Detection and Prevalence of Periodontal Pathogens in a Uruguayan Population with Chronic Periodontitis Using Conventional Methodology and Metagenomics

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Detection and prevalence of periodontal pathogens in a Uruguayan population with chronic periodontitis using conventional methodology and metagenomics

Papone, Virginia*, Verolo, Carolina*, Zaffaroni, Lourdes*, Batlle, Alicia**, Capo, Claudia**, Bueno, Luis**, Gamonal, Jorge***, Silva, Nora***, Soria, Sandra*

Abstract Periodontal diseases are a major problem in human health. Decades of research have shown that the most common disease is chronic periodontitis, characterized by a slow evolution with the formation of periodontal pockets, subsequent alveolar bone resorption, loss and destruction of teeth and bone tissue. While we know the multifactorial origin of the development of periodontitis, the participation of subgingival microbiota is relevant in the etiology of periodontal disease. Some pathogenic bacteria species that have been associated with the development of periodontal disease are Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Fusobacterium nucleatum, among others. In this work we studied which of these five species were present in the periodontal pockets of 51 Uruguayan patients with chronic periodontitis. To achieve the results a conventional microbiological technique and metagenomics (multiplex-PCR) were used. The results of the microbiological conventional technique showed the presence of A. actinomycetemcomitans (33%) and black pigmented anaerobic bacteria (100%) in the samples. From the results obtained in the multiplex-PCR we saw that the most prevalent species were F. nucleatum (100%), T. forsythia (92%) and P. gingivalis (88%). In contrast, lower prevalence species were P. intermedia (39%) and A. actinomycetemcomitans (33%).

Keywords: microbiota, prevalence, periodontal disease, metagenomics, multiplex‑PCR.

* Microbiology Department, School of Dentistry, Universidad de la República. Uruguay ** Cllinic of Periodontics, School of Dentistry, Universidad de la República. Uruguay *** School of Dentistry, Universidad de Chile. Chile Received on: 07.01.15 - Accepted on: 26.03.15 Odontoestomatología / Vol. XVII. Nº 25 / May 2015 23 Introduction periodontopathic patients using different methodologies, as seen in the work of He- Oral cavity infections are a serious public rrera et al. in Colombia, Spain and Chile. In health issue worldwide given their high pre- South America, the F. nucleatum species is the valence in the adult and child population. most prevalent one in Chile (90%) (11); in The etiology of these pathologies includes Colombia, however, two species showed an many factors: the host, the environment and 80% prevalence: P. gingivalis and T. forsythia infectious factors. These pathologies pose the (12, 13). In studies conducted on Brazilian potential risk of tooth loss caused by chronic patients with periodontitis, P. intermediate periodontitis in children and adults, as well and F. nucleatum were the periodontal patho- as the possible development of infective en- gens present, with a detection frequency of docarditis and even oral and colorectal cancer 67%. The least prevalent species were P. gin- (1-6). In the case of chronic periodontitis or givalis (26.7%), T. forsythia (20%) and A. ac- periodontal disease there is loss of attachment tinomycetemcomitans (13%) (14). and bone loss (horizontal and vertical alveolar Patients with oral infections have been mo- bone). This disease has its highest prevalence nitored in Uruguay since the 1990s (Facul- in adults (>40 years), although it can appear ty of Microbiology, School of Dentistry, in people with their primary or permanent Universidad de la República). Pathogens are teeth. It is a slowly evolving disease with the identified using conventional microbiological following symptoms: gingival swelling with methodologies, and the results obtained gui- edema, increase in gingival volume, round de the treatment for each patient. We must re- gingival margins, flattened papilla, accumula- member that conventional methods of direct tion of supragingival and subgingival plaque, diagnosis have serious limitations, such as: formation of calculus, greater tooth mobility a) transport and conservation of the sample; and exfoliation. All these symptoms can affect b) sample processing; d) development time a variable number of functional teeth in each (2–14 days) and the difficulties for culture individual, with variable progression rates (7, growth (15, 16). It is essential to use metage- 8). Over 300 bacterial species associated with nomic methods to reduce the time it takes to periodontitis have been isolated in periodon- obtain results and to increase detection sen- tal pockets. However, only a small percentage sitivity and speciation. Metagenomics is an of them are etiologically relevant, such as the approach to the study of microbial commu- group of Gram-negative anaerobic pigmented nities, defined as the functional and sequence bacilli that belong to the genera Porphyromo- analysis of collective microbial genomes in a nas, Prevotella, Bacteroides and Fusobacterium, sample of the oral cavity. It can be based on of the Bacteroidaceae family. Additionally, expression or sequencing (17, 18). For exam- studies conducted to identify species that are ple, the multiplex-PCR technique (multiplex potential risk indicators in the development polymerase chain reaction) is frequently used of periodontitis have included: Aggregatibac- in the direct detection of the microbiota me- ter actinomycetemcomitans (Aa), Porphyromo- tagenome composition at species and/or sero- nas gingivalis (Pg), Prevotella intermedia (Pi), type level, without having to harvest, isolate Tannerella forsythia (Tf) and Fusobacterium and morphologically characterize pathogens nucleatum (Fn), among others (7, 8, 9, 10). that grow with difficulty and/or slowly. The More studies are conducted globally on molecular technique uses oligonucleotides the composition of the oral microbiota of complementary to regions conserved in the

