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[Objective] To investigate the mechanism of the induced cross resistance of drug-resistant mutants of Escherichia coli to tigecycline in vitro. [Methods] We used doxycycline hydrochloride and the mutation preventive concentration (MPC) method to induce the drug resistance mutation of Escherichia coli ATCC 25922, and the drug resistance spectra of the mutants were determined. Genome-wide next-generation sequencing was utilized to analyze the mutations of key differentially expressed resistance genes of ATCC 25922 and the mutant with the highest resistance index. RT-PCR was used to determine the transcription levels of the key differentially expressed resistance genes of the mutant with the highest resistance index according to the whole genome sequencing results. The expression of key differentially expressed resistance genes in the mutant with the highest resistance index was knocked down by siRNA. [Results] Three drug-resistant E. coli mutants Y3.2-2, Y64, and Y128-2 with different degrees of resistance to tigecycline were obtained after stepwise induction of drug resistance mutation, with the resistance following the order of Y3.2-2<Y64<Y128-2. All the mutants showed multi-drug resistance. Fourteen resistance genes were detected with varying degrees of base mutations and amino acid mutations. In the mutant Y128-2 with the highest resistance index, the expression of acrA, acrE, acrF, acrS, plsC, rpsJ, acrB, and macA was up-regulated, while that of tolC, marA, sdiA, and macB was down-regulated. The resistance genes rpsJ and plsC in Y128-2 were successfully interfered with at tigecycline concentrations of 1×MIC and 1/2×MIC, and the strain regained sensitivity to tigecycline. [Conclusion] Y128-2 develops resistance to tigecycline by the overexpression of the ribosome binding site gene rpsJ and the bacterial cell membrane permeability-related resistance gene plsC.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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