A-027. Single Nucleotide Polymorphisms in the Major Multidrug Efflux Pump, AcrB, of Escherichia coli Isolated from Disparate Animal Reservoirs

L. B. Mullis, C. A. Elkins;
US FDA, Natl. Ctr. for Toxicological Res., Jefferson, AR.

The AcrAB-TolC tripartite efflux pump system of Escherichia coli conveys a strong intrinsic resistance phenotype to a variety of clinically significant molecules. AcrB serves as the pump proper and imparts substrate specificity via two periplasmic loop regions. We assessed the nucleotide sequences of these regions in isolates from two distinct reservoirs to determine the natural plasticity of this gene for potential drug resistance development. E. coli were isolated from nasopharyngeal swabs and intestinal contents of in-house laboratory rodent colonies. Isolates resistant to ceftiofur (a third generation cephalosporin shown here to be a substrate of this efflux system) from fecal samples of dairy calves in Pennsylvania were kindly provided by Dr. Bhushan M. Jayarao (Penn. State Univ.). Thus far, the sequences from a total of 22 strains (7 rodent and 15 clonally distinct bovine isolates) have been obtained and compared with the respective E. coli K-12 sequence. Only four of the bovine but none of the rodent isolates completely matched the K-12 acrB loop domains. A total of 14 and 15 nucleotide variations were observed in loop 1 and loop 2 of acrB, respectively, whilst 7 and 11 of these respective variations were conserved in several members of both isolate pools. Interestingly in all but three cases, the polymorphisms were silent and lacked any predicted amino acid and, hence, functional change. Those resulting in amino acid change were located in loop 2 but were not exclusively confined to one isolate pool: L591H (rodent) and Q733R (two independent instances, bovine). Apart from ceftiofur, MICs of select bovine isolates (including mutants in loop 2) revealed increases in resistance to AMP, CHL, KAN, TET, GEN, and STR when compared to rodent isolates. Both pools exhibited a mixed response to CIP and little difference in resistance to ERY, NOV, EtBr, crystal violet, and bile acids. Further studies of the L591H and Q733R “natural” mutants are planned to determine the significance in drug substrate recognition and relationship to resistance phenotype(s). Our results suggest that efflux pumps may be subject to some mutation-based selective pressure in addition to previously shown expression-based adaptations.