ISSN 1996-0808 ©2012 Academic Journals
Full Length Research Paper
Microbial profiling of dental caries and periodontitis patients using denaturing gradient gel electrophoresis
Ayaz Ahmed
1, Jianjun Liu
1, Yousuf Moosa
2, Li Tang
3, Shizhu Zang
1and Yi Xin
1*
1Department of Biotechnology, Dalian Medical University, Dalian, P. R. China.
2First affiliated Hospital of Dalian Medical University, Dalian, P. R. China.
3Department of Microecology, Dalian Medical University, Dalian, P.R. China.
Accepted 08 February, 2012
Oral cavity contains a diverse group of microorganisms which changes from normal to diseased condition like dental caries and periodontitis. It is hard to depict the complete picture of microbial environment as some species are uncultivable. In this study we detected the microbial diversity of normal, dental caries and periodontal group by using culture independent PCR based denaturing gradient gel electrophoresis (DGGE). DNA was extracted from plaque samples from patients, and PCR was conducted for the 16s rRNA gene with universal primer, followed by DGGE, excision of bands, cloning and sequencing. It was determined that the number of bands detected in normal group (17.06 ± 4.34) was more than dental caries (12.73 ± 3.2) and periodontitis group (10.36 ± 3.5). On clustering they formed mixed clusters. Sequencing results showed that dental caries group contained large proportion of Gram positive bacteria, while periodontitis group contained mostly Gram negative bacteria. Diversity in dental caries and periodontitis groups were less as compared to normal group which suggested that the presence of only cariogenic bacteria and those who tolerate low pH contributes in dental caries and some typical bacteria responsible for causing periodontitis. DGGE is the effective tool to dissect any microbial niche in the body.
Key words: Dental caries, periodontitis, denaturing gradient gel electrophoresis (DGGE), microbial diversity.
INTRODUCTION
Metagenomics allow us to know that microorganisms present within human body are ten times more than the cells in the body and contains in aggregate of 100 times more genes (Gill et al., 2006). Most of the micro- organisms play an important role in human physiological processes while some cause disease. Oral cavity is one of the most complex microbial habitats in the human body that comprises of more than 600 diverse arrays of bacterial species (Kazor et al., 2003). More than 250 oral microbes have been isolated and characterized by the cultivation method but over 450 microbial species have
*Corresponding author. E-mail: jimxin@hotmail.com.
been identified by the culture independent molecular methods (Aas et al., 2008a; Paster et al., 2006). Oral microbiota beside its importance in oral health reflects the health and disease status of the host as it effects many other systemic diseases like bacterial endocarditis, pneumonia, preterm low birth weight and coronary heart disease (Beck et al., 2005a; 2005b; Offenbacher et al., 2006; Paju and Scannapieco, 2007).
Among many oral diseases dental caries and periodontitis are common diseases. Dental caries is characterized by the acid demineralization of the tooth enamel followed by damage to the hard tooth structure, which results in the formation of cavities on tooth surface.
Mutans group Streptococci are closely associated with the caries formation and frequently isolated from dental
caries sites (van Houte, 1994). In the light of ecological plaque hypothesis, dental plaque of healthy individual contains balanced ratio of bacterial community (Marsh, 2003b). On occasion, when balance breaks down, results in significant increase of pathogenic bacteria such as acid producing and aciduric bacteria, which can metabolize dietary sugars to produce acids resulting in pH reduction and enamel dimineralization (Marsh, 1999a; Sbordone and Bortolaia, 2003). Periodontal disease is an inflammatory processes resulting in the destruction of the supporting tissue of the teeth which is a result of mixed microbial infection (Haffajee and Socransky, 1994).
Periodontitis is a progressive disease that affects all supporting tissues of the teeth. Pathogens colonize the subgingival sites, undetected by host’s defense mechanism leading to damage to the whole tooth structure (Houle and Grenier, 2003).
