Identification of keystone taxa in root canals and periapical lesions of post-treatment endodontic infections: Next generation microbiome research.

AIM: The aim of this study was to analyse and compare the microbiome present in root canals and periapical lesions of teeth with post-treatment infections, and to identify the presence of keystone taxa in both habitats using next-generation sequencing analysis. METHODOLOGY: Apices and periapical lesions of patients with post-treatment apical periodontitis were surgically extracted. Specimens were cryo-pulverized, bacterial DNA was extracted, and the V3-V4 hypervariable regions of the 16S rRNA gene were sequenced using the Illumina Miseq platform. Bioinformatic analysis was carried out with Mothur software, whilst diversity indices were obtained using operational taxonomic units (OTUs). The diversity indices were compared with the Kruskal-Wallis test, and community composition differences were explored with Permutational Multivariate Analysis of Variance (PERMANOVA). A bacterial functional study was performed with the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. Co-occurrence network analyses were performed using the Sparse Correlations for Compositional data (SparCC). Eigencentrality, clr-based abundance and ubiquitousness were applied to infer keystone taxa. P values <.05 were considered statistically significant. RESULTS: Thirty-two apices and thirty-nine periapical lesions were sequenced and analysed. A similar alpha-diversity (p < .05) and community composition (p = .91) was observed for apices and lesion samples. The most abundant OTUs identified amongst all samples included Fusobacterium nucleatum, Prevotella loescheii, Streptococcus intermedius, Porphyromonas gingivalis, Parvimonas micra, Synergistetes bacterium, Tannerella forsythia and Peptostreptococcus stomatis. The metabolic pathways with >0.81% abundances included membrane transport, genetic information processing and metabolic pathways. F. nucleatum was identified as a keystone taxon as it showed ubiquitousness, an eigenvector centrality value of 0.83 and a clr-based abundance >4. CONCLUSIONS: The microbiome in apices and periapical lesions of post-treatment endodontic infections showed a similar diversity and taxonomic composition. Co-occurrence network analyses at OTU level identified F. nucleatum as a keystone taxon candidate in these infections.

Correlation between Inflammasomes and Microbiota in Peri-Implantitis.

The activation of inflammasomes is thought to induce the inflammatory process around dental implants. No information is available on the correlation between microbiota and inflammasomes in clinical samples from patients suffering peri-implantitis. For this cross-sectional study, 30 biofilm samples were obtained from 19 patients undergoing surgical treatment for peri-implantitis because of the presence of bleeding on probing, probing depth higher than 6 mm, and radiographic bone loss higher than 3 mm. Then, soft tissue samples from around the implant were also collected. The relative abundance of bacteria and alpha-diversity indexes were calculated after analyzing the 16S rRNA gene using next-generation sequencing. The soft-tissue samples were processed for evaluation of the inflammasomes NLRP3 and AIM2 as well as caspase-1 and IL-1ß. The relative abundance (mean (SD)) of specific species indicated that the most abundant species were Porphyromonas gingivalis (10.95 (14.17)%), Fusobacterium vincentii (10.93 (13.18)%), Porphyromonas endodontalis (5.89 (7.23)%), Prevotella oris (3.88 (4.94)%), Treponema denticola (2.91 (3.19)%), and Tannerella forsythia (2.84 (4.15)%). Several correlations were found between the species and the immunohistochemical detection of the inflammasomes NLRP3 and AIM2 as well as caspase-1 and IL-1ß, both in the epithelium and the lamina propria. A network analysis found an important cluster of variables formed by NLRP3 in the lamina propria and AIM2, caspase-1, and IL-1ß in the lamina propria and the epithelium with Prevotella dentalis, Prevotella tannerae, Tannerella forsythia, or Selenomonas timonae. Thus, it could be concluded that inflammasomes NLRP3 and AIM2 and their downstream effectors caspase-1 and interleukin-1ß can be significantly associated with specific bacteria.

Fermented Goat's Milk Contributes to the Recovery of Iron Deficiency Anemia via Modulation of the Gut Microbiome.

Iron deficiency anemia (IDA) is a global public health concern affecting 1.6 billion people worldwide. The administration of iron supplements during the treatment of IDA adversely affects the intestinal barrier function and the composition and functionality of the intestinal microbiome, both of which are already altered during IDA. For this reason, it is of great interest to develop nutritional strategies aimed at alleviating these gut alterations associated with IDA and its treatment. In this sense, fermented goat's milk (FGM) was studied due to its nutritional quality. Our findings showed that in anemic animals the consumption of a FGM-based diet, compared to a standard diet, had positive modulatory effects on the intestinal microbiome. FGM-based diet restored intestinal dysbiosis, the intestinal barrier functionality, and bacterial translocation, contributing to a more efficient recovery of IDA. Therefore, FGM is a useful nutritional tool to ease intestinal alterations occurring during IDA and during its treatment.

Bacteremia due to Clostridium innocuum: Analysis of four cases and literature review.

