Identification of Clinically Relevant Viridans Streptococci by an Oligonucleotide Array
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微生物临床杂志 2005年第4期
Department of Medical Laboratory Science and Biotechnology, School of Medicine, National Cheng Kung University, Tainan
School of Medical Technology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China
ABSTRACT
Viridans streptococci (VS) are common etiologic agents of subacute infective endocarditis and are capable of causing a variety of pyogenic infections. Many species of VS are difficult to differentiate by phenotypic traits. An oligonucleotide array based on 16S-23S rRNA gene intergenic spacer (ITS) sequences was developed to identify 11 clinically relevant VS. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The method consisted of PCR amplification of the ITS regions by using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species-specific oligonucleotides immobilized on a nylon membrane. After 120 strains of the 11 species of VG and 91 strains of other bacteria were tested, the sensitivity and specificity of the oligonucleotide array were found to be 100% (120 of 120 strains) and 95.6% (87 of 91 strains), respectively. S. pneumoniae cross-hybridized to the probes used for the identification of S. mitis, and simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. In conclusion, identification of species of VS by use of the present oligonucleotide array is accurate and could be used as an alternative reliable method for species identification of strains of VS.
INTRODUCTION
Viridans streptococci (VS) can be isolated as part of the normal flora of the respiratory, genital, and alimentary tracts. However, some species of VS are the most common etiologic agents of subacute infective endocarditis and can cause a variety of pyogenic infections (10). Species of VS are also playing an increasing role in infections among immunocompromised patients (29). On the basis of the 16S rRNA gene sequences, species of VS are divided into five major groups: (i) the mutans group, (ii) the salivarius group, (iii) the anginosus group (also called the milleri group), (iv) the sanguinis group, and (v) the mitis group (10).
Nearly 40 conventional tests have been used to differentiate species of VS, but phenotypic tests do not allow the unequivocal identification of some species of this heterogeneous group of bacteria (2, 11, 20). This is because variability in a common phenotypic trait is common among strains of the same species (5, 19). Identification of species of the strains of S. mutans and the anginosus and mitis groups is the most problematic (13, 16, 28).
The clinical significance of VS may differ between species, and sometimes it may be important to identify the individual species associated with diseases (16). In Taiwan, it was found that VS accounted for 9% of all cases of culture-proven bacterial meningitis in adults and that species of the anginosus group comprised more than 80% of isolates of VS responsible for meningitis in adults (7). Jacobs et al. (17) studied 104 isolates of VS recovered from blood cultures and found that S. oralis and S. mitis were the two species that were the most frequently isolated from patients in the hematology unit, whereas strains of the anginosus group were the dominant species isolated from the general hospital population. Among the VS isolated from patients with significant infections, a study conducted in Taiwan revealed that high-level penicillin resistance (MICs 4 μg/ml) was frequently found in isolates of S. oralis (35%) and S. mitis (20%) (31). In addition, macrolide resistance also occurred most frequently in S. oralis isolates (55%) but in none of the S. mutans isolates (31).
Several molecular biology-based methods have been developed for the identification of VS to the species level. The targets used for molecular diagnosis include the rRNA genes (6, 9, 16, 18, 27); the tRNA gene intergenic spacer (ITS) region (14); and the genes encoding heat shock proteins (groESL) (32), manganese-dependent superoxide dismutase (sodAint) (24), and D-alanine-D-alanine ligase (13). However, strains of S. mitis and S. oralis cannot be differentiated by analyzing either the groESL gene (32) or the sodAint gene (24), as intraspecies sequence variations are higher than interspecies variations.
The 16S-23S rRNA gene ITS region separating the 16S and 23S rRNA genes has been suggested to be a good candidate for bacterial species identification (8, 26, 33) and strain typing (15). Sequences of the ITS regions have been found to have low levels of intraspecies variation and high levels of interspecies divergence (8, 26, 38). In our previous study (8), we demonstrated that species of VS could be effectively differentiated by ITS sequence analysis. On the basis of the results of the previous study, the aim of the present study was to investigate the feasibility of using a panel of oligonucleotide probes designed from the ITS regions to identify 11 clinically relevant VS to the species level by use of an oligonucleotide array.
MATERIALS AND METHODS
Bacterial strains. A total of 211 strains were used in this study (Table 1). Among these strains, 120 (27 reference isolates and 93 clinical isolates) were strains of the 11 target species of VS and 91 (73 reference and 18 clinical isolates) were strains of other species. Reference strains were obtained from the American Type Culture Collection (ATCC; Manassas, Va.), the Bioresources Collection and Research Center (BCRC; Hsichu, Taiwan, Republic of China), and the Culture Collection of the University of Gteborg (CCUG; Gteborg, Sweden). Clinical isolates were obtained from the National Taiwan University Hospital (Taipei, Taiwan) and the National Cheng Kung University Medical Center (Tainan, Taiwan) (Table 1). Most clinical isolates were recovered from blood cultures and deep abscesses. The clinical isolates of VS were initially identified with the Rapid ID 32 STREP system (bioMerieux Vitek, Marcy l'Etoile, France) and by ITS sequence analysis (8). Clinical isolates that produced discrepant identifications with the Rapid ID 32 STREP system and by ITS sequence analysis were further identified by sequencing of their 16S rRNA gene sequences (25). All strains were subcultured 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 subcultured on chocolate agar.