The complete picture of polymicrobial community such as plaque is hard to depict because it consists of numerous different bacterial species amongst them some are uncultivable (Aas et al., 2005b). There are many emerging techniques which focus on 16S rDNA sequences, which broadened the scope of detecting uncultivable bacteria from the oral cavity (Aas et al., 2008a; Aas et al., 2005b; Becker et al., 2002; Munson et al., 2004). Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) is one of them which is useful for analyzing the bacterial diversity profile in different disease conditions (Ledder et al., 2007; Li et al., 2007a; Li et al., 2006b; Liu et al., 2010; Machado de Oliveira et al., 2007). PCR-DGGE allows the analysis of different samples and provides bar coded profiles of microbial communities, in which each bands reflects the community members. Another feature of PCR-DGGE is that the bands can be cut and sequenced to get taxonomic identity (Muyzer and Smalla, 1998b).
The objective of this study was to characterize the microbial diversity profile of patients with dental caries and periodontal disease by PCR-DGGE.
MATERIALS AND METHODS Study design
Fifty patients were included in this study who attended the dental clinic of the first affiliated hospital of Dalian Medical University.
Among them 20 patients had active dental caries, 20 patients had periodontal problems whereas 10 people were normal without any signs of caries lesion. Written informed consents were obtained from the patients. This study was approved by the ethical committee of Dalian Medical University. Dental Caries was characterized with the presence of a lesion, cavity, undermined enamel, decayed softened floor or walls according to the criteria described by the World Health Organization (1997). Periodontal lesions were recorded by measuring the probing pocket depth and probing attachment level by periodontal probes calibrated in millimeters, and bleeding on probing (Lang et al., 1996). All participants were instructed not to brush their teeth in the morning before sampling. Patients who were using antibiotics or have some
other bacterial or viral infections were excluded.
Sample collection
Supragingival plaque samples from dental caries patients and subgingival plaque samples from Periodontitis patients were collected by scrapping the sterile metal loop on the lesions. Sample material was collected in 1ml sterile saline solution and immediately transferred to the lab in an ice container for further processing.
They were stored in -80°C freezer until further use.
DNA isolation
Plaque samples were centrifuged at 10000 × g for 1 min to pallet bacteria. DNA extraction was performed by using the QIAamp DNA Mini Kit (QIAGEN) according to manufacturer protocol. Concen- tration and integrity of the nucleic acids were determined visually by electrophoresis on 1% agarose gel (Invitrogen) containing ethidium bromide, and Bio-Photometer plus (NanoVue) used to determine the exact DNA concentration.
Polymerase chain reaction on 16Sr RNA genes
The V3 region of the 16S rRNA genes were amplified by using universal bacterial primers 341F and 534R as described by Muyzer et al. (1993a). Each PCR mixture (a total of 50 µl) contained 20 pmol of each primer, 20 mM of d NTP mixture, 5 µl 10× Ex Taq buffer (with Mg2+), 5 µl 1% BSA, 2.5 U of EX Taq DNA polymerase (TakaRa, Japan), and 200 ng of DNA template. PCR amplification was performed in an automated thermocycler (Thermo USA) with following program, 94°C for 5 min; followed by 30 cycles of 94°C for 30 s, 54°C for 30 s, 72°C for 30 s and final extension for 72°C for 7 min. The PCR products were evaluated by electrophoresis in 2%
agarose gel containing ethidium bromide at 100V for 45 min. Gels were visualized on UV transilluminator (Jim-X Scientific, China).
Denaturing gradient gel electrophoresis (DGGE)
DGGE was performed on BIO-RAD Dcode System (Hercules, CA, USA). Briefly, samples were loaded on 8% polyacrylamide gel containing 35 to 65% gradient of urea and formamide. The samples were electrophoresed at 200 V for 10 min followed by constant voltage at 85 V for 8 h with a stable temperature of 60°C. The gels were stained with 0.5 µg/ml ethidium bromide solution for 60 min.
Gels were visualized with BIO-RAD Gel Documentation System (Hercules, CA, USA).
