In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa(PA)-induced pulmonary infection,pulmonary infection models were established by intratracheal inje...In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa(PA)-induced pulmonary infection,pulmonary infection models were established by intratracheal injection of K767(wild type),nalB(MexA-MexB-OprM up-regulated mutant),and △m exB(knockout) strains,separately.All mice were treated with Meropenem(intraperitoneal injection,100 mg/kg body weight,twice every day),and strain-related pathology,bacteria count,cytokine level,myeloperoxidase(MPO,indicator of neutrophil recruitment) activity,and macrophage inflammatory protein-2(MIP-2) expression were evaluated at early(3rd day post-infection) and late(7th and 14th day post-infection) stages of infection.E-test showed that △mexB was more significantly sensitive to panipenan(ETP),meropenem(MP) and imipenem(IP) than K767 and nalB strains.There was no significant difference in sensitivity to cefepime(TM) among the three stains.In contrast to the K767 and nalB groups,the △ mexB group showed decreased bacteria burden over time and less extensive pathological change.Additionally,MPO activity and levels of inflammatory cytokines(IL-1b,IL-12,and TNF-α) were increased at the early stage(day 3) and decreased at the later stage(day 14).Serum MIP-2 expression level was steadily increased in all three groups from early to late stages,but significantly higher in △m exB group than in K767 and nalB groups(P<0.05).In conclusion,the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection.High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.展开更多
AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's...AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's blood. The multidrug resistant (MDR) H. pylori were obtained with the inducer chloramphenicol by repeated doubling of the concentration until no colony was seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present study included periods before and after the advent of the EPIs, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine and pantoprazole), and the minimum inhibitory concentrations (MICs) were determined accordingly. In the same way, the effects of 5 proton pump inhibitors (PPIs), used in treatment of H. pylori infection, on MICs of antibiotics were evaluated.RESULTS: Four strains of MDR H. pylori were induced successfully, and the antibiotic susceptibilities of MDR strains were partly restored by CCCP and pantoprazole, but there was little effect of reserpine. Rabeprazole was the most effective of the 5 PPIs which could decrease the MICs of antibiotics for MDR H. pylori significantly.CONCLUSION: In vitro, some EPIs can strengthen the activities of different antibiotics which are the putative substrates of the efflux pump system in H. pylori.展开更多
AIM: To determine whether efflux systems contribute to multidrug resistance of H pylori. METHODS: A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC1...AIM: To determine whether efflux systems contribute to multidrug resistance of H pylori. METHODS: A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC11637) was developed. Multidrug-resistant (MDR) strains were selected and the minimal inhibitory concentration (MIC) of eryth-romycin, metronidazole, penicillin G, tetracycline, and ciprofloxacin in multidrug resistant strains and their parent strains was determined by agar dilution tests. The level of mRNA expression of hefA was assessed by fluorescence real-time quantitative PCR. A H pylori LZ1026 knockout mutant (ΔH pylori LZ1026) for (puta-tive) efflux protein was constructed by inserting the kanamycin resistance cassette from pEGFP-N2 into hefA, and its susceptibility profiles to 10 antibiotics were evaluated. RESULTS: The MIC of six multidrug-resistant strains (including 5 clinical isolates and H pylori NCTC11637) increased signifi cantly (≥ 4-fold) compared with their parent strains. The expression level of hefA gene was significantly higher in the MDR strains than in their parent strains (P = 0.033). A H pylori LZ1026 mutant was successfully constructed and the ΔH pylori LZ1026 was more susceptible to four of the 10 antibiotics. All the 20 strains displayed transcripts for hefA that con-fi rmed the in vitro expression of these genes.CONCLUSION: The efflux pump gene hefA plays an important role in multidrug resistance of H pylori.展开更多
Objective:The efflux pump(EP) is one of the major mechanisms of antibiotic resistance in Klebsiella pneumoniae.However,there are few reports on the effect of the abuse of antibiotic use on the activity of EPs.To deter...Objective:The efflux pump(EP) is one of the major mechanisms of antibiotic resistance in Klebsiella pneumoniae.However,there are few reports on the effect of the abuse of antibiotic use on the activity of EPs.To determine whether the use of low efficacy antibiotics has any effect on the activity of EPs and induces drug resistance in K.pneumoniae,we investigated the effect of ciprofloxacin on the activity of EPs in K.pneumoniae strains.Methods:Sixteen susceptible K.pneumoniae strains were isolated from patients and their minimum inhibitory concentrations(MICs) of ciprofloxacin were measured in the absence and presence of the pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone(CCCP).The strains were then induced with a gradient of ciprofloxacin until the MICs of the strains showed no further increase,to obtain induced resistant strains.The EP activities of the strains before and after induction were compared using EP inhibition and ethidium bromide(EtBr) accumulation assays.Results:The MIC values of the strains were 16 256 times higher after induction than before induction.In the presence of CCCP,the MIC values of 50% of the induced strains were 2 4-fold lower than that in the absence of this inhibitor.The EtBr accumulation assay showed that the fluorescence of EtBr in the induced cells was lower than that in the cells before induction.Conclusions:EPs are widespread in susceptible and drug-resistant K.pneumoniae strains.Induction with ciprofloxacin may increase the activity of EPs in K.pneumoniae.The EtBr accumulation assay is more sensitive than the EP inhibition assay in evaluating the activity of EPs in K.pneumoniae.展开更多
