Resident Oral Microflora Research Articles

Antibiotic resistance among Aerobic Gram-Negative Bacilli isolated from patients with oral inflammatory dysbiotic conditions—a retrospective study

IntroductionAerobic Gram-Negative Bacilli (AGNB) are not part of the resident oral microflora but are occasionally found in high abundance under inflammatory dysbiotic conditions at various oral niches. The aim of the present study was to investigate the identity and antibiotic susceptibility of AGNB isolated from patients in Sweden with mucosal lesions, periodontitis, and peri-implantitis, with special attention to antibiotic resistance and on the presence of phenotypic Extended Spectrum Beta-Lactamase (ESBL) isolates.Materials and methodsMicrobiolgical samples were harvested from 211 patients in total, experiencing mucosal lesions (N = 113), periodontitis (N = 62), or peri-implantitis (N = 36). The growth of AGNB's was semiquantified by selective and non-selective culture and the strains were isolated, identified, and tested for antibiotic susceptibility. A total of 251 AGNB strains, occurring in moderate to heavy growth (>100 CFU/ml sample), indicating a dysbiotic microbiota, were identified. The disc diffusion method was used for screening of the antibiotic susceptibility of the isolates. Phenotypic identification of ESBL isolates was based on resistance to ceftazidime and/or cefotaxime.ResultsThe most commonly detected AGNB isolates in oral inflammatory dysbiotic conditions were fermentative species belonging to Enterobacteriaceae e.g. Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella spp, and the non-fermentative environmental Burkholderia cepacia, Pseudomonas spp., and Stenotrophomonas maltophilia. No clear trends were seen in frequency of the various species in samples from mucosal lesions, severe periodontitis, and peri-implantitis cases. The 138 Enterobacteriaceae isolates and 113 environmental AGNB isolated showed a high antibiotic resistance in general against antibiotics commonly used in dentistry (Amoxicillin, Amoxicillin + Clavulanic acid, Ampicillin, Clindamycin, Doxycycline, Erythromycin, Oxacillin, PenicillinV, and Tetracycline). The majority of these isolates were susceptible to ciprofloxacin. Ten isolates (4.1%) were phenotypically classified as ESBL positive. The ESBL isolates were predominantly found among isolates of S. maltophilia, while only one ESBL positive isolate was found among Enterobacteriaceae.ConclusionsPhenotypically identified ESBL isolates can occasionally be present among oral AGNB strains isolated in abundance from the dysbiotic microbiota occurring in cases with oral mucosal lesions, severe periodontitis, or peri-implantitis.

The place and role of oral microflora in the pathogenesis of oral mucositis in malignant neoplasms (literature review)

Background. Oral mucositis (OM) is one of the most frequent complications of systemic drug therapy of oncological diseases. Clinically, the disease manifests itself in the form of erythema and ulceration of the non-corneating mucous membrane. Although this condition is self-healing, it can affect the conduct of comprehensive antitumor treatment. Currently, the pathophysiological model of the development of OM is supplemented with new data obtained during studies of the oral microflora. Antitumor treatment can lead to changes in the composition of the resident oral microflora and it is quite possible that the changes that occur may affect the development of damage to the oral mucosa. The expansion of knowledge in this area allows researchers to look for new ways to include oral microbiota in OM management strategies.
 Aim. To summarize and update the accumulated data regarding the role of the oral microbiota in the pathogenesis of OM in patients with malignant neoplasms.
 Materials and methods. Literature search was performed in Medline, Cochrane Library, Elibrary and Pubmed, including publications demonstrating the current ability to assess the impact of the oral microbiome on OM, as well as developments in this area relating to OM management.
 Results. In the review the current views on the position of the oral microbiome in the pathogenesis of OM were highlighted and the promising directions for the use of agents influencing the state of the oral microbiota in various strategies to control this disease were emphasised.
 Conclusion. A better understanding of the pathogenesis of OM and the inclusion of new factors, such as the oral microbiome, into the picture of pathogenesis is likely to enable the formation of more effective management strategies for this disease in the future. This direction seems very promising, and developments in this area show promising results.

