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Journal of Clinical Microbiology, December 2006, p. 4414-4424, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01712-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Array-Based Identification of Species of the Genera Abiotrophia, Enterococcus, Granulicatella, and Streptococcus{triangledown} ,{dagger}

Sheng Kai Tung,1 Lee Jene Teng,2 Mario Vaneechoutte,3 Hung Mo Chen,4 and Tsung Chain Chang1*

Department of Medical Laboratory Science and Biotechnology, School of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China,1 School of Medical Technology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China,2 Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium,3 Division of Clinical Microbiology, Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan, Republic of China4

Received 18 August 2006/ Returned for modification 25 September 2006/ Accepted 13 October 2006


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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.


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INTRODUCTION
 
Enterococci, nutritionally variant streptococci, most of which have been allocated to the genera Abiotrophia and Granulicatella, and streptococci are gram-positive and catalase-negative bacteria. Although many species of the above genera are commensals of the human body, some of them cause local or systemic infections, including subacute endocarditis, bacteremia, meningitis, pneumonia, soft-tissue infections, and eye infections (15, 22, 41, 49). Sherman (46) proposed a scheme for dividing the streptococci into four categories: the pyogenic division, the viridans division, the lactic division, and the enterococci. No single system of phenotypic identification suffices for the differentiation of this heterogeneous group of organisms. Instead, classification depends on a combination of features, including patterns of hemolysis on blood agar plates, antigenic composition, growth characteristics, and biochemical reactions (15, 41). Accurate identification of these bacteria would be useful for understanding the pathogenesis of infections and the epidemiology of the increasing antibiotic resistance among some of these microorganisms (50).

In clinical laboratories, phenotypic test kits, such as the Rapid ID 32 STREP system (bioMérieux Vitek, Taipei, Taiwan), the API 20 STREP system (bioMérieux Vitek), or the Vitek GPI card (bioMérieux Vitek), are commonly used for identification of enterococci, streptococci, and related bacteria (16, 23, 28). The inherent problem of the culture-based identification of these microorganisms is the large number of species relative to the limited number of biochemical traits, the poor reproducibility of some tests (4, 41), the variability of some traits within species (6, 24, 41), and the lack of sufficient phenotypic data on more recently described species. The last problem applies to species such as Streptococcus australis, Streptococcus cristatus, Streptococcus infantarius subspecies infantarius, Streptococcus infantis, Streptococcus gallolyticus, and Streptococcus lutetiensis (38, 44).

A variety of molecular methods have been developed for identification of strains of enterococci, viridans group streptococci, and streptococci to the species level. The targets used for molecular diagnosis include genes encoding rRNA (6, 10, 26, 27), the beta subunit of RNA polymerase (rpoB) (14), the D-alanine:D-alanine ligase (18), the RNA subunit of RNase P (rnpB) (25), the elongation factor (tuf) (36), the manganese-dependent superoxide dismutase (sodA) (37, 38), the heat shock proteins (groESL) (51), and the tRNA gene intergenic spacer (2, 3, 12). Recently, nonhemolytic streptococci were successfully identified by phylogenetic sequence analysis of four housekeeping genes (ddl, gdh, rpoB, and sodA) (24). Correct identification of catalase-negative gram-positive cocci is to some extent achievable by sequencing of the 16S rRNA gene (6, 27). However, there are two problems with this approach. First, the method does not allow differentiation of Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae because of significant sequence conservation of the 16S rRNA genes in this group of bacteria (6, 24). Second, many sequences in the public databases are mislabeled, either because of incorrect identification on the source strain or because of nonrecorded revised classification of the strain subsequent to deposition of the sequence (24).

The 16S-23S rRNA gene intergenic spacer (ITS) has been suggested as a good candidate for bacterial identification and strain typing (8, 19, 21, 40). The ITS region is found to have a high degree of sequence and length variation at both the genus and species levels (21, 30, 55). Recently, DNA array technology was found to be a useful tool to identify (or detect) a wide variety of microorganisms (7, 17, 29, 34, 43, 53, 54). In our previous study, the feasibility of using an oligonucleotide array to identify 11 species of viridans group streptococci was established (9). This study aimed to expand the results of the array technique to cover a more comprehensive spectrum including 53 species and 6 subspecies of Abiotrophia, Enterococcus, Granulicatella, and Streptococcus.


