Shotgun metagenomic analysis of the oral microbiome in gingivitis: a nested case-control study.

Background: Gingivitis, i.e. inflammation of the gums, is often induced by dentalplaque. However, its exact link to the oral microbiota remains unclear. Methods: In a case-control study involving 120 participants, comprising 60 cases and 60 controls (mean age (SD) 36.6 (7.6) years; 50% males), nested within a prospective multicentre cohort study, we examined theoral microbiome composition of gingivitis patients and their controlsusing shotgun metagenomic sequencing of saliva samples. Participants underwent clinical and radiographic oral health examinations, including bleeding on probing (BOP), at six tooth sites. BOP ≥33%was considered 'generalized gingivitis/initial periodontitis'(GG/IP), and BOP <33% as 'healthy and localized gingivitis'(H/LG). Functional potential was inferred using HUMANn3. Results: GG/IP exhibited an increase in the abundance of Actinomyces, Porphyromonas, Aggregatibacter, Corynebacterium, Olsenella, and Treponema, whereas H/LG exhibited an increased abundance of Candidatus Nanosynbacter. Nineteen bacterial species and fourmicrobial functional profiles, including L-methionine, glycogen, andinosine-5'-phosphate biosynthesis, were associated with GG/IP. Constructing models with multiple markers resulted in a strong predictive value for GG/IP, with an area under the curve (ROC) of 0.907 (95% CI: 0.848-0.966). Conclusion: We observed distinct differences in the oral microbiome between the GG/IP and H/LG groups, indicating similar yet unique microbial profiles and emphasizing their potential role in progression of periodontal diseases.

Dysbiosis of the oral microbiota composition is associated with oral squamous cell carcinoma and the impact of radiotherapy: a pilot study.

Radiotherapy can potentially influence the diversity and composition of the oral microbiome. We performed a study comparing the composition of oral microbiota in patients with oral squamous cell carcinoma (OSCC) before radiotherapy (n = 6), at three months (n = 6), and six months (n = 6) post-radiotherapy, and controls (n = 6). We profiled the oral microbiome by 16S rRNA gene sequencing using Illumina MiSeq. Alpha diversity (Chao1 index) showed significant differences in species richness between healthy controls and OSCC patients (P = 0.014). Conversely, no noteworthy distinctions were observed in the Chao1 index when comparing the pre-and post-radiation periods at both three and six months. The beta diversity of the oral microbiota differed significantly between the controls and OSCC patients (P = 0.014). However, no significant differences were observed in beta diversity between pre- and post-radiation at three months, whereas a significant difference was observed at six months (P = 0.038). Linear Discriminant Analysis Effect Size (LEfSe) demonstrated lower abundance of Corynebacterium, Actinomyces, Veillonella, and Haemophilus, and higher abundance of Selenomonas and Mycoplasma in OSCC patients than in healthy controls. The oral microbiome composition varied among healthy controls, patients with OSCC, and post-radiation therapy patients with OSCC. The observed recovery in the numerical dominance of specific beneficial oral taxa and the reduction in pathogenic bacteria after radiation therapy highlights the need for further investigations into their clinical implications.

Subgingival microbiome at different levels of cognition.

Oral health and declining cognition may have a bi-directional association. We characterized the subgingival microbiota composition of subjects from normal cognition to severe cognitive decline in two cohorts. Memory and Periodontitis (MINOPAR) include 202 home-living participants (50-80 years) in Sweden. Finnish Oral Health Studies in Older Adults (FINORAL) include 174 participants (≥65 years) living in long-term care in Finland. We performed oral examination and assessed the cognitive level with Mini Mental State Examination (MMSE). We sequenced the 16S-rRNA gene (V3-V4 regions) to analyse the subgingival bacterial compositions. The microbial diversities only tended to differ between the MMSE categories, and the strongest determinants were increased probing pocket depth (PPD) and presence of caries. However, abundances of 101 taxa were associated with the MMSE score. After adjusting for age, sex, medications, PPD, and caries, only eight taxa retained the significance in the meta-analyses of the two cohorts. Especially Lachnospiraceae [XIV] at the family, genus, and species level increased with decreasing MMSE. Cognitive decline is associated with obvious changes in the composition of the oral microbiota. Impaired cognition is accompanied with poor oral health status and the appearance of major taxa of the gut microbiota in the oral cavity. Good oral health-care practices require special deliberations among older adults.

