Diversidad genética de cepas extraintestinales de Escherichia coli productoras de las betalactamasas TEM, SHV y CTX-M asociadas a la atención en salud / Genetic diversity of extraintestinal Escherichia coli strains producers of beta-lactamases TEM, SHV and CTX-M associated with healthcare

RESUMEN Introducción. En Venezuela existen pocos reportes que describan las bases genéticas del potencial patogénico y filogenético de las cepas de Escherichia coli provenientes de hospitales. Objetivo. Determinar la diversidad genética de cepas extraintestinales de E. coli productoras de las betalactamasas TEM, SHV y CTX-M asociadas a la atención de salud. Materiales y métodos. Se estudió una colección de 12 cepas extraintestinales de E. coli con sensibilidad disminuida a las cefalosporinas de amplio espectro. La sensibilidad antimicrobiana se determinó por concentración inhibitoria mínima. La detección de los grupos filogenéticos, de los factores de virulencia y de los genes que codifican la resistencia antimicrobiana se hizo mediante la técnica de reacción en cadena de la polimerasa y la relación clonal se estableció mediante reacción en cadena de la polimerasa de elementos palindrómicos extragénicos repetitivos (Repetitive Element Palindromic-PCR, rep-PCR). Resultados. Todas las cepas analizadas presentaron resistencia a las cefalosporinas, y resistencia conjunta a quinolonas y aminoglucósidos. La distribución filogenética evidenció que los grupos A y B1 fueron los más frecuentes, seguidos por D y B2; en este último, se detectaron todos los factores de virulencia evaluados, y el gen más frecuente fue el fimH. En todas las cepas analizadas, se encontró bla CTX-M, con predominio de las bla CTX-M-8, y en dos de estas cepas se evidenció la presencia simultánea de bla CTX-M-9, variantes bla CTX-M-65 y bla CTX-M-147. Conclusión. Las cepas estudiadas demostraron diversidad genética y albergaron diferentes genes de virulencia y betalactamasas de espectro extendido (BLEE) sin predominio de ningún filogrupo en particular. Este estudio constituye el primer reporte de la variante bla CTX-M-65 en Venezuela y de la variante bla CTX-M-147 en el mundo, en cepas no relacionadas genéticamente aisladas de hospitales, situación que merece atención y la racionalización del uso de los antimicrobianos.

ABSTRACT Introduction: There are few reports from Venezuela describing the genetic basis that sustains the pathogenic potential and phylogenetics of Escherichia coli extraintestinal strains isolated in health care units. Objective: To establish the genetic diversity of extraintestinal E. coli strains producers of beta-lactamases TEM, SHV and CTX-M associated with healthcare. Materials and methods: We studied a collection of 12 strains of extraintestinal E. coli with diminished sensitivity to broad-spectrum cephalosporins. Antimicrobial susceptibility was determined by minimum inhibitory concentration. We determined the phylogenetic groups, virulence factors and genes encoding antimicrobial resistance using PCR, and clonal characterization by repetitive element palindromic-PCR rep-PCR. Results: All strains showed resistance to cephalosporins and joint resistance to quinolones and aminoglycosides. The phylogenetic distribution showed that the A and B1 groups were the most frequent, followed by D and B2. We found all the virulence factors analyzed in the B2 group, and fimH gene was the most frequent among them. We found bla CTX-M in all strains,with a higher prevalence of bla CTX-M-8; two of these strains showed coproduction of bla CTX-M-9 and were genetically identified as bla CTX-M-65 and bla CTX-M-147 by sequencing. Conclusion: The strains under study showed genetic diversity, hosting a variety of virulence genes, as well as antimicrobial resistance with no particular phylogroup prevalence. This is the first report of bla CTX-M alleles in Venezuela and in the world associated to non-genetically related strains isolated in health care units, a situation that deserves attention, as well as the rationalization of antimicrobials use.

Effect of glycemic index on obesity control

Objective Evaluate the effect of glycemic index (GI) on biochemical parameters, food intake, energy metabolism, anthropometric measures and body composition in overweight subjects.Materials and methods Simple blind study, in which nineteen subjects were randomly assigned to consume in the laboratory two daily low GI (n = 10) or high GI (n = 9) meals, for forty-five consecutive days. Habitual food intake was assessed at baseline. Food intake, anthropometric measures and body composition were assessed at each 15 days. Energy metabolism and biochemical parameters were evaluated at baseline and the end of the study.Results Low GI meals increased fat oxidation, and reduced waist circumference and HOMA-IR, while high GI meals increased daily dietary fiber and energy intake compared to baseline. There was a higher reduction on waist circumference and body fat, and a higher increase on postprandial fat oxidation in response to the LGI meals than after high GI meals. High GI meals increased fasting respiratory coefficient compared to baseline and low GI meals.Conclusion The results of the present study showed that the consumption of two daily low GI meals for forty-five consecutive days has a positive effect on obesity control, whereas, the consumption of high GI meals result has the opposite effect. Arch Endocrinol Metab. 2015;59(3):245-51.

