The short-term impact of comprehensive caries treatment on the supragingival microbiome of severe early childhood caries.

BACKGROUND: Children affected by severe early childhood caries (S-ECC) usually need comprehensive caries treatment due to the extensive of caries. How the oral microbiome changes after caries therapy within the short-term warrant further study. AIM: This study aimed to investigate the short-term impact of comprehensive caries treatment on the supragingival plaque microbiome of S-ECC children. DESIGN: Thirty-three children aged 2-4 years with severe caries (dt > 7) were recruited. Comprehensive caries treatment was performed under general anesthesia in one session and included restoration, pulp treatment, extraction, and fluoride application. Supragingival plaque was sampled pre- and 1-month posttreatment. The genomic DNA of the supragingival plaque was extracted, and bacterial 16S ribosomal RNA gene sequencing was performed. RESULTS: Our data showed that the microbial community evenness significantly decreased posttreatment. Furthermore, comprehensive caries treatment led to more diverse microbial structures among the subjects. The interbacterial interactions reflected by the microbial community's co-occurrence network tended to be less complex posttreatment. Caries treatment increased the relative abundance of Corynebacterium matruchotii, Corynebacterium durum, Actinomyces naeslundii, and Saccharibacteria HMT-347, as well as Aggregatibacter HMT-458 and Haemophilus influenzae. Meanwhile, the relative abundance of Streptococcus mutans, three species from Leptotrichia, Neisseria bacilliformis, and Provotella pallens significantly decreased posttreatment. CONCLUSION: Our results suggested that comprehensive caries treatment may contribute to the reconstruction of a healthier supragingival microbiome.

Microbiota present in combined endodontic-periodontal diseases and its risks for endocarditis.

INTRODUCTION: Infective endocarditis (IE) is an inflammatory disease usually caused by bacteria that enter the bloodstream and establish infections in the inner linings or valves of the heart, including blood vessels. Despite the availability of modern antimicrobial and surgical treatments, IE continues to cause substantial morbidity and mortality. Oral microbiota is considered one of the most significant risk factors for IE. The objective of this study was to evaluate the microbiota present in root canal (RC) and periodontal pocket (PP) clinical samples in cases with combined endo-periodontal lesions (EPL) to detect species related to IE using NGS. METHODS: Microbial samples were collected from 15 RCs and their associated PPs, also from 05 RCs with vital pulp tissues (negative control, NC). Genomic studies associated with bioinformatics, combined with structuring of a database (genetic sequences of bacteria reported for infective endocarditis), allowed for the assessment of the microbial community at both sites. Functional prediction was conducted using PICRUSt2. RESULTS: Parvimonas, Streptococcus, and Enterococcus were the major genera detected in the RCs and PPs. A total of 79, 96, and 11 species were identified in the RCs, PPs, and NCs, respectively. From them, a total of 34 species from RCs, 53 from PPs, and 2 from NCs were related to IE. Functional inference demonstrated that CR and PP microbiological profiles may not be the only risk factors for IE but may also be associated with systemic diseases, including myocarditis, human cytomegalovirus infection, bacterial invasion of epithelial cells, Huntington's disease, amyotrophic lateral sclerosis, and hypertrophic cardiomyopathy. Additionally, it was possible to predict antimicrobial resistance variants for broad-spectrum drugs, including ampicillin, tetracycline, and macrolides. CONCLUSION: Microorganisms present in the combined EPL may not be the only risk factor for IE but also for systemic diseases. Antimicrobial resistance variants for broad-spectrum drugs were inferred based on PICRUSt-2. State-of-the-art sequencing combined with bioinformatics has proven to be a powerful tool for conducting studies on microbial communities and could considerably assist in the diagnosis of serious infections. CLINICAL RELEVANCE: Few studies have investigated the microbiota in teeth compromised by combined endo-periodontal lesions (EPL), but none have correlated the microbiological findings to any systemic condition, particularly IE, using NGS techniques. In such cases, the presence of apical periodontitis and periodontal disease can increase IE risk in susceptible patients.

Chemomechanical preparation influences the microbial community and the levels of LPS, LTA and cytokines in combined endodontic-periodontal lesions: A clinical study.

