Porphyromonas Gulae Research Articles

Microbial Composition of Extracted Dental Alveoli in Dogs with Advanced Periodontitis.

Periodontitis is a serious gum infection that damages the soft tissue and destroys the bone supporting the teeth. The aim of the study was to investigate the microbiota using traditional microbiology plating and metagenomic sequencing of extracted tooth alveoli in dogs with severe periodontitis. Isolation of culturable microorganisms was performed as part of bacteriological testing to provide bacteriological diagnosis to veterinary surgeons. Metagenomic sequencing was performed using shotgun sequencing on the Illumina HiSeq system platform. The most prevalent species at sites of periodontal infection detected by metagenomic sequencing were Porphyromonas gulae, Prevotella spp., Tannerella forsythia, Porphyromonas crevioricanis, Porphyromonas cangingivalis, and Bacteroides heparinolyticus. Pasteurella, Streptococcus, and Neisseria were the most frequently isolated culturable bacteria from infected sites detected by traditional microbiologic methods. Metagenomic data revealed that these three genera accounted for only 1.6% of all microbiota at the sites of infection. Antimicrobial resistance patterns of the isolated bacteria included resistance to ampicillin, doxycycline, sulfamethoxazole-trimethoprim, ciprofloxacin, colistin, cefotaxime, and chloramphenicol. Antimicrobial-resistant genes detected using shotgun sequencing also showed resistance to aminoglycosides and macrolides. Dogs with periodontal infections carry bacteria that can cause bite infections in humans as well as multi-resistant isolates. Therefore, treatment and prophylaxis or periodontal disease of dogs is important from a One Health perspective.

Bacterial DNA and serum IgG antibody titer assays for assessing infection of human-pathogenic and dog-pathogenic Porphyromonas species in dogs

Periodontal disease is highly prevalent in both humans and dogs. Although there have been reports of cross-infection of periodontopathic bacteria, methods for assessing it have yet to be established. The actual status of cross-infection remains to be seen. The purpose of this study was to evaluate the utility of bacterial DNA and serum immunoglobulin G (IgG) antibody titer assays to assess infection of human-pathogenic and dog-pathogenic Porphyromonas species in dogs. Four experimental beagles were used for establishing methods. Sixty-six companion dogs at veterinary clinics visiting for treatment and prophylaxis of periodontal disease were used and divided into healthy, gingivitis, and periodontitis groups. Periodontal pathogens such as Porphyromonas gingivalis and Porphyromonas gulae were investigated as target bacteria. DNA levels of both bacteria were measured using species-specific primers designed for real-time polymerase chain reaction (PCR). Serum IgG titers of both bacteria were measured by enzyme-linked immunosorbent assay (ELISA).PCR primers were confirmed to have high sensitivity and specificity. However, there was no relationship between the amount of bacterial DNA and the severity of the periodontal disease. In addition, dogs with periodontitis had higher IgG titers against both bacteria compared to dogs in the healthy and gingivitis groups; there was cross-reactivity between the two bacteria. Receiver operating characteristic (ROC) analysis of IgG titers against both bacteria showed high sensitivity (>90 %) and specificity (>75 %). Since both bacteria were distinguished by DNA assays, the combination of these assays may be useful in the evaluation of cross-infection.

Investigation of periodontal disease development and Porphyromonas gulae FimA genotype distribution in small dogs

In dogs, Porphyromonas gulae is a major periodontal pathogen with 41-kDa proteins polymerizing to form a filamentous structure called fimbriae or pili, termed FimA. FimA is classified into three genotypes: A, B, and C, and there are combinations of types A, B, C, A/B, A/C, B/C, and A/B/C. Periodontal disease is the most common oral disease in small dogs, but the periodontal disease status and P. gulae colonization at each dog age and breed remain unclear. In this study, we stratified 665 small dogs and analyzed the periodontal status and distribution of P. gulae with each FimA genotype. Dogs with periodontal disease and FimA genotype tended to increase with age. The dogs with at least one FimA genotype had significantly more severe periodontal disease compared with P. gulae-negative dogs (P < 0.01). Additionally, periodontal status was significantly associated with specific FimA genotype distribution in Toy Poodles and Chihuahuas (P < 0.05), whereas there was no such association in Dachshunds. These results suggest that the onset of periodontal disease and P. gulae colonization are related and progress with age. The relationship between periodontal disease and FimA genotype may differ depending on the dog breeds.

