Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Potential pathogenic role of aggregative- adhering Corynebacterium diphtheriae of different clonal groups in endocarditis

Invasive diseases caused by Corynebacterium diphtheriae have been described increasingly. Several reports indicate the destructive feature of endocarditis attributable to nontoxigenic strains. However, few reports have dealt with the pathogenicity of invasive strains. The present investigation demonstrates a phenotypic trait that may be used to identify potentially invasive strains. The study also draws attention to clinical and microbiological aspects observed in 5 cases of endocarditis due to C. diphtheriae that occurred outside Europe. Four cases occurred in female school-age children (7-14 years) treated at different hospitals in Rio de Janeiro, Brazil. All patients developed other complications including septicemia, renal failure and/or arthritis. Surgical treatment was performed on 2 patients for valve replacement. Lethality was observed in 40% of the cases. Microorganisms isolated from 5 blood samples and identified as C. diphtheriae subsp mitis (N = 4) and C. diphtheriae subsp gravis (N = 1) displayed an aggregative adherence pattern to HEp-2 cells and identical one-dimensional SDS-PAGE protein profiles. Aggregative-adhering invasive strains of C. diphtheriae showed 5 distinct RAPD profiles. Despite the clonal diversity, all 5 C. diphtheriae invasive isolates seemed to display special bacterial adhesive properties that may favor blood-barrier disruption and systemic dissemination of bacteria. In conclusion, blood isolates from patients with endocarditis exhibited a unique adhering pattern, suggesting a pathogenic role of aggregative-adhering C. diphtheriae of different clones in endocarditis. Accordingly, the aggregative-adherence pattern may be used as an indication of some invasive potential of C. diphtheriae strains.

Emergence of clonal complex 5 (CC5) methicillin-resistant Staphylococcus aureus (MRSA) isolates susceptible to trimethoprim-sulfamethoxazole in a Brazilian hospital

In this study, genotyping techniques including staphylococcal chromosomal cassette mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and restriction-modification tests were used to compare the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered at two times within a 10-year interval (1998 and 2008) from a tertiary Brazilian hospital. In addition, the antimicrobial susceptibility profiles were analyzed. All 48 MRSA isolates from 1998 and 85.7% from 2008 (48/56 isolates) displayed multidrug-resistance phenotypes and SCCmec III. All but one of the 13 representative SCCmec III isolates belonged to CC8 and had PFGE patterns similar to that of the BMB9393 strain (Brazilian epidemic clone of MRSA; BEC). In 2008, we found an increased susceptibility to rifampicin and chloramphenicol among the SCCmec III isolates. In addition, we detected the entrance of diverse international MRSA lineages susceptible to trimethoprim-sulfamethoxazole (SXT), almost all belonging to CC5. These non-SCCmec III isolates were related to the USA 300 (ST8-SCCmec IV; PFGE-type B), USA 800 (ST5-SCCmec IV; subtype D1), USA 100 (ST5-SCCmec II; subtype D2), and EMRSA-3/Cordobes (ST5-SCCmec I, type C) clones. To the best of our knowledge, this is the first report of the emergence of isolates genetically related to the EMRSA-3/Cordobes clone in southeast Brazil. In this regard, these isolates were the most common non-SCCmec III MRSA in our institution, accounting for 8.9% of all isolates recovered in 2008. Thus, despite the supremacy of BEC isolates in our country, significant changes may occur in local MRSA epidemiology, with possible consequences for the rationality of MRSA empiric therapy.

Acumulação de matéria seca pelo cafeeiro conilon

Com o objetivo de caracterizar a acumulação de matéria seca pelo cafeeiro conilon (Coffea canephora Pierre), um experimento foi conduzido em Marilândia/ES, no delineamento de blocos casualizados, com 24 tratamentos (época de amostragem) e três repetições. Utilizou-se a variedade clonal EMCAPA 8111, plantada em outubro de 1995. Constatou­se que o cafeeiro conilon apresentou padrão sigmoidal de crescimento até os seis anos de idade, com acumulação crescente de massa de matéria seca. Houve declínio na taxa de crescimento absoluto após 48 meses de idade e diminuição na taxa de crescimento relativo após o primeiro mês de idade.