The upsurge of multiple drug resistance(MDR)bacteria substantially diminishes the effectiveness of antibiotic arsenal and therefore intensifies the rate of therapeutic failure.The major factor in MDR is efflux pump-me...The upsurge of multiple drug resistance(MDR)bacteria substantially diminishes the effectiveness of antibiotic arsenal and therefore intensifies the rate of therapeutic failure.The major factor in MDR is efflux pump-mediated resistance.A unique pump can make bacteria withstand a wide range of structurally diverse compounds.Therefore,their inhibition is a promising route to eliminate resistance phenomenon in bacteria.Phytochemicals are excellent alternatives as resistance-modifying agents.They can directly kill bacteria or interact with the crucial events of pathogenicity,thereby decreasing the ability of bacteria to develop resistance.Numerous botanicals display noteworthy efflux pumps inhibitory activities.Edible plants are of growing interest.Likewise,some plant families would be excellent sources of efflux pump inhibitors(EPIs)including Apocynaceae,Berberidaceae,Convolvulaceae,Cucurbitaceae,Fabaceae,Lamiaceae,and Zingiberaceae.Easily applicable methods for screening plant-derived EPIs include checkerboard synergy test,berberine uptake assay and ethidium bromide test.In silico highthroughput virtual detection can be evaluated as a criterion of excluding compounds with efflux substrate-like characteristics,thereby improving the selection process and extending the identification of EPIs.To ascertain the efflux activity inhibition,real-time PCR and quantitative mass spectrometry can be applied.This review emphasizes on efflux pumps and their roles in transmitting bacterial resistance and an update plant-derived EPIs and strategies for identification.展开更多
Objective:To evaluate the inhibitory activity of ferulic acid and four of its esterified derivatives(methyl,ethyl,propyl,and butyl)against resistance mechanisms in Staphylococcus aureus strains.Methods:Ferulic acid de...Objective:To evaluate the inhibitory activity of ferulic acid and four of its esterified derivatives(methyl,ethyl,propyl,and butyl)against resistance mechanisms in Staphylococcus aureus strains.Methods:Ferulic acid derivatives were obtained by esterification with methanol,ethanol,propanol,and butanol,and then characterized by hydrogen and carbon-13 nuclear magnetic resonance analysis.The minimum inhibitory concentrations(MIC)of ferulic acid and its esterified derivatives,ethidium bromide,and norfloxacin were obtained using the microdilution test,while the efflux pump inhibition test was conducted by examining reduction in the MICs of norfloxacin and ethidium bromide.Molecular docking was also carried out using the Schrodinger Suite 2015 molecular modeling software.A three-dimensional model of NorA efflux pump was generated using I-TASSER.The best scoring model was used as a receptor for ligand-receptor docking.Results:The methyl and butyl ester derivatives did not demonstrate significant antimicrobial activity.However,a significant synergic effect was evidenced when norfloxacin was combined with the ethyl and propyl esterified derivatives.The docking study demonstrated favorable energy of interaction between ferulate derivatives and NorA,and amino acid residues TYR57,TYR58,and LEU255 were present commonly in stabilizing all complexes.The PCA analysis corroborated the docking hypothesis that the lipophilic character and hydrogen bond interactions were the most relevant characteristics involved with NorA inhibitors.The pharmacokinetic parameters of ferulic acid derivatives showed good ADMET properties,demonstrating that they can be easily absorbed and have no effect or inhibit the cytochrome P450 enzyme complex,revealing their potential as drug candidates.Conclusions:This study provides strong evidence that the molecular basis for this activity is potentially due to the NorA efflux pump.展开更多
Objectives:To identify and test the antibiotic susceptibility of nosocomial coliform bacilli and investigate the presence of oqxA and oqxB genes among the multidrug-resistant(MDR)phenotypes.Methods:One hundred and twe...Objectives:To identify and test the antibiotic susceptibility of nosocomial coliform bacilli and investigate the presence of oqxA and oqxB genes among the multidrug-resistant(MDR)phenotypes.Methods:One hundred and twenty different healthcare-associated infection samples were collected.Coliform bacilli were isolated,identified by conventional methods,and then antibiotic susceptibility tests were done using the VITEK2 system and disk diffusion methods.OqxAB operon was identified using a conventional PCR-based technique.oqxA and oqxB genes were compared between MDR Klebsiella pneumonia(K.pneumonia)phenotypes and MDR Escherichia coli(E.coli)phenotypes.Besides,oqxAB operons were compared between phenotypes of K.pneumonia and E.coli isolates.Results:Seventy coliform bacilli were isolated with the predominance of K.pneumonia and E.coli.Besides,82.1%of K.pneumonia strains and 53.3%of