Contents lists available at ScienceDirect Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio Research Article Pathogenicity and genetic profile of oral Porphyromonas species from canine periodontitis Amanda do Nascimento Silvaa,1, Erica Dorigatti de Avilab,1, Viviane Nakanoa, Mario J. Avila-Camposa,⁎ a Anaerobe Laboratory, Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo – USP, Sao Paulo, SP, Brazil b Department of Dental Materials and Prosthodontics, School of Dentistry of Araraquara, Univ Estadual Paulista – UNESP, Araraquara, SP, Brazil A R T I C L E I N F O Keywords: Porphyromonas spp. Periodontal disease prtC gene fimA gene Genetic diversity A B S T R A C T Objective: In this study, the presence of the prtC and fimA genes involved in the pathogenicity of oral Porphyromonas spp. isolated from dogs with periodontitis and healthy, as well as their genetic diversity was investigated. Design: Thirty-two Beagle dogs, 24 with periodontitis and 8 healthy were evaluated. Subgingival samples from only one gingival site of both groups were collected. Bacteria grown in anaerobiosis were identified by RAPID ID 32A kits. From each strain the respective DNA was obtained and used to genotyping by conventional PCR and AP-PCR. Results: Dogs with periodontitis harbored 28 P. gulae, 2 P. creviocaricanis, 1 P. cangingivalis and 7 P. macacae; and from healthy dogs, 11 P. gulae and 5 P. circumdentaria. In P. gulae isolated from periodontal dogs the gene prtC was observed in 19 (67.85%) and in 7 (63.63%) from healthy dogs. P. gulae strains from periodontal dogs harbored either the gene fimA I or fimA II; while strains from healthy dogs harbored the gene fimA I, fimA II, fimA III or fimA IV, as well as 1 P. circumdentaria the gene fimA II. By AP-PCR strains were grouped in different clusters suggesting heterogeneity of these microorganisms. Conclusions: The results presented herein inform that Porphyromonas spp. isolated from dogs with and without periodontitis harbored the prtC and fimA genes and it could be a role in the establishment of the infectious process. 1. Introduction It is well known that periodontitis is a complex infection, in which bacteria are responsible to initiate the immune inflammatory process and the result is a loss of support of the affected teeth (Meyle & Chapple, 2015). This process is characterized by destruction of the periodontal attachment apparatus, increased bone resorption with loss of crestal alveolar bone, apical migration of the epithelial attachment, and for- mation of periodontal pockets (Graves, Li, & Cochran, 2011). Among all oral pathogenic and non-pathogenic microorganisms, species of the genus Porphyromonas are able to invade and to damage the epithelial layer as well as to induce inflammatory responses leading to attachment loss and periodontal destruction (Paster et al., 2001). Porphyromonas gingivalis has been described as a key pathogen in periodontal infections due to the production of different virulence factors, such as gingipain, amino peptidases, invasin, capsule, collagenases, fimbriae, and lipopolysaccharide (Amano, 2003; Jotwani & Cutler, 2004; Preshaw, Schifferle, &Walters, 1999). Fimbriae-producing P. gingivalis plays an important role in the at- tachment and invasion to periodontal tissues (Hamada et al., 1998) and it is critical for promotion of the bacterial infection (Nakagawa, Amano, & Ohara-Nemoto, 2002). The collagenolytic activity of P. gin- givalis has been associated to the collagen destruction and progression of periodontitis (Kato, Takahashi, & Kuramitsu, 1992). This collagenase produced by prtC gene has been described as responsible for cleavage of type I collagen (Wittstock, Schmidt, Flemmig, & Karch, 2000). Studies have shown that periodontitis is also common in dogs (Weinberg & Bral, 1999), leading to bone resorption and tooth loss. In animals, the anaerobic oral microbiota is represented by others im- portant species of Porphyromonas, such as P. gulae, P. circumdentaria, P. creviocaricanis, P. casulci, P. canoris, P. denticanis, P. salivosa, P. cangin- givalis, P. canis, P. gingivicanis, and P. catoniae (Allaker, de Rosayro, http://dx.doi.org/10.1016/j.archoralbio.2017.07.001 Received 17 May 2017; Received in revised form 30 June 2017; Accepted 3 July 2017 ⁎ Corresponding author at: Av. Prof. Lineu Prestes 1374, 05508-900 Sao Paulo, SP, Brazil. 