Antibiotic Resistance among Fusobacterium, Capnocytophaga, and Leptotrichia Species of the Oral Cavity.

PURPOSE: Antibiotics play an important role in treating periodontal diseases. Due to the effectiveness of antibiotic therapies, their usage in dentistry has significantly increased. The aim of this study focused on the in-vitro susceptibility of different gram-negative oral bacteria species - which are associated with periodontal diseases (Fusobacterium spp., Capnocytophaga spp. and Leptotrichia buccalis) and have different geographical origins (Asia and Europe) - against antimicrobials that are clinically relevant in dental therapy. MATERIALS AND METHODS: A total of 45 strains were tested (29 Fusobacterium spp., 13 Capnocytophaga spp. and 3 L. buccalis) that were either isolated from Chinese patients or were obtained from different strain collections. Their antimicrobial susceptibility to the antimicrobial agents benzylpenicillin, amoxicillin, amoxicillin-clavulanic acid, ciprofloxacin, moxifloxacin, clindamycin, doxycycline, tetracycline and metronidazole was tested using the E-Test. Strains with particular resistance to penicillin, clindamycin and metronidazole were further analysed for resistance genes. RESULTS: All tested bacterial isolates were sensitive to amoxicillin, amoxicillin-clavulanic acid, doxycycline and tetracycline, but showed variable sensitivity towards other antibiotics such as benzylpenicillin, ciprofloxacin, moxifloxacin, clindamycin and metronidazole. CONCLUSION: The results of the present study suggest that certain periodontal disease-related bacterial strains can be resistant towards antimicrobial agents commonly used in adjuvant periodontal therapy.

In vitro versus in situ biofilms for evaluating the antimicrobial effectiveness of herbal mouthrinses.

For centuries, diverse mouthrinses have been applied for medicinal purposes in the oral cavity. In view of the growing resistance of oral microorganisms against conventional antimicrobial agents e.g. chlorhexidine, the implementation of alternative treatments inspired by nature has lately gained increasing interest. The aim of the present study was to compare in vitro biofilm models with in situ biofilms in order to evaluate the antimicrobial potential of different natural mouthrinses. For the in vitro study a six-species supragingival biofilm model containing A. oris, V. dispar, C. albicans, F. nucleatum, S. mutans and S. oralis was used. Biofilms were grown anaerobically on hydroxyapatite discs and treated with natural mouthrinses Ratanhia, Trybol and Tebodont. 0.9% NaCl and 10% ethanol served as negative controls, while 0.2% CHX served as positive control. After 64h hours, biofilms were harvested and quantified by cultural analysis CFU. For the in situ study, individual test splints were manufactured for the participants. After 2h and 72h the biofilm-covered samples were removed and treated with the mouthrinses and controls mentioned above. The biofilms were quantified by CFU and stained for vitality under the confocal laser scanning microscope. In the in vitro study, 0.2% CHX yielded the highest antimicrobial effect. Among all mouthrinses, Tebodont (4.708 ± 1.294 log10 CFU, median 5.279, p<0.0001) compared with 0.9% NaCl showed the highest antimicrobial potential. After 72h there was no significant reduction in CFU after 0.2% CHX treatment. Only Trybol showed a statistically significant reduction of aerobic growth of microorganisms in situ (5.331 ± 0.7350 log10 CFU, median 5.579, p<0.0209). After treatment with the positive control 0.2% CHX, a significant percentage of non-vital bacteria (42.006 ± 12.173 log10 CFU, median 42.150) was detected. To sum up, a less pronounced effect of all mouthrinses was shown for the in situ biofilms compared to the in vitro biofilms.

The antimicrobial effect of Rosmarinus officinalis extracts on oral initial adhesion ex vivo.