Propagação vegetativa de porta-enxertos de pereira por estacas semi-lenhosas

Pesquisou-se a viabilidade técnica da propagação vegetativa dos porta-enxertos de pereira 'Taiwan Nashi-C', 'Taiwan Mamenashi' (Pyrus calleryana Dcne.) e 'Seleção IAC-1' (Pyrus spp.), por estacas semi-lenhosas. O experimento foi conduzido em telado equipado com sistema de nebulização intermitente, pertencente ao Núcleo de Pesquisa e Desenvolvimento do Jardim Botânico, do Instituto Agronômico (IAC), em Campinas, Estado de São Paulo. Adotou-se o fatorial 3 x 4 (porta-enxertos x doses de ácido indolbutírico), com cinco repetições de 20 estacas, em delineamento inteiramente casualizado. Com os resultados obtidos aos 60 dias após a estaquia, conclui-se que: a) é tecnicamente possível a propagação dos porta-enxertos 'Taiwan Nashi-C', 'Taiwan Mamenashi' e 'Seleção IAC-1' por estacas semi-lenhosas; b) dentre os porta-enxertos pesquisados, 'Taiwan Nashi-C' apresenta a maior porcentagem de enraizamento, a menor porcentagem de estacas com calo, além do maior comprimento de raízes e número de raízes por estaca; c) é necessário o uso de ácido indolbutírico para o enraizamento de estacas semi-lenhosas desses porta-enxertos, sendo que, no conjunto das variáveis analisadas, as concentrações de 4.000 mg.L-1 e de 6.000 mg.L-1 proporcionam os maiores benefícios ao enraizamento adventício; d) as concentrações de ácido indolbutírico testadas não são fitotóxicas às estacas semi-lenhosas dos genótipos pesquisados.

Efficiency and response of conilon coffee clones to phosphorus fertilization

Studies on nutritional efficiency of phosphorus in conilon coffee plants are important tools to unravel the high limitation that natural low levels of this nutrient in soil impose to these species cultivars. Therefore, this study aimed at evaluating the nutritional efficiency and the response to phosphorus of conilon coffee clones. Plants were managed during 150 days in pots containing 10 dm³ of soil, in greenhouse. A factorial scheme 13 x 2 was used, with three replications, being the factors: 13 clones constituting the clonal cultivar "Vitória Incaper 8142" and two levels of phosphate fertilization (0% and 150% of the P2O5 usualy recommended), in a completely randomized design (CRD). The results indicate a differentiated response of dry matter production and of phosphorus content on each level of phosphate fertilization for the conilon coffee clones and that CV-04, CV-05 and CV-08 clones are nutritionally efficient and responsive to the phosphate fertilization.

Relações entre caracteres de tubérculo de batata nas primeiras gerações de seleção

O objetivo deste trabalho foi verificar a relação entre caracteres fenotípicos de tubérculo avaliados em progênies de batatas, nas gerações de plântula e primeira geração clonal. Os experimentos foram realizados na Embrapa Clima Temperado, em Pelotas, RS. Foram avaliadas, nas gerações de plântula, 12 progênies de batatas, cultivadas em casa de vegetação na safra de 2010/2011, e primeira geração de campo, cultivadas na safra de 2011/2012. O delineamento, em ambos os experimentos, foi o de blocos ao acaso, com três repetições de 25 plântulas para cada progênie. Após as colheitas, os tubérculos de cada planta foram avaliados para 14 caracteres fenotípicos. Foram realizadas análises de variância individuais e conjunta para as gerações e, análises de correlação entre as gerações, e de trilha, dentro das gerações. Os caracteres aparência e aspereza de tubérculo; curvatura e apontamento; massa e número de tubérculos, proeminência da sobrancelha e profundidade de olhos são fortemente e favoravelmente correlacionados nas duas primeiras gerações de seleção. O grau de correlação entre as duas primeiras gerações para profundidade de olho, proeminência de sobrancelha, massa de tubérculo, número de tubérculos, curvatura de tubérculo e apontamento de tubérculo indica que a seleção para estes caracteres pode ser aplicada na geração de plântula. Aspereza e curvatura de tubérculo são os caracteres que mais influenciam na aparência geral; portanto, sua seleção pode ser utilizada no melhoramento para aparência geral de tubérculo.