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MATERIALS AND METHODS
 
Bacterial strains. A collection of 312 target strains (162 reference strains and 150 clinical isolates) was analyzed (Table 1). Reference strains were obtained from the American Type Culture Collection (ATCC) (Manassas, Va.), the Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan), and the Culture Collection of the University of Göteborg (CCUG, Göteborg, Sweden). Clinical isolates were obtained from the National Taiwan University Hospital (Taipei, Taiwan), the National Cheng Kung University Medical Center (Tainan, Taiwan), and the Ghent University Hospital (Ghent, Belgium). Most clinical isolates were isolated from blood cultures or deep abscesses and identified to the species level with the Rapid ID 32 STREP system. To avoid ambiguous identities of clinical isolates, only those strains having excellent species identification according to the criteria of the Rapid ID 32 STREP system were used for array testing. In addition, species names of clinical isolates of viridans group streptococci were verified by ITS sequence analysis as previously described (8). In addition, a total of 73 nontarget strains (belonging to 51 species other than the species covered by the DNA array) were used for a specificity test of the array (see Table S1 in the supplemental material). All strains were cultured on sheep blood agar, incubated at 35°C for 24 to 48 h, and then used for DNA extraction. Strains of Abiotrophia and Granulicatella were cultured on chocolate agar.


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  		TABLE 1. Reference strains and clinical isolates used in this study

DNA preparation. The boiling method was used to extract DNA from bacteria (31). Briefly, one to several colonies of pure cultures were suspended in 50 µl of sterilized water, heated at 100°C for 15 min in a heating block, and centrifuged in a microcentrofuge (6,000 x g; 10 min). The supernatant containing bacterial DNA was stored at –20°C for further use.

Database of oligonucleotide probes. A total of 88 oligonucleotide probes (16- to 30-mers) (Table 2) were used to construct an oligonucleotide database to identify the bacteria listed in Table 1. Most probes were designed from the ITS regions, except for five that were based on the sequences of the 3' ends of 16S rRNA genes. Reference sequences extracted from GenBank (Table 1) were confirmed by at least one sequence of another reference strain of the same species in the database. If an ITS sequence was determined in this study and used for probe design, the sequence was also confirmed by using the ITS sequence of a second reference strain of the same species, except for a few species that had only one strain in our collection (Table 1).


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  		TABLE 2. Oligonucleotide probes used in this study

Alignments of the ITS sequences of different species were performed by using the PrettyBox algorithm of the Wisconsin Genetics Computer Group package (version 10.3; Accelrys Inc., San Diego, Calif.), and areas displaying sequence divergence among species were used for probe synthesis. The designed probes were checked for internal repeats, self-biding, secondary structure, and GC content by using the software Vector NTI (Invitrogen Corporation, Carlsbad, Calif.) and screened against the databases of the National Center for Biotechnology Information for homology with other bacterial sequences using BLAST (Basic Local Alignment Search Tool). Fifteen, ten, or five additional bases of thymine were added to the 3' (or 5') ends of probes that had Tm values less than 37°C, 45°C, or 50°C, respectively (7) (Table 2). Some probes used for identification of viridans group streptococci were reported previously (9); however, many probes were newly designed or modified to identify several additional species in this group and to meet the change of hybridization stringency used in this study (Table 2).

One probe or multiple probes were designed to identify a single species, depending on the availability of divergent sequences in the ITS regions (Table 2). The 88 probes could be divided into 3 categories: species specific, group specific (i.e., a probe shared by several species), and supplemental probe (i.e., a probe used to differentiate between genetically related streptococci). Supplemental probes were either designed from the ITS regions or from the 3' ends of the 16S rRNA genes. A probe based on a conserved sequence (5'-GTCGTAACAAGGTAGCCGTA-3') at the 3' end of the 16S rRNA gene (GenBank accession no. AB023575) was used as a positive control probe. In addition, the digoxigenin-labeled reverse primer 6R (5'-dig-GGGTTYCCCCRTTCRGAAAT-3' ; Y = C or T, and R = A or G), used to amplify the ITS region, was spotted on the array and used as a position marker after hybridization (Fig. 1 and 2).


Figure 1
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  		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.


