| Summary: | 碩士 === 國立中興大學 === 食品科學系 === 91 === Abstract
Pathogens Escherichia coli which have several virulent factors like heat-labile toxin (LT), heat-stable toxin (ST), shiga-like toxin (SLT) and attaching mechanism (eae) is one of the most important microorganisms causing foodborne illness worldwide. When human was infected with pathogens E. coli, it is potentially fatal and may be associated with serious complication symptoms such as hemolytic-uremic syndrome (HUS), porcine diarrhoea. The curing of these infections are traditionally treated with antibiotics. However, in the past few years, many E. coli strains isolated from human and animals also have seen the development of antibiotic resistance, and multidrug resistance in human and veterinary medicine. For pathogenic bacteria strains, their drug resistance may be obtained by mutation or the transfer of plasmid or transposon. Recently, for Gram negative bacteria, integrons (class I, II, III and IV) which contain the antibiotic resistant genes have be reported. Of them, class I integron is most important. The integrons are mobile DNA elements with a specific structure which acquires or exchanges the antibiotic resistance genes. In recent years, we have collected hundreds of E. coli strains from both the human and animal origins. This study was focused on the analyze of the toxin types, antibiotic susceptibility pattern, and integron of each E. coli strain.
In order to identify the toxin types of 158 E. coli isolates from human and animal origins in Taiwan, the malate dehydrogenase (mdh) gene and several toxin related genes were investigated. The gene was amplified by primer set , i.e. Emdh 1/2, LT I 1/2, LTI 51/31, ST II 1/2, ST Ia 1/2, SLT I 5/3, SLT II 5/3, SK1/2, INV5/3, BFP1/2. For examples, 57 strains (72%) of 79 E. coli human isolates were identified as ETEC; included 31 strains (39%) were LTI toxin, 2 strains (3%) were STII toxin, 9 strains were STIa toxin, 3 strains (4%) were LTI and STII toxins, and 12 strains (15%) were LTI and STIa toxins. From the porcine isolates, i.e., 46 strains (58%) were identified as ETEC; included 9 strains (11%) were of LTI toxin, 9 strains (11%) were STII toxin, 5 strains (6%) were STIa toxin, 2 strains (3%) were LTI and STII toxins, 16 strains (20%) of STII, STIa toxins, and 5 strains (6%) both have the STII, LTI, STIa. One (1%) of 158 isolates was STEC-SLTII toxins.The major type from E. coli human isolates origin in Taiwan is ETEC-LTI type (20%), and the major type from E. coli porcine isolates is ETEC-STIa-STII (10%).
Furthermore, the use of the antibiograms to realize the drugs resistant of E. coli isolates from human and porcine origins. Results demonstrated that both human and porcine origins isolated E. coli strains resistant to multi-drugs, especially from porcine origin (ie., Fluoroquinolones). The major antibiogram type from human isolated is AmCfKGS3SxtETeCb, and the major antibiogram from porcine isolated is AmGmNaETeOtMyNAprSpt. All strains resisted to the first line antibiotics, i.e., cephalothin, erythromycin, and lincomucin. All strains had less resistant to the second and third generation antibiotics, such as cefmetazole (3%) and ceftazidime (5%). Strains had already resistance to fluoroquinolone-like drugs, ie., norfloxacin (22%), ofloxacin (23%), and ciprofloxacin (30%). In addition, E. coli from porcine isolates had still resisted to the banned drugs at a high rate.
In the detection of integron genes for pathogens E. coli isolates, the conserved primers 5’-CS/3’-CS and Int1F/3’-CS were used. The amplification results were 1380 bp,1500 bp, and 2500 bp. 106 strains (67%) had the three amplicons. 61% strains were amplified the 2500 bp products, and 6% strains were amplified two integron products. DNA sequencing analysis showed that the 1380 bp integron PCR product carried the sat-1 gene which resist to streptothricin, 1500 bp integron PCR product carried the aadA gene which responsible to the streptomycin-spectinomycin, and the 2500 bp integron PCR product was carried the dfrA12-orfF-aadA2 gene which resist to the streptomycin-spectinomycin and trimethoprim. Besides, we detected the int gene using Int1F/Int1R primers to confirm the present of integron. Data showed that 134 strains (85%) have the int gene. In addition, the integron related strains were highly resistant to antibiotic drugs.
Multiplex resistance was often found to erythromycin, lincomycin, and sulfisoxazole. These isolates resistant to fluoroquinolones, and the third-cephalothin, i.e. 106 of the 158 (67%) E. coli isolates were found to carry class 1 integrons. The amplified integron fragments are 1380 bp,1500 bp, and 2500 bp. Class 1 integrase gene were also detected, i.e. 85% (134/158) of E. coli isolates. Integrons were strongly associated with multiple-antibiotic-resistant strains, and integron-positive strains, it is demonstrating a greater predilection for antibiotic resistance than integron-native strains.
The data would be useful for developing the monitoring and prevention systems for these E. coli pathogens. In addition, the DNA sequences of integron could be used as the development of biochips, specifilly for the detection of integron related drug-resistant genes in the food pathogens.
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