Simultaneous Genotyping and Species Identification Using Hybridization Pattern Recognition Analysis of Generic Mycobacterium DNA Arrays

Abstract

High-density oligonucleotide arrays can be used to rapidly examine large amounts of DNA sequence in a high throughput manner. An array designed to determine the specific nucleotide sequence of 705 bp of the rpoB gene of Mycobacterium tuberculosis accurately detected rifampin resistance associated with mutations of 44 clinical isolates of M. tuberculosis. The nucleotide sequence diversity in 121 Mycobacterial isolates (comprised of 10 species) was examined by both conventional dideoxynucleotide sequencing of the rpoB and 16S genes and by analysis of the rpoB oligonucleotide array hybridization patterns. Species identification for each of the isolates was similar irrespective of whether 16S sequence, rpoB sequence, or the pattern of rpoB hybridization was used. However, for several species, the number of alleles in the 16S and rpoB gene sequences provided discordant estimates of the genetic diversity within a species. In addition to confirming the array's intended utility for sequencing the region of M. tuberculosis that confers rifampin resistance, this work demonstrates that this array can identify the species of nontuberculous Mycobacteria. This demonstrates the general point that DNA microarrays that sequence important genomic regions (such as drug resistance or pathogenicity islands) can simultaneously identify species and provide some insight into the organism's population structure.[The sequence data described in this paper have been submitted to GenBank under accession nos. AF09766-AF059853 and AF060279-AF060367.]For patients infected with Mycobacteria, especially those coinfected with the human immunodeficiency virus type 1 and type 2 (HIV-1, HIV-2), the identity of the Mycobacterium species and the presence of mutations that confer both biologically and clinically important phenotypes are of critical importance. Both of these issues have implications for the appropriate care and treatment of the infected patient. For example, although M. avium complex (MAC) is the most common cause for both disseminated Mycobacterium disease and death in patients with AIDS in the developed world (∼25%-50% of adults and 10% of children with AIDS are infected [Inderlied et al. 1993], Mycobacterium tuberculosis infections are also found in these patient populations. Important public health and patient management decisions (e.g., the need for clinical isolation and the choice of the appropriate therapeutic regimen) depend on a timely and accurate identification of the infecting agent. (Moore et al. 1997).On the basis of the insights provided by previously characterized RIF resistant mutants in Escherichia coli (Ovchinnikov et al. 1983; Jin and Gross 7 Corresponding author. E-MAIL tom gingeras@affymetrix.com; FAX (408) 481-0422.

Genomic interrogation of ancestral Mycobacterium tuberculosis from south India

“…To identify genomic differences putatively associated with the altered virulence, we performed whole-genome hybridization on a subset of 25 strains belonging to the dominant ancestral lineage using the Affymetrix M. tuberculosis GeneChip™ (Santa Clara, CA, USA) and published methods (Gingeras et al, 1998, Kato-Maeda et al, 2001, Tsolaki et al, 2004. We confirmed putative deletions by PCR and sequencing as previously reported (Chen 2006, Gagneux et al, 2006, Glynn et al, 2002, Gutacker et al, 2006.…”

Tuberculose resistente: revisão molecular

“…Já foram publicados alguns estudos mostrando a sua eficiên-cia. 13,37 Está em desenvolvimento um teste baseado nessa metodologia que objetiva a detecção simultâ-nea de vários determinantes da resistência aos fármacos isoniazida, estreptomicina e fluoroquinolonas. Esse teste contém seqüências dos genes mais freqüentemente associados à resistência.…”

“…Esse teste contém seqüências dos genes mais freqüentemente associados à resistência. 13 Essa estratégia tem potencial para se tornar um método eficiente e rápido para a detecção de TB resistente.…”

Molecular tools for the characterisation of antibiotic-resistant bacteria

“…New technique developments for bacterial identification and genetic profiling of bacterial abundance still use rrs but with specific oligonucleotide arrays [4,20,56], and even high-density DNA probe arrays [65].…”