以Real-time PCR方法检测耐药幽门螺杆菌中HefABC基因的表达
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摘要
为了探讨耐药幽门螺杆菌(Helicobacter. pylori)主动外排系统hefA、hefB、hefC基因表达与菌株克拉霉素耐药、阿莫西林耐药以及多重耐药的关系,随机选择H. pylori克拉霉素耐药株、阿莫西林耐药株、敏感株、多重耐药株各10株,利用Real-time PCR方法检测HefABC系统的表达,以成组t检验法对敏感株与各耐药株之间mRNA表达水平进行比较。结果显示,hefA在敏感组、克拉霉素耐药组、阿莫西林耐药组、多重耐药组中的mRNA表达量以2-ΔΔCt表示,分别为0.903 9±0.154 07、1.482 2±0.378 5、1.278 0±0.206 2、2.255 7±0.289 2,克拉霉素耐药组与敏感组比较:t=4.489,p<0.05;阿莫西林耐药组与敏感组比较:t=4.631,p<0.05;多重耐药组与敏感组比较:t=13.11,p<0.05。hefB在敏感组、克拉霉素耐药组、阿莫西林耐药组、多重耐药组中的m RNA表达量以2-ΔΔCt表示,分别为0.075 9±0.015 7、0.636 1±0.162 3、0.906 4±0.127 6、1.149 2±0.305 8,克拉霉素耐药组与敏感组比较:t=10.86,p<0.05;阿莫西林耐药组与敏感组比较:t=20.42,p<0.05;多重耐药组与敏感组比较:t=11.09,p<0.05。hefC在敏感组、克拉霉素耐药组、阿莫西林耐药组、多重耐药组中的m RNA表达量以2-ΔΔCt表示,分别为0.128 2±0.054 5、1.319 4±0.272 4、0.683 0±0.185 0、3.480 2±0.932 6;克拉霉素耐药组与敏感组比较:t=13.56,p<0.05;阿莫西林耐药组与敏感组比较:t=9.096,p<0.05;多重耐药组与敏感组比较:t=11.35,p<0.05。研究结果证实,H. pylori中外排系统hefA、hefB、hefC的高表达与菌株对克拉霉素耐药、阿莫西林耐药及多重耐药均有关。
This study aimed to explore the role of hefA, hefB and hefC gene expression of H. pylori in amoxicillin resistance, clarithromycin resistance and multiple drug resistance. 10 strains of amoxicillin-resistant strains, 10 strains of clarithromycin resistant strains, 10 strains of susceptible strains and 10 multidrug resistant strains were randomly selected to detect the expression of HefABC system by Real-time PCR method. The m RNA expression levels between susceptible and resistant strains were compared, and statistical analysis was carried out by t test. Expression levels of hefA gene in each group were as follows: susceptible group(0.903 9±0.1540 7), clarithromycin-resistant group(1.482 2±0.378 5), amoxicillin-resistant group(1.278 0±0.206 2) and multidrug-resistant group(2.255 7±0.289 2). The expression of resistant strains of clarithromycin was higher than the susceptible strains(t=4.489,p<0.05). The expression of resistant strains of amoxicillin was higher than the susceptible strains(t=4.631, p<0.05).The expression of resistant strains of multidrug-resistant was significantly higher than the susceptible strains(t=13.11,p<0.05). Expression levels of hefB in each group were as follows: susceptible group(0.075 9±0.015 7), clarithromycin-resistant group(0.636 1±0.162 3), amoxicillin-resistant group(0.906 4±0.127 6) and multidrug-resistant group(1.149 2 ±0.305 8). The expression of resistant strains of clarithromycin was higher than the susceptible strains(t=10.86, p<0.05). The expression of resistant strains of amoxicillin was higher than the susceptible strains(t=20.42,p <0.05). The expression of resistant strains of multidrug-resistant was significantly higher than the susceptible strains(t=11.09, p<0.05)). Expression levels of hefC in each group were as follows: susceptible group(0.128 2±0.054 5),clarithromycin-resistant group(1.319 4±0.272 4), amoxicillin-resistant group(0.683 0±0.185 0) and multidrug-resistant group(3.480 2±0.932 6). The expression of resistant strains of clarithromycin was higher than the susceptible strains(t=13.56, p<0.05)). The expression of resistant strains of amoxicillin was higher than the susceptible strains(t=9.096,p <0.05)). The expression of resistant strains of multidrug-resistant was significantly higher than the susce ptible strains(t=11.35, p<0.05)). The high expression of hefA, hefB, hefC in the efflux system is related to H. pylori resistance to clarithromycin, amoxicillin resistance and multiple drug resistance.
