去甲肾上腺素对大肠杆菌耐药质粒接合转移的影响
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摘要
[目的]本研究旨在探讨生产中普遍存在的急性应激因素对细菌耐药基因水平转移的可能影响。[方法]选取分离自断奶香猪粪便的2株喹诺酮类敏感型大肠杆菌作为试验菌株,通过测定D600值比较菌株在添加不同浓度去甲肾上腺素培养液中的生长曲线,确定去甲肾上腺素对于大肠杆菌的最佳促生长浓度;分离自断奶香猪粪便的7株携带质粒喹诺酮类耐药基因(aac(6')-lb-cr、oqx AB、qnr S)的大肠杆菌,采用滤膜接合法,计算去甲肾上腺素诱导和未诱导的大肠杆菌分离株(供体菌)与大肠杆菌J53(受体菌)的接合转移率;PCR检测接合子及供、受体菌质粒介导抗性基因的携带情况;采用K-B纸片扩散法和琼脂稀释法测定接合子及供、受体菌的多种抗生素最小抑菌浓度(MIC)。[结果]去甲肾上腺素对于大肠杆菌的促生长效果具有浓度依赖性,最佳促生长浓度为100μmol·L-1。7株携带质粒介导的喹诺酮类耐药基因的大肠杆菌中有4株(A、B、C、D)成功获得接合子,经去甲肾上腺素(100μmol·L-1)诱导后与受体菌的接合转移率与去甲肾上腺素未诱导组相比均升高,其中A、B、C 3株供体菌极显著升高(P<0.01)。所有接合子均扩增到恩诺沙星3种耐药基因:aac(6')-lb-cr、oqx AB、qnr S。接合子菌株对6种抗菌药物的MIC值与大部分供体菌株相同。[结论]去甲肾上腺素对于大肠杆菌的促生长效果具有浓度依赖性,且提高了大肠杆菌分离株耐药质粒体外接合转移率。提示:动物养殖生产中应加强应激管理,以减少细菌耐药基因水平传播的发生。
[Objectives]The aim at this paper was to evaluate the potential effects of acute stress factors on the level of the horizontal transfer of bacterial resistant genes in the animal production. [Methods]Two isolates from weaned Xiang pigs were selected as test strains.The optimal growth promotion concentration of norepinepheine( NE) for Escherichia coli was determined by measuring the growth curves of the D600. Four isolates( A,B,C,D) from weaned Xiang pigs were used to carry plasmid-mediated enrofloxacin resistant genes( aac( 6')-lb-cr,oqx AB,qnr S,the transfer rate of the conjugation between the donors( induced by NE or not) and the recipient( E. coli J53) were calculated in filter mating. The minimum inhibitory concentrations( MIC) of the transconjugants,donor strains and the recipient strain to 6 antibiotics were determined by the Kirby·Bauer test and the agar dilution method. The plasmid-mediated enrofloxacin resistant genes of the transconjugants,donor strains and the recipient strain were detected by PCR. [Results]The E. coli growth was promoted by NE dose-dependently,and the optimal growth promotion concentration was determined by the administration of100 μmol·L-1NE. The transfer rates of the conjugation between three donors( A,B,C) induced by NE( 100 μmol·L-1) and the recipient were significantly higher than those of control groups( the donors without exposure to NE)( P<0.01). Three plasmid-mediated enrofloxacin resistant genes( aac( 6')-lb-cr,oqx AB,qnr S) were detected in all transconjugants. The MIC of the transconjugants to 6 antibiotics were the same as those of the donor strains. [Conclusions]NE increased the transfer frequency of resistant plasmids of isolates in vitro,in addition,the E. coli growth was promoted by NE dose-dependently. Suggestion that the management of stress should be strengthened in the animal production and breeding in order to reduce the horizontal transfer of bacterial resistant genes.
