A Study Of Escherichia Coli O157:H7 Strains Isolated From Animal Reservoirs In Henan: Detection Of Toxicity And Determination Of Clonal Relationship

Enterohemorrhagic Escherichia coli (EHEC), an important and emergent pathogen, has been associated with blood and nonblood diarrhea and hemolytic-uremic syndrome (HUS). O157:H7 and O157:H- are the most important serotypes of EHEC. In the period from March to July 2000, an outbreak of O157:H7 occurred in one county of Henan Province in China. Animal reservoirs were suspected to be the cause. So multiplex PCR and cytotoxicity tests were a-pplied to detect the toxicity of 20 strains isolated from animals such as Boer goats, pigs, cows etc. And pulsed-field gel electropho- resis (PFGE) and 16S rDNA fingerprinting were exployed to deter- mine the clonal relationship among the strains. Cluster analysis and principal component analysis were applied to understand the data from PFGE and 16S rDNA fingerprinting.METHODS1 Vero cytotoxicity assays:For each specimen, the polymyxin B extracts of 1 ml of an overnight broth culture was filtered through 0.22-jim membrane filters to be used in cytotoxicity tests; meantime the supernatant after centrifuging another broth culture was also filtered. Both the polymyxin B extracts and the supernatant were inoculated to Vero cells monolayer grown in 96-well microtiter plates in two concentrations. The plates were incubated at 37癈 in 5% COj and cells were examined for cytopathic effect (CPE) using an inverted microscope. The specimen was considered positive only when the two dilutions both produced CPE exceeding 75% of the monolayer in 48 hours.2 Multiplex PCR:The primers for stxl, stx2 and eae were designed online according to the usual pricinples. After determining the optimal program and conditions, the broth cultures were boiled and the molds were amplified. Amplified products were visualized by ethidium bromide staining after electrophoresis.3 Rapid pulsed-field gel electrophoresis:The method of Romesh K. Gautom was used with minor modifications. The DNA plugs were digested with restriction enzyme Xbal. Electrophoresis was performed in a CHEF MAPPER system with pulse times increasing from 2 s to 56 s over a period of 24 h.The results were visualized by ethidium bromide staining after electrophoresis.4 16S rDNA fingerprinting:After the electrophoresis in PFGE, the separated DNA was transferred from the agarose gel to a Hybond-N+ nylon membrane by Southern blotting. The membrane was hybridised with 16S rDNA probe. The probe had been labeled in the PCR with the DIG-dUTR The result was visualized by anti-digoxigenin enzyme-linked immunosorbent assay.5 Statistics:The SAS (V8) software was employed in performing cluster analysis and principal component analysis with the data from PFGE and 16S rDNA fingerprinting.RESULTS1 The cytotoxicity tests:11 strains of the total 20 were identified to be toxic. They all carried two genes: stx2 and eae. In the 11 strains, 10 were isolated from Boer goats and another from a chicken. The other 9 strains from goats, cows, pigs etc were found to be non-toxic.2 The rapid PFGE:11 patterns were identified from the 20 O157:H7 isolates. 5 patterns contained the 11 toxic strains; the other 6 patterns containedall the non-toxic ones.3 16S rDNA fingerprinting:11 patterns were identified from the 20 O157:H7 isolates. 5 patterns contained the 11 toxic strains; the other 6 patterns contained all the non-toxic ones. The results were not same but similar with those of PFGE-RFLP.4 Statistics:The toxic strains appeared to be much nearer one another in the genetic distance than the non-toxic ones. The genetic distances showed that transmittion of O157:H7 existed among different animal reservoirs. 3 indices were found to be significant to differentiate the toxic and non-toxic strains. Some bands important to the colonal relationship were identified in the results of PFGE-RFLP and 16S rDN A fingerprinting.CONCLUSIONS1 Boer goats may be the reservoir which had transmitted the pathogen to persons. But it is important to further study the epidemiology of the non-toxic strains.2 The toxic stra...