DNA preparation. The boiling method was used to extract DNA from the bacteria (34). Briefly, colonies of pure cultures were suspended in 50 μl of sterilized water and heated at 100°C for 15 min in a heating block. After centrifugation in a microcentrifuge (8,000 x g for 10 min), the supernatant containing bacterial DNA was stored at –20°C for further use.
Amplification of ITS fragments for hybridization. The bacterium-specific universal primers 13BF (5'-GTGAATACGTTCCCGGGCCT-3') and 6R (5'-GGGTTYCCCCRTTCRGAAAT-3') (where Y is C or T and R is A or G) were used to amplify a DNA fragment that encompassed a small portion of the 16S rRNA gene region, the ITS region, and a small portion of the 23S rRNA gene region (25). Reverse primer 6R was labeled with digoxigenin at its 5' end and was obtained from MDBio Inc. (Taipei, Taiwan). The 5' end of primer 13BF is located at position 1371 of the 16S rRNA gene, and the 5' end of primer 6R is located at position 108 downstream of the 5' end of the 23S rRNA gene (Escherichia coli numbering). PCR was performed with 5 μl (1 to 5 ng) of template DNA in a total reaction volume of 50 μl consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 0.1 μM (each) primer, 1 U of Taq DNA polymerase, 10 μM digoxigenin-11-dUTP (Roche, Mannheim, Germany), and 50 μl of a mineral oil overlay. The PCR program consisted of 8 cycles of denaturation (94°C for 2 min), annealing (55°C for 1 min), and extension (72°C for 1 min), followed by 30 cycles of denaturation (94°C for 1 min), annealing (60°C for 1 min), and extension (72°C for 1 min) and a final extension step at 72°C for 3 min. An OmniGen thermal cycler (Hybaid Limited, Middlesex, United Kingdom) was used for PCR. DNA extracted from Xanthobacter flavus BCRC 12271 was used as a positive control.
Design of species-specific oligonucleotide probes. The species-specific oligonucleotide probes (20- to 31-mers) used for identification of the 11 species of VS were designed from the ITS sequences determined in our previous study (8), and their GenBank accession numbers are indicated in Table 2. The ITS sequences of different species were aligned by using the PileUp command of the Wisconsin Genetics Computer Group Package (version 10.3; Accelrys Inc., San Diego, Calif.). The probes that were designed were first screened against the sequences in the GenBank database to detect homology with the sequences of other bacteria; and a total of 19 probes, including 2 positive control probes (probes designed from the ITS sequence of X. flavus), were used for fabrication of the oligonucleotide array (Table 2). The 19 probes had a wide range of melting temperatures (Tms; 43.6 to 60°C). Multiple probes were used to identify S. sanguinis (two probes), S. uberis (three probes), S. intermedius (three probes), and S. salivarius (two probes), whereas only one probe was designed for the identification of each of the remaining seven species of VS (Table 2).
Preparation of oligonucleotide arrays. The arrays (3.5 by 8.5 mm) were made in batches of 20. The oligonucleotide probes were diluted 1:1 (final concentration, 10 μM) with a tracking dye solution (30% [vol/vol] glycerol, 40% [vol/vol] dimethyl sulfoxide, 1 mM EDTA [EDTA, disodium salt], 0.15% [wt/vol] bromophenol blue, 10 mM Tris-HCl [pH 7.5]). The probe solutions were drawn into different wells of a round-bottom microtiter plate and spotted onto a positively charged nylon membrane (Roche) with an automatic arrayer (Wittech, Taipei, Taiwan) by use of a solid pin (diameter, 500 μm). The array contained 24 dots, including 17 dots for the 11 target species of VS, 2 dots for positive controls (probes designed from the ITS region of X. flavus), and 5 dots for negative controls (dye only). Once all the probes had been applied, the membrane was dried and exposed to short-wave UV light (Stratalinker 1800; Stratagene, La Jolla, Calif.) for 30 s. Unbound oligonucleotides were removed by two washes (for 2 min each time) at room temperature in 0.5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS). The arrays were air dried and stored at room temperature for further use.
