We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.Modern high-density arrays of DNA probes (microarrays) have proved to be powerful tools to study complex genetic profiles of cellular life. However, only a few experimental assays based on this valuable tool have been introduced for microbiological diagnostics. We present here novel data demonstrating the application of microarray technology combined with broad-range PCR amplification of topoisomerase genes to the diagnosis of bacterial respiratory infections.Broad-range bacterial PCR is based on the use of primers that recognize conserved sequences of bacterial genes encoding essential molecules. The resulting DNA amplicons contain variable regions that provide a basis for the analysis of phylogenetic relationships and identification of different bacterial species. Furthermore, some genetically hypervariable regions provide targets for bacterial-species-specific oligonucleotide sequences. These oligonucleotides that target hypervariable regions may be used in a diagnostic array of probes for detection of bacterial pathogens.The use of broad-range ribosomal DNA (rDNA) PCR technologies for identification of bacterial and fungal culture isolates in the clinical laboratory is well established (3, 28), and there are commercial kits available for that purpose. However, the kits are not widely...