Peculiarities of Periodontal Pocket Microbiome in Patients with Generalized Periodontitis in the Post-COVID Period

The oral cavity, like the lungs, is often referred to as the «ecological niche of commensal, symbiotic, and pathogenic organisms», and the emigration and elimination of microbes between them are constant, ensuring a healthy distribution of saprophytic microorganisms that maintains organ, tissue, and immune homeostasis. The prolonged hospital stays due to COVID-19 complications, cross-infection, oxygenation therapy through the mask or incubation, and long-term intravenous infusions limit the patient’s ability to care about the oral cavity, regularly clean teeth, floss interdental, etc., which creates extremely favorable conditions for colonization by aerobic and anaerobic pathogens of the oral cavity and periodontal pockets and leads to the rapid progression of chronic generalized periodontitis in this category of patients in the future. The goal of the study was to assess the state of the microbiome of the periodontal pockets of dental patients in the post-covid period. Methods. The object of the study was 140 patients with generalized periodontitis of the I and II stages of development in the chronic course (GP), among which 80 patients had coronavirus disease in the closest past. The patients were randomized by age, sex, and stage of GP development. The diagnosis of periodontal disease was established according to the classifi cation by Danilevskyi. The bacteriological material for aerobic and facultative anaerobic microflora and yeast-like fungi was collected from periodontal pockets with a calibrated bacteriological loop and immediately seeded on blood agar. Results. Significant qualitative and quantitative changes in the nature of the oral microbiocenosis were observed in patients with GP after the recent coronavirus disease, compared with similar patients who did not suffer from COVID-19. We have noticed almost complete disappearance of bacteria that belong to the transient representatives of the oral microflora such as Neisseria, corynebacteria (diphtheria), micrococci, and lactobacilli. The main resident representatives of the oral microflora, i.e., α-hemolytic Streptococci of the mitis group, were found in all healthy individuals and patients of groups A and C, but in 30.0±4.58% of patients in group B, α-hemolytic streptococci in the contents of periodontal pockets are present in quantities not available for detection by the applied method (<2.7 lg CCU/mL). In terms of species, Streptococcus oralis and Streptococcus salivarius are more characteristic in gingival crevicular fluid in healthy individuals (93.8% of selected strains). In 68.4±3.32% of patients in group A, 64.0±3.43% of patients in group B, and 67.5±3.76% of patients in group C, the dominant species were Streptococcus gordonii and Streptococcus sanguinis (p<0.01), which increased pathogenic potential as they produce streptolysin-O, inhibit complement activation, bind to fibronectine, actively form biofilms on the surface of tooth enamel and gum epithelial surface, and can act as an initiator of adhesion of periodontal pathogens. The other representatives of the resident microflora of the oral cavity — Stomatococcus mucilaginosus and Veillonella parvula for the patients of group C are also found in periodontal pockets with a signifi cantly lower index of persistence and minimal population level. In the post-covid period, both the population level and the frequency of colonization of periodontal pockets by Staphylococci and β-hemolytic Streptococci decreases rapidly. For these patient groups, unlike for those that did not suff er from COVID-19, we did not find any case of colonization with Staphylococcus aureus, as well as β-hemolytic Streptococci and epidermal Staphylococcus were also absent. The most characteristic in the post-covid period is a decrease in the proportion of α-hemolytic Streptococci, an increase in the proportion of yeast-like fungi of Candida species, as well as the appearance of a significant number of gram-negative rod-shaped bacteria (Enterobacteria and Pseudomonads). In periodontal patients, the microbial count is approximately 2 orders of magnitude lower than in those with GP who did not suffer from COVID-19 (p<0.05). Conclusions. The overpassed coronavirus disease due to intensive antibiotic therapy leads to a marked decrease in the number of viable saprophytic microorganisms in the periodontal pockets of patients with GP. In the post-covid period for the patients with GP, there is a decrease in the level of colonization of periodontal pockets by species of resident oral microflora — α-hemolytic Streptococci, reduction of resident microorganism’s species, and almost complete disappearance of transient microfl ora. On the other hand, the frequency of colonization of periodontal pockets by fungi species, enterobacteria, and pseudomonads significantly increases. There are more expressed disorders in the periodontal pocket’s microbiome for the patients with a severe and complicated course of coronavirus disease, such as post-covid pulmonary fibrosis, which requires reconsideration of approaches to therapeutic and pharmacological treatment in this category of patients.

