Subgingival microbial changes in Down Syndrome adults with periodontitis after chlorhexidine adjunct non-surgical therapy and monthly recalls-A 12-month case series study.

OBJECTIVES: Down Syndrome (DS) adults are at risk for periodontitis. Previous reports indicated difficulties in periodontopathogen reduction or eradication in DS individuals after periodontal treatment. This case series follows the subgingival microbial changes in adult DS individuals with periodontitis who received chlorhexidine adjunct non-surgical therapy plus 12-month recalls. METHODS: Twenty periodontitis DS participants (7 females; 25.5 ± 5.6 years of age; 3 with generalized periodontitis) partook in a study involving non-surgical mechanical periodontal therapy, twice daily chlorhexidine gel toothbrushing, chlorhexidine mouthwash, and monthly recalls. The subgingival microbiota profile was followed at baseline, 6-, and 12-months post-operation. RESULTS: Desulfobulbus, Saccharibacteria (TM7), Tannerella, and Porphyromonas were the major subgingival genera in this DS cohort. Favorable chlorhexidine adjunct non-surgical treatment outcomes were observed, with the relative abundance of Desulfobulbus sp. HMT 041, Saccharibacteria (TM7) [G-1] bacterium HMT 346 or 349, and Tannerella forsythia significantly reduced at the end of the study, but no significant reduction of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans could be observed. Relative abundance of Desulfobulbus sp. HMT 041 and T. forsythia were also found to be significantly associated with plaque, bleeding on probing, and probing pocket depth (PPD, in mm) at a site level, while the relative abundance of Halomonas pacifica was negatively associated with PPD. CONCLUSIONS: Successful chlorhexidine adjunct non-surgical treatment with hygiene care was accompanied by a subgingival microbial shift involving certain periodontopathogenic species, except P. gingivalis and A. actinomycetemcomitans. Further investigations are required to clarify the mechanism underpinning the unchanged relative abundance of the above two pathogens despite favorable clinical responses. CLINICAL SIGNIFICANCE: DS adults face challenges achieving optimal home care or hygiene for periodontal healing and disease prevention. Chemical adjunct mechanical periodontal therapy plus regular recalls appeared promising clinically and microbiologically, with subgingival periodontopathogenic species reduction. The persistence of A. actinomycetemcomitans and P. gingivalis in subgingival niches post-treatment warrants further investigation.

Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola.

The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH) proteins, play central roles in the stringent response. The pathogenic oral spirochete bacterium Treponema denticola lacks a long-RSH homologue but encodes putative small alarmone synthetase (Tde-SAS, TDE1711) and small alarmone hydrolase (Tde-SAH, TDE1690) proteins. Here, we characterize the respective in vitro and in vivo activities of Tde-SAS and Tde-SAH, which respectively belong to the previously uncharacterized RSH families DsRel and ActSpo2. The tetrameric 410-amino acid (aa) Tde-SAS protein preferentially synthesizes ppGpp over pppGpp and a third alarmone, pGpp. Unlike RelQ homologues, alarmones do not allosterically stimulate the synthetic activities of Tde-SAS. The ~180 aa C-terminal tetratricopeptide repeat (TPR) domain of Tde-SAS acts as a brake on the alarmone synthesis activities of the ~220-aa N-terminal catalytic domain. Tde-SAS also synthesizes "alarmone-like" nucleotides such as adenosine tetraphosphate (ppApp), albeit at considerably lower rates. The 210-aa Tde-SAH protein efficiently hydrolyzes all guanosine and adenosine-based alarmones in a Mn(II) ion-dependent manner. Using a growth assays with a ΔrelAΔspoT strain of Escherichia coli that is deficient in pppGpp/ppGpp synthesis, we demonstrate that Tde-SAS can synthesize alarmones in vivo to restore growth in minimal media. Taken together, our results add to our holistic understanding of alarmone metabolism across diverse bacterial species. IMPORTANCE The spirochete bacterium Treponema denticola is a common component of the oral microbiota. However, it may play important pathological roles in multispecies oral infectious diseases such as periodontitis: a severe and destructive form of gum disease, which is a major cause of tooth loss in adults. The operation of the stringent response, a highly conserved survival mechanism, is known to help many bacterial species cause persistent or virulent infections. By characterizing the biochemical functions of the proteins putatively responsible for the stringent response in T. denticola, we may gain molecular insight into how this bacterium can survive within harsh oral environments and promote infection. Our results also expand our general understanding of proteins that synthesize nucleotide-based intracellular signaling molecules in bacteria.