1 These authors contributed equally to this work. E-mail address: mariojac@usp.br (M.J. Avila-Campos). Archives of Oral Biology 83 (2017) 20–24 0003-9969/ © 2017 Elsevier Ltd. All rights reserved. MARK http://www.sciencedirect.com/science/journal/00039969 http://www.elsevier.com/locate/archoralbio http://dx.doi.org/10.1016/j.archoralbio.2017.07.001 http://dx.doi.org/10.1016/j.archoralbio.2017.07.001 mailto:mariojac@usp.br http://dx.doi.org/10.1016/j.archoralbio.2017.07.001 http://crossmark.crossref.org/dialog/?doi=10.1016/j.archoralbio.2017.07.001&domain=pdf Young, & Hardie, 1997; Hardham, Dreier, Wong, Sfintescu, & Evans, 2005; Isogai, Kosako, Benno, & Isogai, 1999). Porphyromonas gulae is a gram-negative, black-pigmented producing, and is considered the commonly species detected in subgingival biofilm of dogs displaying periodontitis (Kato et al., 2011). Similarly to P. gingivalis the fimbriae is an important cell structure involved in the adherence and invasion of host’s cells, and stimulates the production of inflammatory cytokines by macrophages and fibroblasts. This adhesive ability is considered to be a major pathogenic characteristic that causes periodontal tissue destruc- tion (Amano, Nakagawa, Kataoka, Morisaki, & Hamada, 1999; Sasaki, Watanabe, Toyama, Koyata, & Hamada, 2015). On the other hand, little is known about the presence of Porphyromonas spp. isolated from dogs carrying the prtC gene, as well as its relationship with the canine periodontitis. Since pathogenic bacteria from animal origin, particularly dogs, may be transferred to people through bites, for example; and considering their involvement in human and animal periodontal disease; this study, was performed to determine the presence of the prtC and fimA genes in oral Porphyromonas spp. isolated from dogs with and without periodontal disease, as well as, the genetic diversity of these species by using an AP- PCR technique. 2. Materials and methods 2.1. Animal study design Subgingival biofilm samples were collected from thirty-two Beagle dogs of 7-months to 10-years-old at the School of Veterinary Medicine and Zootechny of the University of Sao Paulo (Sao Paulo, SP, Brazil). Dogs were grouped in: (1) Eight dogs without periodontitis, and (2) 24 dogs with periodontitis. As inclusion criteria, only the animals had re- ceived no antibiotic treatment within the previous three months of the sample collection. Prior to collection, the animals with periodontitis were examined in relation to clinical conditions: degree of gingival inflammation, supragingival plaque level, probing pocket depths (5 mm), bleeding on probing, tooth mobility, and alveolar bone loss. The animals underwent anesthesia with a mixture of propofol (2 mg/kg) and diazepam (5.5 mg/kg) administered via intramuscular injection (Senhorinho et al., 2011). Briefly, supragingival biofilms were removed with sterile gauze to avoid any contamination of the paper points. Then, subgingival samples from only one gingival site of both groups were collected with two fine sterile paper points (N. 30, Ta- nariman Ind Ltd, AM, Brazil), introduced into the apical region for 60 s. Paper points were then placed into VMGA III transport medium and processed within 4 h of collection. The protocols used in this study were approved by the Ethics Committee for Animal Experimentation at the Institute of Biomedical Science/USP (116/CEEA), and followed all recommendations of the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines for the execution and submission of studies in animals. 