Clostridium innocuum is a Gram-positive anaerobic spore-forming bacillus that has been identified as part of the normal intestinal microbiota. This bacterium has been rarely associated with human infections, and only few severe infections have been reported until now. In this work, we report on four patients with bacteremia due to C. innocuum, which were well identified by MALDI-TOF MS. Moreover, a review of the previous published cases of bacteremia due to this anaerobic bacterium has been performed.

Comparative evaluation of MALDI-TOF MS and 16S rRNA gene sequencing for the identification of clinically relevant anaerobic bacteria: critical evaluation of discrepant results.

OBJECTIVES: The main study objective was to evaluate the correlation between matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing results for the identification of anaerobes. METHODS: A retrospective study was conducted of all anaerobic bacteria isolated from clinically significant specimens. MALDI-TOF (Bruker Byotyper) and 16S rRNA gene sequencing were performed in all strains. Identifications were considered correct when the concordance with gene sequencing was ≥99%. RESULTS: The study included 364 isolates of anaerobic bacteria: 201 (55.2%) Gram-negative and 163 (44.8%) Gram-positive, mostly belonging to the genus Bacteroides. Isolates were largely obtained from blood cultures (128/35.4%) and intra-abdominal samples (116/32.1%). Overall, 87.3% of isolates were identified at species level using the version 9 database (89.5% of Gram-negative and 84.6% of Gram-positive anaerobic bacteria). All isolates belonging to the species B. fragilis sensu stricto were correctly identified by MALDI-TOF MS, but five cases of Phocaeicola (Bacteroides) dorei were misidentified as Phocaeicola (Bacteroides) vulgatus; all Prevotella isolates were correctly identified at the genus level, and most were correctly identified at the species level. Among Gram-positive anaerobes, 12 Anaerococcus species were not identified by MALDI-TOF MS, while six cases identified as Peptoniphilus indolicus were found to belong to other genera/species. CONCLUSIONS: MALDI-TOF is a reliable technique for identifying most anaerobic bacteria, although the database needs frequent updating to identify rare, infrequent, and newly discovered species.

Differences between bloodstream infections involving Gram-positive and Gram-negative anaerobes.

BACKGROUND: The objectives of this study were to describe differences between bloodstream infections involving Gram-positive (GP) and Gram-negative (GN) anaerobic bacteria. METHODS: Patients with clinically significant anaerobic bacteremia detected between October 2016 and July 2022 in a tertiary hospital in Granada (Spain) were retrospectively included. Species identification was performed by MALDI-TOF MS and/or molecular methods. The association between variables was analyzed using contingency tables, applying the chi-square test when expected frequencies were adequate and the Fisher exact test when not. Variables were gathered at the time of the first positive blood culture. RESULTS: Out of 237 cases of anaerobic bloodstream infections detected, 127 (53.6%) were GN. Crude mortality was 20.3%, corresponding to 48 patients who died of causes directly attributable to bacteremia. The presence of malignant disease (p = 0.011), abdominal and/or pelvic surgery (p = 0.001), and transplantation (p = 0.008) were significantly associated with bacteremia due to GN bacteria, while the presence of diabetes mellitus was significantly associated with bacteremia due to GP bacteria (p = 0.022). The presence of both septic shock and mortality was more frequently associated with bacteremia due to GN versus GP bacteria. CONCLUSIONS: The association of certain variables with the presence of bloodstream infections due to GP or GN anaerobic bacteria may assist in selecting the optimal empirical therapeutic approach and improving the outcome of patients with these types of infection.

Microbiome in paired root apices and periapical lesions and its association with clinical signs in persistent apical periodontitis using next-generation sequencing.

AIM: To assess and compare the microbiome of paired root apices and periapical lesions from cases with failed endodontic treatment and to associate the microbiome and bacterial metabolic pathways in both sites with asymptomatic apical periodontitis (AAP) and symptomatic apical periodontitis (SAP), using next-generation sequencing (NGS). METHODOLOGY: Matched root apices and periapical lesions of patients with failed root canal treatments were surgically extracted. Specimens were cryopulverized, bacterial DNA was extracted and the V3-V4 hypervariable regions of the 16 S rRNA gene were amplified and sequenced using the Illumina Miseq platform. Diversity and community composition were studied in the paired samples, as well as in AAP and SAP cases. Diversity indices were compared in each case by means of the Wilcoxon matched-pairs signed rank and Mann-Whitney U tests. Differences in the community composition were explored with multivariate statistical analysis and Linear discriminant analysis Effect Size (LEfSe). Bacterial functional study was performed through the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis. RESULTS: Twenty-one paired apices and lesions were successfully sequenced and analysed, identifying a total of 21 phyla and 600 genera. A higher alpha-diversity was observed in the periapical lesions, although no global differences in the community composition between the two sites were found (p = .87), the most prevalent genera being Fusobacterium, Porphyromonas and Streptococcus. Prevotella, Clostridiales_vadinBB60_group, Bosea, Phreatobacter, Afipia and Xanthobacteriaceae_unclassified were enriched in SAP samples, while Pseudopropionibacterium, Campylobacter and Peptoniphilus were significantly more abundant in AAP cases (p < .05). Metabolic pathways involved in the amino acid metabolism or degradation and flagellum assembly were more abundant in SAP samples, whereas glucose metabolism-related pathways were associated with AAP. CONCLUSIONS: The bacterial community composition was similar in the apices and periapical lesions. The microbiome was different in AAP and SAP samples, gram-negative bacteria showing higher relative abundances in SAP cases. An association was observed between amino acid degradation and flagellum assembly pathways, and the development of tenderness to percussion or palpation.

New anaerobic bacteria causing infection: Bacteremia due to Murdochiellaasaccharolytica.

Murdochiella asaccharolytica is a Gram-positive anaerobic coccus firstly isolated from wound specimens, since then, it was very rarely isolated from human infections. We report the first case of bloodstream infection due to this bacterium in a 90 years-old-man. The patient had a history of B-cell lymphoma and presented with pain and induration of the base of penis along with fever. A blood culture was obtained yielding M. asaccharolytica in pure culture. Only resistance to clindamycin was demonstrated. Treatment with metronidazole was started and the clinical evolution improved with antimicrobial treatment, being discharged after 6 days.

An uncommon case of bacteremia caused by Lancefieldella parvula in an oncological patient.

We report an uncommon case of bacteremia due to Lancefieldella parvula isolated in pure culture. We present a 72 year-old-man affected with cancer admitted with abdominal pain due to a parietal rupture of the urinary bladder. After surgical treatment, the patient developed fever and two sets of blood cultures yielded a pure culture of an anaerobic microorganism identified as L. parvula (formerly Atopobium parvulum) by MALDI-TOF MS, and confirmed by 16S rRNA gene sequencing. The isolate was susceptible to all antibiotics and the outcome was finally successful. Bacteremia due to L. parvula is an uncommon disease and, in that case, MALDI-TOF MS was an useful tool for the initial identification.

Bacteremia due to Parabacteroides distasonis: Experience with 4 cases.

OBJECTIVE: To report a series of four uncommon cases of bacteremia due to Parabacteroides distasonis. METHODS: We have analyzed the epidemiological characteristics, clinical features, diagnostic methods, treatment and outcome of these patients. RESULTS: Two sets of blood cultures of each patient yielded a pure culture of an anaerobic microorganism identified as P. distasonis by MALDI-TOF MS, and confirmed by 16S rRNA gene sequencing. All patients were male and they had risk factors for anaerobic bacteremia. All isolates were susceptible to metronidazole and the outcome was successful in three patients. CONCLUSIONS: Bloodstream infections due to P. distasonis are still rare. MALDI-TOF MS appear to be an excellent tool for the correct identification.

Curvicollide D Isolated from the Fungus Amesia sp. Kills African Trypanosomes by Inhibiting Transcription.

Sleeping sickness or African trypanosomiasis is a serious health concern with an added socio-economic impact in sub-Saharan Africa due to direct infection in both humans and their domestic livestock. There is no vaccine available against African trypanosomes and its treatment relies only on chemotherapy. Although the current drugs are effective, most of them are far from the modern concept of a drug in terms of toxicity, specificity and therapeutic regime. In a search for new molecules with trypanocidal activity, a high throughput screening of 2000 microbial extracts was performed. Fractionation of one of these extracts, belonging to a culture of the fungus Amesia sp., yielded a new member of the curvicollide family that has been designated as curvicollide D. The new compound showed an inhibitory concentration 50 (IC50) 16-fold lower in Trypanosoma brucei than in human cells. Moreover, it induced cell cycle arrest and disruption of the nucleolar structure. Finally, we showed that curvicollide D binds to DNA and inhibits transcription in African trypanosomes, resulting in cell death. These results constitute the first report on the activity and mode of action of a member of the curvicollide family in T. brucei.

Gallic Acid: A Natural Phenolic Compound Exerting Antitumoral Activities in Colorectal Cancer via Interaction with G-Quadruplexes.

Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC). We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds.

Bacteremia due to Prevotella oris of probable hepatic origin.

Prevotella species are Gram-negative anaerobic bacilli mainly involved in oral cavity infections. We report a rare case of bacteremia due to P. oris of probable hepatic origin. A 70-year-old man with a history of diabetes mellitus was admitted for general malaise, fever and dyspnea. The patient was admitted to the ICU due to septic shock. Two sets of blood cultures yielded a pure culture of an anaerobic microorganism identified as P. oris by MALDI-TOF MS. The same microorganism was isolated from a hepatic abscess drainage. Only resistance to penicillin was documented. Treatment with piperacillin-tazobactam and levofloxacin was administered, and the patient was discharged following his recovery.

Misidentification of Phocaeicola (Bacteroides) dorei in two patients with bacteremia.

Phocaeicola (Bacteroides) dorei is a Gram-negative anaerobic bacillus that is rarely isolated from human specimens. Its accurate identification can be hampered by its close taxonomic relationship with Bacteroides vulgatus. We report on two patients with bacteremia due to P. (B.) dorei, which was initially identified as B. vulgatus by MALDI-TOF MS.