BACKGROUND: This study was conducted to compare the microbiomes, the levels of lipopolysaccharides (LPS), lipoteichoic acid (LTA), and cytokines (interleukin [IL]-1ß and tumor necrosis factor-alpha [TNF-α]), before and after chemomechanical preparation (CMP) of the root canals (RC) and their associated periodontal pockets (PP) in teeth with combined EPL. MATERIALS: Samples were taken from 10 RC and PP, before and after CMP. The microbiomes (next-generation sequencing, V3-V4 hypervariable region of the 16S rRNA gene), microbiome diversity (bioinformatics analyses), LPS (limulus amebocyte lysate), LTA, IL-1ß, and TNF-α (ELISA) were evaluated. A statistical analysis was performed with significance level set at 5%. RESULTS: The most abundant phyla in both sites were Firmicutes and Proteobacteria. Comparative studies of bacterial genera species revealed that some increased and others decreased after CMP at both sites. A 3% reduction in Gram-negative bacteria (RC) and a 4% increase in Gram-positive bacteria (PP) were detected. LPS levels were 4.4 times higher in PP than in the RC. LTA was detected in all samples investigated. Higher levels of IL-1ß and TNF-α were detected in both sites at baseline. After CMP, LPS, LTA, IL-1ß and TNF-α were reduced in both sites. CONCLUSION: The microbial community in the RC and PP in teeth with combined EPL indicated a similarity between both sites. CMP effectively reduced the microbial load and the LPS levels from teeth with EPL, and consequently diminished the cytokine levels. The reduction in LTA levels in the RC and PP proved challenging.

Association between metabolic syndrome and periodontitis: The role of lipids, inflammatory cytokines, altered host response, and the microbiome.

Periodontitis has been associated with many systemic diseases and conditions, including metabolic syndrome. Metabolic syndrome is a cluster of conditions that occur concomitantly and together they increase the risk of cardiovascular disease and double the risk of type 2 diabetes. In this review, we focus on the association between metabolic syndrome and periodontitis; however, we also include information on diabetes mellitus and cardiovascular disease, since these two conditions are significantly intertwined with metabolic syndrome. With regard to periodontitis and metabolic syndrome, to date, the vast majority of studies point to an association between these two conditions and also demonstrate that periodontitis can contribute to the development of, or can worsen, metabolic syndrome. Evaluating the effect of metabolic syndrome on the salivary microbiome, data presented herein support the hypothesis that the salivary bacterial profile is altered in metabolic syndrome patients compared with healthy patients. Considering periodontitis and these three conditions, the vast majority of human and animal studies point to an association between periodontitis and metabolic syndrome, diabetes, and cardiovascular disease. Moreover, there is evidence to suggest that metabolic syndrome and diabetes can alter the oral microbiome. However, more studies are needed to fully understand the influence these conditions have on each other.

Tongue microbiome of smokeless tobacco users.

BACKGROUND: The possibility that smokeless tobacco may contribute to oral carcinogenesis by influencing the oral microbiome has not been explored. This preliminary cross-sectional study sought to assess the effect of using shammah, a form of smokeless tobacco prevalent in Arabia, on the tongue microbiome. Tongue scarping samples were obtained from 29 shammah users (SU; 27.34 ± 6.9 years) and 23 shammah non-users (SNU; 27.7 ± 7.19 years) and analyzed with 16S rRNA gene sequencing (V1-V3). Species-level taxonomy assignment of the high-quality, merged reads was obtained using a previously described BLASTn-based algorithm. Downstream analyses were performed with QIIME, LEfSe, and R. RESULTS: A total of 178 species, belonging to 62 genera and 8 phyla were identified. Genera Streptococcus, Leptotrichia, Actinomyces, Veillonella, Haemophilus, Prevotella and Neisseria accounted for more than 60% of the average microbiome. There were no differences between the two groups in species richness and alpha-diversity, but PCoA showed significant separation (P = 0.015, ANOSIM). LEfSe analysis identified 22 species to be differentially abundant between the SU and SNU. However, only 7 species maintained a false discovery rate of ≤0.2 and could cluster the two groups separately: Rothia mucilaginosa, Streptococcus sp. oral taxon 66, Actinomyces meyeri, Streptococcus vestibularis Streptococcus sanguinis and a potentially novel Veillonella species in association with SU, and Oribacterium asaccharolyticum with SNU. CONCLUSION: These preliminary results indicate that shammah use induces tongue microbiome changes including enrichment of several species with high acetaldehyde production potential, which warrants further investigation.

Antibiotic Korormicin A Kills Bacteria by Producing Reactive Oxygen Species.

Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR.) We show that although korormicin is an inhibitor of Na+-NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O2 and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS). All Pseudoalteromonas contain the nqr operon in their genomes, including Pseudoalteromonas strain J010, which produces korormicin. We present activity data indicating that this strain expresses an active Na+-NQR and that this enzyme is not susceptible to korormicin inhibition. On the basis of our DNA sequence data, we show that the Na+-NQR of Pseudoalteromonas J010 carries an amino acid substitution (NqrB-G141A; Vibrio cholerae numbering) that in other Na+-NQRs confers resistance against korormicin. This is likely the reason that a functional Na+-NQR is able to exist in a bacterium that produces a compound that typically inhibits this enzyme and causes cell death. Korormicin is an effective antibiotic against such pathogens as Vibrio cholerae, Aliivibrio fischeri, and Pseudomonas aeruginosa but has no effect on Bacteroides fragilis and Bacteroides thetaiotaomicron, microorganisms that are important members of the human intestinal microflora.IMPORTANCE As multidrug antibiotic resistance in pathogenic bacteria continues to rise, there is a critical need for novel antimicrobial agents. An essential requirement for a useful antibiotic is that it selectively targets bacteria without significant effects on the eukaryotic hosts. Korormicin is an excellent candidate in this respect because it targets a unique respiratory enzyme found only in prokaryotes, the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR). Korormicin is synthesized by some species of the marine bacterium Pseudoalteromonas and is a potent and specific inhibitor of Na+-NQR, an enzyme that is essential for the survival and proliferation of many Gram-negative human pathogens, including Vibrio cholerae and Pseudomonas aeruginosa, among others. Here, we identified how korormicin selectively kills these bacteria. The binding of korormicin to Na+-NQR promotes the formation of reactive oxygen species generated by the reaction of the FAD and the 2Fe-2S center cofactors with O2.

Resolvin E1 Reverses Experimental Periodontitis and Dysbiosis.

Periodontitis is a biofilm-induced inflammatory disease characterized by dysbiosis of the commensal periodontal microbiota. It is unclear how natural regulation of inflammation affects the periodontal biofilm. Promoters of active resolution of inflammation, including resolvin E1 (RvE1), effectively treat inflammatory periodontitis in animal models. The goals of this study were 1) to compare periodontal tissue gene expression in different clinical conditions, 2) to determine the impact of local inflammation on the composition of subgingival bacteria, and 3) to understand how inflammation impacts these changes. Two clinically relevant experiments were performed in rats: prevention and treatment of ligature-induced periodontitis with RvE1 topical treatment. The gingival transcriptome was evaluated by RNA sequencing of mRNA. The composition of the subgingival microbiota was characterized by 16S rDNA sequencing. Periodontitis was assessed by bone morphometric measurements and histomorphometry of block sections. H&E and tartrate-resistant acid phosphatase staining were used to characterize and quantify inflammatory changes. RvE1 treatment prevented bone loss in ligature-induced periodontitis. Osteoclast density and inflammatory cell infiltration in the RvE1 groups were lower than those in the placebo group. RvE1 treatment reduced expression of inflammation-related genes, returning the expression profile to one more similar to health. Treatment of established periodontitis with RvE1 reversed bone loss, reversed inflammatory gene expression, and reduced osteoclast density. Assessment of the rat subgingival microbiota after RvE1 treatment revealed marked changes in both prevention and treatment experiments. The data suggest that modulation of local inflammation has a major role in shaping the composition of the subgingival microbiota.

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions.

BACKGROUND: Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. RESULTS: A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5'-UTR, 3'-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5'-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3'-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37 °C versus 28 °C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49 % and 51 % similarity, and 16 % and 17 % identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains. CONCLUSION: Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species.

Sublethal concentrations of carbapenems alter cell morphology and genomic expression of Klebsiella pneumoniae biofilms.

