16S rRNA Gene Research Articles

The Bidirectional Effects of Periodontal Disease and Oral Dysbiosis on Gut Inflammation in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) flares can lead to excessive morbidity and mortality. This study aimed to determine whether oral dysbiosis/periodontal disease (PD) is common in IBD and is associated with disease activity in IBD. This single-center, prospective, cross-sectional, proof-of-concept, observational study assessed the frequency of periodontal inflammatory disease and interrogated oral and stool microbiota using 16S rRNA gene amplicon sequencing of active-IBD (aIBD), inactive-IBD (iIBD), and healthy controls (HC). Questionnaires assessed diet, alcohol usage, oral hygiene behavior, and disease activity. A subset of participants underwent comprehensive dental examinations to evaluate PD. PD was severer in aIBD subjects than in HC, as aIBD had poorer quality diets (lower Mediterranean diet scores) than iIBD and HC. Significant differences in microbial community structure were observed in unstimulated saliva, stimulated saliva, gingiva, and stool samples, primarily between aIBD and HC. Saliva from aIBD had higher relative abundances of putative oral pathobionts from the genera Streptococcus, Granulicatella, Rothia, and Actinomyces relative to HC, despite similar oral hygiene behaviors between groups. Our study suggests that patients with aIBD have severer periodontal disorders and higher relative abundances of putative "pro-inflammatory" microbiota in their oral cavity, despite normal oral hygiene behaviors. Our data are consistent with the potential presence of an oral-gut inflammatory-axis that could trigger IBD flare-ups in at-risk patients. Routine dental health assessments in all IBD patients should be encouraged as part of the health maintenance of IBD and as a potential strategy to decrease the risk of IBD flares.

Pooled analysis of oral microbiome profiles defines robust signatures associated with periodontitis.

Oral microbial dysbiosis has been associated with periodontitis in studies using 16S rRNA gene sequencing analysis. However, this technology is not sufficient to consistently separate the bacterial species to species level, and reproducible oral microbiome signatures are scarce. Here, using ultra-deep metagenomic sequencing and machine learning tools, we defined a simple two-factor decision tree, based on Tannerella forsythia and Fretibacterium fastidiosum, that was highly associated with periodontitis. Altogether, we defined robust oral microbiome signatures relevant to the pathophysiology of periodontitis that can help define promising targets for microbiome therapeutic modulation when caring for patients with periodontitis.

Local emergence and global evolution of Neisseria gonorrhoeae with high-level resistance to azithromycin.

Antimicrobial resistance in Neisseria gonorrhoeae (Ng) has severely reduced treatment options, including azithromycin (AZM), which had previously been recommended as dual therapy with ceftriaxone. This study characterizes the emergence of high-level resistance to AZM (HLR-AZM) Ng in Baltimore, Maryland, USA, and describes the global evolution of HLR-AZM Ng. Whole genome sequencing (WGS) of 30 Ng isolates with and without HLR-AZM from Baltimore was used to identify clonality and resistance determinants. Publicly available WGS data from global HLR-AZM Ng (n = 286) and the Baltimore HLR-AZM Ng (n = 3) were used to assess the distribution, clonality, and diversity of HLR-AZM Ng. The HLR-AZM Ng isolates from Baltimore identified as multi-locus sequencing typing sequence type (ST) 9363 and likely emerged from circulating strains. ST9363 was the most widely disseminated ST globally represented in eight countries and was associated with sustained transmission events. The number of global HLR-AZM Ng, countries reporting these isolates, and strain diversity increased in the last decade. The majority (89.9%) of global HLR-AZM Ng harbored the A2059G mutation in all four alleles of the 23S rRNA gene, but isolates with two or three A2059G alleles, and alternative HLR-AZM mechanisms were also identified. In conclusion, HLR-AZM in Ng has increased in the last few years, with ST9363 emerging as an important gonococcal lineage globally. The 23S rRNA A2059G mutation is the most common resistance mechanism, but alternative mechanisms are emerging. Continued surveillance of HLR-AZM Ng, especially ST9363, and extensively drug-resistant Ng is warranted.

