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Tandem amplification of a plasmid-borne tet(A) variant gene confers tigecycline resistance in Escherichia coli.

OBJECTIVES: To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene.

METHODS: E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS).

RESULTS: Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline.

CONCLUSIONS: Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.

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