Capillary electrophoresis-single-strand conformation polymorphism analysisfor rapid identification of Pseudomonas aeruginosa and other gram-negativenonfermenting bacilli recovered from patients with cystic fibrosis

We used capillary electrophoresis-single-strand conformation polymorphism ( CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identi fication of Pseudomonas aeruginosa and other gramnegative nonfermenting bac illi isolated from patients with cystic fibrosis (CF), Target sequences wer e amplified by using forward and reverse primers labeled with various fluor escent dyes, The labeled PCR products were denatured by heating and separat ed by capillary gel electrophoresis with an automated DNA sequencer, Data w ere analyzed with GeneScan 672 software, This program made it possible to c ontrol lane-to-lane variability by standardizing the peak positions relativ e to internal DNA size markers, Thirty-four reference strains belonging to the genera Pseudomonas, Brevundimonas, Burkholderia, Comamonas, Ralstonia, Stenotrophomonas, and Alcaligenes were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism The best results we re obtained with the primer set P11P-P13P, which spans a moderately polymor phic region (Escherichia coli 16S rRNA positions 1173 to 1389 [M. N. Widjoj oatmodjo, A. C. Fluit, and J. Verhoef, J, Clin, Microbiol. 32:3002-3007, 19 94]). This primer set differentiated the main CF pathogens from closely rel ated species but did not distinguish P. aeruginosa from Pseudomonas alcalig enes-Pseudomonas pseudoalcaligenes and Alcaligenes xylosoxidans from Alcali genes denitrificans, Two hundred seven CF clinical isolates (153 of P, aeru ginosa, 26 of Stenotrophomonas maltophilia. 15 of Burkholderia spp., and 13 of A. xylosoxidans) were tested with P11P-P13P, The CE-SSCP patterns obtai ned were identical to those for the corresponding reference strains. Fluore scence-based CE-SSCP analysis is simple to use, gives highly reproducible r esults, and makes it possible to analyze a large number of strains. This ap proach is suited for the rapid identification of the main gram-negative non fermenting bacilli encountered in CF.