OBJECTIVE: In the last few decades, there has been a growing worldwide interest in the use of plant extracts for the prevention of oral diseases. The main focus of this interest lies in the identification and isolation of substances that limit the formation of microbial biofilm which plays a major role in the development of caries, periodontitis, and peri-implantitis. In this clinical ex vivo study, we investigated the antimicrobial effects of Rosmarinus officinalis extract against oral microorganisms within in situ initial oral biofilms. MATERIALS AND METHODS: Initial in situ biofilm samples (2 h) from six healthy volunteers were treated ex vivo with R. officinalis extract at concentrations of 20 mg/ml and 30 mg/ml. The number of viable bacterial cells was determined by counting the colony-forming units. All surviving bacteria were isolated in pure cultures and identified using MALDI-TOF and biochemical testing procedures. Additionally, live/dead staining in combination with epifluorescence microscopy was used for visualizing the antimicrobial effects in the initial biofilms. RESULTS: The number of colony-forming units in the R. officinalis-treated biofilms was significantly lower than in the untreated controls (p < 0.001). The reduction range of log10 was 1.64-2.78 and 2.41-3.23 for aerobic and anaerobic bacteria, respectively. Regarding the bacterial composition, large intra- and interindividual variability were observed. Except for Campylobacter spp., the average amount of all bacterial taxa was lower after treatment with R. officinalis than in the untreated biofilms. A total of 49 different species were detected in the untreated biofilms, while only 11 bacterial species were detected in the R. officinalis-treated biofilms. Live/dead staining confirmed that the R. officinalis-treated biofilms had significantly lower numbers of surviving bacteria than the untreated biofilms. CONCLUSIONS: The treatment with R. officinalis extract has a significant potential to eliminate microbial oral initial biofilms. CLINICAL RELEVANCE: The results of this study encourage the use of R. officinalis extracts in biofilm control and thus in the treatment of caries and periodontitis as a herbal adjuvant to synthetic substances.

Antimicrobial Effects of Inula viscosa Extract on the In Situ Initial Oral Biofilm.

Given the undesirable side effects of commercially used mouth rinses that include chemically synthesized antimicrobial compounds such as chlorhexidine, it is essential to discover novel antimicrobial substances based on plant extracts. The aim of this study was to examine the antimicrobial effect of Inula viscosa extract on the initial microbial adhesion in the oral cavity. Individual test splints were manufactured for the participants, on which disinfected bovine enamel samples were attached. After the initial microbial adhesion, the biofilm-covered oral samples were removed and treated with different concentrations (10, 20, and 30 mg/mL) of an I. viscosa extract for 10 min. Positive and negative controls were also sampled. Regarding the microbiological parameters, the colony-forming units (CFU) and vitality testing (live/dead staining) were examined in combination with fluorescence microscopy. An I. viscosa extract with a concentration of 30 mg/mL killed the bacteria of the initial adhesion at a rate of 99.99% (log10 CFU value of 1.837 ± 1.54). Compared to the negative control, no killing effects were determined after treatment with I. viscosa extract at concentrations of 10 mg/mL (log10 CFU value 3.776 ± 0.831; median 3.776) and 20 mg/mL (log10 CFU value 3.725 ± 0.300; median 3.711). The live/dead staining revealed a significant reduction (p < 0.0001) of vital adherent bacteria after treatment with 10 mg/mL of I. viscosa extract. After treatment with an I. viscosa extract with a concentration of 30 mg/mL, no vital bacteria could be detected. For the first time, significant antimicrobial effects on the initial microbial adhesion in in situ oral biofilms were reported for an I. viscosa extract.

An oral health optimized diet reduces the load of potential cariogenic and periodontal bacterial species in the supragingival oral plaque: A randomized controlled pilot study.

This study aimed to investigate the effects of an oral health optimized diet on the composition of the supragingival oral plaque in a randomized controlled trial. Participants of the standard diet group (n = 5) had a diet high in processed carbohydrates and did not change their dietary behavior during the observation. The healthy diet group (n = 9) had to change the diet after 2 weeks from a diet high in processed carbohydrates to a diet low in carbohydrates, rich in omega-3 fatty acids, rich in vitamins C and D, antioxidants and fiber for 4 weeks. Saliva and supragingival plaque samples were taken at the end of week two and eight of the observation period to investigate the composition of microbiota in saliva and supragingival plaque. Data were subjected to an exploratory analysis to identify significant differences. Statistically significant differences were only found in the healthy diet group between the baseline (week 2) and the final sample (week 8) for specific species in plaque and saliva samples. A reduction of the total counts of Streptococcus mitis group, Granulicatella adiacens, Actinomyces spp., and Fusobacterium spp. was found in plaque samples of the healthy diet group. In saliva samples of the healthy diet group, the total counts of Actinomyces spp. and Capnocytophaga spp. decreased. A diet low in carbohydrates, rich in omega-3 fatty acids, rich in vitamins C and D, and rich in fiber reduced Streptococcus mitis group, Granulicatella adiacens, Actinomyces spp., and Fusobacterium spp. in the supragingival plaque.

Compounds from Olea europaea and Pistacia lentiscus inhibit oral microbial growth.