Safety, beneficial and technological properties of Enterococcus faecium isolated from Brazilian cheeses

This study aimed to characterize the safety and technological properties of Enterococcus faecium strains isolated from Brazilian Coalho cheeses. High levels of co-aggregation were observed between Enterococcus faecium strains EM485 and EM925 and both Escherichia coli and Clostridium perfringens. Both strains presented low levels of hydrophobicity. E. faecium EM485 and EM925 were both able to grow in the presence of 0.5% of the sodium salts of taurocholic acid (TC), taurodeoxycholic acid (TDC), glycocholic acid (GC), and glycodeoxycholic acid (GDC), although they showed the ability to deconjugate only GDC and TDC. Both strains showed good survival when exposed to conditions simulating the gastro intestinal tract (GIT). When tested for the presence of virulence genes, only tyrosine decarboxylase and vancomycin B generated positive PCR results.

Infant and young child feeding in four departments in Haiti: mixed-method study on prevalence of recommended practices and related attitudes, beliefs, and other determinants / Alimentación de los lactantes y niños pequeños en cuatro departamentos de Haití: un estudio con diseño de método mixto sobre la prevalencia de las prácticas recomendadas y las actitudes, las creencias y otros determinantes relacionados

OBJECTIVE: To determine and describe the prevalence and patterns of three recommended practices for infant and young child feeding-exclusive breastfeeding (EB), continued breastfeeding (CB), and achievement of minimum dietary diversity-in four regions in Haiti, and to identify the attitudes and beliefs that inform these practices and any other factors that may facilitate or impede their implementation. METHODS: This study utilized a mixed-methods approach consisting of 1) a cross-sectional survey (n = 310) and 2) 12 focus group discussions among women ≥18 years old with children ≤ 2 years old. Multivariable logistic regression analyses were conducted to identify factors associated with 1) EB during the first six months of life, 2) CB for children ≥ 2 years old, and 3) receipt of a diverse variety of complementary foods. Qualitative data were recorded, transcribed verbatim, and analyzed for common themes. Data were collected in June and July 2013 in four departments in Haiti: Artibonite, Nippes, Ouest, and Sud-Est. RESULTS: Prevalence of EB, CB, and achievement of minimum dietary diversity was 57.0%, 11.9%, and 21.2% respectively. EB was statistically significantly associated with infant's age when controlling for annual household income, location of most recent birth, or receipt of CB counseling (odds ratio (OR) = 0.67 (95% CI: 0.47-0.97)). CB was not statistically significantly associated with rural place of residence, receipt of CB counseling, parity, or infant's age. Meeting minimum dietary diversity was not significantly associated with parity, receipt of postnatal care, rural place of residence, location of most recent birth, receipt of infant and young child feeding counseling, or level of schooling. Beliefs surrounding the relationship between the mother's health and her diet on the quality of breast milk may prohibit EB and CB. Qualitative data revealed that dietary diversity may be low because 1) mothers often struggle to introduce complementary foods and 2) those that are traditionally introduced are not varied and primarily consist of grains and starches. CONCLUSIONS: Prevalence of the three recommended infant and young child feeding practices examined in this study is suboptimal, particularly CB and achievement of minimum dietary diversity. Future communication and programming efforts should address the misunderstandings and concerns identified through the qualitative methods used in this research.

