Molecular Epidemiology ofPseudomonas cepacia Determined byPolymerase ChainReaction Ribotyping

Traditional ribotyping detects genomic restriction fragment length polymorphisms byprobing chromosomal DNA withrRNA.Although itisa powerful methodfordetermining themolecular epidemiology ofbacterial pathogens, technical difficulties limit itsapplication. Asan alternative, polymorphisms were sought inthe 16S-23S spacerregions ofbacterial rRNAgenesbyuseofthepolymerase chain reaction (PCR). Chromosomal DNA fromisolates ofPseudomonas cepacia was usedas a template inthePCRwitholigonucleotide primers complementary tohighly conserved sequencesflanking thespacerregions oftherRNA genes.Length polymorphisms intheamplified DNA distinguished unrelated isolates ofP.cepacia. Isolates ofP.cepacia previously implicated inperson-to-person transmission were showntohaveidentical amplification patterns. Thesedatademonstrate theutility ofthis new PCR ribotyping methodfordetermining themolecular epidemiology ofbacterial species. Determining therelatedness ofisolates ofmicroorganisms hasbecomeincreasingly important asthenumberandspectrumofnosocomial pathogens continue toexpand. Recently, approaches atthemolecular level havebeenusedtoassess therelatedness ofbacterial isolates. DNA analysis, rather thananalysis ofphenotypic parameters suchasouter membraneprotein profiles (19), biochemical profiles (1), orantimicrobial susceptibility patterns (24), ispreferred, since it provides a morestable determination ofisolate identity. However, evengenetic analysis methods mayhavelimitations; forexample, plasmid analysis canonlybeusedfor bacteria thatcontain plasmids (28). Recently, rRNAwasusedasaprobe todetect polymorphisms inbacterial chromosomal DNA (33). Thisribotyping methoddistinguished unrelated isolates ofEscherichia coli frombacteriuric women(18), demonstrated therelatedness ofisolates ofPasteurella multocida inflocks ofturkeys and wildlife whenserotyping could not(29), anddifferentiated mostserovars ofLeptospira species (25). Additionally, traditional ribotyping wasusedtodistinguish isolates of Pseudomonas cepacia incystic fibrosis (CF)patients at different CFcenters (17) anddocumented person-to-person transmission ofthis organism inonesetting (15). Despite thebroadapplicability ofthis method, itsusein clinical microbiology laboratories hasbeenlimited because oftheprolonged timeneeded forSouthern blotanalysis and theuseofradioisotopes. Tocircumvent these problems, we haveusedthepolymerase chainreaction (PCR)(27) in conjunction withribotyping. Ourresults demonstrate that thePCR canbeusedtodetect polymorphisms inthe intergenic spacer regions ofbacterial rRNAgenesandthat it canbeabroadly applicable tool inmolecular epidemiology.