E.coli isolates were MDR phenotypes.Significant more oqxB genes alone were found in MDR E.coli than that in MDR K.pneumoniae phenotypes(χ^(2)=10.160,P=0.003).OqxAB operon was significantly more in MDR phenotypes of E.coli than that in the susceptible phenotypes(P<0.001).There was significantly less of this operon in susceptible E.coli isolates than that in susceptible K.pneumoniae isolates(P<0.001).OqxAB positive isolates that were also resistant to fluoroquinolones,tetracycline,trimethoprim,and chloramphenicol,most probably suggested functional pumps.Conclusions:MDR coliform bacilli are strongly implicated in healthcare-associated infection.Attention should be paid to the presence of oqxAB pump,as an important mechanism in the development of resistance against many antimicrobials because it contributes to co-resistance with other categories;therefore,this pump could be a good target for efflux pump inhibitors.展开更多
Salmonella gallinarum has shown multiple drug resistance(MDR),especially high level fluoroquinolone(FQ)resistance in recent years.To determine whether the active efflux system was responsible for high-level FQ resista...Salmonella gallinarum has shown multiple drug resistance(MDR),especially high level fluoroquinolone(FQ)resistance in recent years.To determine whether the active efflux system was responsible for high-level FQ resistance,this research studied AcrAB efflux pump in Salmonella gallinarum on molecular level.The resistant strains were induced by standard strain C79-13 with ciprofloxacin in vitro.With carbonylcyanide-p-chlorophenyl hydrazone(CCCP)as an energy inhibitor,efflux inhibition test initially showed the potential impact of efflux pump on drug resistance.Sequence analysis of acrA gene indicated that gene mutation of AcrAB efflux pump was not definitely associated with MDR and drug resistance level of Salmonella gallinarum.Detected by competitive RT-PCR,the mRNA expression of acrA and acrB genes in the resistant strains significantly increased(p<0.01)compared with that of the control strain C79-13.The mRNA expression level of acrB gene(increased from 1.6-to 2.9-folds)was consistent with that of acrA gene(increased from 1.6-to 2.8-folds),which increased with the drug resistance level.However,gene mutation of acrA gene showed no correlation with its mRNA expression level,indicating that gene mutation did not affect the expression of AcrAB pump itself.The results suggested that the overexpression rather than the gene mutation of AcrAB efflux pump was an important factor causing the high level drug resistance of Salmonella gallinarum.展开更多
Antimicrobial resistance(AMR)poses a significant threat to public health and is increasingly recognized within the“One Health”framework,which emphasizes the interconnectedness of human,animal,and environmental healt...Antimicrobial resistance(AMR)poses a significant threat to public health and is increasingly recognized within the“One Health”framework,which emphasizes the interconnectedness of human,animal,and environmental health.While extensive research has focused on regulating antibiotic use across healthcare and other sectors,the impact of intensive biocide use on AMR development,particularly in seawater-cooled systems,remains underexplored.In this study,we report the isolation and characterization of a multidrug-resistant Klebsiella quasipneumoniae strain from the cooling water system of a coastal power plant,where continuous chlorination at 0.2 ppm is employed for biofouling control.The isolated strain displayed broad-spectrum resistance to multiple biocides and antibiotics.Interestingly,the strain shown enhances biofilm formation in response to biocides and antibiotics,thereby compounding its resistance profile.Efflux assays with ethidium bromide(EtBr)and whole-genome sequencing revealed that efflux pumps are central to the resistance mechanisms.Additionally,the presence ofβ-lactamase(OKP-A)and FosA genes confers resistance to theβ-lactam and epoxide classes of antibiotics.The strain was found to be salt-tolerant and preferred to grow at normal salinity,indicating a non-marine origin of this isolate.These findings highlight the prevalence of biocide and antibiotic-resistant pathogens in marine cooling water systems that primarily rely on biocides for biofouling control.In line with One Health principles,our research advocates for a reassessment of biocide practices in marine cooling water systems and the implementation of proactive measures to mitigate the spread of antimicrobial resistance(AMR).展开更多
<strong>Background:</strong> Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds f...<strong>Background:</strong> Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. <strong>Aim:</strong> The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). <strong>Methods:</strong> DH5α competent <em>E. coli</em> cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. <strong>Results:</strong> The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. <strong>Conclusion:</strong> The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.展开更多