OBJETIVO: Determinar y describir la prevalencia y los modelos de tres prácticas recomendadas para la alimentación de los lactantes y los niños pequeños (la lactancia materna exclusiva [LME], la lactancia materna continuada [LMC] y el logro de una diversidad alimentaria mínima, en cuatro regiones de Haití, y determinar las actitudes y creencias en las que se basan estas prácticas y otros factores que puedan facilitar o impedir su implantación. MÉTODOS: Este estudio utilizó un diseño de método mixto que consistió en 1) una encuesta transversal (n = 310) y 2) 12 grupos de discusión formados por mujeres de ≥ 18 años de edad o mayores con niños de ≤ 2 años de edad o menores. Se llevaron a cabo análisis de regresión logística multivariable para determinar los factores asociados con 1) la LME durante los seis primeros meses de vida, 2) la LMC en niños de ≥ 2 años de edad o mayores, y 3) el aporte de una amplia variedad de alimentos complementarios. Se registraron, se transcribieron al pie de la letra y se analizaron los datos cualitativos referentes a temas comunes. Estos datos se recopilaron en junio y julio del 2013, en cuatro departamentos de Haití: Artibonite, Nippes, Oeste y Sudeste. RESULTADOS: Las prevalencias de la LME, la LMC y el logro de una diversidad alimentaria mínima fueron de 57,0, 11,9 y 21,2%, respectivamente. La LME se asoció de manera estadísticamente significativa con la edad del lactante si se controlaban las variables de ingresos familiares anuales, ubicación del parto más reciente, o provisión de orientación en materia de LMC (razón de posibilidades [OR] = 0,67 [IC de 95%:0.47-0.97]). La LMC no se asoció de una manera estadísticamente significativa con la residencia en un entorno rural, la provisión de orientación en materia de LMC, la paridad o la edad de lactante. El logro de una diversidad alimentaria mínima no se asoció significativamente con la paridad, la provisión de atención posnatal, la residencia en un entorno rural, la ubicación del parto más reciente, la provisión de orientación en materia de alimentación de los lactantes y los niños pequeños, o el nivel de escolarización. Las creencias con respecto a la relación entre la salud de la madre y su régimen alimentario con la calidad de la leche materna pueden limitar la LME y la LMC. Los datos cualitativos revelaron que la diversidad alimentaria puede ser escasa como consecuencia de que 1) las madres a menudo se esfuerzan por introducir los alimentos complementarios, y 2) los que se introducen tradicionalmente no son variados y consisten principalmente en cereales y féculas. CONCLUSIONES: Las prevalencias de las tres prácticas de alimentación de los lactantes y los niños pequeños recomendadas analizadas en este estudio son subóptimas, en particular las correspondientes a la LMC y al logro de una diversidad alimentaria mínima. Las futuras iniciativas de comunicación y programación deberían abordar los malentendidos y las inquietudes detectadas mediante los métodos cualitativos utilizados en esta investigación.

A Prospective Multi-center Trial of Escherichia coli Extract for the Prophylactic Treatment of Patients with Chronically Recurrent Cystitis

We have assessed the efficacy and safety of Escherichia coli extract (ECE; Uro-Vaxom(R)) which contains active immunostimulating fractions, in the prophylactic treatment of chronically recurrent cystitis. Forty-two patients with more than 2 episodes of cystitis in the proceeding 6 months were treated for 3 months with one capsule daily of ECE and observed for a further 6 months. The primary efficacy criterion was the number of episodes of recurrent cystitis during the 6 months after treatment compared to those during the 6 months before treatment. At the end of the 9-month trial, 34 patients (all women) were eligible for statistical analysis. Their mean age was 56.4 yr (range, 34-75 yr), and they had experienced recurrent urinary tract infections for 7.2+/-5.2 yr. The number of recurrences was significantly lower during the 6-month follow-up period than during the 6 months preceding the trial (0.35 vs. 4.26, P<0.001). During the follow-up, 28 (82.4%) patients had no recurrences and 4 (11.8%) had 1 each. In patients who relapsed, ECE alleviated cystitis symptoms, including painful voiding, frequency and urgency. There were no serious adverse events related to the study drug. Our study demonstrates the efficacy and safety of ECE in the prophylactic treatment of chronically recurrent cystitis.

Doseamento microbiológico de apramicina - desenvolvimento e validação de método empregando leitura cinética em microplacas / Apramycin microbiological assay - method development and validation using kinetic microplate reading