Evaluation of the antibacterial effect of Epigallocatechin gallate on the major pathogens of canine periodontal disease and therapeutic effects on periodontal disease mice.

Periodontal disease (PD) is a prevalent oral affliction in canines, with limited therapeutic options available. The potential transmission of oral bacteria from canines to humans through inter-species contact poses a risk of zoonotic infection. Epigallocatechin gallate (EGCG), the principal catechin in green tea polyphenols, exhibits antibacterial properties effective against human PD. Given the clinical parallels between canine and human PD, this study explores the feasibility of employing EGCG as a therapeutic agent for canine PD. Initially, a survey and statistical analysis of bacterial infection data related to canine PD in China were conducted. Subsequently, the primary pathogenic bacteria of canine PD were isolated and cultivated, and the in vitro antibacterial efficacy of EGCG was assessed. Furthermore, verify the therapeutic effect of EGCG on a mouse PD model in vivo. The high-throughput 16S rRNA gene sequencing identified Porphyromonas, Fusobacterium, Treponema, Moraxella, and Capnocytophaga as the genera that distinguishing PD from healthy canines' gingival crevicular fluid (GCF) samples in China. The anaerobic culture and drug susceptibility testing isolated a total of 92 clinical strains, representing 22 species, from 72 canine GCF samples, including Porphyromonas gulae, Prevotella intermedia, Porphyromonas macacae, etc. The minimum inhibitory concentration (MIC) ranging of EGCG was from 0.019 to 1.25 mg/mL. Following a 7 days oral mucosal administration of medium-dose EGCG (0.625 mg/mL), the abundance of periodontal microorganisms in PD mice significantly decreased. This intervention ameliorated alveolar bone loss, reducing the average cementoenamel junction to the alveolar bone crest (CEJ-ABC) distance from 0.306 mm ± 0.050 mm to 0.161 mm ± 0.026 mm. Additionally, EGCG (0.3125 mg/mL) markedly down-regulated the expression of inflammatory factor IL-6 in the serum of PD mice. Our research demonstrates the significant antibacterial effects of EGCG against the prevalent bacterium P. gulae in canine PD. Moreover, EGCG exhibits anti-inflammatory properties and proves effective in addressing bone loss in a PD mouse model. These findings collectively suggest the therapeutic potential of EGCG in the treatment of canine PD. The outcomes of this study contribute valuable data, laying the foundation for further exploration and screening of alternative antibiotic drugs to advance the management of canine PD.

Fusobacterium necrophorum predomina na microbiota do abscesso dentário mandibular em Blastocerus dichotomus

ABSTRACT: Dental abscess in ruminants is an acute polymicrobial infection, usually resulting from periodontal disease or endodontic infection, with consequences for animal health and welfare. The present study aimed to describe the bacterial microbiota of dental abscesses in Blastocerus dichotomus. Biological material from mandibular dental abscesses, punctured with a sterile syringe and needle during routine veterinary curative procedures or necropsies, was collected from three ex-situ marsh deer. Bacteria were identified using high-throughput sequencing of the 16S ribosomal RNA gene. The three specimens had the presence of facial bulging, and two died because of severe emaciation with a history of progressive weight loss. Bacteroides (38.6%), Fusobacterium (36.65%), and Porphyromonas (7.49%) represented the most abundant genera and Fusobacterium necrophorum (35.69%), Porphyromonas levii (3.12%) and Porphyromonas gulae (1.78%) were among the ten most represented species in the microbiota of mandibular abscess in Blastocerus dichotomus. These molecular findings demonstrate a broader diversity of species in the polymicrobial nature of dental abscesses in B. dichotomus than was previously reported when culture-dependent methods were used in the diagnosis.

