Expression of multidrug efflux pump genes acrAB-tolC, mdfA, and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance

Abstract

In a single quantitative study, we measured acrA, acrB, tolC, mdfA, and norE expression in Escherichia coli clinical isolates by using real-time PCR. acrA and acrB overexpression strongly correlated with fluoroquinolone and multidrug resistance; tolC, mdfA, and norE expression did not. The order of abundance of efflux pump transcripts in all fluoroquinolone-susceptible isolates was tolC (highest), then acrA and acrB, and then mdfA and norE. Our findings suggest acrAB overexpression is an indicator of multidrug resistance.

FIG. 1.

Multidrug efflux pump expression in E. coli clinical isolates. Transcript levels of acrA (A), acrB (B), tolC (C), mdfA (D) and norE (E) were determined by qPCR and are shown normalized to their expression in the standard E. coli strain ATCC 25922, which had a norfloxacin MIC of 0.032 μg/ml. The housekeeping gene rpsL was used to calculate relative expression. Data are displayed relative to the MIC (μg/ml) of the historically relevant fluoroquinolone norfloxacin as measured in our laboratory. Isolates were classified as either susceptible (⋄) or resistant (×) to fluoroquinolones as determined by the hospital. Each point is the average of three experiments. Lines represent the average relative expression values for all of the isolates in the norfloxacin-susceptible (⋄) and -resistant (×) groups. Overexpression was defined as greater than two standard deviations above the mean for the 24 fluoroquinolone-susceptible isolates.

FIG. 2.

Correlation of efflux pump expression levels. For each isolate, the transcript levels of acrB and acrA (A) and tolC and acrA (B) were plotted. Clinical isolates were either susceptible (⋄) or resistant (×) to fluoroquinolones as determined by the hospital. In panel A, isolate ELZ4033 (arrow) was determined to be an outlier by the extreme studentized deviate test statistic and was removed from the best-fit regression. The arrows in panel B denote isolates ELZ4000 and ELZ4001, which significantly overexpressed acrA and tolC relative to the fluoroquinolone-susceptible isolates (P < 0.05).