Molecular diversity of bla genes in Klebsiella pneumoniae and Escherichia coli isolates

Dissertation presented to obtain a Ph.D. degree in Biology, speciality Microbiology, by Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Detection of classic and invasive E. coli and Shigella serotypes in stools by indirect immunofluorescence

IIF in the detection of invasive and classic enteropathogenic E. coli and Shigella serotypes was compared with traditional coproculture methods. IIP results agreed with the coproculture findings in 128 out of 140 cases tested for enteropathogenic E. coli (91%) and in 108 out of 112 for Shigella (96%). All cases with positive reactions by coproculture were confirmed by IIP. In the control group it were obtained by IIF 12 cases with positive reactions for enteropathogenic E. coli and 4 cases for Shigella, including two cases of mixed infection by E. coli 026/Sh. dysenteriae and E. coli 0124/Sh. dysenteriae. It was discussed the high sensitivity and specificity of the IIF when compared with the traditional methods, being suggested that IIF is a valuable tool in epidemiological studies involving these organisms and an important aid in the stablishment of an early presumptive diagnosis of the acute infantile diarrhea.

Incidência de escherichia coli enterotoxigênica (ETEC), rotavirus e Clostridium perfringens de casos de diarréia em crianças, na região de Campinas, SP, Brasil

Foi realizada uma pesquisa na região de Campinas, SP, Brasil, sobre a presença de Escherichia coli enterotoxigênica (ETEC), rotavírus e Clostridium perfringens enterotoxigênico em fezes diarréicas de crianças com até 2 anos de idade. Dos 132 espécimens fecais examinados quanto à presença de ETEC 27 (20,45%) foram positivos. Destes foram isoladas 41 amostras de ETEC, das quais 40 produziram apenas a enterotoxina termolábil (LT) detectada pelo teste de imuno hemólise radial modifi cado. Entre as 183 amostras de fezes examinadas para rotavírus, 29 (15,84%) foram positivas pelas técnicas de eletroforese em gel de poliacrilamida (PAGE) e ensaio imunoenzimático (EIE), sendo que destas, 15 (51,7% ) foram provenientes de materiais coletados nos meses de inverno. Todas as amostras pertenciam ao grupo A e, através da técnica de PAGE, pode-se observar que o tipo eletroforético mais freqüente (9 amostras) foi designado Ib, IIc, Illb, IVa, de acordo com a classificação por nós adotada. Apenas 113 amostras de fezes foram examinadas para a presença de C. perfringens enterotoxigênico. Para a detecção da enterotoxina nos sobrenadantes das culturas foram utilizadas as técnicas de hemaglutinação passiva reversa e inoculação intravenosa em camundongos, sendo encontradas 12 (10,61%) amostras entero-toxigênicas. Diante destes resultados é chamada a atenção sobre o valor apenas relativo de uma coprocultura convencional para fins de diagnóstico, ressaltando-se a importância da criação de métodos simplificados que favoreçam a detecção e identificação dos grupos de agentes enteropatogênicos estudados na presente pesquisa.

Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

Glucose monitoring in vivo is a crucial issue for gaining new understanding of diabetes. Glucose binding protein (GBP) fused to two fluorescent indicator proteins (FLIP) was used in the present study such as FLIP-glu- 3.2 mM. Recombinant Escherichia coli whole-cells containing genetically encoded nanosensors as well as cell-free extracts were immobilized either on inner epidermis of onion bulb scale or on 96-well microtiter plates in the presence of glutaraldehyde. Glucose monitoring was carried out by Förster Resonance Energy Transfer (FRET) analysis due the cyano and yellow fluorescent proteins (ECFP and EYFP) immobilized in both these supports. The recovery of these immobilized FLIP nanosensors compared with the free whole-cells and cell-free extract was in the range of 50–90%. Moreover, the data revealed that these FLIP nanosensors can be immobilized in such solid supports with retention of their biological activity. Glucose assay was devised by FRET analysis by using these nanosensors in real samples which detected glucose in the linear range of 0–24 mM with a limit of detection of 0.11 mM glucose. On the other hand, storage and operational stability studies revealed that they are very stable and can be re-used several times (i.e. at least 20 times) without any significant loss of FRET signal. To author's knowledge, this is the first report on the use of such immobilization supports for whole-cells and cell-free extract containing FLIP nanosensor for glucose assay. On the other hand, this is a novel and cheap high throughput method for glucose assay.

Avaliação de massas específicas de cistos de Giardia duodenalis e Entamoeba coli

Cistos de Giardia duodenalis e Entamoeba coli foram observados quanto à flutuação em soluções de sacarose de massas específicas diversas (1.040, 1.050, 1.060, 1.070,1.080,1.090,1.100, 1.150,1.200 e 1.250 kg/m³), contidas em câmaras de contagem de 0,17 mm de altura. Contaram-se os cistos que flutuaram e os que sedimentaram tendo sido calculadas as porcentagens respectivas. As diferenças de flutuabilidade dos cistos de cada uma das espécies não foram consideráveis. Soluções de massa específica igual a 1.200 kg/m³ fizeram flutuar 88,49% dos cistos de G. duodenalis e 95,71% dos de E. coli. Os maiores valores de flutuabilidade estiveram associados à massa específica 1.250 kg/m³ e foram, respectivamente, 89,15% e 98,59% para cistos de G. duodenalis e de E. coli.