RE-CmeABC is a variant of the efflux pump CmeABC in the major zoonotic pathogen Campylobacter,which is much more potent in conferring resistance to various antibiotics for the treatment of Campylobacter infections in ...RE-CmeABC is a variant of the efflux pump CmeABC in the major zoonotic pathogen Campylobacter,which is much more potent in conferring resistance to various antibiotics for the treatment of Campylobacter infections in both veterinary and medical clinics.This potent multidrug efflux pump RE-CmeABC is widely distributed and disseminated around the world,posing a serious threat to public health.In this study,we targeted the key efflux protein RE-CmeB of RE-CmeABC to explore promising efflux pump inhibitors derived from natural products to improve the multidrug resistance of Campylobacter.We constructed a protein screening model and utilized virtual screening techniques,such as molecular docking,to identify potential efflux pump inhibitors from natural product databases.The activity and safety of these candidates were subsequently evaluated,and ZINC338037 was shown to inhibit antibiotic extrusion and restore the susceptibility of multidrug-resistant Campylobacter.In summary,a potential multidrug efflux pump inhibitor was identified and is expected to be developed as an adjuvant drug for the treatment of clinical infections caused by multidrug-resistant Campylobacter.展开更多
Mycobacterium abscessus,one of the most antimicrobial-resistant bacteria,is increasingly recognized as the cause of infections that are difficult to treat.Novel genetic manipulation tools are required to elucidate the...Mycobacterium abscessus,one of the most antimicrobial-resistant bacteria,is increasingly recognized as the cause of infections that are difficult to treat.Novel genetic manipulation tools are required to elucidate the biology,pathogenesis,and antibiotic resistance mechanisms of M.abscessus.In this study,we modified the method used to prepare M.abscessus electrocompetent cells to achieve efficient transformation,and then optimized the CRISPR-Cas9-assisted genome-editing tools to allow efficient genetic manipulation.Using these tools,we constructed 66 efflux pump mutants of M.abscessus and investigated their roles in drug resistance and virulence.We found that different efflux pumps play distinct roles in drug resistance and survival in Galleria mellonella larvae.Finally,we confirmed that MAB_2806-2807,involved in transportation of triacylglycerides,is vital for the drug resistance and virulence of M.abscessus.The molecular biology tools developed in this study will facilitate molecular research on M.abscessus.In addition,the study on efflux pumps might provide new targets for the development of new drugs and treatment regimens for M.abscessus infection.展开更多
The growing threat of antibiotic resistance calls for novel strategies to enhance antimicrobial efficacy.Blueberries,rich in phenolic compounds,have attracted attention as food-derived agents capable of modulating bac...The growing threat of antibiotic resistance calls for novel strategies to enhance antimicrobial efficacy.Blueberries,rich in phenolic compounds,have attracted attention as food-derived agents capable of modulating bacterial resistance mechanisms.This study explores the potential of phenolic-rich blueberry extracts as antibiotic resistance-modifying agents.To evaluate their modulatory effects,we conducted antibacterial testing,efflux pump inhibition assays,and transcriptomic analyses on Acinetobacter baumannii BAA-1605 treated with blueberry extract,gentamicin,and their combination.While the extract alone showed no direct antibacterial activity,its combination with gentamicin resulted in a twofold reduction in lowering the minimum inhibitory concentration against Acinetobacter baumannii BAA-1605.Mechanistic assays revealed that blueberry extract enhanced gentamicin's effect on inhibiting efflux pump activity.Transcriptomic analysis showed that the combination treatment of blueberry extract and gentamicin modulated genes involved in metabolism,stress response,and virulence,with Kyoto encyclopedia of genes and genomes(KEGG)and gene ontology(GO)enrichment pointing to altered energy production and cellular adaptation pathways.These findings suggest that blueberry phenolics do not act bactericidally but may affect bacterial physiology to potentiate gentamicin action.This study highlights the potential of natural plant phenolic compounds as antibiotic adjuvant and supports further investigation in their fighting against multidrug-resistant pathogens.展开更多
基金supported by grants from the National Natural Science Foundation of China (No. 30873189)the Natural Science Foundation of Hubei Province,China (No.2008CDB165)
文摘In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa(PA)-induced pulmonary infection,pulmonary infection models were established by intratracheal injection of K767(wild type),nalB(MexA-MexB-OprM up-regulated mutant),and △m exB(knockout) strains,separately.All mice were treated with Meropenem(intraperitoneal injection,100 mg/kg body weight,twice every day),and strain-related pathology,bacteria count,cytokine level,myeloperoxidase(MPO,indicator of neutrophil recruitment) activity,and macrophage inflammatory protein-2(MIP-2) expression were evaluated at early(3rd day post-infection) and late(7th and 14th day post-infection) stages of infection.E-test showed that △mexB was more significantly sensitive to panipenan(ETP),meropenem(MP) and imipenem(IP) than K767 and nalB strains.There was no significant difference in sensitivity to cefepime(TM) among the three stains.In contrast to the K767 and nalB groups,the △ mexB group showed decreased bacteria burden over time and less extensive pathological change.Additionally,MPO activity and levels of inflammatory cytokines(IL-1b,IL-12,and TNF-α) were increased at the early stage(day 3) and decreased at the later stage(day 14).Serum MIP-2 expression level was steadily increased in all three groups from early to late stages,but significantly higher in △m exB group than in K767 and nalB groups(P<0.05).In conclusion,the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection.High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.