2.2. DNA extraction, PCR assay and AP-PCR genotyping Bacteria were isolated on Brucella blood agar supplemented with hemine (0.5 μg/ml) and menadione (0.1 μg/ml), at 37 °C, under anae- robic conditions (10% CO2/90% N2), and identified in accordance with Jousimies-Somer et al. (2002) or by RAPID ID 32A kits (bioMérieux) according to the manufacturer’s instructions. The broth bacterial cul- tures were centrifuged at 14.000g for 10 min to spin down the pellet and used to DNA extraction. The total genomic DNA was isolated by Easy-DNA commercial kit (Invitrogen do Brasil, Ltd, Sao Paulo, SP, Brazil) and the concentration of bacterial DNA determined with a spectrophotometer (OD260nm). PCR Amplifications were performed as described: each 25 μL-PCR contained 1 X PCR buffer, 2.5 mM MgCl2, 20 μM dNTP (Invitrogen), 0.5 U Platinum Taq DNA polymerase (Invitrogen), 0.4 μM of each primer (Table 1) (Chen & Slots, 1994; Odell, Baumgartner, Xia, & David, 1999) and 1 μL of DNA. The cycle conditions were optimized according to the gene. For prtC gene, denaturation 1 cycle of 94 °C (5 min); am- plification, for 33 cycles of 94 °C (1 min), 55 °C (1 min), 72 °C (1 min); and 1 cycle of 72 °C (10 min). For fimA gene detection the same PCR conditions were used, but with 30 cycles of 94 °C (30 s), 58 °C (30 s), and 72 °C (30 s). Combinations were used in all PCR reactions and amplifications were performed in a thermal cycler (Perkin Elmer Amp PCR System 2400). In order to investigate the AP-PCR genotyping, four arbitrary pri- mers OPA-03, OPA-05, OPA-13 and OPA-17 (Table 1) and 10 μL of DNA were used, as mentioned above. Conditions were further optimized, as follow: denaturation 1 cycle of 94 °C (5 min); amplification, for 35 cycles of 94 °C (1 min), 42 °C (2 min), 72 °C (2 min); and 1 cycle of 72 °C (10 min). From all the resulting PCR products, twenty microlitres of each amplification product were evaluated by electrophoresis in 1.0% agarose gel. Reference strains P. gingivalis ATCC 33277 and F. nucleatum ATCC 10953 were used as control. 2.3. Statistic analysis Pairwise similarities were computed by the package NTSYS program (Applied Biostatistics, Inc. version 1.7) program using the Dicce coef- ficient of similarity and unweighted pair group method using arithmetic Table 1 Nomenclature, oligonucleotide and PCR conditions. Primer Oligonucleotide 5′ → 3′ Annealing Temperature (°C) Amplicon (bp) References Universal AGA GTT TGA TCC TGG CTC AG GGC TAC CTT GTT ACG ACT T 58 3480 Amano, Nakagawa, Okahashi, and Hamada, 2004 prtC CGA GAT CGG AGT AGA AGT GCA TC CCA CGG TTT GCA GTT CGT ATC G 55 815 Odell et al. (1999) fimA I CTG TGT GTT TAT GGC AAA CTT C AC CCC GCT CCC TGT ATT CCG A 58 392 Amano et al. (2004) fimA II ACA ACT ATA CTT ATG ACA ATG G AAC CCC GCT CCC TGT ATT CCG A 58 257 Amano et al. (2004) fimA III ATT AC ACCT ACA CAG GTG AGG C AAC CCC GCT CCC TGT ATT CCG A 58 247 Amano et al. (2004) fimA IV CTA TTC AGG TGC TAT TCA CCA A AAC CCC GCT CCC TGT ATT CCG A 58 251 Amano et al. (2004) fimA V AAC ACC AGT CTC CTT GAC AGT G TAT TGG GGG TCG AAC GTT ACT GTC 58 462 Nakagawa et al. (2002) OPA-03 AGT CAG CCA C 42 – Chen and Slots (1994) OPA-05 AGG GGT CTTG 42 – Chen and Slots (1994) OPA-13 CGG CAC CCA C 42 – Chen and Slots (1994) OPA-17 GAC CGT CTT GT 42 – Chen and Slots (1994) A. do Nascimento Silva et al. Archives of Oral Biology 83 (2017) 20–24 21 averages (UPGMA) clustering. 