On new genera and species of crack-leg spiders (Araneae, Udubidae) from Madagascar

Two new genera, Tabiboka gen. nov. and Zorascar gen. nov., are described to accommodate five new Udubidae species from Madagascar: Tabiboka milleri gen. et sp. nov. (type species; ♂♀), T. milloti gen. et sp. nov. (♂♀), T. polotowae gen. et sp. nov. (♂♀), Zorascar pasunepipe gen. et sp. nov. (type species; ♂♀) and Z. pasunepomme gen. et sp. nov. (♀). In addition, two new species of Zorodictyna Strand, 1907 are described: Zorodictyna almae sp. nov. (♂♀) and Z. silvadavilae sp. nov. (♂♀). Uduba inhonesta Simon, 1906 is declared to be a junior synonym of Zorodictyna oswaldi (Lenz, 1891). The descriptions of the new species are complemented with a phylogenetic analysis to tentatively place them into an existing phylogenetic framework. Phylogenetic analyses based on COI and 16S rRNA genes and morphological data show that they are separate species and confirm their familial and supraspecific placement within the Udubidae. The new species are further described using high quality colored photographs, drawings, SEM and (for Zorascar gen. nov. ssp.) by the use of X-Ray micro-computed tomography to recreate virtual 3D models without destroying the original specimens. A dichotomous key to identify Malagasy Ububidae is provided. Whereas many undescribed Udubidae remain in Madagascar, the phylogenetic and generic arrangement that we present here succinctly summarizes the higher classification of Madagascar Udubidae.

Effect of the 16S rRNA Gene Hypervariable Region on the Microbiome Taxonomic Profile and Diversity in the Endangered Fish Totoaba macdonaldi

Understanding the intricate dynamics of fish microbiota through 16S rRNA amplicon sequencing is pivotal for ecological insights and effective disease management. However, this approach faces challenges including the co-amplification of host mitochondrial sequences and the variability in bacterial composition influenced by the selected 16S rRNA gene regions. To overcome these limitations, we conducted a comprehensive investigation to identify the most suitable 16S rRNA region for bacterial microbial analysis in endangered fish Totoaba macdonaldi, an endemic species of significant ecological and economic importance in Mexico. Targeting four distinct hypervariable regions (V1–V2, V2–V3, V3–V4, and V5–V7) of the 16S rRNA gene, we determined the microbial composition within the distal intestine. A total of 40 microbiomes were sequenced. Our findings underscore the critical impact of region selection on the accuracy of microbiota analysis. The V3–V4 region detected the highest number of bacterial taxa and exhibited significantly higher alpha diversity indices, demonstrating the highest taxonomic resolution. This study emphasizes the necessity of meticulous 16S rRNA region selection for fish microbiota analysis, particularly in native species of ecological and economic significance such as the endangered T. macdonaldi, where information is limited. Such optimization enhances the reliability and applicability of microbiota studies in fisheries management and conservation efforts.

Assessing antimicrobial resistance in pasture-based dairy farms: a 15-month surveillance study in New Zealand.

