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.

Mechanisms Efflux Pumps of <i>Acinetobacter baumannii</i>(MDR): Increasing Resistance to Antibiotics

Acinetobacter baumannii has greatly increased its degree of resistance to become multidrug resistant (MDR) over the past 30 years and is on the red line of the most widely replicated bacteria according to World Health Organization (WHO). The efflux pumps are the main cause for the increasing antibiotic resistance of A. baumannii originated from nosocomial infection. The progressive resistance of A. baumannii even on the recent drugs (tigecycline and fosfomycin) reduces to very effective antibiotic scale. With attention focused on MDR and pan-drug-resistant (PDR) in A. baumannii multiple works on efflux pumps chemical inhibitor (NMP, PAβN, omeprazole, verapamil, reserpine, CCCP) are still in progress. Certain inhibitors from plants (Biricodar and timcodar, Falvone, Mahonia, Dalea versicolor, Lycopus europaeus, and Rosmarinus officinalis) have the capability to have such compounds according to their very significant synergistic effect with antibiotics. In this review we focused on the growth of antibiotic resistance to explain the mechanism of efflux pumps into these different super families and a comprehensive understanding of the extrusion, regulation and physiology role of drug efflux pumps in the essential development of anti-resistivity drugs. We recapitulated the evolution of the work carried out in these fields during the last years and in the course of elaboration, with the aim of increasing the chances of decreasing bacterial resistivity to antibiotics.

Drug interactions of dipeptidyl peptidase 4 inhibitors involving CYP enzymes and P-gp efflux pump

Dipeptidyl peptidase 4(DPP4) inhibitors are oral antidiabetic drugs approved to manage type 2 diabetes mellitus. Saxagliptin is a substrate of CYP3A4/5 enzymes while other DPP4 inhibitors such as sitagliptin, linagliptin, gemigliptin and teneligliptin are weak substrates of CYP3A4. DPP4 inhibitors have also been identified as substrates of P-gp. Hence, the drugs inhibiting or inducing CYP3A4/5 enzymes and/or P-gp can alter the pharmacokinetics of DPP4 inhibitors. This review is aimed to identify the drugs interacting with DPP4 inhibitors. The plasma concentrations of saxagliptin have been reported to be increased significantly by the concomitant administration of ketoconazole or diltiazem while no significant interactions between various DPP4 inhibitors and drugs like warfarin, digoxin or cyclosporine have been identified.

Mutational and Phylogenetic Analysis of <i>nfxB</i>Gene in Multidrug-Resistant Clinical Isolates of <i>Pseudomonas aeruginosa</i>Hyperexpressing MexCD-OprJ Efflux Pump

The present study focused on MexCD-OprJ efflux pump and its regulatory gene nfxB in multidrug resistant (MDR) clinical isolates of Pseudomonas aeruginosa collected from Kerala, South India. Semi-quantitative reverse transcription-PCR technique was employed to detect hyperexpression of the efflux pump gene, mexD. Amplicons from nfxB gene of isolates hyperexpressing the efflux pump were sequenced for mutational and phylogenetic analysis. Among 29 isolates of MDR P. aeruginosa, increased mexD transcription was detected in 10.3% of the isolates when compared with P. aeruginosa reference strain, PAO (MTCC-3541). Various synonymous and non-synonymous mutations in nfxB regulatory gene sequences were detected. Notably, mutations detected in the strains designate Pa6 and Pa7 have been found to be novel and are hitherto unreported in GenBank data base. The genetic divergence and homogeneity of the nfxB regulatory gene sequences of mexCD-oprJ operon were clearly apparent in the phylogram generated employing similar sequences retrieved from the public database.