24 Papone V, Verolo C, Zaffaroni L, Batlle A, Capo C, Bueno L, Gamonal J, Silva N, Soria S DNA of each species that codifies the small Samples subunit of 16S ribosomal RNA, in this case In each quadrant 4 areas were selected based to detect periodontal pathogens (11, 19-22). on X-rays and clinical study, with pockets ≥ In this study we performed the rapid and si- 5.0 mm deep and ≥ 2.0 mm bone loss. Su- multaneous identification of the microbiota pragingival plaque was removed using sterile involved in oral processes using multiplex- gauze trying to avoid bleeding, and the area PCR, jointly with conventional microbiolo- was dried with sterile cotton rolls. Medium gical methodology, on 51 Uruguayan patients sterile paper points (Nº 25) were placed dee- attending the Clinic of Periodontics of the ply into the pocket and left for 15 s. They were School of Dentistry, Universidad de la Repú- placed in 1.5 ml of RTF (Reduced Transport blica. Fluid) (24). Each sample had 8 paper points that were processed immediately. Samples were then stirred vigorously for 45-60 s, and Methodology serial dilutions in RTF were prepared. 100 µl Patient selection was taken from the sample and placed in an A total of 51 patients who were ≥ 30 years eppendorf tube with 900 µl of RTF. (1st dilu- old were selected (23 women and 28 men) tion: 1:10). 100 µl was streaked, expanding presenting symptoms of mild chronic pe- the sample with a glass rod on a TSVB plate: riodontitis (CAL = 3-4 mm) and advanced tryptic soy serum, bacitracin (75 µg/ml), van- chronic periodontitis (CAL ≥ 5 mm), blee- comycin (5.0 µg/ml), 10 ml of horse serum ding on probing, pathological pockets and (10%) to isolate Aggregatibacter actinomyce- bone loss. Periodontal diagnosis was conduc- temcomitans. The plates were placed in a jar ted following the parameters of the American with a CO2-enriched atmosphere, and incu- Academy of Periodontology (AAP) (23) with bated for 7 days at 37ºC. 100 µl was taken at least 12 teeth present excluding the third from the RTF original sample and placed in molar, without prior periodontal treatment; 900 µl of RTF. 100 µl was taken from this patients free of diabetes, arthritis, ulcerative tube and placed in another tube with 900 µl colitis, HIV, cancer and cardiovascular patho- (2nd dilution: 1:100).100 µl was taken and logy. The following types of patients were also streaked in base agar medium with blood excluded: with initial periodontitis, (patient with menadione and hemin, and incubated with clinical attachment loss of between 1 for 14 days at 37ºC in absolute anaerobiosis. and 3 mm) pregnant women, and those who From the original sample in RTF, 500 µl was had been treated with antibiotics and/or anti- taken, stored at -30ºC and then used in the inflammatories two months before the stu- multiplex-PCR. dy. All patients were notified of the research The following strains were used as positi- and agreed to sign a consent form before a ve controls: Aggregatibacter actinomycete- witness, according to the Regulations of the mcomitans (ATCC 29522), Porphyromonas MERCOSUR. The Ethics Committee of the gingivalis (BAA-308), Prevotella intermedia School of Dentistry, Universidad de la Repú- (ATCC 25611), Tannerella forsythia (ATCC blica, approved the design of the study fo- 43037) and Fusobacterium nucleatum (ATCC llowing the regulations of the MERCOSUR 25586), and as negative control the Escheri- and the Helsinki Declaration on research in- chia coli strain (ATCC 47076). volving human subjects.

