A rapid, sensitive method for testing the activity of Escherichia coli 0157:H7 secreted cytotoxins against epithelial cells from the jejunum and descending colon of cattle

Current methods for assessing Escherichia coli O157:H7 secreted cytotoxin activity are based on exposing human cell lines for 12 to 72 h and monitoring changes in cell morphology, adherence or enzyme release. These methods, although sensitive, use non-ruminant cell lines that may not represent cattle responses to E. coli O157:H7 secreted cytotoxins. The modified lawn assay used in this study was found to be a simple, fast and sensitive method for assessing cattle epithelial cell susceptibility to E. coli O157:H 7 secreted cytotoxins within 4 to 6 h. Key words: Escherichia coli O157:H7, cattle, intestine, cytotoxins, cytotoxicity

Escherichia coli O157:H7-secreted cytotoxins are toxic to enterocytes and increase Escherichia coli O157:H7 colonization of jejunum and descending colon in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157:H7-secreted cytotoxins are toxic to target cells and enhance colonization of intestinal tissues in disease-susceptible animals. It is unclear what role, if any, EHEC O157:H7-secreted cytotoxins play in the colonization of intestinal tissues of healthy reservoir animals. We previously reported that EHEC O157:H7 colonization sites were associated with focal hemorrhages in the jejunum and descending colon of persistent shedding cattle, suggesting a potential role for cytotoxins in EHEC O157:H7 colonization. We have used a traditional EHEC O157:H7 IVOC adherence assay and a novel lawn assay to examine the role of EHEC O157:H7-secreted cytotoxins in EHEC O157:H7 strain colonization of the jejunum and descending colon of non-persistent and persistent shedding cattle. Four EHEC O157:H7 strains that were previously reported to differentially colonize cattle produced cytotoxins that were differentially active against epithelial cells from the jejunum and descending colon. There was a relationship between EHEC O157:H7-secreted cytotoxin activity and strain adherence for epithelial cells from the jejunum and descending colon of cattle. There was also a greater susceptibility of epithelial cells from the jejunum and descending colon to EHEC O157:H7-secreted cytotoxins of persistent shedding cattle compared with non-persistent shedding cattle. Addition of the most active secreted cytotoxins from EHEC O157:H7 R318N to the IVOC adherence assays significantly increased the adhere nce of the most (R318N) and least (H4420N) virulent EHEC O157:H7 strain to intestinal tissues. The current study supports a role for EHEC O157:H7-secreted cytotoxins in enhancing EHEC O157:H7 colonization of intestinal tissues of cattle. Key words: Escherichia coli O157:H7, cattle, intestine, cytotoxins, colonization

The effect of fasting and diet on fecal shedding of Escherichia coli O157:H7 by cattle

Cattle naturally infected with Escherichia coli O157:H7 were used to assess the effects of diet and feed withdrawal on the fecal shedding of E. coli O157:H7. Animals were fed an 80% concentrate diet (80% barley and 20% alfalfa silage), fasted for 48 h, fed a 100% forage diet (alfalfa silage), fasted for 48 h, and subsequently re-fed 100% forage (alfalfa silage). There were no differences in the numbers of animals positive for the shedding of E. coli O157:H7 when fed an 80% barley diet or an all-forage diet (P > 0.05) or during the fasting periods following each diet (P > 0.05). Upon re-feeding an all-forage diet following a 48-h fast, animals positive for E. coli O157:H7 shedding increased (P < 0.05), with 42.5% of the animals shedding the pathogen after 5 d. Re-feeding 100% forage following fasting appeared to have increased the number of animals shedding E. coli O157:H7 in their feces, which may have been influenced by diet in addition to fasting. Key words: Escherichia coli O157:H7, fasting, diet, cattle, fecal shedding

Survival ofEscherichia coliexposed to visible light in seawater: analysis ofrpoS-dependent effects

We investigated the effect of visible light on Escherichia coli in seawater microcosms. Escherichia coli lost its ability to form colonies in marine environments when exposed to artificial continuous visible light. Survival of illuminated bacteria during the stationary phase was drastically reduced in the absence of the σsfactor (RpoS or KatF) that regulates numerous genes induced in this phase. In the stationary phase, double catalase mutants katE katG and mutants defective in the protein Dps (both catalase and Dps are involved in resistance to hydrogen peroxide (H2O2)), were more sensitive to light. In the exponential phase, a mutation in oxyR, the regulatory gene of the adaptive response to H2O2, increased sensitivity to light, further suggesting that deleterious effects might be associated with H2O2production. However, in the stationary phase, the katE katG dps mutant was considerably more resistant to visible light than the rpoS mutant, suggesting rpoS-dependent protection against deleterious effects other than those related to H2O2. The deleterious action of visible light was less important when the salinity decreased. In freshwater, rpoS and katE katG dps mutants did not show a drastic difference in sensitivity to light suggesting that osmolarity sensitizes E. coli to those deleterious effects of visible light that are unrelated to H2O2.Key words: Escherichia coli, stationary phase, RpoS, visible light, seawater.

Effect of incubation of Escherichia coli cells with halophyte extracts on their subsequent survival in seawater

Organic osmolytes are utilized by enteric bacteria for their survival when entering a high-osmolarity medium such as seawater. As these are commonly synthetized by marine organisms (fauna and flora), the purpose of this work was to investigate the effects of aqueous extracts from some marine phytoplanktonic species, alga, and phanerogam on the survival of Escherichia coli cells in seawater. Some of the tested halophyte extracts greatly enhanced survival, whereas others were inhibitory. The efficiency of the extracts could not be correlated to the taxonomic position of the halophyte or to the presence of Dragendorff-positive compounds known to be osmoprotective. Key words: Escherichia coli, survival, seawater, halophytes, osmoprotection.

Assessment of two commercial agglutination kits for detecting Escherichia coli heat-labile enterotoxin

Two commercial agglutination kits, a reserved passive agglutination test (VET-RPLA) and a staphylococcal coagglutination test (Phadebact ETEC-LT Test), were compared with two cell culture assays (Y-1 and Vero) and GM1 ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) for sensitivity in detecting Escherichia coli heat-labile enterotoxin (LT). Of 48 toxigenic strains, 23 were positive by all assays. One strain was negative only by the Phadebact test. Four strains, all LT-II producers, were positive by cell culture only. For LT-I detection, the Phadebact test was the least sensitive but was simple and rapid; VET-RPLA was simple, sensitive, and a good substitute for cell culture or GM1-ELISA. Key words: Escherichia coli, heat-labile enterotoxin, agglutination kits.

Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays

Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin. Key words: Escherichia coli, cytotoxin, ELISA.

Antibiotic resistance indexing of Escherichia coli to identify sources of fecal contamination in water

A total of 202 Escherichia coli isolated from urban and rural water were tested with 11 antibiotics to assess the prevalence of antibiotic resistance from each source. Urban waters harbored higher percentages of resistant E. coli strains than rural waters. Antibiotic-resistant E. coli may offer an index of water quality related to source. Key words: Escherichia coli, antibiotic resistance, indicator.

Aerobactin uptake system, ColV production, and drug resistance encoded by a plasmid from an urinary tract infection Escherichia coli strain of human origin

A study of Escherichia coli strains isolated from patients suffering from urinary tract infections in Ljubljana, Yugoslavia, revealed a plasmid encoding the aerobactin iron uptake system, ColV production, and drug resistance. The plasmid is conjugative and at least 85 kilobases in length. Key words: Escherichia coli, plasmid, aerobactin, ColV, resistance.