Hybridization procedures. Unless indicated otherwise, the hybridization procedures were carried out at room temperature in a hybridization oven with a shaking speed of 60 rpm. All reagents except the buffers used for hybridization were included in the DIG Nucleic Acid Detection kit (catalog no. 1175041; Roche). Each array was prehybridized for 2 h with 1 ml of hybridization solution (5x SSC, 1% [wt/vol] blocking reagent, 0.1% N-laurylsarcosine, 0.02% SDS) in an individual well of a 24-well cell culture plate. The digoxigenin-labeled PCR product amplified from an isolate was heated in a boiling water bath for 5 min and immediately cooled on an ice bath. Ten microliters of denatured PCR product of the test organism and 10 μl of the denatured PCR product amplified from X. flavus (the positive control) were diluted with 0.5 ml of hybridization solution and added to each well. Hybridization was conducted at 50°C for 90 min. After the nonhybridized PCR products were removed, the array was washed four times (for 5 min each time) in 1 ml of washing buffer (0.25x SSC, 0.1% SDS), followed by blocking for 1 h with 1 ml of blocking solution (1% [wt/vol] blocking reagent dissolved in maleic acid buffer [0.1 M maleic acid, 0.15 M NaCl {pH 7.5}]). The blocking solution was then removed, 0.5 ml of alkaline phosphatase-conjugated sheep antidigoxigenin antibodies (diluted 1:2,500 in blocking solution) was added to each well, and the plate was incubated for 1 h. The array was washed three times (for 15 min each time) in 1 ml of washing solution (0.3% [vol/vol] Tween 20 in maleic acid buffer), followed by washing in 1 ml of detection buffer (0.1 M Tris-HCl, 0.15 M NaCl [pH 9.5]) for 5 min. Finally, 0.5 ml of alkaline phosphatase substrate (a stock solution of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate diluted 1:50 in detection buffer) was added to each well and the plate was incubated at 37°C (without shaking). Color development was clearly visible between 30 min and 1 h after the start of the reaction.
Definition of positive reaction, sensitivity, and specificity. A strain was identified as 1 of the 11 target species of VS when the probe (or all probes) designed to be specific for the species and the two positive control probes (Fig. 1, probes 2 and 3) hybridized. Sensitivity was defined as the number of strains of the target species correctly identified by the array (true positives) divided by the total number of strains of that species tested. Specificity was defined as the number of strains of nontarget microorganisms producing negative hybridization reactions (true negatives) divided by total number of strains of that species tested (22).
RESULTS
Hybridization of VS to the oligonucleotide array. Of 120 strains (27 reference strains and 93 clinical isolates) belonging to the 11 target species of VS, all strains hybridized to the respective oligonucleotide probe(s) designed for each species (Table 1). Figure 1 shows the hybridization results for the reference strains of VS and the other bacterial species. Since all target strains were correctly identified, the sensitivity of the oligonucleotide array for the identification of the 11 species of VS was 100% (120 of 120 strains). Although the ITS sequences of S. mitis and S. oralis have rather high degrees of similarity (0.93 to 0.95) (8), S. mitis could be effectively differentiated from S. oralis by the oligonucleotide array (Fig. 1G and H).
Multiple probes were used to identify S. sanguinis (two probes), S. uberis (three probes), S. intermedius (three probes), and S. salivarius (two probes). In general, probes with higher Tms tend to produce stronger hybridization signals. For example, probe 23 (Tm = 46.1°C) and probe 24 (Tm = 52.1°C) were used to identify S. salivarius (Table 2), but the hybridization signal of probe 24 was much stronger than that of probe 23 (Fig. 1K). The same phenomenon was observed for the probes used to identify S. sanguinis (Fig. 1B): the signal of probe 9 (Tm = 51.1°C) was evidently stronger than that of probe 10 (Tm = 48.2°C). However, this generality was not always true. For example, probes 12 (Tm = 49°C), 13 (Tm = 47.3°C), and 14 (Tm = 52.1°C) were designed to identify S. uberis; however, probe 12 produced the most intense hybridization signal (Fig. 1D), although probe 13 still had the weakest signal.
Hybridization of other bacteria to the oligonucleotide array. Ninety-one strains (47 species) of other bacteria, including non-VS, nutritionally variant streptococci (Abiotrophia and Granulicatella), enterococci, staphylococci, and several gram-negative bacteria, were used to test the specificity of the oligonucleotide array. All four reference strains of S. pneumoniae cross-hybridized to probe 17 (Fig. 1N), which was designed to identify S. mitis. However, 87 strains of other bacteria did not cross-hybridize with any of the 17 probes immobilized on the nylon membrane. On the basis of the results obtained with the array, a specificity of 95.6% (87 of 91 strains) for the identification of species of VS by the present array was obtained.
DISCUSSION
Conventional phenotypic tests have already been proven to be difficult for the identification of some species of VS (2, 11, 20). In this study, a reverse hybridization method that could identify 11 species of VS was developed. The 11 target species represented >96% of the identified species of VS isolated from sterile body fluids in the National Taiwan University Hospital (data not shown), with the remaining 2 species being S. sobrinus and S. vestibularis. A pair of universal primers was used to amplify the bacterial ITS regions, followed by hybridization of the digoxigenin-labeled PCR products to the oligonucleotide array. The hybridized spot (diameter, 0.5 mm), which appeared in blue on the white nylon membrane, could easily be read with the naked eye. The whole procedure for the present array method could be finished within a working day (approximately 8 h), starting from the time of colony isolation.
The DNA array (or DNA chip) technology has been found to be a useful tool for the identification of a variety of bacteria, especially those bacteria that are difficult to differentiate by conventional methods and those bacteria for which the identification procedure may take a long time. This methodology has been used to identify Mycobacterium species (12), bacteria in positive blood cultures (3), Campylobacter species (35), Listeria species (36), ammonia-oxidizing bacteria (1), and pathogenic bacteria in cervical swab specimens (23). A variety of target genes encoding the DNA gyrase B subunit (12), the 23S rRNA gene (3, 23), the 16S rRNA gene (1), and other specific genes (35, 36) have been used in hybridization assays. The DNA array generally comprises a solid phase (glass or a membrane) on which multiple DNA probes with known identities are fixed for hybridization with DNA samples.