The Composition and Functional Capacities of Saliva Microbiota Differ Between Children With Low and High Sweet Treat Consumption.

Excess sugar consumption-common in youth-is associated with poor health. Evidence on the relationship between sugar consumption and the oral microbiome, however, remains scarce and inconclusive. We explored whether the diversity, composition, and functional capacities of saliva microbiota differ based on the consumption of select sugary foods and drinks ("sweet treats"). Using 16S rRNA gene sequencing, we characterized saliva microbiota from 11 to 13-year-old children who participated in the Finnish Health in Teens (Fin-HIT) cohort study. The sample comprised children in the lowest (n = 227) and highest (n = 226) tertiles of sweet treat consumption. We compared differences in the alpha diversity (Shannon, inverse Simpson, and Chao1 indices), beta diversity (principal coordinates analysis based on Bray-Curtis dissimilarity), and abundance (differentially abundant operational taxonomic units (OTUs) at the genus level) between these low and high consumption groups. We performed PICRUSt2 to predict the metabolic pathways of microbial communities. No differences emerged in the alpha diversity between low and high sweet treat consumption, whereas the beta diversity differed between groups (p = 0.001). The abundance of several genera such as Streptococcus, Prevotella, Veillonella, and Selenomonas was higher in the high consumption group compared with the low consumption group following false discovery rate correction (p < 0.05). Children with high sweet treat consumption exhibited higher proportions of nitrate reduction IV and gondoate biosynthesis pathways compared with the low consumption group (p < 0.05). To conclude, sweet treat consumption shapes saliva microbiota. Children who consume a high level of sweet treats exhibited different compositions and metabolic pathways compared with children who consume low levels of sweet treats. Our findings reveal novel insights into the relationship between sugary diets and oral microbiota.

Changes in Bacterial Diversity and Composition in Response to Co-inoculation of Arbuscular Mycorrhizae and Zinc-Solubilizing Bacteria in Turmeric Rhizosphere.

In the present study, the impact of co-inoculation of arbuscular mycorrhizal fungi (AM Rhizophagus sp., NCBI-MN710507) and Zinc solubilizing bacteria (ZSB2- Bacillus megaterium, NCBI-KY687496) on plant growth, soil dehydrogenase activity, soil respiration and the changes in bacterial diversity in rhizosphere of turmeric (Curcuma longa) were examined. Our results showed that higher plant height and dry biomass were observed in treatments co-inoculated with AM and ZSB2. Likewise, dehydrogenase activity and soil respiration were more significant in the co-inoculation treatment, indicating abundance of introduced as well as inherent microflora. Bacterial community analysis using 16S rRNA revealed changes in the structure and diversity of various taxa due to co-inoculation of AM and ZSB2. Alpha diversity indexes (Shannon and Chao1) and beta diversity indexes obtained through unweighted unifrac approach also showed variation among the treated samples. Chloroflexi was the dominant phylum followed by Proteobacteria, Actinobacteria and Acidobacteria which accounted for 80% of all treated samples. The composition of bacterial communities at genus level revealed that co-inoculation caused distinct bacterial profiles. The Linear discriminant analysis effect size revealed the dominance of ecologically significant genera such as Bradyrhizobium, Candidatus, Pedomicrbium, Thermoporothrix, Acinetobacter and Nitrospira in treatments co-inoculated with AM and ZSB2. On the whole, co-inoculated treatments revealed enhanced microbial activities and caused significant positive shifts in the bacterial diversity and abundance compared to treatments with sole application of ZSB2 or AM.

