Detection ofActinomycesSpecies Using Nonradioactive Riboprobes Coupled with Polymerase Chain Reaction

BIOCHEMICAL AND MOLECULAR MEDICINE ARTICLE NO.

58, 151–155 (1996)

0043

Detection of Actinomyces Species Using Nonradioactive Riboprobes Coupled with Polymerase Chain Reaction MICHIKO KIYAMA, KOICHI HIRATSUKA, SHIGENO SAITO, TERUAKI SHIROZA, HISASHI TAKIGUCHI, AND YOSHIMITSU ABIKO1 Department of Biochemistry, Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho-Nishi, Matsudo, Chiba 271, Japan Received February 5, 1996, and in revised form March 29, 1996

reported that destructive periodontal disease progressed with alternate periods of exacerbation and remission. Since then, several groups have investigated the correlation between microbial species and the periods of disease activity (2,3). A recent longitudinal study of destructive periodontal diseases indicated that certain Gram-negative species were found more frequently than Gram-positive bacteria during the period of exacerbation where active breakdown has occurred (4,5). Conversely, Gram-positive bacteria were isolated at higher frequency during the inactive state. These findings suggested that if the Gram-positive microorganisms become recessive species compared to Gram-negative pathogens, the inactive site might change into the active site. Thus, identification of these Gram-positive bacteria will be important to determine the period of disease activity on diagnosis of periodontal disease as well as for clinical therapy. Among the Gram-positive bacteria found in the inactive sites, members of the genus Actinomyces are the predominant colonizers of the human oral cavity (6). Adherence of the Actinomyces species to human buccal epithelial cells (7) and other mammalian cell types in vitro (8,9) is facilitated by the enzymatic activity of sialidase. The DNA fragment encoding sialidase has been cloned from Actinomyces viscosus T14V (10) and its complete nucleotide sequence has been determined (11). We have recently developed a DNA – RNA hybridization method using digoxigenin-labeled riboprobes and utilized this technique to identify Gram-negative pathogens (12 – 14) and Actinomyces species (15). In this communication, we report a more sensi-

We have been focusing our attention on the detection and identification of oral bacteria which are frequently associated with periodontal disease. In previous studies, Actinomyces species-specific riboprobes were generated and used to identify this microorganism. However, problems lie in the low sensitivity of this method. We have developed a novel system for the detection of Actinomyces species using nonradioactive riboprobes coupled with polymerase chain reaction (PCR) in this study. This system employs two procedures; initially, DNA fragments specific for the target microorganism are amplified by PCR, and these specific fragments are further hybridized with nonradioactive riboprobes. PCR analysis using chromosomal DNA isolated from Actinomyces species including laboratory strains, clinical isolates, and Actinomyces naeslundii (ATCC 12104) indicated the presence of the predicted common 756-bp fragment, a portion of the sialidase gene. These amplified DNA fragments were effectively visualized by hybridization with the digoxigenin-labeled riboprobes corresponding to the internal region of the amplified sialidase gene. With this system, approximately three orders of magnitude less chromosomal DNA was sufficient for the detection of specific microorganisms compared to the conventional riboprobe systems. q 1996 Academic Press, Inc.

For many decades, periodontal disease was considered to cause continuous slow and irreversible deterioration of the gingival tissue unless treated appropriately. In 1984, however, Socransky et al. (1) 1 To whom correspondence should be addressed. Fax: 81-47361-8880.

