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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 ΔTn As1 -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 ΔTn As1 -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. [ABSTRACT FROM AUTHOR]

To investigate the prevalence of a tet (A) gene variant and its role in developing high-level tigecycline resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates. The mechanism of high-level tigecycline resistance in CRKP mediated by a tet (A) variant was explored by induction experiments, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis. The amplification and overexpression of the tet (A) variant were measured by the determination of sequencing depth, gene copy numbers, and qRT-PCR. A high rate (62.1%, 998/1607) of tet (A) variant carriage was observed among 1607 CRKP clinical isolates from Henan Province, China. High-level tigecycline resistance could rapidly develop by the amplification of the tet (A) variant in these isolates. The analysis of the raw sequencing data and the plasmid mapping depth revealed that the Δ tnpA homologous sequence of Tn 1721 supports the amplification of the region that harbours the tet (A) variant by forming a large number of repeat arrays through translocatable units (TUs). Moreover, the epidemiological analysis of tet (A) variant-carrying structures among 1607 clinical CRKPs showed that the TU structure is widely present. The presence of a tigecycline resistance-mediating tet (A) variant in CRKP clinical isolates represents a greater health concern than initially thought and should be monitored consistently. [ABSTRACT FROM AUTHOR]