Detection and prevalence of periodontal pathogens in a Uruguayan population with chronic periodontitis using conventional methodology and metagenomics 25 Identification and enumeration of colonies centrifuged at 13.500 rpm for 3 min. The Presumptive colonies of A. actinomycete- supernatant was discarded and the pellet was mcomitans were identified given the pre- resuspended again by pipetting in 500 µl of sence of a star-like structure inside the RTF buffer for 5 min at 100ºC. They were colonies (stereo magnifier), Gram stain te- then placed in ice for 5 min and then centri- chnique, catalase test (+) and MUG negati- fuged at 13.500 rpm for 5 min at 4ºC. Finally ve (4-Methylumbelliferyl-β-D-galactoside to the pellet was discarded and the supernatant study lactose fermentation). We calculated with the microbial metagenome was stored at the total number of colony-forming units per -30ºC until multiplex‑PCR was performed. milliliter in each sample (CFU/ml) from the streaked culture media. The relative recovery Extraction of bacterial genomes and selection percentage was calculated from the total CFU of oligonucleotides times the dilution factor. The enumeration of The extraction of the genome of each control colony-forming units of pigmented anaerobic strain was done using the ZymoBead™ Ge- bacteria was performed in each sample. In ge- nomic DNA Kit (as described by manufac- neral, no species of the pigmented colonies turers). DNA concentration (ng/µl) in each were identified, and all pigmented CFU were strain was quantified in NanoDrop 2000. counted by dilution factor. In the selection of species-specific oligonucleotides we consulted bibliographical sou- Control strains rces with information on species-specific Metagenomic DNA extraction from oral mi- oligonucleotides of the 16S ribosomal RNA crobiota in each positive control strain of this study. Samples were defrosted and homogenized Bases were added to the sequences of some for 30 s with a vortex mixer. They were then oligonucleotides to optimize the conditions transferred to another eppendorf tube and of the multiplex-PCR (Table 1 - Figs. 1, 2, 3).

Table 1. Control bacterial strains and sequences of the species-specific oligonucleotides of the 16S ribosomal RNA used in the multiplex-PCR. References Control bacterial strains Oligonucleotides sequences Length (bp) Guillot and Mouton; 1997 Prevotella intermedi 5’- CAG CAC CCA CAA CGA TAT GAT C -3’ 757 (with modifications)* (ATCC 25611) 5’- CTT TCC ATC TTC TCT GCC TGT C -3’ Porphyromonas gingivalis 5’- AGG CAG CTT GCC ATA CTG CG -3’ 404 Figuero et al., 2011 (BAA-308) 5’- ACT GTT AGT AAC TAC CGA TGT GG -3’ Aggregatibacter 5’- ATT GGG GTT TAG CCC TGG TG -3’ 360 Tran and Rudney; 1996 actinomycetemcomitans (ATCC 29522) 5’- ACG TCA TCC CCA CCT TCC TC -3’ Guillot and Mouton; 1997 Tannerella forsythia 5’- GAT GCT CCT GCT GGG TCT GTT -3’ 394 (with modifications)* (ATCC 43037) 5’- CCG ACA CTT CGG ATC GTT TAG -3’ Fusobacterium nucleatum 5’- AGG CGA TGA TGG GTA GCC -3’ 214 Miyagawa et al., 2008 (ATCC 25586) 5’- AGC CGT CAC TTC TTC TGT TG -3

*modifications are written in boldface; for the sequence of the species-specific oligonucleotides selected, modifications were required to optimize the multiplex PCR technique.