151 1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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tive DNA – RNA hybridization method which uses polymerase chain reaction (PCR) to amplify species-specific sequences. We chose the cloned sialidase gene (nanH) as the target Actinomyces species-specific sequence for amplification and used this method to screen for the presence of Actinomyces species in inactive sites during remission periods. MATERIALS AND METHODS Bacterial Strains A. viscosus strains ATCC 19246, T14V, and T14AV and clinically isolated strains 11132 and 13236 were used in this study. Other Gram-positive bacteria tested were A. naeslundii ATCC 12104, Streptococcus mutans GS-5, Streptococcus gordonii Challis, Streptococcus milleri Is57, and Lactobacillus casei 4649. All of the A. viscosus strains and A. naeslundii ATCC 12104 were provided by the Department of Microbiology, Nihon University School of Dentistry at Matsudo, Japan. Gram-negative bacteria including Porphyromonas gingivalis FDC 381, Porphyromonas endodontalis ATCC 35406, Prevotella loescheii ATCC 15930, Actinobacillus actinomycetemcomitans Y4, and Campylobacter rectus ATCC 33238 were also used. All of the bacteria were grown in broth cultures as previously described (16), harvested by centrifugation, and stored at 0207C in Dulbecco’s phosphate-buffered saline (PBS):glycerol (1:1) until use. The bacterial stocks were washed three times by centrifugation (15,000g for 2 min) in a high-speed refrigerated microcentrifuge then used for isolation of chromosomal DNA for PCR. Preparation of DNA Template for PCR Chromosomal DNA from Gram-positive bacteria was prepared using the lysozyme–polyethylene glycol procedure described by Chassy and Giuffrida (17), and Gram-negative bacterial DNA was prepared as described by Smith et al. (18). DNA was purified by two cycles of phenol–chloroform extraction and RNase treatment (0.5 mg/ml; Boehringer Mannheim, Germany). The purified DNA was collected by ethanol precipitation and dissolved in TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8.0) before use as the template for PCR. Primers and PCR Amplification Primers for PCR were designed based on the nucleotide sequence of A. viscosus T14V sialidase

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(nanH) in the GenBank database. As indicated in Table 1, the primers specific for A. viscosus T14V sialidase were designated SIA1 and SIA2. Primers RRN4 and RRN5, specific for eubacterial ribosomal DNA sequences, were prepared as described by Goncharoff et al. (19). PCR amplification was performed in a GeneAmp PCR system 9600 (PerkinElmer). PCR amplification mixtures contained template DNA in 1 1 PCR buffer [50 mM KCl, 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2], 250 nM SIA primers and 62.5 nM RRN primers, 200 mM each of dATP, dGTP, dTTP, and dCTP, and 1 unit of Taq DNA polymerase in a total volume of 25 ml. The reaction mixture was then subjected to 25 cycles of denaturation at 967C for 15 s, primer annealing at 557C for 30 s, and primer extension at 727C for 1 min. An additional 10 min was allowed for completion of primer extension after the last cycle. Amplification products were routinely analyzed by electrophoresis of 5-ml aliquots through 1.5% agarose gels, followed by staining in ethidium bromide solution (1 mg/ml). Preparation of Riboprobe The plasmid pMDAv2 (15) contains the 430-bp DNA fragment from A. viscosus ATCC 19246 which corresponds to the internal region of the 756-bp target sequence of the sialidase gene (from nucleotide position 674 to nucleotide position 1103; see Ref. 11). This plasmid was linearized by digestion with the restriction enzyme SacII, and digoxigenin-labeled riboprobe was prepared using a Dig-RNA labeling kit (Boehringer Mannheim) (15). Hybridization and Detection Five-microliter aliquots of heat-denatured PCR products were blotted onto Hybond-N/ membranes (Amersham) using a vacuum blotting system (Pulphor-SW, Atto Co., Tokyo, Japan). The membranes were hybridized with the digoxigenin-labeled riboprobe and then assayed by an immunoenzymatic method with a DIG nucleic acid detection kit (Boehringer Mannheim). RESULTS The primers SIA1 and SIA2 were designed to hybridize to the A. viscosus T14V nanH and amplify a DNA fragment with a predicted size of 756-bp (Table 1). Primers RRN4 and RRN5 recognized the highly conserved regions of the eubacterial 16S rDNA (19). The RRN-specific amplification predicts the produc-

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DETECTION OF ACTINOMYCES SPECIES BY PCR

TABLE 1 Primers for PCR Primersa

Sequence

Size

Locationb

Directionc

SIA1 SIA2 RRN4 RRN5

5*-CGGCGATGTCATGACCTT-3* 5*-ATGCGGTAGTTGTCGGTG-3* 5*-CAGGATTAGATACCCTGGTAGTCCACGC-3* 5*-GACGGGCGGTGTGTACAAGGCCCGGGAACG-3*