This study aimed to explore the role of hefA, hefB and hefC gene expression of H. pylori in amoxicillin resistance, clarithromycin resistance and multiple drug resistance. 10 strains of amoxicillin-resistant strains, 10 strains of clarithromycin resistant strains, 10 strains of susceptible strains and 10 multidrug resistant strains were randomly selected to detect the expression of HefABC system by Real-time PCR method. The m RNA expression levels between susceptible and resistant strains were compared, and statistical analysis was carried out by t test. Expression levels of hefA gene in each group were as follows: susceptible group(0.903 9±0.1540 7), clarithromycin-resistant group(1.482 2±0.378 5), amoxicillin-resistant group(1.278 0±0.206 2) and multidrug-resistant group(2.255 7±0.289 2). The expression of resistant strains of clarithromycin was higher than the susceptible strains(t=4.489,p<0.05). The expression of resistant strains of amoxicillin was higher than the susceptible strains(t=4.631, p<0.05).The expression of resistant strains of multidrug-resistant was significantly higher than the susceptible strains(t=13.11,p<0.05). Expression levels of hefB in each group were as follows: susceptible group(0.075 9±0.015 7), clarithromycin-resistant group(0.636 1±0.162 3), amoxicillin-resistant group(0.906 4±0.127 6) and multidrug-resistant group(1.149 2 ±0.305 8). The expression of resistant strains of clarithromycin was higher than the susceptible strains(t=10.86, p<0.05). The expression of resistant strains of amoxicillin was higher than the susceptible strains(t=20.42,p <0.05). The expression of resistant strains of multidrug-resistant was significantly higher than the susceptible strains(t=11.09, p<0.05)). Expression levels of hefC in each group were as follows: susceptible group(0.128 2±0.054 5),clarithromycin-resistant group(1.319 4±0.272 4), amoxicillin-resistant group(0.683 0±0.185 0) and multidrug-resistant group(3.480 2±0.932 6). The expression of resistant strains of clarithromycin was higher than the susceptible strains(t=13.56, p<0.05)). The expression of resistant strains of amoxicillin was higher than the susceptible strains(t=9.096,p <0.05)). The expression of resistant strains of multidrug-resistant was significantly higher than the susce ptible strains(t=11.35, p<0.05)). The high expression of hefA, hefB, hefC in the efflux system is related to H. pylori resistance to clarithromycin, amoxicillin resistance and multiple drug resistance.
引文
Bina J.E.,Alm R.A.,Urianickelsen M.,Thomas S.R.,Trust T.J.,and Hancock R.E.,2000,Helicobacter pylori uptake and efflux:basis for intrinsic susceptibility to antibiotics in vitro,Antimicrob.Agents Chemother.,44(2):248-254
Buckner M.M.,Blair J.M.,Ragione R.M.,Newcombe J.,Dwyer D.J.,Ivens A.,and Piddock L.J.,2016,Beyond antimicrobial resistance:evidence for a distinct role of the acrd efflux pump insalmonella biology,m Bio.,7(6):e01916
Belzer C.,Stoof J.,Breijer S.,Kusters J.G.,Kuipers E.J.,and van Vliet A.H.,2009,The Helicobacter hepaticus hefA gene is involved in resistance to amoxicillin,Helicobacter,14(1):72-79
Chai Y.P.,Liu Z.Q.,Zheng Y.P.,Dang J.Y.,and Wu J.J.,2009,Multiple drug resistance of clinical isolates of Helicobacter pylori,Zhongwai Yiliao(The Chinese and Foreign Medical),28(33):64-65(柴玉萍,刘志强,郑鹏远,党君英,吴菁菁,2009,幽门螺杆菌幽门螺杆菌临床分离株多重耐药研究,中外医疗,28(33):64-65)