基金Supported by Henan Distinguished Junior Scholar Grant,No.074100510017
文摘AIM:To evaluate the effect of efflux pump inhibitors (EPIs) on multidrug resistance of Helicobacter pylori (H. pylori).METHODS: H. pylori strains were isolated and cultured on Brucella agar plates with 10% sheep's blood. The multidrug resistant (MDR) H. pylori were obtained with the inducer chloramphenicol by repeated doubling of the concentration until no colony was seen, then the susceptibilities of the MDR strains and their parents to 9 antibiotics were assessed with agar dilution tests. The present study included periods before and after the advent of the EPIs, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), reserpine and pantoprazole), and the minimum inhibitory concentrations (MICs) were determined accordingly. In the same way, the effects of 5 proton pump inhibitors (PPIs), used in treatment of H. pylori infection, on MICs of antibiotics were evaluated.RESULTS: Four strains of MDR H. pylori were induced successfully, and the antibiotic susceptibilities of MDR strains were partly restored by CCCP and pantoprazole, but there was little effect of reserpine. Rabeprazole was the most effective of the 5 PPIs which could decrease the MICs of antibiotics for MDR H. pylori significantly.CONCLUSION: In vitro, some EPIs can strengthen the activities of different antibiotics which are the putative substrates of the efflux pump system in H. pylori.
文摘AIM: To determine whether efflux systems contribute to multidrug resistance of H pylori. METHODS: A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC11637) was developed. Multidrug-resistant (MDR) strains were selected and the minimal inhibitory concentration (MIC) of eryth-romycin, metronidazole, penicillin G, tetracycline, and ciprofloxacin in multidrug resistant strains and their parent strains was determined by agar dilution tests. The level of mRNA expression of hefA was assessed by fluorescence real-time quantitative PCR. A H pylori LZ1026 knockout mutant (ΔH pylori LZ1026) for (puta-tive) efflux protein was constructed by inserting the kanamycin resistance cassette from pEGFP-N2 into hefA, and its susceptibility profiles to 10 antibiotics were evaluated. RESULTS: The MIC of six multidrug-resistant strains (including 5 clinical isolates and H pylori NCTC11637) increased signifi cantly (≥ 4-fold) compared with their parent strains. The expression level of hefA gene was significantly higher in the MDR strains than in their parent strains (P = 0.033). A H pylori LZ1026 mutant was successfully constructed and the ΔH pylori LZ1026 was more susceptible to four of the 10 antibiotics. All the 20 strains displayed transcripts for hefA that con-fi rmed the in vitro expression of these genes.CONCLUSION: The efflux pump gene hefA plays an important role in multidrug resistance of H pylori.
基金supported by the Programme of Zhejiang Scientific Research Fund in Traditional Chinese Medicine,China(No.2011ZA094)the Zhejiang Provincial Natural Science Foundation of China(No.LY13H190008)
文摘Objective:The efflux pump(EP) is one of the major mechanisms of antibiotic resistance in Klebsiella pneumoniae.However,there are few reports on the effect of the abuse of antibiotic use on the activity of EPs.To determine whether the use of low efficacy antibiotics has any effect on the activity of EPs and induces drug resistance in K.pneumoniae,we investigated the effect of ciprofloxacin on the activity of EPs in K.pneumoniae strains.Methods:Sixteen susceptible K.pneumoniae strains were isolated from patients and their minimum inhibitory concentrations(MICs) of ciprofloxacin were measured in the absence and presence of the pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone(CCCP).The strains were then induced with a gradient of ciprofloxacin until the MICs of the strains showed no further increase,to obtain induced resistant strains.The EP activities of the strains before and after induction were compared using EP inhibition and ethidium bromide(EtBr) accumulation assays.Results:The MIC values of the strains were 16 256 times higher after induction than before induction.In the presence of CCCP,the MIC values of 50% of the induced strains were 2 4-fold lower than that in the absence of this inhibitor.The EtBr accumulation assay showed that the fluorescence of EtBr in the induced cells was lower than that in the cells before induction.Conclusions:EPs are widespread in susceptible and drug-resistant K.pneumoniae strains.Induction with ciprofloxacin may increase the activity of EPs in K.pneumoniae.The EtBr accumulation assay is more sensitive than the EP inhibition assay in evaluating the activity of EPs in K.pneumoniae.
基金We are grateful to Chinese Academy of Sciences(CAS)for jointly supports(project No.2018PB0089 to AJS and project No.2019VBA0026 to SDS)under CAS President’s International Fellowship Initiative(CAS-PIFI)projectsthe Major Project for Special Technology Innovation of Hubei Province(Grant No.2017AHB054 to MG).
