In vitro sequential development of gene mutations in fluoroquinolone-resistant salmonellae
Abstract number: 902_p698
(1) To investigate the development of fluoroquinolone-resistant salmonellae in vitro; (2) to characterise the sequential accumulation of target gene mutations leading to high-level fluoroquinolone-resistance.
Fluoroquinolone-resistant mutants of Salmonella enterica serotype Typhimurium and S. Hadar were obtained by plating sensitive strains on agar containing increasing twofold concentrations of ciprofloxacin, gemifloxacin, levofloxacin, moxifloxacin, norfloxacin or ofloxacin. Mutants thus obtained were again subjected to selection to obtain nth-step mutants (where n stands for the number of times the mutants had been subjected to selection). Susceptibilities of the mutants were determined and mutations of target genes were detected by multiplex PCR amplimer conformation analysis and confirmed by direct DNA sequencing.
First-step mutants of S. Typhimurium usually harboured a gyrA mutation (Asp87®Asn/Gly/His/Tyr), the MICs were 416 times higher than those for the parent strains and were below the sensitive breakpoint of fluoroquinolones. One mutant harboured a gyrB (Glu466®Asp) mutation and the MICs were 28 times lower than those for the parent strain. Only three second-step mutants harboured an additional gyrA mutation (Ser83®Phe) while two 2nd-step mutants from the parent with a gyrB mutation harboured a gyrA mutation (Asp87®Gly or Ser83®Phe). Mutations in parC (Glu51®Asp, Gly78®Asp, Ser80®Arg/Ile or Glu84®Gly/Lys) were first observed in third-step mutants. Although most fourth-step mutants were resistant to high concentrations of fluoroquinolones as were third-step mutants, no additional mutation was found.
Fewer mutations were detected in mutants of S. Hadar: First-step mutants harboured a gyrA mutation (Asp87®Asn/Gly/Tyr or Ser83®Phe) and a gyrB (Glu466®Asp) mutation. No additional mutation was found in second-step mutants although MICs were higher than those for the parents.
Low-level fluoroquinolone-resistant salmonellae which evolved by development of a gyrA mutation became high-level resistant by developing additional gyrA and parC mutations. Such sequential development of fluoroquinolone-resistance varied among Salmonella serotypes."
|Session name:||XXIst ISTH Congress|
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