The good performance of the present array might be due to the fact that the ITS sequence is species specific (11, 17, 18, 27, 28). The region was found to have low levels of intraspecies variation and high levels of interspecies divergence (8, 38) and has been suggested to be a good candidate for bacterial species identification (4). The array used in this study may have the potential to be continually extended by adding additional oligonucleotides to the panel without significantly increasing its cost or complexity.
Although the Tms of some of the probes listed in Table 2 were lower than the array hybridization temperature (50°C), clear hybridization signals were obtained with these probes (Fig. 1). For example, probes 1 (Tm = 43.6°C) and 18 (Tm = 44.1°C), used for the identification of S. parasanguinis and S. oralis, respectively, produced signals that were easily recognized. These results might be partially due to the use of a relatively low stringency buffer (which has a high ionic strength [5x SSC] and a low concentration of detergent [0.02% SDS]) for the hybridization reaction. Volokhov et al. (36) also used several probes with Tms (40 to 44°C) lower than the hybridization temperature (45°C) for the identification of Listeria spp. and obtained good hybridization results.
Although a variety of non-VS (91 strains representing 47 species; Table 1) were used for specificity testing, it does not completely exclude the possibility of cross-hybridization with other bacteria. Therefore, strains should be confirmed to be gram-positive and catalase-negative cocci before they are tested with the array. Multiple probes were used to identify S. sanguinis, S. uberis, S. intermedius, and S. salivarius (Table 2 and Fig. 1). Some of these multiple probes (Table 1, probes 13 and 23) produced relatively weak signals (Fig. 1D and K), and in further applications, it may not be necessary to keep these two probes on the array.
Strains of S. oralis and S. mitis, the two most frequently isolated species of VS from patients in the hematology unit, are difficult to differentiate by analysis of their 16S rRNA gene sequences. The 16S rRNA gene sequence identity of type strains of S. oralis (ATCC 35037) and S. mitis (ATCC 49456) is greater than 99% (18). They also cannot be differentiated by sequence analysis of the groESL (32) and sodAint (24) genes because the intraspecies sequence variations may be higher than the interspecies variations. In addition, it was found that the ITS sequences of S. mitis and S. oralis have high degrees of similarity that range from 0.93 to 0.95 (8). However, strains of S. oralis have single nucleotide deletions at positions 199 and 217 of the ITS sequence of S. mitis. In other words, the ITS region of S. mitis strains is 2 nucleotides longer than that of S. mitis strains. The probes used to differentiate the two species were designed from positions 190 to 210 (or 211) (Table 2) of the ITS regions, and this design made the deletion (or insertion) base at about the midpoints of the probes. This design could effectively differentiate these two closely related species (Fig. 1G and H).
In this study, all S. pneumoniae strains cross-hybridized to the probes used for the identification of S. mitis (Table 1; Fig. 1N). Therefore, S. pneumoniae was misidentified as S. mitis by the array method. For this reason, simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. The cross-hybridization was not unexpected, since the two species have almost identical ITS sequences. The ITS sequence similarity between the type strains of S. pneumoniae (ATCC 33400) and S. mitis (ATCC 49456) is 0.99 (8). Phylogenetic trees constructed by using the genes encoding groESL (32), sodAint (24), the 16S rRNA gene (6, 18), and the ITS region (8) grouped S. mitis and S. pneumoniae together.
The anginosus group contains three species: S. anginosus, S. constellatus, and S. intermedius. S. anginosus isolates are commonly isolated from urogenital and gastrointestinal sources, and S. constellatus is often isolated from respiratory and many other sources, while S. intermedius strains are commonly recovered from brain and liver abscesses (10). The classification and identification of members of the anginosus group have caused a lot of confusion. There are beta-hemolytic strains of each of the three species, and the strains may possess one of four different Lancefield group antigens (F, C, A, and G) or have no group antigen (10). Adding to the confusion is the fact that non-beta-hemolytic strains are more common than beta-hemolytic strains (10). Some investigators (30, 37, 39) have proposed the use of identification systems based on several enzymatic activities, carbohydrate fermentation, and biochemical and serological tests to differentiate members of the anginosus group. Limia et al. (21) found that the Rapid ID 32 STREP system was particularly inaccurate when it was used for the identification of isolates in the anginosus group, with an identification rate of only 57% for S. intermedius strains. However, the three species were effectively differentiated by the present oligonucleotide array (Table 1; Fig. 1C, I, and J).
In conclusion, the identification of clinically relevant species of VS by the oligonucleotide array seems to be reliable, and the array method could be used as an accurate alternative for the identification of these microorganisms. The assay may have the potential to be continually extended by adding specific oligonucleotides to the panel to identify more microorganisms.
ACKNOWLEDGMENTS
This project was supported by grants (NSC 93-2323-B006-007 and NSC 93-2323-B006-006) from the National Science Council and, in part, by grants from the Department of Medical Research of the National Taiwan University Hospital, Taipei, Taiwan.
