Species differentiation and antibiotic susceptibility testing with DNA microarrays

1. SUMMARYThere is a need for accurate and rapid species identiﬁcation,strain typing and antibiotic susceptibility testing for severalbacterial genera that contain some of the major humanpathogens like Staphylococcus aureus or Mycobacterium tuber-culosis. Assays based on molecular biology technologies havebeen introduced in recent years and their clinical value is nowwell established, especially for the identiﬁcation of thedifﬁcult-to-grow bacteria, for the epidemiological character-ization of strains involved in nosocomial infections and for thedetectionofmutationsassociatedwithresistancetoantibiotics.Analysis with high density microarrays following multi-plex nucleic acid ampliﬁcation allows for simultaneoustesting of specimens for suspected pathogens. Feasibilitystudies have been conducted on two assays for the Myco-bacterium and Staphylococcus genera, based on the photoli-thography DNA-Chip technology developed by Affymetrix.The Mycobacterium assay combines 16S ribosomal DNAidentiﬁcation of 80 species, strain typing of species inside theM. tuberculosis (MTB) complex and detection of mutationslinked to resistance to rifampin. Spoligotyping can furtherdiscriminate between species inside the TB complex and, fora given species, perform strain typing. A total of 104rifampin mutations were detected in the rpoB gene ofM. tuberculosis. The assay is performed within a working dayon a single smear-positive sputum sample.The Staphylococcus assay combines 16S ribosomal DNAidentiﬁcation of 34 species, typing of S. aureus strains bymulti-locus sequence typing and detection of genes ormutations linked to resistance to methicillin, ﬂuoroquinolo-nes and aminoglycosides. The objective of this assay was toperform the multiplex analysis of these different targetswithin a working day starting from a single colony.Results obtained with these two prototype assays, andwith other similar techniques in the ﬁelds of clinical virologyor food industry testing, show that the microarray technol-ogy is very promising and will soon be part of the laboratorytools available to microbiologists.2. INTRODUCTIONDNA microarrays are expected to take an important place inthe microbiology laboratory. To date, reagents using DNAmicroarrays are mostly used in research laboratories, forexample to look at up- and down-regulated genes. Thisfundamental work yields a set of gene targets that can beused later on as diagnostic tools to routinely serve clinicianneeds. Only few if any DNA microarray are commercializedfor routine in vitro diagnosis of infectious diseases. DNAmicroarrays offer the possibility of multiplex parallelhybridization on DNA probes of nucleic acids eitherdirectly extracted from a specimen or, more often, generatedby an ampliﬁcation step. They can be used for the initialdiagnosis of an infection when several pathogens can be thecause of a disease (panel diagnostic) or for the precisecharacterization of this pathogen, when it is required for amore accurate treatment management. Panel diagnosis can1. Summary, 592. Introduction, 593. Applications, 603.1 Species identiﬁcation, 603.2 Strain typing, 623.3 Antibiotic resistance, 634. Procedures, 654.1 Tiling strategies using high-density DNA micro-arrays, 654.1. 1 4L-tiling, 654.1. 2 2L-tiling, 654.2 Spoligotyping of MTB species, 654.2. 1 Reference spoligotyping, 654.2. 2 Comparison of spoligotypes obtained by dot-blot and by the microarray with a strain ofM. bovis,654.3 Complete Mycobacterium assay, 665. Conclusions, 666. References, 67