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-specific oligonucleotide 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 was monitored 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, gel electrophoretic 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 is ideal 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 of multiple 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 gene expression 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 marker...

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentationnegative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotypespecific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.Rotaviruses are the most important etiological agents of severe diarrhea in infants and young children in both developed and developing countries (15). The rotavirus outer capsid proteins VP4 and VP7 represent neutralization antigens that are the basis for the classification of rotaviruses by P and G serotypes, respectively. VP4-and VP7-specific antibodies provide serotype-specific protection against rotavirus diarrhea (14). Accurate and rapid serological identification is important for the diagnosis of rotavirus infections, host range determination, and global epidemiological surveillance. It is also an important part of molecular epidemiology and can be used for tracing lines of viral transmission, monitoring molecular evolution, identifying new strains, and determining genotype distribution in clinical trials of experimental vaccines. The current classification of rotaviruses is based on neutralization assays with polyclonal or monoclonal antibodies (13). At least 14 G serotypes (VP7 protein) and 13 (not including subtypes) P serotypes (VP4 protein) have been identified by this approach (7,15).Nucleotide sequence analysis of the VP7 and VP4 genes allows rotaviruses to be classified into a number of distinct genotypes. The G genotypes have been shown to consi...

We have developed a rapid microarray-based assay for the reliable detection and discrimination of six species of the Listeria genus: L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi. The approach used in this study involves one-tube multiplex PCR amplification of six target bacterial virulence factor genes (iap, hly, inlB, plcA, plcB, and clpE), synthesis of fluorescently labeled single-stranded DNA, and hybridization to the multiple individual oligonucleotide probes specific for each Listeria species and immobilized on a glass surface. Results of the microarray analysis of 53 reference and clinical isolates of Listeria spp. demonstrated that this method allowed unambiguous identification of all six Listeria species based on sequence differences in the iap gene. Another virulence factor gene, hly, was used for detection and genotyping all L. monocytogenes, all L. ivanovii, and 8 of 11 L. seeligeri isolates. Other members of the genus Listeria and three L. seeligeri isolates did not contain the hly gene. There was complete agreement between the results of genotyping based on the hly and iap gene sequences. All L. monocytogenes isolates were found to be positive for the inlB, plcA, plcB, and clpE virulence genes specific only to this species. Our data on Listeria species analysis demonstrated that this microarray technique is a simple, rapid, and robust genotyping method that is also a potentially valuable tool for identification and characterization of bacterial pathogens in general.

Human rotavirus strains belonging to genotype G9 or P[9] were detected in a collection of stool specimens from children with diarrhea in two cities of the state of Rio de Janeiro, Brazil, between March 1997 and December 1999. G9 strains were first detected in April 1997 and remained prevalent until the end of the study, at a frequency of 15.9% (n ‫؍‬ 157). A high percentage of VP7 nucleotide (99.0 to 99.5%) and deduced amino acid identity (98.6 to 99.1%) was found between three randomly selected Brazilian G9 strains and the American G9 strain US1205. A novel G9:P[4] genotype combination was detected in addition to G9:P[8] and G9:P[6], demonstrating that this G genotype may undergo constant genetic reassortment in nature. The P[9] rotavirus strains constituted 10.2%, the majority of which were detected between April and July 1997. The RNA electrophoretic migration pattern of the G3:P[9] strains resembled that of AU-1 virus (G3:P3[9]), suggesting a genetic similarity between the Brazilian G3:P[9] strains and the Japanese virus, which is similar to a feline rotavirus genetically.Rotaviruses are the major etiologic agents of infantile diarrhea worldwide (15). Epidemiologic studies have demonstrated that rotavirus serotypes G1, G2, G3, and G4 are the mostcommon types associated with disease globally, and therefore they are the targets for vaccine development (13,15). Recently, unusual rotavirus serotypes and genotypes have been described in association with diarrhea in various parts of the world. These include serotype G5 in Brazil (9, 17); G8 in Malawi (5), Kenya (21), South Africa (34), the United Kingdom (34), Nigeria (1), and Australia (24); and G9 in India (27), the United Sates (28), Bangladesh (36), Malawi (5), the United Kingdom, (4), Australia (25), France (2), and Ireland (22). The P specificity of a rotavirus is usually more conservative than its G specificity; P1A[8] is the most common serotype detected worldwide, followed by P1B[4] and P2A[6] (13). A rotavirus strain belonging to serotype P3 [9] was first identified in Japan and was demonstrated to be closely related to feline rotavirus strains genetically (20,35). The P[9] rotavirus strains have been detected more often in Japan (14, 37) than in other parts of the world such as Venezuela (32), Italy (32), Malaysia (29), Brazil (17), Israel (31), South Africa (33), Guinea-Bissau (7), and the United States (12). The P [9] isolates are most commonly associated with either G1 (K8-like) or G3 (AU-1-like) serotypes, except for one isolate from Guinea-Bissau which bears a G4 specificity (7).One hundred fifty-seven (23%; n ϭ 678) rotavirus-positive stool samples from children under 5 years of age with acute diarrhea (32 inpatients and 646 outpatients) were collected between March 1997 and December 1999, at four centers in the city of Rio de Janeiro, Brazil, and one center in the neighboring city of Niterói in the state of Rio de Janeiro. The five centers are located in areas of distinct levels of sanitation and socioeconomic backgrounds. The presence of rotavir...