Estudos de QSAR baseados em dados de atividade biológica obtidos por microcalorimetria: III interação de m-alcoxifenóis e p-hidroxibenzoatos de alquila com Escherichia coli

QSAR studies based on flow microcalorimetric bioassay data for interaction of homologous series of m-alkoxyphenols and p-hydroxybenzoates with E. coli cells were carried out applying factorial design. Results for both series showed a linear relationship between log(dose)max and log Po/w. Analysis of these data allows the identification of contributions toward the derived bioactivity from the parent structures (the molecule minus n-CH2 groups present in the side-chain) and the lipophilic groups, CH2. These results are discussed with respect to drug quantitative structure-relationship.

Inativação por oxidação fotocatalítica de Escherichia coli e Pseudomonas sp.

This study presents the bactericidal activity of titanium dioxide photocatalysis, using as model Escherichia coli and Pseudomonas sp. cells. It was observed that the process efficiency is related to initial cell concentration, light intensity, UV irradiation exposure time, TiO2 concentration increase. The ultimate removal efficiency was above 99.9%.

Combined dielectrophoretic and impedance system for on-chip controlled bacteria concentration: Application to Escherichia coli

The present paper reports a bacteria autonomous controlled concentrator prototype with a user-friendly interface for bench-top applications. It is based on a micro-fluidic lab-on-a-chip and its associated custom instrumentation, which consists in a dielectrophoretic actuator, to pre-concentrate the sample, and an impedance analyser, to measure concentrated bacteria levels. The system is composed by a single micro-fluidic chamber with interdigitated electrodes and a instrumentation with custom electronics. The prototype is supported by a real-time platform connected to a remote computer, which automatically controls the system and displays impedance data used to monitor the status of bacteria accumulation on-chip. The system automates the whole concentrating operation. Performance has been studied for controlled volumes of Escherichia coli (E. coli) samples injected into the micro-fluidic chip at constant flow rate of 10 μL/min. A media conductivity correcting protocol has been developed, as the preliminary results showed distortion of the impedance analyser measurement produced by bacterial media conductivity variations through time. With the correcting protocol, the measured impedance values were related to the quantity of bacteria concentrated with a correlation of 0.988 and a coefficient of variation of 3.1%. Feasibility of E. coli on-chip automated concentration, using the miniaturized system, has been demonstrated. Furthermore, the impedance monitoring protocol had been adjusted and optimized, to handle changes in the electrical properties of the bacteria media over time.

Treated domestic sewage: kinetics of Escherichia coli and total coliform inactivation by oxidation with hydrogen peroxide

Hydrogen peroxide has been used for decades in developed countries as an oxidizing agent in the treatment of water, domestic sewage and industrial effluents. This study evaluated the influence of the concentration of H2O2 and pH on the inactivation of Escherichia coli cells and the disinfection of sewage treated. The results showed that the inactivation rate increased with pH and H2O2. The presence of other contaminants dissolved in the effluent is probably the cause of these differences, because E. coli inactivation in synthetic wastewater was found to be much faster than in the real treated domestic sewage.

Expressão em Escherichia coli da proteína capsidial do Watermelon mosaic virus e produção de anti-soro

Um anti-soro policlonal específico para Watermelon mosaic virus (WMV) foi produzido por meio da imunização de coelhos com proteína capsidial purificada, expressa in vitro em células de Escherichia coli. O gene cp foi amplificado via RT-PCR utilizando oligonucleotídeos específicos, a partir de RNA viral extraído de preparações virais concentradas. O fragmento amplificado foi clonado em pBLUESCRIPT KS+ e completamente seqüenciado para confirmação de sua identidade e integridade. Em seguida, o fragmento foi subclonado no vetor de expressão pRSET-A. Plasmídeos recombinantes foram utilizados para a expressão da proteína capsidial em E. coli BL21::DE3. A proteína foi purificada por meio de cromatografia de afinidade em coluna de Ni+-NTA, a partir de proteínas totais extraídas de E. coli. Uma vez purificada, a proteína foi quantificada e utilizada para imunização dos coelhos. O anti-soro foi testado quanto a sua especificidade e sensibilidade em testes de Western blot, DAS-ELISA, imunodifusão e ELISA indireto. Todos os testes demonstraram que o anti-soro produzido a partir da expressão in vitro da proteína capsidial é altamente específico para a detecção do WMV em extratos foliares, não tendo sido observada nenhuma reação heteróloga interespecífica.

Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Antimicrobial Resistance in Campylobacter jejuni and Campylobacter coli

<b>Antimicrobial Resistance in <i>Campylobacter jejuni</i> and <i>Campylobacter coli</i></b> Campylobacters are a common cause of bacterial gastroenteritis worldwide, with <i>Campylobacter jejuni</i> and <i>C. coli</i> being the most common species isolated in human infections. If antimicrobial treatment is required, the drugs of choice at the moment are the macrolides and fluoroquinolones. In this thesis, the in vitro resistance profiles of the <i>C. jejuni</i> and <i>C. coli</i> strains were evaluated with emphasis on multidrug resistance. The aim was also to evaluate the different resistance mechanisms against the macrolides. Further, the disk diffusion method was compared to agar dilution method and its repeatability was evaluated, since it has been widely used for the susceptibility testing of campylobacters. The results of the present study showed that resistance to the fluoroquinolones is common in strains isolated from Finnish patients, but resistance to the macrolides is still rare. Multidrug resistance was associated with resistance to both ciprofloxacin and erythromycin. Among the available per oral drugs, least resistance was observed to coamoxiclav There was no resistance to the carbapenems. Sitafloxacin and tigecycline were <i>in vitro</i> highly effective towards <i>Campylobacter</i> species. A point mutation A2059G of the 23S rRNA gene was the main mechanism behind the macrolide resistance, whereas the efflux pumps did not seem to play an important role when a strain had A2059G mutation. A five amino acids insertion, which has not been described previously, in the ribosomal protein L22 of one highly-resistant C. jejuni strain without mutation in the 23S rRNA gene was also detected. Concerning the disk diffusion method, there was variation in the repeatability In conclusion, macrolides still appear to be the first-choice alternative for suspected <i>Campylobacter</i> enteritis. The<i> in vitro</i> susceptibilities found suggest that co-amoxiclav might be a candidate for clinical trials on campylobacteriosis, but in life-threatening situations, a carbapenem may be the drug of choice. More studies are needed on whether the disk diffusion test method could be improved or whether all susceptibilities of campylobacters should be done using a MIC based method.

Water quality indicators for environmental and resistance profile of Escherichia coli strains isolated in Rio Cascavel, Paraná, Brazil

In this article it was evaluated the quality of water in the Cascavel river, in the city of Cascavel - Paraná using microbiological indicators, physical and chemical pollution and susceptibility / resistance in strains of Escherichia coli isolated antimicrobial trade. The water sampling was conducted between 2010-July and 2011-June at three points: a) near the source, b) urban area, c) rural area. The samples were analyzed for physical, chemical and microbiological variables: temperature, pH, color, turbidity, electrical conductivity, total nitrogen and total phosphorus, total coliforms (CT), fecal coliform (CTe) and Escherichia coli. Tests were also performed to nine antimicrobial commercial resistances. The variables studied indicated that the Cascavel river water was presented at disagreement with the resolution 357/2005 CONAMA (class I), ranking in the index as regular water quality. The physical, chemical and rainfall did not affect the growth of CT and CTe, with higher counts of E. coli in the urban area. The greatest resistance profiles of the strains of E. coli isolated from Cascavel river water was found in section 2, the urban area as a probable consequence of human influence on water quality.

Influência das operações sobre o baço na distribuição da escherichia coli no sistema mononuclear fagocitário

OBJETIVOS: Para se evitar o estado asplênico, muitas medidas preservadoras do baço têm sido propostas na literatura, como a esplenorrafia, a esplenectomia parcial com preservação dos vasos hilares e o auto-implante de tecido esplênico. A esplenectomia subtotal, com conservação do pólo superior do baço, nutrido apenas pelos vasos esplenogástricos é uma alternativa quando o pedículo esplênico precisa ser ligado. O objetivo deste estudo foi avaliar a influência das esplenectomias parcial, subtotal e total na distribuição da Escherichia coli no sistema mononuclear fagocitário. MÉTODO: Foram estudados 32 ratos divididos em 4 grupos: operação simulada (mantendo todo o baço), esplenectomia parcial, esplenectomia subtotal e esplenectomia total. Após cinco semanas da operação, uma alíquota de Escherichia coli marcada com 99m-tecnécio foi injetada por via venosa. Após 20 minutos, os animais foram mortos, e o baço, os pulmões e o fígado foram retirados para se verificar a distribuição das bactérias marcadas. RESULTADOS: A quantidade de Escherichia coli no tecido esplênico foi maior no grupo com o baço íntegro em comparação com os grupos esplenectomia parcial e subtotal. A distribuição da bactéria marcada pelo baço não diferiu nos grupos com esplenectomia parcial ou subtotal. A quantidade de bactérias no pulmão foi maior no grupo esplenectomia parcial do que a do grupo com esplenectomia subtotal. Após esplenectomia subtotal, a distribuição da bactéria marcada foi maior no fígado em comparação com a captação desse órgão nos demais grupos. CONCLUSÕES: O pólo superior do baço, suprido apenas pelos vasos esplenogástricos, tem capacidade de remover da circulação bactérias vivas, mostrando que, mesmo sem a vascularização pelo pedículo esplênico, há uma eficiente depuração sangüínea. A distribuição da Escherichia coli pelo sistema mononuclear fagocitário apresenta comportamentos diferentes, dependendo do tipo de esplenectomia a que o animal é submetido.

