人肠道微生态恒化器模拟系统建立
|
摘要
残留在动物可食性组织中的抗菌药物被人体摄入后,可能对人体肠道菌群产生诸多不良影响,如选择出耐药的肠道菌群或使肠道细菌耐药性增强,破坏肠道菌群的屏障作用,改变肠道菌群代谢活性等,从而对人类健康构成巨大威胁。在国际上,抗菌药残留对人体肠道菌群影响的安全性评价研究日渐成为热点。但国内由于缺乏被国际组织普遍认可的评价模型,该领域的研究工作进展缓慢。本研究利用细菌连续培养的恒化器原理,通过对稀释率和转入连续培养时间两方面条件的摸索和优化,建立了人肠道微生态恒化器模拟系统。该模拟系统是国际组织推荐的两大类模型之一,其建立将会促进国内外该领域的研究,对保障人类食品和医学安全具有极其重要的意义。
本研究选用拟杆菌—胆汁—七叶苷琼脂培养基、BBL琼脂培养基、伊红美兰琼脂培养基和肠球菌选择性培养基,分别分离计数人粪便中四种优势代表菌(脆弱拟杆菌、双歧杆菌、大肠杆菌和肠球菌)。建立粪便中三种主要短链脂肪酸(SCFA,即乙酸、丙酸和丁酸)浓度测定的气相色谱方法。将50mL(20%)的新鲜粪便悬液接种于发酵罐中,厌氧条件下分批培养,定时取样,采用平板稀释计数法记录培养液中四种优势代表菌的数目,绘制其分批培养的菌体生长曲线,计算对数生长期。在此基础上,以培养液中四种优势代表菌数目和三种主要的短链脂肪酸浓度为监测指标,优化连续培养工艺,包括稀释率(0.05h~(-1)、0.07h~(-1)和0.09h~(-1))和转入连续培养的时间(第5h、第7h和第9h),建立人肠道微生态恒化器模拟系统。在优化后的培养条件下进行为期30 d的试验,进一步评价所建立的模拟系统的稳定性和可靠性。
分批培养显示,四种优势菌的对数生长期不尽相同,但都包含了第3-8h。在0.07h~(-1)的条件下,系统在连续培养的前14d达到了稳态,因此0.07h~(-1)为相对较合适的稀释率。第7h转入连续培养时,系统较其他两个条件更早达到了稳态,且稳态时系统中的菌群数量和SCFA浓度也最高,因此第7h为相对较合适的转入连续培养的时间。30d试验显示,在第9-30d的系统中,四种代表菌的数量和三种SCFA的浓度变化差异均不显著,系统培养条件(如温度、搅拌速度、pH值等)的稳定性也较好。模拟系统在达到稳态后,其培养体系中的四种优势菌数量和三种SCFA浓度与接种前的粪便相比略有下降,但均在报道的健康人正常的生理参数范围内。
上述试验结果表明,本研究通过对稀释率和转入连续培养时间等条件的摸索和优化,成功建立了人肠道微生态恒化器模拟系统,系统具有较好的稳定性和可靠性,能够模拟健康人的肠道微生态环境。本研究首次从菌群数量和代谢物浓度两个角度较全面地对恒化器模拟系统的稳定性、可靠性进行了评价,填补了国内研究的空白。本研究还解决了目前国内外建立同类模拟系统时存在的搅拌不均匀、液位不稳定、操作复杂而导致模拟系统相对不够稳定等问题。
该恒化器模拟系统的建立可以为抗菌药残留对人肠道菌群影响的安全性评价研究构建平台,也可为新药、功能食品等的安全评价研究及药物代谢研究提供更科学的方法和途径,对保障人类食品和医学安全、促进相关学科发展具有极其重要的意义。
The antibiotic residues in food-producing animals may enter the food and be consumed by humans.This would result in a potential risk for human intestinal flora, such as increase of the population of resistant enteric bacteria,disruption of the colonization barrier,alteration of the metabolic activity of intestinal flora,and threaten human health.Nowadays,safety evaluation of residues of veterinary,antibiotics in human food is focusing on influence of human intestinal flora,and many people are interested in the studies gradually.But in China,this research has little progression because of lacking models approved by international organization.The dilution ratio and the time of transferring to continuous cultivation were optimized,and a chemostat system which could simulate human intestinal microecology was established.This system is one of the two models which are recommended by international organization.Development of this system will promote the research mentioned above and be significant to protect human health.
In this study,bacillus-bile-esculin agar culture medium,BBL agar culture medium, eosin methylthioninium chloride agar culture medium and enterococcus element were chosen as the selective medium for Bacteroides fragilis.Bacillus bifidus,Escherichia coli and Enterococci.A gas chromatography method was established to determine the concentrations of three kinds of main short chain fatty acids(SCFA,including ethanoic acid,propionic acid and butyric acid).50ml(20%) fresh excrement suspension was inoculated into fermentor and cultured under anaerobic condition.The viable number of four species of predominant bacterium in fixed time was counted by pour plate method. Then their growth curve in vitro batch culture was drawn and the exponential phase was calculated.On the base of the number of four species of predominant bacteria and the concentration of three kinds of main SCFA in broth culture,the dilution rate of continuous culture(0.05h~(-1),0.07h~(-1) and 0.09h~(-1)) and the time of shifting into continuous culture(5h, 7h and 9h) were optimized,and a chemostat system which could simulate human intestinal microecology was established.The long-term test of 30 days was carried out under the optimal condition,and the stability and reliability of this system were appraised through statistics analysis to the data.
The batch culture indicated that exponential phase of four species of predominant bacteria were different,but they were in the period of 3-8h.The 0.07h~(-1) was the relative appropriate dilution ratio,because it was the suitable condition which could make system reach a steady-state in 14d continuous cultivation.The 7h was the relative appropriate the time of shifting into continuous culture,because system could reach a steady-state earlier than other two conditions,and the flora quantity and the SCFA concentration of system were also highest in the stable state.In the 30 days test,there were no significance on the changes of four species bacterium quantity and three kinds of SCFA concentration in the 9th-30th days system.The stability of culture conditions of system(such as temperature, mixing speed,pH value and so on) is high.When the system achieved the stable state,the numbers of four species of predominant bacterium and the concentration of three kinds of main SCFA in culture solution were slightly decreased comparing with the fresh feces,but within the reported healthy person's normal physiological parameter area.