26 Papone V, Verolo C, Zaffaroni L, Batlle A, Capo C, Bueno L, Gamonal J, Silva N, Soria S ATCC = American Type Culture Collection. Determination of detection limit The lowest detection limit was defined as the smallest number of microorganisms in a sample that could be detected by the multiplex- PCR. This was done through serial dilutions (0 to 1.75x106 cells/ml) of a mixture of pure cultures of all the strains described above.

Multiplex-PCR Programming conditions were optimized according to the parameters of each sequence of oligonucleotides designed in this study (nt, G + C%, Tm). Each reaction mixture Figure 1: PCR products in agarose gel: Lane 1, 100 bp molecular (25 µl) contains 1.0X PCR buffer (10 mM weight marker (DMF-50, sbs); Lane 2, positive control of A. actinomycetemcomitans; Lane 3, positive control of P. gingivalis; Lane Tris-HCl, 50 mM KCl, pH 8.3), 1.5 µl of 2.5 4, positive control of T. forsythia; Lane 5, positive control of F. mM dNTPs, 1 mM MgCl2, 10 pmol of each nucleatum (Agarose gel 1%, GoodView 5%).

Figure 2: PCR products in agarose gel: Lane 1, 100 bp molecular weight marker (DMF-50, sbs); Lane 2, positive control of A. actinomycetemcomitans; Lanes 3-6, samples from different patients that tested positive for A. actinomycetemcomitans; Lane 7, positive control of P. gingivalis; Lane 8, patient sample that tested positive for P. gingivalis; Lane 9, patient sample that tested negative for P. gingivalis; Lanes 10-11, samples from different patients that tested positive for P. gingivalis; Lane 12, positive control of P. intermedia; Lane 13, patient sample that tested positive for P. intermedia; Lane 14, patient sample that tested negative for P. intermediate (Agarose gel 1%, GoodView 5%).

Figure 3. Multiplex-PCR products in agarose gel: Lane 1, positive control of P.gingivalis; Lane 2, patient sample that tested positive for P.gingivalis and P. intermedia; Lane 3, patient sample that tested positive for P.gingivalis and negative for P. intermedia; Lanes 4-5, patient samples that tested positive for P. gingivalis and P. intermedia; Lane 6, patient sample that tested positive for P.gingivalis and negative for P. intermedia; Lane 7, patient sample that tested positive for P.gingivalis and P. intermedia; Lane 8, 100 bp molecular weight marker (DMF-50, sbs); Lane 9, positive control of T. forsythia; Lanes 10-14, patient samples that tested positive for T. forsythia (Agarose gel 1%, GoodView 5%).

