TEM-1, mediated by plasmid and transposon, is the most commonly encountered β-lactamase in Gram-negative bacteria. Four different promoters upstream of bla TEM-related genes have been identified: the weak P3 promoter, and the strong promoters Pa/Pb, P4, and P5. In this study, we investigated the genetic basis of a clinical strain of Escherichia coli (RJ904), which was found to be resistant to BLBLIs (β-lactam/β-lactamase inhibitors), including amoxicillin-clavulanate, ticarcillin-clavulanate (TCC), and piperacillin-tazobactam (TZP) but sensitive to third-generation cephalosporins. The conjugation test and S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) demonstrated that transfer of this resistance was mediated by a ca. 100 kb plasmid. The transformant with TZP resistance was screened out with the shortgun cloning. Sequence analysis revealed that the recombinant plasmid contained a bla TEM-1b gene with the strong promoter Pa/Pb. Different plasmids were cloned based on the clone vector pACYC184 with the insertion of the bla TEM-1b gene with promoters Pa/Pb or P3. Susceptibility to TZP was determined by the E-test, agar dilution, and broth microdilution. The level of bla TEM-1b-specific transcription was determined by quantitative real-time PCR. Substitution of Pa/Pb for P3 resulted in a 128-fold decline of the MIC value of TZP, from >1024 mg/L to 8 mg/L, and a significantly lower bla TEM-1b expression level. Hyperproduction of TEM-1 β-lactamase mediated by the promoter Pa/Pb was responsible for high resistance to TZP in E. coli. Our data show possible risks of resistance development in association with the clinical use of TZP. The bla TEM promoter modifications should be considered for whole genome whole-genome sequencing-inferred bacterial antimicrobial susceptibility testing.
Keywords: Escherichia coli; Pa/Pb; TZP resistance; antimicobial; β-lactamase.