According to the experiment data,a chemostat system which could simulate human intestinal microecology has been established,by groping and optimizing the continuous cultivation dilution ratio and the time of shifting in continuous cultivation.The system has good stability and the reliability.It can simulate the intestinal tract environment of healthy human.The research evaluate the stability and reliability of the chemostat system in the flora quantity and metabolite concentration,filling a domestic gap in this field.This study solves the unstable problem of system induced by uneven stirring,unstable liquid-level and complex operation in the similar system at home and abroad.
The development of this system could provide a platform for safety assessment research of the antibiotics residues on human intestinal microflora and propose a scientific approach to assess the safety of new drugs and functional foods and research of drug metabolism.It will promote the research mentioned above and be significant to protect human health.
本研究选用拟杆菌—胆汁—七叶苷琼脂培养基、BBL琼脂培养基、伊红美兰琼脂培养基和肠球菌选择性培养基,分别分离计数人粪便中四种优势代表菌(脆弱拟杆菌、双歧杆菌、大肠杆菌和肠球菌)。建立粪便中三种主要短链脂肪酸(SCFA,即乙酸、丙酸和丁酸)浓度测定的气相色谱方法。将50mL(20%)的新鲜粪便悬液接种于发酵罐中,厌氧条件下分批培养,定时取样,采用平板稀释计数法记录培养液中四种优势代表菌的数目,绘制其分批培养的菌体生长曲线,计算对数生长期。在此基础上,以培养液中四种优势代表菌数目和三种主要的短链脂肪酸浓度为监测指标,优化连续培养工艺,包括稀释率(0.05h~(-1)、0.07h~(-1)和0.09h~(-1))和转入连续培养的时间(第5h、第7h和第9h),建立人肠道微生态恒化器模拟系统。在优化后的培养条件下进行为期30 d的试验,进一步评价所建立的模拟系统的稳定性和可靠性。
分批培养显示,四种优势菌的对数生长期不尽相同,但都包含了第3-8h。在0.07h~(-1)的条件下,系统在连续培养的前14d达到了稳态,因此0.07h~(-1)为相对较合适的稀释率。第7h转入连续培养时,系统较其他两个条件更早达到了稳态,且稳态时系统中的菌群数量和SCFA浓度也最高,因此第7h为相对较合适的转入连续培养的时间。30d试验显示,在第9-30d的系统中,四种代表菌的数量和三种SCFA的浓度变化差异均不显著,系统培养条件(如温度、搅拌速度、pH值等)的稳定性也较好。模拟系统在达到稳态后,其培养体系中的四种优势菌数量和三种SCFA浓度与接种前的粪便相比略有下降,但均在报道的健康人正常的生理参数范围内。
上述试验结果表明,本研究通过对稀释率和转入连续培养时间等条件的摸索和优化,成功建立了人肠道微生态恒化器模拟系统,系统具有较好的稳定性和可靠性,能够模拟健康人的肠道微生态环境。本研究首次从菌群数量和代谢物浓度两个角度较全面地对恒化器模拟系统的稳定性、可靠性进行了评价,填补了国内研究的空白。本研究还解决了目前国内外建立同类模拟系统时存在的搅拌不均匀、液位不稳定、操作复杂而导致模拟系统相对不够稳定等问题。
该恒化器模拟系统的建立可以为抗菌药残留对人肠道菌群影响的安全性评价研究构建平台,也可为新药、功能食品等的安全评价研究及药物代谢研究提供更科学的方法和途径,对保障人类食品和医学安全、促进相关学科发展具有极其重要的意义。
The antibiotic residues in food-producing animals may enter the food and be consumed by humans.This would result in a potential risk for human intestinal flora, such as increase of the population of resistant enteric bacteria,disruption of the colonization barrier,alteration of the metabolic activity of intestinal flora,and threaten human health.Nowadays,safety evaluation of residues of veterinary,antibiotics in human food is focusing on influence of human intestinal flora,and many people are interested in the studies gradually.But in China,this research has little progression because of lacking models approved by international organization.The dilution ratio and the time of transferring to continuous cultivation were optimized,and a chemostat system which could simulate human intestinal microecology was established.This system is one of the two models which are recommended by international organization.Development of this system will promote the research mentioned above and be significant to protect human health.
In this study,bacillus-bile-esculin agar culture medium,BBL agar culture medium, eosin methylthioninium chloride agar culture medium and enterococcus element were chosen as the selective medium for Bacteroides fragilis.Bacillus bifidus,Escherichia coli and Enterococci.A gas chromatography method was established to determine the concentrations of three kinds of main short chain fatty acids(SCFA,including ethanoic acid,propionic acid and butyric acid).50ml(20%) fresh excrement suspension was inoculated into fermentor and cultured under anaerobic condition.The viable number of four species of predominant bacterium in fixed time was counted by pour plate method. Then their growth curve in vitro batch culture was drawn and the exponential phase was calculated.On the base of the number of four species of predominant bacteria and the concentration of three kinds of main SCFA in broth culture,the dilution rate of continuous culture(0.05h~(-1),0.07h~(-1) and 0.09h~(-1)) and the time of shifting into continuous culture(5h, 7h and 9h) were optimized,and a chemostat system which could simulate human intestinal microecology was established.The long-term test of 30 days was carried out under the optimal condition,and the stability and reliability of this system were appraised through statistics analysis to the data.
The batch culture indicated that exponential phase of four species of predominant bacteria were different,but they were in the period of 3-8h.The 0.07h~(-1) was the relative appropriate dilution ratio,because it was the suitable condition which could make system reach a steady-state in 14d continuous cultivation.The 7h was the relative appropriate the time of shifting into continuous culture,because system could reach a steady-state earlier than other two conditions,and the flora quantity and the SCFA concentration of system were also highest in the stable state.In the 30 days test,there were no significance on the changes of four species bacterium quantity and three kinds of SCFA concentration in the 9th-30th days system.The stability of culture conditions of system(such as temperature, mixing speed,pH value and so on) is high.When the system achieved the stable state,the numbers of four species of predominant bacterium and the concentration of three kinds of main SCFA in culture solution were slightly decreased comparing with the fresh feces,but within the reported healthy person's normal physiological parameter area.
