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Browsing by Subject "multidrug resistance"

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  • Yrjänheikki, Ulla (2019)
    Background: The World Health Organization (WHO) outlined in their report published in 2014 that antimicrobial resistance (AMR) is a real public health threat worldwide and the actions against it should be taken. Otherwise, the post-antibiotic era where common community-acquired infections can lead to death, could hypothetically become true. The discovery and development of novel antibiotics (ATBs) against Gram-negative bacteria (GNB)-related infections is difficult due to a dual defence mechanism: the extra protection barrier called the outer membrane and efflux pumps which GNB utilize to protect themselves against external noxious compounds. Efflux pumps are expressed at the basal level in GNB, such as E. coli, but when exposed to sub-inhibitory concentrations of ATBs and the intrinsic extruding capacity is exceeded, GNB start overexpressing these “so-called” multi-drug resistance (MDR) efflux pumps. The most abundant and studied MDR efflux pump in E. coli is a tripartite protein complex AcrAB-TolC which traverse through the bacterial cell envelope and is capable of extruding a broad range of structurally unrelated compounds, thus leading to cross-resistance against several classes of ATBs. It has been suggested that antibacterial activity of existing ATBs could be restored again by inhibiting increased efflux activity through efflux pump inhibitors (EPIs). Objectives: Define the optimal assay conditions and a positive control (EPI) to be used in high throughput screening (HTS) of novel EPIs. The assay consists of one E. coli strain of clinical relevance with high intrinsic efflux activity, one ATB and one EPI, both of them at specific concentrations defined during this study. Methods: The intrinsic efflux activities of seven E. coli strains were studied by Hoechst 33342 (H33342) accumulation assay, both in the absence and presence of five commercially available EPIs. The same assay was used in the dose-response studies in which an optimal concentration of EPIs was identified for further to be utilized in the checkerboard assays. The minimum inhibitory concentrations (MICs) were determined by broth microdilution method according to Clinical and Laboratory Standards Institute. The synergistic effects of ATB and EPI in terms of decreasing the intrinsic MIC value of the ATB were determined in the checkerboard assays partially performed by the Biomek i7 Automated Workstation. The data was analysed by using Microsoft Excel and IBM SPSS Statistics, version 25. Results and discussion: E. coli ATCC 25922 had statistically significantly the highest efflux activity of all wild-type pathogenic and non-pathogenic E. coli strains. However, when H33342 accumulation assay was carried out in conjunction with EPIs, E. coli BAA1161 (uropathogenic strain) had the highest median increase in the intracellular level of H33342. Mefloquine showed to be the most potent of all EPIs at the tested concentrations. However, mefloquine increased the intracellular H33342 accumulation even in efflux-deficient E. coli JW5503 (ΔtolC), thus possible additional modes of action or inhibitory activity towards other efflux pumps might exist. Dose-response studies carried out in ΔtolC E. coli JW5503 suggested that CCCP at 1.25 g/ml and mefloquine at 0.5 g/ml were the optimal concentrations. However, for mefloquine, when tested at 0.5 g/ml, the intracellular level of H33342 was not increased in six remaining E. coli strains. Therefore higher concentrations up to ½ MIC were tested in the checkerboard assays. In the antibacterial susceptibility testing, E. coli BAA1161 was the only strain showing resistance to tetracycline and piperacillin, resulting in MIC ratios (MIC wild-type/MIC mutant) of 512 to 2048. Piperacillin and ofloxacin, which showed a MIC ratio of 4 in two E. coli strains, were chosen to the checkerboard assays in which mefloquine reduced the intrinsic MIC of piperacillin by 16-fold and CCCP by 32-fold in E. coli BAA1161. Conclusions: E. coli BAA1161 was chosen to be used as a model strain in HTS due to the highest median increase in intracellular H33342 accumulation and also for being the only strain with resistance towards the ATBs tested. Mefloquine (16 g/ml) was the EPI of choice for the positive control in HTS because the synergistic effects observed between piperacillin and mefloquine were most probably explained by efflux pump inhibition and not by antibacterial activity of mefloquine itself. Piperacillin (256 g/ml) was selected to be used as an ATB in HTS because it was the only ATB which was potentiated by the tested EPIs.