Mutant-prevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin

Abstract

The antibacterial activity and selection of resistant bacteria, along with mechanisms of fluoroquinolone resistance, were investigated by integrating the static [MIC or mutant-prevention concentration (MPC)] and in vitro dynamic model approaches using Escherichia coli isolates from diseased dogs. Using the dynamic models, selected E. coli strains and enrofloxacin and marbofloxacin at a range of simulated area under concentration-time curve over a 24 h interval (AUC 24 h )/MIC ratios were investigated. Our results indicated increasing losses in susceptibility of E. coli upon continuous exposure to enrofloxacin and marbofloxacin in vitro. This effect was transferable to other fluoroquinolones, as well as to structurally unrelated drugs. Our results also confirmed an AUC 24 h /MIC (AUC 24 h /MPC)-dependent antibacterial activity and selection of resistant E. coli mutants, in which maximum losses in fluoroquinolone susceptibility occurred at simulated AUC 24 h /MIC ratios of 40-60. AUC 24 h /MPC ratios of 39 (enrofloxacin) and 32 (marbofloxacin) were considered protective against the selection of resistant mutants of E. coli. Integrating our MIC and MPC data with published pharmacokinetic information in dogs revealed a better effect of the conventional dosing regimen of marbofloxacin than that of enrofloxacin in restricting the selection of resistant mutants of E. coli. Target mutations, especially at codon 83 (serine to leucine) of gyrA, and overexpression of efflux pumps contributed to resistance development in both clinically resistant and in vitro-selected mutants of E. coli. We also report here a previously undescribed mutation at codon 116 of parC in two laboratory-derived resistant mutants of E. coli. Additional studies would determine the exact role of this mutation in fluoroquinolone susceptibility, as well as establish the importance of our findings in the clinical setting.

Using In Vitro Dynamic Models To Evaluate Fluoroquinolone Activity against Emergence of Resistant Salmonella enterica Serovar Typhimurium

“…In the present study, AUC/MPC ratios to reduce Salmonella-resistant mutants were 69 (DIF), 62 (ENR), and 39 (MAR). These results were consistent with previous reports (22,24). However, in the current study, no difference of potency was found through conversion from AUC/MIC ratios to AUC/MPC ratios resulting from different MPC/MIC ratios, because the MPCs of all 3 FQs were 4 times higher than their respective MICs.…”

“…This was in agreement with our previous findings using FQs against E. coli and Actinobacillus pleuropneumoniae (16,22,24). Furthermore, the I E showed species specificity that was lower (8 to 41%) than those from reports using non-biofilm-forming bacteria, while it was similar to I E s of FQs against biofilm-forming bacteria (difference of 8 to 10%) (16,22,24). The failure of FQs to suppress Salmonella growth in the culture could result not only from resistant populations but also from biofilm formation.…”

“…MIC increases were transferable between FQs. This is in accordance with our previous findings in which losses in susceptibility of Escherichia coli exposed to one FQ were transferable to other FQs and even to structurally unrelated drugs, partly due to activation of global drug efflux pumps (16,22). To confirm the contribution of efflux pump activity to this resistance, MICs were assessed in the presence of the efflux pump inhibitor phenylalaninearginine-␤-naphthylamide (PA␤N).…”

“…The reliability of FQ pharmacokinetic simulations and the high reproducibility of the time-kill curves provided by the model have been demonstrated previously using several drug-pathogen combinations (9,39). Further, the actual bacterial exposure to the FQ was confirmed by validated high-performance liquid chromatography (HPLC) methods as described earlier (16,22). Pilot studies done using the reference strain and clinical S. Typhimurium strains and two (one high and one low) simulated AUC/MIC ratios found that once the target AUC/MIC was achieved, no strain-specific differences in the kill-regrowth kinetics were observed (data not shown).…”

“…A previously described dynamic model (9,16,22,38,39) was used in this study. In brief, the model consisted of three connected flasks: a dilution compartment containing fresh Mueller-Hinton broth II (MHB II) broth, a central compartment with either a bacterial culture alone (control growth experiment) or a bacterial culture in medium containing drug (killingregrowth experiments), and an elimination compartment containing waste broth and bacteria.…”