In Vitro And In Vivo Studies Of Resistant Mutant Selection For Quinolone Against Staphylococcus Aureus

[Objective] (1 ) to assess the antibacterial activity of a novel des-F(6)-quinolone (NFQ), nemonoxacin, against Staphylococcus aureus in comparison with the activity of ciprofloxacin, levofloxacin, and moxifloxacin; ( 2 )to study the effects of quinolone concentration on the recovery of resistant colonies in terms of both mutant frequency and mutant types; ( 3 ) to determine the primary target of nemonoxacin in Staphylococcus aureus strain ATCC25923 by genomic characterization of first and second step mutants; ( 4 )to analyze susceptibility of Gram-positive and Gram-negative clinical isolates to nemonoxacin and 13 other selected antimicrobial agents.[Methods] S. aureus cells were grown in MH broth. MIC and MPC for nemonoxacin and three fluoroquinolones with Staphylococcus aureus strain ATCC25923, clinical isolates of MSSA and MRSA, either susceptible or resistant to ciprofloxacin, were determined by spreading of various amounts of cells on agar plates containing various concentrations of each drug. The quinolone resistance-determining-regions of target genes, gyrA, gyrB, and grlA of mutants selected were obtained by PCR amplification and sequenced by nucleotide sequence determination. The CLSI agar dilution method was carried out to determine MIC of nemonoxacin and 13 other antimicrobial agents against MSSA (60), MRSA (47), Staphylococcus epidermidis (30 ), Streptococcus pneumoniae (51), Escherichia coli(29),Klebsiella pneumoniae(28), Acinetobacter baumanii (23 ), and Pseudomonas aeruginosa (29) .[Results](1) The MPCs of nemonoxacin, moxifloxacin, levofloxacin, and ciprofloxacin for staphylococcus aureus strain ATCC25923 were 0.2μg /ml, 0.2μg /ml, 0.8μg/ml, and 1.8μg/ml, respectively. The MPC/MIC₉₉ of nemonoxacin, moxifloxacin, levofloxacin, and ciprofloxacin for staphylococcus aureus strain ATCC25923 were 7.1,7.1, 8.0, and 10.6, respectively. (2) Drug concentrations of nemonoxacin and FQs showed strong effects on the frequency of recovery of resistant colonies and the types of resistant mutants selected. (3) All 4 first-step mutants selected using nemonoxacin contained nucleotide changes at codon 86, 439, and 440 that did not result in any amino acid substitution. For the 4 second-step mutants selected from first-step resistance strains, a gyrA mutation (Ser-84→Leu) was found. No mutation was found in grlA region of QRDR. (4)With ciprofloxacin-susceptible MSSA strains, the MPC₉₀ values for ciprofloxacin, levofloxacin, moxifloxacin and nemonoxacin were 8.0μg/ml, 2.0μg/ml, 0.5μg/ml, and 0.25μg/ml, respectively. The rank order of MPC/MIC values was nemonoxacin = moxifloxacin < levofloxacin = ciprofloxacin. With ciprofloxacin-susceptible MRSA isolates, the MPC₉₀ values for ciprofloxacin, levofloxacin, moxifloxacin and nemonoxacin were 16.0μg/ml, 8.0μg/ml, 4.0μg/ml, and 2.0μg/ml, respectively. The rank order of MPC/MIC values was nemonoxacin = moxifloxacin < levofloxacin = ciprofloxacin. With ciprofloxcin-resistant MSSA strains, the MPC90 values for nemonoxacin and moxifloxacin were 4.0μg/ml and 32.0μg/ml, respectively. The ratio of MPC/MIC of nemonoxacin was 8, which was 2-fold lower with nemonoxacin than with moxifloxacin. With ciprofloxcin-resistant MRSA strains, the MPC90 values for nemonoxacin and moxifloxacin were 16.0μg/ml and > 32 ug/ml. (5) The MIC₉₀ for nemonoxacin with MSSA, MRSA, Staphylococcus epidermidis, and Streptococcus pneumoniae were 0.5μg/mL 1.0μg/ml, 0.5μg/ml, and 0.5μg/ml, respectively, exhibiting greater antimicrobial activity than comparator quinolones, such as ciprofloxacin, levofloxacin, moxifloxacin, and other antimicrobials especially when tested against selected multi-drug-resistant Streptococcus pneumoniae and MRSA. Compared with ciprofloxacin, levofloxacin, moxifloxacin, and other antimicrobials, nemonoxacin was less active against all Gram-negative clinical isolates tested, with the MIC90 of nemonoxacin against clinical isolates of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, and Pseudomonas aeruginosa being 8.0μg/mL, 8.0μg/ml, 32μg/ml, and 8.0μg/ml, respectively.