Francesco V.D.,Zullo A.,Hassan C.,Giorgio F.,Rosania R.,and Ierardi E.,2011,Mechanisms of Helicobacter pylori antibiotic resistance:an updated appraisal,World Journal of Gastrointestinal Pathophysiology,2(3):35
Huang Y.Q.,Huang G.R.,Wu M.H.,Tang H.Y.,Huang Z.S.,Zhou X.H.,Yu W.Q.,Su J.W.,Mo X.Q.,Chen B.P.,Zhao L.J.,Huang X.F.,Wei H.Y.,and Wei L.D.,2015,Inhibitory effects of emodin,baicalin,schizandrin and berberine on hef A gene:treatment of Helicobacter pylori-induced mul-tidrug resistance,World J.Gastroenterol.,21(14):4225-4231
Iwamoto A.,Tanahashi T.,Okada R.,Yoshida Y.,Kikuchi K.,Keida Y.,Murakami Y.,Yang L.,Yamamoto K.,Nishiumi S.,Yoshida M.,and Azuma T.,2014,Whole-genome sequencing o f clarithromycin resistant helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes,Gut Pathogens,6(1):27
Kazakos E.I.,Dorrell N.,Polyzos S.A.,Deretzi G.,and Kountouras J.,2017,Comment on“effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics”,World J.Gastroenterol.,23(7):1163-1170
Kaman S.,Guarani M.,Luneburg D.L.,Kubat R.,and Patrauchan M.A.,2017,Calcium induces tobramycin resistance in pseudomonas aeruginosa by regulating RND efflux pumps,Cell Calcium,61:32-43
Kutschke A.,and de Jonge B.L.,2005,Compound efflux in Helicobacter pylori,Antimicrobial Agents&Chemotherapy,49(7):3009-3010
Liu Z.Q.,and Zheng P.Y.,2008,Important role of efflux pump gene hefA in multidrug resistance of Helicobacter pylori,Shijie Huaren Xiaohua Zazhi(World Chinese Journal of Digestology),16(16):1751-1756(刘志强,郑鹏远,2008,主动外排泵外膜蛋白编码基因hefA在幽门螺杆菌多重耐药中的作用,世界华人消化杂志,16(16):1751-1756)
Puzari M.,and Chetia P.,2017,Rnd efflux pump mediated antibiotic resistance in gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa:a major issue worldwide,World J.Microbiol.Biotechnol.,33(2):24
Tian Z.X.,Yi X.X.,Cho A.,O'Gara F.,and Wang Y.P.,2016,Cpxr activates MexAB-oprm efflux pump expression and enhances antibiotic resistance in both laboratory and clinicalnalbtype isolates of pseudomon as aeruginosa,PLo SPathogens,12(10):e1005932
Trainor E.A.,Horton K.E.,Savage P.B.,Testerman T.L.,and Mcgee D.J.,2011,Role of the hefc efflux pump in Helicobacter pylori cholesterol-dependent resistance to ceragenins and bile salts,Infect.Immun.,79(1):88-97
Tseng Y.S.,Wu D.C.,Chang C.Y.,Kuo C.H.,Yang Y.C.,and Jan C.M.,2009,Amoxicillin resistance withβ-lactamase production in helicobacter pylori,Eur.J.Clin.Invest.,39(9):807-812
Wang H.,Bi H.Y.,Pan K.,Qi T.N.,Bi Y.K.,Wang F.,and Chen Z.H.,2015,Expression of the hefA efflux pump gene in MDR Helicobacter pylori isolated from the Guiyang area,Zhongguo Bingyuan Weishengwuxue Zazhi(Journal of Pathogenic Biology),(5):393-396(王欢,毕红燕,潘科,綦廷娜,毕雅坤,王菲,陈峥宏,2015,幽门螺杆菌多重耐药株hefA基因表达的检测与分析,中国病原生物学杂志,(5):393-396)
Zheng Y.P.,Zheng P.Y.,and Liu Z.Q.,2007,Analysis of drug resistance of Helicobacter pylori,Zhengzhou Daxue Xuebao(Yixueban)(Journal of Zhengzhou University(Medical Science)),42(3):512-514(郑云鹏,郑鹏远,刘志强,2007,幽门螺杆菌的耐药性分析,郑州大学学报(医学版),42(3):512-514)
Buckner M.M.,Blair J.M.,Ragione R.M.,Newcombe J.,Dwyer D.J.,Ivens A.,and Piddock L.J.,2016,Beyond antimicrobial resistance:evidence for a distinct role of the acrd efflux pump insalmonella biology,m Bio.,7(6):e01916
Belzer C.,Stoof J.,Breijer S.,Kusters J.G.,Kuipers E.J.,and van Vliet A.H.,2009,The Helicobacter hepaticus hefA gene is involved in resistance to amoxicillin,Helicobacter,14(1):72-79
