AUTHOR=Zhou Kaixin , Tao Ying , Han Lizhong , Ni Yuxing , Sun Jingyong 

TITLE=Piperacillin-Tazobactam (TZP) Resistance in Escherichia coli Due to Hyperproduction of TEM-1 β-Lactamase Mediated by the Promoter Pa/Pb

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.00833

DOI=10.3389/fmicb.2019.00833

ISSN=1664-302X

ABSTRACT=<p>TEM-1, mediated by plasmid and transposon, is the most commonly encountered β-lactamase in Gram-negative bacteria. Four different promoters upstream of <italic>bla</italic><sub>TEM</sub>-related genes have been identified: the weak <italic>P3</italic> promoter, and the strong promoters <italic>Pa/Pb</italic>, <italic>P4</italic>, and <italic>P5.</italic> In this study, we investigated the genetic basis of a clinical strain of <italic>Escherichia coli</italic> (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 <italic>bla</italic><sub>TEM-1b</sub> gene with the strong promoter <italic>Pa/Pb</italic>. Different plasmids were cloned based on the clone vector pACYC184 with the insertion of the <italic>bla</italic><sub>TEM-1b</sub> gene with promoters <italic>Pa/Pb</italic> or <italic>P3</italic>. Susceptibility to TZP was determined by the <italic>E</italic>-test, agar dilution, and broth microdilution. The level of <italic>bla</italic><sub>TEM-1b</sub>-specific transcription was determined by quantitative real-time PCR. Substitution of <italic>Pa/Pb</italic> for <italic>P3</italic> resulted in a 128-fold decline of the MIC value of TZP, from &gt;1024 mg/L to 8 mg/L, and a significantly lower <italic>bla</italic><sub>TEM-1b</sub> expression level. Hyperproduction of TEM-1 β-lactamase mediated by the promoter <italic>Pa/Pb</italic> was responsible for high resistance to TZP in <italic>E. coli</italic>. Our data show possible risks of resistance development in association with the clinical use of TZP. The <italic>bla</italic><sub>TEM</sub> promoter modifications should be considered for whole genome whole-genome sequencing-inferred bacterial antimicrobial susceptibility testing.</p>