RESUMO
The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.
Assuntos
Adulto , Animais , Humanos , Adesinas Bacterianas/fisiologia , Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/fisiologia , Sepse/microbiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/ultraestrutura , Aderência Bacteriana/genética , Chlorocebus aethiops , Células Epiteliais/ultraestrutura , Escherichia coli/genética , Escherichia coli/ultraestrutura , Genótipo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Células VeroRESUMO
Enteric infections caused by the ingestion of contaminated water, especially by Escherichia coli, are important to define the virulence properties of these bacteria. Due to frequent infantile diarrhea in the city of Ouro Preto, Minas Gerais state, Brazil, the phenotypic and genotypic diarrheagenic properties of E. coli isolated from drinking water were studied. The culture supernatants of 39 (40 percent) among a total of 97 E. coli isolates from drinking water were positive by suckling mouse assay and induced cytotoxic effects on Vero cells. The enterotoxic and cytotoxic activities were present in the fraction with less than 10 kDa and were not lost when heated up to 60¨¬C and 100¨¬C for 30 minutes. PCR assays showed that among these 39 Vero cytotoxigenic E. coli, four (10.2 percent) were positive for ST II (estB) and two (5 percent) positive for ¥áHly (hlyA). Gene amplification of SLT (stx 1, stx 2), ST I (estA), LT (eltI, eltII), EAST1 (astA), EHly (enhly) and plasmid-encoded enterotoxin (pet) were not observed. This heat-stable cytotoxic enterotoxin of E. coli is probably a new putative diarrheagenic virulence factor, as a toxin presenting these characteristics has not yet been described.
Assuntos
Animais , Camundongos , Água Potável/análise , Citotoxinas , Escherichia coli Enterotoxigênica , Enterotoxinas , Escherichia coli/isolamento & purificaçãoRESUMO
Sixty strains of Escherichia coli, isolated by hemoculture, from septicemic Brazilian patients were evaluated to determine their serogroup and invasivity to Vero cells. All 60 patients died within 2 days of hospitalization. Furthermore, the molecular study of the following extraintestinal pathogenic E. coli-associated virulence factor (VF) genes was performed by PCR: i) adhesins: type 1 fimbria (fimH), S fimbria (sfaD/E), P fimbria (papC and papG alleles) and afimbrial adhesin (afaB/C); ii) capsule K1/K5 (kpsMTII); iii) siderophores: aerobactin (iucD), yersiniabactin (fyuA) and salmochelin (iroN); iv) toxins hemolysin (hlyA), necrotizing cytotoxic factor type 1 (cnf1) and secreted autotransporter toxin (sat); v) miscellaneous: brain microvascular endothelial cells invasion (ibeA), serum resistance (traT), colicin V (cvaC) and specific uropathogenic protein (usp). Our results showed that isolates are able to invade Vero cells (96.6 percent), differing from previous research on uropathogenic E. coli (UPEC). The O serogroups associated with UPEC were prevalent in 60 percent of strains vs 11.7 percent of other serogroups. The PCR results showed a conserved virulence subgroup profile and a prevalence above 75 percent for fimH, fyuA, kpsMTII and iucD, and between 35-65 percent for papC, papG, sat, iroN, usp and traT. The evasion from the immunological system of the host and also iron uptake are essential for the survival of extraintestinal pathogenic E. coli strains. Interestingly, among our isolates, a low prevalence of VF genes appeared. Therefore, the present study contributes to the identification of a bacterial profile for sepsis-associated E. coli.
Assuntos
Adulto , Animais , Humanos , Escherichia coli/patogenicidade , Sepse/microbiologia , Fatores de Virulência/genética , Chlorocebus aethiops , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Genótipo , Reação em Cadeia da Polimerase , Células Vero/microbiologia , Virulência/genéticaRESUMO
Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 æg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 æg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.