[Conclusion] (1) For Staphylococcus aureus strain ATCC 25923, the ability of nemonoxacin to restrict the selection of next-step resistant mutants is stronger than that of levofloxacin and ciprofloxacin, and is comprarable to that of moxifloxacin.(2) DNA gyrase is the primary target for nemonoxacin with Staphylococcus aureus, in contrast to DNA topoisomerase IV being the primary target for fluoroquinolones. Variations at amino acid position 84 of GyrA subunit may be the most important mutations for conferring resistance to nemonoxacin in Staphylococcus aureus. (3) Compared with FQs, nemonoxacin has excellent antimicrobial activity against clinical isolates (especially with ciprofloxacin-resistant MRSA strains) and hardly selects step-wise resistance mutants. When compared with pharmacokinetic parameters, serum-achievable concentrations of nemonoxacin can restrict the selection of the next-step resistant mutants with ciprofloxacin-susceptible and -resistant MSSA and ciprofloxacin-susceptible MRSA but not with ciprofloxacin-resistant MRSA. (4) Nemonoxacin is a valuable alternative for treatment of quinolone-resistant clinical isolates of MSSA , quinolone-susceptible MRSA, and Streptococcus pneumoniae infections. Nemonoxacin has no advantage in treating infections caused by Gram-negative bacteria.[Objective] To measure the MIC, MIC₉₉ and MPC for levofloxacin with Staphylococcus aureus strain ATCC25923 grown up in vivo, and to study the correlation of levofloxacin pharmacokinetic/pharmacodynamic (PK/PD) parameters with the upper and lower boundaries of the mutant selection window for restricting theselection of resistant mutants in vivo. [Methods] A rabbit tissue-cage infection model was established by surgicalinplantation of hollow plastic balls underneath the skins of rabbits followed by injecting 3×10⁷-3×10⁹ cfu of Staphylococcus aureus strain ATCC25923 into tissue cage. Bacteria amplified inside tissue cages were used to determine MIC, MIC₉₉, and MPC. One group of rabbits were treated orally with different doses and different frequencies of levofloxacin (20mg/kg tid, 30mg/kg bid, 60mg/kg 1 /2d ) for targeted tissue drug concentrations hovering around the upper boundary of the mutant selection window (e.g. the MPC); another group of rabbits were treated orally with different doses and different frequencies of levofloxacin (2.5mg/kg tid, 3.5mg/kg bid, 6.0mg/kg 1/2d ) for targeted tissue drug concentrations fluctuating around the lower boundary of the selection window (e.g. the MIC); a third group of rabbits were treated with saline following the same dosing schedule as controls. All treatment lasted for five days. The concentrations of levofloxacin in the tissue cage fluid were analysized by high-performance liquid chromatographic method. Levofloxacin susceptibility of bacteria was monitored at the site of infection. The correlations between drug exposure and drug resistance were analysized.[Results](1) The MIC, MIC₉₉, and MPC of levofloxacin for ATCC25923 proliferated in vivo were 0.125μg/ml, 0.1μg/ml, and 0.8μg/ml respectively, consistent with values determined with cultures grown up in vitro. (2)Upon changing doses and frequencies, AUC /MPC>25h and C_max/MPC>1.2 were identified as the bestparameters for predicting resistance of the upper boundary of MSW. AUC/MIC≤20h and T> MIC ≤20% (or T_msw≤20%) were found to be the best parameters for resistance prevention of the lower boundary of the MSW. Considering traditional regulations of PK/PD parameters, AUC /MPC>25h and AUC/MIC≤20h were best suited for prediction of the upper and lower boundaries of the MSW, respectively.[Conclusion] (1) The MIC. MPC values measured with cells proliferated in vivo match those determined with bacteria amplified in vitro. (2) Keeping AUC/MPC above 25h or AUC/MIC below 20h can restrict selection of levofloxacin resistance at the site of infection.