文摘The upsurge of multiple drug resistance(MDR)bacteria substantially diminishes the effectiveness of antibiotic arsenal and therefore intensifies the rate of therapeutic failure.The major factor in MDR is efflux pump-mediated resistance.A unique pump can make bacteria withstand a wide range of structurally diverse compounds.Therefore,their inhibition is a promising route to eliminate resistance phenomenon in bacteria.Phytochemicals are excellent alternatives as resistance-modifying agents.They can directly kill bacteria or interact with the crucial events of pathogenicity,thereby decreasing the ability of bacteria to develop resistance.Numerous botanicals display noteworthy efflux pumps inhibitory activities.Edible plants are of growing interest.Likewise,some plant families would be excellent sources of efflux pump inhibitors(EPIs)including Apocynaceae,Berberidaceae,Convolvulaceae,Cucurbitaceae,Fabaceae,Lamiaceae,and Zingiberaceae.Easily applicable methods for screening plant-derived EPIs include checkerboard synergy test,berberine uptake assay and ethidium bromide test.In silico highthroughput virtual detection can be evaluated as a criterion of excluding compounds with efflux substrate-like characteristics,thereby improving the selection process and extending the identification of EPIs.To ascertain the efflux activity inhibition,real-time PCR and quantitative mass spectrometry can be applied.This review emphasizes on efflux pumps and their roles in transmitting bacterial resistance and an update plant-derived EPIs and strategies for identification.
文摘Objective:To evaluate the inhibitory activity of ferulic acid and four of its esterified derivatives(methyl,ethyl,propyl,and butyl)against resistance mechanisms in Staphylococcus aureus strains.Methods:Ferulic acid derivatives were obtained by esterification with methanol,ethanol,propanol,and butanol,and then characterized by hydrogen and carbon-13 nuclear magnetic resonance analysis.The minimum inhibitory concentrations(MIC)of ferulic acid and its esterified derivatives,ethidium bromide,and norfloxacin were obtained using the microdilution test,while the efflux pump inhibition test was conducted by examining reduction in the MICs of norfloxacin and ethidium bromide.Molecular docking was also carried out using the Schrodinger Suite 2015 molecular modeling software.A three-dimensional model of NorA efflux pump was generated using I-TASSER.The best scoring model was used as a receptor for ligand-receptor docking.Results:The methyl and butyl ester derivatives did not demonstrate significant antimicrobial activity.However,a significant synergic effect was evidenced when norfloxacin was combined with the ethyl and propyl esterified derivatives.The docking study demonstrated favorable energy of interaction between ferulate derivatives and NorA,and amino acid residues TYR57,TYR58,and LEU255 were present commonly in stabilizing all complexes.The PCA analysis corroborated the docking hypothesis that the lipophilic character and hydrogen bond interactions were the most relevant characteristics involved with NorA inhibitors.The pharmacokinetic parameters of ferulic acid derivatives showed good ADMET properties,demonstrating that they can be easily absorbed and have no effect or inhibit the cytochrome P450 enzyme complex,revealing their potential as drug candidates.Conclusions:This study provides strong evidence that the molecular basis for this activity is potentially due to the NorA efflux pump.
文摘Objectives:To identify and test the antibiotic susceptibility of nosocomial coliform bacilli and investigate the presence of oqxA and oqxB genes among the multidrug-resistant(MDR)phenotypes.Methods:One hundred and twenty different healthcare-associated infection samples were collected.Coliform bacilli were isolated,identified by conventional methods,and then antibiotic susceptibility tests were done using the VITEK2 system and disk diffusion methods.OqxAB operon was identified using a conventional PCR-based technique.oqxA and oqxB genes were compared between MDR Klebsiella pneumonia(K.pneumonia)phenotypes and MDR Escherichia coli(E.coli)phenotypes.Besides,oqxAB operons were compared between phenotypes of K.pneumonia and E.coli isolates.Results:Seventy coliform bacilli were isolated with the predominance of K.pneumonia and E.coli.Besides,82.1%of K.pneumonia strains and 53.3%of E.coli isolates were MDR phenotypes.Significant more oqxB genes alone were found in MDR E.coli than that in MDR K.pneumoniae phenotypes(χ^(2)=10.160,P=0.003).OqxAB operon was significantly more in MDR phenotypes of E.coli than that in the susceptible phenotypes(P<0.001).There was significantly less of this operon in susceptible E.coli isolates than that in susceptible K.pneumoniae isolates(P<0.001).OqxAB positive isolates that were also resistant to fluoroquinolones,tetracycline,trimethoprim,and chloramphenicol,most probably suggested functional pumps.Conclusions:MDR coliform bacilli are strongly implicated in healthcare-associated infection.Attention should be paid to the presence of oqxAB pump,as an important mechanism in the development of resistance against many antimicrobials because it contributes to co-resistance with other categories;therefore,this pump could be a good target for efflux pump inhibitors.