Diversity of Treponema denticola and Other Oral Treponeme Lineages in Subjects with Periodontitis and Gingivitis.

More than 75 species/species-level phylotypes belonging to the genus Treponema inhabit the human oral cavity. Treponema denticola is commonly associated with periodontal disease, but the etiological roles and ecological distributions of other oral treponemes remain more obscure. Here, we compared the clinical distributions of phylogroup 1 and 2 oral treponemes in subgingival plaque sampled from Chinese subjects with periodontitis (n = 10) and gingivitis (n = 8) via sequence analysis of the highly conserved pyrH housekeeping gene. Two PCR primer sets that targeted oral phylogroup 1 and 2 treponeme pyrH genes were used to construct plasmid clone amplicon libraries for each subject, and the libraries were sequenced for bioinformatic analysis. A total of 1,204 quality-filtered, full-length pyrH gene sequences were obtained from the cohort (median number, 61.5 cloned pyrH sequences per subject; range, 59 to 83), which were assigned to 34 pyrH genotypes (designated pyrH001 to pyrH034; 97% sequence identity cutoff). Eighteen pyrH genotypes (536 pyrH sequences) corresponded to phylogroup 1 treponeme taxa (including Treponema vincentii and Treponema medium). Sixteen pyrH genotypes (668 pyrH sequences) corresponded to T. denticola and other phylogroup 2 treponemes. Samples from periodontitis subjects contained a greater diversity of phylogroup 2 pyrH genotypes than did samples from gingivitis subjects (Mann-Whitney U test). One T. denticola pyrH genotype (pyrH001) was highly prevalent, detected in 10/10 periodontitis and 6/8 gingivitis subjects. Several subjects harbored multiple T. denticola pyrH genotypes. Nonmetric multidimensional scaling and permutational multivariate analysis of variance (PERMANOVA) revealed no significant differences in overall pyrH genotype compositions between periodontitis and gingivitis subjects. Taken together, our results show that subjects with periodontitis and gingivitis commonly harbor highly taxonomically diverse communities of oral treponemes. IMPORTANCE Periodontal diseases, such as periodontitis, are highly complex, multifactorial inflammatory infectious diseases affecting the gums and tooth-supporting structures. They are caused by chronic accumulations of dental plaque below the gum line that typically comprise hundreds of different bacterial species. Certain species of spiral-shaped bacteria known as treponemes, most notably Treponema denticola, are proposed to play key roles in the development and progression of periodontal disease. In our study, we characterized the genetic lineages of T. denticola, Treponema vincentii, Treponema medium, and related species of treponeme bacteria that were present in dental plaque samples from Chinese subjects with periodontal disease. Our results revealed that individual subjects commonly harbored multiple genetic lineages (strains) of T. denticola and other species of treponeme bacteria. Taken together, our results indicate that highly diverse and complex populations of oral treponemes may be present in dental plaque, which may potentially play important roles affecting periodontal health status.

Complete Genome Sequences of Three Human Oral Treponema parvum Isolates.

Treponema parvum is a spirochete associated with human and animal oral/nonoral soft tissue infections. Here, we report the complete genome sequences of three human oral isolates of T. parvum, namely, ATCC 700770T (OMZ 833T), ATCC 700773 (OMZ 842), and OMZ 843, which possess circular chromosomes of a median size of 2.63 Mb.

