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Survey of Oral Microbial Diversity using PCR-based Denaturing Gradient Gel Electrophoresis
Y. Li1,*,
C.Y.S. Ku1,
J. Xu2,
D. Saxena3 and
P.W. Caufield3
1 Department of Basic Science and Craniofacial Biology and
3 Division of Diagnostics, Infectious Disease and Heath Promotion, New York University College of Dentistry, 345 E. 24th Street, New York, NY 10010-4086, USA; and
2 Department of Pediatrics, New York University School of Medicine, New York, NY 10016, USA;
Correspondence: * corresponding author, yihong.li{at}nyu.edu
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ABSTRACT
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Polymicrobial biofilms in the human oral cavity exhibit marked diversity. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) surveys microbial diversity by displaying PCR-generated 16S rDNA fragments that migrate at different distances, reflecting the differences in the base-pair (i.e., % G+C) composition of the fragment. This study examined DGGE-generated diversity profiles of cultivable bacteria from individuals with different caries status. Initially, we developed a set of PCR-DGGE running conditions appropriate to oral bacteria. Next, we assessed migration standards from known oral bacterial reference strains. To test the methods, we profiled 20 bacterial saliva samples cultivated from young adults. The study produced a battery of species-specific 16S rDNA amplicons that could be used as a migration distance standard necessary for computer-assisted profile analysis. From the clinical samples, we found a significantly greater diversity of oral microbes in caries-free individuals compared with caries-active individuals (P = 0.01). These findings suggest thtat a portion of oral microbiota of caries-active individuals may be absent, suppressed, or replaced.
Key Words: microbial diversity • PCR-DGGE • 16S rDNA • dental caries
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INTRODUCTION
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Dental caries is most likely the result of a polymicrobial infection caused by one or more of the over 500 bacterial species that have been identified from the human oral cavity (Moore et al., 1985; Paster et al., 2001). It seems unreasonable, however, to reduce the suspected causative agents to only one or a few species. Previously, studies of micro-organisms associated with caries relied heavily upon cultivation methods. However, the culture method does not allow for the overall bacterial population to be determined, particularly those which cultivation fails to capture (Choi et al., 1994; Kroes et al., 1999; Staley and Gosink, 1999). Thus, the set of bacterial species identified from culture media is not a true representation of the microbial composition of saliva or dental plaque. Because of these limitations, it is also difficult to acquire a complete understanding of the dynamic changes of oral microbes as dental caries progresses.
Alternative molecular techniques—such as polymerase chain-reaction-based denaturing gradient gel electrophoresis (PCR-DGGE)—are capable of surveying entire bacterial communities without cultivation (Muyzer et al., 1993). This method takes advantage of the ubiquity of the 16S rRNA locus in the microbial world, which can be amplified by PCR and sequenced with a set of universal bacterial primers. The elegance of DGGE is that it allows the diverse PCR-amplified gene products of similar lengths, but with different sequences, to be separated in a denaturing gradient gel. The differentiation of bacterial species is based on their differential migration in the gel as a function of percent of guanine plus cytosine (G+C content) and melting behavior. The result is a bar-code-like profile, with each band presumably representing a different micro-organism within the microbial communities (Muyzer et al., 1993; Muyzer, 1999; Bonin et al., 2002). To date, this technique has become an important tool for studying complex bacterial communities in a variety of habitats, including environmental biofilms, food fermentation processes, gastrointestinal tract infections, and periodontal pockets (Zoetendal et al., 1998; Bonin et al., 2002; Peixoto et al., 2002; Meroth et al., 2003; Zijnge et al., 2003). However, a PCR-DGGE diversity profile of bacteria in saliva associated with dental caries status has not yet been reported. Accordingly, we undertook a feasibility study using PCR-DGGE first, to survey the total cultivable microbial diversity in the saliva, followed by a comparative pilot study examining the diversity profiles in subjects with or without dental caries. Ultimately, we will apply this cultivation-independent approach to determine the microbial diversity in saliva and dental plaque for caries risk assessment and for studying caries etiology.
