DNA microarrays for environmental, clinical microbiology, and microbial taxonomy

Abstract number: 1134_04_49

Volokhov D., George J., Anderson C., Sergeev N., Zhang Y., Hitchins A.D., Rasooly A., Chizhikov V.

DNA microarrays have the potential to be excellent tools for environmental, clinical microbiology, and microbial taxonomy due to the capability to significantly improve identification and multilocus typing of target pathogens in a bacterial community. We have developed and evaluated several microarray-based assays for the reliable detection, discrimination, and multilocus typing of the all Listeria species, four Campylobacter species (C. jejuni, C. coli, C. lari, and C. upsaliensis), Bacillus anthracis, most of the Mycoplasma species, and enterotoxins of Staphilococcus spp.; many of which are know as either outbreak related or clinically relevant pathogens for humans and animals. The assay can also detect the presence of bacterial genes responsible for drug resistance in Staphylococcus and Streptococcus spp., as well as those genes related to drug resistance in Mycobacterium tuberculosis. The approach used in this study included: (i) simultaneous PCR amplification of several target bacterial genes; (ii) high-yield synthesis of single-stranded RNA driven by T7 polymerase from the promoter-tagged PCR amplicons; (iii) incorporation of appropriate fluorescent dye into ssRNA; (iv) hybridization of fluorescently labeled sample with the microarray containing multiple individual oligonucleotide probes, and (v) microarray scanning and image data analysis. Microarray analysis of large numbers of bacterial samples from environmental and clinical sources demonstrated that this method enabled unambiguous detection and identification of all target pathogens. The microarray approach is also a promising application in the microbial taxonomy field due to the ability to correctly identify species by a multilocus probing. During this study we found that the occurrence of weak hemolytic or non-hemolytic isolates of Listeria spp. (L. seeligeri), and atypical hemolytic activity of typical non-hemolytic Listeria spp. (L. innocua) can result in false biochemical species differentiation of Listeria as a taxon. How can a microarray improve this? We found seven isolates that initially identified as L. welshimeri has discrepancies with our microarray data. All seven isolates were identified by microarray as L. seeligeri, but lost several genes due to a deletion in the central virulence gene cluster. Thus, our data suggests that microarray technology might be a valuable tool for the identification and accurate characterization of bacterial pathogens and microbial community in general.