Complete Genome Sequence of Human Oral Phylogroup 1 Treponema sp. Strain OMZ 804 (ATCC 700766), Originally Isolated from Periodontitis Dental Plaque.

Host-associated treponeme bacteria play etiological roles in human and animal soft tissue infections. Treponema sp. strain OMZ 804 (ATCC 700766) was isolated from dental plaque sampled from a patient with periodontitis in Switzerland in 1994. We report here the complete genome sequence of its 2.98-Mb circular chromosome.

Species-Level Salivary Microbial Indicators of Well-Resolved Periodontitis: A Preliminary Investigation.

Objective: To profile the salivary microbiomes of a Hong Kong Chinese cohort at a species-level resolution and determine species that discriminated clinically resolved periodontitis from periodontally healthy cases. Methods: Salivary microbiomes of 35 Hong Kong Chinese subjects' under routine supportive dental care were analyzed. All subjects had been treated for any dental caries or periodontal disease with all restorative treatment completed at least 1 year ago and had ≤3 residual pockets. They were categorized based on a past diagnosis of chronic periodontitis into "healthy" (H) or "periodontitis" (P) categories. Unstimulated whole saliva was collected, genomic DNA was isolated, and high throughput Illumina MiSeq sequencing of 16S rRNA (V3-V4) gene amplicons was performed. The sequences were assigned taxonomy at the species level by using a BLASTN based algorithm that used a combined reference database of HOMD RefSeqV14.51, HOMD RefSeqExtended V1.1 and GreenGeneGold. Species-level OTUs were subjected to downstream analysis in QIIME and R. For P and H group comparisons, community diversity measures were compared, differentially abundant species were determined using DESeq2, and disease indicator species were determined using multi-level pattern analysis within the R package "indicspecies." Results: P subjects were significantly older than H subjects (p = 0.003) but not significantly different in their BOP scores (p = 0.82). No significant differences were noted in alpha diversity measures after adjusting for age, gender, and BOP or in the beta diversity estimates. Four species; Treponema sp. oral taxon 237, TM7 sp. Oral Taxon A56, Prevotella sp. oral taxon 314, Prevotella sp. oral taxon 304, and Capnocytophaga leadbetteri were significantly more abundant in P than in the H group. Indicator species analysis showed 7 significant indicators species of P group. Fusobacterium sp oral taxon 370 was the sole positive indicator of P group (positive predictive value = 0.9, p = 0.04). Significant indicators of the H category were Leptotrichia buccalis, Corynebacterium matruchotii, Leptotrichia hofstadii, and Streptococcus intermedius. Conclusion: This exploratory study showed salivary microbial species could discriminate treated, well-maintained chronic periodontitis from healthy controls with similar gingival inflammation levels. The findings suggest that certain salivary microbiome features may identify periodontitis-susceptible individuals despite clinical disease resolution.

The Ps and Qs of alarmone synthesis in Staphylococcus aureus.

During the stringent response, bacteria synthesize guanosine-3',5'-bis(diphosphate) (ppGpp) and guanosine-5'-triphosphate 3'-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp 'alarmones' are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode 'small alarmone synthetase' (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5'-phosphate 3'-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10-fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) and Streptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically modulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species.

Intra-oral single-site comparisons of periodontal and peri-implant microbiota in health and disease.

