Application of different genotyping methods for Pseudomonas aeruginosa in a setting of endemicity in an intensive care unit

Abstract

Colonization with Pseudomonas aeruginosa was studied by taking serial swab specimens from the oropharynges and anuses and tracheal and gastric aspirates from patients in an intensive care unit during a 10-month period in a setting of endemicity. Nineteen (10%) of the 192 patients included in the study were colonized on admission, while another 30 (16%) patients acquired P. aeruginosa while in the hospital. Typing of 353 isolates was performed by random amplified polymorphic DNA (RAPD) analysis, and 56 strains were selected for further typing by RAPD analysis, pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analysis. By these methods, 42, 44, and 44 genotypes were found, respectively. Computer-aided cluster analysis indicated that similar groups of related isolates were obtained by each method. By taking admission periods into account, analysis of the typing results suggested cross-acquisition of P. aeruginosa for five patient pairs. The small number of transfers and the large number of genotypes found indicate that most P. aeruginosa strains were derived from the patients themselves. The numbers of observed typing patterns and band differences between related isolates were counted for each typing method. AFLP analysis with primers without a selective base proved to be the most discriminatory method, followed by PFGE, AFLP analysis (with one selective base), and RAPD analysis. On the basis of a comparison with established strain differentiation criteria for PFGE, the criteria for differentiation of P. aeruginosa by AFLP analysis are presented.

FIG. 1

Digitized PFGE patterns and dendrogram for 56 P. aeruginosa isolates cut with SpeI obtained after selection of 1 isolate of each genotype from each of 44 patients colonized with P. aeruginosa during a 10-month period of study in an ICU. The dendrogram was constructed by cluster analysis by UPGMA with GelCompar 3.1 software (Applied Maths). Percentages of similarity are shown above the dendrogram. Roman numerals indicate clusters with ≥80% homology, which corresponds to the criteria of Tenover et al. (11) except for cluster VII, with isolates with eight or more band differences, and cluster III, with isolates with slightly less than 80% homology with three band differences.

FIG. 2

Digitized AFLP (MseI+C primer) patterns and dendrogram for 56 P. aeruginosa isolates obtained after selection of 1 isolate of each genotype from each of 44 patients colonized with P. aeruginosa during a 10-month period of study in an ICU. The dendrogram was constructed by cluster analysis by UPGMA with GelCompar 3.1 software (Applied Maths). Percentages of similarity and molecular sizes are shown above the dendrogram. Roman numerals indicate clusters with ≥80% homology by PFGE; these correspond to ≥80% homology by AFLP analysis, except that two extra clusters (clusters VIII and IX) were obtained by cluster analysis.

FIG. 3

Digitized RAPD (ERIC2 primer) patterns and dendrogram for 56 P. aeruginosa isolates obtained after selection of 1 isolate of each genotype from each of 44 patients colonized with P. aeruginosa during a 10-month period of study in an ICU. The dendrogram was constructed by cluster analysis by UPGMA with by GelCompar 3.1 software (Applied Maths). Percentages of similarity and molecular sizes are shown above the dendrogram. Roman numerals indicate clusters with ≥80% homology by PFGE and AFLP analysis; these almost correspond to ≥90% homology by RAPD analysis. Isolates from clusters V and VI obtained by PFGE and AFLP analysis are clustered in two other clusters by RAPD analysis, and cluster IV obtained by PFGE and AFLP analysis is not identified by RAPD analysis. Furthermore, an extra cluster (cluster X) is identified by RAPD analysis.

FIG. 4

Overlap in ICU stays of patients colonized with P. aeruginosa isolates clustered as similar types (cluster are given in roman numerals; see also Table 2). Period in the ICU is given as day-month-year.