Klebsiella pneumoniae, a Gram-negative bacterium, is normally associated with pneumonia in patients with weakened immune systems. However, it is also a prevalent nosocomial infectious agent that can be found in infected surgical sites and combat wounds. Many of these clinical strains display multidrug resistance. We have worked with a clinical strain of K. pneumoniae that was initially isolated from a wound of an injured soldier. This strain demonstrated resistance to many commonly used antibiotics but sensitivity to carbapenems. This isolate was capable of forming biofilms in vitro, contributing to its increased antibiotic resistance and impaired clearance. We were interested in determining how sublethal concentrations of carbapenem treatment specifically affect K. pneumoniae biofilms both in morphology and in genomic expression. Scanning electron microscopy showed striking morphological differences between untreated and treated biofilms, including rounding, blebbing, and dimpling of treated cells. Comparative transcriptome analysis using RNA sequencing (RNA-Seq) technology identified a large number of open reading frames (ORFs) differentially regulated in response to carbapenem treatment at 2 and 24 h. ORFs upregulated with carbapenem treatment included genes involved in resistance, as well as those coding for antiporters and autoinducers. ORFs downregulated included those coding for metal transporters, membrane biosynthesis proteins, and motility proteins. Quantitative real-time PCR validated the general trend of some of these differentially regulated ORFs. Treatment of K. pneumoniae biofilms with sublethal concentrations of carbapenems induced a wide range of phenotypic and gene expression changes. This study reveals some of the mechanisms underlying how sublethal amounts of carbapenems could affect the overall fitness and pathogenic potential of K. pneumoniae biofilm cells.

Dermal wound transcriptomic responses to Infection with Pseudomonas aeruginosa versus Klebsiella pneumoniae in a rabbit ear wound model.

BACKGROUND: Bacterial infections of wounds impair healing and worsen scarring. We hypothesized that transcriptome analysis of wounds infected with Klebsiella pneumoniae (K.p.) or Pseudomonas aeruginosa (P.a.) would indicate host-responses associated with the worse healing of P.a.- than K.p.-infected wounds. METHODS: Wounds created on post-operative day (POD) 0 were infected during the inflammatory phase of healing on POD3 and were harvested on POD4 for microarray and transcriptome analysis. Other wounds received topical antibiotic after infection for 24 hours to promote biofilm development, and were harvested on POD6 or POD12. RESULTS: Wounds infected for 24 hours, relative to uninfected wounds, elevated transcripts of immune-response functions characteristic of infiltrating leukocytes. But P.a.-infected wounds elevated many more transcripts and to higher levels than K.p.-infected wounds. Coincidently, suppressed transcripts of both wounds enriched into stress-response pathways, including EIF2 signaling; however, this was more extensive for P.a.-infected wounds, including many-fold more transcripts enriching in the 'cell death' annotation, suggesting resident cutaneous cell toxicity in response to a more damaging P.a. inflammatory milieu. The POD6 wounds were colonized with biofilm but expressed magnitudes fewer immune-response transcripts with no stress-response enrichments. However, elevated transcripts of P.a.-infected wounds were inferred to be regulated by type I interferons, similar to a network unique to P.a.-infected wounds on POD4. On POD12, transcripts that were more elevated in K.p.-infected wounds suggested healing, while transcripts more elevated in P.a.-infected wounds indicated inflammation. CONCLUSIONS: An extensive inflammatory response of wounds was evident from upregulated transcripts 24 hours after infection with either bacterium, but the response was more intense for P.a.- than K.p.-infected wounds. Coincidently, more extensive down-regulated transcripts of P.a.-infected wounds indicated a stronger "integrated stress response" to the inflammatory milieu that tipped more toward cutaneous cell death. Unique to P.a.-infected wounds on POD4 and POD6 were networks inferred to be regulated by interferons, which may result from intracellular replication of P.a. These data point to specific downregulated transcripts of cells resident to the wound as well as upregulated transcripts characteristic of infiltrating leukocytes that could be useful markers of poorly healing wounds and indicators of wound-specific treatments for improving outcomes.

The effect of dental material type and masticatory forces on periodontitis-derived subgingival microbiomes.