O princípio do ensaio turbidimétrico é simples: a solução-teste é adicionada a suspensão do microrganismo-teste em meio de cultura, a mistura é incubada em condições adequadas e o crescimento microbiano é medido através da leitura fotométrica. O emprego de método de microplacas com leitura cínética para a dosagem de antibióticos é de interesse considerável, uma vez que possibilita reduzir quantidade de material e tempo de análise necessários e permite o ensaio de grande número de amostras simultaneamente, com leitura e cálculo automatizados. O objetivo deste trabalho é determinar as condições experimentais ideais para o desenvolvimento de metodologia para a dosagem microbiológica de apramicina empregando microplacas e modo de leitura cinético, e validar o método desenvolvido, através da avaliação dos parâmetros de especificidade e seletividade, linearidade, faixa ou intervalo linear, limite de detecção e quantificação, exatidão e precisão. As condições estabelecidas abrangem curva-padrão de apramicina com concentrações entre 5 e 35 μg/ml, e emprego de meio de cultura caldo de triptona-soja inoculado com Escherichia coli (ATCC 8739) na proporção de 5%. Foram obtidos resultados satisfatórios após 2 horas de incubação. O método desenvolvido apresentou especificidade e seletividade adequadas, linearidade na faixa de 5 a 35 1.19/ml, limite de deteção e quantificação de 0,1 e 0,4 μg/ml, respectivamente, exatidão (recuperação = 98,5%) e precisao (DPR = 6,0%) satisfatórias. O ensaio em microplaca agrega características dos ensaios microbiológicos (avaliação da atividade do antibiótico frente a microrganismo-teste sensível) e físico-químicos (facilidade operacional e major rapidez na obtenção dos resultdos).

The turbidimetric assay principle is simple: the test-solution is added to a suspension of test-microorganism in culture media, the mixture is incubated in appropriate conditions and the microbial growth is measured by photometric reading. Microplate with kinetic reading mode employed in antibiotic assay is considerable interesting, once it allows reduction of material and analysis time and it permits that a great numbers of samples could be analyzed simultaneously, with automated reading and calculating. The aim of this work is to determinate best experimental conditions to development of methodology for microbiological assay of apramycin employing microplate and kinetic reading mode, and to validate the developed method, through evaluation of parameters of specificity and selectivity, linearity, linear range, limit of detection and quantification, accuracy and precision. Established conditions considered standard-curve of apramycin in concentrations from 5 to 35 μg/ml, and tryptic soy broth inoculated with 5% of Escherichia coli (ATCC 8739) suspension. Satisfactory results were obtained with 2 hours incubation. The developed method showed appropriate specificity and selectivity, linearity in the range from 5 to 35 μg/ml, limit of detection and quantification of 0,1 and 0,4 μg/ml, respectively, satisfactory accuracy (recuperation = 98,5%) and precision (RSD = 6,0%). Microplate assay considered characteristics of microbiological assay (evaluation of antibiotic activity against sensible test-microorganism) and physical-chemistry (operational facility and quicker results).

Identification and annotation of promoter regions in microbial genome sequences on the basis of DNA stability.

Analysis of various predicted structural properties of promoter regions in prokaryotic as well as eukaryotic genomes had earlier indicated that they have several common features,such as lower stability, higher curvature and less bendability, when compared with their neighboring regions. Based on the difference in stability between neighboring upstream and downstream regions in the vicinity of experimentally determined transcription start sites, a promoter prediction algorithm has been developed to identify prokaryotic promoter sequences in whole genomes. The average free energy (E) over known promoter sequences and the difference (D) between E and the average free energy over the entire genome (G)are used to search for promoters in the genomic sequences. Using these cutoff values to predict promoter regions across entire Escherichia coli genome,we achieved a reliability of 70% when the predicted promoters were cross verified against the 960 transcription start sites (TSSs) listed in the Ecocyc database. Annotation of the whole E.coli genome for promoter region could be carried out with 49% accuracy. The method is quite general and it can be used to annotate the promoter regions of other prokaryotic genomes.

Studies on structural stability of thermophilic Sulfolobus acidocaldarius ribosomes.

Structural stability of thermophilic archaeon Sulfolobus acidocaldarius ribosomes, with respect their susceptibility to pancreatic RNase A and stability to temperature (deltaTm), on treatment with various stabilizing (polyamines) and destabilizing (sulfhydryl and intercalating) agents were studied and compared with mesophilic E. coli ribosomes, to understand the structural differences between thermophilic and mesophilic ribosomes. Thermophilic archaeal ribosomes and their subunits were 10-times less susceptible to pancreatic RNase A, compared to mesophilic ribosomes, showing the presence of strong and compact structural organization in them. Thermophilic ribosomes treated with destabilizing agents, such as sulfhydryl reagents [5,5'-Dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercurybenzoate) and intercalating agents (ethidium bromide, EtBr) showed higher stability to RNase A, compared to similarly treated mesophilic ribosomes, indicating the unavailability of thiol-reactive groups and the presence of strong solvent inaccessible inner core. Higher stability of thermophilic ribosomes compared to mesophilic ribosomes to unfolding agents like urea further supported the presence of strong inner core particle. Thermophilic ribosomes treated with intercalating agents, such as EtBr were less susceptible to RNase A, though they bound to more reagent, showing the rigidity or resilience of their macromolecular structure to alterations caused by destabilizing agents. Overall, these results indicated that factors such as presence of strong solvent inaccessible inner core and rigidity of ribosome macromolecular structure contributed stability of thermophilic ribosomes to RNase A and other destabilizing agents, when compared to mesophilic ribosomes.