Figure 2
Figure 2
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  		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.

Fabrication of oligonucleotide arrays. The array (0.9 by 1.1 cm) contained 117 dots (9 x 13 dots), including 88 dots for species (or subspecies) identification, 1 dot for a positive control (probe code PC), 8 dots for negative controls (probe code NC, tracking dye only), and 20 dots (probe code M) for position markers (Fig. 1). The oligonucleotide probes were diluted 1:1 (final concentration, 10 µM) with a tracking dye solution, drawn into wells of 96-well microtiter plates, and spotted onto positively charged nylon membranes (Roche, Mannheim, Germany) as described previously (9). The arrays were fabricated with an automatic arrayer (SR-A300; Ezspot, Taipei, Taiwan) by using a solid pin (diameter, 400 µm). Probes on the array were divided by position markers into four areas. Probes in the upper left, upper right, and lower left corner were used for identification of species of nutritionally variant streptococci, enterococci, and streptococci, respectively. The lower right corner contained supplemental probes that helped group-specific probes to differentiate some genetically related streptococci (Fig. 1).

Amplification of the ITS regions for hybridization. The bacterium-specific universal primers 13 BF (5'-GTGAATACGTTCCCGGGCCT-3') and 6R (5'dig-GGGTTYCCCCRTTCRGAAAT3') (Y = C or T, and R = A or G) (39) were used to amplify a DNA fragment that encompassed a small portion of the 16S rRNA gene, the ITS, and a small portion of the 23S rRNA gene. The reverse primer 6R was labeled with a digoxigenin molecule at its 5' end. PCR was carried out as described previously (9), except that digoxigenin-11-dUTP was not included in the PCR mixture.

Hybridization procedures. Unless otherwise indicated, the hybridization procedures were carried out at room temperature in an oven with a shaking speed of 60 rpm. All reagents except buffers were included in the DIG nucleic acid detection kit (Roche). Each array was prehybridized for 2 h with 1 ml of hybridization solution (5x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 1% [wt/vol] blocking reagent, 0.1% N-laurylsarcosine, 0.02% sodium dodecyl sulfate [SDS]) in an individual well of a 12-well cell culture plate. The digoxigenin-labeled PCR product amplified from an isolate was heated on a 100°C heating block for 5 min and immediately cooled in an ice bath. Ten microliters of the denatured PCR product of the test organism was diluted with 0.5 ml of hybridization solution and added to each well. Hybridization was carried out at 45°C for 90 min. The array was then given three washes (5 min each) in 1 ml of washing buffer (2x SSC, 0.1% SDS) and one wash (1 min) in 1 ml of a second washing buffer (0.5x SSC, 0.1% SDS). The array was then blocked with 1% blocking solution supplied in the DIG nucleic acid detection kit (Roche), incubated with alkaline phosphatase-conjugated sheep antidigoxigenin antibodies and then with the substrates of alkaline phosphatase as described previously (9). The hybridized chip was air-dried, and the image of the hybridization pattern was processed by a high-resolution scanner (Powerlook 3000; Umax, Taipei, Taiwan). The hybridized spot (diameter, 400 µm), displaying a blue color on a white nylon membrane, could be easily recognized by the naked eye.

Identification of strains by array hybridization. A strain was identified as one of the species (or subspecies) listed in Table 1 when the probe (or all probes) specified for that species was hybridized (Table 2). Unless otherwise specified, hybridization signals produced by supplemental probes located in the lower right corner of the array were ignored. Some species were identified by their unique hybridization patterns, produced by group-specific or group-specific and supplemental probes, as indicated in Table 2. Supplemental probes were used to differentiate several genetically related streptococci; they had no use for identification of enterococci and nutritionally variant streptococci.