Production of Oligoalginate via Solution Plasma Process and Its Capability of Biological Growth Enhancement.

The objective of the study was to depolymerize alginate into short-length oligoalginates, adopting the simple solution plasma process (SPP) technique, for successful use in free radical scavenging and growth promotion in cell culture and agricultural practices. Alginate at various concentrations was depolymerized to oligoalginates using SPP by discharging for various times. The depolymerization into oligoalginates was proved by DNS, TLC, FT-IR, and HPAEC analyses and caused decrease in viscosity. Oligoalginates derived from 0.5% alginate (100 mg∙mL-1) showed the highest antioxidant activities in vitro. The oligoalginates enhanced growth of the human embryonic kidney (HEK293) cells to significant levels in a concentration-dependent manner without any extent of toxicity. The oligoalginates also promoted growth of lettuce. Thus, SPP is a powerful technique to break down alginate into oligoalginates that can be utilized as a free radical scavenger and as a growth promoter of animal cells and agricultural plants.

High abundance of sugar metabolisers in saliva of children with caries.

Dental caries is a biofilm-mediated, dynamic disease with early onset. A balanced salivary microbiota is a foundation of oral health, while dysbiosis causes tooth decay. We compared the saliva microbiota profiles in children with and without caries. The study consisted of 617 children aged 9-12 years from the Finnish Health in Teens (Fin-HIT) study with available register data on oral health. Caries status was summarised based on Decayed, Missing, and Filled Teeth (DMFT) index in permanent dentition. The children were then classified into the following two groups: DMFT value ≥ 1 was considered as cavitated caries lesions (hereafter called 'caries') (n = 208) and DMFT = 0 as 'cavity free' (n = 409). Bacterial 16S rRNA gene (V3-V4 regions) was amplified using PCR and sequenced by Illumina HiSeq. The mean age (SD) of the children was 11.7 (0.4) years and 56% were girls. The children had relatively good dental health with mean DMFT of 0.86 (1.97). Since sex was the key determinant of microbiota composition (p = 0.014), we focused on sex-stratified analysis. Alpha diversity indexes did not differ between caries and cavity free groups in either sexes (Shannon: p = 0.40 and 0.58; Inverse Simpson: p = 0.51 and 0.60, in boys and girls, respectively); neither did the composition differ between the groups (p = 0.070 for boys and p = 0.230 for girls). At the genus level, Paludibacter and Labrenzia had higher abundances in the caries group compared to cavity free group in both sexes (p < 0.001). Taken together, there were minor differences in saliva microbiota between children with and without caries. Potential biomarkers of caries were the sugar metabolisers Paludibacter and Labrenzia. These bacteria presumably enhance salivary acidification, which contributes to progression of dental caries. The clinical relevance of our findings warrants further studies.

First Report of Pathogenic Bacterium Kalamiella piersonii Isolated from Urine of a Kidney Stone Patient: Draft Genome and Evidence for Role in Struvite Crystallization.

Uropathogenic bacteria are widely distributed in the environment and urinary tract infection is implicated in kidney stone disease. Here, we report on a urease negative bacterium Kalamiella piersonii (strain YU22) isolated from the urine of a struvite stone (MgNH4PO4·6H2O) patient. The closest species, K. piersonii IIIF1SW-P2T was reported from International Space Station samples. However, there are no earlier reports on its human association. Using whole genome and experimental analysis, its involvement in urinary tract colonization and struvite crystallization was explored. The strain YU22 showed many virulence factors that are needed for host cell invasion and colonization including cell adhesion factors, swimming and swarming motilities, biofilm and siderophore among others. In vitro infection studies in HEK-293T cells demonstrated the host cell attachment and killing. It was able to utilize amino acids as sole carbon source and showed growth in synthetic and healthy urine establishing metabolic adaptation to urinary tract. Increased pH and availability of ammonium ions from amino acid breakdown promoted struvite crystallization. The results from this study support the involvement of urease negative uropathogen in the struvite lithogenesis. Further studies on other isolates of K. peirsonii are warranted to assess its health risks.