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School of Medical Technology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China
ABSTRACT
Viridans streptococci (VS) are common etiologic agents of subacute infective endocarditis and are capable of causing a variety of pyogenic infections. Many species of VS are difficult to differentiate by phenotypic traits. An oligonucleotide array based on 16S-23S rRNA gene intergenic spacer (ITS) sequences was developed to identify 11 clinically relevant VS. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The method consisted of PCR amplification of the ITS regions by using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species-specific oligonucleotides immobilized on a nylon membrane. After 120 strains of the 11 species of VG and 91 strains of other bacteria were tested, the sensitivity and specificity of the oligonucleotide array were found to be 100% (120 of 120 strains) and 95.6% (87 of 91 strains), respectively. S. pneumoniae cross-hybridized to the probes used for the identification of S. mitis, and simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. In conclusion, identification of species of VS by use of the present oligonucleotide array is accurate and could be used as an alternative reliable method for species identification of strains of VS.
INTRODUCTION
Viridans streptococci (VS) can be isolated as part of the normal flora of the respiratory, genital, and alimentary tracts. However, some species of VS are the most common etiologic agents of subacute infective endocarditis and can cause a variety of pyogenic infections (10). Species of VS are also playing an increasing role in infections among immunocompromised patients (29). On the basis of the 16S rRNA gene sequences, species of VS are divided into five major groups: (i) the mutans group, (ii) the salivarius group, (iii) the anginosus group (also called the milleri group), (iv) the sanguinis group, and (v) the mitis group (10).
Nearly 40 conventional tests have been used to differentiate species of VS, but phenotypic tests do not allow the unequivocal identification of some species of this heterogeneous group of bacteria (2, 11, 20). This is because variability in a common phenotypic trait is common among strains of the same species (5, 19). Identification of species of the strains of S. mutans and the anginosus and mitis groups is the most problematic (13, 16, 28).
The clinical significance of VS may differ between species, and sometimes it may be important to identify the individual species associated with diseases (16). In Taiwan, it was found that VS accounted for 9% of all cases of culture-proven bacterial meningitis in adults and that species of the anginosus group comprised more than 80% of isolates of VS responsible for meningitis in adults (7). Jacobs et al. (17) studied 104 isolates of VS recovered from blood cultures and found that S. oralis and S. mitis were the two species that were the most frequently isolated from patients in the hematology unit, whereas strains of the anginosus group were the dominant species isolated from the general hospital population. Among the VS isolated from patients with significant infections, a study conducted in Taiwan revealed that high-level penicillin resistance (MICs 4 μg/ml) was frequently found in isolates of S. oralis (35%) and S. mitis (20%) (31). In addition, macrolide resistance also occurred most frequently in S. oralis isolates (55%) but in none of the S. mutans isolates (31).
Several molecular biology-based methods have been developed for the identification of VS to the species level. The targets used for molecular diagnosis include the rRNA genes (6, 9, 16, 18, 27); the tRNA gene intergenic spacer (ITS) region (14); and the genes encoding heat shock proteins (groESL) (32), manganese-dependent superoxide dismutase (sodAint) (24), and D-alanine-D-alanine ligase (13). However, strains of S. mitis and S. oralis cannot be differentiated by analyzing either the groESL gene (32) or the sodAint gene (24), as intraspecies sequence variations are higher than interspecies variations.
The 16S-23S rRNA gene ITS region separating the 16S and 23S rRNA genes has been suggested to be a good candidate for bacterial species identification (8, 26, 33) and strain typing (15). Sequences of the ITS regions have been found to have low levels of intraspecies variation and high levels of interspecies divergence (8, 26, 38). In our previous study (8), we demonstrated that species of VS could be effectively differentiated by ITS sequence analysis. On the basis of the results of the previous study, the aim of the present study was to investigate the feasibility of using a panel of oligonucleotide probes designed from the ITS regions to identify 11 clinically relevant VS to the species level by use of an oligonucleotide array.
MATERIALS AND METHODS
Bacterial strains. A total of 211 strains were used in this study (Table 1). Among these strains, 120 (27 reference isolates and 93 clinical isolates) were strains of the 11 target species of VS and 91 (73 reference and 18 clinical isolates) were strains of other species. Reference strains were obtained from the American Type Culture Collection (ATCC; Manassas, Va.), the Bioresources Collection and Research Center (BCRC; Hsichu, Taiwan, Republic of China), and the Culture Collection of the University of Gteborg (CCUG; Gteborg, Sweden). Clinical isolates were obtained from the National Taiwan University Hospital (Taipei, Taiwan) and the National Cheng Kung University Medical Center (Tainan, Taiwan) (Table 1). Most clinical isolates were recovered from blood cultures and deep abscesses. The clinical isolates of VS were initially identified with the Rapid ID 32 STREP system (bioMerieux Vitek, Marcy l'Etoile, France) and by ITS sequence analysis (8). Clinical isolates that produced discrepant identifications with the Rapid ID 32 STREP system and by ITS sequence analysis were further identified by sequencing of their 16S rRNA gene sequences (25). All strains were subcultured 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 subcultured on chocolate agar.