Longitudinal Analysis of Canine Oral Microbiome Using Whole Genome Sequencing in Aging Companion Dogs.

Aged companion dogs have a high prevalence of periodontal disease and canine cognitive dysfunction syndrome (CCDS) and the two disorders are correlated. Similarly, periodontal disease and Alzheimer's Disease are correlated in people. However, little is known about the oral microbiota of aging dogs. The goal of this project was to characterize the longitudinal changes in oral microbiota in aged dogs. Oral swabs were taken from ten senior client-owned dogs on 2-3 occasions spanning 24 months and they underwent whole genome shotgun (WGS) sequencing. Cognitive status was established at each sampling time. A statistically significant increase in alpha diversity for bacterial and fungal species was observed between the first and last study visits. Bacteroidetes and proteobacteria were the most abundant bacterial phyla. Porphyromonas gulae was the most abundant bacterial species (11.6% of total reads). The species Lactobacillus gasseri had a statistically significant increase in relative abundance with age whereas Leptotrichia sp. oral taxon 212 had a statistically significant positive longitudinal association with cognition score. There is an increased fungal and bacterial alpha diversity in aging dogs over time and nearly universal oral dysbiosis. The role of the oral microbiota, particularly Leptotrichia and P. gulae and P. gingivalis, in aging and CCDS warrants further investigation.

Exploring the genetic and functional diversity of Porphyromonas gingivalis long fimbriae.

Porphyromonas gingivalis is a key pathobiont in periodontitis. Its long fimbriae consist of a single anchor (FimB), a varying number of stalk (FimA), and three accessory (tip-related) proteins (FimC, FimD, and FimE). Based on 133 strains/genomes available, it was our aim to investigate the diversity within FimA and FimB and explain the variety of long fimbriae (super-)structures. Combining the new forward primer fimAnewF with the established fimAunivR, we were able to amplify and sequence fimA including its leader region covering all genotypes and serotypes for phylogenetic analysis. We designed two primer pairs sensing the presence of an internal stop codon in fimB with an impact on fimbrial length. Finally, we examined fimbrial secondary structures by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The phylogeny of fimA/FimA revealed two new subtypes (IIa and IIb) with specific changes in functional domains and thus adding to the current classification scheme (I, Ib, and II-V). Regarding evolution, we confirm that Porphyromonas gulae fimA-type A is closely related to human P. gingivalis strains of cluster Ib and might be its ancestor genotype. A fimB internal stop codon is rare and was found in ATCC 33277 only. Comparing P. gingivalis TEM/SEM pictures of type I ATCC 33277 with type V OMI622 revealed a broad spectrum of fimbrial structures including bundling, cell-cell knotting, and brick-wall formation. In conclusion, FimA forms more distinct subtypes than previously known. The bundling of long fimbriae, a mechanism known from EPEC/EHEC and Salmonella, is proposed and supported by TEM/SEM pictures for the first time here. The role and variations of terminal accessory FimC-E in superstructure formation and/or (co-) adhesion should be investigated more closely next.

The Effect of Subacute Oral Folic Acid Treatment on Growth of Porphyromonas gulae in Dogs.