BACKGROUND: In view of the increasing antibiotic resistance, the introduction of natural anti-infective agents has brought a new era in the treatment of bacterially derived oral diseases. METHODS: The aim of this study was to investigate the antimicrobial potential of five natural constituents of Olea europaea (oleuropein, maslinic acid, hydroxytyrosol, oleocanthal, oleacein) and three compounds of Pistacia lentiscus (24Z-isomasticadienolic acid, oleanolic acid, oleanonic aldehyde) against ten representative oral bacterial species and a Candida albicans strain. After the isolation and quality control of natural compounds, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) assay were performed. RESULTS: Among all O. europaea-derived constituents, maslinic acid was the most active (MIC = 4.9-312 µg mL- 1, MBC = 9.8-25 µg mL- 1) one against oral streptococci and anaerobic pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra), while oleuropein, hydroxytyrosol, oleocanthal and oleacein showed milder, yet significant effects against P. gingivalis and F. nucleatum. Among all P. lentiscus compounds, oleanolic acid was the most effective one against almost all microorganisms with MIC values ranging from 9.8 µg mL- 1 (P. gingivalis) to 625 µg mL- 1 (F. nucleatum, P. micra). In the presence of 24Z-isomasticadienolic acid, a mean inhibitory concentration range of 2.4 µg mL- 1 to 625 µg mL- 1 was observed for strict anaerobia. The MIC value for 24Z-isomasticadienolic acid was estimated between 39 µg mL- 1 (Streptococcus sobrinus, Streptococcus oralis) and 78 µg mL- 1 (Streptococcus mutans). All tested compounds showed no effects against Prevotella intermedia. CONCLUSIONS: Overall, maslinic acid and oleanolic acid exerted the most significant inhibitory activity against the tested oral pathogens, especially streptococci and anaerobic oral microorganisms.

Antimicrobial Photoinactivation Using Visible Light Plus Water-Filtered Infrared-A (VIS + wIRA) and Hypericum Perforatum Modifies In Situ Oral Biofilms.

Due to increasing antibiotic resistance, the application of antimicrobial photodynamic therapy (aPDT) is gaining increasing popularity in dentistry. The aim of this study was to investigate the antimicrobial effects of aPDT using visible light (VIS) and water-filtered infrared-A (wIRA) in combination with a Hypericum perforatum extract on in situ oral biofilms. The chemical composition of H. perforatum extract was analyzed using ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS). To obtain initial and mature oral biofilms in situ, intraoral devices with fixed bovine enamel slabs (BES) were carried by six healthy volunteers for two hours and three days, respectively. The ex situ exposure of biofilms to VIS + wIRA (200 mWcm-2) and H. perforatum (32 mg ml-1, non-rinsed or rinsed prior to aPDT after 2-min preincubation) lasted for five minutes. Biofilm treatment with 0.2% chlorhexidine gluconate solution (CHX) served as a positive control, while untreated biofilms served as a negative control. The colony-forming units (CFU) of the aPDT-treated biofilms were quantified, and the surviving microorganisms were identified using MALDI-TOF biochemical tests as well as 16 S rDNA-sequencing. We could show that the H. perforatum extract had significant photoactivation potential at a concentration of 32 mg ml-1. When aPDT was carried out in the presence of H. perforatum, all biofilms (100%) were completely eradicated (p = 0.0001). When H. perforatum was rinsed off prior to aPDT, more than 92% of the initial viable bacterial count and 13% of the mature oral biofilm were killed. Overall, the microbial composition in initial and mature biofilms was substantially altered after aPDT, inducing a shift in the synthesis of the microbial community. In conclusion, H. perforatum-mediated aPDT using VIS + wIRA interferes with oral biofilms, resulting in their elimination or the substantial alteration of microbial diversity and richness. The present results support the evaluation of H. perforatum-mediated aPDT for the adjunctive treatment of biofilm-associated oral diseases.

Mediterranean herb extracts inhibit microbial growth of representative oral microorganisms and biofilm formation of Streptococcus mutans.