Efeito de extratos proteicos de amendoim sobre o desenvolvimento, a capacidade infectiva e atividade de enzimas proteolíticas de Meloidogyne enterolobii

No Brasil, Meloidogyne enterolobii vem causando perdas significativas na produção de goiabeiras e, no submédio do Vale do São Francisco, por exemplo, o impacto negativo decorrente da infecção e morte de goiabeiras tem refletido diretamente na qualidade de vida dos agricultores. Até o momento, não existem métodos de controle efetivos: os nematicidas avaliados experimentalmente não têm sido eficientes e não há produtos registrados no Ministério da Agricultura, Pecuária e Abastecimento para aplicação em goiabeiras. Assim, há necessidade de realizar pesquisa básica nesta área. A prospecção de proteínas relacionadas com a resistência em espécies resistentes pode ser uma alternativa para o desenvolvimento de medidas para seu controle. Para isso, foram realizados ensaios biológicos de toxicidade para a análise do efeito de extratos proteicos de amendoim sobre o desenvolvimento, a capacidade infectiva e atividade de enzimas proteolíticas de M. enterolobii visando à identificação de proteínas com potencial para controle desse nematoide. Os resultados obtidos indicaram que o extrato proteico total de amostras de raízes de plantas de amendoim inoculadas não tem efeito sobre as três características supracitadas. Sendo assim, são necessários estudos dessa natureza com outras espécies resistentes ao patógeno visando à identificação de proteínas que apresentem potencial para o seu controle.

Correlação fenotípica entre caracteres agronômicos em população segregante de mandioca de mesa

Um dos principais entraves do melhoramento genético de mandioca é a dificuldade de seleção precoce de indivíduos superiores nas primeiras gerações do melhoramento. Dessa forma, o objetivo deste trabalho foi estimar correlações fenotípicas entre caracteres agronômicos de interesse em uma população segregante de mandioca nas gerações de propagações vegetativa e sexual e entre as duas, a fim de contribuir na geração de indicadores fenotípicos para elevar a eficiência do processo de seleção de genótipos superiores de mandioca de mesa em populações segregantes. Na população segregante obtida por meio do cruzamento entre as variedades de mandioca de mesa IAC 576-70 e BRS Japonesa, foi estimada a correlação fenotípica entre sete caracteres agronômicos avaliados individualmente na população de propagação sexual e na população clonal e entre as duas populações. Nenhum caráter na geração de propagação sexual apresentou correlação de elevada magnitude com a produtividade de raízes na geração de propagação clonal. Essa fraca associação entre caracteres na geração de propagação por sementes e o seu potencial produtivo, quando da propagação clonal, revelaram a dificuldade de se prever com acurácia, por meio de avaliações fenotípicas, o potencial produtivo de determinado genótipo precocemente, mesmo quando da sua avaliação a partir de sementes. Entretanto, a associação elevada observada entre o tempo para a cocção na geração de propagação por sementes e na geração de propagação clonal é um indicativo da possibilidade da seleção precoce de clones com baixo tempo para o cozimento.

Modelagem de probabilidade de churn

Clientes podem abandonar uma organização ainda que altos investimentos em prospecção e retenção sejam realizados, o que requer diagnóstico e compreensão. Este artigo objetiva modelar a probabilidade de clientes abandonarem o relacionamento com uma organização, fenômeno conhecido como churn, utilizando dados do histórico de relacionamento cliente/empresa, validar o modelo em uma segunda amostra e descrever as possíveis variáveis que influenciam o abandono/permanência do cliente. Utilizou-se o modelo de regressão logística em uma amostra de calibração de 70.000 clientes que possuíam cartão de crédito próprio de uma grande rede varejista. Dezesseis variáveis explicativas (14 características individuais e duas variáveis comportamentais) foram usadas e o modelo foi validado em uma amostra de 30.000 clientes, usando-se o teste de KS (Kolmogorov-Smirnov) e a curva ROC (Receiver Operating Characteristic), que demonstraram a boa adequação do modelo à amostra de validação. Implicações da pesquisa e sugestões para futuras investigações são discutidas à luz da gestão do relacionamento com o cliente.