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MATERIALS & METHODS
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Clinical Sample Collection
This study was approved by the Institutional Review Board of the University of Alabama at Birmingham and the New York University Committee on Activities Involving Human Subjects. Twenty healthy women, from 18 to 22 yrs old, were randomly selected from a Birmingham mother-child cohort study (Li and Caufield, 1995; Li et al., 2003). Written informed consent was obtained at the initial clinical visit after the study protocol was explained to each subject. Stimulated whole salivary samples (2–5 mL) were obtained; a 100-µL quantity of 1/1000 and 1/10,000 diluted saliva samples was plated on the selective medium, mitis-salivarius with bacitracin (MSB) (Gold et al., 1973), and the non-selective MM10 sucrose blood agar plates (MM10) (Syed and Loesche, 1973), respectively, with the Spiral Autoplate® 4000 (Spiral Biotech, Inc., Bethesda, MD, USA). The individual’s caries status was measured by DMFT/DMFS indices (decayed, missing, and filled teeth/tooth surfaces) according to the criteria defined by the National Institute of Dental and Craniofacial Research for caries diagnosis and recording (USDHHS, 1989). ‘Caries-active’ (N = 11) was defined as an individual who had at least one untreated decayed tooth surface (D score greater than or equal to 1). ‘Caries-free’ (N = 9) was defined as an individual who had a DMFS score equal to zero.
Total Cultivable Bacterial Genomic DNA Isolations
After a 72-hour incubation at 37°C in an anaerobic atmosphere of 85% N2, 10% H2, and 5% CO2, the levels of total cultivable bacteria and S. mutans in the saliva were determined by the enumeration of colony-forming units (CFU) on MM10 and MSB media, respectively. All colonies on the MM10 plate were collected with a cotton swab, washed in 1 mL of 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA (TE) buffer, and then saved at –80°C. In this study, the bacterial samples were dissolved at 4°C. Total genomic DNA of each bacterial sample was isolated by means of the MaterPureTM DNA purification kit (EPICENTRE, Madison, WI, USA), following a phenol/chloroform/isoamyl alcohol extraction (Zoetendal et al., 1998). DNA quality and quantity were measured by a UV spectrophotometer at 260 nm and 280 nm (DU®640, Beckman Instruments, Inc., Fullerton, CA, USA). The DNA samples were used for DGGE analysis.
Bacterial Strains and DNA Isolation
A set of 25 ATCC type strains originating from the oral cavities of humans (except ATCC 19645, S. ratti) was selected based on the known G+C content of their genomic DNA. The strains represented a variety of oral bacterial species with different G+C contents, ranging from 27% to 71% (Table 1 ). The genomic DNA of those strains was obtained from pure cultures by use of the Qiagen genomic DNA purification kit (Qiagen, Hilden, Germany). The DNA samples were used for the initial assessment of the migration behavior of their 16S rDNA PCR amplicons in the presence of a denaturing gradient.
PCR Assay
PCR was performed with use of the GeneAmp PCR SystemTM 9700 (PE Applied Biosystems, Foster, CA, USA). A set of universal bacterial 16S rDNA primers (Bac1 and Bac2) (Rupf et al., 1999) was used to generate an approximately 300-bp amplicon. A 40-nucleotide GC-clamp was added to the 5' end of Bac1 (Sheffield et al., 1989). Each PCR reaction mixture (a total volume of 50 µL) contained: a standardized 100 ng of the total genomic DNA; 200 µM of each dNTP; 40 pmol of each primer; 4.0 mM MgCl2; 5 µL of 10X PCR buffer II; and 2.5 U of Taq DNA polymerase (PE Applied Biosystems, Foster, CA, USA). PCR conditions were as follows: initial denaturation at 94°C for 3 min, and 35 cycles consisting of 1 min at 94°C, 1 min at 56°C, 2 min at 72°C, and an additional cycle of 5 min at 72°C for chain elongation. The PCR products were evaluated by electrophoresis in 1.0% agarose gels run at 100V for 60 min.