Detection of the Mex Efflux Pumps in <i>Pseudomonas</i><i>aeruginosa</i>by Using a Combined Resistance-Phenotypic Markers and Multiplex RT-PCR

The aim of this study was to detect the expression of 4 clinically-important efflux pumps in the Resistance-Nodulation-Cell Division (RND) family including MexAB-OprM, MexXY, MexCD-OprJ and MexEF-OprN in Pseudomonas aeruginosa using a combination of resistance-phenotypic markers and multiplex RT-PCR (mRT-PCR). The antibiotic substrates specific for each Mex systems were used as phenotypic markers including carbenicillin, MexAB-OprM, erythromycin, MexCD-OprJ, norfloxacin and imipenem, MexEF-OprN and gentamicin, MexXY-OprM. The methods were validated with reference strains with known genotypes of the Mex systems and the potential applicability in clinical practice was tested with clinical isolates. The results for the reference strains support that the combination of resistance phenotype and mRT-PCR is a potential-attractive method for diagnosis of efflux-mediated resistance in P. aeruginosa. Further development to make it more practical for clinical use and study in a larger number of clinical isolates is required.

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.

Low nutrient levels as drinking water conditions can reduce the fitness cost of efflux pump-mediated ciprofloxacin resistance in Pseudomonas aeruginosa

The long-term persistence of antibiotic resistance in the environment, especially in drinking water, is a public health concern. Expression of an efflux pump, an important mechanism of resistance to antibiotics, usually confers a fitness cost in bacteria. In this study, we aimed to determine why antibiotic resistance conferred by overexpression of an efflux pump persisted in low-nutrient environments(TOC < 10 mg/L) such as drinking and source water in which antibiotic selective pressure might be very low or even absent.Competition experiments between wild-type Pseudomonas aeruginosa and ciprofloxacinresistant mutants revealed that the fitness cost of ciprofloxacin resistance significantly decreased(p < 0.05) under low-nutrient(0.5 mg/L total organic carbon(TOC)) relative to high-nutrient(500 mg/L TOC) conditions. Mechanisms underlying this fitness cost were analyzed. The mexD gene expression in resistant bacteria(cip3 strain) was significantly lower(p < 0.05) in low-nutrient conditions, with 10 mg/L TOC((8.01 ± 0.82)-fold), than in high-nutrient conditions, with 500 mg/L TOC((48.89 ± 4.16)-fold). Moreover, rpoS gene expression in resistant bacteria((1.36 ± 0.13)-fold) was significantly lower(p < 0.05) than that in the wild-type strain((2.78 ± 0.29)-fold) under low-nutrient conditions(10 mg/L TOC),suggesting a growth advantage. Furthermore, the difference in metabolic activity between the two competing strains was significantly smaller(p < 0.05) in low-nutrient conditions(5 and 0.5 mg/L TOC). These results suggest that nutrient levels are a key factor in determining the persistence of antibiotic resistance conferred by efflux pumps in the natural environment with trace amounts or no antibiotics.

