Influence of efflux pump inhibitors on the multidrug resistance of Helicobacter pylori

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.

Efflux pump gene hefA of Helicobacter pylori plays an important role in multidrug resistance

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.

Plant-derived secondary metabolites as the main source of efflux pump inhibitors and methods for identification

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.

Role of MexA-MexB-OprM Efflux Pump System in Chronic Pseudomonas Aeruginosa Pulmonary Infection in Mice

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.

Antibacterial activity and inhibition against Staphylococcus aureus NorA efflux pump by ferulic acid and its esterified derivatives

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.

Detection of oqxA and oqxB efflux pump genes among nosocomial coliform bacilli:An observational cross-sectional study

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.

AcrAB Efflux Pump in Fluoroquinolone Resistant Salmonella gallinarum Induced by Ciprofloxacin Selective Pressure

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.

A Fluorescent Cell-Based Technique for Monitoring Efflux of MRP4

Background: 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. Aim: 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). Methods: DH5α competent E. coli 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. Results: 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. Conclusion: 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.

Biogeochemical cyclic activity of bacterial arsB in arsenic-contaminated mines

Biogeochemical cyclic activity of the ars (arsenic resistance system) operon is arsB influx/effux encoded by the ecological of Pseudomonas putida.This suggests that studying arsenite-oxidizing bacteria may lead to a better understanding of molecular geomicrobiology,which can be applied to the bioremediation of arsenic-contaminated mines.This is the first report in which multiple arsB-binding mechanisms have been used on indigenous bacteria.In ArsB (strains OS-5; ABB83931; OS-19; ABB04282 and RW-28; ABB88574...

Whole Genome Analysis Reveals New Insights into Macrolide Resistance in Mycoplasma pneumoniae

Objective Mutations in 23 S rRNA gene are known to be associated with macrolide resistance in Mycoplasma pneumoniae（M. pneumoniae）. However, these mutations alone do not fully explain the high resistance rates in Asia. The aim of this study was to investigate other possible mutations involved in macrolide resistance in M. pneumoniae. Methods The whole genomes of 10 clinical isolates of M. pneumoniae with macrolide resistance were sequenced by Illumina Hi Seq2000 platform. The role of the macrolide-specific efflux transporter was assessed by efflux-pump inhibition assays with reserpine and carbonyl cyanide m-chlorophenyl-hydrazone（CCCP）. Results A total of 56 single nucleotide polymorphisms（SNPs） were identified in 10 clinical isolates in comparison to the reference strains M129 and FH. Strikingly, 4 of 30 SNPs causing non-synonymous mutations were clustered in macrolide-specific efflux system gene mac B encoding macrolide-specific efflux pump protein of the ATP-binding cassette transporter family. In assays of the minimal inhibitory concentrations（MIC） of macrolide antibiotics in the presence of the efflux pump inhibitors caused a significant decrease of MICs, even under detectable levels in some strains. Conclusion Our study suggests that macrolide efflux pump may contribute to macrolide resistance in M. pneumoniae in addition to the common point mutations in 23 S r RNA gene.

Reversal of Tetracycline Resistance by Cepharanthine,Cinchonidine,Ellagic Acid and Propyl Gallate in a Multi-drug Resistant Escherichia coli

Bacterial resistance to antibiotics is an increasing threat to global healthcare systems.We therefore sought compounds with potential to reverse antibiotic resistance in a clinically relevant multi-drug resistant isolate of Escherichia coli(NCTC 13400).200 natural compounds with a history of either safe oral use in man,or as a component of a traditional herb or medicine,were screened.Four compounds;ellagic acid,propyl gallate,cinchonidine and cepharanthine,lowered the minimum inhibi-tory concentrations(MICs)of tetracycline,chloramphenicol and tobramycin by up to fourfold,and when combined up to eightfold.These compounds had no impact on the MICs of ampicillin,erythromycin or trimethoprim.Mechanistic studies revealed that while cepharanthine potently suppressed efflux of the marker Nile red from bacterial cells,the other hit com-pounds slowed cellular accumulation of this marker,and/or slowed bacterial growth in the absence of antibiotic.Although cepharanthine showed some toxicity in a cultured HEK-293 mammalian cell-line model,the other hit compounds exhibited no toxicity at concentrations where they are active against E.coli NCTC 13400.The results suggest that phytochemicals with capacity to reverse antibiotic resistance may be more common in traditional medicines than previously appreciated,and may offer useful scaffolds for the development of antibiotic-sensitising drugs.

