BACTERIAL ANTIBIOTIC RESISTANCE IN AGRICULTURE

农业中的细菌抗生素耐药性

• 批准号: 7720447
• 负责人: MANUEL F VARELA
• 金额: $ 9.46万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720447
• 关键词: Agar; Agriculture; Amino Acids; Amoxicillin; Ampicillin; Antimicrobial Resistance; Bacteria; Bacterial Antibiotic Resistance; Cefotaxime; Cells; Cloaca Chamber; Cloning; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Sequence Analysis; Enterobacter cloacae; Enterobacteriaceae; Environment; Erythromycin; Escherichia coli; Ethidium; Exhibits; Family; Farming environment; Fosfomycin; Frequencies; Funding; Gatifloxacin; Genes; Grant; Guidelines; Guns; Hand; Homidium Bromide; Incubated; Infection; Institution; Measures; Mediating; Membrane; Methods; Milk; Multi-Drug Resistance; Nosocomial Infections; Open Reading Frames; Pharmaceutical Preparations; Predisposition; Proteins; Regulon; Reporting; Research; Research Personnel; Resistance; Resources; Rifampin; Source; Testing; Tetracycline; Tetracyclines; Trimethoprim; United States National Institutes of Health; Vesicle; Water; antimicrobial drug; antiporter; efflux pump; foodborne infection; microorganism; novel; resistance mechanism

