A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli.
OBJECTIVE
Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR.


METHODS
The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format.


RESULTS
The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins.


CONCLUSIONS
The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.
Coldham
Nick G
NG
Department of Food and Environmental Safety, Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB, UK. n.g.coldham@vla.defra.gsi.gov.uk
Webber
Mark
M
Woodward
Martin J
MJ
Piddock
Laura J V
LJ
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001562:Benzimidazoles / Q000378:metabolism; D002463:Cell Membrane Permeability; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D017353:Gene Deletion; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests / Q000379:methods; D012486:Salmonella typhimurium / Q000187:drug effects* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001562:Benzimidazoles; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; C017807:bisbenzimide ethoxide trihydrochloride