Identification of non-fermenting Gram-negative bacteria of clinical importance by an oligonucleotide array

Abstract

Many species of non-fermenting Gram-negative bacilli (non-fermenters) are important opportunistic and nosocomial pathogens. Identification of most species of non-fermenters by phenotypic characteristics can be difficult. In this study, an oligonucleotide array was developed to identify 38 species of clinically relevant non-fermenters. The method consisted of PCR-based amplification of 16S–23S rRNA gene intergenic spacer (ITS) regions using bacterial universal primers, followed by hybridization of the digoxigenin-labelled PCR products with oligonucleotide probes immobilized on a nylon membrane. A total of 398 strains, comprising 276 target strains (i.e. strains belonging to the 38 species to be identified) and 122 non-target strains (i.e. strains not included in the array), were analysed by the array. Four target strains (three reference strains and one clinical isolate) produced discrepant identification by array hybridization. Three of the four discordant strains were found to be correctly identified by the array, as confirmed by sequencing of the ITS and 16S rRNA genes, with the remaining one being an unidentified species. The sensitivity and specificity of the array for identification of non-fermenters were 100 and 96.7 %, respectively. In summary, the oligonucleotide array described here offers a very reliable method for identification of clinically relevant non-fermenters, with results being available within one working day.