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Antimicrobial Agents and Chemotherapy, July 2004, p. 2665-2672, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2665-2672.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Detection and Susceptibility Testing of Hypermutable Pseudomonas aeruginosa Strains with the Etest and Disk Diffusion

Servicio de Microbiología, Hospital Son Dureta, Palma de Mallorca, Spain

Received 26 November 2003/ Returned for modification 31 December 2003/ Accepted 24 March 2004

Resistance development in Pseudomonas aeruginosa from chronically colonized cystic fibrosis (CF) patients has been linked to the presence of a high proportion of mismatch repair-deficient hypermutable strains. The detection of hypermutable strains by microbiology laboratories may be useful for establishing adequate antimicrobial therapies. In this work, we find that the Etest and disk diffusion can be used as simple methods for the detection and susceptibility testing of hypermutable P. aeruginosa isolates. Strain PAO1 and its hypermutable derivative strain PAOmutS were used to standardize the procedure, which was tested with 35 P. aeruginosa isolates from 21 CF patients. Mutation frequencies were estimated by standard methods, and 29% of the isolates were found to be hypermutable. MICs and inhibition zone diameters were determined for ceftazidime, imipenem, meropenem, ciprofloxacin, and tobramycin by using Etest strips and conventional disks, respectively. The presence (or absence) of resistant mutant subpopulations, as well as their relative numbers and the highest MICs for them (or smallest inhibition zone diameters), was recorded. The presence of resistant mutant subpopulations within the inhibition zones of three or more antibiotics clearly identified the strains as hypermutable (they were present in 10 of 10 hypermutable strains and 0 of 25 nonhypermutable strains) with both methods. Additionally, these methods allowed us to differentiate between dual effects of hypermutation in antibiotic resistance, namely, that (i) hypermutable isolates were substantially more resistant than nonhypermutable isolates and that (ii) the resistance of hypermutable isolates was dramatically increased by the presence of resistant mutant subpopulations. This differentiation may be relevant for the design of adequate treatments, since the second effect, in contrast to the first, may be overcome by antibiotic combinations.

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