Restorative dental materials can frequently extend below the gingival margin, serving as a potential haven for microbial colonization, and altering the local oral microbiome to ignite infection. However, the contribution of dental materials on driving changes of the composition of the subgingival microbiome is under-investigated. This study evaluated the microbiome-modulating properties of three biomaterials, namely resin dental composites (COM), antimicrobial piezoelectric composites (BTO), and hydroxyapatite (HA), using an optimized in vitro subgingival microbiome model derived from patients with periodontal disease. Dental materials were subjected to static or cyclic loading (mastication forces) during biofilm growth. Microbiome composition was assessed by 16S rRNA gene sequencing. Dysbiosis was measured in terms of subgingival microbial dysbiosis index (SMDI). Biomaterials subjected to cyclic masticatory loads were associated with enhanced biofilm viability except on the antibacterial composite. Biomaterials held static were associated with increased biofilm biomass, especially on HA surfaces. Overall, the microbiome richness (Chao index) was similar for all the biomaterials and loading conditions. However, the microbiome diversity (Shannon index) for the HA beams was significantly different than both composites. In addition, beta diversity analysis revealed significant differences between composites and HA biomaterials, and between both loading conditions (static and cyclic). Under static conditions, microbiomes formed over HA surfaces resulted in increased dysbiosis compared to composites through the enrichment of periopathogens, including Porphyromonas gingivalis, Porphyromonas endodontalis, and Fretibacterium spp., and depletion of commensals such as Granulicatella and Streptococcus spp. Interestingly, cyclic loading reversed the dysbiosis of microbiomes formed over HA (depletion of periopathogenes) but increased the dysbiosis of microbiomes formed over composites (enrichment of Porphyromonas gingivalis and Fusobacterim nucleatum). Comparison of species formed on both composites (control and antibacterial) showed some differences. Commercial composites enriched Selenomonas spp. and depleted Campylobacter concisus. Piezoelectric composites effectively controlled the microbiome viability without significantly impacting the species abundance. Findings of this work open new understandings of the effects of different biomaterials on the modulation of oral biofilms and the relationship with oral subgingival infections.

Subgingival microbial profiles in pre- and postmenopausal women: Associations with serum estradiol levels.

BACKGROUND: Subgingival dental plaque is an ecosystem playing a key role in supporting both oral health and systemic health. Menopause-related changes have the potential to disrupt its balance, which is crucial to postmenopausal well-being. Our study explored how circulating estradiol levels correlate with subgingival microbial composition using checkerboard DNA-DNA hybridization in premenopausal and postmenopausal women. We also demonstrated that combining this method with 16S ribosomal RNA (rRNA) sequencing insights remains valuable for examining subgingival ecology. METHODS: We assessed 40 bacterial species in 77 premenopausal and 81 postmenopausal women using checkerboard DNA-DNA hybridization and measured serum estradiol with enzyme-linked immunosorbent assay (ELISA). Women were categorized by subgingival dysbiosis severity using a modified Subgingival Microbial Dysbiosis Index (mSMDI). Six women from each normobiotic and dysbiotic subgroup across premenopausal and postmenopausal women underwent 16S rRNA sequencing analysis. RESULTS: DNA checkerboard analysis revealed that most observed variability in individual bacterial proportions is associated with periodontitis. Two species, Leptotrichia buccalis and Streptococcus constellatus, exhibited differences related to estradiol levels within the premenopausal group (p = 0.055 and p = 0.009, respectively). 16S rRNA sequencing confirmed the mSMDI's validity in categorizing normobiotic and dysbiotic states. Menopausal status was not associated with a dysbiotic shift in the subgingival microbiome despite significantly more attachment loss in postmenopausal compared to premenopausal women. CONCLUSIONS: Our results indicate that decreased estradiol levels or increased attachment loss during menopause are not associated with changes in species abundance or dysbiotic shifts in women. The mSMDI may be a useful tool for classifying subgingival ecology based on its normobiotic or dysbiotic inclination.

Quantifying periodontitis-associated oral dysbiosis in tongue and saliva microbiomes-An integrated data analysis.