Evidence on the Use of Mouthwash for the Control of Supragingival Biofilm and Its Potential Adverse Effects.

Antimicrobial mouthwash improves supragingival biofilm control when used in conjunction with mechanical removal as part of an oral hygiene routine. Mouthwash is intended to suppress bacterial adhesion during biofilm formation processes and is not aimed at mature biofilms. The most common evidence-based effects of mouthwash on the subgingival biofilm include the inhibition of biofilm accumulation and its anti-gingivitis property, followed by its cariostatic activities. There has been no significant change in the strength of the evidence over the last decade. A strategy for biofilm control that relies on the elimination of bacteria may cause a variety of side effects. The exposure of mature oral biofilms to mouthwash is associated with several possible adverse reactions, such as the emergence of resistant strains, the effects of the residual structure, enhanced pathogenicity following retarded penetration, and ecological changes to the microbiota. These concerns require further elucidation. This review aims to reconfirm the intended effects of mouthwash on oral biofilm control by summarizing systematic reviews from the last decade and to discuss the limitations of mouthwash and potential adverse reactions to its use. In the future, the strategy for oral biofilm control may shift to reducing the biofilm by detaching it or modulating its quality, rather than eliminating it, to preserve the benefits of the normal resident oral microflora.

Late-onset Endophthalmitis Caused by Streptococcus Cristatus after Trabeculectomy

Purpose: To report a case of late-onset bleb-associated endophthalmitis caused by <i>streptococcus cristatus</i> (<i>S. cristatus</i>) after trabeculectomy.Case summary: A 59-year-old woman presented with left ocular pain and visual disturbance. She had undergone trabeculectomy of the left eye 2 years ago. At the initial visit, she was only able to count fingers from the left eye. Whitish thin bleb, conjunctival injection, and inflammation were observed in the left anterior chamber, and the fundus could not be visualized. Despite administering topical and systemic antibiotics, the chamber reaction worsened, and hypopyon and inflammatory membrane were noticed the next day. Vitrectomy with phacoemulsification and intravitreal antibiotic injection was performed, and the intravitreal antibiotic injections were repeated over the next two days. Bacterial culture of vitreous fluid showed a growth of <i>S. cristatus</i> 5 days after the surgery, and inflammation improved after 7 days. One month later, she was still only able to count fingers from the left eye, but there were no signs of endophthalmitis.Conclusions: Postoperative endophthalmitis after glaucoma filtering surgeries caused by <i>S. cristatus</i> has not been reported previously from Korea. To the best of our knowledge, this is the first Korean case of late-onset bleb-associated endophthalmitis caused by <i>S. cristatus</i>. If gram-positive cocci are grown in samples from the patients with endophthalmitis after trabeculectomy, resident oral microflora such as <i>S. cristatus</i> should be considered as the causative pathogen.

Contemporary perspective on plaque control

The aim of this review article is to provide a scientific platform that will enable the dental team to develop a rational approach to plaque control based on the latest knowledge of the role of the oral microflora in health and disease. The resident oral microflora is natural and forms spatially-organised, interactive, multi-species biofilms on mucosal and dental surfaces in the mouth. These resident oral microbial communities play a key function in the normal development of the physiology of the host and are important in preventing colonisation by exogenous and often undesirable microbes. A dynamic balance exists between the resident microflora and the host in health, and disease results from a breakdown of this delicate relationship. Patients should be taught effective plaque control techniques that maintain dental biofilms at levels compatible with oral health so as to retain the beneficial properties of the resident microflora while reducing the risk of dental disease from excessive plaque accumulation. Antimicrobial and antiplaque agents in oral care products can augment mechanical plaque control by several direct and indirect mechanisms that not only involve reducing or removing dental biofilms but also include inhibiting bacterial metabolism when the agents are still present at sub-lethal concentrations.

Controlling the oral biofilm with antimicrobials

The aim of this article is to review the properties of compounds available for the control of dental plaque biofilms, and describe their mode of action. The mouth is colonised by a diverse but characteristic collection of micro-organisms, which confer benefit to host. Numerous antiplaque (e.g. surfactants, essential oils) and antimicrobial agents (e.g. bisbiguanides, metal ions, phenols, quaternary ammonium compounds, etc.) have been successfully formulated into toothpastes and mouthrinses to control plaque biofilms. At high concentrations, these agents can remove biofilm and/or kill disease-associated bacteria, while even at sub-lethal levels they can inhibit the expression of pathogenic traits. Successful antimicrobial agents are able to meet the apparently contradictory requirements of maintaining the oral biofilm at levels compatible with oral health but without disrupting the natural and beneficial properties of the resident oral microflora.

