Microarray-based identification of thermophilic Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis

Abstract

DNA microarrays are an excellent potential tool for clinical microbiology, since this technology allows relatively rapid identification and characterization of microbial and viral pathogens. In the present study, an oligonucleotide microarray was developed and used for the analysis of thermophilic Campylobacter spp., the primary food-borne pathogen in the United States. We analyzed four Campylobacter species: Campylobacter jejuni, C. coli, C. lari, and C. upsaliensis. Our assay relies on the PCR amplification of specific regions in five target genes (fur, glyA, cdtABC, ceuB-C, and fliY) as a first step, followed by microarray-based analysis of amplified DNAs. Alleles of two genes, fur and glyA, which are found in all tested thermophilic Campylobacter spp., were used for identification and discrimination among four bacterial species, the ceuB-C gene was used for discrimination between C. jejuni and C. coli, and the fliY and cdt genes were used as additional genetic markers specific either for C. upsaliensis and C. lari or for C. jejuni. The array was developed and validated by using 51 previously characterized Campylobacter isolates. All isolates were unambiguously identified on the basis of hybridization patterns with 72 individual species-specific oligoprobes. Microarray identification of C. jejuni and C. coli was confirmed by PCR amplification of other genes used for identification (hipO and ask). Our results demonstrate that oligonucleotide microarrays are suitable for rapid and accurate simultaneous differentiation among C. jejuni, C. coli, C. lari, and C. upsaliensis.

FIG. 1.

Microarray-based detection of Campylobacter spp. using fur-specific oligoprobes. (A) PCR amplification of fur gene. Genomic DNAs from four reference strains were amplified by using the universal fur gene primers, CmpfurF and CmpfurR (Table 1). The resulting PCR products were separated on a 1.5% agarose gel. Lanes: M, 100-bp DNA Ladder Mix (MBI Fermentas); 1, C. jejuni (ATCC 33560); 2, C. coli (ATCC 43485); 3, C. lari (ATCC 35222); 4, C. upsaliensis (D1673). (B) Microarray-based detection of Campylobacter spp. by using the fur-specific oligoprobes. The fur-derived Cy5-labeled ssRNA transcripts were hybridized to the microchip. Each row of the array contains six individual species-specific probes (Table 2) as follows: a, C. jejuni; B, C. coli; C, C. lari; and D, C. upsaliensis. The image labeled QC is the microarray QC Cy3 image.

FIG. 2.

PCR amplification of ceuB-C genes. Genomic DNAs from four reference strains were amplified by using the ceuB-C primers (Table 1). The resulting products were separated by using a 1% agarose gel. Lanes: M, 1-kb DNA ladder mix (MBI Fermentas); 1, C. jejuni (ATCC 33560); 2, C. coli (ATCC 43485); 3, C. lari (ATCC 35222); 4, C. upsaliensis (D1673).

FIG. 3.

PCR amplification of the cdtABC gene cluster from the C. jejuni and the lctP-cydA region of C. coli. Genomic DNAs from seven reference strains were amplified by using the cdtABC primers (Table 1). The resulting products were separated by using a 1% agarose gel. Lanes: M, 1-kb DNA ladder mix (MBI Fermentas); 1, C. jejuni (ATCC 33560); 2, C. jejuni (ATCC 35918); 3, C. jejuni (CDC1420); 4, C. jejuni (DENVER-2); 5, C. jejuni (GH18401); 6, C. coli (ATCC 43485); 7, C.coli (ATCC 43473).

FIG. 4.

Composite microarray for Campylobacter spp. identification. The QC image shows the layout of the array. The assay was composed of five subarray panels labeled from I to V. Each of four rows (a to d) of the subarray I contains six oligoprobes complementary to species-specific alleles of the fur gene. Subarrays from II to V contain oligoprobes for the glyA, ceuB-C, cdts, and fliY gene alleles, respectively. Microarray hybridization patterns of each of four Campylobacter species—C. jejuni (A), C. coli (B), C. lari (C), and C. upsaliensis (D)—are indicated.

FIG. 5.

Hybridization patterns of sixteen Campylobacter isolates. The composite microarray (Fig. 4) was used for the analysis of 16 Campylobacter isolates: C. jejuni (ATCC 35919, ATCC 29428, ATCC 33560, and DENVER-1) (A to D, respectively); C. coli (ATCC 33559, ATCC 43481, ATCC 43478, and 92B4QA) (E to H, respectively); C. lari (ATCC 35222, ATCC 35221, ATCC 43675, and 3125) (I to L, respectively); and C. upsaliensis (D2237, 5613, 5512, and 5502) (M to P, respectively).

FIG. 6.

Microarray hybridization patterns of bacterial samples containing mixtures of different Campylobacter species are shown. The composite microarray (Fig. 4) was used for six analyses of mixed Campylobacter isolates. Panels A to F show hybridization patterns for mixtures of C. lari and C. upsaliensis (A), C. jejuni and C. coli (B), C. jejuni and C. upsaliensis (C), C. jejuni and C. lari (D), C. coli and C. lari (E), and C. coli and C. upsaliensis (F).