Sublethal ciprofloxacin treatment leads to rapid development of high-level ciprofloxacin resistance during long-term experimental evolution of Pseudomonas aeruginosa

Abstract

The dynamics of occurrence and the genetic basis of ciprofloxacin resistance were studied in a long-term evolution experiment (940 generations) in wild-type, reference strain (PAO1) and hypermutable (PAOΔmutS and PAOMY-Mgm) P. aeruginosa populations continuously exposed to sub-MICs (1/4) of ciprofloxacin. A rapid occurrence of ciprofloxacin-resistant mutants (MIC of ≥12 μg/ml, representing 100 times the MIC of the original population) were observed in all ciprofloxacin-exposed lineages of PAOΔmutS and PAOMY-Mgm populations after 100 and 170 generations, respectively, and in one of the PAO1 lineages after 240 generations. The genetic basis of resistance was mutations in gyrA (C248T and G259T) and gyrB (C1397A). Cross-resistance to beta-lactam antibiotics was observed in the bacterial populations that evolved during exposure to sublethal concentrations of ciprofloxacin. Our study shows that mutants with high-level ciprofloxacin resistance are selected in P. aeruginosa bacterial populations exposed to sub-MICs of ciprofloxacin. This can have implications for the long-term persistence of resistant bacteria and spread of antibiotic resistance by exposure of commensal bacterial flora to low antibiotic concentrations.

Fig 1

Characterization of the susceptibility to ciprofloxacin of the bacterial population and assignment of population MIC values. To distinguish between different sizes of the resistant subpopulations that grew in the inhibition zone, four descriptive entities were defined: (i) “double zone” if the resistant colonies exhibit a confluent growth, (ii) +++ if the number of colonies was higher than 100, (iii) ++ if the number of colonies was between 10 and 100, and (iv) + if fewer than 10 colonies were present. The descriptions of the two bacterial populations in this figure are thus as follows: in the left panel, the population MIC is 1.5 μg/ml, there is no double zone (dz−), and the size of the resistant subpopulation is +++; in the right panel, the population MIC is 1.5 μg/ml, a double zone is present until 12 μg/ml (dz 12), and resistant colonies in the inhibition zone are also present (+).

Fig 2

Development of resistance to ciprofloxacin in ciprofloxacin-treated (top three rows) and untreated (bottom three rows) PAO1, PAOΔmutS, and PAOMY-Mgm during the evolution experiment. Resistant mutants occurred faster in the mutator populations than in PAO1, with an especially drastic increase in MICs for the ciprofloxacin-treated PAOΔmutS strains. Black bars, MICs of population; white bars, MICs of double zone; gray bars, MICs of resistant colonies. Notice the difference in the numerical size of the ordinates in the top three and bottom three rows.