Antimicrobial resistance is a global public and animal health concern. Antimicrobial resistance genes (ARGs) have been detected in dairy farm environments globally; however, few longitudinal studies have utilized shotgun metagenomics for ARG surveillance in pasture-based systems. This 15-month study aimed to undertake a baseline survey using shotgun metagenomics to assess the relative abundance and diversity of ARGs in two pasture-based dairy farm environments in New Zealand with different management practices. There was no statistically significant difference in overall ARG relative abundance between the two dairy farms (P = 0.321) during the study period. Compared with overseas data, the relative abundance of ARG copies per 16S rRNA gene in feces (0.08-0.17), effluent (0.03-0.37), soil (0.20-0.63), and bulk tank milk (0.0-0.12) samples was low. Models comparing the presence or absence of resistance classes found in >10% of all feces, effluent, and soil samples demonstrated no statistically significant associations (P > 0.05) with "season," and only multi-metal (P = 0.020) and tetracycline (P = 0.0003) resistance were significant at the "farm" level. Effluent samples harbored the most diverse ARGs, some with a recognized public health risk, whereas soil samples had the highest ARG relative abundance but without recognized health risks. This highlights the importance of considering the genomic context and risk of ARGs in metagenomic data sets. This study suggests that antimicrobial resistance on pasture-based dairy farms is low and provides essential baseline ARG surveillance data for such farming systems.IMPORTANCEAntimicrobial resistance is a global threat to human and animal health. Despite the detection of antimicrobial resistance genes (ARGs) in dairy farm environments globally, longitudinal surveillance in pasture-based systems remains limited. This study assessed the relative abundance and diversity of ARGs in two New Zealand dairy farms with different management practices and provided important baseline ARG surveillance data on pasture-based dairy farms. The overall ARG relative abundance on these two farms was low, which provides further evidence for consumers of the safety of New Zealand's export products. Effluent samples harbored the most diverse range of ARGs, some of which were classified with a recognized risk to public health, whereas soil samples had the highest ARG relative abundance; however, the soil ARGs were not classified with a recognized public health risk. This emphasizes the need to consider genomic context and risk as well as ARG relative abundance in resistome studies.

Assessing the Occurrence of Carbapenemase Producers Using Marine Animals as Sentinel Species

Carbapenemase-producing (CP) strains represent a substantial global threat, deactivating carbapenems and conferring resistance to β-lactam antibiotics. They can spread across various environments, yet data on their presence in marine animals are sparse. This study aimed to assess the occurrence of carbapenemase-producing strains in wild marine animals and to analyse their antimicrobial resistance (AMR) profiles to crucial antimicrobials and heavy metals frequently encountered in the marine environment due to anthropogenicactivity. A total of 28 samples were obtained from a fish auction in the Centre Region of Portugal. Non-fermenting bacilli (NFB) was isolated from the visceral content of wild marine animals. Identification of isolates was achieved through PCR-based amplification of the 16S rRNA gene, followed by sequencing. Antimicrobial susceptibility testing was conducted according to EUCAST guidelines, covering nine antimicrobials. Research of carbapenemases and metal tolerance genes was conducted by PCR, and statistical analysis utilized the Fisher’s exact test. Pseudomonas spp. and Aeromonas spp., among other isolates were identified (n=47/9/7, respectively). Susceptibility profiles showed 100% resistance or intermediate resistance to ticarcillin, piperacillin, piperacillin-tazobactam, ceftazidime, ciprofloxacin, and imipenem (n=63/63), while 27% were resistant to meropenem (n=17/63) and 13% to tobramycin (n=8/63). All of them exhibited susceptibility to amikacin and carried multidrug resistance (MDR) profiles, including heavy metal genes (merA and silA). None harboured the carbapenemase genes searched (blaKPC, blaGES, blaIMP, blaNDM or/and blaVIM). In this study, MDR profiles to clinically important antimicrobials were observed, including to carbapenems. However, no carbapenemase-producing strains were identified, suggesting the presence of other genes or alternative mechanisms of resistance. These findings underscore the importance of monitoring AMR in marine ecosystems, particularly given its close ties to the food chain.

Comparative genomic analyses of the genus Robertmurraya and proposal of the novel species Robertmurraya mangrovi sp. nov., isolated from mangrove soil.

