| In recent years, with the increasing use of antibiotics, the resistance of bacteria is gradually enhancing, meanwhile, the speed of bacteria acquiring drug resistance is gradually increasing. Even "super bacteria" which is able to tolerate most of the antibiotics have appeared. People pay more attention to the role of antibiotics in killing pathogenic bacteria, however, the effect of the subinhibitory concentrations of antibiotics on the dissemination efficiency of drug resistance among bacteria is neglected.The aims of the study are to provide the information for evaluating the potential risk of drug resistance spread caused by artificial domestication then release of this wild pheasant, as well as for the influence of subinhibitory concentrations of antibiotics on the resistance genes horizontal spread. For the aims above, we explored the drug resistance and the status of carrying mobile genetic elements in Escherichia coli form this kind of pheasant in wild; Meanwhile, the relationship between subinhibitory concentrations of antibiotics and the drug resistance dissemination efficiency among bacteria was also explored.We collected the Syrmaticus humiae fresh feces in natural distribution region of wild Syrmaticus humiae in Tianlin county, Baise city, Guangxi provence and screened E. coli from the feces using chromogenic medium. Furtherly, the screened strains were identified by the specific primers of malic dehydrogenase gene (mdh) and verified by amplifying their 16S rRNA. Enterobacterial repetitive intergenic consensus PCR(ERIC-PCR) in combination with the software of SPSS 19.0 were used to eliminate the overlapping strains from the isolated E. coli. Kirby-Bauer method (K-B paper) was used to detect drug resistances of the isolated strains. We performed polymerase chain reaction (PCR) to detect the mobile genetic elements in the isolated strains. Then, the type and location of tetracycline resistance genes in one of the strains was detected by the PCR and in situ hybridization. Effects of different antibiotics in subinhibitory concentrations on the dissemination efficiency of drug resistance genes were explored by mixed culturing of Escherichia coli and Klebsiella oxytoca in culture medium containing different subinhibitory concentrations of antibiotics. The results are as follow:(1) In this study,100 strains of E. coli were isolated from 5 stool samples using the Escherichia coli chromogenic medium.2 strains that were not E.coli were detected in the 100 strains. This result shows that the accuracy rate of the chromogenic medium used in the study was 98%.(2) 17 different ERIC fingerprints strains were obtained by the statistical results of ERIC-PCR. And they could be divided into 4 groups at level 15 of rescaled distance cluster combine showing that the population structure of the wild Syrmaticus humiae fecal E. coli was complex.(3) The drug sensitivity test showed that the inhibitory effect of the β-lactam and tetracycline antibiotics on the isolated E.coli was poor. These strains could not be inhibited by penicillin G.11.76% of the strains were resistant to ceftriaxone, and all the strains were not sensitive to tetracycline. On the contrary, quinolones, lipopeptides and aminoglycoside antibiotics had a better effect on antibacteria. The amount of strains sensitive to ciprofloxacin and levofloxacin belonging to quinolone antibiotics accounted for 88.24% and 76.47% separately. Meanwhile, the rate of strains sensitive to polymyxin B was 82.35%.(4) In the 17 different ERIC fingerprints strains, the rate of tolerant to 4 kinds of antibiotics was 35.30%(6/17), tolerant again 3 kinds of antibiotics was 29.40%(5/17), again 2 or less kinds of antibiotics was only 35.30%(6/17). And all strains had at least an antibiotic resistance. Regardless of the natural resistance of G- to penicillin G, among all the drug-resistant strains, there was no "super bacteria"(ie bacteria tolerate 3 or more classes of the antibiotics).(5) F plasmid genetic marker traA and integron genetic marker intl were detected separately in all the 17 different ERIC fingerprints strains, and 5 different size fragments of variable region of class I integron carrying no drug resistance gene cassette were also detected. But genetic markers traF、intⅡ、intⅢ and Int-Tn were not checked out. Although the species of mobile genetic elements in the isolated stains was not various, there was potential risk of their obtain or spread of exogenous drug resistance genes.(6) NO.15 E. coli strain E15 containing doxycycline resistance gene tetB which was on the plasmid but not the F one. This gene could be disseminated to the mutant strain TR-M30-1 of Klebsiella oxytoca by co-culture method. The dissemination efficiency was positively related to the subinhibitory concentrations of kanamycin and ampicillin (P<0.05). When they were cocultured in the medium, there was no significant correlation between the dissemination efficiency and the subinhibitory concentrations of sulfamethazine, doxycycline and Cu2+. Some antibiotics in subinhibitory concentrations had certain promotion on the spread of drug resistance genes. However, when they were cocultured the simulated natural environmen, there was no significant correlation between dissemination efficiency and concentrations of the drugs above. |
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