High-resolution taxonomic examination of the oral microbiome after oil pulling with standardized sunflower seed oil and healthy participants: a pilot study.

OBJECTIVES: We aimed at the high-resolution examination of the oral microbiome depending on oil pulling, compared it with saline pulling, and analyzed whether the method is capable of reducing the overall microbial burden of the oral cavity. MATERIALS AND METHODS: The study was a cohort study with three healthy subjects. Oil pulling samples, saline pulling samples, and saliva samples were microscoped and cultured under microaerophilic and anaerobic conditions; colony-forming units were counted; and cultivated bacteria were identified employing MALDI-TOF MS. The oral microbiomes (saliva) and the microbiota incorporated in oil and saline pulling samples were determined in toto by using 16S rDNA next-generation sequencing (NGS) and bioinformatics. RESULTS: Microscopy revealed that oral epithelial cells are ensheathed with distinct oil droplets during oil pulling. Oil pulling induced a higher production of saliva and the oil/saliva emulsion contained more bacteria than saline pulling samples. Oil pulling resulted in a significant and transient reduction of the overall microbial burden in comparison to saliva examined prior to and after pulling. Both oil and saline pulling samples mirrored the individual oral microbiomes in saliva. CONCLUSIONS: Within the limitations of this pilot study, it might be concluded that oil pulling is able to reduce the overall microbial burden of the oral cavity transiently and the microbiota in oil pulling samples are representative to the oral microbiome. CLINICAL RELEVANCE: Within the limitations of this pilot study, it might be concluded that oil pulling can be considered as an enlargement of standard oral hygiene techniques since it has the characteristic of an oral massage, enwrapping epithelial cells carrying bacteria in oil vesicles and reaching almost all unique habitats in oral cavity.

Depletion of Lipocalin 2 (LCN2) in Mice Leads to Dysbiosis and Persistent Colonization with Segmented Filamentous Bacteria.

Lipocalin 2 (LCN2) mediates key roles in innate immune responses. It has affinity for many lipophilic ligands and binds various siderophores, thereby limiting bacterial growth by iron sequestration. Furthermore, LCN2 protects against obesity and metabolic syndrome by interfering with the composition of gut microbiota. Consequently, complete or hepatocyte-specific ablation of the Lcn2 gene is associated with higher susceptibility to bacterial infections. In the present study, we comparatively profiled microbiota in fecal samples of wild type and Lcn2 null mice and show, in contrast to previous reports, that the quantity of DNA in feces of Lcn2 null mice is significantly lower than that in wild type mice (p < 0.001). By using the hypervariable V4 region of the 16S rDNA gene and Next-Generation Sequencing methods, we found a statistically significant change in 16 taxonomic units in Lcn2-/- mice, including eight gender-specific deviations. In particular, members of Clostridium, Escherichia, Helicobacter, Lactococcus, Prevotellaceae_UCG-001 and Staphylococcus appeared to expand in the intestinal tract of knockout mice. Interestingly, the proportion of Escherichia (200-fold) and Staphylococcus (10-fold) as well as the abundance of intestinal bacteria encoding the LCN2-sensitive siderphore enterobactin (entA) was significantly increased in male Lcn2 null mice (743-fold, p < 0.001). This was accompanied by significant higher immune cell infiltration in the ileum as demonstrated by increased immunoreactivity against the pan-leukocyte protein CD45, the lymphocyte transcription factor MUM-1/IRF4, and the macrophage antigen CD68/Macrosialin. In addition, we found a higher expression of mucosal mast cell proteases indicating a higher number of those innate immune cells. Finally, the ileum of Lcn2 null mice displayed a high abundance of segmented filamentous bacteria, which are intimately associated with the mucosal cell layer, provoking epithelial antimicrobial responses and affecting T-helper cell polarization.

Recombinant Porphyromonas gingivalis W83 FimA alters immune response and metabolic gene expression in oral squamous carcinoma cells.

OBJECTIVES: The Gram-negative anaerobic rod Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in periodontitis and expresses a multitude of virulence factors iincluding fimbriae that are enabling adherence to and invasion in cells and tissues. The progression of periodontitis is a consequence of the interaction between the host immune response and periodontal pathogens. The aim of this study was to investigate the genome-wide impact of recombinant fimbrial protein FimA from P. gingivalis W83 on the gene expression of oral squamous carcinoma cells by transcriptome analysis. MATERIALS AND METHODS: Human squamous cell carcinoma cells (SCC-25) were stimulated for 4 and 24 h with recombinant FimA. RNA sequencing was performed and differential gene expression and enrichment were analyzed using gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and REACTOME. The results of transcriptome analysis were validated using quantitative real-time polymerase chain reaction (PCR) with selected genes. RESULTS: Differential gene expression after 4 and 24 h revealed upregulation of 464 (4 h) and 179 genes (24 h) and downregulation of 69 (4 h) and 312 (24 h) genes. GO, KEGG, and REACTONE enrichment analysis identified a strong immunologic transcriptomic response signature after 4 h. After 24 h, mainly those genes were regulated, which belonged to cell metabolic pathways and replication. Real-time PCR of selected genes belonging to immune response and signaling demonstrated strong upregulation of CCL20, TNFAIP6, CXCL8, TNFAIP3, and NFkBIA after both stimulation times. CONCLUSIONS: These data shed light on the RNA transcriptome of human oral squamous carcinoma epithelial cells following stimulation with P. gingivalis FimA and identify a strong immunological gene expression response to this virulence factor. The data provide a base for future studies of molecular and cellular interactions between P. gingivalis and oral epithelium to elucidate basic mechanisms that may provide new prospects for periodontitis therapy and give new insights into the development and possible treatments of cancer.

Odontogenic Cervicofacial Necrotizing Fasciitis: Microbiological Characterization and Management of Four Clinical Cases.