昆明地区耐碳青霉烯铜绿假单胞菌10种膜蛋白编码基因表达的实验研究

目的 掌握昆明地区碳青霉烯耐药铜绿假单胞菌(CRPA)膜蛋白表达的分子流行情况,为临床合理用药及外排泵抑制剂的应用提供依据。方法 收集昆明地区四所医院2022年10月~2023年8月分离的铜绿假单胞菌,使用SYBR-PCR法定量检测10种膜蛋白编码基因mRNA相对表达量(RE),包括mexA,B,C,D,E,F,X,Y及oprD,M。根据头孢他啶(CAZ)、头孢吡肟(CFP)、亚胺培南(IPM)、美罗培南(MEM)耐药表型组合将菌株分为5组,包括全敏感组(Ⅰ组),全耐药组(Ⅱ组),IPM,MEM耐药、CAZ及CFP敏感组(Ⅲ组),IPM耐药、MEM非耐药(敏感或中介)组(Ⅳ组)和IPM,MEM耐药、CAZ和CFP非耐药组(Ⅴ组),分析不同耐药表型组间各膜蛋白编码基因的RE中位数情况。结果 共收集108株铜绿假单胞菌,Ⅰ组24株作为对照,84株为碳青霉烯耐药组,包括Ⅱ组32株,Ⅲ组22株,Ⅳ组13株和Ⅴ组17株。耐药组mexD,mexE,mexF,mexX和mexY表达高于对照组,差异具有统计学意义(U=409.5~661.0,均P<0.05);mexA,mexB,mexC,oprD,oprM与对照组比较,差异无统计学意义(U=767.0~1 004.5,均P>0.05)。各膜蛋白编码基因RE在不同医院来源菌株间的表达,差异无统计学意义(H=0.914~7.407,均P>0.05)。四组不同表型中,mexA和oprM RE在各组无规律分布与对照组比较差异无统计学意义(U_(mexA)=95.0~264.0,U_(oprM)=143.0~331.0);各组mexC RE均低于对照组,但差异无统计学意义(U=134.0~344.5,均P>0.05);mexE和mexY RE均高于对照组(U_(mexE)=48.0~230.0,U_(maxY)=83.0~184.0),mexB在Ⅳ组中低于对照组(U=72.0),差异具有统计学意义(均P<0.05)。mexD和mexF表现一致,在Ⅲ,Ⅳ,Ⅴ组表达高于对照组(U_(mexD)=34.0~102.0,U_(mexF)=65.0~113.0),mexX在Ⅱ,Ⅳ,Ⅴ组表达高于对照组(U=164.0,58.0,111.0),oprD仅在Ⅲ组表达低于对照组(U=140.0),差异具有统计学意义(均P<0.05);oprD在Ⅱ,Ⅳ,Ⅴ组中表达虽低于对照组,但差异无统计学意义(U=381.0,102.0,144.0,均P>0.05)。结论 mexCD,mexEF,mexXY是昆明地区CRPA主要外排泵的膜蛋白组合,通过mexD,E,F,X,Y膜蛋白表达上调加强外排,mexAB-oprM外排泵与该地区CRPA碳青霉烯耐药相关性低,oprD低表达在不产β-内酰胺酶的菌株中与外排机制共同发挥作用,但在产酶菌株中则未见显著的低表达差异。

多重耐药鲍曼不动杆菌对替加环素不敏感的耐药机制研究

目的探讨多重耐药鲍曼不动杆菌(MDR-AB)对替加环素不敏感的耐药机制,为临床合理用药及医院感染防控提供参考。方法收集2022年4月—2023年5月广州医科大学附属第一医院临床分离的替加环素不敏感MDR-AB(TIS-MDR-AB)及替加环素敏感MDR-AB(TS-MDR-AB)各22株。应用外排泵抑制剂羰基氰化物间氯苯腙(CCCP)进行外排泵表型抑制试验,采用聚合酶链式反应(PCR)技术对主要外排泵基因(ade B、ade G、ade J)和替加环素耐药基因tet(X)进行筛选,并应用实时荧光定量PCR检测其mRNA表达水平;Sanger测序分析外排泵调控基因ade RS的突变。结果两组MDR-AB外排泵基因ade B、ade G、ade J的检出率均>95%,未检测到tet(X)基因。外排泵抑制剂试验显示,TIS-MDR-AB菌株在加入CCCP后最低抑菌浓度(MIC)下降,且有3株菌株外排泵表型阳性。TIS-MDR-AB组MDR-AB ade B的mRNA表达水平高于TS-MDR-AB组(P<0.01),ade G和ade J基因的表达差异无统计学意义。ade R基因和ade S基因中发现多个突变,且有2株菌株ade S基因中插入ISA ba 1,3株菌株插入ISA ba 13。结论外排泵系统ade ABC过度表达可能在MDR-AB对替加环素敏感性下降机制中起重要作用,且其过度表达可能与外排泵调控基因ade RS中出现插入序列或突变有关。