Discrepant analysis. When a strain produced discrepant identification by the array, the near-complete-length 16S rRNA gene of the strain was amplified by PCR and sequenced for species clarification. To amplify the near-complete-length 16S rRNA gene by PCR, the primer pair of 8FPL (5'-GTTTGATCCTGGCTCAG-3') and 1492RPL (5'-GGTTACCTTGTTACGACTT-3') was used (39). PCR products were purified by a PCR-M Clean Up kit (Viogene, Taipei, Taiwan) and sequenced in both directions by using the above two primers and an additional primer, 1055r (5'-CACGAGCTGACGACAGCCAT-3') with the BigDye Terminator cycle sequencing kit (Applied Biosystems, Taipei, Taiwan) and the ABI 377 sequencing system (Applied Biosystems). The determined sequences were compared to known sequences of 16S rRNA genes in the databases of the National Center for Biotechnology Information using the BLASTN algorithm. The following criteria were used for identification of a strain to the genus or species level by 16S rRNA gene sequencing: (i) when the comparison of the determined sequence with a best-scoring reference sequence of a classified species yielded an identity of ≥99%, the unknown isolate was assigned to that species; and (ii) when the identity was <99% and ≥95%, the unknown isolate was assigned to the corresponding genus (6).

Nucleotide sequence accession numbers. Sequences of accession numbers with a prefix of "DQ" in Table 2 were determined in this study and have been submitted to GenBank.


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RESULTS
 
Construction of oligonucleotide probe database. A total of 88 probes (Table 2) were used to construct an oligonucleotide database, which was then used for fabrication of the array shown in Fig. 1. Among the 88 probes, 70 were species specific, 8 were group specific, and 10 were supplemental probes. A group-specific probe (such as the probes Ecas and Salbe) could hybridize with the PCR products of two or more species due to high sequence similarities among these species (Table 2). Supplemental probes were designed from the ITS or the 16S rRNA gene regions that were coamplified by PCR during ITS amplification and were used to differentiate genetically related species of streptococci.

An individual species was identified by one to four probes, depending on the availability of divergent sequences in the ITS region. For example, Enterococcus avium was identified by a single probe (Eav), while a strain was identified as Enterococcus dispar if all three probes (Edi1, Edi2, and Edi3) were simultaneously hybridized (Table 2). Some species were identified by a combination of group-specific probes. For example, Streptococcus bovis was identified by its hybridization to two group-specific probes, Salbe and Sbec (Table 2; Fig. 2). Although Streptococcus alactolyticus also hybridized to the probe Salbe and S. lutetiensis hybridized to the probe Sbec, neither species could simultaneously hybridize with both probes. Furthermore, some species were identified by their unique patterns of hybridization to group-specific and supplemental probes. For example, both S. gallolyticus subsp. gallolyticus and S. gallolyticus subsp. macedonicus hybridized with the group-specific probe Sgalgm and were differentiated by hybridization to two supplemental probes, S16SC and S16ST. S. gallolyticus subsp. gallolyticus produced positive hybridization with the probe S16ST, whereas S. gallolyticus subsp. macedonicus was positive with the probe S16SC (Table 2; Fig. 2). Another example was S. mitis and S. oralis; both species hybridized to the group-specific probe Smg and the supplemental probe Smgmo1. The two species were distinguished by hybridization of S. mitis to an additional supplemental probe, Smgmp1, and S. oralis to another supplemental probe, Smgor1 (Table 2; Fig. 2). Species that required group-specific and supplemental probes for identification are indicated in Table 2.

Identification of reference strains by the oligonucleotide array. Of 162 target reference strains tested, 154 hybridized to their respective oligonucleotide probes and were correctly identified. The hybridization patterns of 53 species and 6 subspecies, a majority of them being type strains, on the arrays are alphabetically shown in Fig. 2. Reference strains of the different taxa in the S. bovis complex, i.e., S. bovis, S. gallolyticus subsp. gallolyticus, S. gallolyticus subsp. macedonicus, and S. gallolyticus subsp. pasteurianus, were successfully differentiated by the array. Species that have high sequence similarities in their 16S rRNA genes were also accurately identified by the present method. These species were S. mitis and S. pneumoniae, Streptococcus gordonii and S. mitis, Enterococcus durans and Enterococcus faecium, and Enterococcus gallinarum and Enterococcus casseliflavus (27, 35).