Assuntos
Animais , Masculino , Camundongos , Toxinas Bacterianas , Eritrócitos , Escherichia coli , Células Eucarióticas , Toxinas Bacterianas , Membrana Celular , Colesterol , Eletroforese em Gel de Poliacrilamida , Hemólise , Ligação ProteicaRESUMO
Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 æg/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli
Assuntos
Animais , Cricetinae , Humanos , Camundongos , Citotoxinas , Serratia marcescens , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Haplorrinos , Hemólise , Peso MolecularRESUMO
Tamm-Horsfall glycoprotein (THP) contains manno-oligosaccharides that are recognized by type 1 fimbriae (F1) of Escherichia coli. In the present study, we examined the in vivo phagocytic activity of mouse peritoneal macrophages after treatment of bacteria with THP. At low THP concentrations (12.5 æg/ml and 50 æg/ml) no significant difference was observed in the phagocytosis of E. coli F1+. However, at high THP concentrations (500 æg/ml and 1250 æg/ml) we obtained a reduction of bacterial phagocytosis by mouse peritoneal macrophages
Assuntos
Humanos , Animais , Camundongos , Adjuvantes Imunológicos/farmacologia , Escherichia coli/citologia , Fímbrias Bacterianas/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Escherichia coli/efeitos dos fármacosRESUMO
Semipurified K99 and F41 fimbrial antigens were used to prepare an oil-emulsified vaccine against bovine enterotoxigenic colibacillosis. Nine Nelore cows about 7 months pregnant were divided into 3 groups (A, B and C) of 3 animals each, which received different doses of vaccine (1,500 HU, 750 HU and 380 HU, respectively) 8 and 2 weeks before delivery, in the neck by the subcutaneous route. As a control (group D), 3 pregnant cows of the same breed were not vaccinated for later challange of their calves. Vaccine efficiency was measured by the serological tests double diffusion and ELISA. Challenge of calves from the vaccinated and from the three control unvaccinated cows was carried out with the virulent Escherichia coli B41 strain (0101), STa+, K99+, F41+). Two of the 3 calves from unvaccinated cows died within 48 h with acute diarrhea. E. coli B4 was recovered as pure culture from their stools. In contrast, none of the calves born from vaccinated cows presented diarrhea. These data suggest that the antibody transfer to calves through colostrum gave full protection aginst the challenge. This semipurified fimbrial vaccine against K99-F41-harboring strains is the first oil-emulsified immunogen prepared in Brazil, which was not only efficient, but also had no adverse effects on vaccinated pregnant cows
Assuntos
Bovinos , Animais , Feminino , Gravidez , Antígenos de Superfície/farmacologia , Vacinas Bacterianas/imunologia , Fímbrias Bacterianas/imunologia , Infecções por Escherichia coli/imunologia , Doenças dos Bovinos/prevenção & controle , Ensaio de Imunoadsorção EnzimáticaRESUMO
Os antígenos K99-F41 foram extraídos da amostra de Escherichia coli B41 por aquecimento e semi-purificados pela precipitaçäo com sulfato de amônio e tratamento com desoxicolato de sódio (DOC). Os antígenos semi-purificados foram utilizados na produçäo de uma vacina oleosa contra a colibacilose bovina. Foram preparadas vacinas contendo em cada dose 1.500 HU (Unidades Hemaglutinantes), 750 HU e 380 HU. A eficiência da vacina foi avaliada através do ensaio de imunodifusäo dupla, ensaio imunoenzimático (ELISA) e por um desafio, em que a amostra de Escherichia coli virulenta foi inoculada nos bezerros nascidos de vacas vacinadas e näo vacinadas. Observamos que a vacina contendo 750 HU foi a que melhor induziu a produçäo de anticorpos nas vacas vacinadas, e que estes mostratam-se protetores, uma vez que os bezerros nascidos de vacas vacinadas e que mamaram o colostro, nada sofreram no desafio. Näo se verificou nenhum efeito colateral nas vacas vacinadas
Assuntos
Animais , Antígenos/imunologia , Bovinos , Infecções por Escherichia coli/imunologia , Vacinas/análiseRESUMO
Foram estudadas 165 amostras de Escherichia coli isoladas de 41 bezerros provenientes de 11 rebanhos leiteiros do Estado de Minas Gerais. Sete amostras produziram toxina termoestável. Destas, duas aglutinaram hemácias de cavalo na presença de manose e foram sorologicamente positivas para K99 (FS), uma apresentou aglutinaçäo de hemácias de cavalo e sorologia negativa para K99 (FS) e duas näo aglutinaram hemácias de cavalo e foram sorologicamente positivas para K99 (FS). Vinte e cinco amostras, isoladas de 18 bezerros com idade entre sete e 51 dias, foram produtoras de citotoxinas. Estes resultados indicam maior importância das amostras citotoxigênicas de E. coli sobre as enterotoxigênicas, no Estado de Minas Gerais