Virulence factors in Escherichia coli strains isolated from urinary tract infection and pyometra cases and from feces of healthy dogs

The aim of this study was to compare the prevalence of virulence genes in 158 Escherichia coli strains isolated from 51 clinical cases of UTIs, 52 of pyometra and from 55 fecal samples from healthy dogs by PCR. papC was found in 12 (23.5%) strains isolated from UTIs, 19 (36.5%) from pyometra and 10 (18.2%) from feces. papGII was observed in 3 (5.8%) strains from pyometra, and papGIII in 10 (19.6%) from UTIs, 15 (28.8%) from pyometra and 9 (16.4%) from feces. sfaS was detected in 22 (43.1%) strains from UTIs, 24 (46.1%) from pyometra and 19 (34.5%) from feces. hlyA was observed in 17 (33.3%) strains from UTIs, 18 (34.6%) from pyometra and 7 (12.7%) from feces, while cnf-1 was detected in 11 (21.6%) from UTIs, 21 (40.4%) from pyometra and 9 (16.4%) from feces. iucD was observed in 12 (23.5%) strains from UTIs, 9 (17.3%) from pyometra and 1 (1.8%) from feces. usp was found 17 (33.3%) isolates from UTIs and 36 (69.9%) from pyometra. (c) 2008 Elsevier Ltd. All rights reserved.

An unusual gangrenous goat mastitis caused by Staphylococcus aureus, Clostridium perfringens and Escherichia coli co-infection

Relata-se, pela primeira vez no Brasil, a ocorrência de mastite gangrenosa caprina atípica causada pela co-infecção por Staphylococcus aureus, Clostridium perfringens e Escherichia coli em uma cabra da raça Boer, na segunda semana de lactação. Descrevem-se os achados clínicos, os procedimentos de diagnóstico microbiológico e a conduta terapêutica.

Endotoxemia por lipopolissacarídeo de Escherichia coli, em eqüinos: efeitos de antiinflamatórios nas concentrações sérica e peritoneal do fator de necrose tumoral alfa (TNF-alfa)

Avaliou-se a inibição da produção do fator de necrose tumoral alfa (TNF-alfa) devido ao pré-tratamento com antiinflamatório esteroidal (dexametasona) e não esteroidal (diclofenaco sódico) em eqüinos com endotoxemia induzida experimentalmente. Foram utilizados 15 cavalos machos não castrados, distribuídos em três grupos de cinco animais: controle (C), diclofenaco sódico (DS) e dexametasona (DM). A endotoxemia subletal foi induzida pela infusão intravenosa (IV) de 0,1mg/kg/pv de lipopolissacarídeo (LPS) de Escherichia coli 055:B5, administrado em 250ml de solução estéril de cloreto de sódio a 0,9%, durante 15min. Os cavalos do grupo-controle foram tratados com solução de cloreto de sódio a 9% IV. Nos animais do grupo DS, administraram-se, por via oral, 2,2mg/kg de diclofenaco sódico e, nos do grupo DM, 1,1mg/kg de dexametasona IV, respectivamente, 60 e 30min antes da infusão da endotoxina. Mensurou-se, por meio de ensaio de toxicidade com células da linhagem L929, a concentração de TNF-alfa no soro e no líquido peritoneal às 0, 1¼, 3 e 6 horas após injeção do LPS. No grupo-controle, observou-se aumento significativo de TNF-alfa sérico, em relação ao valor basal e aos grupos DS e DM, 1,15 horas após a indução da endotoxemia. No líquido peritoneal, as concentrações observadas estavam abaixo daquelas da curva padrão de TNF-alfa, não havendo diferença entre os grupos (P>0,05).

A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-kappa B ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide

Background and Objective: Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-kappa B ligand (RANKL) expression by murine periodontal ligament cells.Material and Methods: Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression.Results: Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL.Conclusion: Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.

In vitro antibacterial activity of medicinal plant extracts against Escherichia coli strains from human clinical specimens and interactions with antimicrobial drugs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reduced etiological role for enteropathogenic Escherichia coli in cases of diarrhea in Brazilian infants

Previously common in Brazil, enteropathogenic Escherichia coli (EPEC) strains of serogroups O55, O111, and O119 are now rare, while enteroadherent strains other than EPEC, belonging to serogroups such as O125, were prevalent among 126 diarrheic infants less than 1 year old who were surveyed. None of these strains had the EPEC bundle-forming pilus (bkA) gene.

Atividade antibacteriana de óleos essenciais de plantas frente a linhagens de Staphylococcus aureus e Escherichia coli isoladas de casos clínicos humanos

A ação antibacteriana in vitro de óleos essenciais de seis plantas foi verificada por meio da Concentração Inibitória Mínima (CIM=%v/v) pela diluição dos óleos em meio de cultura Mueller Hinton Agar, frente a linhagens de Staphylococcus aureus (n=16) e Escherichia coli (n=16) isoladas de casos clínicos humanos, além de 1 amostra padrão ATCC para cada espécie (Sa ATCC 25923 e Ec ATCC 25922), e determinação de curvas de sobrevivência em concentrações equivalentes a CIM90% dos respectivos óleos. O óleo essencial de canela foi o mais eficiente, com valores de CIM90% de 0,047 e 0,09 para S. aureus e E. coli respectivamente, enquanto gengibre (0,09), cravo da índia (0,095) e capim cidreira (0,1) apresentaram eficiências semelhantes para S. aureus. Frente a E. coli, os óleos de gengibre (0,52) e capim cidreira (0,55) foram equivalentes quanto à eficiência. de acordo com as curvas de sobrevivência, foi possível verificar também que os valores de CIM90% obtidos podem ser tanto bactericidas ou bacteriostáticas de acordo com a bactéria testada. em conclusão, verificou-se que os óleos essenciais testados foram efetivos no controle do desenvolvimento bacteriano, sendo o potencial antimicrobiano diferente em função da espécie bacteriana testada, sendo que a bactéria Gram positiva (S. aureus) mostrou-se mais susceptível aos óleos testados que a Gram negativa (E. coli).