According to the experiment data,a chemostat system which could simulate human intestinal microecology has been established,by groping and optimizing the continuous cultivation dilution ratio and the time of shifting in continuous cultivation.The system has good stability and the reliability.It can simulate the intestinal tract environment of healthy human.The research evaluate the stability and reliability of the chemostat system in the flora quantity and metabolite concentration,filling a domestic gap in this field.This study solves the unstable problem of system induced by uneven stirring,unstable liquid-level and complex operation in the similar system at home and abroad.
The development of this system could provide a platform for safety assessment research of the antibiotics residues on human intestinal microflora and propose a scientific approach to assess the safety of new drugs and functional foods and research of drug metabolism.It will promote the research mentioned above and be significant to protect human health.
引文
白秀峰.发酵工艺学.北京:中国医药科技出版社,2003,177-180
曹郁生,刘兰,洪亦武,黄筱萍.鸡盲肠菌群的厌氧连续培养及培养物的生理生化特性.中国微生态学杂志,2000,12:208-210
曹军卫,马辉文,张甲耀.微生物工程.北京:科学出版社,2007,175-182
陈坚.堵国成.李寅,华兆哲.发酵工程试验技术.北京:化学工业出版社,2003.170-191
陈宝安,杜鹃,张春秀,程坚,高峰,陆祖宏.应用蛋白质芯片检测多药耐药蛋白表达水平.中华肿瘤杂志,2005,9(27):528-530
程殿林.微生物工程技术原理.北京:化学工业出版社,2007,7:108-114
邓旭明,李乾学,于录,周学章,张艳萍.金黄色葡萄球菌NorA蛋白免疫检测方法的建立.中国抗生素杂志,2005,30(9):546-548
高延玲,陈杖榴,刘健华,曾振灵.人体肠道菌群的离体恒化器模型的建立.中国畜牧兽医学会兽医药理毒理学分会第九次学术讨论会论文与摘要集,2006,269
何晓青,冉陆,付萍,姚景会.GB/T 4789.6-2003.食品卫生微生物学检验致泻大肠埃希氏菌检验
胡丽霞.体外模型中金霉素对人源粪肠球菌和志贺氏菌耐药诱导研究.[硕士学位论文].武汉:华中农业大学图书馆,2005
蒋红霞,陈杖榴,曾振灵.寡核苷酸芯片检测兽医病原菌耐药性的研究.中国农业科学,2004,37(9):1385-1389
李友荣,马辉文.发酵生理学.长沙:湖南科学技术出版社,1988,322-350
李影林.临床微生物学及检验.北京:人民卫生出版社,1995,83-96
李燕.耐药结核分枝杆菌检测方法的研究进展.国外医学临床生物化学与检测学分册,2000,21(4):190-192
邱志鹏.恒化器系统的建模与稳定性分析.[博士学位论文].南京:南京理工大学图书馆,2003
钱铭镛.发酵工程最优化控制.南京:江苏科学技术出版社,1998,105-130
冉陆,陈稚峰,付萍,杨宝兰,李志刚,姚景会,赵熙.北京地区184例健康人肠道菌群值的调查.中国微生态学杂志,1999,11(1):10-12
田洪涛.现代发酵工艺原理与技术.北京:化学工业出版社,2007,1:33-36
王照华,侯先志,李鹏飞.利用双外流连续培养系统研究不同稀释率对真菌生长及发酵参数的影响.饲料工业,2006,18:21-27
肖东光.微生物工程原理.北京:中国轻工业出版社,2004,7:180-213
杨宝兰,冉陆,李志刚,王淑真,杨大进,贾珍珍.GB/T 4789.34-2003.食品卫生微生物学检验双歧杆菌检验
倪语星,韩立中.分子生物学方法在细菌耐药性检测方面的应用.上海医学检验杂志,2001,16(6):330-333
周德庆.微生物学实验手册.上海:上海科学技术出版社,1986,116-123
张可煜.低浓度环丙沙星对体外模型中人大肠杆菌和粪肠球菌敏感性影响研究.[硕士学位论文].武汉:华中农业大学图书馆,2004
赵虎.厌氧菌和微需氧菌感染与实验室诊断.上海:上海科学技术出版社,2005,91-94
张敏,范小兵,杨虹,李壁宝,奚万艳,王一鸣.人体肠道菌群构成及重要代谢产物的研究.微生物生态学研究进展—第五届微生物生态学术研讨会论文集,2003.125-130
Agnes P G,Cottin S,Corpet D E,Boisseau J and Poul J M.Evaluation of residual and therapeutic doses of tetracycline in the human-flora-associated(HFA) mice model.Regulatory Toxicology and Pharmacology,2001,34:125-136
Agnes P G,Poul J M,Corpet D E,Sanders P,Fernandez A H and Bartholomew M.Impact of residual and therapeutic doses of ciprofloxacin in the human-flora-associated mice model.Regulatory Toxicology and Pharmacology,2005,42:151-160
Agnes P G,Poul J M,Laurentie M,Sanders P,Fernandez H and Bartholomew M.Impact of ciprofloxacin in the human-flora-associated(HFA) rat model:comparison with the HFA mouse model.Regulatory Toxicology and Pharmacology,2006,45:66-78
Anna M D,Ida L and Alessandra C.Identification of ciprofloxacin-resistant Campylobacter jejuni and analysis of the gyrA gene by the Light Cycler mutation assay.Molecular and Cellular Probes,2004,18:255-261
Arlet G,Brami D,Decre A,Flippo O and Gaillot P H.Molecular characterization by PCR-rstriction fragmented length polymorphism of TEM β-lactamases. FEMS Microbiol Let, 1995,134: 203-208
Backhed F, Ley R E. Sonnenburg J L, Peterson D A and Gordon J I. Host-bacterial mutualism in the human intestine. Science, 2005, 307: 1915-1921
Brien T F. The global epidemic nature of antimicrobial resistance and the need to monitor and manage it locally. Clinical Infectious Diseases, 1997, 24(1): 2-8
Carman R J, Van T and Wilkins T D. The normal intestinal microflora: ecology, variability and stability. Veterinary and Human Toxicology, 1993, 35: 11-14
Carman R J and Woodburn M A. Effects of low levels of ciprofloxacin on a chemostat model of the human colonic microflora. Regulatory Toxicology and Pharmacology, 2001,33:276-284
Carman R J and Silmon M A. Ciprofloxacin at low levels disrupts colonization resistance of human fecal microflora growing in chemostats. Regulatory Toxicology and Pharmacology, 2004,40: 319-326
Carman R J, Simon M A, Petzold H E, Wimmer R F, Batra M R, Fernandez A H, Miller M A and Bartholomew M. Antibiotics in the human food chain: establishing no effect levels of tetracycline, neomycin, and erythromycin using a chemostat model of the human colonic microXora. Regulatory Toxicology and Pharmacology, 2005, 43: 168-180
Cai H Y, Archambault M, Gyles C L and Prescott J F. Molecular genetic methods in the veterinary clinical bacteriology laboratory: current usage and future applications. Animal Health and Research Reviews, 2003,4(2): 73-93
Call D R, Bakko M K, Krug M J and Roberts M C. Identifying antimicrobial resistance genes with DNA microarrays. Antimicrobial Agents and Chemotherapy, 2003, 47: 3290-3295