DNA preparation. The boiling method was used to extract DNA from the bacteria (34). Briefly, colonies of pure cultures were suspended in 50 μl of sterilized water and heated at 100°C for 15 min in a heating block. After centrifugation in a microcentrifuge (8,000 x g for 10 min), the supernatant containing bacterial DNA was stored at –20°C for further use.
Amplification of ITS fragments for hybridization. The bacterium-specific universal primers 13BF (5'-GTGAATACGTTCCCGGGCCT-3') and 6R (5'-GGGTTYCCCCRTTCRGAAAT-3') (where Y is C or T and R is A or G) were used to amplify a DNA fragment that encompassed a small portion of the 16S rRNA gene region, the ITS region, and a small portion of the 23S rRNA gene region (25). Reverse primer 6R was labeled with digoxigenin at its 5' end and was obtained from MDBio Inc. (Taipei, Taiwan). The 5' end of primer 13BF is located at position 1371 of the 16S rRNA gene, and the 5' end of primer 6R is located at position 108 downstream of the 5' end of the 23S rRNA gene (Escherichia coli numbering). PCR was performed with 5 μl (1 to 5 ng) of template DNA in a total reaction volume of 50 μl consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 0.1 μM (each) primer, 1 U of Taq DNA polymerase, 10 μM digoxigenin-11-dUTP (Roche, Mannheim, Germany), and 50 μl of a mineral oil overlay. The PCR program consisted of 8 cycles of denaturation (94°C for 2 min), annealing (55°C for 1 min), and extension (72°C for 1 min), followed by 30 cycles of denaturation (94°C for 1 min), annealing (60°C for 1 min), and extension (72°C for 1 min) and a final extension step at 72°C for 3 min. An OmniGen thermal cycler (Hybaid Limited, Middlesex, United Kingdom) was used for PCR. DNA extracted from Xanthobacter flavus BCRC 12271 was used as a positive control.
Design of species-specific oligonucleotide probes. The species-specific oligonucleotide probes (20- to 31-mers) used for identification of the 11 species of VS were designed from the ITS sequences determined in our previous study (8), and their GenBank accession numbers are indicated in Table 2. The ITS sequences of different species were aligned by using the PileUp command of the Wisconsin Genetics Computer Group Package (version 10.3; Accelrys Inc., San Diego, Calif.). The probes that were designed were first screened against the sequences in the GenBank database to detect homology with the sequences of other bacteria; and a total of 19 probes, including 2 positive control probes (probes designed from the ITS sequence of X. flavus), were used for fabrication of the oligonucleotide array (Table 2). The 19 probes had a wide range of melting temperatures (Tms; 43.6 to 60°C). Multiple probes were used to identify S. sanguinis (two probes), S. uberis (three probes), S. intermedius (three probes), and S. salivarius (two probes), whereas only one probe was designed for the identification of each of the remaining seven species of VS (Table 2).
Preparation of oligonucleotide arrays. The arrays (3.5 by 8.5 mm) were made in batches of 20. The oligonucleotide probes were diluted 1:1 (final concentration, 10 μM) with a tracking dye solution (30% [vol/vol] glycerol, 40% [vol/vol] dimethyl sulfoxide, 1 mM EDTA [EDTA, disodium salt], 0.15% [wt/vol] bromophenol blue, 10 mM Tris-HCl [pH 7.5]). The probe solutions were drawn into different wells of a round-bottom microtiter plate and spotted onto a positively charged nylon membrane (Roche) with an automatic arrayer (Wittech, Taipei, Taiwan) by use of a solid pin (diameter, 500 μm). The array contained 24 dots, including 17 dots for the 11 target species of VS, 2 dots for positive controls (probes designed from the ITS region of X. flavus), and 5 dots for negative controls (dye only). Once all the probes had been applied, the membrane was dried and exposed to short-wave UV light (Stratalinker 1800; Stratagene, La Jolla, Calif.) for 30 s. Unbound oligonucleotides were removed by two washes (for 2 min each time) at room temperature in 0.5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS). The arrays were air dried and stored at room temperature for further use.