Distribuição de Escherichia coli nos órgãos do sistema mononuclear fagocitário após esplenectomia total isolada ou combinada com auto-implante esplênico em rato

OBJETIVO: O auto-implante esplênico parece constituir a única alternativa para preservação de tecido esplênico, após esplenectomia total. O objetivo deste trabalho foi analisar a depuração de Escherichia coli pelos órgãos do sistema mononuclear fagocitário (SMF) após esplenectomia total e auto-implante esplênico. MÉTODO: Utilizou-se um modelo experimental com ratos Wistar jovens e adultos, de ambos os sexos, submetidos a esplenectomia total e auto-implante esplênico. O método de avaliação foi a inoculação intravenosa de suspensão de Escherichia coli marcada com tecnécio - 99m. Analisou-se a captação desta bactéria pelos órgãos do SMF e o remanescente bacteriano na corrente sangüínea. RESULTADOS: Dentro de cada grupo, não foi encontrado diferença entre animais jovens e adultos no que se refere à captação de bactérias pelos órgãos do SMF. Na comparação entre os grupos verificou-se que o percentual médio de captação pelo baço e pelo fígado de animais do Grupo-Controle foi maior que o dos auto-implantes. Embora a captação de bactérias pelo baço de animais do Grupo-Controle tenha sido maior que o dos auto-implantes esplênicos, o remanescente bacteriano no sangue não foi diferente. Animais submetidos a esplenectomia total isolada apresentam maior remanescente de bactérias na corrente sangüínea que animais do Grupo-Controle ou do grupo submetido a esplenectomia total combinada com auto-implante esplênico. CONCLUSÃO: Nossos resultados indicam que o auto-implante esplênico é eficaz na depuração de bactérias, em rato, mediante a fagocitose por seus macrófagos, e não interfere na função de remoção bacteriana do fígado e do pulmão.

Occurrence of F42 colonization factor in Escherichia coli strains isolated from piglets with diarrhea

The objective of this study was to determine the presence of the colonization factor F42 in 168 strains of Escherichia coli isolated from diarrheic stools of newborn piglets. The presence of F42 in 12 (7.1%) strains was detected with the agglutination test. Through the Polymerase Chain Reaction (PCR) of F42 positive strains, gene encoding enterotoxins (ST-I, ST-II, LT-I and LT-II) were detected. The finding of ST-I/ST-II genes in 50% of the strains, ST-I (16%) and ST-II (25%) indicates a strong association of FC F42 with heat-stable enterotoxins (91%). In contrast, the thermolabile enterotoxin (LT-I and LT-II) genes were not detected. Serogroups of F42 positive strains were determined, serogroup O8 being the most prevalent (41,7%). Other serogroups, as there are O9, O11, O18, O32, O35, O98 and O101, were also identified. Thus, FC F42 was confirmed as an additional factor of virulence in the pathogenesis of porcine colibacillosis.

Caracterização epidemiológica, molecular e perfil de resistência aos antimicrobianos de Escherichia coli isoladas de criatórios suínos do sul do Brasil

A colibacilose é a enfermidade entérica de maior impacto na suinocultura, sendo ocasionada por cepas enterotoxigênicas de Escherichia coli. Quarenta isolados clínicos de suínos com diarréia e 13 isolados ambientais foram analisados quanto ao perfil genotípico, relação genética e resistência antimicrobiana. O gene que codifica para a toxina Stb foi identificado em 50% dos isolados clínicos, seguido por Sta e Lt, com 35%. Dentre os fatores de adesinas pesquisados, a F18 foi encontrada em 27,5% das amostras. A técnica de ERIC-PCR utilizada para caracterização epidemiológica dos isolados, não demonstrou poder discriminatório esperado, e apesar de permitir a separação dos isolados em grupos, estes não evidenciaram grupos relacionados aos fatores de virulência. No teste de susceptibilidade antimicrobiana a maior resistência foi observada à tetraciclina, em 88,6%. O índice de resistência múltipla aos antimicrobianos (IRMA), variou entre 0 a 0,69.

Typing of avian pathogenic Escherichia coli strains by REP-PCR

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Genes associated with pathogenicity of avian Escherichia coli (APEC) isolated from respiratory cases of poultry

The virulence mechanisms of avian pathogenic Escherichia coli (APEC) have been continually studied and are believed to be multi-factorial. Certain properties are primarily associated with virulent samples and have been identified in avian isolates. In this study a total of 61 E. coli, isolates from chicken flocks with respiratory symptomatology, were probed by Polimerase Chain Reation (PCR) for the presence of genes responsible for the adhesion capacity, P fimbria (papC) e F11 fimbria (felA), colicin production (cvaC), aerobactin presence (iutA), serum resistance (iss), temperature-sensitive hemagglutinin (tsh), and presence of K1 and K5 capsular antigens (kpsII). The iss gene was detected in 73,8%, tsh in 55,7%, iutA in 45,9%, felA in 39,3%, papC in 24,3%, cvaC in 23% and kpsII in18%.