Demineralization of tooth enamel following radiation therapy; An in vitro microstructure and microhardness analysis.

OBJECTIVE: The objective of this study is to evaluate the effects of radiotherapy doses on mineral density and percentage mineral volume of human permanent tooth enamel. MATERIALS AND METHODS: Synchrotron radiation Xray microcomputed tomography (SRµCT) and microhardness testing were carried out on 8 and 20 tooth samples, respectively. Enamel mineral density was derived from SRµCT technique using ImageJ software. Microhardness samples were subjected to Vickers indentations followed by calculation of microhardness and percentage mineral volume values using respective mathematical measures. Data were analyzed using paired t-test at a significance level of 5%. Qualitative analysis of the enamel microstructure was done with two-dimensional projection images and scanned electron micrographs using µCT and field emission scanning electron microscopy, respectively. RESULTS: Vickers microhardness and SRµCT techniques showed a decrease in microhardness and an increase in mineral density, respectively, in postirradiated samples. These changes were related to mineral density variation and alteration of hydroxyapatite crystal lattice in enamel surface. Enamel microstructure showed key features such as microporosities and loss of smooth homogeneous surface. These indicate tribological loss and delamination of enamel which might lead to radiation caries. CONCLUSIONS: Tooth surface loss might be a major contributing factor for radiation caries in head-and-neck cancer patients prescribed to radiotherapy. Such direct effects of radiotherapy cause enamel abrasion, delamination, and damage to the dentinoenamel junction. Suitable measures should, therefore, be worked out to protect nontarget oral tissues such as teeth while delivering effective dosages to target regions.

Microstructural diversity of gallstones revealed by spectral microanalyses and their association with hepatic parameters.

BACKGROUND: Gallstones (GS) are formed as a result of impaired metabolic regulation and can be reflected in serum parameters. This study was focused on classifying GS based on spectral microanalysis and identifying the possible role of serum hepatic parameters on GS of different compositions. METHODS: The study included a total of 126 GS from 80 consecutive patients who underwent cholecystectomy for GS diseases in a single center. The composition and microstructure of GS were analyzed using Fourier-transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), and energy dispersive X-ray spectroscopy (EDS). The serum hepatic parameters were studied in order to establish a possible etiologic relationship with GS composition. RESULTS: In the study group, the incidence of GS was higher in females 62 (77.5%) compared with males 18 (22.5%). The mean age was 42.81 ± 13.01 and 43.78 ± 14.4 years for female and male patients, respectively. Based on composition, the GS were assigned to four major groups: cholesterol, pigment, phosphate, and mixed stones. Mixed composition stones totally represented the majority 53 (66.3%), followed by pure cholesterol 23 (28.8%), pigment stones 2 (2.5%), and phosphate stones 2 (2.5%), respectively. Elemental composition revealed the presence of carbon (C), oxygen (O), calcium (Ca), and phosphorus (P) to be major elements along with traces of sodium (Na), magnesium (Mg), aluminum (Al), iron (Fe), copper (Cu), bromine (Br), manganese (Mn), and zinc (Zn). Among serum parameters, total bilirubin, direct bilirubin, indirect bilirubin, serum glutamic-oxaloacetic transaminase, and total protein were higher in patients with pigment GS than cholesterol GS. CONCLUSIONS: Characterization of GS indicates that different types of stones have different characteristics in terms of microstructure, elemental composition, and distribution. Serum hepatic function test profiles showed an association with the compositions of GS.

Morphological characteristics and microstructure of kidney stones using synchrotron radiation µCT reveal the mechanism of crystal growth and aggregation in mixed stones.

