Expression analysis of the native ampC gene in Norwegian clinical isolates of Escherichia coli with defined ampC promoter mutations and an AmpC-resistance profile
Abstract number: P1222
Aasnæs B., Haldorsen B., Hegstad Dahl K., Skov Simonsen G., Sundsfjord A., Lundblad E.
Objective: The objective was to examine native ampC mRNA levels by quantitative RT-PCR (qRT-PCR) in Norwegian clinical isolates of Escherichia coli with defined ampC promoter-attenuator mutations and relate those to minimum inhibition concentrations (MICs) of cefoxitin, third generation cephalosporins, piperacillin-tazobactam, and aztreonam.
Methods: The Norwegian Reference Centre for Detection of Antimicrobial Resistance (K-res) received 106 clinical isolates of E. coli from different Norwegian laboratories with reduced susceptibility to third-generation cephalosporins without clavulanic acid synergy, from 2003 to 2005. Of these, twelve were non-plasmid mediated blaampC strains, with defined ampC promoter and attenuator alterations compared to the E. coli wild-type ampC promoter-attenuator. Expression of the native ampC gene was examined by qRT-PCR. ampC mRNA levels, normalised to the expression of the reference gene gapA, were compared to those in six cefoxitin-susceptible isolates of E. coli, including ATCC 25922. MICs were determined by Etest.
Results: The 12 isolates showed a 27 to 380-fold increase in ampC mRNA levels compared to the ampC mRNA level in E. coli ATCC 25922. The ampC mRNA levels varied within the different promoter-attenuator mutation groups. A single strain had an ISEc10 element inserted between the -35 and -10 boxes, providing an almost perfect E. coli-35 box 17 bp upstream of the endogenous -10 box. This isolate showed a 54-fold increase in ampC mRNA level. The ampC mRNA levels in the five cefoxitin-susceptible clinical isolates of E. coli varied from 0.7 to 2.2 fold, suggesting that the mutations observed in these isolates have no or minor effect on transcription of the ampC gene. For the isolates with reduced susceptibility to third-generation cephalosporins we did not observe any significant correlation between ampC mRNA levels and MICs of FOX, CPD, CTX, CAZ, TZP, and ATM.
Conclusions: (i) Clinical isolates of E. coli with defined ampC promoter-attenuator alterations overexpressed ampC 27380-fold compared to E. coli ATCC 25922. (ii) We did not find any significant correlation between ampC mRNA levels and MICs of cefoxitin, third generation cephalosporins, aztreonam and piperacillin-tazobactam.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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