Mechanisms of tigecycline resistance in Gram-negative bacteria:A narrative review

Tigecycline serves as a critical“final-resort”antibiotic for treating bacterial infections caused by multidrug-resistant bacteria for which treatment options are severely limited.The increasing prevalence of tigecycline resistance,particularly among Gram-negative bacteria,is a major concern.Various mechanisms have been iden-tified as contributors to tigecycline resistance,including upregulation of nonspecific Resistance Nodulation Divi-sion(RND)efflux pumps due to mutations in transcriptional regulators,enzymatic modification of tigecycline by monooxygenase enzymes,and mutations affecting tigecycline binding sites.This review aims to consolidate our understanding of tigecycline resistance mechanisms in Gram-negative bacteria and offer insights and perspectives for further drug development.

Influence of induced ciprofloxacin resistance on efflux pump activity of Klebsiella pneumoniae

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.

Metal nanoparticles as inhibitors of enzymes and toxins of multidrug-resistant Staphylococcus aureus

Staphylococcus aureus is an aerobic Gram-positive spherical bacterium known to cause a broad range of infections worldwide.It is a major cause of infective skin and soft infections and severe and life-threatening conditions,such as pneumonia,bloodstream infections,and endocarditis.The emergence of drug-resistant strains of S aureus,par-ticularly methicillin-resistant S aureus(MRSA),has become a significant concern in the healthcare community.Antibiotic-resistant S aureus is commonly acquired in hospitals and long-term care facilities.It often affects pa-tients with weakened immune systems,those undergoing invasive medical procedures,or those who have been hospitalized for extended periods.In the US,S aureus is known to cause potentially fatal illnesses,such as toxic shock syndrome(TSS)and acute-onset toxic shock syndrome(TSS),which are characterized by fever and hy-potension.It develops resistance to antibiotics through several mechanisms,such as the production of enzymes that inactivate antibiotics,target site modification,efflux pumps,and plasmid-mediated resistance.Therefore,preventing the spread of drug-resistant S aureus is needed,and there is an urgent need to explore novel ap-proaches in the development of anti-staphylococcal agents.This article reviews the principal infections caused by S aureus,major virulence factors,mechanisms of resistance development,and nanotechnology-based solutions for the control of drug-resistant S aureus.

Antibacterial and Antibiotic-Potentiation Activities of Some Cameroonian Food Plants against Multi-Drug Resistant Gram-Negative Bacteria

Objective： To evaluate the in vitro antibacterial properties and the ability to potentiate some common antibiotics effects of the methanol extracts of 11 Cameroonian food plants on 29 Gram-negative bacteria expressing multidrug resistant （MDR） phenotypes. Methods： The antimicrobial activity of the extracts was performed using the broth microdilution method. The phytochemical screening of these extracts was also performed using standard methods. Resalts： Ocimum basilicum, Gnetum africanum and Eucalyptus robusta extracts possessed an antibacterial activity against all the 29 studied bacteria. The extracts from G. africanum and E. robusta were the most active with the lowest minimal inhibitory concentration of 64 p, g/mL on Escherichia coil AG100A for both extracts and also against Klebsiella pneumoniae K24 for G. africanum. When tested in the presence of phenylalanine-arginine 13 -Naphtylamide （PA 13 N）, an efflux pump inhibitor, the extract of Thymus vulgafis and E. Robusta showed the best activities on most tested strains. E. Robusta extract showed good synergistic effects, improving the activity of commonly used antibiotics in about 85% of cases. Coaclasion： The overall results obtained provide the baseline information for the use of the tested plants in the treatment of bacterial infections.

Genome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysis

Background Candida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression.Methods Candida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations （MICs, 4 mg/L） for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）. Results A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette （ABC） or major facilitator supeffamily （MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1）, genes involved in stress response and detoxification （CDR1, AGP2, HOL3）, and gene involved in the ergosterol biosynthesis pathway （ERG12）. The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis. Conclusions The up-regulation of the gene encoding the multidrug resistance efflux pump CDR1 may contribute to the terbinafine resistance in Candida albicans. However, the precise roles of other affected genes remain unclear, further studies of these genes and their respective products that play roles in the context of antifunqal resistance are warranted.