The metabolism of gluconate in Escherichia coli: Physiological evidences of a regulatory effect of idnR on the expression of the gntR regulon operons

Uptake and phosphorylation initiate the catabolism of gluconate in E. coli. Such activities conform two systems,GntI and GntII, encoded by two sets of genes differently located on the E. coli chromosome and under different regulation. gntT, gntU and gntK (minute 76) encode for high and low affinity gluconate transports and for a thermoresistant gluconokinase respectively, that conform GntI; the mentioned genes and those of the edd-eda operon (minute 41) are negatively regulated by the gntR gene product conforming the gntR regulon. idnT and gntV (minute 96), encode for another gluconate transport and a thermosensitive gluconokinase, conforming GntII. These genes are presumably positively controlled by IdnR. IdnT also functions as a permease for idonate; the corresponding gene is included in the idnDOTR operon, responsible for idonate metabolism, in which gluconate is an intermediary. Here we report a regulatory action of IdnR on the operons of the gntR regulon; i.e., gntT, gntKU and edd-eda. The expression of these operons, was diminished in a gntR mutant complemented with a clone of idnR and also in E. coli mutants in which the idnDOTR operon is expressed in a gluconate dependent inducibility. This is the first report of a regulatory effect of IdnR on edd-eda operon expression.

El transporte y la fosforilación inician el catabolismo del gluconato en E. coli. Estas actividades conforman dossistemas, GntI y GntII, codificados por dos grupos de genes, diferentemente regulados y ubicados en distintos sitios del cromosoma. Los genes gntT, gntU y gntK (minuto 76), codifican distintas proteínas para transportes de alta y baja afinidad para gluconato y una gluconoquinasa termoresistente respectivamente, que forman el sistema GntI; los genes respectivos junto con los del operón edd-eda (minuto 41), son regulados negativamente por el producto de gntR (minuto76) constituyendo el regulón gntR. Los genes IdnT y gntV (minuto 96), codifican otra permeasa para gluconato y una gluconoquinasa termosensible que forman GntII. IdnT funciona también como permeasa para idonato; el gen correspondiente es parte del operón idnDOTR, regulado positivamente por IdnR y responsable del metabolismo del idonato en el que gluconato es un intermediario. Se reporta un efecto regulatorio de IdnR sobre los operones del regulón gntR; i.e., gntT, gntKU and edd-eda. La expresión de estos operones resultó disminuida en una mutante gntR complementada con un clon de idnR y también en mutantes de E. coli en la que la expresión del operón idnDOTR se induce en presencia de gluconato. Este es el primer reporte de la acción regulatoria de IdnR sobre la expresión del operón eddeda.

The effects of Porphyromonas gingivalis LPS and Actinobacillus actinomycetemcomitans LPS on human dendritic cells in vitro, and in a mouse model in vivo.

Interaction between different bacterial plaque pathogens and dendritic cells may induce different types of T helper (Th) cell response, which is critical in the pathogenesis of periodontitis. In this study we investigated the effects of lipopolysaccharide (LPS) from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans on human monocyte-derived dendritic cells (Mo-DCs) with respect to co-stimulatory molecule expression, cytokine production and Th cell differentiation. Unlike Escherichia coli and A. actinomycetemcomitans LPS, P. gingivalis LPS induced only low levels of CD40, CD80, HLA-DR and CD83 expression on Mo-DCs. LPS from both bacteria induced considerably lower TNF-alpha and IL-10 than did E. coli LPS. LPS from all three bacteria induced only negligible IL-12 production. In a human mixed-leukocyte reaction, and in an ovalbumin-specific T cell response assay in mice, both types of LPS suppressed IFN-gamma production. In conclusion, stimulation by P. gingivalis LPS and A. actinomycetemcomitans LPS appears to bias Mo-DCs towards Th2 production.

