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.

幽门螺杆菌Acr AB-Tol C外排泵相关基因hefA/D/G在克拉霉素耐药性患者中的表达

目的观察幽门螺杆菌的Acr AB-Tol C外排泵相关基因hef A/D/G m RNa在克拉霉素耐药性患者的表达水平。方法选取2018年3月-2019年12月因消化不良于佛山市第五人民医院就诊的患者303例,筛选纳入幽门螺旋杆菌阳性患者100例,且行胃镜检查。分析就诊患者的幽门螺旋杆菌的阳性的感染率,分析其对克拉霉素的耐药性,使用RNAiso Reagent Kit RNA提取组织中的总RNA,RNA逆转录为c DNA,Real-time PCR检测hef A/D/G m RNA的表达水平。结果303例因消化不良就诊患者中,幽门螺旋杆菌阳性100例(33.00%),男女幽门螺旋杆菌感染率比较差异无统计学意义(P>0.05);100例幽门螺旋杆菌阳性患者中,耐药率为39.00%。克拉霉素耐药组hef A、hef D和hef G m RNA表达水平显著高于克拉霉素敏感组(P<0.01)。结论幽门螺旋杆菌的耐药患者中外排泵的同源基因hef A、hef D、hef G表达水平升高,因此幽门螺旋杆菌对克拉霉素的耐药性与幽门螺旋杆菌的外排泵关系极为密切,可能成为幽门螺旋杆菌耐克拉霉素发生机制之一。

幽门螺杆菌多重耐药株hefA基因表达的检测与分析

目的了解hefA外排基因表达与贵阳市幽门螺杆菌(Hp)多重耐药的关系。方法提取耐3种抗生素的多重耐药菌株(10株)、敏感菌株(6株)及质控菌株SS1的基因组DNA,PCR扩增hefA基因片段,PCR产物经2%琼脂糖凝胶电泳检测后测序,将测序基因片段序列输入NCBI中进行BLAST比对;利用TRIzol试剂提取上述细菌的RNA并逆转录成cDNA,以16sRNA为内参基因,通过实时荧光定量PCR(Real time PCR)检测hefA基因和内参基因的CT值,通过比较2-ΔΔCT判断多重耐药菌株和敏感菌株hefA基因表达的差异,以两小样本t检验进行统计学分析。结果 2%琼脂糖凝胶电泳显示,6株敏感菌和10株多耐药菌及SS1hefA基因PCR产物均约140bp,测序显示受试菌hefA基因序列与SS1菌株序列的一致性大于96%;RT-PCR检测hefA外排基因的表达,敏感菌株的2-ΔΔCT为0.895±0.540,多重耐药菌株的2-ΔΔCT为3.387±1.597,差异无统计学意义(t=9.399,P<0.05)。结论 hefA外排基因高表达是贵阳地区Hp多重耐药的机制之一。

Effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics

AIM To evaluate the role of biofilm formation on the resistance of Helicobacter pylori(H. pylori) to commonly prescribed antibiotics, the expression rates of resistance genes in biofilm-forming and planktonic cells were compared.METHODS A collection of 33 H. pylori isolates from children and adult patients with chronic infection were taken for the present study. The isolates were screened for biofilm formation ability, as well as for polymerase chain reaction(PCR) reaction with HP1165 and hp1165 efflux pump genes. Susceptibilities of the selected strains to antibiotic and differences between susceptibilities of planktonic and biofilm-forming cell populations were determined. Quantitative real-time PCR(qP CR) analysis was performed using 16 S r RNA gene as a H. pylori-specific primer, and two efflux pumps-specific primers, hp1165 and hefA.RESULTS The strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidalconcentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qP CR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones.CONCLUSION The present work demonstrated an association between H. pylori biofilm formation and decreased susceptibility to all the antibiotics tested. This decreased susceptibility to antibiotics was associated with enhanced functional activity of two efflux pumps: hp1165 and hefA.

张志坚后辛汤和法缓治内科杂病

张志坚教授认为郁证治疗以怡郁开怀,心理疏导为上。用药以轻扬和解为宜,偏寒偏热均不适当。无论郁证或其他内伤杂病,具备时时叹息,胸闷不舒,口苦或兼咽干胸胁胀痛等症均可使用后辛汤。

和解法防治原发性肝癌化疗栓塞综合征的疗效研究

目的:观察和解法防治原发性肝癌化疗栓塞综合征的临床疗效。方法:治疗组予以和解法辨证中药配合西医对症处理,对照组仅以西医对症处理,2组对照观察。结果:中医症状积分、KPS评分、肝功能改善方面,治疗组较对照组有显著性差异(P<0.05)。结论:和解法能有效治疗化疗栓塞综合征,提高患者生活质量,提高介入治疗效果。

生物喷气燃料应用进展

生物喷气燃料在全生命周期内能有效降低二氧化碳排放,是航空业实现减排目标的重要手段。2020年,催化热解工艺生产的煤油组分(CHJ-SPK)和烃类、脂类和脂肪酸类加氢改质工艺生产的煤油组分(HC-HEFA SPK)两类调合组分纳入了ASTM D7566规范中,拓宽了生物喷气燃料的来源,有利于生物喷气燃料的大规模应用。

赵文霞教授“和法”治疗胃脘痛经验介绍

胃脘痛主要由于外邪犯胃、饮食伤胃、情志不畅和脾胃素虚等导致胃气郁滞,胃失和降,不通则痛。其病理因素主要有寒凝、气滞、湿阻、食积、热郁、血瘀。是消化内科常见病,多发病,赵文霞教授行医40余载,对胃脘痛的治疗灵活运用理气和胃的治疗大法,从广义的角度去理解和运用和法,即“温而和之”“清而和之”“泄而和之”“消而和之”“润而和之”“行而和之”“补而和之”等,用于临床效果卓著。