Periodontitis is one of the most prevalent infectious diseases in humans and animals. It is a multifactorial disease resulting in attachment loss and tooth loss. Therefore, preventive dentistry, such as daily teeth cleaning or providing dental chews from puppyhood is essential. This study aimed to find an alternative option for preventive dentistry by examining both in vitro and clinically, the antibacterial, antihalitosis, and anti-inflammatory effects of folic acid (FA) in dogs with periodontal disease. The antibacterial and antihalitosis responses of FA were evaluated in vitro using Porphyromonas gulae, a bacterium that plays a significant role in the development of periodontal disease in dogs. Anti-inflammatory responses, such as secretion of IL-1β, IL-6, and IL-8 induced by P. gulae infection in human gingival epithelium have been studied. This study used dogs with P. gulae-associated periodontal diseases and was conducted by providing a dental chew containing 0.13% FA for 28 days. The viability and halitosis production (hydrogen sulfide and methyl mercaptan) of P. gulae was significantly inhibited by FA in a dose and time-dependent manner. IL-1β, IL-6, and IL-8 secretion were also significantly suppressed by FA treatment in a dose-dependent manner. In vitro bactericidal, antihalitosis, and anti-inflammatory effects of FA were confirmed in dogs with P. gulae-associated periodontal disease. One month of oral treatment with 0.13% FA-containing dental chews significantly reduced halitosis as well as P. gulae activity. This study suggests that oral treatment with FA can be a preventive option for periodontal disease in dogs as well as humans.

Bioinformatic mining for RiPP biosynthetic gene clusters in Bacteroidales reveals possible new subfamily architectures and novel natural products.

The Bacteroidales order, widely distributed among diverse human populations, constitutes a key component of the human microbiota. Members of this Gram-negative order have been shown to modulate the host immune system, play a fundamental role in the gut's microbial food webs, or be involved in pathogenesis. Bacteria inhabiting such a complex environment as the human microbiome are expected to display social behaviors and, hence, possess factors that mediate cooperative and competitive interactions. Different types of molecules can mediate interference competition, including non-ribosomal peptides (NRPs), polyketides, and bacteriocins. The present study investigates the potential of Bacteroidales bacteria to biosynthesize class I bacteriocins, which are ribosomally synthesized and post-translationally modified peptides (RiPPs). For this purpose, 1,136 genome-sequenced strains from this order were mined using BAGEL4. A total of 1,340 areas of interest (AOIs) were detected. The most commonly identified enzymes involved in RiPP biosynthesis were radical S-adenosylmethionine (rSAM), either alone or in combination with other biosynthetic enzymes such as YcaO. A more comprehensive analysis of a subset of 9 biosynthetic gene clusters (BGCs) revealed a consistent association in Bacteroidales BGCs between peptidase-containing ATP-binding transporters (PCATs) and precursor peptides with GG-motifs. This finding suggests a possibly shared mechanism for leader peptide cleavage and transport of mature products. Notably, human metagenomic studies showed a high prevalence and abundance of the RiPP BGCs from Phocaeicola vulgatus and Porphyromonas gulae. The mature product of P. gulae BGC is hypothesized to display γ-thioether linkages and a C-terminal backbone amidine, a potential new combination of post-translational modifications (PTM). All these findings highlight the RiPP biosynthetic potential of Bacteroidales bacteria, as a rich source of novel peptide structures of possible relevance in the human microbiome context.

Porphyromonas gulae and PPAD antibodies are not related to citrullination in rheumatoid arthritis

IntroductionPorphyromonas gulae have the enzyme PPAD, as P. gingivalis, which is responsible for citrullination related to the pathophysiology of rheumatoid arthritis and periodontitis; this implies the presence of two species of PPAD-producing bacteria in the mouth as well as the presence of citrullinated proteins. There are no previous reports or studies investigating an association between P. gulae PPAD in rheumatoid arthritis (RA).ObjectiveTo assess the presence of P. gulae and anti-citrullinated peptide antibodies of P. gulae PAD in patients with RA and their possible relationship with clinical activity markers.Subjects and methodsA total of 95 patients with RA and 95 controls were included. Erythrocyte sedimentation rate (ESR), C-reactive protein, anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) were measured. Activity index-28 (DAS28) and SCDAI. The periodontal diagnosis was established. Presence of P. gulae and P. gingivalis. An ELISA was used to determine antibodies against citrullinated peptides of P. gulae PAD.ResultsA P. gulae frequency of 15.8% was observed in the RA group and 9.5% in the control group. Higher levels of ACPA were found in the P. gulae-positive patients of the RA group, finding no significant difference, but if in patients positive for P. gingivalis with statistical significance (p = 0.0001). The frequency of anti-VDK-cit and anti-LPQ-cit9 antibodies to PPAD of P. gulae was higher in the RA group than in the control group without significant difference. No relationship was found with the clinical variables despite the presence of P. gulae and anti-citrullinated peptide antibodies of P. gulae PPAD in patients with RAConclusionsIt was not possible to establish a connection with clinical variables in RA and P. gulae; as a result, the presence of P. gingivalis continues to contribute significantly to the increase in antibodies against citrullinated proteins/peptides from exogenous sources of citrullination in RA and periodontitis.