A Gram-positive, aerobic, motile, rod-shaped bacterial strain, designated 31A1RT, was isolated from the mangrove soil of Xilian village, Zhanjiang, China. Strain 31A1RT thrives at temperatures ranging from 15 to 45°C (optimum at 30°C), pH 6.5-10 (optimum at 8.5), and in the presence of 0-5% (w/v) NaCl (optimum at 1.5%). The strain shares the highest 16S rRNA gene sequence similarity with Robertmurraya crescens (97.24%) and Robertmurraya dakarensis (97.18%). The complete genome of strain 31A1RT spans 4.71 Mbp with a genomic DNA G + C content of 35.9 mol%. The average nucleotide identity and DNA-DNA hybridization values between strain 31A1RT and type strains of other species of the genus Robertmurraya were 71.24-72.11% and 19.90-21.40%, respectively. The amino acid identity values and percentage of conserved proteins ranged from 66.94 to 68.10% and from 58.34 to 61.62%, respectively, aligning with intrageneric cutoff values. The major fatty acids (≥ 5.0%) were iso-C14:0 (5.0%), iso-C15:0 (41.4%), iso-C16:0 (12.6%), C16:1ω7c alcohol (12.2%), and iso-C17:1 ω10c (6.5%). The polar lipids profile was mainly composed of diphosphatidyl glycerol, phosphatidyl glycerol, and phosphatidyl ethanolamine. We also profiled the pan-genome and metabolic features of genomic assemblies of strains belonging to the genus Robertmurraya, which indicated functional capacities and metabolic similarities. Consequently, we propose that strain 31A1RT represents a new species in the genus Robertmurraya, for which the name Robertmurraya mangrovi sp. nov. is proposed, with the type strain being 31A1RT (= GDMCC 1.4378T = JCM 36937T).

Flavobacterium capsici sp. nov., isolated from the rhizospheric soils of bell pepper (Capsicum annuum L.).

Two separate bacterial strains, PMTSA4T and PMR2A8, were isolated from the rhizospheric soils of bell pepper plants grown in a plant nursery. These strains are Gram-negative, non-motile and rod-shaped and grow in aerobic conditions. They exhibit a positive reaction for catalase activity but negative results for oxidase activity. Phylogenetic analysis of the 16S rRNA gene sequences revealed that the strains PMTSA4T and PMR2A8 are closely related to Flavobacterium piscinae ICH-30T (95.6%, respectively), Flavobacterium ahnfeltiae 10Alg 130T (95.5%) and Flavobacterium maris KMM 9535T (95.3%), aligning them within the genus Flavobacterium. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identities (ANIs) of the whole-genome sequences for the two strains and related Flavobacterium species were significantly below the established thresholds for prokaryotic species delineation (<70% for dDDH and <95% for ANI). The observed values were as follows: Flavobacterium aquatile LMG 4008T (dDDH: 19.8% and ANI: 75.5%), F. piscinae ICH-30T (dDDH: 18.6% and ANI: 73.3%) and F. stagni WWJ 16T (dDDH: 18.5% and ANI: 72.0%). The strains have genome sizes of 3 068 185 bp and 3 068 330 bp, with a G+C content of 32.5 mol%. In phenotypic characterization, the new strains grew at 10-35 °C and tolerated up to 4% NaCl at pH 5-9 (optimum pH 8). The predominant cellular fatty acids were observed to be iso-C15:0, iso-C17:0 3-OH and iso-C15:0 3-OH. Menaquinone-6 was the predominant quinone. Considering the results from phenotypic, chemotaxonomic, phylogenetic and genomic analyses, it is proposed that the strains PMTSA4T and PMR2A8 represent a novel species within the genus Flavobacterium.

Streptomyces yaizuensis sp. nov., a berninamycin C-producing actinomycete isolated from sponge.

While screening for antibiotics in a marine sample, we discovered a berninamycin C-producing actinomycete, designated YSPA8T, isolated from a sponge. A polyphasic approach was used to determine the taxonomic position of the strain. Strain YSPA8T formed sympodially branched aerial mycelia that ultimately segment into chains of spores. Comparative and phylogenetic analyses of the 16S rRNA gene sequence showed that strain YSPA8T were closely related to Streptomyces clavuligerus ATCC 27064T (99.66%), Streptomyces amakusaensis NRRL B-3351T (98.69%), Streptomyces inusitatus NBRC 13601T (98.48%), and 'Streptomyces jumonjinensis' JCM 4947 (98.41%). The phylogenetic tree using the 16S rRNA gene sequences, and both phylogenomic trees suggested that the closest relative of strain YSPA8T was S. clavuligerus ATCC 27064T. The average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values between strain YSPA8T and S. clavuligerus ATCC 27064T were 84.1%, 28.9%, and 82.5%, respectively, which were below the thresholds of 95%, 70%, and 95% for a prokaryotic conspecific assignment. The G + C of the strain YSPA8T was 72.6%. Whole-cell hydrolysates of strain YSPA8T contained LL-diaminopimelic acid. The predominant menaquinones were MK-9(H6) (49%) and MK-9(H8) (48%), and the major fatty acids were C16:0 (26.8%), C16:1 ω7c/ω6c (17.2%), iso-C16:0 (16.0%), and iso-C15:0 (12.5%). The major phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, and other unidentified phospholipids. Based on the phenotypic, phylogenetic, genomic, and chemotaxonomic data, strain YSPA8T represents a novel species of the genus Streptomyces, and the proposed name for this species is Streptomyces yaizuensis sp. nov. The type strain is YSPA8T (=NBRC 115866T = TBRC 17196T).