OBJECTIVE: Periodontitis and peri-implantitis are oral infectious-inflammatory diseases that share similarities in their pathology and etiology. Our objective was to characterize the single-site subgingival and submucosal microbiomes of implant-rehabilitated, partially dentate Chinese subjects (n = 18) presenting with both periodontitis and peri-implantitis. MATERIALS AND METHODS: Subgingival/submucosal plaque samples were collected from four clinically distinct sites in each subject: peri-implantitis submucosa (DI), periodontal pocket (DT), clinically healthy (unaffected) peri-implant submucosa (HI), and clinically healthy (unaffected) subgingival sulcus (HT). The bacterial microbiota present was analyzed using Illumina MiSeq sequencing. RESULTS: Twenty-six phyla and 5,726 operational taxonomic units (OTUs, 97% sequence similarity cutoff) were identified. Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes, Actinobacteria, Synergistetes, TM7, and Spirochaetes comprised 99.6% of the total reads detected. Bacterial communities within the DI, DT, HI, and HT sites shared high levels of taxonomic similarity. Thirty-one "core species" were present in >90% sites, with Streptococcus infantis/mitis/oralis (HMT-070/HMT-071/HMT-638/HMT-677) and Fusobacterium sp. HMT-203/HMT-698 being particularly prevalent and abundant. Beta-diversity analyses (PERMANOVA test, weighted UniFrac) revealed the largest variance in the microbiota was at the subject level (46%), followed by periodontal health status (4%). Differing sets of OTUs were associated with periodontitis and peri-implantitis sites, respectively. This included putative "periodontopathogens," such as Prevotella, Porphyromonas, Tannerella, Bacteroidetes [G-5], and Treponema spp. Interaction network analysis identified several putative patterns underlying dysbiosis in periodontitis/peri-implantitis sites. CONCLUSIONS: Species (OTU) composition of the periodontal and peri-implant microbiota varied widely between subjects. The inter-subject variations in subgingival/submucosal microbiome composition outweighed differences observed between implant vs. tooth sites, or between diseased vs. healthy (unaffected) peri-implant/periodontal sites.

Salivary microbiome in non-oral disease: A summary of evidence and commentary.

OBJECTIVE: The advent of high-throughput sequencing and 'omic' technologies is facilitating an 'open-ended' understanding of the human microbial community and its interplay with health. This commentary aims to present key perspectives and summarize current evidence from metagenomic studies of salivary microbiota in relation to general health and systemic diseases. DESIGN: A narrative review of studies that described salivary microbiome composition in relation to various general health conditions was conducted and the main results were summarized. RESULTS: Currently available evidence shows salivary microbial patterns and fingerprints as related to a range of metabolic, autoimmune and immunodeficiency associated conditions, similar to albeit at a far lower scale than similar studies in the gut microbiome. CONCLUSIONS: Considering the relative ease of collection, emerging evidence of association with non-oral diseases may imply that saliva microbiome research may have potential diagnostic or prognostic value.

Multilocus Sequence Analysis of Phylogroup 1 and 2 Oral Treponeme Strains.

More than 75 "species-level" phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet-uncultivated taxa or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly conserved 16S rRNA, pyrH, recA, and flaA genes. We utilized this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n = 71) of diverse geographical origins. This comprises phylogroup 1 (n = 23) and phylogroup 2 (n = 48) treponeme strains, including all relevant American Type Culture Collection reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450-bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074-nucleotide [nt]), recA (1,377-nt), and pyrH (696-nt) gene sequence data sets. Our data confirmed the species differentiation between Treponema denticola (n = 41) and Treponema putidum (n = 7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into five distinct "species-level" phylotypes. These respectively corresponded to "Treponema vincentii" (n = 11), Treponema medium (n = 1), "Treponema sinensis" (Treponema sp. IA; n = 4), Treponema sp. IB (n = 3), and Treponema sp. IC (n = 4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence. IMPORTANCE: Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although Treponema denticola is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.

Laribacter hongkongensis anaerobic adaptation mediated by arginine metabolism is controlled by the cooperation of FNR and ArgR.

Laribacter hongkongensis is a fish-borne pathogen associated with invasive infections and gastroenteritis. Its adaptive mechanisms to oxygen-limiting conditions in various environmental niches remain unclear. In this study, we compared the transcriptional profiles of L. hongkongensis under aerobic and anaerobic conditions using RNA-sequencing. Expression of genes involved in arginine metabolism significantly increased under anoxic conditions. Arginine was exploited as the sole energy source in L. hongkongensis for anaerobic respiration via the arginine catabolism pathway: specifically via the arginine deiminase (ADI) pathway. A transcriptional regulator FNR was identified to coordinate anaerobic metabolism by tightly regulating the expression of arginine metabolism genes. FNR executed its regulatory function by binding to FNR boxes in arc operons promoters. Survival of isogenic fnr mutant in macrophages decreased significantly when compared with wild-type; and expression level of fnr increased 8 h post-infection. Remarkably, FNR directly interacted with ArgR, another regulator that influences the biological fitness and intracellular survival of L. hongkongensis by regulating arginine metabolism genes. Our results demonstrated that FNR and ArgR work in coordination to respond to oxygen changes in both extracellular and intracellular environments, by finely regulating the ADI pathway and arginine anabolism pathways, thereby optimizing bacterial fitness in various environmental niches.

