| Since penicillin was discovered at 1940, antibiotics have a period of rapid development. A variety of antibiotics were used in clinical treatment of human as well as animal husbandry, fisheries and other fields. However, the overuse or misuse of antibiotics has resulted in an increasingly serious situation of antibiotic resistance. One of the main causes of the widespread of antimicrobial resistance is Horizontal transfer of antimicrobial resistance genes.As the common recombinant expression systems in bacteria, integron plays an important role in the spread of antimicrobial resistance. Nowadays, more than 80 different gene cassettes have been identified in integrons and most of them are antimicrobial resistance gene cassettes, which are related to antimicrobial resistance to nearly all class of antibiotics. However, the role of ISCR (Insertion sequence common region) elements which is more powerful than integrons should not be ignored. Both integrons and ISCR elements provide an efficient medium for the transfer of resistance genes.Quinolones and beta-lactam antibiotics are antimicrobial angents which are the most frequently used in clinical therapy. And, as the most common clinical opportunistic pathogens, Enterobacteriaceae may cause a variety of respiratory system infections or urinary tract infections. Therefore, antimicrobial resistant Enterobacteriaceae should be paid enough attention.In this thesis, we performed the research on the dissemination of integrons and associated gene cassettes and the mechanisms about the generation and spread of antimicrobial resistance among 463 antimicrobial resistant Enterobacteriaceae from a commercial broiler slaughter plant. It has a great influence not only on the ratioonal use of antibiotics and the decrease of the resistance level and occrence of antimicrobial isolates in animal husbandry and aquaculture industry, but also on the development and production of new antibiotics.1. Identification of Physiology groups, integron and gene cassettes of drug-resistant in Escherichia coli strainsOf 373 tested E. coli isolates,95.71%(n= 357) were multi-drug resistant. According to phylogenetic assays, E. coli strains could be classified into four phylogenetic groups (A, B1, B2, and D), of which group B1 was predominant, accounting for 33.51% of the tested isolates. A high percentage of the E. coli isolates were resistant to amoxicillin-clavulanic acid (99.20%, n= 370), doxycycline (92.23%, n= 344), sulfamethoxazole-trimethoprim (90.88%, n= 339), ciprofloxacin, (64.61%, n= 241), sulbactam-cefoperazone (51.21%, n= 191), and amikacin (33.78%, n= 126). Furthermore, among the 373 isolates, class 1 and 2 integrons were identificated in 292 (78.28%) and 49 (13.14%) isolates, respectively, while no class 3 integrons were detected.11 types of gene cassette arrays were identificated in the variable region of class 1 integrons:dfrA17-aadA5, dfrA12-orfF-aadA2, catB3-arr3, dfrA1-aadA1, aacA4-cmlA1, dfrA16-aadA2, dfrA17, arr3-dfrA27-aadA16, aacA4, aadA2 and empty. While, only one type of class 2 integrons (dfrA1-sat2-aadA1) was identified. Class 1 integrons were distributed in various physiological subtypes. The presence of class 2 integrons in the B21 subtype was significantly higher than in the other subtypes, and it coexisted with the class 1 integron.2. The idenfication of antibiotic-resistant genes and the analysis of molecular characteristics of integrons and ISCR elements in Klebsiella spp. strainsOut of the 90 Klebsiella spp. strains,96.67%(n=87) were multi-drug resistant isolates, and 87 (96.67%) were ESBL (Extended-spectrum beta-lactamase)-producing isolates. Besides,89 isolates were identified as phylogenetic group KpI which was mainly responsible for Klebsiella spp. infections. And, the other one isolates was a relatively rare K. variicola. A high percentage of the Klebsiella spp. strains were resistant to CTX (100.00%), AMP (98.89%), CHL (92.22%), KAN (91.10%), CAZ (82.22%), CIP (80.00%), TET (78.89%), CPE (73.33%) and CFP (48.89%), respectively. Furthermore, class 1 and 2 integrons were detected in 85 and 5 isolates respectively, while no class 3 integron was detected. Among 85 class 1 integron-positive isolates, three types of class 1 integrons containing different gene cassette arrays were identified:dfrA12-orfF-aadA2, dfrA17-aadA5 and empty. Only one gene cassette array (dfrAl-sat2-aadAl) was detected in the variable region of class 2 integrons. Furthermore, four different types of ISCR1-mediated downstream structures were successfully identified in 46 class 1 complex integron-positive isolates, among which ISCR1-sapA-like-qnrB2-qacEAl was the most commonly observed structure. According to the prevalence of ESBL genes, PMQR (plasmid-mediated quinolone resistance) genes and class 1 integrons, Chi-square test revealed the significant association (p<0.01) between the three kind of genes, respectively. Additional conjugation experiments confirmed this relationship (p<0.01) in transconjugants by finding that a high percentage of PMQR genes (74.02%) and class 1 integrons (73.68%) were co-transferred with ESBL genes. Furthermore, multilocus sequence typing (MLST) was performed to determine that isolates from chicken has been widely distributed in human and that antibiotic resistance is not only disseminated by clonal spreading, but largely by horizontal gene transfer. These results suggest that horizontal transfer of antibiotic resistance genes by mobile genetic elements, such as integrons, plays a major role in the spread of antibiotic resistance. |
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