[Objectives]The aim at this paper was to evaluate the potential effects of acute stress factors on the level of the horizontal transfer of bacterial resistant genes in the animal production. [Methods]Two isolates from weaned Xiang pigs were selected as test strains.The optimal growth promotion concentration of norepinepheine( NE) for Escherichia coli was determined by measuring the growth curves of the D600. Four isolates( A,B,C,D) from weaned Xiang pigs were used to carry plasmid-mediated enrofloxacin resistant genes( aac( 6')-lb-cr,oqx AB,qnr S,the transfer rate of the conjugation between the donors( induced by NE or not) and the recipient( E. coli J53) were calculated in filter mating. The minimum inhibitory concentrations( MIC) of the transconjugants,donor strains and the recipient strain to 6 antibiotics were determined by the Kirby·Bauer test and the agar dilution method. The plasmid-mediated enrofloxacin resistant genes of the transconjugants,donor strains and the recipient strain were detected by PCR. [Results]The E. coli growth was promoted by NE dose-dependently,and the optimal growth promotion concentration was determined by the administration of100 μmol·L-1NE. The transfer rates of the conjugation between three donors( A,B,C) induced by NE( 100 μmol·L-1) and the recipient were significantly higher than those of control groups( the donors without exposure to NE)( P<0.01). Three plasmid-mediated enrofloxacin resistant genes( aac( 6')-lb-cr,oqx AB,qnr S) were detected in all transconjugants. The MIC of the transconjugants to 6 antibiotics were the same as those of the donor strains. [Conclusions]NE increased the transfer frequency of resistant plasmids of isolates in vitro,in addition,the E. coli growth was promoted by NE dose-dependently. Suggestion that the management of stress should be strengthened in the animal production and breeding in order to reduce the horizontal transfer of bacterial resistant genes.
引文
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[20]吴村.去甲肾上腺素对空肠弯曲菌的促生长作用鉴定及表达谱分析[D].泰安:山东农业大学,2014.Wu C.Effects of norepinephrine on growth promotion and gene expression profiling of Campylobacter jejuni[D].Tai'an:Shandong Agricultural University,2014(in Chinese with English abstract).
[21]徐福洲,吴村,林军.微生物内分泌学:揭示微生物与激素相互作用对感染的影响机制[J].微生物学报,2013,53(9):901-907.Xu F Z,Wu C,Lin J.Microbial endocrinology:impact of interactions between microbes and neuroendocrine hormones on infection[J].Acta Microbiologica Sinica,2013,53(9):901-907(in Chinese with English abstract).
[22]Freestone P P,Williams P H,Haigh R D,et al.Growth stimulation of intestinal commensal Escherichia coli by catecholamines:a possible contributory factor in trauma-induced sepsis[J].Shock,2002,18(5):465-470.
[23]Martinez J L,Baquero F,Andersson D I.Predicting antibiotic resistance[J].Nat Rev Micro,2007,5(12):958-965.
[24]Davies J.Inactivation of antibiotics and the dissemination of resistance genes[J].Science,1994,264(5157):375-382.
[25]Halling-Srensen B,Nors Nielsen S,Lanzky P F,et al.Occurrence,fate and effects of pharmaceutical substances in the environment:a review[J].Chemosphere,1998,36(2):357-393.
[2]Reacher M H,Shah A,Livermore D M,et al.Bacteraemia and antibiotic resistance of its pathogens reported in England and Wales between 1990and 1998:trend analysis[J].British Medical Journal,2000,320(7229):213-216.
[3]郭蓉,张峰,郑卫江,等.撤除教槽料抗生素和无机铜对哺乳仔猪粪便大肠杆菌耐药性的影响[J].南京农业大学学报,2016,39(3):448-454.DOI:10.7685/jnau.201601057.Guo R,Zhang F,Zheng W J,et al.Effects of withdrawing antibiotic and copper in creep feed on the resistance of Escherichia coli isolated from feces of sucking piglets[J].Journal of Nanjing Agricultural University,2016,39(3):448-454(in Chinese with English abstract).
[4]Thomas C M.Paradigms of plasmid organization[J].Molecular Microbiology,2000,37(3):485-491.
[5]Burrus V,Pavlovic G,Decaris B,et al.Conjugative transposons:the tip of the iceberg[J].Molecular Microbiology,2002,46(3):601-610.
[6]Nakamura Y,Itoh T,Matsuda H,et al.Biased biological functions of horizontally transferred genes in prokaryotic genomes[J].Nat Genet,2004,36(7):760-766.
