Search

OBJECTIVES: Knowledge about the regulatory mechanisms of CTX-M β-lactamase-encoding genes in Escherichia coli is limited. The objectives of this study were to determine the growth response of CTX-M-1-producing E. coli exposed to cefotaxime and to investigate how blaCTX-M-1 expression at mRNA and protein levels is influenced by cefotaxime concentration, growth phase and gene location (chromosome versus plasmid).METHODS: Two isogenic E. coli strains, MG1655/CTX-M-1 and MG1655/IncI1/CTX-M-1, containing blaCTX-M-1 on the chromosome and on a wild-type IncI1 plasmid, respectively, were constructed and the MIC of cefotaxime was determined. Growth of the two strains was studied in the presence of increasing concentrations of cefotaxime ranging from 0 to 512 mg/L. The levels of mRNA and protein in different growth phases and at different cefotaxime concentrations were studied by qPCR and selected-reaction-monitoring MS, respectively.RESULTS: The MICs of cefotaxime were 168 and 252 mg/L for MG1655/CTX-M-1 and MG1655/IncI1/CTX-M-1, respectively. Both strains displayed a prolonged lag phase when exposed to cefotaxime. The mRNA of blaCTX-M-1 and CTX-M-1 protein levels increased in the presence of high cefotaxime concentrations and varied with growth phase. Higher mRNA expression levels were detected for MG1655/CTX-M-1 compared with MG1655/IncI1/CTX-M-1, but a higher protein level was found for MG1655/IncI1/CTX-M-1 compared with MG1655/CTX-M-1, the latter corresponding well with the higher MIC for this strain.CONCLUSIONS: blaCTX-M-1 mRNA expression and CTX-M-1 protein levels were dependent on cefotaxime concentration, growth phase and gene location. These results provide insight into the expression of cephalosporin resistance in CTX-M-1-producing E. coli, improving our understanding of the relationship between antimicrobial therapy and the expression of resistance mechanisms.

BACKGROUND: β-lactams target the peptidoglycan layer in the bacterial cell wall and most β-lactam antibiotics cause filamentation in susceptible Gram-negative bacteria at low concentrations. The objective was to determine the initial morphological response of cephalosporin resistant CTX-M-1-producing E. coli to cefotaxime and to determine whether the response depended on the growth phase of the bacterium and the concentration of antibiotic.RESULTS: Two antibiotic resistant strains carrying bla CTX-M-1 on the chromosome and on an IncI1 plasmid and three sensitive strains were used in this study. The resistant strains displayed elongated cells when exposed to cefotaxime at sub-inhibitory as well as therapeutic concentrations (1 to 512 mg/L of cefotaxime) in both lag and early exponential phase, suggesting that the elongation was an initial response mechanism to the antibiotic. Normal sized cells were the dominant cell type in exponential and stationary growth phase. No elongated cells were seen in cultures without cefotaxime. In cultures with high concentrations of cefotaxime (128-512 mg/L), no growth other than initial filamentation was observed, but spheroplats appeared after 14-17 hours in cultures of the resistant strains. Filaments were also observed in sensitive control strains with sub-inhibitory concentrations of cefotaxime.CONCLUSIONS: We showed that E. coli resistant to β-lactams by an extended-spectrum β-lactamase, bla CTX-M-1, produced filaments when exposed to cefotaxime. The filament formation was restricted to early growth phases and the time the cells grew as filaments was antibiotic concentration dependent. This indicates that antibiotic resistant E. coli undergo the same morphological changes as sensitive bacteria in the presence of β-lactam antibiotic. It was showed that the filament formation was an initial response to the antibiotics.