DGGE Profile of the Type Strains
To generate an initial approximation for predicting migration distances in DGGE gels, we retrieved, from GenBank, the gene sequences of the 16S rDNA of the 25 ATCC strains (Table 1 ). Based on their sequences, we calculated the melting profile of each strain, using the WinMeltTM program (Bio-Rad Laboratories, Hercules, CA, USA). DGGE was performed by use of the Bio-Rad DCode System (Hercules, CA, USA) for all DGGE experiments. A 30% to 70% linear DNA denaturing gradient (100% denaturant is equivalent to 7 mol/L urea and 40% de-ionized formamide) was formed in 8% (w/v) polyacrylamide gels. PCR products were directly loaded in each lane, and electrophoresis was performed at a constant 60 V at 58°C for 16 hrs in 1X Tris-acetate-EDTA (TAE), pH 8.5, buffer. After electrophoresis, the gels were rinsed and stained for 15 min in water containing 0.5 µg/mL ethidium bromide, followed by 15 min of de-staining in water. DGGE profile images were digitally captured and recorded by means of the AlphaImagerTM 3300 System (Alpha Innotech Corporation, San Leandro, CA, USA). To confirm the species affiliations of the 16S rDNA amplicons, we excised PCR fragments from the DGGE gel, eluted them by electrophoresis with dialysis tubing, and recovered them with the Ultrafree®-MC filter unit (Millipore, Bedford, MA, USA). The recovered DNA samples were re-amplified with the universal primers minus the GC-clamp. The PCR products were then purified and sequenced for confirmation of their identities. Their phylogenetic affiliation and similarity to archived 16S rDNA sequences were determined with the use of a Web-based program, Ribosomal Database Project II (RDP) (Maidak et al., 1999).
Analysis of DGGE Profile from Clinical Samples
The same set of universal primers and PCR conditions were applied to the 20 genomic DNA samples purified from the MM10 medium. The characterization of the total cultivable microbes presenting in the saliva was analyzed based on PCR-generated profiles of the 16S rDNA fragments on DGGE gels. We used the Diversity Fingerprint Software (BioRad) to assess the diversity by comparing the number of DGGE bands detected per lane and the relative distribution of DGGE bands according to the DGGE reference markers created in this study. We measured the degree of intensity of the detected bands to estimate the relative quantity of each band on the DGGE gels. Marked differences in the DGGE profiles of caries-active vs. caries-free individuals indicated the presence or absence of bacterial species in the MM10 culture samples. The differences were analyzed by the non-parametric Mann-Whitney test.
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RESULTS
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The primers used in this study produced a 320-bp PCR amplicon from the 16S rDNA locus for all type strains (Fig. 1A). These amplicons, however, exhibited different migrating behaviors in the presence of a denaturing gradient on the DGGE gel, despite being indistinguishable on a non-denaturing gel (Fig. 1B). Sequencing analysis of each fragment excised from the DGGE gel indicated that their migration behavior was directly correlated to their G+C content. The RDP II search of their sequences revealed identical matches between the retrieved DGGE fragments and the 16S rDNA of the original type strains (data not shown). Of the 25 ATCC type strains surveyed, we selected 10 that yielded distinct and well-delineated migrating patterns for inclusion in the migration standard (Fig. 2A, Lane R). These 10 reference markers were used for all of the DGGE experiments.
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Figure 1. PCR-DGGE analysis of 16S rDNA gene fragments of ATCC type strains. (A) The PCR-amplified 16S rDNA of the ATCC type-strains shows the same 350-bp amplicons for various oral bacteria on a 1% agarose gel. (B) Denaturing gradient gel electrophoresis profiles of PCR-amplified 16S rDNA of various oral bacteria arranged from low to high %GC content. The denaturing gradient ranged from 30% to 70%.