18-mer 18-mer 28-mer 30-mer

555–572 1293–1310 783–810 1378–1407

Forward Reverse Forward Reverse

Amplified fragment size 756 bp 625 bp

a

SIA1–SIA2 and RRN4–RRN5 are primer pairs. Positions of the first and last nucleic acids of the primer in the targeted fragment. Nucleotide sequence of sialidase gene obtained from Genbank (L06898). RRN4 and RRN5 were designed as described by Goncharoff et al. (19). c Direction relative to the coding strands of target genes. b

tion of the 625-bp DNA fragment in all eubacteria which provides an internal positive control for PCR when testing for the presence of nanH-specific sequences. To assess the availability of these four primers, we first carried out simple PCR using chromosomal DNAs isolated from various oral bacteria as templates. As shown in Fig. 1, PCR products from A. viscosus T14V yielded two major bands corresponding to the 756-bp nanH-specific fragment and 625-bp 16s rDNA-specific fragment (A, lane 2). Four other A. viscosus strains and A. naeslundii ATCC 12104 also yielded a 756-bp fragment (Fig. 1A, lane 1, and lanes 3 through 5, and Fig. 1B, lane 1). PCR amplification of Gram-positive microorganisms other than the Actinomyces species (Fig. 1B, lanes 2 through 5) and all Gram-negative bacteria (Fig. 1C) yielded only the latter fragment. These results indicated that the amplified region is specific for the eubacterial 16s rDNA. Using the A. viscosus nanH-specific riboprobe previously prepared from the plasmid pMDAv2 containing a portion of the nanH gene from A. viscosus

ATCC 19246 (15), we next attempted to identify PCR-generated products from five A. viscosus strains and A. naeslundii ATCC 12104 along with various representative oral bacteria. The PCR-amplified DNAs were immobilized on a nylon membrane, followed by hybridization with the digoxigenin-labeled A. viscosus nanH-specific riboprobe. As shown in Fig. 2, all PCR-amplified products from five A. viscosus strains hybridized with the nanHspecific riboprobe (Fig. 2A). PCR product from A. naeslundii ATCC 12104 DNA also hybridized with the riboprobe (Fig. 2B, lane 1). No other Gram-positive or Gram-negative microorganisms examined exhibited detectable positive signals (Fig. 2B, lanes 2 through 5, and Fig. 2C, lanes 1 through 5). Finally, to determine the amount of template necessary for detection, chromosomal DNA of A. viscosus ATCC 19246 was serially diluted with PCR buffer and amplified. Aliquots of PCR products were dot-blotted onto a nylon membrane and hybridized with the riboprobe as described above. As shown in Fig. 3, 1003 mg of chromosomal DNA seemed to be

FIG. 1. PCR amplification of nanH-specific and 16s rDNAspecific sequences from various representative oral microorganisms. PCR products from reactions containing primers SIA 1, SIA2, RRN4, and RRN5 are shown separated on a 1.5% agarose gel. The numbers to the left of the gel indicated DNA fragment sizes in bp. (A) Lane 1, A. viscosus ATCC 19246; 2, A. viscosus T14V; 3, A. viscosus T14AV; 4, A. viscosus 11132; 5, A. viscosus 13236. (B) Lane 1, A. naeslundii ATCC 12104; 2, S. mutans GS5; 3, S. gordonii Challis; 4, S. milleri Is57; 5, L. casei 4649. (C) Lane 1, P. gingivalis FDC 381; 2, P. endodontalis ATCC 35406; 3, P. loescheii ATCC 15930; 4, A. actinomycetemcomitans Y4; 5, C. rectus ATCC 33238.

FIG. 2. Specificity of PCR-amplified products analyzed by dotblot hybridization. (Row A) Lane 1, A. viscosus ATCC 19246; 2, A. viscosus T14V; 3, A. viscosus T14AV; 4, A. viscosus 11132; 5, A. viscosus 13236. (Row B) Lane 1, A. naeslundii ATCC 12104; 2, S. mutans GS-5; 3, S. gordonii Challis; 4, S. milleri Is57; 5, L. casei 4649. (Row C) Lane 1, P. gingivalis FDC 381; 2, P. endodontalis ATCC 35406; 3, P. loescheii ATCC 15930; 4, A. actinomycetemcomitans Y4; 5, C. rectus ATCC 33238.