Limited Variation in Bacterial Communities of Scaphoideus titanus (Hemiptera: Cicadellidae) Across European Populations and Different Life Stages

The Nearctic leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae) is the primary vector of ‘Candidatus Phytoplasma vitis’, the causative agent of Flavescence doreé in Europe. Although microorganisms play an important role in the ecology and behavior of insects, knowledge about the interaction between S. titanus and microbes is limited. In this study, we employed an amplicon metabarcoding approach for profiling the V4 region of the 16S rRNA gene to characterize the bacterial communities of S. titanus across several populations from four European localities. Additionally, we investigated changes in bacterial communities between nymphal and adult stages. In total, we identified 7,472 amplicon sequence variants (ASVs) in adults from the European populations. At the genus level, ‘Candidatus Karelsulcia’ and ‘Candidatus Cardinium’ were the most abundant genera, with both being present in every individual. While we found significant changes in the microbial composition of S. titanus across different European populations, no significant differences were observed between nymphal and adult stages. Our study reveals new insights into the microbial composition of S. titanus and highlights the role of geography in influencing its bacterial community.

Bioremediation of nitrate in agricultural drainage ditches: Impacts of low-grade weirs on microbiomes and nitrogen cycle gene abundance

Artificial drainage is essential for the success of modern agriculture, but it can also accelerate the movement of nutrients, especially nitrate, from soil to surrounding and downstream water bodies. Removal of nitrate from agricultural drainage by using controlled drainage systems, such as ditches installed with low-grade weirs, has been shown to help reduce nutrient loading into watersheds. However, the effect of low-grade weirs varies greatly, likely due to the differences in climate, system designs (e.g., hydraulic characteristics), and the resulting variation in microbial structures and functions in the ditch. In this study, we analyzed the temporal and spatial dynamics of microbiomes in a paired ditch system with weir-installed and uninstalled (control) channels over two years by using the 16S rRNA gene amplicon sequencing and the high-throughput quantitative PCR targeting various N cycle-associated genes [the Nitrogen Cycle Evaluation (NiCE) chip]. The installation of the low-grade weir had a significant impact on the microbiome structure and the distribution of denitrifiers. Microbiome structures also differed significantly between the ditch inlets and the outlets. Denitrification functional genes were more abundant in the inlets than in the other locations and in the channel installed with a low-grade weir. Additionally, oxygenic denitrifiers that utilize nitric oxide dismutase (nod) to produce N2 and O2 gases from nitric oxide were detected in the ditch channels, suggesting the occurrence of nitrate removal process that bypasses the production of nitrous oxide (N2O). The ditch microbiomes sampled during high-flow seasons (i.e., spring and fall) exhibited greater similarity to each other than microbiomes sampled during low-flow seasons (i.e., summer). Taken together, this study indicates that the low-grade weirs have the potential to foster a more favorable environment for denitrifiers, resulting in an increase in the abundance of denitrification functional genes. These findings could offer valuable insights into system management and optimization strategies.

