Identification of bacteria has traditionally been by morphologic features and phenotypic testing. Identification of some common organisms (e.g., Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli) can be rapid (e.g., less than one hour), exploiting their characteristic morphology and a few selected phenotypic tests. However, the identification of most organisms is slow, requiring hours to days for a definitive answer. More recently the use of genomics such as sequencing ribosomal RNA genes has proved to be a useful tool. Although sequencing specific genes is a powerful discriminatory tool, the current methodology requires one or more days before a result is available. A logical extension of sequencing genes is to use the gene products for bacterial identification. Significant variations in a structural gene sequence would result in variations in the protein product. In the last 5 years, preliminary work in the analysis of bacterial proteins for identification has been performed using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF MS). These studies demonstrated that whole microorganisms can provide distinct and reproducible mass spectra of proteins. The advantage of this approach is the results are available in minutes. However, MALDI-ToF MS produces extremely complex spectra of low molecular weight proteins, which may be unsuitable for applications in the clinical microbiology laboratory. During the past 6 months, we have explored using an alternative method, Surface-Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-ToF MS). The advantage of this technique is ease of use, relative low cost for the instrumentation and consumable supplies, and the use of """"""""protein chips"""""""" for selective capture of proteins from the bacterial cell lysate. The protein chips are available in a variety of chromatographic surfaces that allow binding proteins with different affinities (e.g., cationic, anionic, and metal affinity). Bacterial lysates can be exposed to one or more protein chips and then the bound proteins analyzed by SELDI-ToF MS. In our preliminary experiments we have (1) defined the conditions required for lysing both gram-positive and gram-negative bacteria, and (2) identified the appropriate protein chips for analysis. We believe we have completed the first phase of the experiments and are currently assessing the reproducibility of the protein spectra and the discriminatory power of the profiles. If these results appear to be satisfactory, we will expand the study to include a large number of well-characterized, clinically significant organisms selected from our culture collection and the American Type Cell Culture (ATCC) collection. Current work on another project (CL010326-01 DLM) has revealed that phenotypic identification of culture collection isolates (including the ATCC collection) is not precise. That is, genomic studies have demonstrated that a number of collection isolates are misidentified by phenotypic tests. Therefore, the identification of all isolates used in these studies will be by comprehensive phenotypic and genotypic methods.