Understanding the mechanisms of kidney stone formation, development patterns and associated pathological features are gaining importance due to an increase in the prevalence of the disease and diversity in the presentation of the stone composition. Based on the microstructural characteristics of kidney stones, it may be possible to explain the differences in the pathogenesis of pure and mixed types of stones. In this study, the microstructure and distribution of mineral components of kidney stones of different mineralogy (pure and mixed types) were analyzed. The intact stones removed from patients were investigated using synchrotron radiation X-ray computed microtomography (SR-µCT) and the tomography slice images were reconstructed representing the density and structure distribution at various elevation planes. Infrared (IR) spectroscopes, X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used to confirm the bulk mineral composition in the thin section stones. Observations revealed differences in the micro-morphology of the kidney stones with similar composition in the internal 3-D structure. Calcium oxalate monohydrate stones showed well-organised layering patterns, while uric acid stones showed lower absorption signals with homogenous inner structure. Distinct mineral phases in the mixed types were identified based on the differential absorption rates. The 3-D quantitative analysis of internal porosity and spatial variation between nine different types of stones were compared. The diversity among the microstructure of similar and different types of stones shows that the stone formation is complex and may be governed by both physiological and micro-environmental factors. These factors may predispose a few towards crystal aggregation and stone growth, while, in others the crystals may not establish stable attachment and/or growth.

Electron probe micro-analysis reveals the complexity of mineral deposition mechanisms in urinary stones.

Urinary stones are complex mineralogical formations in the urinary system often impairing the kidney function. Several studies have attempted to understand the mechanisms of stone formation and growth; however, it remains to be fully explored. Here, we present a detailed investigation on the morphological and mineralogical characterizations of urinary stones. Structural properties of different types of urinary stones were done by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and field-emission scanning electron microscope (FE-SEM) analyses. X-ray maps of major and the trace elements were obtained using electron microprobe (EPMA) technique. Basic metabolic panel and urinary parameters of the patients were used for comparing mineral compositions among stone types. The study included five major types of stones identified based on the FTIR spectra. FTIR and XRD helped in identifying the major components of these stones. FE-SEM images revealed distinct microstructure and morphology of the stones among the stone types. EPMA analysis showed the presence of many metals other than calcium and certain non-metals within the urinary stone matrix at measurable levels, sometimes with distinct distribution patterns. The study demonstrates the characteristic micro-structure, morphology, distribution, and composition of elements in different stone types. Findings of the study provide scope for understanding the complex mechanisms involved in the urolithogenesis and association of trace elements in it.

Vitamin C inhibits crystallization of struvite from artificial urine in the presence of Pseudomonas aeruginosa

ABSTRACT Background: Formation of struvite stones is associated with urinary tract infection by urease-producing bacteria. Biogenic crystal growth in natural and synthetic materials is regulated by the action of inhibitors, ranging from small ions, molecules to large macromolecules. Materials and Methods: We report the dynamics of in vitro crystallization of struvite in presence of vitamin C in synthetic urine using single diffusion gel growth technique. Sodium metasilicate gel of specific gravity 1.05 and the aqueous solution of ammonium dihydrogen phosphate were used as the medium for growing the struvite crystals. The crystallization process was induced by a urease positive struvite stone associated Pseudomonas aeruginosa to mimic the infection leading to stone formation. The grown crystals were characterized by ATR-FTIR and powder XRD. The surface morphology was analysed through FE-SEM for comparison between treatments. Results: We observed decrease in number, dimension, and growth rate of struvite crystals with the increasing concentrations of vitamin C. Crystals displayed well-defined faces and dendritic morphology of struvite in both control and biogenic systems. Conclusion: The results strongly suggest that, vitamin C can modulate the formation of struvite crystals in the presence of uropathogenic bacteria.

Morphological and micro-tomographic study on evolution of struvite in synthetic urine infected with bacteria and investigation of its pathological biomineralization.