Localización molecular mediante PCR inverso del sitio de inserción de un transposon TN 10 ligado a gntS en mutantes de Escherichia coli / Molecular location by inverse PCR of the insertion site of a Tn10 transposón ligated to gntS in Escherichia coli mutants

La vecindad del sitio de inserción del transposón Tn10, portado por la mutante de Escherichia coli DF601, contiene el gene gntS, un presunto regulador positivo involucrado en el metabolismo del gluconato en E. coli. Aunque el análisis molecular de la región del minuto 95.3, señalado originalmente como el sitio de inserción del transposón, reveló el ORF f251 con características de regulador, transformaciones con éste y otros ORFs de la región, una vez clonados, no complementaron la función perdida en mutantes gntS. El presente trabajo racionaliza la causa de tales resultados. Con base a la secuencia nucleotídica suministrada por GenBank y la aplicación de la técnica de PCR inverso, se encontró que el sitio exacto de inserción del transposón Tn10, portado por la mencionada mutante y sus derivadas TetR, es la posición 4442377, la cual interrumpe el ORF ytfN en la región del minuto 95.8 del mapa genético y no en la del minuto 95.3, como fue originalmente establecido. Los resultados, además de señalar sin ambigüedad la región cromosómica a investigar para lograr los fines propuestos, indican la conveniencia de aplicar la técnica sencilla de PCR inverso, para ubicar elementos genéticos antes de emplearlos en estudios moleculares.

The vicinity of the Tn10 transposon insertion site, carried by the Escherichia. coli mutant DF601, contains the genegntS, a putative positive regulator involved in the metabolism of the gluconate in E. coli. Although the molecular analysis of the 95.3 minute region, originally reported as the transposon insertion site, revealed the ORF f251 as one with regulator characteristics, transformations with this and other ORFs associated with the region, once cloned, did not complement the lost function in gntS mutants. The present work rationalizes on the cause of such results. Based on the nucleotide sequence provided by GenBank and application of the inverse PCR technique, it was found that the exact site of the Tn10 transposon insertion is in the position 4442377, interrupting the ytfN ORF at the minute 95.8 of the E. coli genetic map and not at minute 95.3, as it was originally established. The results indicate the precise chromosomal region to investigate in order to obtain the initially proposed aims and the convenience of applying the simple technique of inverse PCR to locate genetic elements as well.

Comparative protein profiles of Salmonella and Escherichia coli.

Protein profiles of selected Salmonella serovars were compared with E. coli to identify genus specific protein(s) for Salmonella. The PDP formed of different Salmonella serovars were compared with E. coli O78 when subjected to SDS-PAGE yielded 11, 15, 15, 11 and 14 bands in S. Bareilly, S. Gallinarum, S. Typhimurium and S. Weltevreden and E. coli O78 respectively. The bands produced were compared with each other. It was found that S. Weltevreden shared 7 bands with E. coli O78, A protein of molecular weight 20.89 kDa was found in all Salmonella serovars, but not in E. coli O78 suggesting its genus specific attribute.

Validación de una técnica de PCR múltiple para la detección de Escherichia coli productor de toxina Shiga / Validation of a multiplex PCR for detection of Shiga toxin-producing Escherichia coli

La infección por Escherichia coli productor de toxina Shiga (STEC) es causa de diarrea con o sin sangre, colitis hemorrágica y síndrome urémico hemolítico (SUH) en humanos. El objetivo de este trabajo fue validar una técnica de PCR múltiple para el diagnóstico de STEC basado en la detección de los genes stx1, stx2 y rfbO157. La validación de la técnica se realizó en dos laboratorios independientes, en forma paralela. Se determinó rango de trabajo, selectividad y robustez. Se evaluó el desempeño de la técnica al combinar distintas concentraciones de dos cepas con diferentes factores de virulencia. El rango de trabajo dependió de la cepa analizada, los valores máximos y mínimos fueron 6,6 x 107 y 1,0 x 104 UFC/50 µl. El límite de detección fue de 1,0 x 104 UFC/50 µl y el límite de corte de 1,0 x 105 UFC/50 µl. La robustez fue óptima al modificar diferentes variables. Se obtuvo 100% de inclusividad, exclusividad, precisión analítica, valor predictivo positivo y valor predictivo negativo. No se observó interferencia al combinar distintas concentraciones de los factores de virulencia blanco de la reacción. La técnica validada es una alternativa apropiada para la detección y confirmación de STEC O157 y no-O157 a partir de cultivos bacterianos.

