Invitro selection and resistant mechanism investigation of flouroquinolone resistant Staphylococcus aureu ATCC 25213

Abstract

Application of antibiotics with concentrations lower than the minimal inhibitory concentration (MIC) is widely believed to be the main reason for antibiotic resistance development. This work aims to provide more understanding on this phenomenon. Pure strain of Staphylococcus aureus ATCC 25213 was serially and separately exposed to Ciprofloxacin, Levofloxacin and Ofloxacin below its MIC for 12 days then cultured for another 10 days without antibiotics. DNA of naïve S. aureus and three selected strains (CIP-, LEV- and OFL- resistant S. aureus) after exposure were investigated for 16S rRNA sequencing as well subjected to gyrA and grlA sequencing. Four bacterial strains were also tested for susceptibility to 12 different antibiotics. There was a strong and irreversible increase of MIC of S. aureus to all FQs used in the exposures (increasing 32 to 128 times higher after exposure; remaining 16 to 32 times higher after antibiotic-free culture). Sequencing results showed no mutation in gyrA gene and one mutation in grlA (Ser80->Phe in both CIP- and OFL-resistant strains, Ala64->Ala (GCG->GCA) in LEV-resistant one). Additionally, each of FQ-resistant S. aureus strains became cross-resistant to three other FQs and also resistant to other unrelated antibiotics. In summary, the serial exposure to sub-MIC concentrations could irreversibly turn FQ-sensitive S. aureus into FQ-resistant phenotype which seemed to be primarily affected by genetic modification rather than gene regulation. Key words: Ciprofloxacin, Levofloxacin, Ofloxacin, minimal inhibitory concentration (MIC), antibiotic resistance mechanisms, serial exposure, Staphylococcus aureus, gyrA, grlA.