A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli
A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli
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DOI：
10.1093/jac/dkq169
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发表时间：
2010-08-01
影响因子：
5.2
通讯作者：
Piddock, Laura J. V.
中科院分区：
文献类型：
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作者：
Coldham, Nick G.;Webber, Mark;Piddock, Laura J. V.
大肠杆菌和肠道沙门氏菌的多重耐药（MAR）突变体的特征是对几种不相关的抗生素、杀生物剂和其他外源性物质的敏感性降低。本研究建立了一种快速检测MAR的方法，用Hoechst（H）33342荧光探针检测MAR的细胞内蓄积，并与对照组比较，结果显示，MAR的细胞内蓄积量与对照组相比有显著性差异（P < 0.05）。在96孔板格式中测定MAR突变体和具有明确的外排泵和孔蛋白基因破坏的突变体对H33342（双苯甲酰亚胺）的蓄积。（P < 0.0001）。肠道血清型鼠伤寒沙门氏菌（n = 4）和E.大肠杆菌（n = 3）与其亲本菌株相比分别降低了41 ± 8%和17.3 ± 7.2%，这种情况可被跨膜质子梯度破坏剂羰基氰化物-间氯苯腙（CCCP）和外排泵抑制剂苯丙氨酸-精氨酸-β-萘酰胺（PA β N）逆转。在编码孔蛋白OmpC、OmpF、OmpX和OmpW的基因中具有确定破坏的鼠伤寒沙门氏菌突变体中，H33342的积累显著减少，但在外排泵组分TolC、AcrB和AcrF中具有破坏的突变体中增加。H33342在鼠伤寒沙门氏菌的其他三种MAR突变体中的积累减少与孔蛋白和外排泵蛋白的表达相关。对CCCP和PA β N的不同敏感性提供了进一步的方法来表型鉴定MAR突变体和每种菌株中主动外排的作用。
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.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.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.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.