Eight reference strains (4.9%), including one enterococcal and seven streptococcal species, produced discrepant identification by the array. Determination of the 16S rRNA gene sequences of these strains showed that six of the eight strains had been given wrong species names (Table 3). Enterococcus pseudoavium CCUG 44888 was identified as E. avium by array hybridization. A BLAST search revealed that the 16S rRNA gene sequence of the strain CCUG 44888 had identities of 99.1 and 98.8% with GenBank reference sequences of E. avium and E. pseudoavium, respectively (Table 3). Therefore, E. pseudoavium CCUG 44888 should be a strain of E. avium, as identified by the array and confirmed by 16S rRNA gene sequencing. Similarly, Streptococcus mutans BCRC 15255, S. mutans BCRC 15256, Streptococcus sobrinus CCUG 35254, Streptococcus uberis ATCC 13386, and Streptococcus vestibularis CCUG 32749 were found to be misidentifications of Streptococcus ratti, S. ratti, Streptococcus mutans, Streptococcus parauberis, and Streptococcus salivarius, respectively, as revealed by array hybridization and further confirmed by their 16S rRNA gene sequences (Table 3). S. cristatus CCUG 35233 and S. mitis ATCC 15914 were identified only to the genus level (Streptococcus) by sequencing of the 16S rRNA genes (Table 3), since the identities between the query sequences and the best-scoring reference sequences in the public databases were only 98%. However, a BLAST search of the ITS sequences of S. cristatus CCUG 35233 and S. mitis ATCC 15914 against GenBank revealed that the both best-scoring sequences were from S. oralis, with sequence identities of 99.6% and 99.1%, respectively. To further clarify the identities of strains CCUG 35233 and ATCC 15914, the manganese-dependent superoxide dismutase genes (sodA) of both strains were amplified (38) and sequenced. A BLAST search of the sodA sequences of strains CCUG 35233 and ATCC 15914 revealed that the best-scoring reference sequences were S. oralis (GenBank accession no. Z99195; sequence identity, 96%) and S. oralis (GenBank accession AB200066; sequence identity, 96%), respectively. Since the hybridization results for strains CCUG 35233 and ATCC 15914 were supported by sequence analysis of the ITS region and sodA gene, the two strains were considered to be correctly identified as S. oralis by the array. Since all eight discordant reference strains were proved to be correctly identified by array hybridization, the test sensitivity of the array for reference strains was 100% (162/162).


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  		TABLE 3. Strains that produced discrepant identification by array hybridization and results of discrepant analysis

Identification of clinical isolates by the oligonucleotide array. Of 150 target clinical isolates tested, E. avium 2510 and E. durans 790 yielded discrepant identifications by array hybridization (Table 3). E. avium 2510 and E. durans 790 were identified as, respectively, Enterococcus raffinosus and Enterococcus hirae by the array, and this identification was confirmed by sequence analysis of the 16S rRNA genes. Since these two discordant isolates were proved to be correctly identified by hybridization, the test sensitivity of the array for clinical isolates was 100% (150/150). If reference strains and clinical isolates were considered together, an overall sensitivity of 100% (312/312) was obtained by the present method.

Hybridization of nontarget strains to the oligonucleotide array. Of 73 nontarget strains (51 species) tested by the array (see Table S1 in the supplemental material), one strain (Vibrio alginolyticus BCRC 12829) hybridized to the probe Saga and was misidentified as Streptococcus agalactiae (Fig. 2). The remaining 72 strains did not produce any hybridization signals with probes on the array, except for the positive control probe. Therefore, the test specificity of the array was 98.6% (72/73).


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DISCUSSION
 
In this study, an oligonucleotide array was developed to identify 53 species and six subspecies of the genera Abiotrophia, Enterococcus, Granulicatella, and Streptococcus. A sensitivity of 100% (312/312) and a specificity of 98.6% (72/73) were obtained by the array. The present method used a standardized protocol encompassing DNA extraction, PCR amplification of the ITS regions, and hybridization of the PCR products to the array. The hybridized spot (400 µm in diameter), displaying a blue color on a white nylon membrane, could be easily recognized by the naked eye. The whole procedure can be finished within approximately 8 h, starting from isolated colonies. The present method might be useful for conditions that necessitate identification of enterococci, streptococci, and related bacteria to the species (and subspecies) level.

In this study, an individual species was identified by either one or multiple probes, depending on the availability of divergent sequences in the ITS region. The advantage of using multiple probes is the increase in specificity, since the chance for a cross-reacting strain to hybridize to all probes designed for a species is very low. However, the use of multiple probes to identify a species may potentially decrease sensitivity, due to the possibility of mutations that occur at the regions used for probe design. The successful design of different probes, including group-specific and supplemental probes, was based on the known sequences in regions of the ITS and the 3' ends of 16S rRNA genes. Multiple sequence alignment (interspecies and intraspecies) plays an essential role in finding out the regions that could be used for probe design.

