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.

Prevalence of Candida albicans and Candida dubliniensis in caries-free and caries-active children in relation to the oral microbiota-a clinical study.

OBJECTIVES: The correlation between caries and the oral prevalence of Candida spp. in children is contradictory in literature. Thereby, authors focused on Candida albicans as the most isolated Candida species from the oral cavity. Therefore, the aim of the present study was to compare caries-free and caries-bearing children regarding their oral carriage of Candida spp. MATERIAL AND METHODS: Twenty-six caries-free (CF group) and 26 caries-active children (CA group) were included into this study. Three different types of specimens were assessed, saliva and plaque, and in the case of caries, infected dentine samples were microbiologically analyzed for aerobic and anaerobic microorganisms and their counts. Special attention was given to the differentiation between C. albicans and Candida dubliniensis. Additionally, different biochemical tests, VITEK 2 (VITEK®2, bioMérieux, Marcy-l'Etoile, France) and 16S and 18S ribosomal DNA (rDNA) sequencing, were applied for identification. RESULTS: The detection of C. albicans did not differ between the CF and CA groups. C. dubliniensis was never detected in any specimen of the CF group, but occurred in one quarter of the CA group (27 % in plaque, 23 % in saliva), thus leading to a statistically significant difference between the two groups (p < 0.05). In six of these cases, C. dubliniensis was detected concomitantly in saliva and plaque and once only in plaque. CA group harbored statistically more Streptococcus mutans than the control group revealing a correlation between S. mutans and C. dubliniensis regarding the caries group. CONCLUSIONS: This is the first study reporting a frequent detection of C. dubliniensis in caries-active children, which could have been underestimated so far due to difficulties in differentiation between this yeast species and C. albicans. CLINICAL RELEVANCE: Microbiological diagnostic-especially of oral Candida species-is an important determinant for identifying etiological factors of dental caries in children.

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

Recently, growing attention has been paid to antimicrobial photodynamic therapy (aPDT) in dentistry. Changing the microbial composition of initial and mature oral biofilm by aPDT using visible light plus water-filtered infrared-A wavelengths (VIS + wIRA) has not yet been investigated. Moreover, most aPDT studies have been conducted on planktonic bacterial cultures. Therefore, in the present clinical study we cultivated initial and mature oral biofilms in six healthy volunteers for 2 hours or 3 days, respectively. The biofilms were treated with aPDT using VIS+wIRA (200 mW cm(-2)), toluidine blue (TB) and chlorine e6 (Ce6) for 5 minutes. Chlorhexidine treated biofilm samples served as positive controls, while untreated biofilms served as negative controls. After aPDT treatment the colony forming units (CFU) of the biofilm samples were quantified, and the surviving bacteria were isolated in pure cultures and identified using MALDI-TOF, biochemical tests and 16S rDNA-sequencing. aPDT killed more than 99.9% of the initial viable bacterial count and 95% of the mature oral biofilm in situ, independent of the photosensitizer. The number of surviving bacterial species was highly reduced to 6 (TB) and 4 (Ce6) in the treated initial oral biofilm compared to the 20 different species of the untreated biofilm. The proportions of surviving bacterial species were also changed after TB- and Ce6-mediated aPDT of the mature oral biofilm, resulting in a shift in the microbial composition of the treated biofilm compared to that of the control biofilm. In conclusion, aPDT using VIS + wIRA showed a remarkable potential to eradicate both initial and mature oral biofilms, and also to markedly alter the remaining biofilm. This encourages the clinical use of aPDT with VIS + wIRA for the treatment of periimplantitis and periodontitis.

Decreased UV absorbance as an indicator of micropollutant removal efficiency in wastewater treated with ozone.

Ozone transforms various organic compounds that absorb light within the UV and visible spectra. UV absorbance can therefore be used to detect the transformation of chemicals during ozonation. In wastewater, decolourisation can be observed after ozonation. This study investigates the correlation of the UV absorbance difference between the ozonation inlet and outlet and the removal efficiency of micropollutants in wastewater. The absorbance at 254 and 366 nm was measured at the ozonation inlet and outlet, as was the concentration of 24 representative micropollutants and the dissolved organic carbon (DOC). The results clearly showed that the relative decrease of absorbance (ΔAbs) is positively correlated with the relative removal efficiency of micropollutants. We therefore suggest that UV absorbance can be used as a feedback control parameter to achieve optimal ozone dosage in wastewater treatment plants and to gain a fast insight into the process efficiency and stability of the ozonation.

Is Helicobacter pylori resident or transient in the human oral cavity?