Caracterização preliminar da bioenergética do efluxo pelo sistema AcrAB-TolC em Escherichia coli

A resistência aos antibióticos em bactérias Gram-negativas pode ser aumentada pela extrusão de antibióticos através de sistemas de efluxo. Em Escherichia coli, o principal sistema de efluxo é o AcrAB-TolC o qual tem como principal fonte energética a força proto-motriz. Este trabalho pretendeu estudar alguns aspectos essenciais da bioenergética na actividade de efluxo de E. coli usando três estirpes bem caracterizadas genotipica e fenotipicamente. Foi utilizado um método fluorimétrico semi-automático no qual a fluorescência do fluorocromo brometo de etídeo, substrato de bombas de efluxo foi seguida, permitindo a medição em tempo real da actividade de efluxo e acumulação de fluorocromo (inibição do efluxo). A utilização de brometo de etídeo é particularmente vantajosa pois emite baixa fluorescência no exterior da célula bacteriana tornando-se extremamente fluorescente no seu interior. Este método é uma nova aplicação do termociclador em tempo real RotorGeneTM 3000 que permite o cálculo da cinética de transporte reflectindo o balanço entre acumulação de substrato por difusão passiva através da membrana e a sua extrusão/efluxo, proporcionando uma detecção rápida e económica de inibidores de efluxo. Os resultados obtidos mostram, para todas as estirpes, que a GLU e o pH afectam a acumulação e o efluxo do brometo de etídeo. De todos os inibidores de vias biossintéticas testados, o ortovanadato de sódio, foi o que demonstrou maior actividade inibitória, a qual é revertida na presença de GLU. Em conclusão, este estudo mostra que a actividade de efluxo de E. coli depende não só da fosforilação oxidativa por via da força proto-motriz mas também da energia proveniente da hidrólise de ATP pelas ATPases. O ortovanadato de sódio tem potencial para ser um novo inibidor de bombas de efluxo de largo espectro. A tecnologia utilizada neste trabalho demonstrou ser apropriada para a caracterização bioenergética da actividade de bombas de efluxo e permite a selecção de novos inibidores de bombas de efluxo em bactérias.

Purification and parcial characterization of a hemagglutinating factor (HAF): a possible adhesive factor of the diffuse adherent of Escherichia coli (DAEC)

The mannose-resistant hemagglutinating factor (HAF) was extracted and purified from a diffuse adherent Escherichia coli (DAEC) strain belonging to the classic enteropathogenic E. coli (EPEC) serotype (0128). The molecular weight of HAF was estimated to be 18 KDa by SDS-PAGE and 66 KDa by Sephadex G100, suggesting that the native form of HAF consists of 3-4 monomeric HAF. Gold immunolabeling with specific HAF antiserum revealed that the HAF is not a rigid structure like fimbriae on the bacterial surface. The immunofluorescence test using purified HAF on HeLa cells, in addition to the fact that the HAF is distributed among serotypes of EPEC, suggests that HAF is a possible adhesive factor of DAEC strains

The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

PLOS ONE, 4(8):ARTe6820

Asymptomatic infections by diarrheagenic Escherichia coli in children from Misiones, Argentina, during the first twenty months of their lives

Diarrheagenics Escherichia coli are the major agents involved in diarrheal disease in developing countries. The aim of this study was to evaluate the time of appearance of the first asymptomatic infection by the different categories of diarrheagenic E. coli in 44 children since their birth and during the first 20 months of their lives. In all of the children studied, we detected at least one category of diarrheagenic E. coli through the 20 months of the study. 510 diarrheagenic E. coli (33.5%) were obtained from the 1,524 samples collected from the 44 children during the time of the study (31.4% EAggEC, 28.8% EPEC, 27.1% DAEC, and 12.7% ETEC). Neither EHEC nor EIEC were identified. The median age for diarrheagenic E. coli colonization was 7.5 months. The mean weaning period was 12.8 months and the mean age for introduction of mixed feeding (breast fed supplemented) was 3.8 months. A significantly lower incidence of diarrheal disease and asymptomatic infections was recorded among the exclusively breast-fed rather than in the supplemented and non breast-fed infants. For ETEC, EPEC and EAggEC the introduction of weaning foods and complete termination of breast-feeding were associated with an increase of asymptomatic infections.

Pesquisa de factores de virulência em estirpes de Escherichia coli em resíduos de produção animal

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Hybrid systems biology: application to Escherichia coli

Dissertation presented to obtain a Master degree in Biotechnology

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.

Glucose consume and growth of E. coli under electromagnetic field

E. coli was submitted to a 5G electromagnetic field generated by a alternate 60 Hz voltage source. The differences on growth and glucose consume in control and exposed groups were evaluated using the non-parametric Mann-Whitney U-test. There was a significant difference in glucose consume and growth in E. coli after 8 hours of exposition to electromagnetic field. It can be concluded that electromagnetic field had a positive effect in consume of glucose and growth of E. coli. The cause of these results can be explained by an increasing of glucose entrance through membrane due to the stimulated transport system via Facility Diffusion or cyclotron resonance. The growth can be caused by shortening of lag phase and excitement of log phase.

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins (OMPs) was developed. Two single stranded DNA sequences were tested as recognition elements and compared. The aptamer capture probes were immobilized, with and without 6-mercapto-1-hexanol (MCH) on a gold electrode. Each step of the modification process was characterized by Faradaic impedance spectroscopy (FIS). A linear relationship between the electron-transfer resistance (Ret) and E. coli OMPs concentration was demonstrated in a dynamic detection range of 1 × 10−7–2 × 10−6 M. Moreover, the aptasensor showed selectivity despite the presence of other possible water contaminates and could be regenerated under low pH condition. The developed biosensor shows great potential to be incorporated in a biochip and used for in situ detection of E. coli OMPs in water samples.

Stochastic model of transcription initiation of closely spaced promoters in escherichia coli

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica