The oral microbiota is a reservoir for antimicrobial resistance: resistome and phenotypic resistance characteristics of oral biofilm in health, caries, and periodontitis.

BACKGROUND: Antimicrobial resistance (AMR) is an ever-growing threat to modern medicine and, according to the latest reports, it causes nearly twice as many deaths globally as AIDS or malaria. Elucidating reservoirs and dissemination routes of antimicrobial resistance genes (ARGs) are essential in fighting AMR. Human commensals represent an important reservoir, which is underexplored for the oral microbiota. Here, we set out to investigate the resistome and phenotypic resistance of oral biofilm microbiota from 179 orally healthy (H), caries active (C), and periodontally diseased (P) individuals (TRN: DRKS00013119, Registration date: 22.10.2022). The samples were analysed using shotgun metagenomic sequencing combined, for the first time, with culture technique. A selection of 997 isolates was tested for resistance to relevant antibiotics. RESULTS: The shotgun metagenomics sequencing resulted in 2,069,295,923 reads classified into 4856 species-level OTUs. PERMANOVA analysis of beta-diversity revealed significant differences between the groups regarding their microbiota composition and their ARG profile. The samples were clustered into three ecotypes based on their microbial composition. The bacterial composition of H and C samples greatly overlapped and was based on ecotypes 1 and 2 whereas ecotype 3 was only detected in periodontitis. We found 64 ARGs conveying resistance to 36 antibiotics, particularly to tetracycline, macrolide-lincosamide-streptogramin, and beta-lactam antibiotics, and a correspondingly high prevalence of phenotypic resistance. Based on the microbiota composition, these ARGs cluster in different resistotypes, and a higher prevalence is found in healthy and caries active than in periodontally diseased individuals. There was a significant association between the resistotypes and the ecotypes. Although numerous associations were found between specific antibiotic resistance and bacterial taxa, only a few taxa showed matching associations with both genotypic and phenotypic analyses. CONCLUSIONS: Our findings show the importance of the oral microbiota from different niches within the oral cavity as a reservoir for antibiotic resistance. Additionally, the present study showed the need for using more than one method to reveal antibiotic resistance within the total oral biofilm, as a clear mismatch between the shotgun metagenomics method and the phenotypic resistance characterization was shown.

Antimicrobial efficiency of mouthrinses versus and in combination with different photodynamic therapies on periodontal pathogens in an experimental study.

BACKGROUND AND OBJECTIVE: In the therapy of destructive periodontal disease, chemical antimicrobial agents and increasingly photodynamic therapy (PDT) play an important adjunctive role to standard mechanical anti-infective treatment procedures. However, both antiseptic methods have their shortcomings in terms of eliminating periodontal pathogens. The aim of the study was to compare the antibacterial efficacy of different antiseptic mouthrinses, of a conventional and a new, modified PDTplus as well as of the different antiseptic mouthrinses combined with either the conventional or the modified PDTplus against periopathogens. MATERIAL AND METHODS: Six representative periodontitis-associated bacterial strains were grown for 24 h under anaerobic conditions. After mixing the individual cell pellets they were exposed to 10 different antiseptic mouthrinse formulations: chlorhexidine (0.2%, 0.06%, CHX); CHX + cetylpyridinium chloride (each 0.05%); sodium hypochlorite (0.05%); polyhexanide (0.04%, PHMB1; 0.1%, PHMB2); octenidine dihydrochloride (0.1%); fluoride (250 ppm); essential oils; povidone iodine (10%); and saline (0.9%, NaCl) as control. Furthermore, the bacteria were treated with conventional PDT based on light-emitting diodes and a new modified photodisinfection combining photosensitizer with hydrogen peroxide to PDTplus also based on light-emitting diodes. In addition to the single treatments, a combined application of antiseptic exposure followed by use of PDT or PDTplus was performed. The microbial viability was characterized by analyzing colony growth and fluorescence-based vitality proportions. RESULTS: Nearly all mouthrinses caused a statistically significant growth inhibition. The most effective antiseptics, CHX (0.2%), CHX/cetylpyridinium chloride and octenidine dihydrochloride, inhibited bacterial growth completely. Conventional PDT resulted in moderate reduction of colony growth. The modified PDTplus achieved maximum antimicrobial effect. The combination of antiseptic exposure and PDT against periopathogens predominantly increased antibacterial efficacy compared to the single applications. The mouthrinse containing essential oil seemed to interfere with PDT. CONCLUSION: A combination therapy of preceding chemotherapeutical exposure and subsequent photodisinfection may be a more effective and promising antibacterial treatment than single applications of the antiseptic methods. The modified PDTplus using oxygen-enriched toluidine showed a superior antibacterial effect on periodontal pathogens to conventional PDT and to the majority of the investigated mouthrinses.