Genetic structure of Neisseria meningitidis serogroup C epidemic strains in South Brazil

In the present study we report the results of an analysis, based on serotyping, multilocus enzyme electrophoresis (MEE), and ribotyping of N. meningitidis serogroup C strains isolated from patients with meningococcal disease (MD) in Rio Grande do Sul (RS) and Santa Catarina (SC) States, Brazil, as the Center of Epidemiology Control of Ministry of Health detected an increasing of MD cases due to this serogroup in the last two years (1992-1993). We have demonstrated that the MD due to N.meningitidis serogroup C strains in RS and SC States occurring in the last 4 years were caused mainly by one clone of strains (ET 40), with isolates indistinguishable by serogroup, serotype, subtype and even by ribotyping. One small number of cases that were not due to an ET 40 strains, represent closely related clones that probably are new lineages generated from the ET 40 clone referred as ET 11A complex. We have also analyzed N.meningitidis serogroup C strains isolated in the greater São Paulo in 1976 as representative of the first post epidemic year in that region. The ribotyping method, as well as MEE, could provide useful information about the clonal characteristics of those isolates and also of strains isolated in south Brazil. The strains from 1976 have more similarity with the actual endemic than epidemic strains, by the ribotyping, sulfonamide sensitivity, and MEE results. In conclusion, serotyping with monoclonal antibodies (C:2b:P1.3), MEE (ET 11 and ET 11A complex), and ribotyping by using ClaI restriction enzyme (Rb2), were useful to characterize these epidemic strains of N.meningitidis related to the increased incidence of MD in different States of south Brazil. It is mostly probable that these N.meningitidis serogroup C strains have poor or no genetic corelation with 1971-1975 epidemic serogroup C strains. The genetic similarity of members of the ET 11 and ET 11A complex were confirmed by the ribotyping method by using three restriction endonucleases.

Meningococcal Disease Caused by Neisseria meningitidis Serogroup B Serotype 4 in São Paulo, Brazil, 1990 to 1996

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988.21 A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Genetic diversity and antimicrobial resistance of enterococcal isolates from Southern region of Brazil

In the present study, a total of 455 enterococcal isolates, recovered from patients living in the city of Porto Alegre, State of Rio Grande do Sul, Brazil, during the period from July 1996 to June 1997, were identified to the species level by conventional biochemical and microbiological tests, and assayed for their susceptibilities to antimicrobial agents. The genetic diversity of antimicrobial resistant strains was evaluated by pulsed-field gel electrophoresis (PFGE) analysis of SmaI restricted chromosomal DNA. The most frequent species was Enterococcus faecalis (92.8%). Other species identified were: E. faecium (2.9%), E. gallinarum (1.5%), E. avium (1.1%), E. hirae (0.7%), E. casseliflavus (0.4%), E. durans (0.4%) and E. raffinosus (0.2%). The overall prevalence of isolates with high-level resistance (HLR) to aminoglycosides was 37.8%. HLR to gentamicin was found in 24.8%. No strains with acquired resistance to vancomycin were found. PFGE analysis showed the predominance of clonal group A, comprising strains isolated from different clinical specimens obtained from patients in three hospitals. These results suggest intra and inter-hospital dissemination of one predominant clonal group of E. faecalis isolates with HLR to gentamicin in the hospitals included in this study.

Molecular characterization of vancomycin-resistant Enterococci strains eight years apart from its first isolation in São Paulo, Brazil

E. faecium was the first reported VRE species, carrying the vanA gene in Brazil. In spite of this, vancomycin-resistant E. faecalis has become the predominant species in Brazilian hospitals. The aim of this study was to evaluate the genetic relatedness of VREs isolated in a Brazilian teaching hospital eight years apart from its first isolation. We analyzed 38 VRE strains obtained from 81 surveillance cultures of patients admitted to the four largest intensive care units in Hospital São Paulo in February, 2006. Presence of the vanA gene was assayed by PCR and PFGE analysis was used for molecular characterization. All VRE strains carried the vanA gene. Two distinct clonal groups were observed among vancomycin-resistant E. faecalis. Vancomycin-resistant E. faecium belonged to five distinct clones were demonstrated by molecular typing. All of these clones were different from the first vancomycin-resistant enterococci clone isolated eight years ago in our hospital.