[7]van den Eede G,Aarts H,Buhk H J,et al.The relevance of gene transfer to the safety of food and feed derived from genetically modified(GM)plants[J].Food and Chemical Toxicology,2004,42(7):1127-1156.
[8]Furness J B.Types of neurons in the enteric nervous system[J].J Auton Nerv Syst,2000,81(1/2/3):87-96.
[9]Thomas C M,Nielsen K M.Mechanisms of,and barriers to,horizontal gene transfer between bacteria[J].Nat Rev Micro,2005,3(9):711-721.
[10]Lyte M,Ernst S.Catecholamine induced growth of gram negative bacteria[J].Life Sci,1992,50(3):203-212.
[11]Freestone P P E,Lyte M,Neal C P,et al.The mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial growth in the presence of transferrin or lactoferrin[J].Journal of Bacteriology,2000,182(21):6091-6098.
[12]Bearson B L,Bearson S M D.The role of the QseC quorum-sensing sensor kinase in colonization and norepinephrine-enhanced motility of Salmonella enterica serovar Typhimurium[J].Microbial Pathogenesis,2008,44(4):271-278.
[13]Dowd S E.Escherichia coli O157∶H7 gene expression in the presence of catecholamine norepinephrine[J].FEMS Microbiology Letters,2007,273(2):214-223.
[14]Chen C,Brown D R,Xie Y,et al.Catecholamines modulate Escherichia coli O157∶H7 adherence to murine cecal mucosa[J].Shock,2003,20(2):183-188.
[15]Clarke M B,Sperandio V.Events at the host-microbial interface of the gastrointestinal tractⅢ.Cell-to-cell signaling among microbial flora,host,and pathogens:there is a whole lot of talking going on[J].American Journal of Physiology,2005,288(6):1105-1109.
[16]Peterson G,Kumar A,Gart E,et al.Catecholamines increase conjugative gene transfer between enteric bacteria[J].Microbial Pathogenesis,2011,51(1/2):1-8.
[17]Watts J L.Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals[C]//Approved Standard,Third Edition.CLSI Document M31-A3.Wayne,PA:Clinical Laboratory Standards Institute,2008.
[18]Patel J B,Cockerill F R,Alder J,et al.Performance standards for antimicrobial susceptibility testing;twenty-fourth informational supplement[C]//CLSI Standards for Antimicrobial Susceptibility Testing.Wayne,PA:Clinical Laboratory Standards Institute,2014:1-226.
[19]Black P H.Central nervous system-immune system interactions:psychoneuroendocrinology of stress and its immune consequences[J].Antimicrobial Agents and Chemotherapy,1994,38(1):1-6.
[20]吴村.去甲肾上腺素对空肠弯曲菌的促生长作用鉴定及表达谱分析[D].泰安:山东农业大学,2014.Wu C.Effects of norepinephrine on growth promotion and gene expression profiling of Campylobacter jejuni[D].Tai'an:Shandong Agricultural University,2014(in Chinese with English abstract).
[21]徐福洲,吴村,林军.微生物内分泌学:揭示微生物与激素相互作用对感染的影响机制[J].微生物学报,2013,53(9):901-907.Xu F Z,Wu C,Lin J.Microbial endocrinology:impact of interactions between microbes and neuroendocrine hormones on infection[J].Acta Microbiologica Sinica,2013,53(9):901-907(in Chinese with English abstract).
[22]Freestone P P,Williams P H,Haigh R D,et al.Growth stimulation of intestinal commensal Escherichia coli by catecholamines:a possible contributory factor in trauma-induced sepsis[J].Shock,2002,18(5):465-470.
[23]Martinez J L,Baquero F,Andersson D I.Predicting antibiotic resistance[J].Nat Rev Micro,2007,5(12):958-965.
[24]Davies J.Inactivation of antibiotics and the dissemination of resistance genes[J].Science,1994,264(5157):375-382.
[25]Halling-Srensen B,Nors Nielsen S,Lanzky P F,et al.Occurrence,fate and effects of pharmaceutical substances in the environment:a review[J].Chemosphere,1998,36(2):357-393.