Bestatin as a treatment modality in experimental periodontitis.

Chronic periodontitis (CP), the most prevalent dysbiotic bacteria-driven chronic inflammatory disease, is an underestimated global health problem in itself, and due to a causative relationship with other disorders such as cardiovascular diseases or Alzheimer disease. The CP pathogenesis is primarily driven by Porphyromonas gingivalis in humans, and Porphyromonas gulae in dogs. These microorganisms initiate a pathogenic shift in the composition of the tooth-surface microflora. Our objective was to evaluate antimicrobial effects of bestatin, a potential CP drug candidate. We evaluated bestatin bacteriostatic efficiency against periodontopathogens in planktonic cultures via microplate assay, and mono- and multispecies oral biofilm models. Neutrophil bactericidal activities, such as phagocytosis, were investigated in vitro using granulocytes isolated from the peripheral blood. The therapeutic efficacy and the immunomodulatory function of bestatin was assessed in a murine model of CP. Bestatin exhibited bacteriostatic activity against both P. gingivalis and P. gulae, and controlled the formation and species composition of the biofilm. We demonstrated that bestatin promotes the phagocytosis of periodontopathogens by neutrophils. Finally, we found that providing bestatin in the animal feed prevented alveolar bone resorption. We show that in a murine model of CP bestatin not only shifted the biofilm species composition from pathogenic to a commensal one, but also promoted bacteria clearance by immune cells and alleviated inflammation. Taken together, these results suggest that bestatin is a promising drug choice for the treatment and/or prevention of periodontitis and clinical trials are required to fully evaluate its potency.

Porphyromonas gulae infection in canines, pet owners and veterinarians in China: an epidemiological study and risk factor analysis

Porphyromonas gulae is a clinically prevalent, anaerobic, oral bacteria in canines, that may be a causative agent of canine periodontal disease, and a potential threat to human oral health. Research on P. gulae pathogenicity in canines, their owners, and veterinarians is lacking in China. This study aimed to determine the isolation and detection rates of P. gulae in gingival crevicular fluid (GCF) samples from 101 canines in Beijing, using anaerobic culture techniques and 16S rRNA gene sequencing. The main risk factors for the transmission of P. gulae from canines to humans were also analyzed through analyzing the statistical data on risk factor variables from 103 canine owners and 60 veterinarians in Beijing who tested positive for P. gulae detection in GCF samples. The isolation and detection rates of P. gulae in canines were 31.5% (29/92) and 92.1% (93/101), respectively, compared with detection rates of 24.3% (25/103) in canine owners, 43.3% (26/60) in veterinarians, and 52.0% (13/25) in dentists. The degree of contact with canines (P = 0.001, P < 0.01) and smoking (P = 0.021, P < 0.05) were significant risk factors for P. gulae detection in owners. Moreover, the degree of contact during ultrasonic scaling (P = 0.065, 0.05 < P < 0.1) was the most important risk factor for the positive detection of P. gulae in veterinarians. These findings suggest that P. gulae may colonize the human oral cavity through intimate contact with canines or participation in dental ultrasonic scaling operations.Graphical