BACKGROUND: Periodontitis is primarily driven by subgingival biofilm dysbiosis. However, the quantification and impact of this periodontal dysbiosis on other oral microbial niches remain unclear. This study seeks to quantify the dysbiotic changes in tongue and salivary microbiomes resulting from periodontitis by applying a clinically relevant dysbiosis index to an integrated data analysis. METHODS: The National Center for Biotechnology Information (NCBI) database was searched to identify BioProjects with published studies on salivary and tongue microbiomes of healthy and periodontitis subjects. Raw sequence datasets were processed using a standardized bioinformatic pipeline and categorized by their ecological niche and periodontal status. The subgingival microbial dysbiosis index (SMDI), a dysbiosis index originally developed using the subgingival microbiome, was computed at species and genus levels and customized for each niche. Its diagnostic accuracy for periodontitis was evaluated using receiver operating characteristic curves. RESULTS: Four studies, contributing 328 microbiome samples, were included. At both species and genus levels, periodontitis samples had a higher SMDI, but the differences were only significant for subgingival biofilm and saliva (p < 0.001). However, SMDI showed good diagnostic accuracy for periodontitis status for all three niches (area under curve ranging from 0.76 to 0.90, p < 0.05). The dysbiosis index of subgingival biofilm was positively correlated with saliva consistently (p < 0.001) and with the tongue at the genus level (p = 0.036). CONCLUSIONS: While the impact on the tongue microbiome requires further investigation, periodontitis-associated dysbiosis affects the salivary microbiome and is quantifiable using the dysbiosis index. The diagnostic potential of salivary microbial dysbiosis as a convenient periodontal biomarker for assessing periodontal status has potential public health and clinical applications. PLAIN LANGUAGE SUMMARY: Periodontitis, a severe inflammation of the gums which causes bone loss, is a disease caused by an imbalance of good and bad bacteria under the gums. However, it is unclear how this bacterial imbalance in the gums affects the bacterial balance of other distinct parts of the mouth, such as the saliva and tongue. This study uses bacteria datasets of four previously published studies, contributing a total of 328 bacterial samples. The data were processed using a uniform data analysis workflow, and a bacterial score, the subgingival microbial dysbiosis index (SMDI), previously shown to capture periodontitis-associated bacteria imbalance, was calculated separately for samples from under the gums, the saliva, and the tongue. The SMDI was able to distinguish between health and periodontitis within each oral location, and in general, the scores were higher for periodontitis samples, though this difference was significant only for bacteria under the gums and in saliva. Saliva scores were also consistently correlated with bacteria under the gums. This study shows that periodontitis-associated bacterial imbalances are observed in oral locations beyond just under the gums, particularly the saliva. Thus, saliva bacteria may be used as a convenient biomarker for assessing gum disease, allowing for potential public health and clinical applications.

The nucleoid-associated protein HUß affects global gene expression in Porphyromonas gingivalis.

HU is a non-sequence-specific DNA-binding protein and one of the most abundant nucleoid-associated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUα (PG1258) and the HUß (PG0121) subunit. We have previously reported that PG0121 encodes a non-specific DNA-binding protein and that PG0121 is co-transcribed with the K-antigen capsule synthesis operon. We also reported that deletion of PG0121 resulted in downregulation of capsule operon expression and produced a P. gingivalis strain that is phenotypically deficient in surface polysaccharide production. Here, we show through complementation experiments in an E. coli MG1655 hupAB double mutant strain that PG0121 encodes a functional HU homologue. Microarray and quantitative RT-PCR analysis were used to further investigate global transcriptional regulation by HUß using comparative expression profiling of the PG0121 (HUß) mutant strain to the parent strain, W83. Our analysis determined that expression of genes encoding proteins involved in a variety of biological functions, including iron acquisition, cell division and translation, as well as a number of predicted nucleoid associated proteins were altered in the PG0121 mutant. Phenotypic and quantitative real-time-PCR (qRT-PCR) analyses determined that under iron-limiting growth conditions, cell division and viability were defective in the PG0121 mutant. Collectively, our studies show that PG0121 does indeed encode a functional HU homologue, and HUß has global regulatory functions in P. gingivalis; it affects not only production of capsular polysaccharides but also expression of genes involved in basic functions, such as cell wall synthesis, cell division and iron uptake.

Phages are unrecognized players in the ecology of the oral pathogen Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis (hereafter "Pg") is an oral pathogen that has been hypothesized to act as a keystone driver of inflammation and periodontal disease. Although Pg is most readily recovered from individuals with actively progressing periodontal disease, healthy individuals and those with stable non-progressing disease are also colonized by Pg. Insights into the factors shaping the striking strain-level variation in Pg, and its variable associations with disease, are needed to achieve a more mechanistic understanding of periodontal disease and its progression. One of the key forces often shaping strain-level diversity in microbial communities is infection of bacteria by their viral (phage) predators and symbionts. Surprisingly, although Pg has been the subject of study for over 40 years, essentially nothing is known of its phages, and the prevailing paradigm is that phages are not important in the ecology of Pg. RESULTS: Here we systematically addressed the question of whether Pg are infected by phages-and we found that they are. We found that prophages are common in Pg, they are genomically diverse, and they encode genes that have the potential to alter Pg physiology and interactions. We found that phages represent unrecognized targets of the prevalent CRISPR-Cas defense systems in Pg, and that Pg strains encode numerous additional mechanistically diverse candidate anti-phage defense systems. We also found that phages and candidate anti-phage defense system elements together are major contributors to strain-level diversity and the species pangenome of this oral pathogen. Finally, we demonstrate that prophages harbored by a model Pg strain are active in culture, producing extracellular viral particles in broth cultures. CONCLUSION: This work definitively establishes that phages are a major unrecognized force shaping the ecology and intra-species strain-level diversity of the well-studied oral pathogen Pg. The foundational phage sequence datasets and model systems that we establish here add to the rich context of all that is already known about Pg, and point to numerous avenues of future inquiry that promise to shed new light on fundamental features of phage impacts on human health and disease broadly. Video Abstract.