3. Results Fifty-four strains were isolated and identified from 24 dogs with periodontitis (38 strains) and from eight healthy dogs (16 strains) (Table 2). The prtC gene was detected in 19 (67.86%) of 28 P. gulae strains, and in 1 P. cangingivalis isolated from dogs with periodontitis. Neither two P. creviocaricanis nor seven P. macacae strains harbored that gene. Of the 16 strains from healthy dogs, 7 (63.64%) P. gulae and 1 P. circumdentaria harbored the prtC gene. The fimA I gene was ob- served only in eight P. gulae strains from periodontitis and in four strains from healthy dogs. The presence of fimA II gene was noted in 1 P. gulae from periodontitis, and in 4 P. gulae and 1 P. circumdentaria from healthy dogs. Interestingly, the fimA III and fimA IV were not observed in P. gulae strain from periodontal dogs, but both genes were noted in 2 P. gulae, and fimA IV in another strain isolated from dogs without periodontitis. Moreover, neither periodontal nor healthy dogs harbored strains with the fimA V gene (Table 2). Double or triple bac- terial associations with fimbriae types were observed only in strains isolated from healthy dogs. Reference strains P. gingivalis ATCC 33277 harbored the prtC and fimA I genes, and F. nucleatum ATCC 10953 did not harbor both genes. In Fig. 1 is observed the genetic analysis of the 38 Porphyromonas strains. A High degree of DNA polymorphism was detected with OPA- 03 primer, which produced clear bands and a good bacterial distribu- tion showing the presence of 15 clusters with 50% of similarity. In addition, the primers OPA-05, OPA-13 and OPA 17, revealed weak amplification bands. These bands were not constantly detected by vi- sual inspection, and were not considerate in this analysis. The presence or absence of prtC and fimA genes did not show any relationship with the clusters. The genotyping of the strains by AP-PCR produced from 4000 to 400 bp DNA fragments, with 15 different genetic profiles in strains isolated from dogs with and without periodontitis. In addition, in Fig. 1 can also be observed three main groups: group A, harbored only strains isolated from periodontitis including P. gingivalis ATCC 33277 (P.g ATCC); group B, strains isolated from dogs with and without disease, and group C, containing only strains from healthy dogs. The cluster IX was formed for two sub-group containing P. macacae strains isolated from dogs with periodontitis. These strains showed 100% of similarity. Strains isolated from healthy dogs, were grouped from XI to XV. The cluster XIV grouped three P. circumdentaria (P.c) with 100% of similarity. Some strains of Porphyromonas and F. nucleatum ATCC 10953 did not amplify fragments. 4. Discussion Dog periodontitis-related bacterial species have been found in human oral cavity and are involved with aggressiveness of the disease. Similarly to the human periodontal disease, species of Porphyromonas are also important etiological agents in dog oral infections (Pihlstrom, Michalowicz, & Johnson, 2005), and among them, P. gulae is considered the most prevalent (Kato et al., 2011). In order to investigate the pre- sence of black-pigmented anaerobic bacteria from animal origin and their virulence factors involved in periodontal disease, we determined the presence of Porphyromonas species in dogs with and without peri- odontal disease. The genetic diversity of these species isolated from periodontal and healthy dogs were demonstrated by AP-PCR analysis. Porphyromonas gulae from dogs with periodontitis were significantly present in higher number than those of the healthy group. Furthermore, we demonstrated that bacterial species isolated from periodontal dogs displayed both prtC and fimA genes, that promote the adherence and colonization to the host’s tissues. Importantly, we also found a high level of polymorphism among the Porphyromonas spp., which might reflect the potential of virulence and its involvement with periodontal disease. This is an important finding since competition among clones might select more resistant clones able to survival in the human host. Fimbriae are also considered to be critical factors that mediate bacterial interactions in the adherence and invasion to the host’s cells. FimA is encoded by the fimA gene and its virulence is related to the genetic variation. The presence of P. gingivalis fimA genotypes II and IV have been shown to be significantly more prevalent than other geno- types in human chronic marginal periodontitis processes (Amano et al., 2000; Feng et al., 2014). The