Helicobacter pylori colonizes the stomachs of at least half of the world's human population. The role of the oral cavity in this colonization is not clear and there are, to date, no comprehensive data that clearly demonstrate the isolation of this bacterium from the oral cavity. The aim of this study was to evaluate the prevalence of H. pylori in the oral cavity of 15 patients who tested positive for H. pylori. A comprehensive dental examination of all patients was conducted. Samples were taken from supragingival and subgingival plaque, saliva, periapical exudates and tongue swabs. All samples were taken before the application of antibiotics. A total of 163 oral samples were investigated by PCR using two different H. pylori-specific primer pairs. A PCR inhibition control using a modified plasmid was always included for the most specific primer pair. In addition, a culture technique was used to confirm PCR results. Despite a PCR detection limit of 10(2) bacteria ml(-1), out of 14 patients, H. pylori could not be detected in any of the samples taken. In one patient, H. pylori-positive PCR signals were obtained in two samples using only one primer pair. H. pylori could not be cultivated from these two PCR-positive samples; therefore, no correlation to oral colonization status could be established. This study challenges the misleading preconception that H. pylori resides in the human oral cavity and suggests that this bacterium should be considered transient and independent of the oral status. To date, positive PCR results for H. pylori in the oral cavity have been overestimated and not critically interpreted in literature.

Airborne microbes in different dental environments in comparison to a public area.

OBJECTIVE: The aim of this study was to investigate quantitatively and qualitatively the airborne microbial load in a multi-chair dental clinic, a normal dental practice and a non-dental public area over a time period of four days and at different time points to estimate the risk of infections during dental surgery. METHODS: A multi-chair and a single chair treatment room each were examined in comparison to a non-medical public area over a period of four days. The colony forming units m(-3) (CFUs) were determined and isolated bacteria were characterised by morphological and biochemical analysis, gas chromatography and by 16S rRNA-gene sequencing. In the analyses enterococci were selectively searched for. RESULTS: The CFUs in the multi-chair treatment room were between 20 and 1050 CFU m(-3). During treatment the maxima reached were below 800 CFU m(-3). The values in the dental practice were between 200 and 600 CFU m(-3) and remain slightly but not significantly below the levels of the clinic (p > 0.05). In the common area, the CFUs were between 200 and 800 CFU m(-3). The proportion of micrococci was 56.8% in the clinic, 56.07% in the practice and 69.67% in the public area Coagulase-negative staphylococci constituted 35% at the dental clinic, 25% at the bank and 38% at the dental practice. No significant differences amongst the units were detected in the microbial composition of their dental aerosols (p > 0.05). CONCLUSION: Although, the bacterial counts in dental room were not significantly higher than the bacterial counts in a public area, the risk from dental clinic might be higher than a public area due to the type of micro-organisms, host susceptibility and the exposure time.

Comparison of bacterial adhesion and cellular proliferation on newly developed three-dimensional scaffolds manufactured by rapid prototyping technology.

Scaffolds used in the field of tissue engineering should facilitate the adherence, spreading, and ingrowth of cells as well as prevent microbial adherence. For the first time, this study simultaneously deals with microbial and tissue cell adhesion to rapid prototyping-produced 3D-scaffolds. The cell growth of human osteosarcoma cells (CAL-72) over a time period of 3-11 days were examined on three scaffolds (PLGA, PLLA, PLLA-TCP) and compared to the adhesion of salivary microorganisms and representative germs of the oral flora (Porphyromonas gingivalis, Prevotella nigrescens, Candida albicans, Enterococcus faecalis, Streptococcus mutans, and Streptococcus sanguinis). Scanning electron microscopy (SEM), cell proliferation measurements, and determination of the colony forming units (CFU) were performed. The cell proliferation rates on PLLA and PLLA-TCP after 3, 7, and 11 days of cultivation were higher than on PLGA. On day 3 the proliferation rates on PLLA and PLLA-TCP, and on day 5 on PLLA-TCP, proved to be significantly higher compared to that of the control (culture plate). The strain which showed the most CFUs on all of the investigated scaffolds was P. gingivalis, followed by E. faecalis. No significant CFU differences were determined examining P. gingivalis among the biomaterials. In contrast, E. faecalis was significantly more adherent to PLGA and PLLA compared to PLLA-TCP. The lowest CFU values were seen with C. albicans and P. nigrescens. Salivary born aerobic and anaerobic microorganisms adhered significantly more to PLGA compared to PLLA-TCP. These results supported by SEM point out the high potential of PLLA-TCP in the field of tissue engineering.

