Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

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.

Putative new heat-stable cytotoxic and enterotoxic factors in culture supernatant of Escherichia coli isolated from drinking water

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.

Serogroups and virulence genotypes of Escherichia coli isolated from patients with sepsis

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.

Thiol-independent activity of a cholesterol-binding enterohemolysin produced by enteropathogenic Escherichia coli

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.

Biological activity of Serratia marcescens cytotoxin

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

Role of Tamm-Horsfall protein in the binding and in vivo phagocytosis of type 1 fimbriated Escherichia coli by mouse peritoneal macrophages

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

Chemotherapy for advanced non-small cell lung cancer.

Less than 20% of all cases of non-small cell lung cancer are operable. The treatment option available for such patients is either radiotherapy or chemotherapy. Various combinations of chemotherapeutic agents have been tried. The combination of cisplatin and 5-fluorouracil (5-FU) has been proved to have a synergistic antitumour effect in many experimental and clinical studies. In this paper the authors report the results of a trial using tegafur (which is a prodrug of 5-FU; 1-[2-tetrahydrofuryl]-5-FU) and uracil along with cisplatin in inoperable non-small cell lung cancer. Thirty-one patients were entered into the study, all of whom were less than 75 years old (mean age 61 years). The patients (except for 2) had either stage IIIB (12) or stage IV (17) disease with adenocarcinoma and squamous cell carcinoma in equal proportions (15 each). A combination of uracil and tegafur (400 mg/m2) in 100 mg capsules (100 mg tegafur and 224 mg of uracil), was given orally for 21 days. Most of the patients received 300 mg of the combination tablets twice a day. Cisplatin (80 mg/m2) was given as an infusion over 90 minutes on day 8 after adequately hydrating the patients. This cycle was repeated every 4 weeks. At least 2 treatment cycles were given, unless there was disease progression or toxicity. The response and survival rates were assessed after a follow up period which ranged between 9 and 30 months. The patients received 1 to 4 cycles of therapy and the response rates were: complete response--nil,partial response--11, no change--11 and disease progression--9. The overall response rate was 35% (95% CI: range 19-52%). Five patients achieved at least 50% tumour reduction after 2 cycles. Of the 11 patients who showed partial response, 5 (35%) had stage IV disease and 6 (36%) had other stages; 6 (40%) had squamous cell carcinomas and 5 (31%) had tumours of other histological types. The median duration of response was 6 months (3-13 months). In stage III disease, the 1-year survival rate was 31%, with a median survival time of 11 months, while in stage IV disease it was 29% with a median survival time of 8 months. There was a low incidence of toxicity, with anaemia (10%), leukopenia (6%) and thrombocytopenia (6%) being the most common side-effects. There were also no treatment-related deaths. The authors concluded that oral uracil and tegafur were as effective as other combinations with cisplatin. They also caused very few side-effects. They suggest that a larger trial needs to be carried out.

Determination of the efficiency of K99-F41 fimbrial antigen vaccine in newborn calves

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

Evaluation of an oil emulsified vaccine against bovine colibacillosis using semi-purified K99-F41 adhesins

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

Toxigenic Escherichia coli in calves with diarrhea in Minas Gerais, Brazil

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