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis.

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, ß = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.

Distributions of Synergistetes in clinically-healthy and diseased periodontal and peri-implant niches.

Bacterial taxa belonging to the phylum Synergistetes are commonly detected within diseased periodontal niches, but are rarely found within healthy oral sites. However, as they typically constitute a minor fraction of the oral microbiota, their precise distributions and disease-associations remain to be fully established. Here, we surveyed the Synergistetes taxa present within individual periodontal/subgingival and peri-implant/submucosal sites, within Chinese subjects (n = 18) affected by both peri-implantitis and periodontitis. Four individual, clinically-distinct sites were analyzed in each patient: healthy sulcus; periodontitis lesion; healthy peri-implant space; peri-implantitis lesion. We employed a clone library-based approach, using PCR-primers that specifically amplified ca. 650bp regions of the 16S rRNA gene from oral cluster A and B Synergistetes taxa. Twenty-one of the 72 sites (from 12/18 subjects) yielded Synergistetes 16S rRNA PCR products. Sequencing of cloned amplicon libraries yielded 1338 quality-filtered 16S rRNA sequences, which were assigned to 26 Synergistetes operational taxonomic units (OTUs; oral taxon SH01-SH26) using a 98.5% identity cut-off. We identified 25 Synergistetes oral cluster A OTUs (genus Fretibacterium; corresponding to Human Oral Taxon (HOT) numbers 358, 359, 360, 361, 362, 363, 452, and 453), and one oral cluster B OTU (Pyramidobacter piscolens oral taxon SH04, HOT-357). Three OTUs predominated: Fretibacterium oral taxon SH01 (HOT-360), Fretibacterium oral taxon SH02 (HOT-452), and Fretibacterium fastidiosum oral taxon SH03 (HOT-363). The Synergistetes community compositions within the respective periodontal and peri-implant sites were variable and complex, and no statistically-significant correlations could be established. However, the detection frequency of F. fastidiosum SH03 and Fretibacterium oral taxon SH01 were both positively associated with plaque index at healthy subgingival sites. Taken together, our results show that diverse Synergistetes populations inhabit both diseased and healthy periodontal and peri-implant niches, with considerable site-to-site variations in composition occurring within the same oral cavity.

In-depth snapshot of the equine subgingival microbiome.