Routine procedures based on phenotypic tests do not allow unequivocal identification of some streptococci. Hoshino et al. (24) examined a collection of 115 strains of nonhemolytic streptococci isolated from bacteremic patients and 33 reference strains by using 2 commercial kits (rapid ID 32 STREP and STREPTOGRAM [Wako Pure Chemicals, Osaka, Japan]). The correct identification rates for strains by both commercial kits were below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. They concluded that phenotypic characterization is of limited value for identification of many species of nonhemolytic streptococci and is not a valid approach at the present time. Recently, Bosshard et al. (6) tested 171 strains of aerobic catalase-negative gram-positive cocci with the API 20 STREP system and found that less than 60% of isolates could be identified to the species or genus level. To solve the problems of phenotypic identification, the feasibility of using an oligonucleotide array for species identification was investigated here and favorable results were obtained. The array technology has been used to identify a spectrum of microorganisms, including Mycobacterium (17, 34, 43), bacteria in positive blood cultures (7), bacteria from cervical swab specimens (32), Campylobacter (53), Listeria (54), and food-borne bacterial pathogens (29).

Clinical isolates used in this study were identified by the Rapid ID 32 STREP system. To avoid as much as possible discrepant identification results, thus avoiding sequence analysis of the 16S rRNA gene as much as possible, only those strains having excellent species identification (i.e., percent identification of ≥99.9%) according to the criteria of the ID 32 STREP system were used for array testing. Strains with good identification (percent identification of ≥90%) or acceptable identification (percent identification of ≥ 80%) were not included in this study, since Bosshard et al. (6) observed that such identifications were unreliable. In addition, clinical isolates of viridans group streptococcus were verified by ITS sequence analysis before hybridization (8). The primary screening and verification of clinical isolates may partially explain why only 2 out of 150 isolates yielded discrepant identifications by array hybridization. This screening largely limited the numbers of strains and species that could be tested in this study. Moreover, the database of the Rapid ID 32 STREP system does not include many recently described species listed in Table 1, such as S. australis, S. cristatus, S. infantarius subspecies infantarius, S. infantis, S. gallolyticus, and others. Therefore, clinical isolates of these recently described species were not available for array testing. The accuracy of the array for identification of species listed in Table 1 could be regarded as being mainly based on testing of reference strains.

Pairwise comparison of two given streptococcal species revealed a lower level of sequence similarity between their ITS sequences than between their 16S rRNA gene sequences (8, 27). These results indicate that the ITS region might constitute a more discriminative target sequence than the 16S rRNA gene for differentiating closely related species. By 16S rRNA gene sequencing, Bosshard et al. (6) found that as much as 19% of isolates of aerobic catalase-negative gram-positive cocci could be identified only to the genus level. The ITS region has been suggested as a suitable target for bacterial identification by some investigators (20, 40, 47). A major disadvantage of this approach is the limited number of ITS sequences in public databases. However, the numbers of ITS sequences in public databases have increased in recent years. In 2006, software was developed to analyze bacterial ITS sequences (11).

Although most probes used in this study were designed from the ITS regions, advantage was taken to utilize divergent regions at the 3' ends of the 16S rRNA genes that were coamplified with the ITS regions by PCR. The following sets of type strains display high ITS sequence identities: E. casseliflavus and Enterococcus flavescens (99.6%); the three subspecies (subsp. gallolyticus, subsp. macedonicus, and subsp. pasteurianus) of S. gallolyticus (94 to 99.6%); S. infantis and S. pneumoniae (96%); S. infantis and S. mitis (96%); S. mitis and S. pneumoniae (99%); and S. salivarius, Streptococcus thermophilus, and S. vestibularis (98 to 99%) (our unpublished data). For this reason, a combination of group-specific and supplemental probes was used to differentiate these closely related species. An extreme example was the differentiation of the three members in the salivarius group (S. salivarius, S. thermophilus, and S. vestibularis). All three species hybridized to two group-specific probes, Sinfsa1 and Ssal, and unequivocal identification of the three species was based on their differential hybridizations to three supplemental probes, S16SC, S16ST, and Stv (Table 2; Fig. 2), creating a unique hybridization pattern for each of the three species.