Formation and photocatalytic decomposition of a pellicle on anatase surfaces.

The acquired dental pellicle plays a critical role in the adhesion and detachment of dental plaque bacteria. It has been reported that titanium dioxide biomaterials decompose single-protein films by photocatalysis. However, it is not known whether this can also be achieved with complex structured pellicle films. This in vitro study investigated in real-time the formation and photocatalytic decomposition of human pellicle at anatase-saliva interfaces. Nanostructured polycrystalline anatase layers were deposited on titanium-coated quartz crystals by magnetron-sputtering, serving as a model for titanium implant surfaces. The quartz crystals were used as acoustic sensors in a quartz-crystal microbalance (QCM) system with dissipation. In situ UV irradiation of pellicle-covered anatase caused a statistically significant decrease of the adsorbed salivary mass. In contrast, photocatalytic decomposition of pellicle could not be observed on reference titanium surfaces. Wettability characterization revealed superhydrophilicity of anatase upon UV irradiation, whereas titanium was unaffected. XPS measurements provide further information concerning the decomposition of the salivary films. The results suggest that the photocatalytic activity of polycrystalline anatase-modified biomaterial surfaces is able to decompose complex structured macromolecular pellicle films. Therefore, this study opens the way to surface modifications supporting therapeutic approaches of biofilm removal.

Multifunctional nature of UV-irradiated nanocrystalline anatase thin films for biomedical applications.

Anatase is known to decompose organic material by photocatalysis and to enhance surface wettability once irradiated by ultraviolet (UV) light. In this study, pulse magnetron-sputtered anatase thin films were investigated for their suitability with respect to specific biomedical applications, namely superhydrophilic and biofilm degrading implant surfaces. UV-induced hydrophilicity was quantified by static and dynamic contact angle analysis. Photocatalytic protein decomposition was analyzed by quartz crystal microbalance with dissipation. The surfaces were characterized by X-ray diffraction, atomic force microscopy, scanning electron microscopy and X-ray photoelectron spectroscopy. The radical formation on anatase, responsible for photocatalytic effects, was analyzed by electron spin resonance spectroscopy. Results have shown that the nanocrystalline anatase films, in contrast to reference titanium surfaces, were sensitive to UV irradiation and showed rapid switching towards superhydrophilicity. The observed decrease in carbon adsorbents and the increase in the fraction of surface hydroxyl groups upon UV irradiation might contribute to this hydrophilic behavior. UV irradiation of anatase pre-conditioned with albumin protein layers induces the photocatalytic decomposition of these model biofilms. The observed degradation is mainly caused by hydroxyl radicals. It is concluded that nanocrystalline anatase films offer different functions at implant interfaces, e.g. bedside hydrophilization of anatase-coated implants for improved osseointegration or the in situ decomposition of conditioning films forming the basal layer of biofilms in the oral cavity.

Prevalence of oral pathologic findings in an ancient pre-Columbian archeological site in the Atacama Desert.