This study explored the range of bacterial taxa present within healthy subgingival (below the gum-line) niches in the horse oral cavity using 16S rRNA gene amplicon pyrosequencing. Pooled subgingival plaque samples were collected from approximately 200 sulcus sites from two horses (EQ1, EQ2) for analysis. A total of 14,260 quality-filtered pyrosequencing reads were obtained, which were assigned to 3875 operational taxonomic units (OTUs; 99% identity cut-off); 1907 OTUs for EQ1 and 2156 OTUs for EQ2. Diverse taxa from 12 phyla were identified, including Actinobacteria (3.17%), Bacteroidetes (25.11%), Chloroflexi (0.04%), Firmicutes (27.57%), Fusobacteria (5.15%), Proteobacteria (37.67%), Spirochaetes (0.15%), Synergistetes (0.22%), Tenericutes (0.16%), GN02 (0.19%), SR1 (0.01%) and TM7 (0.37%). Many OTUs were not closely related to known phylotypes, and may represent 'equine-specific' taxa. Phylotypes corresponding to Gammaproteobacteria were abundant, including Actinobacillus spp. (8.75%), unclassified Pasteurellaceae (9.90%) and Moraxella spp. (9.58%). PCR targeting the Synergistetes and Spirochaetes phyla was performed, and resultant plasmid libraries of 16S rRNA gene amplicons (ca. 1480 bp) were Sanger sequenced. Twenty-six Spirochaetes OTUs, and 16 Synergistetes OTUs were identified (99% identity cut-off). These 'species-level' OTUs were assigned Equine Oral Taxon (EOT) numbers, whose phylogenies and taxonomy were comprehensively investigated, in conjunction with corresponding Synergistetes and Spirochaetes OTUs identified by pyrosequencing. The vast majority of Spirochaetes taxa belonged to the genus Treponema, which corresponded to 7 of the 10 human oral treponeme phylogroups. Other Spirochaetes taxa belonging to the Leptospiraceae family were observed; but many treponemes commonly implicated in animal hoof/foot and non-oral soft tissue infections; e.g. Treponema phagedenis, Treponema pedis, Treponema refringens, Treponema calligyrum; were not identified here. Diverse Synergistetes taxa corresponding to oral clusters A and B were identified, which included Fretibacterium fastidiosum and Pyramidobacter piscolens. Taken together, our data reveals that equine subgingival plaque microbiota shares many similarities with the human, canine and feline oral microbiomes.

Periodontal and peri-implant microbiota in patients with healthy and inflamed periodontal and peri-implant tissues.

OBJECTIVE: To compare the prevalence and levels of six bacterial pathogens within the subgingival/submucosal microbiota at teeth versus implants with various clinical conditions. MATERIAL AND METHODS: Twenty-two Chinese were included. Four subgingival/submucosal sites were selected for microbiological sampling within each subject, that is, (1) healthy peri-implant tissues; (2) peri-implantitis [PPD ≥ 5 mm, presence of bleeding on probing (BOP) and confirmed radiographic bone loss]; (3) healthy gingiva; and (4) periodontitis (PPD ≥4 mm). Subgingival/submucosal plaque was sampled using paper points. Quantitative real-time polymerase chain reaction (q-PCR) was used to quantify six pathogens, including Porphyromonas gingivalis (P.g.), Treponema denticola (T.d.), Aggregatibacter actinomycetemcomitans (A.a.), Fusobacterium nucleatum (F.n.), Prevotella intermedia (P.i.), and Staphylococcus aureus (S.a.). Counts were log10-transformed. RESULTS: The most commonly detected species were S. a. and F. n., while A. a. and. P. i. had the lowest detection frequency. The detection frequencies of diseased tooth or implant sites for each of the six target species were either equal to or higher than the respective frequencies at the corresponding healthy sites. There were no statistically significant differences for any of the species or clinical sites (P > 0.05, Cochran's Q test). No statistically significant differences in the bacterial loads were found among the four clinical sites; with the exception of F. nucleatum. This was more abundant in periodontitis sites (P = 0.023, Friedman's 2-way anova). Both periodontal and peri-implant sites, irrespective of their health status, were revealed to harbor S. aureus cells. The log10-transformed loads of S. aureus were approximately 3.5 within each of the clinical sites (P = 0.232). This was the highest of the six species analyzed. CONCLUSIONS: Within the same subjects, putative periodontal pathogens were common to both periodontal and peri-implant sites irrespective of health status. The prevalence and levels of P. gingivalis and F. nucleatum were significantly associated with periodontitis, but not with peri-implantitis. A. actinomycetemcomitans was associated with both disease conditions, periodontitis and peri-implantitis, but not with either gingival or mucosal health.

Salivary IL-1ß and red complex bacteria as predictors of the inflammatory status in sub-peri-implant niches of subjects with peri-implant mucositis.

