This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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