Bacteriome analysis of Aggregatibacter actinomycetemcomitans-JP2 genotype-associated Grade C periodontitis in Moroccan adolescents.

Background: Grade C (previously aggressive) periodontitis (GCP) in adolescents is prevalent in certain parts of Africa where it is associated with JP2 genotype, a highly virulent strain of Aggregatibacter actinomycetemcomitans. The aim of this study was to characterize the subgingival bacteriome in Moroccan subjects with GCP positive to A. actinomycetemcomitans JP2 genotype. Methods: Subgingival plaque samples were collected from shallow and deep pockets of 8 subjects with GCP (17.2 ± 1.5 years) and from gingival sulci of 13 controls with no periodontitis (14.6 ± 1.1 years). Identification and genotyping of A. actinomycetemcomitans was performed using PCR analysis of the ltx operon, while bacteriome profiling was done by 16S rRNA gene sequencing (V1-V3 region). Groups were compared in terms of microbial diversity, abundances, and dysbiosis. Results: The shallow and deep pocket sites from GCP cases had a significantly altered microbial composition compared to controls. Species associated with health included Haemophilus parainfluenzae, Lautropia mirabilis, Streptococcus spp., Gemella spp., and Rothia spp. While known periodontal pathogens, including Porphyromonas gingivalis, Tannerella forsythia, Treponema spp. and Fretibacterium spp., were significantly enriched in GCP, non-conventional taxa, including Pseudomonas oral taxon C61 and Enterobacter cloacae were more abundant and showed stronger association with the disease. Less significant differences in abundances of individual taxa were observed between shallow and deep pockets. Overall dysbiosis measured in terms of Subgingival Microbial Dysbiosis Index (SMDI) differentiated between GCP and no-periodontitis with 95% accuracy. Conclusions: The results suggest that several periodontal pathogens involved in the adult-type periodontitis also play a role in JP2 genotype-associated GCP. The potential role of non-conventional taxa in the pathogenesis of GCP warrants further investigation.

Integrative Metatranscriptomic Analysis Reveals Disease-specific Microbiome-host Interactions in Oral Squamous Cell Carcinoma.

Studies on the microbiome of oral squamous cell carcinoma (OSCC) have been limited to 16S rRNA gene sequencing. Here, laser microdissection coupled with brute-force, deep metatranscriptome sequencing was employed to simultaneously characterize the microbiome and host transcriptomes and predict their interaction in OSCC. The analysis involved 20 HPV16/18-negative OSCC tumor/adjacent normal tissue pairs (TT and ANT) along with deep tongue scrapings from 20 matched healthy controls (HC). Standard bioinformatic tools coupled with in-house algorithms were used to map, analyze, and integrate microbial and host data. Host transcriptome analysis identified enrichment of known cancer-related gene sets, not only in TT versus ANT and HC, but also in the ANT versus HC contrast, consistent with field cancerization. Microbial analysis identified a low abundance yet transcriptionally active, unique multi-kingdom microbiome in OSCC tissues predominated by bacteria and bacteriophages. HC showed a different taxonomic profile yet shared major microbial enzyme classes and pathways with TT/ANT, consistent with functional redundancy. Key taxa enriched in TT/ANT compared with HC were Cutibacterium acnes, Malassezia restricta, Human Herpes Virus 6B, and bacteriophage Yuavirus. Functionally, hyaluronate lyase was overexpressed by C. acnes in TT/ANT. Microbiome-host data integration revealed that OSCC-enriched taxa were associated with upregulation of proliferation-related pathways. In a preliminary in vitro validation experiment, infection of SCC25 oral cancer cells with C. acnes resulted in upregulation of MYC expression. The study provides a new insight into potential mechanisms by which the microbiome can contribute to oral carcinogenesis, which can be validated in future experimental studies. Significance: Studies have shown that a distinct microbiome is associated with OSCC, but how the microbiome functions within the tumor interacts with the host cells remains unclear. By simultaneously characterizing the microbial and host transcriptomes in OSCC and control tissues, the study provides novel insights into microbiome-host interactions in OSCC which can be validated in future mechanistic studies.

