TABLE 5.

Antibiotic susceptibility profile and resistance mechanisms of in vitro- and in vivo-selected PAO1- or PAOΔmutS-resistant mutants

Origin of resistant mutants and treatmentaMIC range (μg/ml)bβ-Lactamase sp act (mean ±SD)cQRDR or effluxd
CAZFEPIMPMERATMGENTOBCIPTETCHLBasalInduced
PAO1 (wild type)111.50.51.5210.122432150 ± 13
PAOΔmutS (wild type)111.50.751.5310.1224323.5 ± 0.958 ± 16
PAO1 (in vitro expt)
    CAZ (4; n = 5)24-3212-321.5-20.5-0.758-122-31-1.50.064-0.1216-3216-3218 ± 2-50 ± 763 ± 24-149 ± 63
    CAZ (16; n = 5)24-3224-3220.75-1122-310.094-0.123232-4833 ± 18-67 ± 28147 ± 62-513 ± 251
PAOΔmutS (in vitro expt)
    CAZ (4; n = 5)24-6424-481.5-20.5-1.512-322-40.75-1.50.064-0.1216-2416-3255 ± 25-230 ± 90261 ± 157-817 ± 340
    CAZ (16; n = 5)48-6424-481.5-20.75-124-482-40.75-20.1216-2424-3241 ± 6-92 ± 27167 ± 73-817 ± 255
PAO1 (in vivo expt)
    CAZ (4; n = 5)24-4816-481.5-20.75-212-162-31-20.12-0.1924-4832-6426 ± 7-62 ± 8119 ± 61-189 ± 73
    CAZ (16; n = 1)24161.5112210.12326434 ± 11391 ± 98
PAOΔmutS (in vivo expt)
    CAZ (4; n = 5)32-9624-481.5-21.5-324-323-41.5-20.094-0.1224-4832-48123 ± 37-237 ± 120336 ± 82-426 ± 38
    CAZ (16; n = 5)64-96481.5-21-2323-41.5-20.1224-3224-4870 ± 7-162 ± 82367 ± 22-654 ± 248
PAOΔmutS (in vitro expt, double mutants)
    CAZ-TOB (4, 4; n = 5)12-484-321.5-20.75-1.58-3212-486-240.12-0.2516-3212-3228 ± 8-60 ± 18256 ± 95-426 ± 204mexY (no change)
    CAZ-CIP (5, 0.5; n = 1)331.51.5341.51.5243256 ± 23131 ± 45GyrA(Thr83Ile)
    CAZ-CIP (4, 0.5; n = 2)32-6424-321.5-21-1.58-163-41.5216-24481.6 ± 0.2-2.1 ± 144 ± 3-46 ± 15GyrB(Ser466Phe)
    CAZ-CIP (4, 0.5; n = 1)8323648212>256>2561.9 ± 0.252 ± 18mexB (19-fold)
    CAZ-CIP (4, 0.5; n = 1)3232111221.5396>25652 ± 23136 ± 77mexD (25-fold)
  • a Up to five mutants of each strain (PAO1 or PAOΔmutS), for each condition (in vitro or in vivo), and for each antibiotic concentration (CAZ at concentrations 4- and 16-fold higher than the MICs and CAZ-CIP and CAZ-TOB at concentrations 4-fold higher than the MICs) were characterized. Values in parentheses indicate the n-fold increase(s) in drug MICs and the number of mice. CAZ-TOB and CAZ-CIP mutants were obtained only from in vitro PAOΔmutS experiments, and mutants were obtained from only one mouse in in vivo experiments with mice inoculated with PAO1 and treated with CAZ at a concentration 16-fold higher than the MIC; therefore, results for just one mutant are shown.

  • b FEP, cefepime; IMP, imipenem; MER, meropenem; ATM, aztreonam; GEN, gentamicin; TET, tetracyclines; CHL, chloramphenicol.

  • c Relative values of basal and induced (cefoxitin at 50 μg/ml) specific β-lactamase activities, with 1 being considered the obtained basal activity for the reference strain PAO1 (19 nmol of nitrocefin hydrolyzed per min per mg of protein).

  • d Mutation in the topoisomerase (numbering corresponds to that of the published PAO1 sequence) or increase (relative to the level in wild-type PAO1 or PAOΔmutS) in mRNA level for the genes mexB, mexD, mexF, and mexY, coding for efflux pumps. QRDR, quinolone resistance-determining region.