Anchorage independent colony growth of fetal hamster lung epithelial cells after treatment with diepoxybutane.

To test the reliability of a new cell transformation assay, a cloned fetal Syrian hamster lung epithelial cell line (M3E3/C3) was used. The target cells originating from the respiratory tract were treated in vitro over a concentration range of 0-10(-5) M/l with diepoxybutane, cultured during the expression period of 28 or 35 days and then transferred into soft agar. Anchorage independent colony growth in soft agar occurs only if cells are transformed. Growth and number of colonies were taken as a score of the carcinogenic potential of the test substance. Under the conditions of this cell transformation assay it was possible to detect the carcinogenic potential of diepoxybutane unequivocally.

Occurrence of Shiga toxin-producing Escherichia coli in fecal samples from children with diarrhea and from healthy zebu cattle in Uganda.

Fecal samples collected from 237 diarrheic infants in Kampala, Uganda and from 159 healthy cattle from a ranch in the Central Region of Uganda were investigated for the presence of Escherichia coli O157 and other types of Shiga toxin-producing E. coli (STEC). E. coli O157 were not detected in 150 stool samples from children which were cultivated on sorbitol MacConkey agar. A search for all types of STEC performed on 87 additional human stool samples with an enzyme-immunoassay for Shiga toxins (Premier EHEC) was also negative. Forty-two stool samples from infants were additionally investigated for enteropathogenic E. coli (EPEC) by DNA-hybridization with an eae-specific gene probe. Compared to STEC, EPEC were frequent and found in six (14.3%) of these 42 randomly selected stool specimens. We were further interested in the role of cattle as a reservoir for STEC in Uganda. STEC were isolated from 45 of 159 cattle from a herd in the Central Region of Uganda. STEC strains from cattle belonged to 16 different O- and nine different H-types and nine O:H types were identical to those found in bovine STEC from other continents. Only one bovine STEC strain was positive for the eae-gene, and O-groups associated with enterohemorrhagic E. coli (EHEC) types (O26, O103, O111, O145 and O157) were not found. Our report demonstrates that STEC are not frequent in urban children in Uganda, but domestic cattle were identified as an important natural reservoir for these organisms in this country.

Serotypes of Escherichia coli isolated from healthy infants in Berlin, Germany and Melbourne, Australia.

The characterization of Escherichia coli strains isolated from healthy infants under one year of age with respect to O:H serotype, K1 and K5 antigens in two disparate parts of the developed world was the purpose of this investigation. A total of 450 strains were examined, 264 from Berlin and 186 from Melbourne. Of all the 220 different O:H serotypes found, 179 were only isolated once, 90 in Berlin and 89 in Melbourne. However, 30 of the 41 O:H serotypes (73.2%) found more than once were isolated in both centers. The most commonly identified serotypes were found in both centers and included O1:H-; O1:H7; O2:H2; O2:H4; O2:H7; O4:H5; O6:H-; O6:H1; O15:H1; O18:H7; O25:H1; and 075:H-. Potentially pathogenic serotypes were found in both cities. Enteropathogenic E. coli (EPEC)-associated serotypes (O18:H7; O26:H-; O44:H34; O86:H-; O128:H2) were present in 11 cases and enterohaemorrhagic E. coli (EHEC)-associated types including O26:H11; O128:H2) were present in four cases. The distributions of serotypes found were similar in the two cities, strongly suggesting the wider applicability of these results.

Effects of nitroso compounds and aromatic amines on fetal tracheal explants.

Tracheas were excised from fetal Syrian golden hamsters on the 15th day of gestation. Tracheal explants were cultured in vitro and exposed to different dose-levels of well known carcinogens. We chose two nitroso compounds, N-Methyl-N-nitro-N-nitrosoguanidine (MNNG) and Diethylnitrosamine (DEN) and two aromatic amines, Aminofluorene (AF) and Acetylaminofluorene (AAF). The tracheal explants were treated for 24 h in vitro, then the carcinogens were washed off and the tracheas were kept for 21, 28 or 35 days in culture. After fixation tracheal explants were transversely cut with serial section techniques and scored for morphological changes of the epithelium by light microscopy. Most of the control explants completed differentiation and had a normal morphology at the end of the in vitro culture period. Occasionally we found a decrease of the number of ciliated cells and some areas with squamous metaplasia in the respiratory epithelium. Carcinogen treatment with nitroso compounds led to a significant increase of the morphologic changes of the epithelium. These effects were especially obvious after DEN treatment. Morphologic changes of the epithelium such as metaplasia and hyperplasia were discussed as carcinogen-related events. In vitro exposure with aromatic amines did not induce marked metaplastic or hyperplastic changes in the respiratory epithelium of tracheal explants.

Rapid detection and isolation of shiga-like toxin (verocytotoxin)-producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay.