Detection and prevalence of periodontal pathogens in a Uruguayan population with chronic periodontitis using conventional methodology and metagenomics 27 oligonucleotide (Table 1, Figs. 1, 2, 3) and gical cultures. The results of this study showed 1.2 U of Taq DNA polymerase and 5 ng of that 23% of patients had A. actinomycetemco- the metagenoma extracted from each sample mitans in values of 3 x 105 UFC/ml, and 10% and 2 ng of the genome of the control strains in a range of 2-7 x 104 UFC/ml. It was deter- (NanoDrop 2000). mined that 100% of the samples had black- In the detection by multiplex-PCR of the pigmented anaerobic bacteria (BPAB) in the P. gingivalis and P. intermedia species, the fo- following percentages: 14% had counts lower llowing conditions were applied: 1 cycle of than 105 UFC/ml, 74% between 105 - 106 denaturation at 95ºC for 3 min, 35 cycles of UFC/ml, and finally 12% had counts higher 94ºC for 1 min, 55ºC for 1 min, 72ºC for 2 than 106 UFC/ml of the sample. These results min, and 1 cycle of final extension of 72ºC showed that there was confirmed presence of for 5 min. BPAB in the periodontal pockets of all the The following conditions were applied in the patients. If we remember that in the group of detection by multiplex-PCR of the species the BPAB quantified in the cultures we can A. actinomycetemcomitans, T. forsythia and F. probably find some genera ofPorphyromonas, nucleatum: 1 cycle of denaturation at 95ºC Prevotella and Fusobacterium, then, there may for 2 min, 35 cycles of 94ºC for 30 s, 55ºC be adhesion, aggregation, and coaggregation for 1 min, 72ºC for 2 min, and 1 cycle of among these genera that form the gingival mi- final extension of 72ºC for 5 min. crobiota. For example, it has been shown that The results of amplifications (bands) were F. nucleatum can participate in coaggregation viewed in 1.2% agarose gel, 0.5 X TBE (Tris- through the interaction of certain lipopolysac- borate-EDTA) buffer, 5% of GoodView, 1% charides which recognize and join the surface of Cyan/Orange loading buffer; 2 µl of the O-galactosides of A.actinomycetemcomitans, P. PCR product for 2 h at 80 V. The results were gingivalis and P. intermedia (25, 26). This is of recorded using a photodocumentation device great importance given the possible “bridge” (PHOTO/Phoresis; Fotodyne, UV). role played by F. nucleatum in the colonization of the subgingival area. F. nucleatum can survive high concentrations of oxygen and is Statistical analysis one of the most important bacterial species The results of the multiplex-PCR were obtai- that works as a bridge between aerobes and ned using absolute frequencies in percenta- obligate anaerobes, actively participating in ges, and the clinical conditions were compa- the link and establishment of the microbial red by means of the Student’s-T Test with a consortium (27). significance level of < 0.05. The results obtained by simultaneous detection of six oral bacterial pathogenic species Results from 51 patients by multiplex-PCR showed that 100% of the samples tested positive for A total of 51 samples collected from patients F. nucleatum; this shows that it is the most with chronic periodontitis were processed prevalent species in a Uruguayan population with the conventional method of microbiolo- with chronic periodontitis (Fig. 4).

28 Papone V, Verolo C, Zaffaroni L, Batlle A, Capo C, Bueno L, Gamonal J, Silva N, Soria S were detected less frequently: 49% and 33%, respectively (Fig. 4). Depending on prevalence percentages, Gajardo and colleagues used the following criterion: the higher the percentage of detection of a pathogenic species, the higher the link between the pathogen and the development of chronic periodontitis. Accor- ding to this criterion, in this study we con- sider F. nucleatum, T. forsythia and P. gingivalis as critical pathogens in connection with chronic periodontitis, and P. intermedia and A. actinomycetemcomitans as bacteria associated with chronic periodontitis with lower prevalence. We must highlight the strong evidence regar- Figure 4. Graph with the incidence percentages of periodontal pathogens in Uruguayan patients with chronic periodontitis. ding the potential association between periodontal diseases and the development of car- These results indicate that the most preva- diovascular complications in both men and lent species is F. nucleatum, the same as in the women (28-30). Therefore it is important to studies conducted in Chile (11). This infor- implement fast detection and treatment of mation is very relevant as it has been shown patients, thus avoiding the action of these in- that F. nucleatum can colonize the colon and vasive pathogens that can favor the adhesion become a prominent biological factor in the of leukocytes to vascular epithelium, which development of colorectal carcinoma. This might have a pro-coagulant effect among type of cancer causes approximately 610.000 others, as has been reported in the bibliogra- deaths annually around the world and is con- phy (3). nected with infection by F. nucleatum, which When comparing our results with the results occurs mainly orally (6). Additionally, T. for- achieved Chile (1, 11), Spain, Colombia (31) sythia and P. gingivalis pathogens were also and Brazil (14), we can observe that the most highly prevalent with 92% and 88% respecti- prevalent species vary in each country. This vely. This shows that these are high-frequency might be due to various factors such as the pathogens in a Uruguayan population with existence of geographical, racial, etiological, chronic periodontitis. When comparing this genetic, environmental, habit-related and with the studies conducted in Colombia and oral health behavior differences.