Necrotizing fasciitis of the head and neck is a rare, very severe disease, which, in most cases, originates from odontogenic infections and frequently ends with the death of the patient. Rapid surgical intervention in combination with a preferably pathogen-specific antibiotic therapy can ensure patients' survival. The question arises concerning which pathogens are causative for the necrotizing course of odontogenic inflammations. Experimental 16S-rRNA gene analysis with next-generation sequencing and bioinformatics was used to identify the microbiome of patients treated with an odontogenic necrotizing infection and compared to the result of the routine culture. Three of four patients survived the severe infection, and one patient died due to septic multiorgan failure. Microbiome determination revealed findings comparable to typical odontogenic abscesses. A specific pathogen which could be causative for the necrotizing course could not be identified. Early diagnosis and rapid surgical intervention and a preferably pathogen-specific antibiotic therapy, also covering the anaerobic spectrum of odontogenic infections, are the treatments of choice. The 16S-rRNA gene analysis detected significantly more bacteria than conventional methods; therefore, molecular methods should become a part of routine diagnostics in medical microbiology.

Microbiome of Odontogenic Abscesses.

Severe odontogenic abscesses are regularly caused by bacteria of the physiological oral microbiome. However, the culture of these bacteria is often prone to errors and sometimes does not result in any bacterial growth. Furthermore, various authors found completely different bacterial spectra in odontogenic abscesses. Experimental 16S rRNA gene next-generation sequencing analysis was used to identify the microbiome of the saliva and the pus in patients with a severe odontogenic infection. The microbiome of the saliva and the pus was determined for 50 patients with a severe odontogenic abscess. Perimandibular and submandibular abscesses were the most commonly observed diseases at 15 (30%) patients each. Polymicrobial infections were observed in 48 (96%) cases, while the picture of a mono-infection only occurred twice (4%). On average, 31.44 (±12.09) bacterial genera were detected in the pus and 41.32 (±9.00) in the saliva. In most cases, a predominantly anaerobic bacterial spectrum was found in the pus, while saliva showed a similar oral microbiome to healthy individuals. In the majority of cases, odontogenic infections are polymicrobial. Our results indicate that these are mainly caused by anaerobic bacterial strains and that aerobic and facultative anaerobe bacteria seem to play a more minor role than previously described by other authors. The 16S rRNA gene analysis detects significantly more bacteria than conventional methods and molecular methods should therefore become a part of routine diagnostics in medical microbiology.

Clinical Relevance of the Microbiome in Odontogenic Abscesses.

Odontogenic abscesses are usually caused by bacteria of the oral microbiome. However, the diagnostic culture of these bacteria is often prone to errors and sometimes fails completely due to the fastidiousness of the relevant bacterial species. The question arises whether additional pathogen diagnostics using molecular methods provide additional benefits for diagnostics and therapy. Experimental 16S rRNA gene analysis with next-generation sequencing (NGS) and bioinformatics was used to identify the microbiome of the pus in patients with severe odontogenic infections and was compared to the result of standard diagnostic culture. The pus microbiome was determined in 48 hospitalized patients with a severe odontogenic abscess in addition to standard cultural pathogen detection. Cultural detection was possible in 41 (85.42%) of 48 patients, while a pus-microbiome could be determined in all cases. The microbiomes showed polymicrobial infections in 46 (95.83%) cases, while the picture of a mono-infection occurred only twice (4.17%). In most cases, a predominantly anaerobic spectrum with an abundance of bacteria was found in the pus-microbiome, while culture detected mainly Streptococcus, Staphylococcus, and Prevotella spp. The determination of the microbiome of odontogenic abscesses clearly shows a higher number of bacteria and a significantly higher proportion of anaerobes than classical cultural methods. The 16S rRNA gene analysis detects considerably more bacteria than conventional cultural methods, even in culture-negative samples. Molecular methods should be implemented as standards in medical microbiology diagnostics, particularly for the detection of polymicrobial infections with a predominance of anaerobic bacteria.

A New Type of Chronic Wound Infection after Wisdom Tooth Extraction: A Diagnostic Approach with 16S-rRNA Gene Analysis, Next-Generation Sequencing, and Bioinformatics.

Delayed-onset infections are rare postoperative complications of lower third molar extractions. This article presents a case of a chronic combined hard and soft tissue infection after the extraction of a third molar, where the causative organisms could only be elucidated by molecular methods. Experimental 16S-rRNA gene analysis with next-generation sequencing and bioinformatics was used to identify the bacterial spectrum of the infection. 16S-rRNA gene analysis delivered the microbiome of the abscessing inflammation while standard culture and laboratory examinations were all sterile. The microbiome showed a mixed bacterial infection with a dominance of Delftia and Alcanivorax (spp.) besides other bacteria of the normal oral flora. Using 16S-rRNA-gene analysis, next-generation sequencing, and bioinformatics, a new type of chronic wound infection after wisdom tooth extraction was found. The property of Delftia and Alcanivorax (spp.) as water-affine environmental bacteria raises suspicion of infection from contaminated water from a dental unit. Thus, osteotomies of teeth should only be done with sterile cooling water. The 16S-rRNA gene analysis should become a part of the routine diagnostics in medical microbiology.

Draft Genome Sequence of Lactobacillus delbrueckii Strain #22 Isolated from a Patient with Short Bowel Syndrome and Previous d-Lactic Acidosis and Encephalopathy.

d-Lactic acidosis with associated encephalopathy caused by overgrowth of intestinal lactic acid bacteria is a rarely diagnosed neurological complication of patients with short bowel syndrome. Here, we report the draft genome sequence of Lactobacillus delbrueckii strain #22 isolated from a patient with short bowel syndrome and previous d-lactic acidosis/encephalopathy.