BACTERIAL ANTIBIOTIC RESISTANCE IN AGRICULTURE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肠杆菌科细菌与食源性感染有关。我们测试了这样一个假设，即牛奶降低了含有marCRAB调节子的大肠杆菌细胞对多种抗菌剂的敏感性。我们用大肠杆菌GC4460(野生型marCRAB)、JHC1096(野生型marCRAB对照)或AG112(Marr)接种巴氏灭菌全脂牛奶，并在37℃下培养过夜，随后从牛奶培养物中回收所有菌株，并根据建立的CLSI指南，通过E-test试纸法测定临床和marCRAB相关抗菌药的敏感性水平。GC4460菌株在牛奶中培养前，对甲氧苄氨嘧啶、加替沙星、头孢噻肟和四环素敏感。然而，在巴氏杀菌牛奶中培养GC4460后，加替沙星、头孢替辛、四环素和甲氧苄啶的MIC显著增加(P<0.026)，分别是1.5倍、2.0倍、1.4倍和1.4倍。而在牛奶琼脂上培养GC4460后，甲氧苄氨嘧啶、头孢噻肟、四环素和加替沙星的MIC分别提高3.4倍、7.1倍、40.5倍和10倍(P<0.05)。对照细胞JHC1096在巴氏杀菌全脂奶中培养后的MIC值与在同类型牛奶中培养前的MIC值无显著差异(P>0.05)。因此，携带marCRAB基因的大肠杆菌细胞在巴氏杀菌全脂牛奶中培养后，对多种抗菌剂的敏感性降低。因此，我们得出结论，巴氏杀菌牛奶中大肠杆菌对抗菌剂敏感性的降低是由marCRAB基因座介导的。在另一个项目中，我们测试了这一假设，即主动外排代表着农业环境中细菌对抗菌剂耐药性的重要机制，如奶牛场。阴沟肠杆菌是引起医院感染的主要原因之一。近年来，耐多药阴沟肠杆菌感染的频率呈上升趋势。到目前为止，只分析了该细菌的三种耐药机制。这些机制本身不足以解释报告的多重耐药阴沟肠杆菌，以及在乳制品水分离株DFW9阴沟肠杆菌中测量的结果。因此，了解这种微生物的潜在耐药机制是至关重要的。我们用鸟枪克隆染色体DNA的方法克隆了一个基因，命名为emrF，它与阴沟肠杆菌的多重耐药有关。根据CLSI指南，携带emrF基因的大肠杆菌KAM32的宿主细胞对磷霉素、利福平、氨苄西林、阿莫西林、红霉素和溴化乙锭的耐药性增加。DNA测序和分析揭示了一个开放阅读框架(ORF)，编码一个由120个氨基酸组成的新蛋白质。TMHMM和GenomeNet TBLASTN分析发现，推导出的EmrF蛋白有四个跨膜片段，与多药外排泵SMR家族相似。我们用含有EmrF的外翻膜囊检测磷霉素的能量依赖性外流，发现EmrF是一种H+/药物逆向转运蛋白。我们还通过比较有无EmrF的细胞中溴化乙锭的蓄积来观察乙锭的外排活性。这些结果表明EmrF在阴沟肠杆菌中作为SMR家族的多药外排泵发挥作用。

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.