By combining the enterohemolysin test and the VTEC-RPLA test (specific for the detection of Shiga-like toxin I [SLT-I], SLT-II, and SLT-IIc), single colonies of SLT-producing Escherichia coli were found to constitute between 0.03 and 68.1% of the coliform flora in human stool cultures and were isolated and characterized within 72 to 96 h.

Human infections with Shiga toxin-producing Escherichia coli other than serogroup O157 in Germany.

We investigated different types of Shiga toxin-producing Escherichia coli (STEC) not belonging to serogroup O157 for their role as human pathogens. Non-O157 STEC isolated from 89 human patients in Germany were characterized according to serotypes, virulence markers, and association with human illness. EaeA-positive STEC were isolated from 54 (60.7%) of the patients and were frequently associated with severe diarrheal disease, hemolytic uremic syndrome, and young age. EaeA-negative STEC were found in 35 (39.3%) of the patients and were more associated with clinically uncomplicated cases and adult patients. For pediatric patients, a serotype-independent diagnosis of STEC is recommended.

Virulence factors and phenotypical traits of verotoxigenic strains of Escherichia coli isolated from human patients in Germany.

Fecal isolates of Escherichia coli which were collected from human patients in different parts of Germany between 1985 and 1992 were examined for production of verotoxins (VT). Among 2165 isolates 54 (2.5%) verotoxigenic E. coli (VTEC) were found. The 54 VTEC belonged to 13 different serotypes, 46 (85.2%) of these were enterohemorrhagic E. coli (EHEC) types as O157:H7, O157:H-, O145:H-, O111:[H8] and O26:[H11]. Of the 54 VTEC 50 (92.6%) hybridized with one or both of the DNA probes specific for VT1 and VT2. The 4 VTEC strains which were negative for VT1 and VT2 differed from all other VTEC by many phenotypical trains such as serotype, production of alpha-hemolysin and absence of EHEC-plasmid and "attaching and effacing" (eae)-specific DNA sequences. In contrast, VTEC which were positive for VT1, VT2 or both were frequently positive for eae sequences (92.0%), EHEC-plasmids (90.0%) and for production of enterohemolysin (88.0%). With enterohemolysin as an epidemiological marker more VTEC strains (81.5%) could be identified than with others such as the absence of beta-glucuronidase activity (61.1%) or non-fermentation of sorbitol (48.1%). Case reports were available for 42 of the 54 VTEC strains. The clinical presentation of 42 cases with VTEC ranged from uncomplicated diarrhea to severe diseases as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). However, bloody diarrhea, HC and HUS were more associated with the O157 group than with other VTEC groups.

Origin and characteristics of enteroinvasive strains of Escherichia coli (EIEC) isolated in Germany.

Thirty-five E. coli strains belonging to O-serogroups with enteroinvasive types of Escherichia coli (EIEC) isolated in Germany between 1989 and 1995 were investigated for invasivity-associated DNA sequences. Only 11 strains were positive for ipaH and thus confirmed as EIEC. All 11 EIEC isolates originated from human infections which were imported to Germany from Eastern Europe. EIEC O124 were most frequent and originated from asymptomatic Romanians arriving at Rostock, Germany in 1992 and 1993. In January 1993, EIEC O124 were isolated from faeces of a laboratory technician with diarrhoea working at the enteric pathogen department of the Institute of Hygiene in Rostock. By comparing her E. coli O124 isolate with recently imported O124 strains for Xba I restriction fragment length polymorphisms (RFLP) the probable source of infection could be determined. Four major RFLP patterns were found in the group of O124 strains. O124 strains with identical RFLP patterns were found in the group of 0124 strains. 0124 strains with identical RFLP patterns were isolated from people who were in close contact to each other.

Investigation of human infections with verocytotoxin-producing strains of Escherichia coli (VTEC) belonging to serogroup O118 with evidence for zoonotic transmission.

Twenty verocytotoxigenic Escherichia coli (VTEC) O118 strains isolated between 1996 and 1998 from human patients in Germany were analysed for their serotypes, their virulence markers and their epidemiological relatedness. Three strains were typed as O118:H12, these carried only the VT2d-Ount variant gene and were not associated with diarrhoea or haemolytic uraemic syndrome (HUS). Seventeen strains were serotyped as O118:H16 or O118:non-motile (NM). These carried all the genes for VTI, eae and EHEC-haemolysin. The O118:H16/NM strains were from diarrhoea (13 cases) and HUS (2 cases). Sixteen of the patients were young infants and most infections were associated with a rural environment. Evidence for zoonotic transmission from cattle to humans was found in two cases. The epidemiological relationship between the human and bovine O118:H16/NM isolates was indicated by homogeneous plasmid patterns and by very similar XbaI restriction patterns obtained by pulsed-field gel electrophoresis. VTEC O118:H16/NM are emerging pathogens in Germany and should be classified as new enterohaemorrhagic E. coli (EHEC) types.