| Background andObjectiveDiarrheagenic Escherichia coli (DEC) can be categorized into six pathotypesby its virulence genes and pathogenicity, including enteropathogenic E. coli (EPEC), enterohemorrhagic (or Shiga toxin-producing) E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC). Global epidemics and outbreaks of all types of DEC, except DAEC, have been frequently reported. A serious outbreak caused by Shiga toxin-producing Escherichia coli (STEC) O104:H4 occurred in Germany from May to July 2011. And the outbreak of the culprit as stx encoding Stx, can cause HUS, even death. Reported stx gene can be carried in a variety of bacteria. Duo to it’s unique geographical location and tourist attractions, Hangzhou has always been a high incidence of diarrhea city. So we have to strengthen the monitoring and control of DEC, master of its popular features and response measures to establish a fast and accurate method for stx gene detection, timely report to clinical doctors the results in order to follow their designated treatment program. It is important to epidemic early warning and effective in preventing that detection of DEC or bacteria earring stx.Methods1. Consecutive fecal specimens from outpatients with acute diarrhea in The First Affiliated Hospital, College of Medicine, Zhejiang University were collected from January 2014 to December 2014. Bacterial and viral pathogens were detected and identified by stool culture and RT-PCR, respectively.2. DEC isolates were further classified into enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shiga toxin (Stx)-producing E. coli (STEC), and enteroinvasive E. coli (EIEC) by a multiplex PCR.3. Datas of patients with acute diarrhea and DEC testing from 2011 to 2014 in the First Affiliated Hospital of Zhejiang Universitywere analyzed;4. To establish the single, double fluorescence quantitative PCR method to detect the stxl, stx2.5. To identify the suspicious clinical isolates collected from inside and outside the gut and feces collected from patients with acute diarrhea by fluorescent PCR method, and positive isolates were be sent to sequencing, then perform sequence alignment and analysis.Results1.735 samples of acute diarrhea by isolation, culture, nucleic acid detection, immunological detection methods to detect pathogen, which positive samples (at least one kind of detection of bacteria, fungi, viruses) were 338, the positive rate was 45.99%. diarrhea caused by E. coli 144, the detection rate was 19.59%, the first column of acute diarrhea pathogen, followed by Norovirus and Vibrio parahaemolyticus.2.11 of the total Acute diarrhea samples were simultaneously detected two or more pathogen, one of which three kinds of pathogens was detected simultaneously.3.144of DECwere detected among 735 cases of acute diarrhea, the detection rate was 19.59%(144/735). Among them,73 of EAEC, the detection rate was 9.93% (73/735); 32 of ETEC, the detection rate was 4.35%(32/735); 20 of EPEC, which thedetection rate was 2.72%(20/735); and the isolates of STEC and EIEC were 2, the detection rate was 0.27%(2/735). In addition,9 of DEC isolates were simultaneouslycarried virulence genes of EAEC, ETEC and EPEC. DEC isolates, were mainly for EAEC, accounting for 50.69%(73/144), followed by ETEC (22.22%) and EPEC (13.89%), respectively.4. DEC were isolated in alltheyear round, mainly in summer and autumn. Through data analysis, there was a seasonal difference (P= 0.027,0.025 and 0.016) in 2011,2012, 2013, but in 2014, the detection rate was no significant seasonal difference. But the detection rate of EAEC/ETEC was highest in summer and autumn, there was a seasonal difference, P= 0.025,0.001 (P<0.05).5. DEC was detected in Male and female patients was 21.69%,17.87%, respectively. The difference was statistically insignificant. ETEC/EAEC which male and female detection rates were 2.41%,0.50%, P= 0.049(P<0.05), with significant difference.In addition, according to statistical analysis of surveillance datasfrom 2011,2012 and 2013, there were no statistically significant difference in terms of gender distribution in DEC and subtypes.6. DEC was detected mainly in patients among 19 to 60 years old with acute diarrhea in 2014, accounting for 77.78%(112/144), followed by the> 60 years old group, accounting for 16.67%; butthe detection rateof DEC and subtypes in patients of different ages with acute diarrhea was not statistically different. Like in 2014, DEC isolates were mainly detected from patients between 19 and 60 years old with acute diarrhea in Hangzhou in 2011,2012,2013.Excepting 2012, the detection rates of DECs did not exist statistical differencein the different age groups. The rate of group> 60 years of age was the highest (20.43%) and there was significant difference, P= 0.024 (P <0.05).7. DEC was mainly isolated from watery feces in 2014, accounting for 78.47% (113/144), followed by Migan stools (13.89%); however, the highest detection rate was Migan-like stool mixed bloody, about 50%. There were statistical difference within different character of feces in 2011,2012,2013.8. There are 40,15isolates of DEC emergence of multi-type virulence genes in 2013and 2014, respectively, such as 24 of astA+estlb,6 of astA+escV,2 of astA+elt+estlb,1 of astA+elt, astA+elt+estla, astA+elt+estlb+pic, astA+estlb+escV, astA+estIb, astA+elt+estla+escV, invE+escV, elt+escV in 2013, respectively. In 2014,1 of aggR+escV,1 of astA+escV,1 ofpic+bjpB,1 of astA+elt,2 of astA+estIa,7 of astA+estIa,1 of astA+estIb+escV,1 of bfpB+stx2.9. The specificity of the single/double fluorescence PCR method to stx gene was better, there is no cross reaction among other bacteria isolated and intestinal fungi; detection limit of 1×103～1×1012 copies/ml; stability at room temperature for 7 days had no obvious effect; the repeatability between batches, coefficient of variation were less than 10%.10. To screen the clinical isolates by the double fluorescence PCR method established, the result show that four of E. colipositive, including two ofstx1 positive, two of positive stx2.11. Directly detect acute diarrhea samples by double fluorescence PCR method showing 8 positive samples in 55.Conclusions1. Studies have demonstrated that DEC was the most prevalent pathogen in the fecal specimens from patients with acute diarrhea in areas of Hangzhou. And the most prevalent is EAEC, followed by ETEC, EPEC, STEC and EIEC.2. DEC have certain distribution characters in different season and feces.3. We prove that the single/double fluorescence quantitative PCR method is reliable and can be applied by specificity, sensitivity, stability, repeatability experiment.4. Through clinical isolates screening, STEC positive rate was 0.54%.5. According to acute diarrhea stool samples for fluorescence PCR results, PCR assay established can directly detect fecal specimens, thereby greatly reducing the testing cycle. |
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