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. 2001 Apr;45(4):1126-36.
doi: 10.1128/AAC.45.4.1126-1136.2001.

Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes

M C Sulavik  1 , C Houseweart, C Cramer, N Jiwani, N Murgolo, J Greene, B DiDomenico, K J Shaw, G H Miller, R Hare, G Shimer
Affiliations

Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes

M C Sulavik et al. Antimicrob Agents Chemother. 2001 Apr.

Abstract

The contribution of seven known and nine predicted genes or operons associated with multidrug resistance to the susceptibility of Escherichia coli W3110 was assessed for 20 different classes of antimicrobial compounds that include antibiotics, antiseptics, detergents, and dyes. Strains were constructed with deletions for genes in the major facilitator superfamily, the resistance nodulation-cell division family, the small multidrug resistance family, the ATP-binding cassette family, and outer membrane factors. The agar dilution MICs of 35 compounds were determined for strains with deletions for multidrug resistance (MDR) pumps. Deletions in acrAB or tolC resulted in increased susceptibilities to the majority of compounds tested. The remaining MDR pump gene deletions resulted in increased susceptibilities to far fewer compounds. The results identify which MDR pumps contribute to intrinsic resistance under the conditions tested and supply practical information useful for designing sensitive assay strains for cell-based screening of antibacterial compounds.

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Figures

FIG. 1
FIG. 1
Location of primers for (i) the design of the overlap extension method and (ii) for verification of allelic exchange. Three amplification products were constructed into one product by an overlap extension method for subsequent use in MDR pump gene replacement with the Kmr-FRT fragment. Primers 1A and 1B amplify Fragment 1 from the chromosome. Primers 2A and 2B amplify the Kmr-FRT fragment from pCP15. Primers 3A and 3B amplify Fragment 3 from the chromosome. Primers “outside A” and “outside B” amplify chromosomal DNA to verify those strains for which the Kmr-FRT fragment has replaced the MDR pump gene.
FIG. 2
FIG. 2
Evidence for successful allelic exchange of the Kmr-FRT determinant for the genes or operons used in this study, as assessed by ethidium bromide-stained PCR products following electrophoresis in a 0.7% agarose gel. PCRs using oligonucleotides located outside the operons deleted (outside primers in Table 1) were used with genomic DNA templates of the wild type (W3110) or of the specific mutant Kmr strain. PCR products were amplified using tolC primers with genomic W3110 (lane 1) and with HS151 (lane 2); yjcP primers with W3110 (lane 3) and HS154 (lane 4); yohG primers with W3110 (lane 5) and HS157 (lane 6); ylcB primers with W3110 (lane 7) and HS208 (lane 8); acrAB primers with W3110 (lane 9) and HS832 (lane 10); acrEF primers with W3110 (lane 11) and HS212 (lane 12); yhiUV primers with W3110 (lane 13) and HS193 (lane 14); acrD primers with W3110 (lane 15) and HS215 (lane 16); b2074yegNO primers with W3110 (lane 17) and HS199 (lane 18); emrD primers with W3110 (lane 19) and HS196 (lane 20); mdfA primers with W3110 (lane 21) and HS221 (lane 22); emrAB primers with W3110 (lane 23) and HS833 (lane 24); emrKY primers with W3110 (lane 25) and HS205 (lane 26); emrE primers with W3110 (lane 27) and HS218 (lane 28); tehAB primers with W3110 (lane 29) and HS202 (lane 30); and b0878ybjC primers with W3110 (lane 31) and HS273 (lane 32). The two tehAB 5.5-kbp bands seen for both wild-type and mutant strains in lanes 29 and 30 do not distinguish mutant from wild-type alleles. The wild-type product was distinguished from the ΔtehAB::Kmr PCR product upon digestion with HindII; the ΔtehAB::Kmr PCR product seen in lane 30 was digested to two fragments of size 2 and 3.5 kbp, whereas the wild-type product seen in lane 29 remained a 5.5-kbp undigested product as expected (data not shown).
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