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Figure 2. PCR-DGGE profiles and profile analysis of 16S rDNA gene fragments of clinical samples. (A) PCR-DGGE profiles of oral bacterial 16S rDNA from the total cultivated bacteria of 20 clinical patients with different caries status. The number of detected and dominant fragments is more pronounced in Lanes 1 to 9 (caries-free individuals) than in Lanes 10 to 20 (caries-active individuals). Lane R consists of the DGGE reference markers created according to the %GC content of the 16S rDNA amplicons and their migrating behaviors. 1, F. nucleatum vincentii (ATCC49256); 2, F. nucleatum nucleatum (ATCC25586); 3, S. sanguinis (ATCC10556); 4, S. oralis (ATCC35037); 5, S. salivarius (ATCC7073); 6, S. mutans (ATCC700610); 7, L. paracasei subsp (ATCC25598); 8, P. gingivalis (ATCC33277); 9, A. odontolyticus (ATCC17929); and 10, A. naeslundii genospecies-1 (ATCC12104). (B) A standard graph generated by the Diversity Database software for comparison of the relative distributions and the intensity profiles of multiple lanes from the DGGE image. The X-axis of the graph is the retention factor (Rf) value (from 0.0 to 1.0), and the Y-axis is the pixel intensity value at each point along the lane. The graph demonstrates the differences in the distribution and the degree of intensity of the detected bands representing S. mutans between a caries-active (CA #12) and a caries-free (CF #6) individual.
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Based on conventional culturing methods, the study found that the total cultivable bacterial counts were significantly higher in the caries-free group (independent t test; P = 0.013) (Table 2). The total number of detectable bands on the DGGE gels was also significantly higher in the caries-free individuals compared with the caries-active individuals (53 vs. 33; P = 0.018). The higher counts corresponded to the higher number of amplicons displayed on the DGGE gels (Fig 2A). Overall, caries-free individuals exhibited a greater diversity in the overall cultivable microbial population than did caries-active individuals.
Moreover, the study also observed differences in the relative distribution of DGGE profiles and in the intensity profiles of S. mutans amplicons presented in DGGE gels for caries-active individuals compared with caries-free individuals (Fig. 2B). Although S. mutans was detected in 19 of the 20 subjects (95%), there was no significant difference in the S. mutans levels between the two groups. Interestingly, there was as high as a five-fold difference in the relative intensities of the detected amplicons representing S. mutans between the two groups. The ratio of the number of detected S. mutans fragments to the total detected fragments in the DGGE was higher for caries-active individuals than for caries-free individuals (non-parametric Mann-Whitney test; P = 0.008) (Table 2).
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DISCUSSION
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DGGE was originally developed in the 1980s to detect single mutations in genomic DNA (Borresen et al., 1988; Cariello et al., 1988; Sheffield et al., 1989). Recently, microbial ecologists applied this technique for analyzing whole bacterial communities and for studying the microbial diversity in various bacterial populations. The advantages of DGGE include its ability to amplify regions of 16S rDNA directly from environmental samples, without cultivation of the samples (Muyzer et al., 1993; Santegoeds et al., 1996; Zoetendal et al., 1998; Bonin et al., 2002; Peixoto et al., 2002; Randazzo et al., 2002; Meroth et al., 2003). Thus, more bacterial species, particularly those of strict anaerobes and non-cultivable microbes, can be detected, and their sequences can be determined and compared with various DNA databases, such as the RDP II.