Perturbed saliva microbiome is gender-specific in patients with oral lichen planus

ObjectiveTo understand the gender characteristics of oral lichen planus (OLP) by identifying the gender-specific salivary microbiome and its potential biomarkers. MethodsA gender-based study was undertaken, commencing with the collection of saliva samples, followed by 16S rRNA gene sequencing, to explore the differences in the composition of saliva microbiome in OLP patients (40 males and 56 females) and healthy controls (40 males and 56 females), respectively. ResultsBoth male and female OLP patients had significant differences in saliva microbiome composition from healthy controls, especially in female patients. Notably, Pseudomonas was only enriched in female patients. Rhodococcus (AUC: 0.91) and Pseudomonas (AUC: 0.97) had great potential as diagnostic biomarkers in male and female patients, respectively. The KEGG results showed metabolic dysfunction was more pronounced in female patients and a high level of microbial metabolism in diverse environments, ABC transporters, Quorum sensing and Two-component system. Capnocytophaga was negatively correlated with the erosion area in male patients. Neisseria was negatively correlated with the erosion area and Rothia was positively correlated with the pain level in female patients. ConclusionsOur study revealed gender-specific perturbation in salivary microbiome within OLP patients, suggesting that the male and female patients with OLP may have different pathogenesis.

Biofilm Formation on Excavation Damaged Zone Fractures in Deep Neogene Sedimentary Rock.

Deep underground galleries are used to access the deep biosphere in addition to mining and other engineering applications, such as geological disposal of radioactive waste. Fracture networks developed in the excavation damaged zone (EDZ) are concerned with accelerating mass transport, where microbial colonization might be possible due to the availability of space and nutrients. In this study, microbial biofilms at EDZ fractures were investigated by drilling from a 350-m-deep gallery and subsequent borehole logging at the Horonobe Underground Research Laboratory (URL). By using microscopic and spectroscopic techniques, the dense colonization of microbial cells was demonstrated at the surfaces of the EDZ fractures with high hydraulic conductivity. 16S rRNA gene sequence analysis revealed the dominance of gammaproteobacterial lineages, the cultivated members of which are aerobic methanotrophs. The near-complete genomes from Horonobe groundwater, affiliated with the methanotrophic lineages, were fully equipped with genes involved in aerobic methanotrophy. Although the mediation of aerobic methanotrophy remains to be demonstrated, microbial O2 production was supported by the presence of genes in the near-complete genomes, such as catalase and superoxide dismutase that produce O2 from reactive oxygen species and a nitric oxide reductase gene with the substitutions of amino acids in motifs. It is concluded that the EDZ fractures provide energetically favorable subsurface habitats for microorganisms.

Using fungal–bacterial community analysis to explore potential microbiomes to manage Meloidogyne incongnita

Rhizosphere microbial communities strongly affect outbreaks of root-knot nematode (RKN) disease. However, little is known about the interactions among fungi, bacteria and RKN. The bacterial and fungal community compositions in the rhizospheres of four representative tobacco varieties, both resistant and susceptible to RKN, were characterized using 16S rRNA gene sequencing for bacteria and internal transcribed spacer gene sequencing for fungi. Our findings revealed that the fungi played crucial roles in facilitating the cross-kingdom and symbiotic fungal–bacterial interactions to suppress RKN. Moreover, our investigation suggested Microbacterium as a potential microbial antagonist against RKN based on its enhanced presence in RKN-resistant tobacco genotypes, and the relative abundance of Microbacterium was 34.49% greater in the rhizosphere of resistant tobacco than that of susceptible tobacco significantly. Notably, the richness of fungal community enhanced tobacco’s microbe-associated resistance to RKN through the positive regulation of the richness and diversity of bacterial community and the relative abundance of Microbacterium. This study underscores the critical role of the fungus–dominated fungal–bacterial community in bolstering tobacco resistance against RKN. The potential antagonistic role of Microbacterium presents promising avenues for innovative RKN management strategies.

Growing older, growing more diverse: Sea turtles and epibiotic cyanobacteria.