pathogenicity of the various fimA geno- types has also been evaluated in animal models. Strains fimA genotypes II, Ib or IV appear to cause severe infectious symptoms and in- flammatory changes, when compared to strains fimA genotypes I and III (Amano et al., 1999; Nakano et al., 2004). Additionally, studies on the pathogenic potential of Porphyromonas spp. have shown that fimA genotypes II are prevalent in patients with aggressive periodontitis (Miura, Hamachi, Fujise, &Maeda, 2005). These results support the proposal that P. gulae could be considered as a possible risk factor for the periodontitis development (Senhorinho et al., 2011). In line with previous studies, our findings show the presence of Porphyromonas spp. fimA genotype I in both groups of dogs with peri- odontitis and healthy, suggesting no association between that genotype and the disease evolution. On the other hand, fimA genotype II asso- ciated with human aggressive oral diseases was found in only one dog with periodontitis. These results suggest that Porphyromonas spp. har- boring genes, such as prtC and fimA can be pathogenic to animals, but it might not be true to produce an oral disease in humans, in accordance with (Amano et al., 2000). Some studies affirm that it is difficult for oral bacteria from canine origin to survive and colonize the human oral Table 2 Presence of the prtC and fimA genes in Porphyromonas strains from dogs with and without periodontitis. Genes Origin Strains (no.) prtC fimA I fimA II fimA III fimA IV fimA V Dogs with Periodontitis P. gulae (n = 28) D1, D4, D7, D20, D22 + + − − − − D3 + − + − − − D2, D8, D12, D21, D25, D26, D27, D28, D30, D32, D33, D34, D35 + − − − − − D9, D23, D29 − + − − − − D5, D6, D10, D11, D24, D36 − − − − − − P. creviocaricanis (n = 2) D37, D38 − − − − − − P. cangingivalis (n = 1) D31 + − − − − − P. macacae (n = 7) D13, D14, D15, D16, D17, D18, D19 − − − − − − Healthy Dogs P. gulae (n = 11) S2 + + − + + − S1 + − − + + − S3 + + + − + − S4 + − + − − − S5, S6 + + + − − − S7 + − − − − − S8, S10, S12, S13 − − − − − − P. circumdentaria (n = 5) S11 − − + − − − S16 + − − − − − S9, S14, S15 − − − − − − Reference strains P. gingivalis ATCC 33277 + + − − − − F. nucleatum ATCC 10953 − − − − − − Presence (+); absence (−). A. do Nascimento Silva et al. Archives of Oral Biology 83 (2017) 20–24 22 cavity (Oh et al., 2015) due to the physiological differences between the two environments, such as: difference of pH (Li & Bowden, 1994) and by the fact that humans brush their teeth removing the supra- and sub- gingival bacterial biofilms. In addition, when canine oral bacteria are transmitted to the human oral cavity, these microorganisms begin an ecological competition with the human resident oral microbiota, and it can influence the expression of some virulence factor for the initiation of a disease (Senhorinho et al., 2012). The bacterial dynamic relation- ship in different microbial ecosystem needs more studies. Our results show that periodontal dogs harbored strains fimA I and fimA II, and healthy dogs harbored fimA I, fimA II, fimA III, and fimA IV. This result suggests that species of Porphyromonas with genotypes fimA III and fimA IV are not implicated in periodontal disease of dogs. Since in humans, the presence of P. gingivalis harboring the fimA gene may be associated with the severity of the periodontitis; it is possible that P. gulae with the genotype fimA may also be associated with the patho- genesis of periodontitis in dogs. Studies have shown that the presence of long fimbriae observed in P. gingivalis serve to mediate the co-adhesion with other bacteria, such as Actinomyces viscosus, Treponema denticola and Streptococcus oralis (Goulbourne & Ellen, 1991; Hashimoto, Ogawa, Asai, Takai, & Ogawa, 2003; Maeda et al., 2004). The FimA protein produced by P. gulae has been classified into genotypes A–C. The bacterial detection with different fimA genotypes is considered a risk factor to the