Cerniglia C E, Kotarski S. Evaluation of veterinary drug residues in food for their potential to affect human intestinal microflora. Regulatory Toxicology and Pharmacology, 1999, 29: 238-261
CVM. CVM update: CVM plans to modify guidance on microbiological safety on drug residues in food. www.fda.gov/cvm/CVM_Updates/52up.html. March, 3, 2000
Christian M, Daniel L and Jorg MS. A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon. Journal of Biochemical and Biophysical Methods, 2004, 59: 217-227
Corpet D E. Current status of models for testing antibiotic residues. Veterinary and Human Toxicology, 1993, 35: 37-46.
Deborah J E, Ernesto L, Martin J W and Laura J V P. Detection of gyrA mutations in quinlone-resistant Salmonella enterica by denaturing high-performance liquid chromatography. Journal of Clinical Microbiology, 2002,40( 11): 4121 -4125
Deivecchio V G. Rapid detection of enterococci and antibitic resistance genes in urine proaching real time capability. Clinical Microbiology Newsletter, 1998, 20: 192-195
Erwin E S, Marco G J and Emery E M. Volatile fatty acids analysis of blood and rumen fluid by gas chromatography. Journal of Dairy Science, 1961,44: 1768-1771
EMEA, Studies to assess the potential for resistance resulting from the use of antimicrobial veterinary medicinal products. EMEA/CVMP/234/01, 2002
FDA, Studies to evaluate the safety of residues of veterinary drugs in human food: general approach to estabilish a microbiological ADI. Final guidance #159. 2005
Freter R, Stauffer E, Cleven D, Holdeman V and Walter E C. Continuous-flow cultures as in vitro models of the ecology of large intestinal flora. Infection and Immunity, 1983, 39: 666-675
Franklin R C. Genetic methods for assessing antimicrobial resistance. Antimicrobial Agents and Chemotherapy, 1999,43(2): 199-212
Frye J G, Jesse T, Long F, Rondeau G, Porwollik S, McClelland M, Jackson C R, Englen M and Fedorka-Cray P J. DNA microarray detection of antimicrobial resistance genes in diverse bacteria. International Journal of Antimicrobial Agents, 2006, 27(2): 138-151
Glenn G R, Cummings J C and Macfarlane G T. Use of a three-Stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria. Applied and Environmental Microbiology, 1988,9: 2750-2755
Grimm V, Ezaki S, Susa M, Knabbe C, Schmid R D and Bachmann T T. Use of DNA microarrays for rapid genotyping of TEM beta-lactamases that confer resistance. Journal of Clinical Microbiology, 2004,42: 3766 3774
Guarner F and Malagelada J R. Gut flora in health and disease. The Lancet, 2003, 361(9356): 512-519
Hecker K H, Asea A, Kobayashi K, Green S, Tang D and Calderwood S K. Mutation detection in the human HSP7OB' gene by denaturing high-performance liquid chromatography. Cell Stress Chaperones, 2000, 5(5): 415-424
Hoek A H, Scholtens I M, Cloeckaert A, Aarts H J. Detection of antibiotic resistance genes in different Salmonella serovars by oligonucleotide microarray analysis. Journal of Micobiological Methods, 2005, 62: 13-23
Hoverstad T, Fausa O, Bjorneklett A and Bohmer T. Short-chain fatty acids in the normal human feces. Scandinavian Journal of Gastroenterology, 1984, 19(3): 375-381
Joint FAO/WHO Expert Committee on Food Additives (JECFA), Evaluation of certain veterinary drug residues in food: fifty eight report of the joint FAO/WHO Expert Committee on Food Additives. In WHO Technical Report Series, 911. WHO Geneva, 2002
Johnson J R, Kuskowski M A, Menard M, Gajewski A, Xercavins M and Garau J. Similarity between Human and Chicken Escherichia coli Isolates in Relation to Ciprofloxacin Resistance Status. Journal of Infectious Diseases, 2006, 194(1): 71-79
Karam R, Maria B, Peter J and Baldwin T. Detection of methicillin resistance in coagulase-negative staphylococci initially reported as methicillin susceptible using automated methods. Diagnostic Microbiology and Infectious Disease, 1998, 30: 267-273
Kim J Y, Kim S H, Kwon N H, Bae W K, Lim J Y, Koo H C, Kim J M, Noh K M, Jung W K, Park K T and Park Y H. Isolation and identification of Escherichia coli O157:H7 using different detection methods and molecular determination by multiplex PCR and RAPD. Journal of Veterinary Science, 2005,6(1): 7-19
Kohner P C, Patel R, Uhl J R, Garin K M, Hopkins M K, Wegener L T and Cockerill F R. Comparison of agar dilution, broth microdilution, E-test, disk diffusion, and automated Vitek methods for testing susceptibilities of Enterococcusspp to vancomycin. Journal of Clinical Microbiology, 1997, 35: 3258-3263
Krul C, Kumblot C, Philippe C, Vermeulen M, Nuenen M, Havenaar R and Rabot S. Metabolism of sinigrin (2-propenyl glucosinolate)by the human colonic microflora in a dynamic in vitro large-intestinal model. Carcinogenesis, 2002, 23: 1009-1016
Lee Y, Lee EK, Cho YW, Matsui T, Kang IC, Kim TS and Han MH. Proteo Chip: a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-potein interaction studies. Proteomics, 2003,3(12): 2289-2304
Lorelei W, Carolyn M, Dana V, David C, Ray R, Micheal N and James P. Antimicrobial: resistance and bacterial identification utilizing a microelectronic chip array. Journal of Clinical Microbiology, 2001, 39(3): 1097-1104.