Cyanobacteria are known for forming associations with various animals, including sea turtles, yet our understanding of cyanobacteria associated with sea turtles remains limited. This study aims to address this knowledge gap by investigating the diversity of cyanobacteria in biofilm samples from loggerhead sea turtle carapaces, utilizing a 16S rRNA gene amplicon sequencing approach. The predominant cyanobacterial order identified was Nodosilineales, with the genus Rhodoploca having the highest relative abundance. Our results suggest that cyanobacterial communities become more diverse as sea turtles age, as we observed a positive correlation between community diversity and the length of a sea turtle's carapace. Since larger and older turtles predominantly utilize neritic habitats, the shift to a more diverse cyanobacterial community aligned with a change in loggerhead habitat. Our research provides detailed insights into the cyanobacterial communities associated with loggerhead sea turtles, establishing a foundation for future studies delving into this fascinating ecological relationship and its potential implications for sea turtle conservation.

Prognostic Impact of Microbiome Dysbiosis: A Prospective Study.

To determine the relationship between microbiome dysbiosis indices and biofilm immunogenicity and their prognostic implications on periodontal treatment response. Thirty periodontally healthy controls and 30 periodontitis cases (stage III) were recruited. Cases received non-surgical periodontal therapy (NSPT), and their treatment response at 6 months was evaluated using a treat-to-target endpoint (≤ 4 sites with probing depths ≥ 5 mm). Pooled subgingival biofilm samples were obtained from controls and cases. The V3-4 hypervariable region of the 16S rRNA gene was sequenced and two compositional indices (subgingival microbiome dysbiosis index, SMDI, and dysbiosis ratio, DR) were calculated. Nuclear factor kappa-B (NF-κB) activation elicited by biofilm samples in monocytic reporter cells was quantified to assess biofilm immunogenicity. SMDI, DR and biofilm immunogenicity were highly diagnostic for periodontitis (area under curves [AUC] > 0.90, p < 0.001). Among periodontitis cases, all three microbial parameters were significantly reduced after NSPT (p < 0.001). Cases achieving the treat-to-target endpoint had lower pre-treatment SMDI and biofilm immunogenicity (p < 0.05) and different microbial recolonization patterns from poor responders. Both measures predicted treatment response (AUC of 0.767 and 0.835, respectively, p < 0.05). Subgingival biofilm dysbiosis quantified using SMDI and biofilm immunogenicity was diagnostic of periodontitis and predictive of NSPT outcomes.

Optimal 16S rRNA gene amplicon sequencing analysis for oral microbiota to avoid the potential bias introduced by trimming length, primer, and database.

16S rRNA gene amplicon sequencing analysis is used to investigate bacterial communities; however, the estimated bacterial composition can differ from the original due to experimental and analytical biases. Therefore, this study determines the optimal conditions to minimize the potential biases in 16S rRNA gene amplicon sequencing analysis using different DNA samples, trimming lengths, primers, and databases. The results of the mock1 community with 250 and 300 bp paired-end (PE), comprising 15 bacteria from various environments, showed the highest similarity between the theoretical value and the data using the Greengenes2 and the V3 region at the genus level and V5-V6 at the species level. In the 300 bp PE sequencing analysis of the mock2 community, comprising six major oral bacteria, the data using the V3-V4, V4, and V5-V6 regions with the SILVA, Greengenes2, and the Human Oral Microbiome Database (HOMD) showed the highest similarity to the theoretical values at the genus level. At the species level, the data using the V3-V4 and V4 regions with Greengenes2 and the data using the V1-V2 with HOMD exhibited the highest alignment with the theoretical values. In the species analysis of the dental calculus samples with 300 bp PE, the Shannon index value was higher with the V1-V2 region and HOMD than with others. Our results suggest that the optimal conditions for oral microbiome analysis are the combinations of 300 bp PE and the V3-V4 or V4 region with the Greengenes2 and the V1-V2 with HOMD.IMPORTANCEThe 16S rRNA gene amplicon sequencing analysis is frequently used in oral microbiome research. However, this method can have biases that distort the experimental data, which depend on the methodological steps, including sequencing length, trimming length, selected amplification regions, and referenced databases. In this study, a combination of 300 bp PE and the primer targeting the V3-V4 or V4 regions with the Greengenes2 and the V1-V2 region with the HOMD was the most bias-minimizing condition for oral microbiota analysis. In addition, this is the first report of such analyses in modern Japanese dental calculus. The methods used in this study will aid in setting appropriate conditions for sequence analysis of microbiota obtained not only from the oral cavity but also from any environment.

