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Genetic and phenotypic variations of a resistant Pseudomonas aeruginosa epidemic clone
Didier Hocquet1, Xavier Bertrand, Thilo Köhler
1Laboratoire de Bactériologie, Hôpital Jean Minjoz, Besançon, France.
Abstract:
From May 1997 to December 2001, a serotype O:6 multidrug-resistant strain of Pseudomonas aeruginosa colonized or infected 201 patients in the University Hospital of Besançon (France). The susceptibility profile of this epidemic clone to fluoroquinolones and aminoglycosides was relatively stable during the outbreak but showed important isolate-to-isolate variations (up to 64-fold) in the MICs of beta-lactams. Analysis of 18 genotypically related isolates selected on a quarterly basis demonstrated alterations in the two DNA topoisomerases II and IV (Thr83-->Ile in GyrA and Ser87-->Leu in ParC) and production of an ANT(2")-I enzyme. Although constitutively overproduced in these bacteria, the MexXY efflux system did not appear to contribute significantly to aminoglycoside resistance. beta-Lactam resistance was associated with derepression of intrinsic AmpC beta-lactamase (with isolate-to-isolate variations of up to 58-fold) and sporadic deficiency in a 46-kDa protein identified as the carbapenem-selective porin OprD. Of the 18 isolates, 14 were also found to overproduce the efflux system MexAB-OprM as a result of alteration of the repressor protein MexR (His107-->Pro). However, complementation experiments with the cloned mexR gene demonstrated that MexAB-OprM contributed only marginally to beta-lactam and fluoroquinolone resistance. Of the four isolates exhibiting wild-type MexAB-OprM expression despite the MexR alteration, two appeared to harbor secondary mutations in the mexA-mexR intergenic region and one harbored secondary mutations in the putative ribosome binding site located upstream of the mexAB oprM operon. In conclusion, this study shows that many mechanisms were involved in the multiresistance phenotype of this highly epidemic strain of P. aeruginosa. Our results also demonstrate that the clone sporadically underwent substantial genetic and phenotypic variations during the course of the outbreak, perhaps in relation to local or individual selective drug pressures.
Insights
A multidrug-resistant Pseudomonas aeruginosa strain caused an outbreak, exhibiting stable resistance to some antibiotics but variable resistance to beta-lactams. Genetic analysis revealed multiple resistance mechanisms, including topoisomerase mutations and efflux pump alterations, with ongoing variations during the epidemic.
Area of Science:
- Clinical Microbiology
- Molecular Biology
- Infectious Diseases
Background:
- An outbreak of multidrug-resistant (MDR) serotype O:6 Pseudomonas aeruginosa occurred between 1997 and 2001, affecting 201 patients.
- The epidemic clone displayed stable resistance to fluoroquinolones and aminoglycosides but significant variability in beta-lactam minimum inhibitory concentrations (MICs).
Purpose of the Study:
- To investigate the genetic and phenotypic mechanisms underlying the multiresistance of this epidemic Pseudomonas aeruginosa strain.
- To analyze the evolution of resistance mechanisms during the prolonged outbreak.
Main Methods:
- Genotypic and phenotypic analysis of 18 selected isolates over the outbreak period.
- Susceptibility testing, including MIC determination for beta-lactams, fluoroquinolones, and aminoglycosides.
- Analysis of DNA topoisomerases (GyrA, ParC), beta-lactamase production (AmpC), porin expression (OprD), and efflux systems (MexXY, MexAB-OprM).
Main Results:
- Resistance mechanisms identified include alterations in DNA topoisomerases II and IV, production of ANT(2")-I enzyme, derepression of AmpC beta-lactamase, and sporadic deficiency in OprD.
- Overproduction of MexAB-OprM due to MexR alterations was common but contributed marginally to resistance; secondary mutations were observed in some isolates.
- The MexXY efflux system did not significantly contribute to aminoglycoside resistance.
Conclusions:
- The multiresistance phenotype of this epidemic Pseudomonas aeruginosa strain resulted from a combination of diverse genetic and biochemical mechanisms.
- The clone exhibited significant genetic and phenotypic plasticity throughout the outbreak, likely influenced by selective drug pressures.