Chai Y.P.,Liu Z.Q.,Zheng Y.P.,Dang J.Y.,and Wu J.J.,2009,Multiple drug resistance of clinical isolates of Helicobacter pylori,Zhongwai Yiliao(The Chinese and Foreign Medical),28(33):64-65(柴玉萍,刘志强,郑鹏远,党君英,吴菁菁,2009,幽门螺杆菌幽门螺杆菌临床分离株多重耐药研究,中外医疗,28(33):64-65)
Francesco V.D.,Zullo A.,Hassan C.,Giorgio F.,Rosania R.,and Ierardi E.,2011,Mechanisms of Helicobacter pylori antibiotic resistance:an updated appraisal,World Journal of Gastrointestinal Pathophysiology,2(3):35
Huang Y.Q.,Huang G.R.,Wu M.H.,Tang H.Y.,Huang Z.S.,Zhou X.H.,Yu W.Q.,Su J.W.,Mo X.Q.,Chen B.P.,Zhao L.J.,Huang X.F.,Wei H.Y.,and Wei L.D.,2015,Inhibitory effects of emodin,baicalin,schizandrin and berberine on hef A gene:treatment of Helicobacter pylori-induced mul-tidrug resistance,World J.Gastroenterol.,21(14):4225-4231
Iwamoto A.,Tanahashi T.,Okada R.,Yoshida Y.,Kikuchi K.,Keida Y.,Murakami Y.,Yang L.,Yamamoto K.,Nishiumi S.,Yoshida M.,and Azuma T.,2014,Whole-genome sequencing o f clarithromycin resistant helicobacter pylori characterizes unidentified variants of multidrug resistant efflux pump genes,Gut Pathogens,6(1):27
Kazakos E.I.,Dorrell N.,Polyzos S.A.,Deretzi G.,and Kountouras J.,2017,Comment on“effect of biofilm formation by clinical isolates of Helicobacter pylori on the efflux-mediated resistance to commonly used antibiotics”,World J.Gastroenterol.,23(7):1163-1170
Kaman S.,Guarani M.,Luneburg D.L.,Kubat R.,and Patrauchan M.A.,2017,Calcium induces tobramycin resistance in pseudomonas aeruginosa by regulating RND efflux pumps,Cell Calcium,61:32-43
Kutschke A.,and de Jonge B.L.,2005,Compound efflux in Helicobacter pylori,Antimicrobial Agents&Chemotherapy,49(7):3009-3010
Liu Z.Q.,and Zheng P.Y.,2008,Important role of efflux pump gene hefA in multidrug resistance of Helicobacter pylori,Shijie Huaren Xiaohua Zazhi(World Chinese Journal of Digestology),16(16):1751-1756(刘志强,郑鹏远,2008,主动外排泵外膜蛋白编码基因hefA在幽门螺杆菌多重耐药中的作用,世界华人消化杂志,16(16):1751-1756)
Puzari M.,and Chetia P.,2017,Rnd efflux pump mediated antibiotic resistance in gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa:a major issue worldwide,World J.Microbiol.Biotechnol.,33(2):24
Tian Z.X.,Yi X.X.,Cho A.,O'Gara F.,and Wang Y.P.,2016,Cpxr activates MexAB-oprm efflux pump expression and enhances antibiotic resistance in both laboratory and clinicalnalbtype isolates of pseudomon as aeruginosa,PLo SPathogens,12(10):e1005932
Trainor E.A.,Horton K.E.,Savage P.B.,Testerman T.L.,and Mcgee D.J.,2011,Role of the hefc efflux pump in Helicobacter pylori cholesterol-dependent resistance to ceragenins and bile salts,Infect.Immun.,79(1):88-97
Tseng Y.S.,Wu D.C.,Chang C.Y.,Kuo C.H.,Yang Y.C.,and Jan C.M.,2009,Amoxicillin resistance withβ-lactamase production in helicobacter pylori,Eur.J.Clin.Invest.,39(9):807-812
Wang H.,Bi H.Y.,Pan K.,Qi T.N.,Bi Y.K.,Wang F.,and Chen Z.H.,2015,Expression of the hefA efflux pump gene in MDR Helicobacter pylori isolated from the Guiyang area,Zhongguo Bingyuan Weishengwuxue Zazhi(Journal of Pathogenic Biology),(5):393-396(王欢,毕红燕,潘科,綦廷娜,毕雅坤,王菲,陈峥宏,2015,幽门螺杆菌多重耐药株hefA基因表达的检测与分析,中国病原生物学杂志,(5):393-396)
Zheng Y.P.,Zheng P.Y.,and Liu Z.Q.,2007,Analysis of drug resistance of Helicobacter pylori,Zhengzhou Daxue Xuebao(Yixueban)(Journal of Zhengzhou University(Medical Science)),42(3):512-514(郑云鹏,郑鹏远,刘志强,2007,幽门螺杆菌的耐药性分析,郑州大学学报(医学版),42(3):512-514)