Prevalence of diarrheogenic Escherichia coli and rotavirus among children from Botucatu, São Paulo State, Brazil

In a one-year prospective study carried out to define the role of rotavirus and Escherichia coli in local childhood diarrhea, we determined the prevalence of both agents in 54 diarrheic children attending a health center in Botucatu. Diarrheogenic E. coli (DEC) strains were characterized by O:H serotyping, a search for virulence genetic markers, and assays of adherence to HEp-2 cells. Except for enteroaggregative E. coli (EAEC), no other DEC category was detected in the children's stools. Both EAEC and rotavirus were isolated from 22 of the 54 (41.0%) diarrheic children as single agents or in combination with other enteropathogens. However, when considering the presence of a single agent, EAEC was dominant and isolated from 20.4% of the patients, whereas rotavirus was detected in 14.8%. These results indicate that rotavirus and EAEC play a significant role as agents of childhood diarrhea in the local population.

Mucosa-associated but not luminal Escherichia coli is augmented in Crohn's disease and ulcerative colitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sinergismo entre óleos essenciais e drogas antimicrobianas sobre linhagens de Staphylococcus aureus e Escherichia coli isoladas de casos clínicos humanos

Estudos com plantas e utilização em terapias combinatórias têm sido estimulados. Verificou-se as possíveis interações entre óleos essenciais de plantas [canela (Cinnamomum zeylanicum Blume Lauraceae), capim-cidreira (Cymbopogon citratus (DC.) Stapf, Poaceae), hortelã-pimenta (Mentha piperita L. Lamiaceae), gengibre (Zingiber officinale Roscoe Zingiberaceae), cravo-da-índia (Caryophillus aromaticus L. Myrtaceae) e alecrim (Rosmarinus officinalis L. Lamiaceae)] combinados a oito drogas antimicrobianas frente a doze linhagens de Staphylococcus aureus e doze de Escherichia coli isoladas de humanos. Após determinação da Concentração Inibitória Mínima (CIM) para os óleos pelo método da diluição foram realizados ensaios para verificação de sinergismo entre os óleos essenciais e os antimicrobianos pela metodologia de Kirby & Bauer. S. aureus foi mais suscetível às interações óleos e drogas, tendo o óleo de capim cidreira apresentado sinergismo com as oito drogas testadas, seguido pelo óleo de hortelã com sete drogas. Nos ensaios com E. coli, houve sinergismo apenas para os óleos de alecrim (três drogas) e capim-cidreira (duas drogas). Não ocorreram casos de antagonismo e os resultados de sinergismo foram influenciados pelos microrganismos estudados.

Studies on the electrochemical disinfection of water containing Escherichia coli using a Dimensionally Stable Anode

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Refolding and purification of bothropstoxin-1, a Lys49 - Phospholipase A(2) homologue, expressed as inclusion bodies in Escherichia coli

Hydrolysis of phospholipids by Group II phospholipase A(2) enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region, Bothropstoxin-I (BthTX-I) is a PLA, variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E, coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes, (C) 2001 Academic Press.

Separation and partitioning of Green Fluorescent Protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems

The partitioning of Green Fluorescent Protein (GFP) in poly(ethylene glycol)/Na-poly(acrylate) aqueous two-phase systems (PEG/NaPA-ATPS) has been investigated. The aqueous two-phase systems are formed by mixing the polymers with a salt and a protein solution. The protein partitioning in the two-phase system was investigated at 25 degrees C. The concentration of the GFP was measured by fluorimetry. It was found that the partitioning of GFP depends on the salt type, pH and concentration of PEG. The data indicates that GFP partitions more strongly to the PEG phase in presence of Na2SO4 relative to NaCl. Furthermore, the GFP partitions more to the PEG phase at higher pH. The partition to the PEG phase is strongly favoured in systems with larger tie-line lengths (i.e. systems with higher polymer concentrations). The molecular weight of PEG is important since the partition coefficient (K) of GFP gradually decreases with increasing PEG size, from K ca. 300-400 for PEG 400 to K equal to 1.19 for PEG 8000. A separation process was developed where GFP was separated from a homogenate in two extraction steps: the GFP is first partitioned to the PEG phase in a PEG 3000/NaPA 8000 system containing 3 wt% Na2SO4, where the K value of GFP was 8. The GFP is then re-extracted to a salt phase formed by mixing the previous top-phase with a Na2SO4 solution. The K-value of GFP in this back-extraction was 0.22. The total recovery based on the start material was 74%. (c) 2008 Elsevier B.V. All rights reserved.