Malorny B, Guerra B, Zeltz P, Rissler K and Helmuth R. Typing of Salmonella by DNA-microarrays. Berl Munch Tierarztl Wochenschr, 2003, 116(11-12): 482-488
Martineau F, Picard F J, Roy P H, Ouellette M and Bergeron M G. Species 2 specific and ubiquitous DNA based assays forrapid identification of Staphylococcusaureus. Journal of Clinical Microbiology, 1998, 36(3): 618-623
Minekus M, Smeets-Peeters M, Bernalier A, Marol B S, Havenaar R, Marteau P, Alric M and Fonty G. A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products. Applied Microbiology and Biotechnology, 1999, 53: 108-114
Moore W E and Holdeman L V. Human fecal flora: the normal flora of 20 Japanese- Hawaiians. Applied Microbiology and Biotechnology, 1974, 27: 961-979
Nawaz M S, Erickson B D, Khan A A, Khan S A, Pothuluri J V, Rafii F, Sutherland J B, Wagner R D and Cerniglia C E. Human health impact and regulatory issues involving antimicrobial resistance in the food animal production environment. regulatory research perspectives. Impact on Public Health, 2001, 1(1): 1-9
Nicola C E, Andrew J L and Robert J O. Application of 3'-mismatched reverse primer PCR compared with real-time PCR and PCR-RFLP for the rapid detection of 23SrDNA mutations associated with clarithromycin resistance in Helicobacter pylori. International Journal of Antimicrobial Agents, 2004, 23: 349-355
Niwa H, Chuma T, Okamoto K and Itoh K. Rapid detection of mutations associated with resistance to erythromycin in Campylobacter jejuni/coli by PCR and line probe assay. International Journal of Antimicrobial Agents. 2001, 18 : 359-364
Niwa H, Chuma T, Okamoto K and Itoh K. Simultaneous detection of mutations associated with resistance to macrolides and quinolones in Campylobacter jejuni and E.coli using a PCR-line probe assay. InternationalJournal of Antimicrobial Agents, 2003,22: 374-379
Radu S, Toosa H, Rahim R A, Reezal A, Ahmad M, Hamid A N, Rusul G and Nishibuchi M. Occurrence of the vanA and vanC2/C3 genes in Enterococcus species isolated from poultry sources in Malaysia. Diagnostic Microbiology and Infectious Disease, 2001, 39(3): 145-153
Ribeiro M L, Gerrits M M, Benvengo Y H, Berning M, Godoy A P, Kuipers E J, Mendonca S, Vliet A H, Pedrazzoli J and Kusters J G Detection of highlevel tetracycline resistance in clinical isolates of Helciobacter pylori using PCR-RFLP. FEMS Immunol Med Microbiol, 2004,40(1): 57-61
Sidjabat H E, Townsend K M, Hanson N D, Bell J M, Stokes H W, Gobius K S, Moss S M and Trott D J. Identification of bla(CMY-7) and associated plasmid-mediated resistance genes in multidrug-resistant Escherichia coli isolated from dogs at a veterinary teaching hospital in Australia. Journal of Antimicrobial Chemotherapy, 2006, 57(5): 840-848
Susana D, Patricia R, Adolfo S and Baltasar M. Interindividual differences in microbial counts and biochemical-associated variables in the feces of healthy Spanish adults. Digestive Diseases and Sciences, 2006, 51(4): 737-743
Sunde M, Fossum K, Solberg A and Sorum H. Antibiotic resistance in Escherichia coli of the normal intestinal flora of swine. Microbial Drug Resistance, 1998,4(4): 289-299
Surbhi M K, Christine L, Jasper P and Herman G Multiplex PCR for simultaneous detection of macrolide and tetracycline resistance determinants in streptococci. Antimicrob Agents Chemother, 2005,49(11): 4798-4800
Veilleux B G and Rowland I. Simulation of the rat intestinal ecosystem using a two-stage continuous culture system. Journal of General and Applied Microbiology, 1981, 123(1): 103-115
VICH. VICH safety working group studies to evaluation the safety of residues of veterinary drugs in human food. General approach to establish a microbiological ADI. http://www.mhlw. go.jp /public /bosyuu /p0627-1c. html, April 30, 2003
Visser M R and Fluit A C. Amplification methods for the detection of bacterial resistance genes. Journal of Microbiological Methods, 1995, 23: 105-116
Weller T M A, Crook D W, Crow M R, Ibrahim W, Pennington T H and Selkon J B. Methicillin susceptibility testing of staphylococci by E-test and comparison with agar dilution and mecA detection. Journal of Antimicrobial Chemotherapy, 1997, 39: 251-253
Woodmansey E J, Mcmurdo M E T, Macfarlane G T and Macfarlane S. Comparison of compositions and metabolic activities of fecal microbiotas in young adults and in antibiotic-treated and non-antibiotic-treated elderly subjects. Applied and Environmental Microbiology, 2004, 70: 6113-6122
Wu B, Xia C, Du X, Cao X and Shen J. Influence of anti-FloR antibody on florfenicol accumulation in florfenicol-resistant Escherichia coli and enzyme linked immunosorbent assay for detection of florfenicol-resistant E.coli isolates. Journal of Clinical Microbiology, 2006,44(2): 378-382
Yu X, Susa M, Knabbe C, Schmid R D and Bachmann T T. Development and validation of a diagnostic DNA microarray to detect quinolone-resistant Escherichia coli among clinical isolates. Journal of Clinical Microbiology, 2004,42: 4083-4091