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FIG. 3. Comparison of sensitivity of the riboprobe. Target DNA for DNA–RNA hybridization analysis without (lane 1) or with (lane 2) PCR amplification. The amounts of DNA used as template in each sample are shown to the left of the lane.

sufficient to detect the presence of the nanH using this riboprobe system. DISCUSSION Sialidase from a number of microorganisms has been characterized, and more recently, the genes that encode several of these proteins were cloned and their nucleotide sequences were determined (20–22). Comparison of the predicted amino acid sequences of the sialidase from various bacteria revealed the presence of the short amino acid sequence referred to as the ‘‘Asp box’’ (23). Based on the nucleotide sequence of the A. viscosus T14V sialidase gene, five 12-amino-acid stretches containing this motif have been localized within the central domain of the predicted protein (11). In addition, significant sequence homology has been observed by comparing the predicted amino acid sequences of the sialidase from A. viscosus T14V (11) and that from A. viscosus DSM 47798 (22). Since the enzyme sialidase is widely distributed among bacteria, it is likely that certain microorganisms other than Actinomyces species might possess these conserved regions. Therefore, this region (from nucleotide position 1423 to nucleotide position 2202; see Ref. 11) was excluded form the target sequence for amplification, and the 756-bp fragment (555 to 1301) close to the domain specifying the signal sequence was chosen for amplification as the Actinomyces species-specific region. The present study indicated that the SIA primers can accurately identify the nanH-specific sequence resident in the A. viscosus T14V chromosome. These

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findings also demonstrated that all of the A. viscosus strains tested, including some clinical isolates, contained nanH sequences. Furthermore, analysis of PCR-amplification product (Fig. 1B, lane 1) and dotblot hybridization analysis using the nanH-specific riboprobe (Fig. 2B, lane 1) suggested that some Actinomyces species, at least A. naeslundii ATCC 12104, might have the nanH. This is consistent with previous results (11), which showed the presence of the sialidase gene in the genomes of 18 strains of five Actinomyces species, including A. naeslundii, indicating that the sialidase genes are highly conserved among divergent groups of Actinomyces species. Detection of periodontal pathogens is crucial for the diagnosis and treatment of periodontal diseases. A number of diagnostic methods have been developed such as microbiological assays using anaerobic culture techniques, direct microscopic evaluation, enzyme-linked immunoassays, and DNA probe hybridization. Among these, DNA probe analysis is reliable because it utilizes only the homologous region of DNA sequences irrespective of the viability of bacteria. On the other hand, it is possible to use whole chromosomal DNA as a probe to detect the target microorganism (24). This method provides a high degree of sensitivity but it is likely to result in nonspecific hybridization leading to misidentification. Conversely, if DNA fragments from specific regions are used as probes, there seems to be a tendency for specific hybridization to the target sequence with relatively low sensitivity (25). PCR allows the amplification and identification of specific genes using only trace amounts of template DNA. It is thus an ideal system for the identification of microorganisms when strain-specific regions are utilized for amplification. However, both nonspecific amplification and sensitivity limitations in visualization of DNA fragments by staining with ethidium bromide make it difficult to identify the presence of certain bacteria. Therefore, we developed a novel method by combining use of digoxigenin-labeled riboprobes and PCR amplification. We conclude that the application of this region using the riboprobe system coupled with PCR might facilitate identification of Actinomyces species in the inactive sites during periods of remission in periodontal disease. ACKNOWLEDGMENTS This work was supported in part by a grant-in-aid for Developmental Scientific Research from the Ministry of Education, Sci-

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DETECTION OF ACTINOMYCES SPECIES BY PCR ence, and Culture of Japan (05557086) and by a research grant from Tuchiya Foundation. This study was also partly supported by Funds for Comprehensive Research on Aging and Health (93A2311) by the Ministry of Health and Welfare of Japan.

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