基金Supported by the Application Technology Research and Development Project in Heilongjiang Province of China(PC13S03)the Foundation of Heilongjiang Province Educational Committee of China(11541030)。
文摘Salmonella gallinarum has shown multiple drug resistance(MDR),especially high level fluoroquinolone(FQ)resistance in recent years.To determine whether the active efflux system was responsible for high-level FQ resistance,this research studied AcrAB efflux pump in Salmonella gallinarum on molecular level.The resistant strains were induced by standard strain C79-13 with ciprofloxacin in vitro.With carbonylcyanide-p-chlorophenyl hydrazone(CCCP)as an energy inhibitor,efflux inhibition test initially showed the potential impact of efflux pump on drug resistance.Sequence analysis of acrA gene indicated that gene mutation of AcrAB efflux pump was not definitely associated with MDR and drug resistance level of Salmonella gallinarum.Detected by competitive RT-PCR,the mRNA expression of acrA and acrB genes in the resistant strains significantly increased(p<0.01)compared with that of the control strain C79-13.The mRNA expression level of acrB gene(increased from 1.6-to 2.9-folds)was consistent with that of acrA gene(increased from 1.6-to 2.8-folds),which increased with the drug resistance level.However,gene mutation of acrA gene showed no correlation with its mRNA expression level,indicating that gene mutation did not affect the expression of AcrAB pump itself.The results suggested that the overexpression rather than the gene mutation of AcrAB efflux pump was an important factor causing the high level drug resistance of Salmonella gallinarum.
文摘Antimicrobial resistance(AMR)poses a significant threat to public health and is increasingly recognized within the“One Health”framework,which emphasizes the interconnectedness of human,animal,and environmental health.While extensive research has focused on regulating antibiotic use across healthcare and other sectors,the impact of intensive biocide use on AMR development,particularly in seawater-cooled systems,remains underexplored.In this study,we report the isolation and characterization of a multidrug-resistant Klebsiella quasipneumoniae strain from the cooling water system of a coastal power plant,where continuous chlorination at 0.2 ppm is employed for biofouling control.The isolated strain displayed broad-spectrum resistance to multiple biocides and antibiotics.Interestingly,the strain shown enhances biofilm formation in response to biocides and antibiotics,thereby compounding its resistance profile.Efflux assays with ethidium bromide(EtBr)and whole-genome sequencing revealed that efflux pumps are central to the resistance mechanisms.Additionally,the presence ofβ-lactamase(OKP-A)and FosA genes confers resistance to theβ-lactam and epoxide classes of antibiotics.The strain was found to be salt-tolerant and preferred to grow at normal salinity,indicating a non-marine origin of this isolate.These findings highlight the prevalence of biocide and antibiotic-resistant pathogens in marine cooling water systems that primarily rely on biocides for biofouling control.In line with One Health principles,our research advocates for a reassessment of biocide practices in marine cooling water systems and the implementation of proactive measures to mitigate the spread of antimicrobial resistance(AMR).
文摘<strong>Background:</strong> Overexpression of efflux pumps is the drug resistance and adaptation mechanism employed by some eukaryotes and bacteria to transport endogenous and chemotherapeutic compounds from the intracellular to the extracellular environment. <strong>Aim:</strong> The study aimed at establishing a fluorescent cell-based assay to monitor the efflux activities of an ABC-transporter, multi-drug resistance protein 4 (MRP4). <strong>Methods:</strong> DH5α competent <em>E. coli</em> cells were transformed with pcDNA-MRP4 by the heat-shock process. The presence of the MRP4 gene was analyzed by the digestion of plasmid using EcoRI and analyzed on a 1% agarose gel. HEK 293 cells were transfected with purified pcDNA-MRP4 under optimized conditions using a Polyethylenimine (PEI) protocol. The level of MRP4 in the HEK 293 cells was characterized by western blotting analysis using M4I-10 anti-MRP4 and anti-Rat IgG (whole molecule)-Alkaline phosphatase antibodies. The fluorescent uptake study was performed by the incubation of 0.02 mM 8-[fluo-cAMP] with the MRP4-transfected and control HEK 293 cells for 1 h. The level of fluorescence was analyzed using fluorescence microscopy and spectrometer. <strong>Results:</strong> The agarose gel analysis showed a plasmid of 9.4 kb and restriction product of 5 kb, which correspond with the pcDNA and MRP4 sizes respectively. The western blot results of the transfection showed 4 μg pcDNA-MRP4 and the N/P ratio of 9 was the optimized condition to transfect our HEK 293 cells as it showed the broadest band. In the efflux studies, the fluorescence images of the MRP4-transfected HEK 293 cells were very low compared to the untransfected control. The level of fluorescence accumulation was significantly (P ≤ 0.0001) higher 228.6 ± 13.1 RFU in the untransfected cells than the MRP4-transfected cells 8.6 ± 1.8 RFU. <strong>Conclusion:</strong> The higher levels of fluorescence detected in the control in both the fluorescent microscopy and spectrophotometer showed that MRP4-transfected cells had effluxed the 8-[fluo-cAMP] substrate out of the cell. This method could be employed in the detection of MRP4 functions in bacteria and cancer cells.