Pathological biomineralization in the urinary system leads to urolithiasis. Formation of kidney stones involves a series of events during which they undergo morphological and mineralogical changes. We investigated the mineralization of biogenic struvite (in vitro) and examined the transformation of distinct interior and exterior structure of struvite. In vitro crystallization of struvite was performed in the presence of two bacteria that were originally isolated from the kidney stone patients. Morphological evaluation was carried out using SR-µCT as well as FESEM, XRD and FT-IR. Characteristic internal 3-D morphology and porosity of the stones were studied. For comparison, patient derived struvite stones were used. From the results obtained, we report that the presence of bacteria enhances the crystallization process of struvite in vitro. A series of time-resolved experiments revealed that struvite crystals experienced a significant morphologic evolution from pin pointed structure to X-shaped and tabular morphologies. These X-shaped and unusual tabular habits of struvite resembled biogenic morphologies of struvite. SR-µCT showed similarities between the patient derived and the in vitro derived struvite crystals. In conclusion, these experiments revealed that the bacteria play a major role in the specific morphogenesis of struvite and can able to control the nucleation, modulate crystalline phases, and shape of the growing crystal.

Vitamin C inhibits crystallization of struvite from artificial urine in the presence of Pseudomonas aeruginosa.

BACKGROUND: Formation of struvite stones is associated with urinary tract infection by urease-producing bacteria. Biogenic crystal growth in natural and synthetic materials is regulated by the action of inhibitors, ranging from small ions, molecules to large macromolecules. MATERIALS AND METHODS: We report the dynamics of in vitro crystallization of struvite in presence of vitamin C in synthetic urine using single diffusion gel growth technique. Sodium metasilicate gel of specific gravity 1.05 and the aqueous solution of ammonium dihydrogen phosphate were used as the medium for growing the struvite crystals. The crystallization process was induced by a urease positive struvite stone associated Pseudomonas aeruginosa to mimic the infection leading to stone formation. The grown crystals were characterized by ATR-FTIR and powder XRD. The surface morphology was analysed through FE-SEM for comparison between treatments. RESULTS: We observed decrease in number, dimension, and growth rate of struvite crystals with the increasing concentrations of vitamin C. Crystals displayed well-defined faces and dendritic morphology of struvite in both control and biogenic systems. CONCLUSION: The results strongly suggest that, vitamin C can modulate the formation of struvite crystals in the presence of uropathogenic bacteria.

Characteristics of bacterial colonization after indwelling double-J ureteral stents for different time duration.

BACKGROUND: Indwelling Double-J ureteral stenting is commonly used in urological practice and has various complications. This study aimed to assess the frequency of bacterial stent colonization and stent-associated bacteriuria after indwelling it for different time durations and to evaluate the significance of urinary cultures for identification of colonizing microorganisms. MATERIALS AND METHODS: A prospective cross-sectional study was conducted. Midstream urine from 72 patients undergoing J stent insertion was investigated microbiologically before stent insertion and on the day of stent removal. The stents were removed by aseptic manipulation, and 1-3 cm of the tip located in the bladder was collected for microbiological study. The urine and stent samples were cultured, and the bacterial pathogens were identified using standard microbiological methods followed by Phoenix automated system. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method. RESULTS: Bacterial colonies were found in 47.2% (34 of 72) of the stents. Of the multiple pathogens identified, Escherichia coli (20%) was the most common, followed by Streptococcus sp. (17.5%) and Pseudomonas sp. (12.5%). The bacteria did not colonize within the first 2 weeks of stent placement. Results showed that 55% of the isolates were resistant to erythromycin, 52.5% to ampicillin, 42.5% to piperacillin, and least resistant being 17.5% for tetracycline and imipenem. However, 81.3% and 66.7% of the stents were colonized when placed for 90-120 days and 60-90 days, respectively. CONCLUSION: High prevalence of bacterial isolates and risk of bacteriuria and colonization was found in the DJ stent tips, with E. coli being dominant colonizer. Most of the bacteria were resistant to different classes of antibiotics. Bacteriuria and stent colonization gradually increases with the duration of stent retention in the body.