BACKGROUND AND OBJECTIVES: Salivary biomarkers may enhance diagnostic sensitivity for peri-implant disease assessment. This study aimed to investigate the association of salivary periodontopathogen count and salivary interleukin-1beta (IL-1ß) level with the peri-implant crevicular fluid IL-1ß response at peri-implant mucositis (PM) sites among subjects with differing periodontal disease susceptibility. MATERIALS AND METHODS: Eighty-seven partially edentulous subjects having at least one implant with peri-implant mucositis were included: 40 with history of chronic periodontitis (P) and 47 with no history of periodontitis (NP). Salivary IL-1ß, peri-implant crevicular fluid (PICF) IL-1ß, and salivary red complex pathogen counts were recorded. Subjects were scored according to a threshold salivary pathogen level of more than 5log (10) counts and assigned a "red complex score." Quartiles of salivary and PICF IL-1ß levels were also scored. Area under receiver operating curve (AUC) was computed to predict the highest PICF IL-1ß score using salivary biomarker as predictors and age-adjusted logistic regression performed for the significant predictors. RESULTS: In the NP group, red complex score (AUC = 0.758 P = 0.010) (odds ratio = 1.377) and salivary IL-1ß (AUC = 0.708 P = 0.038) (odds ratio = 2.506) were significant predictors of highest PICF IL-1ß quartile score. In the P group, no significant associations were noted. CONCLUSIONS: Salivary biomarkers could distinguish the "high" pro-inflammatory responders at PM sites only in subjects without inherent periodontal disease susceptibility. Periodontal susceptibility may impact the immuno-inflammatory response in sub-peri-implant niches of those with peri-implant mucositis.

Molecular characterization of arginine deiminase pathway in Laribacter hongkongensis and unique regulation of arginine catabolism and anabolism by multiple environmental stresses.

The betaproteobacterium Laribacter hongkongensis is associated with invasive bacteremic infections and gastroenteritis. Its genome contains two adjacent arc gene cassettes (arc1 and arc2) under independent transcriptional control, which are essential for acid resistance. Laribacter hongkongensis also encodes duplicate copies of the argA and argB genes from the arginine biosynthesis pathway. We show that arginine enhances the transcription of arcA2 but suppresses arcA1 expression. We demonstrate that ArgR acts as a transcriptional regulator of the two arc operons through binding to ARG operator sites (ARG boxes). Upon temperature shift from 20°C to 37°C, arcA1 transcription is upregulated while arcA2, argA2, argB2 and argG are downregulated. The transcription of arcA1 and arcA2 are augmented under anaerobic and acidic conditions. The transcription levels of argA1, argA2, argB1, argB2 and argG are significantly increased under anaerobic and acidic conditions but are repressed by the addition of arginine. Deletion of argR significantly decreases bacterial survival in macrophages, while expression of both arc operons, argR and all five of the anabolic arg genes increases 8 h post-infection. Our results show that arginine catabolism in L. hongkongensis is finely regulated by controlling the transcription of two arc operons, whereas arginine anabolism is controlled by two copies of argA and argB.

Complete Genome Sequence for Treponema sp. OMZ 838 (ATCC 700772, DSM 16789), Isolated from a Necrotizing Ulcerative Gingivitis Lesion.

The oral treponeme bacterium Treponema sp. OMZ 838 was originally isolated from a human necrotizing ulcerative gingivitis (NUG) lesion. Its taxonomic status remains uncertain. The complete genome sequence length was determined to be 2,708,067 bp, with a G+C content of 44.58%, and 2,236 predicted coding DNA sequences (CDS).

Complete Genome Sequence of the Oral Spirochete Bacterium Treponema putidum Strain OMZ 758T (ATCC 700334T).

The oral spirochete bacterium Treponema putidum inhabits human periodontal niches. The complete genome sequence of the OMZ 758(T) (ATCC 700334(T)) strain of this species was determined, revealing a 2,796,913-bp chromosome, with a G+C content of 37.30% and a single plasmid (pTPu1; 3,649 bp) identical to pTS1 from Treponema denticola.

pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis.

BACKGROUND: The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. RESULTS: A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. CONCLUSIONS: We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.