基金supported by grants from the National Key Research and Development Program of China(no.2023YFD1801000)the National Natural Science Foundation of China(no.32473090)+2 种基金the Natural Science Foundation of Henan Province(no.232300421037)the Henan Province University Young Backbone Teachers Training Program(no.2023GGJS031)Graduate Education Reform Project of Henan Agricultural University(no.NDYJS2023-26).
文摘RE-CmeABC is a variant of the efflux pump CmeABC in the major zoonotic pathogen Campylobacter,which is much more potent in conferring resistance to various antibiotics for the treatment of Campylobacter infections in both veterinary and medical clinics.This potent multidrug efflux pump RE-CmeABC is widely distributed and disseminated around the world,posing a serious threat to public health.In this study,we targeted the key efflux protein RE-CmeB of RE-CmeABC to explore promising efflux pump inhibitors derived from natural products to improve the multidrug resistance of Campylobacter.We constructed a protein screening model and utilized virtual screening techniques,such as molecular docking,to identify potential efflux pump inhibitors from natural product databases.The activity and safety of these candidates were subsequently evaluated,and ZINC338037 was shown to inhibit antibiotic extrusion and restore the susceptibility of multidrug-resistant Campylobacter.In summary,a potential multidrug efflux pump inhibitor was identified and is expected to be developed as an adjuvant drug for the treatment of clinical infections caused by multidrug-resistant Campylobacter.
基金supported by the National Key R&D Program of China(2022YFC2303200)the National Natural Science Foundation of China(32370199)+1 种基金the CAMS Initiative for Innovative Medicine(2021-I2M-1-035)the Beijing Jishuitan Research Funding(ZR-202520).
文摘Mycobacterium abscessus,one of the most antimicrobial-resistant bacteria,is increasingly recognized as the cause of infections that are difficult to treat.Novel genetic manipulation tools are required to elucidate the biology,pathogenesis,and antibiotic resistance mechanisms of M.abscessus.In this study,we modified the method used to prepare M.abscessus electrocompetent cells to achieve efficient transformation,and then optimized the CRISPR-Cas9-assisted genome-editing tools to allow efficient genetic manipulation.Using these tools,we constructed 66 efflux pump mutants of M.abscessus and investigated their roles in drug resistance and virulence.We found that different efflux pumps play distinct roles in drug resistance and survival in Galleria mellonella larvae.Finally,we confirmed that MAB_2806-2807,involved in transportation of triacylglycerides,is vital for the drug resistance and virulence of M.abscessus.The molecular biology tools developed in this study will facilitate molecular research on M.abscessus.In addition,the study on efflux pumps might provide new targets for the development of new drugs and treatment regimens for M.abscessus infection.
基金support for HPLC-QTOF-MS analysissupported by the PhD scholarship of School of Agriculture,Food and Ecosystem Sciences,Faculty of Science,University of Melbourne awarded to Mr Ze Liangsupported by the Researcher Development Grant,Faculty of Science,University of Melbourne awarded to Dr Pangzhen Zhang.
文摘The growing threat of antibiotic resistance calls for novel strategies to enhance antimicrobial efficacy.Blueberries,rich in phenolic compounds,have attracted attention as food-derived agents capable of modulating bacterial resistance mechanisms.This study explores the potential of phenolic-rich blueberry extracts as antibiotic resistance-modifying agents.To evaluate their modulatory effects,we conducted antibacterial testing,efflux pump inhibition assays,and transcriptomic analyses on Acinetobacter baumannii BAA-1605 treated with blueberry extract,gentamicin,and their combination.While the extract alone showed no direct antibacterial activity,its combination with gentamicin resulted in a twofold reduction in lowering the minimum inhibitory concentration against Acinetobacter baumannii BAA-1605.Mechanistic assays revealed that blueberry extract enhanced gentamicin's effect on inhibiting efflux pump activity.Transcriptomic analysis showed that the combination treatment of blueberry extract and gentamicin modulated genes involved in metabolism,stress response,and virulence,with Kyoto encyclopedia of genes and genomes(KEGG)and gene ontology(GO)enrichment pointing to altered energy production and cellular adaptation pathways.These findings suggest that blueberry phenolics do not act bactericidally but may affect bacterial physiology to potentiate gentamicin action.This study highlights the potential of natural plant phenolic compounds as antibiotic adjuvant and supports further investigation in their fighting against multidrug-resistant pathogens.
