Discriminatory power of three DNA-based typing techniques for Pseudomonas aeruginosa

Abstract

We assessed the capacity of three DNA typing techniques to discriminate between 81 geographically, temporally, and epidemiologically unrelated strains of Pseudomonas aeruginosa. The methods, representing powerful tools for hospital molecular epidemiology, included hybridization of restricted chromosomal DNA with toxA and genes coding for rRNA (rDNA) used as probes and macrorestriction analysis of SpeI-digested DNA by pulsed-field gel electrophoresis. The probe typing techniques were able to classify all strains into a limited number of types, and the discriminatory powers were 97.7 and 95.6% for toxA and rDNA typing, respectively. Strains that were indistinguishable on the basis of both toxA and rDNA types defined 12 probe type homology groups. Of these, one contained five strains, three contained three strains each, and eight groups were represented by two strains each. Strains in 10 of the homology groups had the same O serotype. SpeI macrorestriction patterns discriminated between all strains with at least four band differences, which corresponded to a similarity level of 85%. Fifteen pairs of strains were similar at a level of >75% and differed by only four to seven bands. Of these pairs, 11 belonged to the same probe type homology group, indicating their clonal relatedness. We conclude that macrorestriction analysis of P. aeruginosa with SpeI provides the best means of discrimination between epidemiologically unrelated strains. However, DNA probe typing with either toxA or rDNA reveals information on the strain population structure and evolutionary relationships.

Molecular epidemiology provides evidence of genotypic heterogeneity of multidrug-resistant Pseudomonas aeruginosa serotype O:12 outbreak isolates from a pediatric hospital

“…While the antibiotic susceptibility patterns appeared more discriminative than the genotypic methods, antibiotic susceptibility testing has relatively limited utility in epidemiological studies because antibiotic resistance is affected by extraordinary selective pressure in contemporary hospitals. Our results providing evidence of genotypic heterogeneity of multidrug-resistant P. aeruginosa O:12 outbreak isolates are in agreement with those reported by Grundmann et al (10). However, these authors did not look at random PCR (10).…”

Outbreaks of Multidrug-Resistant Pseudomonas aeruginosa in Community Hospitals in Japan

“…Pulsed-field gel electrophoresis (PFGE). Chromosomal DNA was prepared by the procedure of Grundmann et al (10) and digested overnight with 10 U SpeI (Takara Bio, Inc., Shiga, Japan). The DNA fragments were separated on 1.0% agarose gels in 0.5ϫ Tris-borate-EDTA buffer with a CHEF Mapper system (Bio-Rad Laboratories, Hercules, CA) at 6 V/cm for 20 h. The obtained fingerprinting patterns, normalized to the molecular weight markers, were analyzed by the unweighted-pair-group method with Molecular Analyst Fingerprinting Plus software, version 1.6 (Bio-Rad Laboratories, Inc.), to obtain average linkagebased dendrograms.…”

Ribotyping of Pseudomonas aeruginosa from infected patients: evidence of common strain types

“…As we have found in our studies (data not shown), most strains from the same hospital generally share the same biochemical profiles and similar antibiograms. In contrast, molecular typing methods including ribotyping have contributed significantly to a better understanding of the epidemiology of P. aeruginosa infections (9,12,16,26).…”

“…Diverse DNA-based typing methods have recently been used to fingerprint P. aeruginosa isolates (9), particularly, restriction endonuclease analysis of genornic DNA (21), macrorestriction analysis of digested DNA by pulsefield gel electrophoresis (29), and AP-PCR (12,17,20), and several comparisons of phenotypic and genotypic methods for typing clinical (16,26) or environmental (1 3) strains of P. aeruginosa have been published. Ribotyping has also been successfully used for the typing of various medically important microorganisms (3), including P. aeruginosa (2, 8, 10).…”