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bacteria from the Enterobacteriaceae family have been implicated in food-borne infections. We tested the hypothesis that milk confers reduced susceptibilities to multiple antimicrobial agents in E. coli cells harboring the marCRAB regulon. We inoculated pasteurized whole milk with E. coli strain GC4460 (wild-type marCRAB), strain JHC1096 (¿ marCRAB control), or strain AG112 (¿ marR), and incubated each overnight at 37¿C, subsequently recovered all strains from the milk cultures, and determined susceptibility levels to clinically- and marCRAB-relevant antimicrobial agents by the E-test strip method, according to established CLSI guidelines. Cells of strain GC4460, prior to culturing in milk, were susceptible to trimethoprim, gatifloxacin, cefotaxime and tetracycline. After culturing GC4460 in pasteurized milk, however, the MICs significantly increased (P d 0.026) by 1.5-fold for gatifloxacin, by 2.0-fold for cefotaxime, by 1.4-fold for tetracycline and by 1.4-fold for trimethoprim (P d 0.05). On the other hand, the MICs after culturing GC4460 on milk agar were enhanced (P0.05) by 3.4-fold for trimethoprim, by 7.1-fold for cefotaxime, by 40.5-fold for tetracycline, and by 10-fold for gatifloxacin. The MICs of the antimicrobial agents for the control cell JHC1096 after culturing in pasteurized whole milk were indistinguishable (P ¿ 0.05) from MICs measured before culturing in the same type of milk. Thus, E. coli cells harboring the marCRAB locus exhibit reduced susceptibilities to multiple antimicrobial agents after culturing in pasteurized whole milk. Therefore, we conclude that the reduction of antimicrobial agent susceptibility of E. coli in pasteurized milk is mediated by the marCRAB locus. In another project, we tested the hypothesis that active efflux represents an important mechanism for bacterial resistance to antimicrobial agents in an agricultural environment, such as dairy farms. Enterobacter cloacae are one of the leading causes of nosocomial infections. Recently, the frequency of infections by multidrug resistant E. cloacae has been reported to be increasing. To date, only three antimicrobial resistance mechanisms in this bacterium have been analyzed. These mechanisms alone are not sufficient to explain the reported multidrug resistant E. cloacae, and that measured in a dairy water isolate strain DFW9 of E. cloacae. Therefore, an understanding of the underlying resistance mechanisms of this microorganism is vital. We cloned a gene, designated emrF, which is responsible for multidrug resistance in E. cloacae using shot-gun cloning of chromosomal DNA. Host cells of E. coli KAM32 possessing the emrF gene showed elevated resistances to fosfomycin, rifampicin, ampicillin, amoxicilline, erythromycin and ethidium bromide, as measured using CLSI guidelines. DNA sequencing and analysis revealed one open reading frame (ORF) encoding a novel protein of 120 amino acids. The deduced EmrF protein was predicted to have four transmembrane segments and found to be similar to the SMR family of multidrug efflux pumps using TMHMM and GenomeNet TBLASTN analyses. We detected energy-dependent efflux of fosfomycin with everted membrane vesicles harboring EmrF, and found that EmrF is an H+/drug antiporter. We also observed ethidium efflux activity by comparing ethidium bromide accumulation in cells with and without EmrF. These results indicate that EmrF functions as an SMR family multidrug efflux pump in E. cloacae.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 来自肠杆菌科的细菌与食源性感染有关。 我们检验了牛奶降低大肠杆菌对多种抗菌剂敏感性的假设。大肠杆菌细胞的marCRAB调节子。 我们用E.大肠杆菌菌株GC 4460（野生型marCRAB）、菌株JHC 1096（marCRAB对照）或菌株AG 112（马尔R），并在37 ℃下孵育过夜，随后从乳培养物中回收所有菌株，并根据已建立的CLSI指南，通过E-测试条方法确定对临床和marCRAB相关抗菌剂的敏感性水平。 菌株GC 4460的细胞在牛奶中培养之前，对甲氧苄啶、加替沙星、头孢噻肟和四环素敏感。 然而，在巴氏消毒奶中培养GC 4460后，加替沙星的MIC显著增加（P d 0.026），增加了1.5倍，头孢噻肟增加了2.0倍，四环素增加了1.4倍，甲氧苄啶增加了1.4倍（P d 0.05）。 另一方面，在乳琼脂上培养GC 4460后，甲氧苄啶、头孢噻肟、四环素和加替沙星的MIC分别增加了3.4倍、7.1倍、40.5倍和10倍（P 0.05）。 在巴氏灭菌全脂奶中培养后，对照细胞JHC 1096的抗菌剂MIC与在相同类型牛奶中培养前测量的MIC无差异（P <$0.05）。 因此，E.携带marCRAB基因座的大肠杆菌细胞在巴氏灭菌的全脂乳中培养后，对多种抗微生物剂表现出降低的亲合性。 因此，我们得出结论，抗菌药物敏感性的降低是大肠杆菌。巴氏杀菌乳中的大肠杆菌是由marCRAB基因座介导的。在另一个项目中，我们测试了主动外排是农业环境（如奶牛场）中细菌对抗菌剂耐药性的重要机制的假设。 阴沟肠杆菌是医院感染的主要原因之一。 近年来，多药耐药大肠埃希菌感染的发生率不断上升。据报告，霍乱正在增加。 迄今为止，仅分析了该细菌中的三种抗生素耐药机制。 这些机制本身并不足以解释报道的多药耐药大肠杆菌。在乳用水分离菌株DFW 9中测定的，clothing.因此，了解这种微生物的潜在耐药机制至关重要。我们克隆了一个名为emrF的基因，该基因与大肠杆菌的多药耐药性有关。使用染色体DNA的鸟枪克隆进行克隆。 大肠杆菌的宿主细胞。具有emrF基因的大肠杆菌KAM 32显示出对磷霉素、利福平、氨苄青霉素、阿莫西林、红霉素和溴化乙锭的升高的抗性，如使用CLSI指南测量的。 DNA序列分析表明，该基因含有一个开放阅读框（ORF），编码120个氨基酸的蛋白质。 推测EmrF蛋白具有四个跨膜片段，并使用TMHMM和GenomeNet TBLAYPINE分析发现其与SMR家族的多药外排泵相似。 我们检测到能量依赖性外排磷霉素与外翻膜囊泡窝藏EmrF，并发现EmrF是一个H+/药物逆向转运蛋白。 我们还观察了乙锭外排活性，通过比较溴化乙锭在细胞中的积累与EmrF。 这些结果表明EmrF在E. clothing.