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...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。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
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 ...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.METHODSA 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 (qPCR) analysis was performed using 16S rRNA gene as a H. pylori-specific primer, and two efflux pumps-specific primers, hp1165 and hefA.RESULTSThe strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidal concentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qPCR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones.CONCLUSIONThe 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.展开更多
文摘目的了解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。
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.
文摘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.METHODSA 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 (qPCR) analysis was performed using 16S rRNA gene as a H. pylori-specific primer, and two efflux pumps-specific primers, hp1165 and hefA.RESULTSThe strains were resistant to amoxicillin, metronidazole, and erythromycin, except for one strain, but they were all susceptible to tetracycline. Minimum bactericidal concentrations of antibiotics in the biofilm-forming cells were significantly higher than those of planktonic cells. qPCR demonstrated that the expression of efflux pump genes was significantly higher in the biofilm-forming cells as compared to the planktonic ones.CONCLUSIONThe 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.
