| Avian colibacillosis is frequent primary or sequential bacterial disease in domestic poultry, which can result in the significant threats and economic loss. Antimicrobials play a very important role in controlling diseases caused by E. coli. At present, overwhelming majority of the research data about avian colibacillosis focus on the chickens and pigs. However, as a country with the biggest number of raised ducks in the world, along with the wide use of antibiotics, there exists significant increasing tendency of multi-resistance in E. coli originated from ducks. The multi-resistance in E. coli has raised several concerns related to human health and the development of poultry industry. So far, systematic research data about resistance in E. coli originated from ducks remains lack of. Therefore, it is necessary to investigate the multi-resistance phenotypes, and the relationship between the presence of specific and nonspecific mechanism and their individual contribution to the multi-resistance in E. coli originated from ducks, which has the significance in exploring the mechanism of resistance development and its regulation, and in controlling further resistance development and even the development of the novel drugs.1. 108 ducks E. coli isolates were identified with the Vitek-32 automatic microbe system, which were isolated from different duck farms located in Henan, Fujian, Guangdong, Liaoning, Anhui, and Shandong province. The MDR profiles of 108 clinical isolates were determined using the microbroth dilution method as the CLSI recommended. 19 antimicrobial agents or its combinations consisted of fluoroquinolones ,β-lactam antibiotics, aminoglycosides, tetracyclines, chloramphenicols, etc. The results showed that, 62.0%～72.2% of E. coli isolates presented resistance to fluoroquinolones, 31.5%～64.8% to third generation cephalosporins, 92.6% to classicalβ-lactam, and 41.7%～70.4% to the combinations of classicalβ-lactam and enzyme inhibitor. However, only imipenem belonging to the carbapenems is exceptional, which all isolates were susceptible to. As for the resistance profiles, 96.3% of isolates were resistant to more than 7 antimicrobial agents, just 4 strains resistant to 1～5 antimicrobial agents. As for the regional difference, the isolates in Henan and Shandong province were more resistant than those in other districts. The ESBLs detection showed that 36.1%(39/108)of isolates were ESBL-positive, the resistant rates of ESBL-producing isolates to third generation cephalosporins, fluoroquinolones were 33.1%～89.7% and 74.4%～84.6%, respectively. However, the resistant rate of non-ESBL-producing isolates to the above antimicrobials were 30.4%～37.7% and 53.6%～62.3%, respectively.2. To explore whether active efflux mechanism exist in the clinical E.coli or not, the change of MIC of doxycycline, Levofloxacin were detected with or without CCCP and reserpine. The result showed that if the efflux inhibitor was reserpine, the positive rate of efflux phenotype of the isolates were 55.6%、24.1%, with regard to CCCP, the positive rate were 45.4%、41.7%. All of the clinical isolates can detected acrA、acrB、tolC gene. To analysis the relationship between drug-resistant degree and the expression level of active efflux system, the mRNA expression level of acrA, acrB, acrD, acre, acrF, mdtA were determined by real time fluorescence quantitative PCR of different E.coli. The result showed that the mRNA expression level of above genes and drug-resistant degree are positive correlation, the relative expression level of above genes in 3 high level drug-resistant strains were 2.88～4.53, 2.28～2.63, 10.11～24.26, 6.22～11.93, 7.03～11.26, 2.90～4.00. The relative expression level of above genes in 3 middle level drug-resistant strains were higher than the expression of ATCC25922, the expression of susceptive strain was similar to that of ATCC25922.3. A series of similar methods described in part 2 were adopted to determine the relative expression levels of active efflux system regulatory genes marA, soxS and robA in clinical E. coli isolates with different degree of resistance, the results showed that the relative expression level of marA and soxS gene have a positive correlation with the drug-resistant degree in clinical E. coli isolates. The relative expression quantities of marA gene in 3 isolates with high level of resistance were 2.58, 2.83 and 1.85, respectively, while soxS gene 3.89, 3.05 and 2.10, respectively. However, the expression level of robA gene was lower than that in the standard strain ATCC25922.4. O73 strains were induced in broth containing amikacin, ciprofloxacin or ceftriaxone with 1/2MIC subinhibitory concentrations to the 30th generation. The results showed that under the pressure of the above drugs, the O73 (standard susceptible strain) could evolve into multiple drug-resistant strains gradually. The induced resistance was most easily produced when ceftriaxone acted as an inducer. The susceptible strain could firstly produce resistant to amoxicillin, levofloxacin, ciprofloxacin and doxycycline, then to florfenicol, gatifloxacin, amikacin and ampicillin, and finally to ceftriaxone, ceftiofur, cefotaxime and fosfomycin. Cefepime and neomycin were the slowest to generate induced resistance. Imipenem couldn’t generate induced resistance by the above three antimicrobial inducer. The resistant genes were detected by PCR, and the results showed that TEM, OXA-1, rmtB, qnrD, qnrS and aac(6’)-Ib-cr genes were located in partial induced strains.The expression level of the active efflux genes in most of induce-strains increased compared with the parent strain, except amikacin-induced strain in 20th generation. Under the influence of the drug inducer, the expression level of marA and SoxS declined firstly, and then rose. The mRNA expression level of marA in CIP20 was highest, which was 2.30 times compared with the parent strain. Other strains with the higher expression level of marA than that in the parent were CIP10, CRO30, CIP30 and AMK20; The expression level of soxS gene in AMK20 was the highest, which was 5.98 times that in parent strain. The expression level of other induced strains were also higher than the parent strain, except CRO10 and AMK30, which were slightly lower than that in parent strain; The expression of robA gene in all the induced strains decreased compared with the parent strain, except CIP10, which was higher than that in the parent strain.The mutation in bases and amino acids of acrA, acrB and tolC could happen under the induction of the above antimicrobials, but the deletion of these genes couldn’t. Sequencing analysis revealed that there was one base mutation in the nucleotide sequence of acrA gene after induction with ciprofloxacin. It had seven mutations at the 10th generation after induction with ceftriaxone, of them there was also one amino acid substitution T103A. One base mutation could also occur in the 20th and 30th generation, which was same when induced with ciprofloxacin; As for the gene acrB, 10 base mutations and one amino acid substitution occurred in its nucleotide sequence after induction with amikacin. It has the same 10 base mutations in the 10th, 20th and 30th generation if induced with ciprofloxacin. Interestingly, it had the identical base mutation in the 20th and 30th generation whether induced with ceftriaxone or with ciprofloxacin. But it had different base mutation site in the 10th generation; As for the gene tolC, there were 9 base mutations in the 20th and 30th generation after induction with amikacin, and only 4 with ciprofloxacin. There were seven base mutations and one amino acid substitution A245T in the 10th generation when induced with ceftriaxone, but the base mutation site is same in the 20th and 30th whether induced with ceftriaxone or with ciprofloxacin. |
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