Yu Z, Michel F C J, Hansen G, Wittum T and Morrison M. Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance. Applied and Environmental Microbiology, 2005, 71(11): 6926-6933
曹郁生,刘兰,洪亦武,黄筱萍.鸡盲肠菌群的厌氧连续培养及培养物的生理生化特性.中国微生态学杂志,2000,12:208-210
曹军卫,马辉文,张甲耀.微生物工程.北京:科学出版社,2007,175-182
陈坚.堵国成.李寅,华兆哲.发酵工程试验技术.北京:化学工业出版社,2003.170-191
陈宝安,杜鹃,张春秀,程坚,高峰,陆祖宏.应用蛋白质芯片检测多药耐药蛋白表达水平.中华肿瘤杂志,2005,9(27):528-530
程殿林.微生物工程技术原理.北京:化学工业出版社,2007,7:108-114
邓旭明,李乾学,于录,周学章,张艳萍.金黄色葡萄球菌NorA蛋白免疫检测方法的建立.中国抗生素杂志,2005,30(9):546-548
高延玲,陈杖榴,刘健华,曾振灵.人体肠道菌群的离体恒化器模型的建立.中国畜牧兽医学会兽医药理毒理学分会第九次学术讨论会论文与摘要集,2006,269
何晓青,冉陆,付萍,姚景会.GB/T 4789.6-2003.食品卫生微生物学检验致泻大肠埃希氏菌检验
胡丽霞.体外模型中金霉素对人源粪肠球菌和志贺氏菌耐药诱导研究.[硕士学位论文].武汉:华中农业大学图书馆,2005
蒋红霞,陈杖榴,曾振灵.寡核苷酸芯片检测兽医病原菌耐药性的研究.中国农业科学,2004,37(9):1385-1389
李友荣,马辉文.发酵生理学.长沙:湖南科学技术出版社,1988,322-350
李影林.临床微生物学及检验.北京:人民卫生出版社,1995,83-96
李燕.耐药结核分枝杆菌检测方法的研究进展.国外医学临床生物化学与检测学分册,2000,21(4):190-192
邱志鹏.恒化器系统的建模与稳定性分析.[博士学位论文].南京:南京理工大学图书馆,2003
钱铭镛.发酵工程最优化控制.南京:江苏科学技术出版社,1998,105-130
冉陆,陈稚峰,付萍,杨宝兰,李志刚,姚景会,赵熙.北京地区184例健康人肠道菌群值的调查.中国微生态学杂志,1999,11(1):10-12
田洪涛.现代发酵工艺原理与技术.北京:化学工业出版社,2007,1:33-36
王照华,侯先志,李鹏飞.利用双外流连续培养系统研究不同稀释率对真菌生长及发酵参数的影响.饲料工业,2006,18:21-27
肖东光.微生物工程原理.北京:中国轻工业出版社,2004,7:180-213
杨宝兰,冉陆,李志刚,王淑真,杨大进,贾珍珍.GB/T 4789.34-2003.食品卫生微生物学检验双歧杆菌检验
倪语星,韩立中.分子生物学方法在细菌耐药性检测方面的应用.上海医学检验杂志,2001,16(6):330-333
周德庆.微生物学实验手册.上海:上海科学技术出版社,1986,116-123
张可煜.低浓度环丙沙星对体外模型中人大肠杆菌和粪肠球菌敏感性影响研究.[硕士学位论文].武汉:华中农业大学图书馆,2004
赵虎.厌氧菌和微需氧菌感染与实验室诊断.上海:上海科学技术出版社,2005,91-94
张敏,范小兵,杨虹,李壁宝,奚万艳,王一鸣.人体肠道菌群构成及重要代谢产物的研究.微生物生态学研究进展—第五届微生物生态学术研讨会论文集,2003.125-130
Agnes P G,Cottin S,Corpet D E,Boisseau J and Poul J M.Evaluation of residual and therapeutic doses of tetracycline in the human-flora-associated(HFA) mice model.Regulatory Toxicology and Pharmacology,2001,34:125-136
Agnes P G,Poul J M,Corpet D E,Sanders P,Fernandez A H and Bartholomew M.Impact of residual and therapeutic doses of ciprofloxacin in the human-flora-associated mice model.Regulatory Toxicology and Pharmacology,2005,42:151-160
Agnes P G,Poul J M,Laurentie M,Sanders P,Fernandez H and Bartholomew M.Impact of ciprofloxacin in the human-flora-associated(HFA) rat model:comparison with the HFA mouse model.Regulatory Toxicology and Pharmacology,2006,45:66-78
Anna M D,Ida L and Alessandra C.Identification of ciprofloxacin-resistant Campylobacter jejuni and analysis of the gyrA gene by the Light Cycler mutation assay.Molecular and Cellular Probes,2004,18:255-261
Arlet G,Brami D,Decre A,Flippo O and Gaillot P H.Molecular characterization by PCR-rstriction fragmented length polymorphism of TEM β-lactamases. FEMS Microbiol Let, 1995,134: 203-208
Backhed F, Ley R E. Sonnenburg J L, Peterson D A and Gordon J I. Host-bacterial mutualism in the human intestine. Science, 2005, 307: 1915-1921
Brien T F. The global epidemic nature of antimicrobial resistance and the need to monitor and manage it locally. Clinical Infectious Diseases, 1997, 24(1): 2-8
Carman R J, Van T and Wilkins T D. The normal intestinal microflora: ecology, variability and stability. Veterinary and Human Toxicology, 1993, 35: 11-14
Carman R J and Woodburn M A. Effects of low levels of ciprofloxacin on a chemostat model of the human colonic microflora. Regulatory Toxicology and Pharmacology, 2001,33:276-284
Carman R J and Silmon M A. Ciprofloxacin at low levels disrupts colonization resistance of human fecal microflora growing in chemostats. Regulatory Toxicology and Pharmacology, 2004,40: 319-326
Carman R J, Simon M A, Petzold H E, Wimmer R F, Batra M R, Fernandez A H, Miller M A and Bartholomew M. Antibiotics in the human food chain: establishing no effect levels of tetracycline, neomycin, and erythromycin using a chemostat model of the human colonic microXora. Regulatory Toxicology and Pharmacology, 2005, 43: 168-180
Cai H Y, Archambault M, Gyles C L and Prescott J F. Molecular genetic methods in the veterinary clinical bacteriology laboratory: current usage and future applications. Animal Health and Research Reviews, 2003,4(2): 73-93
Call D R, Bakko M K, Krug M J and Roberts M C. Identifying antimicrobial resistance genes with DNA microarrays. Antimicrobial Agents and Chemotherapy, 2003, 47: 3290-3295
Cerniglia C E, Kotarski S. Evaluation of veterinary drug residues in food for their potential to affect human intestinal microflora. Regulatory Toxicology and Pharmacology, 1999, 29: 238-261
CVM. CVM update: CVM plans to modify guidance on microbiological safety on drug residues in food. www.fda.gov/cvm/CVM_Updates/52up.html. March, 3, 2000
Christian M, Daniel L and Jorg MS. A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon. Journal of Biochemical and Biophysical Methods, 2004, 59: 217-227
Corpet D E. Current status of models for testing antibiotic residues. Veterinary and Human Toxicology, 1993, 35: 37-46.