DGGE gel analysis
The difference in microbial diversity was evaluated by comparing the DGGE profile among the defined groups. The degree of correla- tion in above stated diseases was determined by nonparametric Mann Whitney U test. The analysis was performed using SPSS software version 11.5. The similarity score and cluster analysis of DGGE profiles were performed using the Ward’s method based on the Dice similarity coefficient by using phoretix 1D Pro Multiple Gel Dendrogram (Totallab, UK) which generated a similarity matrix between DGGE profiles. The Shannon–Weaver index of diversity (H’) was used to determine species richness i.e the number of different distinct bands in any individual sample, and evenness in samples (Silva and Russo, 2000).
Sequence analysis
30 bands were excised from the gel (12 bands from dental caries group, 9 from periodontitis group and 9 from normal group) and sequenced (Figure 3). On the basis of gray intensity and clarity bands were excised from the gel with the sterile scalpel in 0.5 ml Eppendorf tube, washed with deionized water, and incubated in 35 µl TE buffer at 4°C overnight. Four microliter of this fragment served as a template for PCR re-amplification with the same set of primer and program stated above but without GC clamp. The PCR product was electrophoresed on 2% agarose gel, purified and then cloned into the PMD18-T Easy vector (TaKaRa, Japan), transformed into Competent E. coli Nova blue cells, and screened for positive plasmid insertions according to the manufacturer protocol. 2 to 3 clones per band were selected and sequenced. Plasmid DNA was extracted from positive clones and sequenced using ABI PRISM cycle sequencing kit (Applied Biosystem, USA) and Universal primer. Taxonomic affiliations of the obtained sequences were determined by using Human Oral Microbiome Database (HOMD).
RESULTS
DGGE banding patterns
Each band in DGGE profile showed the presence of single or more than one bacterial species in sample.
Samples from 20 periodontitis patients, 20 dental caries patients and 10 normal people (n = 50) were analyzed in this study. DGGE profiles which were obtained from the plaque samples of disease and normal group are shown in Figure 1. It is clearly observed that number of bands detected in periodontitis groups were less than dental caries and normal groups. Total numbers of bands detected in periodontitis groups were 10.36 ± 3.5, in dental caries groups were 12.73 ± 3.2, and in normal individuals were 17.06 ± 4.34. Number of bands were rich in normal group as compared to the other two groups and was statistically significant with the p value of 0.002, 0.001 among diseased and normal group, respectively.
The diversity of bacteria in periodontitis group and dental caries group (Table 1) were statistically significant with P value 0.002, the diversity between normal group and dental caries group were statistically significant with P value 0.004, while no significant difference was observed among the diversities of normal and periodontitis groups.
Cluster analysis
Cluster analysis was performed by Ward's analysis in which Dice coefficient for measuring similarity algorithm in banding pattern was applied. This method provides further understanding to analyze the data. Figure 2 illustrates that two clusters were formed. Cluster 1 mostly contains periodontitis group, with a sub cluster containing samples from all three groups, while in cluster 2 there are 3 sub clusters, sub cluster 1 contain normal group, sub cluster 2 contains dental caries group while sub cluster 3
contains samples from all three groups. Samples from the three groups were failed to form significant different clusters.
Sequence analysis
Only the intense or clear bands were cut (Figure 3). In order to certify the resolution capability of DGGE, bands in the same position but in different lanes were also selected. Twenty three genera were identified from the 80 sequences as shown in Table 2. Dental caries samples contains predominantly Gram positive bacterial species then Gram negative bacteria, Periodontitis group contains mostly Gram negative bacteria and normal group contains Mixed proportion of Gram negative and Gram positive bacterial species. Interestingly, we identified three genera of uncultivable bacteria including Kocuria species, Clostridiales (F-2) (G-1) sp. and TM7 (G-1) sp.
Most of the genera identified were common in all three groups except Porphyromonas gingivalis which was only present in periodontitis group and Dialister invisus was only present in normal group.