Safety Assessment of ProBiora3, a Probiotic Mouthwash: Subchronic Toxicity Study in Rats

Streptococcus viridans are commensal bacteria that constitute a significant portion of the resident oral microflora. The objective of the present study is to investigate adverse effects, if any, of a blend of 3 natural strains, Streptococcus uberis KJ2, Streptococcus oralis KJ3, and Streptococcus rattus JH145 (probiotic mouthwash, ProBiora(3)). The blend is administered to rats orally once daily (5 days per week) at doses of 0, 10(6), or 10(9) colony-forming units of each strain for 14 weeks. No treatment-related adverse effects are observed in the physiological parameters during the study or in the evaluation of blood and tissue samples taken from the animals at the end. Results of an in vitro antibiotic susceptibility study demonstrate that all 3 ProBiora(3) strains are susceptible to commonly used therapeutic antibiotics. The results of these investigations reveal that the no-observed-adverse-effect level of the probiotic mouthwash is 2.16 x 10(9) colony-forming units per strain per kilogram of body weight per day, the highest dose used.

Modeling Shifts in Microbial Populations Associated with Health or Disease

Stable microbial communities associated with health can be disrupted by altered environmental conditions. Periodontal diseases are associated with changes in the resident oral microflora. For example, as gingivitis develops, a key change in the microbial composition of dental plaque is the ascendancy of Actinomyces spp. and gram-negative rods at the expense of Streptococcus spp. We describe the use of an in vitro model to replicate this population shift, first with a dual-species model (Actinomyces naeslundii and Streptococcus sobrinus) and then using a microcosm model of dental plaque. The population shift was induced by environmental changes associated with gingivitis, first by the addition of artificial gingival crevicular fluid and then by a switch to a microaerophilic atmosphere. In addition to the observed population shifts, confocal laser scanning microscopy also revealed structural changes and differences in the distribution of viable and nonviable bacteria associated with the change in environmental conditions. This model provides an appropriate system for the further understanding of microbial population shifts associated with gingivitis and for the testing of, for example, antimicrobial agents.

Do dental diseases resemble ecological catastrophes?

Ecological catastrophes can take many forms, and can come in all shapes and sizes! Nitrogenous fertilisers can be washed off farm-land into lakes and ponds, resulting in overgrowth by algae. Such an overgrowth can lead to secondary effects to the ecosystem; the algae can consume dissolved oxygen in the water leading to the loss of aerobic microbial, plant and insect life (eutrophication). Similarly, atmospheric pollution with sulphur dioxide and nitrogen oxides can produce acid rain causing damage to plants and trees and loss of aquatic life. On a larger scale, it has been postulated that the extinction of the dinosaurs followed climate change resulting from the impact of a meteorite. At the other end of the spectrum, it will be argued in this article that the key to a more complete understanding of the role of micro-organisms in dental diseases depends on a paradigm shift away from concepts that have evolved from studies of diseases with a simple and specific (e.g. single species) aetiology to an appreciation of ecological principles similar to those outlined above, where a substantial change to a key parameter influencing the habitat can disrupt the natural balance of the resident oral microflora. Acceptance of such principles can more readily explain the transition of the oral microflora from having a benign to a pathogenic relationship with the host, while opening up new opportunities for the control of dental plaque-mediated diseases.

Plaque as a biofilm: pharmacological principles of drug delivery and action in the sub- and supragingival environment.