Bacterial and Candida albicans adhesion on rapid prototyping-produced 3D-scaffolds manufactured as bone replacement materials.

Rapid prototyping (RP)-produced scaffolds are gaining increasing importance in scaffold-guided tissue engineering. Microbial adhesion on the surface of replacement materials has a strong influence on healing and long-term outcome. Consequently, it is important to examine the adherence of microorganisms on RP-produced scaffolds. This research focussed on manufacturing of scaffolds by 3D-bioplotting and examination of their microbial adhesion characteristics. Tricalciumphosphate (TCP), calcium/sodium alginate, and poly(lactide-co-glycolic acid) (PLGA) constructs were produced and used to study the adhesion of dental pathogens. Six oral bacterial strains, one Candida strain and human saliva were used for the adhesion studies. The number of colony forming units (CFU) were determined and scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were performed. Microorganisms adhered to all scaffolds. All strains, except for Streptococcus oralis, adhered best to PLGA scaffolds. Streptococcus oralis adhered to each of the biomaterials equally. Streptococcus mutans and Enterococcus faecalis adhered best to PLGA scaffolds, followed by alginate and TCP. Prevotella nigrescens, Porphyromonas gingivalis, Streptococcus sanguis, and Candida albicans showed the highest adherence to PLGA, followed by TCP and alginate. In contrast, the microorganisms of saliva adhered significantly better to TCP, followed by PLGA and alginate. SEM observations correlated with the results of the CFU determinations. CLSM detected bacteria within deeper sheets of alginate. In conclusion, because of the high adherence rate of oral pathogens to the scaffolds, the application of these biomaterials for bone replacement in oral surgery could result in biomaterial-related infections. Strategies to decrease microbial adherence and to prevent infections due to oral pathogens are discussed.

Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance.

There is cumulative resistance against antibiotics of many bacteria. Therefore, the development of new antiseptics and antimicrobial agents for the treatment of skin infections is of increasing interest. We have screened six plant extracts and isolated compounds for antimicrobial effects on bacteria and yeasts with dermatological relevance. The following plant extracts have been tested: Gentiana lutea, Harpagophytum procumbens, Boswellia serrata (dry extracts), Usnea barbata, Rosmarinus officinalis and Salvia officinalis (supercritical carbon dioxide [CO2] extracts). Additionally, the following characteristic plant substances were tested: usnic acid, carnosol, carnosic acid, ursolic acid, oleanolic acid, harpagoside, boswellic acid and gentiopicroside. The extracts and compounds were tested against 29 aerobic and anaerobic bacteria and yeasts in the agar dilution test. U. barbata-extract and usnic acid were the most active compounds, especially in anaerobic bacteria. Usnea CO2-extract effectively inhibited the growth of several Gram-positive bacteria like Staphylococcus aureus (including methicillin-resistant strains - MRSA), Propionibacterium acnes and Corynebacterium species. Growth of the dimorphic yeast Malassezia furfur was also inhibited by Usnea-extract. Besides the Usnea-extract, Rosmarinus-, Salvia-, Boswellia- and Harpagophytum-extracts proved to be effective against a panel of bacteria. It is concluded that due to their antimicrobial effects some of the plant extracts may be used for the topical treatment of skin disorders like acne vulgaris and seborrhoic eczema.

Differences in the fatty acid composition of KB-cells and gingival keratinocytes is culture medium additive dependent.

The influence of culture medium additives foetal bovine serum (FBS), serum effective substitutes (SES) and human autologous serum on the fatty acid profile of KB-cells and human gingival keratinocytes was examined. The KB-cells were cultivated in RPMI medium added with FBS or SES and the gingival keratinocytes in D-MEM added with FBS or human autologous serum. Two days before the cells were prepared for gas chromatography (GC), the media were changed to serum- and antibiotic-free media. Whole fatty acids of the cells were analysed using GC and the fatty acid profiles were compared. KB-cells as well as gingival keratinocytes changed their fatty acid composition, according to the medium additive used. Significant differences were observed. In the case of KB-cells cultivated with SES the fatty acid changes suggest an increase of the membrane fluidity. Corresponding and significant differences were observed with gingival keratinocytes cultivated in medium added with human autologous serum: the membrane fluidity of the gingival keratinocytes was increased. It is supposed that an increased membrane fluidity caused by a different fatty acid spectrum of the host cell may relate to mechanisms of bacterial adhesion. Consequently, in vitro studies on invasion and adhesion of bacteria or virus are dependent on the medium used. Further analyses are necessary of the functional effects caused by differences in the content of specific FAs, especially with regard to the application of cultivated cells in the field of tissue engineering.