Chile (1, 11) we can state that P. gingivalis The significant differences detected between also has a high prevalence in Uruguay. The men and women might be due to the diffe- high prevalence of both species is important rent factors and mechanisms involved in the because these are endogenous pathogens, that events of colonization of the periodontal poc- is to say that they can invade the epithelial kets in both sexes. Besides, it is highly likely tissue and allow entry to other opportunistic for the establishment and multiplication of a microorganisms such as Herpes virus, Candi- pathogenic species to be different from that das spp., among others. On the other hand, of another pathogenic species; or a bacterial P. intermedia and A. actinomycetemcomitans species can be favored by the environmental

Detection and prevalence of periodontal pathogens in a Uruguayan population with chronic periodontitis using conventional methodology and metagenomics 29 conditions of a host but not of other hosts. bacterial coaggregations, perhaps especially Klinger and colleague established that nu- favored by the hormonal microenvironment, trients available to the bacterial consortium different from that of women. inside periodontal pockets are very different in men than in women, which is mainly de- termined by hormonal levels (32). Ethnic origin can probably influence the selection of bacterial species in the gingival microbiota. Besides, reports have shown that some have a predilection for a specific ethnic group, for example, P. gingivalis is found mainly in Afri- can-American patients with periodontitis; while F. nucleatum seems to be more frequent Figure 5. Percentages of multiple detection of pe- in Caucasian patients. riodontal pathogens in men and women Figure 5 shows the values of simultaneous and combined detection by order of incidents of the five periodontal pathogens present in Conclusions both female and male patients. Interestingly, The samples analyzed using the conventional men had higher simultaneous detection va- microbiological technique showed a 33% pre- lues than women. Besides, among men, the sence of A. actinomycetemcomitans and a 100% co-detection of F. nucleatum and T. forsythia presence of black-pigmented anaerobic bacte- reached 100%, while among women it was ria. The analysis of the species-specific sequen- significantly lower (83%). The same trend ces of 16S ribosomal RNA with mutiplex-PCR was observed in the simultaneous detection allowed for the fast detection, in each species, of the three pathogens F. nucleatum, T. fors- of five periodontal pathogens in samples taken ythia and P. gingivalis: 93% among men and from 51 patients of both sexes. Among the 70% among women. As simultaneous detec- bacterial species identified, F. nucleatum was tion increases (F. nucleatum, T. forsythia, P. the only periodontal pathogen detected in all gingivalis and P. intermedia), the percentages male and female patients. The most prevalent of detection diminish; nevertheless, they re- pathogenic species in a Uruguayan population main significantly higher among men (39%) with chronic periodontitis were F. nucleatum, than among women (26%). Finally, the si- T. forsythia and P. gingivalis. multaneous detection of all the periodontal pathogens of this study (F. nucleatum, T. forsythia, P. gingivalis, P. intermedia and A. ac- Acknowledgments tinomycetemcomitans) showed values of 18% among men and 13% among women (Fig. We would like to thank the technical support 5). These results suggest that subgingival mi- of the School of Graduates of the School of crobiota greatly differs between men and wo- Dentistry, Universidad de la República, as men of a Uruguayan population with chronic well as Paul Gaytán, Jorge Yáñez and Eugenio periodontitis. In men there are multigeneric López for the synthesis of oligonucleotides, IBT- UNAM (Mexico).

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32 Papone V, Verolo C, Zaffaroni L, Batlle A, Capo C, Bueno L, Gamonal J, Silva N, Soria S Normativa MERCOSUR. Estudios clí- Nº 129/96. June 2007. Available from: nicos con medicamentos. Resolución http://www.msp.gub.uy/. Cited: 2014 October

Virginia Papone: [email protected]

Detection and prevalence of periodontal pathogens in a Uruguayan population with chronic periodontitis using conventional methodology and metagenomics 33