Deborah J E, Ernesto L, Martin J W and Laura J V P. Detection of gyrA mutations in quinlone-resistant Salmonella enterica by denaturing high-performance liquid chromatography. Journal of Clinical Microbiology, 2002,40( 11): 4121 -4125
Deivecchio V G. Rapid detection of enterococci and antibitic resistance genes in urine proaching real time capability. Clinical Microbiology Newsletter, 1998, 20: 192-195
Erwin E S, Marco G J and Emery E M. Volatile fatty acids analysis of blood and rumen fluid by gas chromatography. Journal of Dairy Science, 1961,44: 1768-1771
EMEA, Studies to assess the potential for resistance resulting from the use of antimicrobial veterinary medicinal products. EMEA/CVMP/234/01, 2002
FDA, Studies to evaluate the safety of residues of veterinary drugs in human food: general approach to estabilish a microbiological ADI. Final guidance #159. 2005
Freter R, Stauffer E, Cleven D, Holdeman V and Walter E C. Continuous-flow cultures as in vitro models of the ecology of large intestinal flora. Infection and Immunity, 1983, 39: 666-675
Franklin R C. Genetic methods for assessing antimicrobial resistance. Antimicrobial Agents and Chemotherapy, 1999,43(2): 199-212
Frye J G, Jesse T, Long F, Rondeau G, Porwollik S, McClelland M, Jackson C R, Englen M and Fedorka-Cray P J. DNA microarray detection of antimicrobial resistance genes in diverse bacteria. International Journal of Antimicrobial Agents, 2006, 27(2): 138-151
Glenn G R, Cummings J C and Macfarlane G T. Use of a three-Stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria. Applied and Environmental Microbiology, 1988,9: 2750-2755
Grimm V, Ezaki S, Susa M, Knabbe C, Schmid R D and Bachmann T T. Use of DNA microarrays for rapid genotyping of TEM beta-lactamases that confer resistance. Journal of Clinical Microbiology, 2004,42: 3766 3774
Guarner F and Malagelada J R. Gut flora in health and disease. The Lancet, 2003, 361(9356): 512-519
Hecker K H, Asea A, Kobayashi K, Green S, Tang D and Calderwood S K. Mutation detection in the human HSP7OB' gene by denaturing high-performance liquid chromatography. Cell Stress Chaperones, 2000, 5(5): 415-424
Hoek A H, Scholtens I M, Cloeckaert A, Aarts H J. Detection of antibiotic resistance genes in different Salmonella serovars by oligonucleotide microarray analysis. Journal of Micobiological Methods, 2005, 62: 13-23
Hoverstad T, Fausa O, Bjorneklett A and Bohmer T. Short-chain fatty acids in the normal human feces. Scandinavian Journal of Gastroenterology, 1984, 19(3): 375-381
Joint FAO/WHO Expert Committee on Food Additives (JECFA), Evaluation of certain veterinary drug residues in food: fifty eight report of the joint FAO/WHO Expert Committee on Food Additives. In WHO Technical Report Series, 911. WHO Geneva, 2002
Johnson J R, Kuskowski M A, Menard M, Gajewski A, Xercavins M and Garau J. Similarity between Human and Chicken Escherichia coli Isolates in Relation to Ciprofloxacin Resistance Status. Journal of Infectious Diseases, 2006, 194(1): 71-79
Karam R, Maria B, Peter J and Baldwin T. Detection of methicillin resistance in coagulase-negative staphylococci initially reported as methicillin susceptible using automated methods. Diagnostic Microbiology and Infectious Disease, 1998, 30: 267-273
Kim J Y, Kim S H, Kwon N H, Bae W K, Lim J Y, Koo H C, Kim J M, Noh K M, Jung W K, Park K T and Park Y H. Isolation and identification of Escherichia coli O157:H7 using different detection methods and molecular determination by multiplex PCR and RAPD. Journal of Veterinary Science, 2005,6(1): 7-19
Kohner P C, Patel R, Uhl J R, Garin K M, Hopkins M K, Wegener L T and Cockerill F R. Comparison of agar dilution, broth microdilution, E-test, disk diffusion, and automated Vitek methods for testing susceptibilities of Enterococcusspp to vancomycin. Journal of Clinical Microbiology, 1997, 35: 3258-3263
Krul C, Kumblot C, Philippe C, Vermeulen M, Nuenen M, Havenaar R and Rabot S. Metabolism of sinigrin (2-propenyl glucosinolate)by the human colonic microflora in a dynamic in vitro large-intestinal model. Carcinogenesis, 2002, 23: 1009-1016
Lee Y, Lee EK, Cho YW, Matsui T, Kang IC, Kim TS and Han MH. Proteo Chip: a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-potein interaction studies. Proteomics, 2003,3(12): 2289-2304
Lorelei W, Carolyn M, Dana V, David C, Ray R, Micheal N and James P. Antimicrobial: resistance and bacterial identification utilizing a microelectronic chip array. Journal of Clinical Microbiology, 2001, 39(3): 1097-1104.