Shiga toxin-producing Escherichia coli (STEC) cause non-bloody or bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The aim of the present study was to validate a multiplex PCR for the STEC diagnosis based on the detection of stx1, stx2 and rfbO157 genes. The multiplex PCR validation was carried out in two independent laboratories in a parallel way. Work range, selectivity and robustness were established. The PCR performance was evaluated using different concentrations of two STEC strains harboring different target genes. The work range depended on the strain analyzed, the maximum and the minimum values were 6.6 x 107 and 1.0 x 104 CFU/50 µl. The detection limit was 1.0 x 104 CFU/50 µl and the cut limit 1.0 x 105 CFU/50 ml. A good robustness was observed when different variables were introduced. Inclusivity, exclusivity, positive predictivity, negative predictivity and analytical accuracy were of 100%. Interference was not shown when different concentrations of STEC strains, carrying different genes, were used. The validated technique is an appropriate alternative for detection and confirmation of STEC O157 and non-O157 strains from bacterial cultures.

Medida da atividade da Fosfatase Alcalina de E. coli em pH=11: potencial uso na avaliação da qualidade microbiológica da água de abastecimento / E. coli alkalyne phosphatase assay at pH 11: potencial use in the microbiological evaluation of drinking water quality

Objetivo. Um dos problemas fundamentais da Saúde Pública consiste na qualidade microbiológica da água de abastecimento. Garantir que a população seja servida por água de boa qualidade depende de diversos aspectos. Um deles fundamenta-se na avaliação da qualidade microbiológica da água para consumo humano. A detecção rápida e eficiente de microorganismos é uma necessidade constante para que medidas adequadas sejam tomadas e que a comunidade abastecida seja protegida. Este estudo tem como objetivo testar a reprodutibilidade de uma condição experimental específica (1M CAPS, pH 11) que teria potencial para ser utilizada como modelo para o I desenvolvimento de novas técnicas na detecção de coliformes fecais na água de abastecimento, de maneira simples, rápida e com baixo custo. Métodos. Ensaios de atividade da fosfatase alcalina da bactéria E.coli foram realizados em tampões diferentes com valores variáveis de pH utilizando o método colorimétrico Resultados. Os resultados dos ensaios foram reprodutíveis. Os ensaios realizados neste estudo em pH 11 indicaram que esta condição experimental pode levar a atividades enzimáticas maiores do que aquelas descritas na literatura. Conclusões.Os resultados poderiam ser utilizados como ponto de partida para um estudo com o objetivo de se estabelecer uma metodologia enzimática para a determinação imediata de coliformes na água de abastecimento.

A Preliminary Investigation on the Chemical Composition of the Cell Surface of Five Enteropathogenic Escherichia coli Serotypes

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100oC for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction

Chemical characterization of the lipopolysaccharides from enteropathogenic Escherichia coli O142 and O158.

Lipopolysaccharides (LPS) from two enteropathogenic strains of E. coli O142 and O158 were isolated by hot phenol-water extraction procedure. Polyacrylamide gel electrophoretic pattern of the LPS showed the typical ladder like pattern of smooth type of LPS. The LPS of E. coli O158 was found to contain L-rhamnose, D-glucose and N-acetyl-D-galactosamine as major constituents together with D-galactose, N-acetyl-D-glucosamine, L-glycero-D-manno-heptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) whereas LPS from E. coli O142 contained L-rhamnose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as major constituents together with D-glucose, D-galactose, N-acetyl-D-glucosamine, L-glycero-D-mannoheptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO). LPS was degraded by mild acid hydrolysis to yield a degraded polysaccharide fraction and an insoluble lipid-A fraction. The main fatty acids of the lipid-A fraction of the LPS were C12:O, C14:O, and 3-OH C14:O for O158 strain whereas E. coli O142 lipid-A consisted of C12:O, C14:O, 3-OH C14:O, and C16:O. The degraded polysaccharide fraction on gel permeation chromatography gave a high moleculer weight O-chain fraction and a core oligosaccharide and a fraction containing degraded sugars. The chemical composition of LPS and its fragmented products are reported in this communication.

Asp 280 residue is important in the activity of the Escherichia coli leader peptidase

Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.

