Abstract
TEM-1, mediated by plasmid and transposon, is the most commonly encountered β-lactamase in Gram-negative bacteria. Four different promoters upstream of blaTEM-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 blaTEM-1b gene with the strong promoter Pa/Pb. Different plasmids were cloned based on the clone vector pACYC184 with the insertion of the blaTEM-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 blaTEM-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 blaTEM-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 blaTEM promoter modifications should be considered for whole genome whole-genome sequencing-inferred bacterial antimicrobial susceptibility testing.
Highlights
The production of β-lactamases is the predominant cause of resistance to β-lactam antibiotics in Gram-negative bacteria (Bonnet, 2004), including the hyperproduction of plasmid-mediated TEM1 β-lactamases, production of extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC enzymes (Caroff et al, 1999) and carbapenem-hydrolyzing β-lactamasesTEM-1 β-Lactamase Hyperproduction and PTZ Resistance (Wu et al, 1994; Jacoby and Munoz-Price, 2005)
The short gun method was used to screen out a strain that was highly resistant to TZP, and sequence analysis revealed that the plasmid harbored a 3.9kb insertion embedded in the blaTEM−1b gene with the strong promoter Pa/Pb
Of TZP resistance was due to the hyperproduction of TEM-1b β-lactamases mediated by the strong promoter Pa/Pb
Summary
The production of β-lactamases is the predominant cause of resistance to β-lactam antibiotics in Gram-negative bacteria (Bonnet, 2004), including the hyperproduction of plasmid-mediated TEM1 β-lactamases, production of extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC enzymes (Caroff et al, 1999) and carbapenem-hydrolyzing β-lactamases (carbapenemases)TEM-1 β-Lactamase Hyperproduction and PTZ Resistance (Wu et al, 1994; Jacoby and Munoz-Price, 2005). The production of β-lactamases is the predominant cause of resistance to β-lactam antibiotics in Gram-negative bacteria (Bonnet, 2004), including the hyperproduction of plasmid-mediated TEM1 β-lactamases, production of extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC enzymes (Caroff et al, 1999) and carbapenem-hydrolyzing β-lactamases (carbapenemases). The subgroups were defined and designated a, b, and c for a given blaTEM gene derivative, because of their relation to a certain number of nucleotide differences in their structural gene sequence (Leflon-Guibout et al, 2000). BlaTEM−1c gene differs from blaTEM−1a by the nucleotide substitution C436T, which is silent. P3 corresponds to the promoter of the blaTEM gene located in a Tn2 or Tn3 transposon (Sutcliffe, 1978; Lartigue et al, 2002; Partridge and Hall, 2005). An updated blaTEM gene nomenclature was proposed on the basis of the sequences of structural blaTEM genes and their promoters (Goussard and Courvalin, 1999)
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