| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
The ampC and ampR gene of Enterobacter cloacae GN7471 were cloned into. pMW218 to yield pKU403. Four mutant plasmids derived from pKU403(pKU404, pKU405, pKU406PP and pKU407) were isolated in an AmpD mutant of Escherichia coli ML4953 by selection with CAZ ot AZT. The β- lactamase activities expressed by pKU404,pKU405,pKU406 and pKU407 were about 450, 95, 160 and 160 times higher respectively than expressed by the original plasmid, pKU403. These mutant pilsmids all carried point mutations in the ampR gene. In pKU404 and pKU405, Asp135 was changed to Asu and Val, respectively. In both pKU406 and pKU407, Arg86 was changed to Cys. The each of selection of AmpR mutation at a frequency of about 10^<-6> in this study strongly suggests that derepressed strains, such as AmpD or ampR mutants, could frequently emrge in the clinical setting.
The disk screening methods for extended-spectrum β-lactamase producing strains were evaluated the confirming work is reduced significantly in settings much as t
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hose study, by changing the cefpodoxime break point to <20mm and by not testing cefoxitin resistant isoaltea. Cefotaxime and ceftazidime disk screening is reliable, and the laboratory prepared cefotaxime and ceftazidime-clavulanic acid disks are stable at -20 C for 12 weeks.
A mutant of E.cloacae KU69, derived from KU4925, produced AmpC constitutively at very low basal levels(0.02 u/mg protein) and it was susceptible to cefotaxime. High amount of AmpC mutants were isolated by selection with cephems from E.cloacae KU69 at a frequency 10^<-7>. Intorduction of ampG clone, derived from E. coli, by transformation into E.cloacae KU69 restored high amount of AmpC β-lactamase and resistance to Gefotaxime. From DNA sequences, It indicated that E.cloacae KU69 carries point mutation in the ampG gene. The ampG gene of tryptophan (TGG)-132 in E.cloacae KU4925 was changed to stop codon(TAG). In three reversion mutants(Mut-31 -49, 56) of E.cloacae KU69 ampG, the stop codon on these mutants was changed to tyrosine(TGG).Moreover remaining two revertants(Mut-51 and -14) isolated from KU69 ampG, ths stop codon was changed to either glutamine(CAG) or lysine(TGG). These mutants showed two times higher activities than those expressed by original strain of.KU4925. Less
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