Species differentiation and antibiotic susceptibility testing with DNA microarrays

There is a need for accurate and rapid species identification, strain typing and antibiotic susceptibility testing for several bacterial genera that contain some of the major human pathogens like Staphylococcus aureus or Mycobacterium tuberculosis. Assays based on molecular biology technologies have been introduced in recent years and their clinical value is now well established, especially for the identification of the difficult-to-grow bacteria, for the epidemiological characterization of strains involved in nosocomial infections and for the detection of mutations associated with resistance to antibiotics. Analysis with high density microarrays following multiplex nucleic acid amplification allows for simultaneous testing of specimens for suspected pathogens. Feasibility studies have been conducted on two assays for the Mycobacterium and Staphylococcus genera, based on the photolithography DNA-Chip technology developed by Affymetrix. The Mycobacterium assay combines 16S ribosomal DNA identification of 80 species, strain typing of species inside the M. tuberculosis (MTB) complex and detection of mutations linked to resistance to rifampin. Spoligotyping can further discriminate between species inside the TB complex and, for a given species, perform strain typing. A total of 104 rifampin mutations were detected in the rpoB gene of M. tuberculosis. The assay is performed within a working day on a single smear-positive sputum sample. The Staphylococcus assay combines 16S ribosomal DNA identification of 34 species, typing of S. aureus strains by multi-locus sequence typing and detection of genes or mutations linked to resistance to methicillin, fluoroquinolones and aminoglycosides. The objective of this assay was to perform the multiplex analysis of these different targets within a working day starting from a single colony. Results obtained with these two prototype assays, and with other similar techniques in the fields of clinical virology or food industry testing, show that the microarray technology is very promising and will soon be part of the laboratory tools available to microbiologists.