Hybridization procedures. Unless indicated otherwise, the hybridization procedures were carried out at room temperature in a hybridization oven with a shaking speed of 60 rpm. All reagents except the buffers used for hybridization were included in the DIG Nucleic Acid Detection kit (catalog no. 1175041; Roche). Each array was prehybridized for 2 h with 1 ml of hybridization solution (5x SSC, 1% [wt/vol] blocking reagent, 0.1% N-laurylsarcosine, 0.02% SDS) in an individual well of a 24-well cell culture plate. The digoxigenin-labeled PCR product amplified from an isolate was heated in a boiling water bath for 5 min and immediately cooled on an ice bath. Ten microliters of denatured PCR product of the test organism and 10 μl of the denatured PCR product amplified from X. flavus (the positive control) were diluted with 0.5 ml of hybridization solution and added to each well. Hybridization was conducted at 50°C for 90 min. After the nonhybridized PCR products were removed, the array was washed four times (for 5 min each time) in 1 ml of washing buffer (0.25x SSC, 0.1% SDS), followed by blocking for 1 h with 1 ml of blocking solution (1% [wt/vol] blocking reagent dissolved in maleic acid buffer [0.1 M maleic acid, 0.15 M NaCl {pH 7.5}]). The blocking solution was then removed, 0.5 ml of alkaline phosphatase-conjugated sheep antidigoxigenin antibodies (diluted 1:2,500 in blocking solution) was added to each well, and the plate was incubated for 1 h. The array was washed three times (for 15 min each time) in 1 ml of washing solution (0.3% [vol/vol] Tween 20 in maleic acid buffer), followed by washing in 1 ml of detection buffer (0.1 M Tris-HCl, 0.15 M NaCl [pH 9.5]) for 5 min. Finally, 0.5 ml of alkaline phosphatase substrate (a stock solution of nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate diluted 1:50 in detection buffer) was added to each well and the plate was incubated at 37°C (without shaking). Color development was clearly visible between 30 min and 1 h after the start of the reaction.
Definition of positive reaction, sensitivity, and specificity. A strain was identified as 1 of the 11 target species of VS when the probe (or all probes) designed to be specific for the species and the two positive control probes (Fig. 1, probes 2 and 3) hybridized. Sensitivity was defined as the number of strains of the target species correctly identified by the array (true positives) divided by the total number of strains of that species tested. Specificity was defined as the number of strains of nontarget microorganisms producing negative hybridization reactions (true negatives) divided by total number of strains of that species tested (22).
RESULTS
Hybridization of VS to the oligonucleotide array. Of 120 strains (27 reference strains and 93 clinical isolates) belonging to the 11 target species of VS, all strains hybridized to the respective oligonucleotide probe(s) designed for each species (Table 1). Figure 1 shows the hybridization results for the reference strains of VS and the other bacterial species. Since all target strains were correctly identified, the sensitivity of the oligonucleotide array for the identification of the 11 species of VS was 100% (120 of 120 strains). Although the ITS sequences of S. mitis and S. oralis have rather high degrees of similarity (0.93 to 0.95) (8), S. mitis could be effectively differentiated from S. oralis by the oligonucleotide array (Fig. 1G and H).
Multiple probes were used to identify S. sanguinis (two probes), S. uberis (three probes), S. intermedius (three probes), and S. salivarius (two probes). In general, probes with higher Tms tend to produce stronger hybridization signals. For example, probe 23 (Tm = 46.1°C) and probe 24 (Tm = 52.1°C) were used to identify S. salivarius (Table 2), but the hybridization signal of probe 24 was much stronger than that of probe 23 (Fig. 1K). The same phenomenon was observed for the probes used to identify S. sanguinis (Fig. 1B): the signal of probe 9 (Tm = 51.1°C) was evidently stronger than that of probe 10 (Tm = 48.2°C). However, this generality was not always true. For example, probes 12 (Tm = 49°C), 13 (Tm = 47.3°C), and 14 (Tm = 52.1°C) were designed to identify S. uberis; however, probe 12 produced the most intense hybridization signal (Fig. 1D), although probe 13 still had the weakest signal.
Hybridization of other bacteria to the oligonucleotide array. Ninety-one strains (47 species) of other bacteria, including non-VS, nutritionally variant streptococci (Abiotrophia and Granulicatella), enterococci, staphylococci, and several gram-negative bacteria, were used to test the specificity of the oligonucleotide array. All four reference strains of S. pneumoniae cross-hybridized to probe 17 (Fig. 1N), which was designed to identify S. mitis. However, 87 strains of other bacteria did not cross-hybridize with any of the 17 probes immobilized on the nylon membrane. On the basis of the results obtained with the array, a specificity of 95.6% (87 of 91 strains) for the identification of species of VS by the present array was obtained.
DISCUSSION
Conventional phenotypic tests have already been proven to be difficult for the identification of some species of VS (2, 11, 20). In this study, a reverse hybridization method that could identify 11 species of VS was developed. The 11 target species represented >96% of the identified species of VS isolated from sterile body fluids in the National Taiwan University Hospital (data not shown), with the remaining 2 species being S. sobrinus and S. vestibularis. A pair of universal primers was used to amplify the bacterial ITS regions, followed by hybridization of the digoxigenin-labeled PCR products to the oligonucleotide array. The hybridized spot (diameter, 0.5 mm), which appeared in blue on the white nylon membrane, could easily be read with the naked eye. The whole procedure for the present array method could be finished within a working day (approximately 8 h), starting from the time of colony isolation.
The DNA array (or DNA chip) technology has been found to be a useful tool for the identification of a variety of bacteria, especially those bacteria that are difficult to differentiate by conventional methods and those bacteria for which the identification procedure may take a long time. This methodology has been used to identify Mycobacterium species (12), bacteria in positive blood cultures (3), Campylobacter species (35), Listeria species (36), ammonia-oxidizing bacteria (1), and pathogenic bacteria in cervical swab specimens (23). A variety of target genes encoding the DNA gyrase B subunit (12), the 23S rRNA gene (3, 23), the 16S rRNA gene (1), and other specific genes (35, 36) have been used in hybridization assays. The DNA array generally comprises a solid phase (glass or a membrane) on which multiple DNA probes with known identities are fixed for hybridization with DNA samples.
