Investigation Of The Mechanism For Artesunate Increasing Susceptibility Of Escherichia Coli To ?-lactam Antibiotics

Objective:Antibiotic plays a key role in the treatment of diseases caused by bacterial infection in clinic. However, after the long-term exposure to antibiotics, the bacteria alter their metabolic pathways, produce inactivated enzyme, and regulate the expressions of efflux pump proteins, which result in the bacterial resistance against some antibiotics in some degree. A study on gram-negative bacteria resistance have documented that the most common gram-negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli (E. coli) became resistant against many antibiotics including ceftazidime, cefoperazone/sulbactam, ciprofloxacin, gentamicin and etc. those were widely used in clinic.By now there was no such a successful strategy except?-lactamase inhibitors to restore antibacterial activities of antibiotics. In the previous study, we found that 256?g/ml Artesunate (AS), a derivative of artemisinin (ART), which was widely used against falciparum malaria and cerebral malaria, could decrease the MIC of Escherichia coli ATCC35218 to gentamicin from 4?g/ml to 0.25?g/ml (16-fold). Expanding the combined range of AS, we found that AS also increased the susceptibility of Escherichia coli ATCC35218 to ampicillin sodium-sulbactam sodium and cefpiramide. Excitedly, the synergetic antibacterial effect was also observed in Escherichia coli isolates and methicillin resistant staphylococcus aureus (MRSA). Meanwhile, AS increased the accumulation of daunorubicin within ATCC35218. Importantly, ART could significantly decrease the mortality of mice challenged with live E. coli when it was used with ampicillin together.However, how AS potentiate the antibacterial effects of various antibiotics in vitro is still not clarified. In the present study we first measured the AS's antibacterial potentiation on various?-lactam antibiotics against E. coli ATCC35218. Then we investigated the role of the main efflux pump related genes in the artesunate's antibacterial potentiation effect against E. Coli. Based on these, we tried to illuminate the possible mechanism for AS's antibacterial potentiation effect and provide an efficient approach to reduce the resistance of gram -negative bacteria in the treatment of infectious diseases in clinic.Methods:1.Two-fold diluted method was used to measure the susceptibility of E. Coli ATCC35218 and clinical isolates to various?-lactam antibiotics in the absence or presence of AS;2.Dynamic growth assay of effects of various?-lactam antibiotics alone or combined with AS on E. coli ATCC35218 and clinical isolates;3.RT-PCR analysis of the mRNA levels of the main efflux pump related gene(s) and upstream regulator(s) within E. coli ATCC35218 after the treatments of AS with different concentrations;4.qRT-PCR analysis of the mRNA level(s) of the differential gene(s) and and upstream regulator(s) within E. coli ATCC35218 after the treatment of 256?g/ml AMP alone or in combination with 256?g/ml AS;5.Two-fold diluted method was used to measure the susceptibility of E. Coli AG100A lacking AcrA and AcrB in the absence or presence of AS treatment.6.Antisense oligonucleotide technique was employed to knockdown the main differential genes to investigate its or their role of the differential gene(s) in the antibacterial potentiation effect produced by AS;7.Western blot analysis of protein(s) expression levels of the differential gene(s) within E. coli ATCC35218 after the treatment of AMP alone or in combination with AS;8.The accumulation of AMP in E. coli ATCC35218 by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS).Results:1. Although AS alone had no significant antibacterial effect, it almost increased the susceptibility of E. Coli to all tested?-lactam antibiotics including ampicillin, ampicillin sodium-sulbactam sodium, penicilin G, oxacillin, cefpiramide, and ect.;2. Single AS had little inhibitory effect on E. coli ATCC35218, while AS in combination with various?-lactam antibiotics significantly suppressed the bacterial growth compared with single antibiotics treatments;3. RT-PCR results showed that different concentrations AS (512-32?g/ml) could not significantly reduce the expression of AcrA, MdtA, MdtC, and MdtF. In fact, 512?g/ml AS markedly down-regulated the expressions of AcrB and MdtE. In addition, AS (512-32?g/ml) had no obvious effects on the common outer membrane channel, TolC;4. qRT-PCR results demonstrated that 256?g/ml AS in combination with 256?g/ml AMP significantly reduced the mRNA levels of AcrB compared with single AS or AMP treatment. However, the expressions of AcrA, MdtA, MdtC, MdtE, and MdtF were not significantly changed in the presence or absence of AS. Meanwhile, the expression of TolC was not significantly changed;5. E. Coli AG100A lacking AcrA and AcrB genes became very sensitive to various?-lactam antibiotics. However, the additional AS treatment could not further increase the bacterial susceptibility;6. The transformation of as-ODNs targeting AcrA and AcrB significantly increased the the bacterial susceptibility to AMP. However, the bacterial resistance was not further decreased even after the additional AS adding when AcrB gene was knockdown.7. Except MarA gene, AS (512-32?g/ml) had no significant effect on the upstream regulators of AcrAB-TolC. However, AS (256?g/ml) in combination with AMP (256?g/ml) significantly down-regulated expressions of the positive regultors including SoxS, and MarA compared with single AS or AMP treatment. However, AS had no significant effects on the negative regulators such as AcrR and MppA;8. High concentration AS (512?g/ml) significantly down-regulted the expression of AcrB protein within ATCC35218. However, AS with lower concentration (32-256?g/ml) had no significant inhibitory effect on AcrB protein;9. AS could significantly increase the accumulation of AMP in the bacteria. The largest accumulation was found after the additional AS treatment at a concentration of 128?g/ml. Then the accumulation product was gradually decreased accompanied by increasing concentration of AS.10. Artesunate could not increase the accumulation of daunorubicin in E. coli AG100A, suggesting that artesunate lost its main effect when AcrB gene was knockout. Also, artesunate could not increase the the accumulation of daunorubicin when AcrB was overexpressed. We speculated it was due to the limited artesunate that could not play its antibacterial potentiation effect in front of the overexpression of AcrB. Conclusions:1. AS widely increased the susceptibility of E. coli to?-lactam antibiotics;2. AS inhibited the expression of a major multi-drug efflux pump system AcrAB-TolC;3. AcrB played a major role in the antibacterial potentiation produced by AS, and AcrA played a minor role in this event;4. AS alone or in combination with AMP significantly down-regulated the positiveregulators and up-regultated the negative regulator on the upstream of AcrAB-TolC, thereby reducing the expression of AcrAB-TolC, which decreased the bacterial resistance against AMP;5. AS increased the accumulation of AMP within E. coli ATCC35218, which might be associated with the decreased the efflux output of antibiotics by the inhibition of the main efflux pump system AcrAB-TolC. While, the accumulation of the antibiotics was reduced when AS was over some certain concentration.6. AS could not increase the accumulation of DNR in E. coli AG100A (?AcrB), suggesting AS lost its main target when AcrB was knockout. And, AS could not increase the accumulation of DNR in E. coli AG100A transfected with pQE30-AcrB, indicating AS could not play its inhibitory effect on efflux pumps in the presence of the AcrB overexpression.