Comparative analysis of biofilm bacterial communities developed on different artificial reef materials.

Artificial reefs play a vital role in restoring and creating new habitats for marine species by providing suitable substrates, especially in areas where natural substrates have been degraded or lost due to declining water quality, destructive fishing practices, and coral diseases. Artificial reef restoration aimed at coral larval settlement is gaining prominence and initially depends on the development of biofilms on reef surfaces. In this study, we hypothesized that different artificial reef materials selectively influence the composition of biofilm bacterial communities, which in turn affected coral larval settlement and the overall success of coral rehabilitation efforts. To test this hypothesis, we evaluated the impact of six different reef made materials (porcelain, granite, coral-skeleton, calcium-carbonate, shell-cement, and cement) on the development of biofilm bacterial communities and their potential to support coral larval settlement. The biofilm bacterial communities were developed on different artificial reef materials and studied using 16S rRNA gene amplicon sequencing and analysis. The bacterial species richness and evenness were significantly (P<0.05) low in the seawater, while these values were high in the reef materials. At the phylum level, the biofilm bacterial composition of all materials and seawater was majorly composed of Pseudomonadota, Cyanobacteria, and Bacteroidetes, however, significantly (P<0.05) low Bacteroidetes were found in the seawater. At the genus level, Thalassomonas, Glaciecola, Halomicronema, Lewinella, Hyphomonas, Thalassospira, Polaribacter, and Tenacibaculum were significantly (P<0.05) low in the coral-skeleton and seawater, compared to the other reef materials. The genera Pseudoaltermonas and Thalassomonas (considered potential inducers of coral larval settlement) were highly abundant in the shell-cement biofilm, while low values were found in the biofilm of the other materials. The biofilm bacterial community composition can be selective for different substrate materials, such as shell-cement exhibited higher abundances of bacteria known to facilitate coral larval settlement, highlighting their potential in enhancing restoration outcomes.

Microbial and Transcriptomic Landscape Associated With Neutrophil Extracellular Traps in Perianal Fistulizing Crohn's Disease.

Perianal fistulizing Crohn's disease (pfCD) poses significant healing challenges, closely associated with neutrophil extracellular traps (NETs). This study aimed to investigate the microbe-host interactions influencing NETs in pfCD. From January 2019 to July 2022, patients with pfCD were screened at Ren Ji Hospital. Patients in remission following comprehensive treatment were recruited. We documented clinical characteristics, medication regimens, healing outcomes, and infliximab levels in fistula tissues. NET positivity was confirmed by positive results in citrullinated histone H3 (CitH3) enzyme-linked immunosorbent assay (ELISA) and dual immunofluorescence staining for myeloperoxidase and CitH3. Microbial and transcriptomic profiles from fistula tissues, obtained during surgery, were analyzed using 16S rRNA gene sequencing and RNA sequencing. Differences in microbiome and transcriptomic profiles were evaluated, and their relationships were assessed using Mantel's and Spearman's coefficients. Significant differences in microbial communities were found between groups (P = .007). Representatively differential microbes such as Prevotella bivia, Streptococcus gordonii, and Bacteroides dorei were enriched in NETs-positive fistulas (P < .05). Functional analysis of microbes revealed reduced ubiquinol biosynthesis and butanoate production in NETs-negative fistulas (P < .05). Transcriptomic analysis indicated increased neutrophil and monocyte infiltration in NETs-positive fistulas, associated with pathways involving bacterial response, neutrophil chemotaxis, secretory processes, and peptidase activity (P < .05). Species prevalent in NETs-positive fistulas correlated positively with immune responses and wound healing pathways, whereas bacteria in NETs-negative fistulas correlated negatively. NETs were negatively associated with tissue infliximab levels (P = .001) and healing outcomes (P = .025). Our findings reveal unique microbial and transcriptomic signatures associated with NETs in pfCD, highlighting their profound influence on clinical outcomes.