Presence of zoonotic black-pigmented periodontal pathogens in the oral microbiota of pet and stray cats

Black-pigmented bacteria are one of the neglected species to cause periodontal disease in cats, and they are also zoonotic agents that pose an infection risk to humans. In this study, we aimed to determine the presence of Porphyromonas gingivalis, Porphyromonas gulae and Prevotella nigrescens in the oral microbiota of pet and stray cats. Dental swab samples were taken from 25 pet cats and 25 stray cats with symptoms of periodontal disease and then investigated by multiplex polymerase chain reaction using 16S rRNA species-specific primers. As a result of the multiplex PCR analysis, P. gingivalis 3/25 (12%), P. nigrescens 1/25 (4%), P. gingivalis + P. gulae 7/25 (28%), P. gingivalis + P. nigrescens 1/25 (4%), P. gulae + P. nigrescens 1/25 (4%), and P. gingivalis + P. gulae + P. nigrescens 2/25 (8%) were molecularly typed in the pet cats. In addition, 1/25 (4%) of P. gulae and 21/25 (84%) of P. gingivalis + P. gulae were typed in the stray cats. In 10/25 (40%) pet and 3/25 (12%) stray cat samples, no bacteria were detected by molecular typing. In summary, the results provide strong evidence that black-pigmented zoonotic pathogens are associated with cat periodontal disease.

Possible association of fimA genotype of Porphyromonas gulae with the severity of periodontal disease and the number of permanent teeth in dogs.

Previous research has demonstrated that Porphyromonas gulae (P. gulae) significantly contributes to the development of periodontal disease in dogs. Porphyromonas gulae is divided into three subtypes according to the 41-kDa filamentous appendage (fimA), defined as types A, B, and C. This study aimed to elucidate the association between fimA type of P. gulae with the number of permanent teeth, reflecting the severity of periodontal disease. Two hundred twenty-five dogs were categorized by P. gulae fimA type as negative, type A dominant, type B dominant, and type C dominant. The stage of periodontal disease in P. gulae-positive dogs increased with age, particularly in type C dominant dogs. Correspondingly, the number of permanent teeth in P. gulae fimA type C-dominant dogs was significantly lower than that of P. gulae-negative dogs, suggesting there is a significant association between fimA type of P. gulae and the number of permanent teeth resulting from the development of periodontal disease.

The association between gingivitis and oral spirochetes in young cats and dogs.

Although gingivitis frequently occurs in young cats, spirochetes are often found in the early stages of periodontal disease. This study was conducted to determine the association between gingivitis and oral spirochetes in young cats and dogs. The degree of gingivitis was evaluated in a total of 68 cats and 31 dogs under one year of age, and plaques were collected from each carnassial. To detect spirochetes or Porphyromonas gulae in plaque samples, 16S rRNA gene was amplified by polymerase chain reaction (PCR) using specific primers. All data were analyzed using Fisher's exact probability test and odds ratio (OR) with a 95% confidence interval (95% CI). The prevalence of gingivitis was significantly higher in young cats (92.6%) than in young dogs (45.2%). The positive rate of spirochetes by PCR in gingivitis cases was 85.4% in young cats and 15.4% in young dogs, and the positive rate of P. gulae was 66.7% in young cats and 15.4% in young dogs. Both results were significantly higher in young cats than in young dogs. In young cats, spirochetes were significantly associated with gingivitis (OR = 7.95; 95% CI = 1.17, 53.83; P < 0.05), but P. gulae was not (OR = 2.44; 95% CI = 0.38, 15.66; P = 0.23). These results suggest that spirochetes may be associated with the early stages of periodontal disease in cats.

Prevalence of Periodontal Pathogens in Slovak Patients with Periodontitis and Their Possible Aspect of Transmission from Companion Animals to Humans.