Malorny B, Guerra B, Zeltz P, Rissler K and Helmuth R. Typing of Salmonella by DNA-microarrays. Berl Munch Tierarztl Wochenschr, 2003, 116(11-12): 482-488
Martineau F, Picard F J, Roy P H, Ouellette M and Bergeron M G. Species 2 specific and ubiquitous DNA based assays forrapid identification of Staphylococcusaureus. Journal of Clinical Microbiology, 1998, 36(3): 618-623
Minekus M, Smeets-Peeters M, Bernalier A, Marol B S, Havenaar R, Marteau P, Alric M and Fonty G. A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products. Applied Microbiology and Biotechnology, 1999, 53: 108-114
Moore W E and Holdeman L V. Human fecal flora: the normal flora of 20 Japanese- Hawaiians. Applied Microbiology and Biotechnology, 1974, 27: 961-979
Nawaz M S, Erickson B D, Khan A A, Khan S A, Pothuluri J V, Rafii F, Sutherland J B, Wagner R D and Cerniglia C E. Human health impact and regulatory issues involving antimicrobial resistance in the food animal production environment. regulatory research perspectives. Impact on Public Health, 2001, 1(1): 1-9
Nicola C E, Andrew J L and Robert J O. Application of 3'-mismatched reverse primer PCR compared with real-time PCR and PCR-RFLP for the rapid detection of 23SrDNA mutations associated with clarithromycin resistance in Helicobacter pylori. International Journal of Antimicrobial Agents, 2004, 23: 349-355
Niwa H, Chuma T, Okamoto K and Itoh K. Rapid detection of mutations associated with resistance to erythromycin in Campylobacter jejuni/coli by PCR and line probe assay. International Journal of Antimicrobial Agents. 2001, 18 : 359-364
Niwa H, Chuma T, Okamoto K and Itoh K. Simultaneous detection of mutations associated with resistance to macrolides and quinolones in Campylobacter jejuni and E.coli using a PCR-line probe assay. InternationalJournal of Antimicrobial Agents, 2003,22: 374-379
Radu S, Toosa H, Rahim R A, Reezal A, Ahmad M, Hamid A N, Rusul G and Nishibuchi M. Occurrence of the vanA and vanC2/C3 genes in Enterococcus species isolated from poultry sources in Malaysia. Diagnostic Microbiology and Infectious Disease, 2001, 39(3): 145-153
Ribeiro M L, Gerrits M M, Benvengo Y H, Berning M, Godoy A P, Kuipers E J, Mendonca S, Vliet A H, Pedrazzoli J and Kusters J G Detection of highlevel tetracycline resistance in clinical isolates of Helciobacter pylori using PCR-RFLP. FEMS Immunol Med Microbiol, 2004,40(1): 57-61
Sidjabat H E, Townsend K M, Hanson N D, Bell J M, Stokes H W, Gobius K S, Moss S M and Trott D J. Identification of bla(CMY-7) and associated plasmid-mediated resistance genes in multidrug-resistant Escherichia coli isolated from dogs at a veterinary teaching hospital in Australia. Journal of Antimicrobial Chemotherapy, 2006, 57(5): 840-848
Susana D, Patricia R, Adolfo S and Baltasar M. Interindividual differences in microbial counts and biochemical-associated variables in the feces of healthy Spanish adults. Digestive Diseases and Sciences, 2006, 51(4): 737-743
Sunde M, Fossum K, Solberg A and Sorum H. Antibiotic resistance in Escherichia coli of the normal intestinal flora of swine. Microbial Drug Resistance, 1998,4(4): 289-299
Surbhi M K, Christine L, Jasper P and Herman G Multiplex PCR for simultaneous detection of macrolide and tetracycline resistance determinants in streptococci. Antimicrob Agents Chemother, 2005,49(11): 4798-4800
Veilleux B G and Rowland I. Simulation of the rat intestinal ecosystem using a two-stage continuous culture system. Journal of General and Applied Microbiology, 1981, 123(1): 103-115
VICH. VICH safety working group studies to evaluation the safety of residues of veterinary drugs in human food. General approach to establish a microbiological ADI. http://www.mhlw. go.jp /public /bosyuu /p0627-1c. html, April 30, 2003
Visser M R and Fluit A C. Amplification methods for the detection of bacterial resistance genes. Journal of Microbiological Methods, 1995, 23: 105-116
Weller T M A, Crook D W, Crow M R, Ibrahim W, Pennington T H and Selkon J B. Methicillin susceptibility testing of staphylococci by E-test and comparison with agar dilution and mecA detection. Journal of Antimicrobial Chemotherapy, 1997, 39: 251-253
Woodmansey E J, Mcmurdo M E T, Macfarlane G T and Macfarlane S. Comparison of compositions and metabolic activities of fecal microbiotas in young adults and in antibiotic-treated and non-antibiotic-treated elderly subjects. Applied and Environmental Microbiology, 2004, 70: 6113-6122
Wu B, Xia C, Du X, Cao X and Shen J. Influence of anti-FloR antibody on florfenicol accumulation in florfenicol-resistant Escherichia coli and enzyme linked immunosorbent assay for detection of florfenicol-resistant E.coli isolates. Journal of Clinical Microbiology, 2006,44(2): 378-382
Yu X, Susa M, Knabbe C, Schmid R D and Bachmann T T. Development and validation of a diagnostic DNA microarray to detect quinolone-resistant Escherichia coli among clinical isolates. Journal of Clinical Microbiology, 2004,42: 4083-4091
Yu Z, Michel F C J, Hansen G, Wittum T and Morrison M. Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance. Applied and Environmental Microbiology, 2005, 71(11): 6926-6933