[Endoscopic removal of large colorectal polyps]. / Ist die endoskopische Polypektomie grosser kolorektaler Polypen gerechtfertigt?

BACKGROUND: Due to the potential risk of malignancy and technical difficulties in achieving complete removal, large colorectal polyps represent a special problem for the endoscopist. The aim of this study was to evaluate the capabilities and risks of endoscopy in complete removal of large colorectal polyps. METHODS: Endoscopic polypectomy of 189 colorectal polyps (141 sessile, 48 pedunculated) larger than 3 cm in diameter (range 3-13 cm) was carried out. Sessile polyps were removed using the piecemeal technique. RESULTS: Histology showed an adenoma in 173 cases, and invasive carcinoma was present in the adenoma in 16 patients. Complete endoscopic removal was achieved in one session in 129 sessile polyps and all pedunculated polyps; the other patients required two to ten sessions. Bleeding occurred in 20 patients during polypectomy, and after polypectomy in four. Except for two cases, definitive hemostasis was immediately achieved by endoscopic treatment. Perforation occurred in four patients. 3 were treated conservatively, in one patient who showed an invasive carcinoma of the cecum, resection was performed. There was no mortality due to polypectomy. During a median follow-up period of 77 months (6-107 months), six patients presented with recurrence of a benign adenoma, which was treated endoscopically, and one patient presented with a recurrent invasive carcinoma, which was treated surgically. CONCLUSIONS: Endoscopic polypectomy is a safe and effective method of treating large colorectal polyps, associated with a low complication rate, reduced hospital stay and lower costs in comparison to surgical procedure.

[Cancer and Crohn's disease]. / Das Karzinom beim M. Crohn.

We report 12 cases, complicating Crohn's disease by cancer origin. 8 patients who had chronic anorectal fistula, developed mucinous adenocarcinoma, 4 patients colorectal cancer. Features like early onset of disease, duration of disease for more than 10 years, chronic (pan-)colitis with high inflammatory activity and persistence of chronic fistulas and stenosis seem to trigger malignant transformation. Submucosal growth, overlap-syndromes with Crohn's disease and absence of sufficient clinical control of stenosis or fistula lead to advanced tumour stages at time of diagnosis combined with poor prognosis. Within four years after diagnosis 7 of 10 patients died related to metastatic disease or local recurrence. Absence of sufficient possibilities for clinical control indicates radical and early surgical resection of both endoscopically not surveilable stenosis and longstanding anorectal fistulas.

Gas chromatographic detection of Mycobacterium tuberculosis complex in pure cultures from respiratory specimens.

It is important to identify mycobacteria to the species level in order to establish their clinical significance and to take the appropriate therapeutical decision. Biochemical tests on primary cultures take time (3-6 weeks) until the report of results; that's why more rapid techniques are needed. We have used gas chromatography with flame-ionisation detection (GC-FID) as an alternative identification method for 53 mycobacterial strains isolated from respiratory specimens. We have extracted fatty acids from whole mycobacterial cells, then derivatized them into methylesters, detectable by GC-FID. All the strains were identified as M. tuberculosis complex (MTC), using the Microbial Identification System (MIS) software. The specificity of the identification by GC-FID of MTC is 100%. In conclusion, pulmonary mycobacteriosis are dominated by MTC; GC-FID is a rapid and accurate method for the identification of MTC.

Quantitative detection of lac-Z-transfected CC531 colon carcinoma cells in an orthotopic rat liver metastasis model.

Disseminated colon carcinoma metastases in the liver are associated with low cure rates and constitute a serious therapeutic problem. Appropriate experimental models which mimic metastases development and outgrowth can provide insight into the mechanism of this lethal process and facilitate the finding of new approaches for its control. We established an orthotopic liver metastases model based on CC531 rat colon adenocarcinoma cells which were transfected with a beta-galactosidase gene as marker to facilitate their detection. Intraportal injection of CC531-lac-Z cells resulted in a rapid and locally aggressive growth within the liver and was characterised by a tumour volume doubling time of 20 h and abundant angiogenesis. A commercially available chemi-luminescence assay allowed rapid, quantitative and sensitive detection of the diffusely growing tumour cells. Immunogenicity of CC531-lac-Z cells induced by the marker gene was significantly reduced by co-administering the tumour cells with matrigel. Within an observation period of three weeks following tumour cell injection only 6% of the animals showed lung involvement, thus indicating a specific homing of CC531-lac-Z cells to the liver. This period appears long enough to allow therapeutic manipulations at various stages of tumour growth in the liver. It is envisaged that the model will have applications for various therapeutic strategies.