| Escherichia coli (E.coli) ,Salmonella and Campylobacter jejuni are three major food-borne pathogens which were most frequently reported to cause disease outbreak worldwide. The identification and tracing the source of the pathogens are the keys for preventing deterioration of the epidemic, and economic loss. Because of its high discrimination ability and repeatability, simplicity of analyzing the result and comparing through internet, Pulsed-field gel electrophoresis (PFGE) was named the"gold method"for subtyping pathogens. Multidrug-resistant pathogens are being isolated at an increasing rate in hospital settings and are having a significant impact on clinical practice and overall treatment costs.Because of the high concentration, it is easy to acquire the resistance genes by horizontal transfer between Enterobacteriaceae. As the indicator bacteria of dejecta contamination, E.coli is regarded as the reservoir of resistance genes. Study on the antibiotic resistance, the antibiotic resistance emergence, the resistance genes transfers and related influencing factors of E.coli have significant impact on controlling the producing of multidrug-resistance pathogens, and clinical practice. We studied the prevalence of E.coli among meats and Chinese salads, the virulence genes, class I integron and antimicrobial susceptibility in these E.coli, and subtyping 43 E.coli O157:H7, 4 serotypes salmonella(114) and 43 Campylobacter jejuni with 6 enzymes PFGE. The main results as following:1,575 E.coli were isolated from meats (100%) and Chinese salads (86.7%) sampled from Xi'an and Yangling in Shannxi Province. The detection rate of E.coli was unrelated with the species of meat, but resources. The flavorings maybe the main reason in inhibitting E.coli in Chinese salads. The detection rate of fimA genes encoding adhesion toxin was 56.1%(273). Furthermore, we detected the stxI,stxII,st and lt genes encoding intestinotoxin. The detection rate of STEC was 0.62%, and the detection rate of ETEC was 14.4%. This means that the food safety in Xi'an and Yingling is potentially at risk.2,575 food-borne E.coli isolates were highly resistant to Tetracycline (99.3%), Streptomycin(65.6%), Amoxicillin(61.3%), Nalidixic acid(57.7%), Ampicilllin(55.1%), whose antibiotic resistance rate were above 50%. The others'ordering were Ciprofloxacin (38.7%), Chloramphenicol(38.0%), Kanamycin(34.6%), Gentamicin(30.8%), Cefoperazone(23.4%), Cefoxitin(12.4%). Only 8.8% isolates were resistance to Amikacin.3,In 575 food-borne E.coli,2.1%(12)isolates were susceptible to all 12 antibiotics used in the study. 563(97.9%)isolates were resistant to one agent at least. Multidrug resistance (≥3)rate was 62.3%(358),and 37.0% E.coli were resistant to 7 or more agents. In 358 multidrug resistance isolates(≥3), 58 were resistant to 9 agents, isolates resistant to 6,10,8,3,4,7,5,11,7 agents were 49,49,43,35,34,31,27,25,7, respectively. All data indicates that the multidrug resistance food-borne E.coli are prevalent in Shaanxi province。4,304 isolates from Chicken displayed 100% resistance,85.5%(260)isolates resistant to 3 or more antibiotics. 7 isolates with the highest level of antibiotic resistance were resistant to all 12 agents used in the study. 56 isolates were resistant to 9 agents. Isolates resistant to 10,8,7,11,6,4,5,3 antibiotics were 47,40,26,25,21,14,14,11, respectively. 66.1%(201)isolates from chicken were resistant to 7 or more agents. Isolates from different resources had different antimicrobial susceptibility. The isolates from chicken displayed higher resistance level than isolates from other resources.5,49.1% isolates were PCR positive for class I integron among 118 meat E.coli isolates. 55.1% chicken isolates were PCR positive for class I integron. 59.1% isolates were PCR positive for class I integron among multidrug resistance(≥3) isolates. Isolates resistant to 1 or 2 agents only displayed 16.7% PCR positive for class I integron. Among the isolates resistant to more than 7 agents, 66.7% isolate were PCR positive for class I integron. It demonstrates that antibiotic resistance is associated with class I integron in E.coli.6,29 isolates had 2.0kb class I integron among 58 class I integrons PCR positive isolates . 19 isolates were 750bp class I integron positive. 12 isolates were 1.5kb class I integron positive. One isolates displayed PCR positive for both 750bp and 1.5kb class I integrons,and two isolates displayed PCR positive for 750bp and 2.0kb class I integron.7,Enzyme SpeI was most suitable for subtyping STEC O157:H7. There were 21 to 32 bands appeared in the PFGE gel. 43 O157:H7 were divided into 22 subtypes. The dendrograms that resulted from three enzyme combination BlnI/SpeI/PacI and six enzyme combination XbaI/BlnI/NheI/SpiI/SpeI/PacI showed same discrimination, produced 26 subtypes, yielded an average 4.4 isolates per unresolved cluster, 52% isolates unresolved, a node-to-isolates ratio of 0.67, and a SID of 0.902.8,For 4 serotypes Salmonella -S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, 6 enzymes subtyping results were different in difference serotypes. The enzyme which was most suitable for PFGE subtyping were PacI, SpeI, SpiI and NotI for S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, respectively. For S. Heidelberg, S.Kentucky, S.SaintPaul and S.Hadar, the most suitable three enzyme combination were SpeI/PacI/BlnI, SpeI/NotI/SfiI, SpeI/BlnI/SfiI和SpeI/NotI/SfiI, respectively. 6 enzyme combination PFGE yielded different discrimination for 4 Salmonella serotypes. The simultaneous analysis of 3 enzyme combination proved to be a beneficial scheme for the PFGE-based differentiation of closely related isolates of the four Salmonella serotypes.9,BamHI was the most suitable Enzyme for Campylobacter jejuni in PFGE subtyping. In order to get higher discrimination ability for subtyping Campylobacter jejuni , PFGE should be used with other methods together.10,Analyzing all data of PFGE subtyping, the discrimination ability of PFGE subtyping for STEC O157:H7 and Salmonella was better than Campylobacter jejuni. |
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