PepO and CppA modulate Streptococcus sanguinis susceptibility to complement immunity and virulence.

Streptococcus sanguinis is a ubiquitous commensal species of the oral cavity commonly involved as an opportunistic pathogen in cardiovascular infections. In this study, we investigated the functions of endopeptidase O (PepO) and a C3-degrading protease (CppA) in the systemic virulence of S. sanguinis. Isogenic mutants of pepO and cppA obtained in strain SK36 showed increased susceptibility to C3b deposition and to opsonophagocytosis by human polymorphonuclear neutrophils (PMN). These mutants differ, however, in their profiles of binding to serum amyloid P component (SAP) and C1q, whereas both showed reduced interaction with C4b-binding protein (C4BP) and/or factor H (FH) regulators as compared to SK36. The two mutants showed defects in ex vivo persistence in human blood, serum-mediated invasion of HCAEC endothelial cells, and virulence in a Galleria mellonella infection model. The transcriptional activities of pepO and cppA, assessed by RT-qPCR in nine wild-type strains, further indicated strain-specific profiles of pepO/cppA expression. Moreover, non-conserved amino acid substitutions were detected among the strains, mostly in CppA. Phylogenetic comparisons with homologues of streptococcal species of the oral and oropharyngeal sites suggested that S. sanguinis PepO and CppA have independent ancestralities. Thus, this study showed that PepO and CppA are complement evasion proteins expressed by S. sanguinis in a strain-specific manner, which are required for multiple functions associated with cardiovascular virulence.

Longitudinal changes in subgingival biofilm composition following periodontal treatment.

BACKGROUND: Current periodontal treatment involves instrumentation using hand and/or ultrasonic instruments, which are used either alone or in combination based on patient and clinician preference, with comparable clinical outcomes. This study sought to investigate early and later changes in the subgingival biofilm following periodontal treatment, to identify whether these changes were associated with treatment outcomes, and to investigate whether the biofilm responded differently to hand compared with ultrasonic instruments. METHODS: This was a secondary-outcome analysis of a randomized-controlled trial. Thirty-eight periodontitis patients received full-mouth subgingival instrumentation using hand (n = 20) or ultrasonic instrumentation (n = 18). Subgingival plaque was sampled at baseline and 1, 7, and 90 days following treatment. Bacterial DNA was analyzed using 16S rRNA sequencing. Periodontal clinical parameters were evaluated before and after treatment. RESULTS: Biofilm composition was comparable in both (hand and ultrasonics) treatment groups at all time points (all genera and species; p[adjusted] > 0.05). Large-scale changes were observed within groups across time points. At days 1 and 7, taxonomic diversity and dysbiosis were reduced, with an increase in health-associated genera including Streptococcus and Rothia equating to 30% to 40% of the relative abundance. When reassessed at day 90 a subset of samples reformed a microbiome more comparable with baseline, which was independent of instrumentation choice and residual disease. CONCLUSIONS: Hand and ultrasonic instruments induced comparable impacts on the subgingival plaque microbiome. There were marked early changes in the subgingival biofilm composition, although there was limited evidence that community shifts associated with treatment outcomes.

Specific host metabolite and gut microbiome alterations are associated with bone loss during spaceflight.

Understanding the axis of the human microbiome and physiological homeostasis is an essential task in managing deep-space-travel-associated health risks. The NASA-led Rodent Research 5 mission enabled an ancillary investigation of the gut microbiome, varying exposure to microgravity (flight) relative to ground controls in the context of previously shown bone mineral density (BMD) loss that was observed in these flight groups. We demonstrate elevated abundance of Lactobacillus murinus and Dorea sp. during microgravity exposure relative to ground control through whole-genome sequencing and 16S rRNA analyses. Specific functionally assigned gene clusters of L. murinus and Dorea sp. capable of producing metabolites, lactic acid, leucine/isoleucine, and glutathione are enriched. These metabolites are elevated in the microgravity-exposed host serum as shown by liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomic analysis. Along with BMD loss, ELISA reveals increases in osteocalcin and reductions in tartrate-resistant acid phosphatase 5b signifying additional loss of bone homeostasis in flight.