烟曲霉外排泵唑类耐药研究进展

烟曲霉是一种广泛存在于自然界的机会性病原体,可引起侵袭性曲霉病,在免疫功能低下的患者中死亡率很高。三唑类药物是侵袭性曲霉病的一线治疗药物,然而近年来全球范围内烟曲霉唑类耐药分离株不断增多,极大地威胁到患者的生命健康。外排泵由跨膜蛋白质组成,可以将不需要的物质排出细胞,对微生物的存活有着重要的保护作用,与此同时,研究表明它还介导了微生物的耐药。文章综述了近年来关于烟曲霉药物外排泵的功能及调控机制的研究进展,为深入理解烟曲霉耐药机制及筛选抗烟曲霉感染药物新靶点提供理论基础。

微生物降解苯扎氯铵系统构建、优化及调控机制研究

为消除苯扎氯铵(BAC)对环境和人类健康带来的威胁,构建了微生物降解BAC系统,优化了系统的运行条件,探讨了微生物降解BAC的调控机制。结果表明,在曝气和添加10 g/L葡萄糖时,系统对初始质量浓度为50 mg/L的BAC的7 d去除率可达98.1%;初始BAC质量浓度若降为2.8 mg/L,2 d去除率接近100%。优化后,系统菌团中无色杆菌(Achromobacter sp.)占比达71.16%,是菌团降解BAC的主要功能菌。宏基因组分析和主要功能抑制剂实验验证结果表明,群体感应和外排泵等机制在菌团降解BAC中发挥着重要调控作用。研究结果有助于深入理解菌团对BAC的降解调控机制,为BAC污染的微生物治理提供新思路。

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.

鲍曼不动杆菌多重耐药性与外排泵及生物膜形成相关性研究

目的探讨临床感染性疾病所分离鲍曼不动杆菌(acinetobacter baumarmii,AB)的生物膜基因、外排泵基因及生物膜形成能力与耐药性的相关性。方法收集非重复鲍曼不动杆菌共119株(多重耐药鲍曼不动杆菌MDR-AB菌株74株,非多重耐药菌株45株),PCR检测鲍曼不动杆菌外排泵基因(adeB、adeJ、adeG、adeS和adeR)和生物膜相关基因(bap、ompA、csuA、csuB、csuC、csuD、csuE、abaI、bfmR、bfmS),应用24孔细胞培养板体外构建细菌生物膜,通过结晶紫染色法定性观察所有菌株是否形成生物膜。结果鲍曼不动杆菌对常用抗菌药物均多数为耐药,对替加环素、多粘菌素B、米诺环素较为敏感;生物膜相关基因的总体检出率约80%左右,多重耐药菌中bap、csuB、csuC、csuD基因检出率显著高于非多重耐药菌(P<0.05);主动外排泵基因的检出率不到70%,其中adeB、adeJ、adeS和adeR基因在多重耐药菌中的检出率明显高于非多重耐药菌(P<0.05);而adeG携带率相反。有3株多重耐药菌株及5株非多重耐药菌株未携带有上述外排泵相关基因,所有菌株均未检出abeM基因;95.80%菌株可观察到生物膜的形成。结论医院内检出的鲍曼不动杆菌多呈现出多重耐药特点,多数具有较强的生物膜形成能力,多重耐药菌生物膜基因和外排泵基因携带率更高,可能参与调控形成其多重耐药性。

基于分子对接与分子动力学模拟的NorA抑制剂虚拟筛选

目的 基于计算机的方法筛选天然产物数据库中潜在的新型NorA抑制剂。方法 利用分子对接、ADMET性质预测、分子动力学模拟、结合自由能计算等方法进行虚拟筛选。结果 筛选得到4个化合物,它们通过与NorA蛋白发生特异性非共价相互作用而结合,分子动力学模拟也表明这些复合物体系在模拟时间内保持稳定。结论 这4个化合物具有潜在的NorA抑制活性,本研究为发现新的NorA抑制剂提供了新思路,并为进一步实验验证和药物开发提供了潜在候选化合物。