The good performance of the present array might be due to the fact that the ITS sequence is species specific (11, 17, 18, 27, 28). The region was found to have low levels of intraspecies variation and high levels of interspecies divergence (8, 38) and has been suggested to be a good candidate for bacterial species identification (4). The array used in this study may have the potential to be continually extended by adding additional oligonucleotides to the panel without significantly increasing its cost or complexity.
Although the Tms of some of the probes listed in Table 2 were lower than the array hybridization temperature (50°C), clear hybridization signals were obtained with these probes (Fig. 1). For example, probes 1 (Tm = 43.6°C) and 18 (Tm = 44.1°C), used for the identification of S. parasanguinis and S. oralis, respectively, produced signals that were easily recognized. These results might be partially due to the use of a relatively low stringency buffer (which has a high ionic strength [5x SSC] and a low concentration of detergent [0.02% SDS]) for the hybridization reaction. Volokhov et al. (36) also used several probes with Tms (40 to 44°C) lower than the hybridization temperature (45°C) for the identification of Listeria spp. and obtained good hybridization results.
Although a variety of non-VS (91 strains representing 47 species; Table 1) were used for specificity testing, it does not completely exclude the possibility of cross-hybridization with other bacteria. Therefore, strains should be confirmed to be gram-positive and catalase-negative cocci before they are tested with the array. Multiple probes were used to identify S. sanguinis, S. uberis, S. intermedius, and S. salivarius (Table 2 and Fig. 1). Some of these multiple probes (Table 1, probes 13 and 23) produced relatively weak signals (Fig. 1D and K), and in further applications, it may not be necessary to keep these two probes on the array.
Strains of S. oralis and S. mitis, the two most frequently isolated species of VS from patients in the hematology unit, are difficult to differentiate by analysis of their 16S rRNA gene sequences. The 16S rRNA gene sequence identity of type strains of S. oralis (ATCC 35037) and S. mitis (ATCC 49456) is greater than 99% (18). They also cannot be differentiated by sequence analysis of the groESL (32) and sodAint (24) genes because the intraspecies sequence variations may be higher than the interspecies variations. In addition, it was found that the ITS sequences of S. mitis and S. oralis have high degrees of similarity that range from 0.93 to 0.95 (8). However, strains of S. oralis have single nucleotide deletions at positions 199 and 217 of the ITS sequence of S. mitis. In other words, the ITS region of S. mitis strains is 2 nucleotides longer than that of S. mitis strains. The probes used to differentiate the two species were designed from positions 190 to 210 (or 211) (Table 2) of the ITS regions, and this design made the deletion (or insertion) base at about the midpoints of the probes. This design could effectively differentiate these two closely related species (Fig. 1G and H).
In this study, all S. pneumoniae strains cross-hybridized to the probes used for the identification of S. mitis (Table 1; Fig. 1N). Therefore, S. pneumoniae was misidentified as S. mitis by the array method. For this reason, simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. The cross-hybridization was not unexpected, since the two species have almost identical ITS sequences. The ITS sequence similarity between the type strains of S. pneumoniae (ATCC 33400) and S. mitis (ATCC 49456) is 0.99 (8). Phylogenetic trees constructed by using the genes encoding groESL (32), sodAint (24), the 16S rRNA gene (6, 18), and the ITS region (8) grouped S. mitis and S. pneumoniae together.
The anginosus group contains three species: S. anginosus, S. constellatus, and S. intermedius. S. anginosus isolates are commonly isolated from urogenital and gastrointestinal sources, and S. constellatus is often isolated from respiratory and many other sources, while S. intermedius strains are commonly recovered from brain and liver abscesses (10). The classification and identification of members of the anginosus group have caused a lot of confusion. There are beta-hemolytic strains of each of the three species, and the strains may possess one of four different Lancefield group antigens (F, C, A, and G) or have no group antigen (10). Adding to the confusion is the fact that non-beta-hemolytic strains are more common than beta-hemolytic strains (10). Some investigators (30, 37, 39) have proposed the use of identification systems based on several enzymatic activities, carbohydrate fermentation, and biochemical and serological tests to differentiate members of the anginosus group. Limia et al. (21) found that the Rapid ID 32 STREP system was particularly inaccurate when it was used for the identification of isolates in the anginosus group, with an identification rate of only 57% for S. intermedius strains. However, the three species were effectively differentiated by the present oligonucleotide array (Table 1; Fig. 1C, I, and J).
In conclusion, the identification of clinically relevant species of VS by the oligonucleotide array seems to be reliable, and the array method could be used as an accurate alternative for the identification of these microorganisms. The assay may have the potential to be continually extended by adding specific oligonucleotides to the panel to identify more microorganisms.
ACKNOWLEDGMENTS
This project was supported by grants (NSC 93-2323-B006-007 and NSC 93-2323-B006-006) from the National Science Council and, in part, by grants from the Department of Medical Research of the National Taiwan University Hospital, Taipei, Taiwan.
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