A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

Coldham, N. G.; Webber, Mark A.; Woodward, Martin J.; Piddock, Laura J. V.

doi:10.1093/jac/dkq169

Cited by 142 publications

(159 citation statements)

References 35 publications

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“…The H33342 accumulation assay was introduced to estimate the contribution of active efflux pumps to antimicrobial resistance in Salmonella Typhimurium strains [6]. The H33342 accumulation assay was also used to assess the roles of efflux pumps in antimicrobial resistance in A. baumannii clinical isolates [9].…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…The H33342 accumulation assay is a cheap, relatively rapid, sensitive and specific test for MDR [6]. It has been used for the assessment of cellular permeability and efflux activity in Salmonella enterica and Escherichia coli [6,7]. H33342 accumulation has also been used in A. baumannii to assess the contribution of efflux activity to antibiotic accumulation [8,9].…”

Section: Introductionmentioning

confidence: 99%

“…Although it was used effectively to assess efflux activity between several strains of bacteria including S. enterica, E. coli and A. baumannii [6,9], it is hard to find out studies using it as an assessing tool of efflux activity in a lot of clinical strains of A.…”

Section: Introductionmentioning

confidence: 99%

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Assessment of Efflux Activity Using H33342 Accumulation in Tigecycline-ResistantAcinetobacter baumanniiClinical Isolates

et al. 2017

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Background: Tigecycline resistance has emerged recently and has shown diverse mechanisms. The aim of this study was to assess the role of efflux activity in tigecycline resistance in 120 clinical isolates of A. baumannii using two methods: the H33342 accumulation assay and adeB real-time reverse transcriptase polymerase chain reaction. In addition, we analyzed the correlation between the expression level of adeB and H33342 accumulation level. Methods: A. baumannii clinical isolates was divided into tigecycline-resistant (49 strains), intermediate (40 strains), and susceptible (31 strains) groups. The H33342 accumulation was measured in the absence or presence of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Real-time RT-PCR was performed to determine the relative expression of the adeB gene in A. baumannii clinical isolates. Results:The level of H33342 accumulation in the resistant group was relatively lower than those in the other groups. The addition of CCCP caused a significantly increased fold change in H33342 accumulation in the tigecycline-resistant group. Significant difference in the fold change level in H33342 accumulation was found between tigecycline-susceptible and resistant isolates. Those findings support the role of efflux pumps of which substrates are H33342 in the resistance of tigecycline. Significant differences in the relative expression levels of adeB were shown between tigecycline-susceptible and resistant groups also. Conclusion:The results showed that several efflux pumps of which substrates were H33342 can contribute to tigecycline resistance. The adeB overexpression can also contribute to tigecycline resistance. It is possible that efflux pumps other than adeB efflux pumps contribute to tigecycline resistance because there was no correlation between fold change level in H33342 accumulation and adeB expression level.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Assessment of Efflux Activity Using H33342 Accumulation in Tigecycline-ResistantAcinetobacter baumanniiClinical Isolates

et al. 2017

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“…coli TG1 and its ∆acrB-∆tolC mutant, E. amylovora and its ∆acrB -∆tolC mutant were used in intercellular EtBr accumulation assay according to Coldham et al [36]. Bacterial strains were grown in LB medium, 250 rpm until it reaches to an OD 600 of 1, and centrifuged at 4000 rpm for 30min.…”

Section: Intercellular Accumulation Of Ethidium Bromide (Etbr)mentioning

confidence: 99%

Detection of potential AcrAB-TolC multidrug efflux pump inhibitor in calyces extract of Hibiscus sabdariffa

Al‐Karablieh¹,

Abu-Qatouseh²,

Aburjai³

2017

J Intercult Ethnopharmacol

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Aim: The aim of this study is to investigate the occurrence of potential efflux pump inhibitor (EPI) against AcrAB-TolC efflux pump in the methanol extract of Hibiscus sabdariffa. Materials and Methods: Calyces of H. sabdariffa were purchased from the local market in April 2014, used in methanol extraction. The methanol extract of H. sabdariffa was subjected to agar plate diffusion against Escherichia coli TG1 and its ∆acrB-∆tolC followed by a thin layer chromatography (TLC) bioassay. The fraction corresponding to EPI fraction was eluted from the silica gel by methanol. The synergistic effect of antimicrobials and EPI fraction was measured by minimum inhibitory concentration (MIC) determination for E. coli and Erwinia amylovora strains. The ability of EPI fraction to enhance ethidium bromide (EtBr) accumulation was conducted. Results: E. coli TG1 was more sensitive to the methanol extracts of H. sabdariffa than E. coli ∆acrB-∆tolC. Inhibition zone corresponding to flavones on TLC bioassay plate has been formed which might be related to the fraction of potential EPI. The MIC values revealed that EPI fraction enhanced the activity of the used antimicrobials by 4-8 folds in E. coli TG1 and by 4-10 folds in E. amylovora 1189. Addition of EPI fraction in a dose-dependent manner increased the intercellular accumulation of EtBr in the wild type stains of E. coli TG1 and E. amylovora 1189. Conclusion: An EPI fraction behaves like a multidrug EPI, and further investigation should be conducted for determination the structure of chemical constituents in EPI fraction.

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Bacterial Efflux Pumps and Their Inhibitors

Zgurskaya

Rybenkov

Walker

et al. 2021

Burger's Medicinal Chemistry and Drug Discovery

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Bacterial multidrug efflux transporters have distinct molecular features and mechanisms that enable their impressive substrate polyspecificity. These transporters are involved in all physiological processes where nonspecific but active transport across membranes is critical for survival and proliferation. Importantly, efflux pumps contribute to bacterial virulence and pathogenesis, as well as the spread of antibiotic resistance in clinics. Transporters belonging to several protein superfamilies have been selected for polyspecificity by evolution and augmented with accessory proteins that extend and couple transport reactions across the inner and outer membranes of the bacterium. As a result, bacteria are equipped with diverse efflux machines that protect cells from a broad variety of toxic compounds including antibiotics and other small molecule therapeutics. The inhibition of such polyspecific biomolecular machines is a challenging task, which still awaits an efficient solution. Here, we review the molecular mechanisms of efflux pumps from Gram‐negative bacteria and recent advances and challenges associated with the discovery of effective inhibitors of these pumps.

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A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

Abstract: 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.

Cited by 142 publications

References 35 publications

Assessment of Efflux Activity Using H33342 Accumulation in Tigecycline-ResistantAcinetobacter baumanniiClinical Isolates

Assessment of Efflux Activity Using H33342 Accumulation in Tigecycline-ResistantAcinetobacter baumanniiClinical Isolates

Detection of potential AcrAB-TolC multidrug efflux pump inhibitor in calyces extract of Hibiscus sabdariffa

Bacterial Efflux Pumps and Their Inhibitors

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