Simple SummaryPeriodontal disease represents a worldwide health problem. Human periodontal pathogens such as Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans are the cause of inflammatory response resulting in periodontitis. Porphyromonas gulae is mostly involved in periodontitis in dogs; however, it is not a common pathogen in humans. This study deals with the prevalence of periodontal pathogens in Slovak patients with periodontitis. Furthermore, based on the previous findings of animal-to-human transmission of periodontal pathogens, this study also assesses the possible bacterial transmission between animals and their owners. The highest prevalence in Slovak patients amongst the monitored periodontal pathogens had T. forsythia. In regard to the limited information available on T. forsythia, antibiotic sensitivity of this bacterium was evaluated. Most of the T. forsythia isolates were susceptible to antibiotics, namely amoxicillin-clavulanic acid, clindamycin and moxifloxacin, while they were resistant to metronidazole. Moreover, the transmission of P. gulae between animals and their owners was confirmed. Based on the similarity of P. gulae with human P. gingivalis, there arises the question as to whether P. gulae can also be involved in the periodontitis pathogenesis in humans. However, more studies are required for further clarification.Oral health and diseases are greatly influenced by oral bacteria. During dysbiosis, bacterial composition changes, which can lead to periodontitis. Periodontitis in humans is associated with periodontal pathogens such as Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans. Animal-to-human transmission of some of these pathogens has also been reported. The aim of this study was to evaluate the prevalence of periodontal pathogens in Slovak patients and to assess the possible risk of transmission of these pathogens from animals to their owners. The presence of periodontal pathogens in dental plaque was monitored by PCR. Amplified products were analysed using Sanger sequencing. T. forsythia isolates were assessed for the susceptibility to different antibiotics using the disk diffusion method. In humans, T. denticola, P. gingivalis, T. forsythia and A. actinomycetemcomitans were present in 69.23%, 69.23%, 100% and 84.62%, respectively. Most isolates of T. forsythia were susceptible to amoxicillin-clavulanic acid, clindamycin and moxifloxacin, but they were resistant to metronidazole. The transmission of T. forsythia from animals to their owners was not proven based on sequence analysing. On the other hand, transmission of Porphyromonas gulae was confirmed, but the risk of its involvement in the pathogenesis of periodontitis in humans must be further investigated.

Inhibitory Effect of Bacillus licheniformis Strains Isolated from Canine Oral Cavity.

Bacillus licheniformis is used in a broad spectrum of areas, including some probiotic preparations for human and veterinary health. Moreover, B. licheniformis strains are known producers of various bioactive substances with antimicrobial and antibiofilm effects. In searching for new potentially beneficial bacteria for oral health, the inhibitory effect of B. licheniformis strains isolated from canine dental biofilm against pathogenic oral bacteria was evaluated. The antimicrobial effect of neutralized cell-free supernatants (nCFS) was assessed in vitro on polystyrene microtiter plates. Furthermore, molecular and morphological analyses were executed to evaluate the production of bioactive substances. To determine the nature of antimicrobial substance present in nCFS of B. licheniformis A-1-5B-AP, nCFS was exposed to the activity of various enzymes. The nCFS of B. licheniformis A-1-5B-AP significantly (p < 0.0001) reduced the growth of Porphyromonas gulae 3/H, Prevotella intermedia 1/P and Streptococcus mutans ATCC 35668. On the other hand, B. licheniformis A-2-11B-AP only significantly (p < 0.0001) inhibited the growth of P. intermedia 1/P and S. mutans ATCC 35668. However, enzyme-treated nCFS of B. licheniformis A-1-5B-AP did not lose its antimicrobial effect and significantly (p < 0.0001) inhibited the growth of Micrococcus luteus DSM 1790. Further studies are needed for the identification of antimicrobial substances.