OBJECTIVE: To determine the prevalence of oral pathologic findings in an ancient culture that inhabited the Atacama Desert. MATERIALS AND METHODS: A systematic examination was performed on the remains of 83 individuals unearthed from a prehistoric burial ground. A total of 57 skeletal remains achieved appropriate inclusion criteria, from which estimated age at death, gender, ante- and postmortem tooth loss, prevalence and location of caries, apical periodontitis sequela, alveolar bone resorption and attrition were recorded. RESULTS: From the analyzed skeletal remains (13 male, 22 female and 22 not identifiable), the mean age estimated was 29.9 +/- 13.8 years. A total of 89.4% of them presented permanent dentition with a mean ante-mortem tooth loss of 9.0 teeth and a postmortem mean tooth loss of 14.4 teeth per subject. In all, 46.4% of the postmortem remaining permanent teeth (n = 237) showed caries lesions. Interproximal caries was most frequently observed (31.5%), followed by occlusal (25.9%) and cervical caries (19.4%). Root remnants were found in 23.1% of the cases. In addition, 58.0% of the adults presented attrition, 26.0% signs of apical periodontitis and 44.0% loss of alveolar bone support >5 mm. CONCLUSION: The remains of jaws and teeth of the individuals examined in this study presented sequelae of severe oral health damage due to caries and periodontal disease.

A synergistic chlorhexidine/chitosan combination for improved antiplaque strategies.

BACKGROUND: The minor efficacy of chlorhexidine (CHX) on other cariogenic bacteria than mutans streptococci such as Streptococcus sanguinis may contribute to uneffective antiplaque strategies. METHODS AND RESULTS: In addition to CHX (0.1%) as positive control and saline as negative control, two chitosan derivatives (0.2%) and their CHX combinations were applied to planktonic and attached sanguinis streptococci for 2 min. In a preclinical biofilm model, the bacteria suspended in human sterile saliva were allowed to attach to human enamel slides for 60 min under flow conditions mimicking human salivation. The efficacy of the test agents on streptococci was screened by the following parameters: vitality status, colony-forming units (CFU)/ml and cell density on enamel. The first combination reduced the bacterial vitality to approximately 0% and yielded a strong CFU reduction of 2-3 log(10) units, much stronger than CHX alone. Furthermore, the first chitosan derivative showed a significant decrease of the surface coverage with these treated streptococci after attachment to enamel. CONCLUSIONS: Based on these results, a new CHX formulation would be beneficial unifying the bioadhesive properties of chitosan with the antibacterial activity of CHX synergistically resulting in a superior antiplaque effect than CHX alone.

Susceptibility of planktonic versus attached Streptococcus sanguinis cells to chlorhexidine.

The effect of chlorhexidine (CHX) on the viability of Streptococcus sanguinis was investigated in a preclinical biofilm model separately on cells in the planktonic or attached life form. Saliva-coated human enamel and glass slides were exposed to the streptococci suspended in sterile saliva for 30 min and 60 min in the flow chamber system. The CHX exposition was performed in two parts: pretreatment of the planktonic bacteria before their attachment to enamel or glass, and treatment of bacteria already attached to enamel. The susceptibility measured by vitality percentages was determined by fluorescence microscopy using vital/dead cells. After CHX pretreatment of planktonic cells, the mean values of the vitality percentages after adhesion were 14-18% (enamel) and 24-25% (glass). In contrast, the mean vitality percentages of untreated attached streptococci reached 70-75% (enamel) and 68% (glass). The vitality percentages of CHX-exposed bacteria dropped markedly to 2-5%, whereas those of untreated attached cells remained at 65-66%. The exposure of initially attached streptococci to CHX resulted in greater reduction of bacterial viability than with the planktonic counterparts. This preclinical biofilm model allows the investigation of various bacterial life forms and can furthermore be used to select efficient antiplaque therapeutics which might be beneficial for clinical plaque control.