Expresión del gen de la penicilino acilasa de la cepa recombinante escherichia coli JM101/pPACII en un sistema de cultivo exponencial de lote alimentado / Expression of the acylasa penicillin gene of the recombinant strain escherichia coli JM101/pPACII in a exponentially culture system of the feed part

La penicilino acilasa es una enzima inducible que cataliza la hidrólisis de las penicilinas generando, entre otros, el intermediario más importante en la producción de las penicilinas semisintéticas de nueva generación, el ácido 6-amino penicilánico (6-Apa). Esta enzima es codificada por el gen estructural pac, el cual, al igual que su producto polipeptídico, se encuentra modulado por diferentes elementos de regulación. En la cepa recombinante Escherichia coli JM101, transformada con el plásmido pPACII, se incluyó este gen con la región reguladora exógena con IPTG. Tradicionalmente, los estudios para evaluar los efectos de los factores ambientales y condiciones de crecimiento sobre la expresión y estabilidad de plásmidos en cepas recombinantes se han realizado en sistemas de escalaminetos por lote o por cultivo contínuo, dificultándose, en primer caso, el control de las condiciones ambientales y, en el segundo, la inherente complejidad operativa del sistema; por lo tanto, en este trabajo se evaluaron los efectos de la concentración del inductor y de la tasa de crecimiento sobre la expresión y estabilidad de la cepa recombinante JM101/pPACII, utilizándose un medio de cultivo balanceado y el sistema de cultivo exponencial de lote alimentado de volumen variable, iniciando la alimentación con un volumen correspondiente al 20 por ciento del volumen final. Un incremento en la expresión del plásmido pPACII para la producción de penicilino acilasa fue observado a medida que disminuyó la tasa de crecimiento y el tiempo de inducción. Se determinó en cultivos por lote, un comportamiento tipo saturación en la expresión del plásmido pPACII a concentraciones superiores de 50 My del indutor, reflejado a través de la actividad máxima de la penicilino acilasa. Por otra parte se observó un incremento en la actividad máxima de la enzima, así como una disminución no significativa en la cinética de crecimiento, a medida que el tiempo de inducción era cercano a la inoculación. Estos resultados demostraron que el cultivo exponencial de lote alimentado de volumen variable puede ser utilizado como una alternativa simple para el estudio de los microorganismos recombinantes

Kinetics of a recombinant protein production by E. coli BL21

Troponina C, TnC, uma proteína do músculo esquelético de galinha, foi produzida em células de Escherichia coli BL21 (DE3) pLysS contendo o cDNA no plasmídio pET sob controle do promotor lac UV5. Neste sistema, a expressäo do gene da TnC depende da presença de uma molécula indutora, o que permitiu a realizaçäo de ensaios com uma fase de crescimento independente da fase de produçäo. O balanço gasoso aplicado à fase de produçäo da TnC em cultivos em biorreator, resultou em um fator de conversäo de oxigênio a células, Yx/o, entre6.5 e 142 mg/mmol, e porcentagem de TnC na célula com relaçäo ao teor de proteína celular, TnC (porcentagem), entre 11.7 e 26.1 por cento, sendo que para os maiores valores de TnC (porcentagem) correspondem aos menores valores de Yx/o. Além disso, para o maior o valor de y no início da fase de induçäo, obteve-se maior valor de TnC (porcentagem). Os dados apresentados indicam que na produçäo de TnC sob controle do gene lac UV5 e promotor de T7 em E.coli, a energia do metabolismo aeróbico é direcionada principalmente à síntese da proteína recombinante com conseqüente reduçäo da atividade de crescimento.

Ausencia de actividad antimicrobiana de un extracto acuoso liofilizado de Aloe vera (Sabila) / Absence of antimicrobial activity of lyophilized aqueous extract of Aloe vera (sabila)

Se estudio la actividad antimicrobiana de dos concentraciones (10 y 50 mg/mL) de un extracto acuoso liofilizado de hojas de Aloe vera (sabila), mediante el sistema de ensayo de difusion en agar, con una bateri aminima de cepas de microorganismos compuesta por cuatro bacterias (Staphylococcus aureus, Bacillus subtilils, Escherichia coli y Pseudomonas aeruginosa) y una levadura (Candida albicans). Los resultados indican que solo frente al Staphylococcus aureus se obteine una ligera actividad inhibitoria, al compararla con la que produce el control positivo (estreptomicina). Para el resto de los microorganismos estudiados la respuesta es negativa. Estos resultados permiten desestimar el uso del extracto acuoso liofilizado de Aloe vera como antimicrobiano, en tanto que sugiere explorar este efecto con otro tipo de extracto con el objetivo de avalar o no la utilizacion de esta planta como antimicrobiano