Erythritol inhibits the growth of periodontal-disease-associated bacteria isolated from canine oral cavity

Periodontal disease (PD) is the most common oral disease that is caused by infection with periodontal-disease-associated bacteria (PDAB) such as Porphyromonas gulae and Porphyromonas macacae in dogs as well as in humans. Unlike humans, most dogs do not follow daily oral hygiene routine, and this results in many dogs being affected by PD. Thus, to prevent PD, it is important to control PDAB. Xylitol is a sugar alcohol that inhibits the growth of oral bacteria in humans. However, xylitol is poisonous to dogs and can lead to hypoglycemia and hepatic failure. Herein, we show the inhibitory effect of erythritol, a sugar alcohol that can be used safely in dogs, on the growth of PDAB isolated from dogs with PD. Oral bacteria were isolated from the oral cavities of dogs with PD, and the distribution of PDAB was evaluated. Interestingly, Porphyromonas gingivalis, a bacterium typically responsible for PD in humans, was not isolated from dog samples. The bacteriostatic effect of erythritol supplementation was investigated on isolated PDAB in vitro. Our results show that erythritol exert bacteriostatic effects on PDAB comparable to xylitol. Thus, application of erythritol can be suggested to control PDAB in dogs in the future.

In vitro effectiveness of pomegranate extract present in pet oral hygiene products against canine oral bacterial species.

Background and Aim:Pomegranate is known to possess antibacterial properties, partly because of its punicalagin content. However, its effect on canine oral bacterial species has not yet been elucidated. In this study, we evaluated the effect of pomegranate extract present in pet dental products on the growth and survival of five canine oral bacterial species in biofilms.Materials and Methods:Five bacterial species, Neisseria shayeganii, Neisseria canis, Porphyromonas gulae, Porphyromonas macacae, and Porphyromonas crevioricanis, were individually cultured for biofilm formation and exposed to pomegranate extract (or control) for 15 min. Cell survival was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and was compared between different conditions using a student’s t-test. In addition, the individual strains were grown in planktonic suspensions and exposed to serial dilutions of the extract to determine the minimum inhibitory concentration.Results:At a concentration of 0.035% w/v, the extract significantly reduced the survival of P. gulae (−39%, p < 0.001) and N. canis (−28%, p = 0.08) in biofilms. At similar concentrations, the extract also completely or partially inhibited the growth of N. canis and Porphyromonas spp. in planktonic suspensions, respectively.Conclusion:The pomegranate extract found in some pet dental products can limit bacterial growth and survival in the biofilms formed by N. canis and P. gulae in vitro. As P. gulae is involved in periodontal disease progression, limiting its proliferation using products containing pomegranate extract could contribute to disease prevention. Further studies on dogs receiving such products are necessary to confirm these effects.

Green tea catechins inhibit Porphyromonas gulae LPS-induced inflammatory responses in human gingival epithelial cells

ObjectivesTo determine the anti-inflammatory effects of green tea catechins in immortalized human gingival epithelial cells (Ca9-22) stimulated with Porphyromonas gulae lipopolysaccharide (LPS). MethodsCa9-22 cells were incubated with P. gulae LPS (10 μg/ml) with or without green tea catechins, epigallocatechin-3-gallate (EGCg), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC) (each at 50 μM), for 6 or 24 h. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay were used to determine the induction of cyclooxygenase 2 (COX2), tumor necrosis factor alpha (TNF-ɑ), interleukin 6 (IL-6), and IL-8. Furthermore, the expression of toll-like receptors (TLRs) 2 and 4 was examined using real-time PCR and western blotting analysis, and phosphorylation of the p38 and ERK1/2 was examined using western blotting analysis. ResultsAt the mRNA and protein levels, EGCg, EGC, ECG, and EC were found to significantly inhibit COX2, TNF-ɑ, IL-6, and IL-8. Furthermore, the levels of ERK1/2 and p38 phosphorylation induced by P. gulae LPS were decreased following the addition of each of the catechins, as well as TLR2 and 4 mRNA and protein. ConclusionsThese findings indicate that green tea catechins are potent inhibitors of inflammatory responses induced by P. gulae LPS, and may also be useful for prevention and/or attenuation of periodontitis.