Vital microorganisms in early supragingival dental plaque and in stimulated human saliva.

The aim of this study was to compare the percentage of vital microorganisms (= microbial vitality) of saliva with that of supragingival plaque both collected at various times during the early phases of de novo plaque formation. Between intervals of optimal oral hygiene, 14 healthy participants refrained from all oral hygiene measures for periods of 1, 4, 8 and 72 h. Stimulated whole saliva was collected at the beginning (= baseline) and the end of each period. Vestibular plaque was removed from teeth 13-16, and 23-26. Analysis of the pooled plaque (p) and saliva (s) samples comprised the total number of bacterial counts and colony-forming units to estimate the percentage of viable microorganisms (PEp; PEs). The microbial vitality (VFp; VFs) was determined by using a fluorescence staining to differentiate vital from dead bacterial cells. The bulk of the PEs values reached 5-30%. At baseline VFs ranged between 70% and 90%. The VFs values recorded at baseline or in the presence of 1 h and 4 h-old plaque, were significantly (alpha = 0.05) higher than the corresponding VFp values ranging from 5% to 30%. It was concluded that there is a considerable discrepancy between the microbial vitality of a very early dental plaque and that of whole surrounding saliva sampled at the same time. Unfavourable local environmental conditions prevailing at cervical tooth surfaces are suggested to restrain the survival of the majority of the first bacteria adhering to a particular tooth area during the early phases of supragingival plaque formation.

Microbial vitality of supragingival dental plaque during initial stages of experimental gingivitis in humans.

Although vital plaque micro-organisms are part of the natural ecosystem in the oral cavity they are also the key factor in the development of diseases induced by the human dental plaque. In a previous study (9) the portion of vital bacteria related to the total number of plaque micro-organisms (i.e. the microbial vitality) appeared low in small plaque samples. The objective of this investigation was to determine the exact relationship of microbial vitality and age of supragingival plaque during the early phases of human dental plaque formation. Between intervals of optimal oral hygiene, thirteen participants refrained from all oral hygiene measures for periods of 1, 2, 4, 8, 24 and 72 h. Plaque was completely sampled from a defined area situated on the vestibular surface of the teeth 13, 14, 15, 23, 24 and 25. The pooled plaque from these areas was immediately processed. Total bacterial counts (BC) as enumerated by darkfield microscopy, and colony-forming units (CFU) were recorded. The microbial vitality was calculated indirectly as plating efficiency (PE = CFU per BC) and directly assessed using a vital fluorescence (VF) technique. In the 1 h old plaque samples the median values of PE and VF were 29% and 18%, respectively. Thereafter, the microbial vitality increased significantly with plaque age. The 24 h old plaque samples yielded values of 77% (PE) and 62% (VF). It was concluded that the microbial vitality of the early dental plaque investigated was considerably lower compared to that of a more mature plaque.

Microbial generation time during the early phases of supragingival dental plaque formation.

The objective of this investigation was to determine the microbial generation time during early human supragingival plaque formation. On 2 occasions, 13 participants refrained from all oral hygiene measures for periods of 1, 2, 4, 8, 24 and 72 h. Plaque was sampled from defined areas of the teeth 13, 14, 15, 23, 24 and 25. Two independent methods were used to estimate the total number of viable cells: colony-forming units BCVF, total bacterial counts (BC) as enumerated by microscopic examination, and the percentage of vital bacteria using a vital fluorescence technique (VF) were recorded. The total number of vital microorganisms was calculated by BCVF (= BC x VF). The generation time based on either colony-forming units or BCVF increased significantly with plaque age. During the first interval (1-4 h), the microbial multiplication took slightly less than 1 h. After the establishment of a more mature plaque (24-72 h), the replicating time was between 12.5 h (BCVF) and 14.8 h (colony-forming units). It was concluded that the microbial generation time recorded at the initial stages of plaque formation is considerably lower than that found in older plaque.