Array-based identification of species of the genera Abiotrophia, Enterococcus, Granulicatella, and Streptococcus

Abstract

Some species of enterococci and streptococci are difficult to differentiate by phenotypic traits. The feasibility of using an oligonucleotide array for identification of 11 viridans group streptococci was previously established. The aim of this study was to expand the array to identify species of Abiotrophia (1 species), Enterococcus (18 species), Granulicatella (3 species), and Streptococcus (31 species and 6 subspecies). The method consisted of PCR amplification of the ribosomal DNA intergenic spacer (ITS) regions, followed by hybridization of the digoxigenin-labeled PCR products to a panel of oligonucleotide probes (16- to 30-mers) immobilized on a nylon membrane. Probes could be divided into three categories: species specific, group specific, and supplemental probes. All probes were designed either from the ITS regions or from the 3' ends of the 16S rRNA genes. A collection of 312 target strains (162 reference strains and 150 clinical isolates) and 73 nontarget strains was identified by the array. Most clinical isolates were isolated from blood cultures or deep abscesses, and only those strains having excellent species identification with the Rapid ID 32 STREP system (bioMérieux Vitek, Taipei, Taiwan) were used for array testing. The test sensitivity and specificity of the array were 100% (312/312) and 98.6% (72/73), respectively. The whole procedure of array hybridization took about 8 h, starting from isolated colonies, and the hybridization patterns could be read by the naked eye. The oligonucleotide array is accurate for identification of the above microorganisms and could be used as a reliable alternative to phenotypic identification methods.

FIG. 1.

Layout of oligonucleotide probes on the array (0.9 by 1.1 cm). The probe “PC” (A9) (a positive control) was designed from a conserved region at the 3′ end of the 16S rRNA gene. Probes coded “NC” were negative controls (tracking dye only). Probes coded “M” were digoxigenin-labeled primer 6R and were used as position markers. Probes in the upper left, upper right, and lower left corners were used to identify species of nutritionally variant streptococci, enterococci, and streptococci, respectively. Probes in the lower right corner were supplemental probes. The corresponding sequences of all probes are listed in Table 2.

FIG. 2.

Hybridization results for species of enterococci (18 species), streptococci (31 species and 6 subspecies), and nutritionally variant streptococci (4 species). All strains, except two, were type strains and were alphabetically arranged according to their species names. The corresponding probes hybridized on the arrays are indicated in Fig. 1, and the corresponding sequences of the hybridized probes are shown in Table 2. The hybridized probe on the uppermost right corner on each array was the positive control. Hybridization signals produced by supplemental probes (located at the lower right corner) were used to differentiate genetically related species of streptococci and had no use in identification of enterococci and nutritionally variant streptococci.

FIG. 2.

Hybridization results for species of enterococci (18 species), streptococci (31 species and 6 subspecies), and nutritionally variant streptococci (4 species). All strains, except two, were type strains and were alphabetically arranged according to their species names. The corresponding probes hybridized on the arrays are indicated in Fig. 1, and the corresponding sequences of the hybridized probes are shown in Table 2. The hybridized probe on the uppermost right corner on each array was the positive control. Hybridization signals produced by supplemental probes (located at the lower right corner) were used to differentiate genetically related species of streptococci and had no use in identification of enterococci and nutritionally variant streptococci.