na cernelha, na décima segunda vértebra torácica e na garupa, menores temperaturas no testículo e na décima segunda vértebra torácica, bem como as menores frequência respiratória e temperatura retal.

Topological analysis of carbon flux during multi-stress adaptation in Halomonas sp. AAD12

Background Osmolytes with their effective stabilizing properties are accumulated as protectants not only against salinity but also against denaturing harsh environmental stresses such as freezing, drying, high temperatures, oxygen radicals and radiation. The present work seeks to understand how Halomonas sp. AAD12 cells redirect carbon flux specifically to replenish reactions for biomass and osmolyte synthesis under changing salinity and temperature. To accomplish this goal, a combined FBA-PCA approach has been utilized. Results Experimental data were collected to supply model constraints for FBA and for the verification of the model predictions, which were satisfactory. With restrictions on the various combinations of selected anaplerotic paths (reactions catalyzed by phosphoenolpyruvate carboxylase, pyruvate carboxylase or glyoxylate shunt), two major phenotypes were found. Moreover, under high salt concentrations, when the glucose uptake rate was over 1.1 mmoL DCW- 1 h- 1, an overflow metabolism that led to the synthesis of ethanol caused a slight change in both phenotypes. Conclusions The operation of the glyoxylate shunt as the major anaplerotic pathway and the degradation of 6-phosphogluconate through the Entner-Doudoroff Pathway were the major factors in causing a distinction between the observed phenotypes.

Condições higienicossanitárias da água de coco fornecida por ambulantes

O coco verde é um fruto de natureza litorânea muito apreciado pela população e, portanto, houve um crescimento no consumo da água de coco em regiões não litorâneas, dando origem ao comércio de água de coco em carrinhos refrigerados como forma de atividade ambulante. Porém, esta tem sido apontada como uma das causas do aumento de Doenças Transmitidas por Alimentos (DTA). O presente trabalho teve como objetivo avaliar a qualidade microbiológica da água de coco fornecida por ambulantes, em equipamentos de resfriamento, no centro da cidade de Teresina - PI. Foram feitas 30 coletas em diferentes pontos da cidade para a pesquisa de coliformes totais e termotolerantes, presença de Salmonella spp., contagem de bactérias aeróbias mesófilas e contagem de bolores e leveduras, seguindo-se a Resolução RDC n° 12, de 2 de janeiro de 2001, da ANVISA. As contagens de bactérias foram relativamente altas, variando entre 0 e 2,52x 105 UFC/mL. Para coliformes totais e termotolerantes variaram de 11 a >2400 e de 2400 (NMP/mL), respectivamente. Houve ausência de Salmonella spp. em 100% das amostras. Em 16 amostras (53,3%), houve crescimento fúngico, cuja contagem variou de <10 a 3,2x104 UFC/mL. Ao fim dos experimentos, concluiu-se que 37% das amostras se encontravam impróprias para consumo por apresentar valores de coliformes a 45°C acima dos limites estabelecidos pela legislação vigente.

The coconut is a fruit naturally found on the coastal region and it is very valued by the local population. There was an increase on the consummation of coconut water on non coastal regions which originated mobile cooling equipment as a street vending business. However, the street vending activity has been pointed as a source of increased cases of Diseases Transmitted by Food (DTA). The current research paper aimed to evaluate the microbiological quality of coconut water provided by street vendors in mobile cooling equipments downtown Teresina city, Piaui. Thirty samples were collected in different spots of the city for the search of total and thermotolerant coliforms, presence of Salmonella ssp, aerobic mesophilic bacteria, yeast and mold count, following the Resolution - RDC n° 12 from January 2nd, 2001 of ANVISA/MS. The bacteria counts were relatively high ranging between 0 and 2,52x105 CFU/mL. Total and thermotolerant coliforms ranged between 11 to >2400 and between 2400 MPN/mL respectively. There was absence of Salmonella ssp in 100% of the samples. There was fungal growth in 16 samples (53,3%) which count ranged between <10 to 3,2x104 CFU/mL. By the end of the experiments it was concluded that 37% of the samples were improper to consume because they showed values of 45º coliforms over the established limits by current legislation.

Greenhouse evaluation on the performance of heat tolerant transgenic broccoli and genetic diversity analysis using inter simple sequence repeat (ISSR) markers

Background: Broccoli, Brassica oleracea subsp. italica is one of the many valuable Brassica species which is still less cultured under in vitro condition. Heat tolerant transgenic and non-transgenic broccoli cv. Green Marvel plantlets with well-developed root system obtained through in vitro culture were transferred into disposable plastic pots containing sterilized potting mixture consisting of (peatgroTM) + coconut dust (2:1) and maintained in a growth chamber. Results: After one month, the hardened plantlets were transferred and maintained in a transgenic greenhouse. After four months of acclimatization in the transgenic greenhouse, the efficacy of HSP101 gene in increasing the heat tolerance of the transgenic broccoli was evaluated. Results showed that the transgenic plants could survive and performed normally, producing flower heads even at the highest tested temperature of 34ºC. Seven transgenic broccoli lines with different gene copy number of the AtHSP101 gene as well as the control plant were assessed for genetic diversity using inter simple sequence repeat (ISSR) markers. Conclusions: ISSR results showed polymorphism and phylogenetic relationship between the transgenic and non-transgenic (control) Brassica oleracea cv. Green Marvel.