periodontitis development (Maeda et al., 2004). In addition, differences in the fimbriae protein from P. gulae are related to the level of aggressiveness determined by their bone de- struction capacity (Nomura et al., 2012). The 41-kDa fimbriae protein is able to induce the osteoclast differentiation as well as the inflammatory cytokine production by macrophages, in murine model, as well as it was similar to LPS in the osteoclast activation in periodontal diseases (Nomura et al., 2012). Thus, the presence of specific fimbriae proteins in P. gulae can explain the divergent results observed in different stu- dies, and it might be useful to understand the association of the pre- sence of fimA genotypes with either oral health or disease. In order to examine the potential role of the bacterial collagenases in oral diseases, we investigated the presence of the prtC gene in oral Porphyromonas isolated from dogs with and without periodontal dis- ease. Collagenase is a potential virulence factor, which is expressed by P. gingivalis associated with periodontal disease (Odell et al., 1999), and it is suggested that prtC gene-positive Porphyromonas spp. plays an important role in the progression of periodontitis; however, studies of this enzyme produced by P. gulae on host’s cells need to be performed. A limitation of this study was the sample size and the differences on number of dogs in both with and without disease groups. Furthermore, we selected dogs with a large age range, and since the total number of bacterial species detected in these animals was positively correlated with their chronological age, this variation could be interfered our findings (Harvey, Shofer, & Laster, 1994). Further studies focusing the understanding of the relationship among the bacterial transmission, bacterial genes and protein expres- sion in oral environment, and the relationship with oral disease of pa- thogenic oral bacteria from dogs to humans might be important to help us to better understand the complex oral microbial ecosystem. In con- clusion, the high level of Porphyromonas spp. from animal origin takes many paths to a reinfection of humans, due to colonized or infected pets might influence the pathogenesis of disease in humans. Our findings strongly suggest that the colonization and adaptation of Porphyromonas spp. from dogs in human oral cavity is a possible fact. Also, these results allow us to correlate the presence of the prtC and fimA genes, particu- larly fimA I and fimA II, with periodontal disease in dogs. Conflict of interests The authors declare that they have no competing interests. Acknowledgements The authors would like to thank Mrs. Marcia Harumi Fukugaiti for her technical support. This study was supported by FAPESP No. 2013/ 13652-6. References Allaker, R. P., de Rosayro, R., Young, K. A., & Hardie, J. (1997). Prevalence of Porphyromonas and Prevotella species in the dental plaque of dogs. Veterinary Record, 140, 147–148. Amano, A., Nakagawa, I., Kataoka, K., Morisaki, I., & Hamada, S. (1999). Distribution of Fig. 1. Dendrogram showing the genetic profile of oral Porphyromonas strains isolated from dogs with and without periodontitis. (D) Strains from dogs with periodontitis; (S) strains from healthy dogs; (−/+) absence/presence of genes; (prtC and fimA) presence of the genes; (P. gul) P. gulae; (P. m) P. macacae; (P. c) P. circumdentaria; and (ATCC) P. gingivalis ATCC 33277. A. do Nascimento Silva et al. 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http://refhub.elsevier.com/S0003-9969(17)30213-3/sbref0170 http://refhub.elsevier.com/S0003-9969(17)30213-3/sbref0175 http://refhub.elsevier.com/S0003-9969(17)30213-3/sbref0175 Pathogenicity and genetic profile of oral Porphyromonas species from canine periodontitis Introduction Materials and methods Animal study design DNA extraction, PCR assay and AP-PCR genotyping Statistic analysis Results Discussion Conflict of interests Acknowledgements References