| It is the major mechanism of resistance to the third generation cephalosporins that the bacteria could produce extended spectrum beta-lactamases(ESBLs). The major ESBL producer is E.coli worldwide. The ESBLs contain many genotypes(for example TEM, SHV, CTX-M, et al), recently the CTX-M genotype has been the most popular ones in the world. The purpose of this experiment is to explore the genetic environment and background of bla CTX-M of different subtypes in different sources. It is based on to detect the molecular epidemiological characteristics of CTX-M-type ESBLs of chicken E.coli from different sources wihich isolated from healthy chicken in retail and diseased chicken livers which dead suspected by E.coli, The results will provide a method to prevention and control disseminations and infections of CTX-M-producing chicken E.coli.The MICs of ampicillin, ceftiofur, cefotaxime, gentamycin, amikacin, doxycycline, florfenicol and enrofloxacin in 106 chicken E.col(18 isolates of chicken and 88 isolates of diseased) were determined by a broth microdilution method. The result showed that the isolates from different sources have similar characteristics at drug resistance and the drug resistance rates in both of them range from 18.2% to 94.4%. The results of ESBLs of the 76 isolates, third-generation Cephalosporins resistant avian E.coli, showed that five of ten chicken isolates carried bla CTX-M and the detection rate was 50%, four enzyme-producing strains carrying CTX-M of which belonged to CTX-M-9 subgroup(three bla CTX-M-14 and one bla CTX-M-65), one of which belonged to CTX-M-1 subgroup(one bla CTX-M-55). Sixty-three of sixty-six isolates of diseased carried bla CTX-M and the detection rate was 95%, 25 of which belonged to CTX-M-9 subgroup(twenty-one bla CTX-M-65 and four bla CTX-M-14), twenty-three of which were CTX-M-1 subgroup(twenty-two bla CTX-M-55, one bla CTX-M-15), seven of which were two different subgroup hybrid(four bla CTX-M-64 and three bla CTX-M-123). Besides, the specific gene subtype of the rest eight strains have not yet been determined. In a word, the E.coli mainly belonged to CTX-M-1-type and CTX-M-9-type extended-spectrum-lactamases.Upstream and downstream of bla CTX-M genes from different subgroups were analysed by PCR mapping. Results showed that upstream of bla CTX-M genes from different subgroups all containing ISEcp1, while different insertion sequence in downstream where ORF477 was identified downstream of bla CTX-M-1, bla CTX-M-64 and bla CTX-M-123. and IS903 was identified downstream of bla CTX-M-9. Furthermore, the Results showed that, ISEcp1 was truncated by other insertion sequences in 80.5% of bla CTX-M-9 isolates, while only 12.5% of ISEcp1 in bla CTX-M-1 isolates were truncated by IS26, It illustrated that it is easier to truncated by IS26, IS10 and other elements for the upstream and downstream of bla CTX-M-9, and it is more complex of the structure for downstream insertion sequence in which the ends of IS903 was also containing a inverted repeats(IRL) of 18 bp length.Results of plasmid incompatibility group showed that, 13 kinds of plasmid replicon types were identified in these tested isolates, in which 67(91.8%, 67/73) were not less than three kind. Different CTX-M-types carrying plasmid replicon types are basically the same, and no significant difference in carrying plasmid replicon between chicken isolates and diseased isolates. The detection rate of Inc FII, Inc K and Inc FIB were highest which up from 82% to 86%, and the replicons of Inc X, Inc L/M, Inc W, Inc Y and Inc Frep B were not identified in both of them. It is illustrated that the majority were approved by carrying multiple plasmid replicons, which help to improve the host adaptability of plasmids and the spread of resistance genes. But it is obvious difference in carrying replicon types between 13 isolates of previous diseased saved and 55 isolates of new diseased, in which Inc FIA, Inc A/C and Inc T were only detected in diseased isolates, while the detection rate of Inc I1 and Inc B was significantly higher in the former. Results of plasmid conjugation test showed that, 63.0%(46/73) isolates of CTX-M-producing could be successfully delivered plasmid which resistance to third-generation cephalosporin to the recipient isolate J53, in which five isolates of chicken and nine of thirteen isolates of previous diseased and thirty-two of fifty-five isolates of new diseased were successfully transmited their plasmid. No significant difference among the three. Explained that transconjuganted plasmid has played an important role in spread of bla CTX-M.Results of phylogenetic group showed that the percent of 73 isolates of bla CTX-M resistant isolates for four phylogenetic Group A, B1, B2 and D is respectively 31.5%, 46.6%, 4.11% and 17.8%, while the percent of 20 isolates of non-ESBLs sensitive isolates is respectively 30%, 65%, 0% and 5%. Among these tested isolates, there were 21.9% of them with virulence in which CTX-M-producing, While only 5% of non-ESBLs susceptible isolates having virulence, and highly pathogenic strains appeared only in CTX-M-producing isolates, indicating that the virulence in CTX-M-producing of chicken E.coli might be stronger than isolates of non-ESBLs. However, it need further tests to be confirmed. MLST analysis results revealed that 73 bla CTX-M isolates belonged to 26 different STs, while 20 non-ESBLs isolates belonged to 10 different STs. ST155 and ST359 are both detected in bla CTX-M isolates and none ESBLs isolates, and ST155 in the detection rate of two types isolates are both the first. 10 new ST types were detected in this study, in which two new alleles including fum C612 of ST4753 and mdh382 of ST4752 were detected and their related sequences has also been deposited in MLST database. It not only illustrated the complexity genetic relationships of these tested isolates which belonging to different clones and its rapid mutation rate, but also showed that cloning only played a minor role in the dissemination of CTX-M clusters.In summary, the main CTX-M clusters were CTX-M-1 and CTX-M-9 cluster of Henan province, and a few hybrid variants were identified. It was complex of genetic relationships in CTX-M-producing isolates which sources also diversified; Insertion sequence ISEcp1 and conjugative plasmid played a key role in the dissemination of CTX-M clusters, while the cloning only played a minor role. |
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