DISCUSSION
In this study microbial etiology of dental caries and periodontitis disease were detected by using PCR- DGGE. We demonstrated that a total of 23 genera of bacteria including three uncultured bacterial species were present. Our results indicated that Streptococcus spp., Neisseria spp., Haemophilus spp., Veillonella spp.
contribute in the microbiota of dental caries whereas Porphyromonas spp., Corynebacterium spp., Capnocytophaga spp. and Actinomyces spp. constituted the major microbiota in case periodontitis. In agreement with previous report, we observed increased bacterial diversity in normal people as compared to dental caries and periodontitis group (Li et al., 2006b). The results also verify the ecological plaque hypothesis which states that disease results after imbalance in the normal microflora.
It is already known that cariogenic bacteria can be present in healthy oral cavity but at less clinically signi- ficant levels. Sometime a substantial change in a habitat results in breakdown of the microbial balance which leads to disease condition or community dominance (Wei et al., 2011).
The relationship among pathogenicity and microbial species richness must need clarification as community behavior is dependent on its diversity. At present, PCR- DGGE is the most frequently applied techniques in such studies (Fujimoto et al., 2003; Ledder et al., 2007; Li et al., 2006b; Siqueira et al., 2008). It is one of the molecular fingerprinting methods that targets 16s rDNA hypervariable region which allow to survey bacterial
Figure 1. PCR-DGGE profile of dental plaque samples from periodontitis, dental caries and normal group.
Table 1. Microbial diversity index analysis of dental caries, periodontitis and normal groups.
Shannon-Weiner Index [H’] = - pilnpi Evenness [E] = H‘/ Hmax. Hmax = natural log of total number of samples. *= Mean±Standard Error of mean.
communities without cultivation. Among many features of DGGE, the most important is bands were excised from the gels and sequenced to get taxonomic identity. This study represents microecological analysis of oral flora studied by PCR-DGGE method in combination with cloning and sequencing. In this study, less numbers of bands were detected which might be due to sample collection, site specific sample, DNA extraction method and due to the hypervariable region of 16s rRNA selected. Less diversity in dental caries patients as dental caries lesion create niches of cariogenic microorganisms only which is supported by many other people (van Houte et al., 1996). Becker et al showed that richness of the bacterial species involved in caries initiation and development as well as other bacteria which survives acidic environment increase at the caries site then intact
enamel surfaces (Becker et al., 2002). Periodontitis is the progressive inflammatory processes results in the destruction of the supporting tissues and a result of mixed microbial infection (Zijnge et al., 2003), Clinical studies have revealed the presence of 10 to 15 bacterial species that are potential periodontal pathogens in adults (Frisken et al., 1990) of these, the three most cited are Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Tannerella forsythensis (formerly Bacteroides forsythus), which have been implicated in the progress of periodontal diseases (Tanner et al., 2002). In our studies we detected Actinomyces, Porphromonas and Capnocytophaga spp.
which constitute most in periodontal samples. After sequencing we were able to dissect our data like distribution of Gram positive versus Gram negative,
Parameter Dental caries group Periodontitis group Normal group
Number of bands 12.73 ± 3.2* 10.36 ± 3.5 17.06 ± 4.34
H’ 3.12 ± 0.12 2.82 ± 0.10 3.26 ± 0.17
E 0.83 0.98 0.99
Figure 2. Cluster analysis.
Figure 3. Number of bands cut and sequenced form dental caries, periodontitis and normal groups.
Table 2. Genera or species identified after sequence analysis.
