Molecular Epidemiology Research Of Shiga Toxin-producing E.Coli Isolated From Different Sources

BackgroundEschericha coli that producs one or more types of Shige toxin(STX)is refered to as Shiga toxin-producing E.coli(STEC).STEC is a well-known pathogen as a cause of diarrhea,hemorrhagic colitis(HC)and hemolytic uremic syndrome(HUS).Domestic ruminants,especially cattle and sheep,are the major reservoirs of STEC.0157.H7 STEC was known as the new type of food poisoning pathogenic bacteria since 1982 in USA.there were more than 400 kinds of serotypes had been recognized.Serious threats to human health and life.Bacterial resistance is a serious public health problem in the world.STEC strains resistant situation is relatively serious in China.Through the study of drug resistance and molecular typing of STEC,we can acquire knowledge of epidemiological characteristics and help revising better clinical drug usage,and finally provide evidences to know well the rule of the strains.Objective1.Collect STEC strains from different sources,to understand the epidemiological characteristics of STEC and stablish a simple STEC database,in order to lay a foundation for the future long-term surveillance.2.To analyze the drug resistance of STEC through drug sensitivity test.To provide a suggestion for clinical medicine.3.To compare the difference between different sources of STEC by molecular technology,in order to explore the genetic relationship between these strains and discuss the relations between phenotyping and molecular typing.Method1.Sample source:Collected fresh animal feces samples from the rural areas in Weishan county and Laizhou city;Collected food source and foodborne disease source Escherichia coli from the strain reservoir of the Shandong CDC.And separated STEC from the samples and strains.2.Isolated strains:Isolated STEC strains from animal feces samples by using immune magnetic beads capture and ECC color plate culture method.Identified the virulence genes and adhesion gene by PCR.Recovered Escherichia coli strains from food and foodborne diseases patians,amplified and identify the virulence genes by PCR.3.Biochemical characteristics:A series of biochemical characteristics recognized by using DBI-09.4.Serotypes:Through specific primer amplification method and glass plate agglutination method confirmed the serotypes of STEC strains.5.Drug sensitivity test:According to the M100-S20 standard recommended by the Clinical Laboratory Standards Institute took antibiotics senstivity test of STEC.Took the test by using the broth microdilution method,got the MIC values of 16 kinds of clinical common use antibiotics.Result showed in sensitive(S),intermediate(I)and resistant(R).6.Molecular typing:The PFGE molecular typing and multilocus sequence typing(MLST)were used to analyze the genetic evolution of the strains.Result1.A total of 1022 samples of animal feces were collected;And a total of 26 STEC strains were isolated.157 strains of Escherichia coli isolated from food,from which no STEC positive strains were detected and 10 strains of diarrheagenic Escherichia coli were detected;726 strains of Escherichia coli isolated from patients with foodborne diseases,from which 3 STEC strains were detected;6 STEC strains from animals isolated from 2007 to 2012.2.Among 35 STEC strains,23 only carried STX2 genes,2 strains only carried STX1 gene;9 carried two genes of STX2 and attachment gene eae;and 1 carried three genes of STX1,STX2 and attachment gene eae.3.Among 35 STEC strains,8 were identified as 0157:H7 serotype strains,accounting for 22.86%,and 27 as non-0157 STEC serotype strains,no dominant serotypes.The serotypes of 10 strains of Escherichia coli isolated from food were different,so dominant serotypes could not be identified.4.Drug resistance rates of 35 STEC strains to 16 antibiotics of 7 categories differed.The resistance rate to Sulfafurazole was the highest(65.71%),followed by tetracycline(60.00%),compound sulfamethoxazole(57.14%)and nalidixic acid(54.29%).Among 35 STEC strains,there were 24 multi-drug resistant strains,accounting for 68.57%.Among 10 diarrheogenic Escherichia coli strains isolated from food,there were 2 multidrug-resistant strains.accounting for 20.00%.5.45 strains were classified into 30 genotypes,and the homology of fingerprints of 36 PFGE genes ranged from 57.6%to 100%.There were 4 SD025 genotypes and 4 SD028 genotypes,topping among all genotypes and accounting for 8.89%,followed by SD011,SD021 and SD023.all of which were 3 and accounted for 6.67%.There are 14 MLST genotype strains in 35 STEC strains,among which there were at most 8 ST11 genotype strains(22.86%),followed by 4 ST5133 strains and 4 ST540 strains(11.43%).MLST typing of 10 Escherichia coli strains isolated from food differed,which showed high degree of polymorphism.Conclusion1.The distribution of serotypes is dispersal,no dominant serotypes,reflecting a high degree of variability.2.STEC stains show a low level of resistance to beta lactam antibiotics and a high level of resistance to sulfonamides.3.Molecular types of STEC present polymorphism.In general,PFGE has a higher profile capability than MLST,and strains STs are associated with serotypes.