**4.1 Genus** *Porphyromonas*

*Bacterial Biofilms*

**4. Periodontal pathogens**

attributable to chronic periodontitis [38].

process [32]. In case of chronic periodontitis usually have abundance of plaque and calculus, which match with the amount of periodontal destruction. On the other hand, in case of aggressive periodontitis, there is usually a mismatch between the amount of local factors and the periodontal destruction [33]. Periodontal disease affects more frequently and more severely regions of premolars and molars than regions of maxillary and mandibular incisors. Missing of teeth is observed at a high and increasing incidence with age. The tooth most commonly lost is the first premolar, followed by the other premolars and molars, where severe periodontitis is frequently found [34]. Periodontitis is a serious infection that can have medical consequences such as anorexia and weight loss, chronic pain, swollen gums, dental caries, breakage or loss of teeth and breakage of the maxillary or mandibular bone [35]. Unfortunately, the damage from periodontal disease is not confined to just loss of teeth. Oral infection, especially periodontitis, may affect the course and pathogenesis of a number of systemic diseases, such as chronic bronchitis, pulmonary fibrosis, endocarditis, interstitial nephritis, glomerulonephritis and hepatitis [1].

Although there is sufficient evidence that biofilm accumulation and maturation is essential for initiation and progression of periodontal disease, studies show that bacterial species colonizing periodontal pocket have different roles in the pathogenesis of this disease [36]. Microbial density is considered to be critical for the development of gingivitis, and some types of chronic periodontitis, while the species of the microorganisms may be of greater importance in the initiation of aggressive periodontitis [35]. Subgingival microbiota in periodontitis may contain hundreds of bacterial species, but only a small number is associated with disease progression, and is considered to be of importance etiologically [37]. The presence of *Mycobacterium tuberculosis* is an indication of tuberculosis, and *Treponema pallidum* a positive diagnosis of syphilis, but there is no single microorganism, which is

As with any other infection, identification of the microbial pathogens associated with the etiology of periodontitis is the first step towards the development of effective therapeutic approaches. The establishment of a microorganism as a true pathogen should be based on two main levels of evidence: (1) the organism should be present in higher prevalence and/or levels in disease than in health, and (2) its suppression or elimination should reduce or stop disease progression [39]. In human, the presence of three species of Gram-negative anaerobic bacteria within subgingival biofilm, *Porphyromonas gingivalis*, *Treponema denticola* and *Tannerella forsythia*, described by Socransky et al. as the "red complex," show a strong association to periodontitis, and some studies have indicated their involvement also in dogs [40]. There are many others candidates as human periodontal pathogens, including *Aggregatibacter actinomycetemcomitans*, *Prevotella intermedia*, *Prevotella nigrescens*, *Fusobacterium nucleatum*, *Parvimonas micra*, *Eikenella corrodens*, *Capnocytophaga gingivalis*, *Treponema socranskii*, *Eubacterium nodatum* and *Campylobacter rectus* [41]. Some of them are also associated with canine periodontal disease. *Tannerella forsythia*, *Campylobacter rectus* and *Porphyromonas gulae* were detected in almost all dogs with periodontitis. *Prevotella intermedia* and *Eikenella corrodens* were detected only in few dogs with periodontitis, *Treponema denticola*, *Capnocytophaga ochracea* and *Porphyromonas gingivalis* were detected in only one specimen. In addition, *Prevotella nigrescens* and *Aggregatibacter actinomycetemcomitans* were not detected in any of the specimens [42]. *Actinomyces canis* and *Corynebacterium canis* are significantly more prevalent in dogs with periodontitis than in healthy dogs [23, 24].

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The genus *Porphyromonas* is phylogenetically classified in the family *Porphyromonadaceae*, order *Bacteroidales*, class *Bacteroides* and phylum *Bacteroidetes*. Representatives of this genus are Gram-negative, obligate anaerobic, non-motile and non-spore-forming rods. Several species of *Porphyromonas*, including *P. endodontalis*, *P. circumdentaria* and *P. gingivalis* were isolated from the biofilm of adult dogs, but not from any oral sites of puppies and adolescent dogs [45]. Also, several new *Porphyromonas* species (*P. gulae*, *P. macacae*, *P. cangingivalis*, *P. cansulci*, *P. creviocanis*, *P. gingivacanis*, *P. canoris*, *P. denticanis*) associated with periodontal disease have been described [23]. In humans, the major periodontal pathogen is *P. gingivalis* [46]. There are several differences between isolates *Porphyromonas* from humans and from dogs. For example, *P. gingivalis* isolates of canine origin are catalase positive, but isolates of human origin are catalase negative. These catalase positive organisms like *P. gingivalis* may represent the species *P. gulae* [47].

*P. gulae* is rarely found in humans and healthy animals, usually is isolated from animals, including dogs, with active periodontitis [48]. From dogs with periodontal disease are most frequently isolated three *Porphyromonas* species: *P. gulae*, *P. salivosa* (current name, *P. macacae*) and *P. denticanis* [49]. Of these only, *P. gulae* exhibits virulence characteristics similar to those of the human periodontal pathogen *P. gingivalis* such as lysyl- and arginyl-specific proteolytic activity of the gingipains. This finding suggested that *P. gulae* may play a key role in the development of periodontitis in dogs [50]. In addition, fimbrial protein with the same size and antigenicity similar the 41-kDa fimbrial subunit protein (fimbrillin, FimA) of *P. gingivalis* was identified in *P. gulae* [51]. The fimbria 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 of *Porphyromonas* that causes periodontal tissue destruction [52].

*P. cangingivalis* is the most prevalent canine oral bacterial species in both plaque from healthy gingiva and plaque from dogs with early periodontitis. The ability of *P. cangingivalis* to predominate in both health and disease environments suggests that it is both metabolically flexible enough to colonize in health and also able to compete against other *Porphyromonas* spp. in a disease environment [24]. *P. cangingivalis* has a complete protoporphyrin IX synthesis pathway potentially allowing it to synthesize its own heme unlike pathogenic Porphyromonads such as *P. gingivalis* that acquire heme predominantly from blood. The ability to synthesize siroheme and vitamin B12 point to enhanced metabolic flexibility for *P. cangingivalis*, which may underlie its prevalence in the canine oral cavity [53].