No. of Bands Genus or species Similarity (%)
Dental caries
1 Streptococcus sanguinis Haemophillus spp. 98.1 98.7
2 Streptococcus oralis Niesseria spp. Corynebacterium spp. 98.7 96.2 95.5
3 Corynebacterium spp., Uncultured TM7 [G-1] spp. 95.5 97.8
4 Capnocytophaga spp. Bacteriodetes spp. 97.4 92.7
5 Moraxella spp. Cardiobacterium spp. 90.7 88.0
6 Klebsiella spp. Selemonas spp. 87.0 98.8
7 Streptococcus mutans Aggregatibacter segnis 99.0 98.1
8 Niesseria spp. Corynebacterium matruchchotii 97.2 95.5
9 Staphylococcus spp. Veillonella spp. 98.1 98.1
10 Rothia spp. Haemophillus spp. Actinomyces spp. 99.3 99.4 98.1
11 Turicella spp. Campylobacter spp. 87.3 96.4
12 Uncultured Kocuria spp. Neisseria spp. 93.5 97.2
Periodontitis disease
13 Corynebacterium spp. Porphyromonas spp. 95.5 90.0
14 Rothia dentiocariosa Microbacterium spp. 99.3 93.6
15 Capnocytophaga spp. Haemophillus spp. 96.8 98.8
16 Streptococcus spp., Corynebacterium matruchchotii 98.1 95.5
17 Kingella spp. , Neisseria spp. 95.1 94.4
18 Porphyromonas spp.spp. Clostridiales [F-2] [G-1] 99.0 98.4
19 Enterobacter spp. Proteus spp. 91.9 92.5
20 Yersenia spp. Capnocytophaga spp. 95.7 96.8
21 Veillonella spp. Selemonas spp. 97.5 98.1
Normal
22 Streptococcus spp. Staphylococcus spp. 99.4 98.1
23 Corynebacterium spp. Veillonella spp. 95.5 98.1
24 Corynebacterium matruchchotii Niesseria spp. 95.5 98.1
25 Streptococcus mitis streptococcus infantis 99.3 99.3
26 Dialister invisus 98.1
27 Staphylococcus spp. 96.3
28 Rothia spp. Streptococcus spp. 99.3 99.4
29 Actinomyces spp. Veillonella spp. 98.1 93.8
30 Niesseria spp. 99.4
identification at genus level and number of phylotypes in one amplicon. Our results show that the number of amplicons which were present in one lane doesn’t mean that only these species are present in the samples because some bacterial species have similar migration distances (Muyzer and Smalla, 1998b). To overcome this problem we do cloning and then sequenced positive clone to detect phylotypes, we detected 1-3 phylotypes per band. With different populations and different methodology Li et al. (2007) detected 26 genera among caries lesion in which Gram negative bacteria dominates.
Munson et al. (2004) found 32 phylotypes within caries lesion in which Gram positive were dominant. In our results we detect 23 genera among dental caries, periodontitis and normal groups. Difference of phylotypes among different studies might be the reason of sampling sites as well as number of clones sequenced.
Cluster Analysis was done on the basis of detected bands in the DGGE profiles. In our studies mixed clusters was formed. Samples are failed to form significant cluster which is contradictory with previous studies (Li et al., 2007a; 2006b). The differentiation in clusters was reflected by the number of bands, intensity and migration distribution of the amplicons. Molecular fingerprinting profiles even though did not provide immediate discrimination within microbial species but it allows the analysis of multiple samples and facilitates direct comparison (Muyzer, 1993a; Muyzer and Smalla, 1998b).
Conclusion
In conclusion, we observed variation in the DGGE profiles of dental caries, periodontitis and normal group.
Microbial diversity were less in periodontitis patients as compared to dental caries which in turn less diverse than normal group. Still further work need to dissect periodontitis microbiota. PCR DGGE is a valuable tool to detect the microbial composition and diversity in the oral plaque as well we can detect the predominant bacteria and uncultivable bacteria present in disease condition which can be used to target caries and periodontitis intervention and prevention.
ACKNOWLEDGMENTS
I would like to thank the National Basic Research Program of China with 973 Project (2007CB513006). I would also like to thank Li Qiu Hong, Department of Stomatology, First Affiliated Hospital Dalian Medical University for providing plaque samples. No conflict of interest is declared among the authors regarding this manuscript.
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