OLIGONUCLEOTIDE PRIMERS DESIGNED TO DIFFERENTIATE PATHOGENIC PSEUDOMONADS ON THE BASIS OF THE SEQUENCING OF GENES-CODING FOR 16S-23S RIBOSOMAL-RNA INTERNAL TRANSCRIBED SPACERS

Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 235 rRNA genes (rDNAs) were used to amplify the 16S-23S rDNA internal transcribed spacers (ITS) from eight species of pseudomonads which have been associated with human infections. Amplicons from reference strains of Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas maillei, Pseudomonas mendocina, Pseudomonas pickettii, Pseudomonas pseudomallei, and Xanthomonas maltophilia were cloned from each species, and sequence analysis revealed a total of 19 distinct ITS regions, each defining a unique sequevar with ITS sizes ranging from 394 (P. cepacia) to 641 (P. pseudomallei) bp. Five distinct ITS sequevars in P. cepacia, four in P. mendocina, three in P. aeruginosa, two each in P. gladioli end P. pseudomallei, and one each in P. mallei, P. pickettii, and X maltophilia were identified. With the exception of one P. cepacia ITS, all TTS regions contained potential tRNA sequences for isoleucine and/or alanine. On the basis of these ITS sequence data, species-specific oligonucleotide primers were designed to differentiate P. aerugiposa, P. cepacia, and P. pickettii. The specificities of these primers were investigated by testing 220 clinical isolates, including 101 strains of P. aeruginosa, 103 strains of P. cepacia, and 16 strains of P. pickettii, in addition to 24 American Type Culture Collection (ATCC) Pseudomonas strains. The results showed that single primer pairs directed at particular ITSs were capable of specifically identifying the ATCC reference strains and all of the clinical isolates of P. aeruginosa and P. pickettii, but this was not the case with several ITS-based primer pairs tested for P. cepacia. This pathogen, on the other hand, could be specifically identified by primer pairs directed against the 235 rDNA.