Emergence of resistant Streptococcus pneumoniae in an in vitro dynamic model that simulates moxifloxacin concentrations inside and outside the mutant selection window: related changes in susceptibility, resistance frequency and bacterial killing

ABSTRACT Recent clinical failures associated with levofloxacin treatment for Streptococcus pneumoniae infections and growing evidence of frequent mutations in the isolate population have led to increased concerns regarding fluoroquinolone resistance. Our objective was to characterize the efficacies of levofloxacin and moxifloxacin against various genotypes of S. pneumoniae after simulated bronchopulmonary exposures. An in vitro model was used to simulate a levofloxacin concentration of 500 mg and a moxifloxacin concentration of 400 mg, which were previously determined to be the concentrations in the epithelial lining fluid of older adults receiving once-daily dosing. The effects of the drugs were tested against six S. pneumoniae containing various mutations. Bacterial density and resistance were quantitatively assessed over 48 h. The S. pneumoniae isolate with no mutation displayed a 4-log reduction in CFU after treatment with both agents and did not develop resistance. Isolates containing the parC or parE mutation or both mutations regrew and developed resistance when they were exposed to levofloxacin, despite an unbound area under the concentration-time curve (AUC):MIC ratio of ∼100. When the isolate containing the parC and gyrA mutations was exposed to levofloxacin, there was a half-log reduction in the number of CFU compared to that for the control, but the isolate subsequently regrew. Likewise, levofloxacin did not kill the isolate containing the parC, gyrA, and parE mutations. Moxifloxacin sustained the killing of all bacterial isolates tested without the development of resistance. Levofloxacin did not sustain bacterial killing and did not prevent the emergence of further resistance in mutants with the parC or parE mutation or both mutations, even though an unbound AUC:MIC ratio for exposure well above the breakpoint of 30 to 40 established in the literature for S. pneumoniae was maintained. Moxifloxacin was effective against all isolates tested, despite the presence of isolates with two- and three-step mutations, for which the MICs were increased.

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Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1129 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1129-1136 ◽

Cited By ~ 85

Author(s):

Xiao-Su Pan ◽

L. Mark Fisher

Keyword(s):

Streptococcus Pneumoniae ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Topoisomerase Iv ◽

T7 Promoter ◽

Bacterial Killing ◽

E Coli ◽

Apparent Homogeneity ◽

Comparable Activity

ABSTRACT Streptococcus pneumoniae gyrA and gyrBgenes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity againstS. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

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Comparative pharmacodynamics of the new fluoroquinolone ABT492 and levofloxacin with Streptococcus pneumoniae in an in vitro dynamic model

International Journal of Antimicrobial Agents ◽

10.1016/j.ijantimicag.2005.02.004 ◽

2005 ◽

Vol 25 (5) ◽

pp. 409-413 ◽

Cited By ~ 7

Author(s):

Alexander A. Firsov ◽

Irene V. Alferova ◽

Maria V. Smirnova ◽

Irene Yu. Lubenko ◽

Yury A. Portnoy ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Dynamic Model

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Pharmacodynamic Modeling of Clarithromycin against Macrolide-Resistant [PCR-Positive mef(A) or erm(B)] Streptococcus pneumoniae Simulating Clinically Achievable Serum and Epithelial Lining Fluid Free-Drug Concentrations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.12.4029-4034.2002 ◽

2002 ◽

Vol 46 (12) ◽

pp. 4029-4034 ◽

Cited By ~ 33

Author(s):

Ayman M. Noreddin ◽

Danielle Roberts ◽

Kim Nichol ◽

Aleksandra Wierzbowski ◽

Daryl J. Hoban ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Compartment Model ◽

Free Drug ◽

Pharmacodynamic Modeling ◽

Epithelial Lining ◽

Bacterial Killing ◽

Epithelial Lining Fluid ◽

Initial Inoculum ◽

Drug Concentrations

ABSTRACT The association between macrolide resistance mechanisms and clinical outcomes remains understudied. The present study, using an in vitro pharmacodynamic model, assessed clarithromycin (CLR) activity against mef(A)-positive and erm(B)-negative Streptococcus pneumoniae isolates by simulating free-drug concentrations in serum and both total (protein-bound and free) and free drug in epithelial lining fluid (ELF). Five mef(A)-positive and erm(B)-negative strains, one mef(A)-negative and erm(B)-positive strain, and a control [mef(A)-negative and erm(B)-negative] strain of S. pneumoniae were tested. CLR was modeled using a one-compartment model, simulating a dosage of 500 mg, per os, twice a day (in serum, free-drug Cp maximum of 2 μg/ml, t 1/2 of 6 h; in ELF, C ELF(total) maximum of 35μg/ml, t 1/2 of 6 h; CELF(free) maximum of 14 μg/ml, t 1/2 of 6 h). Starting inocula were 106 CFU/ml in Mueller-Hinton broth with 2% lysed horse blood. With sampling at 0, 4, 8, 12, 20, and 24 h, the extent of bacterial killing was assessed. Achieving CLR T/MIC values of ≥90% (AUC0-24/MIC ratio, ≥61) resulted in bacterial eradication, while T>MIC values of 40 to 56% (AUC0-24/MIC ratios of ≥30.5 to 38) resulted in a 1.2 to 2.0 log10 CFU/ml decrease at 24 h compared to that for the initial inoculum. CLR T/MIC values of ≤8% (AUC0-24/MIC ratio, ≤17.3) resulted in a static effect or bacterial regrowth. The high drug concentrations in ELF that were obtained clinically with CLR may explain the lack of clinical failures with mef(A)-producing S. pneumoniae strains, with MICs up to 8 μg/ml. However, mef(A) isolates for which MICs are ≥16 μg/ml along with erm(B) may result in bacteriological failures.

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Influence of Inoculum Size on the Selection of Resistant Mutants of Escherichia coli in Relation to Mutant Prevention Concentrations of Marbofloxacin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00156-07 ◽

2007 ◽

Vol 51 (11) ◽

pp. 4163-4166 ◽

Cited By ~ 31

Author(s):

Aude Ferran ◽

Véronique Dupouy ◽

Pierre-Louis Toutain ◽

Alain Bousquet-Mélou

Keyword(s):

Escherichia Coli ◽

Bacterial Population ◽

Inoculum Size ◽

Pharmacodynamic Model ◽

Initial Size ◽

Mutant Selection ◽

Selection Window ◽

Resistant Mutants ◽

Selection Of

ABSTRACT We demonstrate using an in vitro pharmacodynamic model that the likelihood of selection of Escherichia coli mutants resistant to a fluoroquinolone was increased when the initial size of the bacterial population, exposed to fluoroquinolone concentrations within the mutant selection window, was increased.

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