S. pneumoniae, S. oralis, and S. mitis are related species and difficult to be differentiated by sequencing of the 16S rRNA gene (6, 27). Phylogenetic trees constructed by using the genes encoding 16S rRNA (5, 27), sodA (37, 38), groESL (51), and the ITS region (8) grouped S. mitis and S. pneumoniae together. The interspecies similarities of the ITS sequences between S. mitis and S. pneumoniae could be higher than intraspecies similarities of both species (8). In our previous study, strains of S. pneumoniae cross-hybridized to probes used to identify S. mitis (9). However, differentiation of S. mitis and S. pneumoniae was achieved here by their differential hybridization to two supplemental probes, Smgmo1 and Smgp1 (Table 2; Fig. 2). S. mitis produced positive hybridization with the probe Smgmo1, while S. pneumoniae was positive with another probe, Smgp1.

E. casseliflavus and E. flavescens shared a group-specific probe, Ecas, and could not be differentiated from each other by array hybridization (Table 2; Fig. 2). Strains of E. casseliflavus and E. flavescens are motile, intrinsically vancomycin resistant (possessing the vanC genotype), and able to produce pigments (49). E. casseliflavus and E. flavescens also could not be distinguished from each other by using the tRNA intergenic spacer PCR technique (2). The two species are most probably synonymous, as is also apparent from several other studies (13, 48).

The S. bovis complex comprises a heterogeneous group of bacteria that belong to group D of the Lancefield classification. Phenotypic characterization leads to a further subdivision of S. bovis strains based on their biotype. Biotype I comprises mannitol-positive strains, whereas biotype II comprises mannitol-negative strains (45). S. bovis biotype I is responsible for human endocarditis associated with colonic cancer (33, 52), and this was highly correlated with an underlying colonic neoplasm compared with bacteremia due to S. bovis biotype II (42). Therefore, careful identification of streptococcal bacteremic isolates as S. bovis biotype I provides clinically important information. Recently, S. gallolyticus of the S. bovis complex was proposed to contain three subspecies: S. gallolyticus subsp. gallolyticus (S. bovis biotype I), S. gallolyticus subsp. macedonicus, and S. gallolyticus subsp. pasteurianus (S. bovis biotype II) (44). Members of the S. bovis complex were successfully identified to the species or subspecies level in this study.

The Tm values of probes used in this study varied to a large degree (from 27.7 to 60.5°C), and many probes had Tm values lower than the hybridization temperature (45°C) (Table 2). Although some probes produced relatively weak hybridization, clear signals were obtained for almost all species tested (Fig. 2). This might be partially due to the use of relatively low-stringency washing buffer (2x SSC-0.1% SDS) after hybridization. Volokhov et al. (54) also reported the successful use of probes having Tm values significantly lower than the hybridization temperature for identification of Listeria species. It has been found that the addition of low numbers of 3' thymine bases can improve the hybridization signal of oligonucleotide probes that failed to show detectable hybridization to target DNA or gave weak hybridization signals. Although the mechanisms of the effect of adding low numbers of thymine bases are not clear, it was proposed that this could increase the binding of probes to the nylon membrane and thus the hybridization intensity (1, 7).

In conclusion, species identification of Abiotrophia, Enterococcus, Granulicatella, and Streptococcus by the present array is highly reliable. With the exception of beta-hemolytic streptococci, the method could be used as an accurate alternative to the conventional methods if adequate species identification is of concern.


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ACKNOWLEDGMENTS
 
This project was supported by grants (NSC 95-2323-B006-007 and NSC 95-2320-B-006-034) from the National Science Council and in part by the Department of Medical Research in National Taiwan University Hospital, Taiwan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Laboratory Science and Biotechnology, School of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan, Republic of China. Phone: 886-6-2353535, ext. 5790. Fax: 886-6-2363956. E-mail: tsungcha{at}mail.ncku.edu.tw. Back

{triangledown} Published ahead of print on 25 October 2006. Back

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/. Back


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Journal of Clinical Microbiology, December 2006, p. 4414-4424, Vol. 44, No. 12
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