In light of the growing antibiotic resistance, the usage of plant-derived antimicrobial agents could serve as an effective alternative treatment against oral infections. The aim of this study was to investigate the antimicrobial and antibiofilm activity of Mediterranean herb extracts against representative oral microorganisms. The extraction procedures and the analysis of the obtained extracts were performed under established experimental conditions. The minimum inhibitory (MIC) and bactericidal (MBC) concentrations of the methanol extracts of Cistus creticus ssp. creticus, Cistus monspeliensis, Origanum vulgare, Rosmarinus officinalis, Salvia sclarea and Thymus longicaulis against eight typical oral bacteria and the fungus Candida albicans were determined. The antibiofilm activity against Streptococcus mutans was also quantified using the microtiter plate test. Overall, all tested extracts inhibited effectively the screened obligate anaerobic microorganisms and in concentrations ≥0.3 mg ml-1 had moderate to high antibiofilm activity comparable to that of chlorhexidine (CHX) against S. mutans. In particular, R. officinalis (MIC: 0.08-5.00 mg ml-1) and S. sclarea (MIC: 0.08-2.50 mg ml-1) showed the highest antibacterial activity, while Cistus spp., R. officinalis and S. sclarea significantly inhibited S. mutans biofilm formation at 0.60, 1.25 and 2.50 mg ml-1, respectively. Porphyromonas gingivalis and Parvimonas micra were high susceptible to O. vulgare (MIC = 0.30 mg ml-1), whereas T. longicaulis eradicated all oral bacteria (MBC: 0.15-2.50 mg ml-1). Nevertheless, C. albicans showed no sensitivity to the tested extracts. In conclusion, the tested plant extracts could serve as alternative natural antibacterial and antibiofilm components against oral infections.

Natural Antimicrobials and Oral Microorganisms: A Systematic Review on Herbal Interventions for the Eradication of Multispecies Oral Biofilms.

Oral diseases such as caries and periodontitis are mainly caused by microbial biofilms. Antibiotic therapy has reached its limits with regard to antimicrobial resistance, and new therapeutic measures utilizing natural phytochemicals are currently a focus of research. Hence, this systematic review provides a critical presentation of the antimicrobial effects of various medicinal herbs against in vitro, ex vivo, and in situ formed multispecies oral biofilms. Searches were performed in three English databases (PubMed, EMBASE, CAMbase) and the electronic archives of five German journals from the times of their establishment until October 10th, 2014, with the search terms "(plant extracts OR herbal extracts OR plant OR herb) AND (oral biofilm OR dental biofilm OR dental plaque OR oral disease OR dental disease)." The pooled data were assessed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines (PRISMA). Initially, 1848 articles were identified, out of which 585 full-text articles were screened, 149 articles were reevaluated for eligibility and finally, 14 articles met all inclusion criteria. The data of 14 reports disclosed enhanced antiadhesive and antibiofilm activity by the plant extracts obtained from Vitis vinifera, Pinus spp., Coffea canephora, Camellia sinensis, Vaccinium macrocarpon, Galla chinensis, Caesalpinia ferrea Martius, Psidium cattleianum, representative Brazilian plants and manuka honey. Overall, a positive correlation was revealed between herb-based therapies and elimination rates of all types of multispecies oral biofilms. In that context, integrating or even replacing conventional dental therapy protocols with herbal-inspired treatments can allow effective antimicrobial control of oral biofilms and thus, dental diseases.

High-level antimicrobial efficacy of representative Mediterranean natural plant extracts against oral microorganisms.

Nature is an unexplored reservoir of novel phytopharmaceuticals. Since biofilm-related oral diseases often correlate with antibiotic resistance, plant-derived antimicrobial agents could enhance existing treatment options. Therefore, the rationale of the present report was to examine the antimicrobial impact of Mediterranean natural extracts on oral microorganisms. Five different extracts from Olea europaea, mastic gum, and Inula viscosa were tested against ten bacteria and one Candida albicans strain. The extraction protocols were conducted according to established experimental procedures. Two antimicrobial assays--the minimum inhibitory concentration (MIC) assay and the minimum bactericidal concentration (MBC) assay--were applied. The screened extracts were found to be active against each of the tested microorganisms. O. europaea presented MIC and MBC ranges of 0.07-10.00 mg mL(-1) and 0.60-10.00 mg mL(-1), respectively. The mean MBC values for mastic gum and I. viscosa were 0.07-10.00 mg mL(-1) and 0.15-10.00 mg mL(-1), respectively. Extracts were less effective against C. albicans and exerted bactericidal effects at a concentration range of 0.07-5.00 mg mL(-1) on strict anaerobic bacteria (Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Parvimonas micra). Ethyl acetate I. viscosa extract and total mastic extract showed considerable antimicrobial activity against oral microorganisms and could therefore be considered as alternative natural anti-infectious agents.