Here, we present a study using the DGGE of PCR-amplified 16S rDNA fragments to examine bacterial diversity cultivated from human saliva and its association with dental caries. A greater diversity of bacterial populations was observed in caries-free compared with caries-active individuals. This result corresponds to the findings achieved by conventional culturing methods, in which total cultivable bacterial levels are significantly higher in the caries-free group. Although the study detected a maximum of 53 PCR amplicons on the DGGE gels from any given sample, we cannot conclude that only 53 bacterial species are present in the saliva, because: (1) unrelated bacterial species may have similar or identical migration distances (Muyzer, 1999); (2) two or more phylogenetically related bacterial species may display close or overlapping band positions on the DGGE gel (Hayes et al., 1999); (3) levels of certain microbes in the sample may have been at or below detectable levels for PCR; or (4) although over 500 bacterial species have been identified in the human oral cavity (Moore et al., 1985; Paster et al., 2001), not all the species have been detected in a single individual’s oral cavity. In fact, only a substantially small group of bacterial species may be associated with dental caries (Becker et al., 2002). Furthermore, bacterial samples used for this study were obtained from the MM10 medium, a partially selective medium that may not reflect all of the possible cultivable bacterial species in the saliva. The 53 amplicons most likely represent the predominant bacterial species obtained from caries-free individuals. In contrast, only 33 amplicons were detected from caries-active individuals. This suggests a significant difference in the composition of microbial communities between the two groups. We hypothesized that a greater diversity of indigenous bacteria inhabits a caries-free environment, and that a significant proportion of oral biota may be absent, suppressed, or replaced in a caries-prone environment. Because PCR-DGGE can detect bacterial species within a wide spectrum of G+C content, it can be a powerful tool, not only for determining the presence or absence of cariogenic organisms such as S. mutans in the oral cavity, but also for evaluating total microbial diversity, and the presence of other oral microbes that might contribute to caries.
One limitation of the DGGE technique is that the analyses of the high-quality diversity patterns are restricted to a visual comparison and interpretation. Most studies report the relative position of each amplicon (de Medeiros Muniz et al., 2001), while others provide an estimated denaturing percentage for comparisons (Kowalchuk et al., 1997). Only a few computer programs have the ability to acquire DGGE gel images, transfer the images to specially designed analytical software, and record the banding patterns. To complete an analysis, these programs require a standardized marker present with each gel. Because the principles of obtaining DGGE diversity profiles are based on the DNA melting domains of each 16S rDNA amplicon, as a function of the G+C content, we developed a reference standard containing a set of predominant oral bacterial species with G+C content ranging from 27% to 70%. Based on this standard, the Diversity Database Fingerprinting program not only detected the presence or absence of a particular band and reported band frequency comparing multiple gels, it also provided an estimated G+C content and relative densities of the PCR fragments, and generated a similarity index for paired comparison. Consequently, we were able to complete the profile comparisons between caries-active and caries-free individuals. The use of computer-assisted data analysis provides a significant advancement for an objective and unambiguous interpretation of the association between the microbial community and oral health. Currently, identification of DGGE amplicons is ascertained by extraction from gels, cloning, and sequencing, followed by searching for similarity with known bacterial 16S rDNAs in the RDP II (Muyzer et al., 1995; Nielsen et al., 1999; Casamayor et al., 2000; Favier et al., 2002). This approach, however, is laborious. Reference markers of known G+C content not only allow for standardization of gels, but also provide an initial approximation of G+C content and species identification. Further exploration of the sensitivity and specificity of this novel method for rapid and accurate determination of microbial diversity in the oral cavity is under way.
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ACKNOWLEDGMENTS
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This study was supported by the New York University College of Dentistry Dean’s Student Research Award and the USPHS Research Grants P50 DE11147 and DE13937 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892. The authors thank to Dr. John Ruby, an Associate Professor at the University of Alabama at Birmingham, for his invaluable assistance during the start of the project, and Ms. Janice Wu for proofreading this manuscript.
Received for publication April 9, 2004.
Revision received January 13, 2005.
Accepted for publication February 24, 2005.
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Journal of Dental Research, Vol. 84, No. 6,
559-564 (2005)
DOI: 10.1177/154405910508400614
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ABSTRACT
INTRODUCTION