Dental plaque is the diverse microbial community, embedded in a matrix of host and bacterial polymers, growing on teeth as a biofilm. Dental plaque develops naturally, and contributes to the host defences by preventing colonization by exogenous species. The composition of dental plaque varies at distinct surfaces as a result of the inherent biological and physical properties at these sites; the balance of the predominant bacterial populations shifts in disease. Plaque has an open architecture, with channels traversing from the biofilm surface through to the enamel. This structure affects the movement of molecules within plaque, and gradients in key determinants develop. Bacteria growing on a surface display a novel phenotype; one consequence of which is an increased resistance to antimicrobial agents. Resistance can result from restricted inhibitor penetration (diffusion-reaction theory), slower bacterial growth rates, transfer of resistance genes, suboptimal environmental conditions for inhibitor activity, and the expression of a resistant phenotype. Such biofilm-associated traits, coupled with the pharmacokinetic profile of orally delivered antimicrobial agents (high concentrations for short periods/lower concentrations for longer periods), affect the mode of action and efficacy of antimicrobials. Agents with a broad spectrum of activity in laboratory studies may display a far narrower inhibitory profile under the conditions prevailing in the mouth. This may result in a selective inhibition of species implicated in disease, or reduced production of virulence factors, while preserving the benefits associated with a resident oral microflora.

Role of the Oral Microflora in Health

The mouth contains both distinct mucosal (lips, cheek, tongue, palate) and, uniquely, non-shedding surfaces (teeth) for microbial colonisation. Each surface harbours a diverse but characteristic microflora, the composition and metabolism of which is dictated by the biological properties of each site. The resident oral microflora develops in an orderly manner via waves of microbial succession (both autogenic and allogenic). Pioneer species (many of which are sIgA protease-producing streptococci) colonise saliva-coated surfaces through specific stereo-chemical, adhesin-receptor interactions. The metabolism of these organisms modifies local environmental conditions, facilitating subsequent attachment and growth by later, and more fastidious, colonisers. Eventually, a stable biofilm community develops, that plays an active role in (a) the normal development of the physiology of the habitat, and (b) the innate host defences (colonisation resistance). Thus, when considering treatment options, clinicians should be aware of the need to maintain the beneficial properties of the resident oral microflora.

Physiological approaches to the control of oral biofilms.

Evidence that physiological strategies may be potential routes for oral biofilm control has come from (i) observations of the variations in the intra-oral distribution of members of the resident oral microflora, (ii) changes in plaque composition in health and disease, and (iii) data from laboratory model systems. Key physiological factors that were identified as significant in modulating the microflora included the local pH, redox potential (Eh), and nutrient availability. Increases in mutans streptococci and lactobacilli occur at sites with caries; growth of these species is selectively enhanced at low pH. In contrast, periodontal diseases are associated with plaque accumulation, followed by an inflammatory host response. The increases in Gram-negative, proteolytic, and obligately anaerobic bacteria reflect a low redox potential and a change in nutrient status due to the increased flow of gingival crevicular fluid (GCF). Consequently, physiological strategies for oral biofilm control should focus on reducing the frequency of low pH in plaque by (i) inhibiting acid production, (ii) using sugar substitutes, and (iii) promoting alkali generation from arginine or urea supplements. Similarly, strategies to make the pocket environment less favorable to periodonto-pathogens include (i) anti-inflammatory agents to reduce the flow of (and hence nutrient supply by) GCF, (ii) bacterial protease inhibitors, and (iii) redox agents to raise the Eh locally. Most laboratory and clinical findings support the concept of physiological control. However, some data suggest that the ordered structure and metabolically interactive organization of mature dental plaque could generate a community with a high level of homeostasis that is relatively resistant to deliberate external manipulation.

Review of chemosystematics: multivariate approaches to oral bacteria and yeasts.

There are several problems related to the classification and identification of bacterial and yeast species assigned to the genera Actinobacillus, Haemophilus, Pasteurella, Bacteroides, Prevotella, Porphyromonas, Campylobacter, Wolinella, Treponema, Candida, Torulopsis, and Saccharomyces, most of which belong to the resident oral microflora. The present review was written to demonstrate how multivariate analyses of data on cellular fatty acids, sugars, enzyme activities, and lysis kinetics during ethylenediaminetetraacetic acid (EDTA) and EDTA plus lysozyme treatment can be used to distinguish closely related species of these bacterial and yeast genera. With the exception of the Actinobacillus-Haemophilus-Pasteurella group, fatty acids were more discriminating than sugars. Enzymes from whole cells and outer membrane vesicles also contributed to taxonomic distinction. Apparently, chemosystematics, involving multivariate analyses, is a useful adjunct in oral microbial taxonomy.