Factores de virulencia articulo

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2001, p. 3258–3263 0099-2240/01/$04.00 0 DOI: 10.1128/AEM.67.7.3258–3263.2001

Vol. 67, No. 7

Microarray Analysis of Microbial Virulence Factors
VLADIMIR CHIZHIKOV,1 AVRAHAM RASOOLY,2* KONSTANTIN CHUMAKOV,1
AND

DAN D. LEVY2

Food and Drug Administration Center for Biologics Evaluation and Research, Rockville, Maryland,1 and Food and DrugAdministration Center for Food Safety and Applied Nutrition, Washington, D.C.2
Received 1 December 2000/Accepted 22 March 2001

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specificoligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains wasmonitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gelelectrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.

In recent years, DNA and oligonucleotide microchip (microarray)technology has played an increasingly important role in genomic studies, drug discovery, and toxicological research. Unlike other hybridization formats (hybridization with microplates or dot blot hybridization with membrane-bound probes), glass microchips allow significant miniaturization so that thousands of individual probes can be arranged on one glass slide. As a result, this technology isideal for an extensive parallel identification of nucleic acids and analysis of gene expression. Simultaneous analysis for the presence of multiple markers makes it possible to determine a complete genetic profile of a single strain or distinguish one strain from a very large collection of possible alternatives in one experiment. Therefore, this approach is potentially useful for the screening ofmultiple microbial isolates in a diagnostic assay. Oligonucleotide microchips containing multiple oligonucleotides are spotted on the chip surface. DNA samples for analysis are labeled with fluorescent dyes and hybridized with the oligonucleotide spots on the chip. The fluorescence pattern is then recorded by a scanner, quantified, and analyzed. While DNA microchips have been used mostly for geneexpression studies, the technique has great potential to be used for the discrimination of genotypes, point mutants, and other closely related sequences by employing oligonucleotides specific for each sequence variant. Microarray technology has great potential for use in diagnostic microbiology. Microbial pathogens are currently identified by using surrogate biochemical and immunological markers. Analternative approach developed in recent years makes use of ribosomal DNA (rDNA) as surrogate markers for bacterial identification. These conventional approaches are well
* Corresponding author. Mailing address: Food and Drug Administration, 200 C St. SW, Washington, DC 20204. Phone: (202) 2054192. Fax: (202) 401-7740. E-mail: axr@vm.cfsan.fda.gov. 3258

established and effective; however, they are...