A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

Aerococcus kribbianus sp. nov., a facultatively anaerobic bacterium isolated from pig faeces.

Two cocci-shaped, facultatively anaerobic, Gram-positive bacteria isolated from the faeces of a pig were designated as strains YH-aer221T and YH-aer222. Analysis of the 16S rRNA gene sequences revealed that the isolates were most closely related to Aerococcus suis JCM 18035T with 96.6 % similarity. The multi-locus sequence tree revealed that the isolates formed a sub-cluster adjacent to A. suis JCM 18035T. The average nucleotide identity values for the isolates and their most closely related strains were 71.8 and 71.7 %, respectively; and the digital DNA-DNA hybridization values for the isolates and their most closely related strains were 25.6 and 25.5 %, respectively. The main fatty acids were C18 : 1ω9c, C16 : 0 and C18 : 0. The cell wall contained the meso-diaminopimelic acid-based peptidoglycan. The two isolates shared the same metabolic pathways. Isolates YH-aer221T and YH-aer222 harboured the same CRISPR array with 33 and 46 spacers, respectively. Single-genome vs. metagenome analysis showed that the genomes of the isolates were not found in the available metagenome database. Given their chemotaxonomic, phenotypic and phylogenetic properties, YH-aer221T (= KCTC 25571T=JCM 35699T) and YH-aer222 (=KCTC 25573=JCM 35700) represent a novel taxon. The name Aerococcus kribbianus sp. nov. is proposed.

Description of Fusibacillus kribbianus gen. nov., sp. nov., a fusiform anaerobe isolated from pig feces.

OBJECTIVE: The family Lachnospiraceae is affiliated with the order Clostridiales and was originally contained within Clostridial cluster XIVa. The members of Lachnospiraceae inhabiting the gut comprise the chemoorganotrophic genera, generating sundry short-chain fatty acids to supply energy to the host, and are considered to be related to obesity and gut health. METHODS: The polyphasic taxonomic approach was used to characterize the isolate YH-rum2234T. A detailed metabolic analysis was conducted to compare the novel isolate with related strains within the family Lachnospiraceae. RESULTS: A fusiform, obligately anaerobic, Gram-stain-negative bacterium, YH-rum2234T, was isolated from pig feces. Analysis of the 16S rRNA gene sequence revealed that the similarities between the isolate and the familiarly interrelated strain Lientehia hominis KCTC 25345T was 94.3%. The average nucleotide identities and genome-to-genome distances of YH-rum2234T and its closely related strains were below 85.5% and 32.5%, respectively. The G + C content of the genomic DNA was 49.2 mol%. The main fatty acids were C16:0, C14:0, and C14:0 DMA. The major polar lipids were aminophospholipids. The cell wall did not contain the peptidoglycan meso-diaminopimelic acid. CONCLUSION: Given the chemotaxonomic, phenotypic, and phylogenetic properties, YH-rum2234T (=KCTC 25710T = DSMZ 116041T) represents a new genus and species in the family Lachnospiraceae. Fusibacillus kribbianus gen. nov., sp. nov. is the proposed name.

Limosilactobacillus kribbianus sp. nov., isolated from pig faeces.

A rod-shaped, aerotolerant, Gram-stain-positive bacterium isolated from pig faeces was designated as strain YH-lim2214T. Analysis of the 16S rRNA gene sequence revealed that the isolate was most closely related to Limosilactobacillus pontis KCTC 25258T with 98.0 % similarity. The average nucleotide identity and average amino acid identity values between YH-lim2214T and the most closely related strain Lm. pontis KCTC 25258T were 81.4 and 81.3 %, respectively. The major fatty acids were C18 : 1 ω9c, summed feature 7 and C16 : 0. The cell-wall peptidoglycan type was A4α l-Lys-d-Asp. The genomic DNA G+C content was 51.1 mol%. The chemotaxonomic, phenotypic and phylogenetic properties of YH-lim2214T (=KCTC 25572T=JCM 35701T) suggest that it represents a novel taxon, for which the name Limosilactobacillus kribbianus sp. nov. is proposed.

Characteristics of the Salivary Microbiota in Periodontal Diseases and Potential Roles of Individual Bacterial Species To Predict the Severity of Periodontal Disease.

The purposes of this study were to examine the compositional changes in the salivary microbiota according to the severity of periodontal disease and to verify whether the distribution of specific bacterial species in saliva can distinguish the severity of disease. Saliva samples were collected from 8 periodontally healthy controls, 16 patients with gingivitis, 19 patients with moderate periodontitis, and 29 patients with severe periodontitis. The V3 and V4 regions of the 16S rRNA gene in the samples were sequenced, and the levels of 9 bacterial species showing significant differences among the groups by sequencing analysis were identified using quantitative real-time PCR (qPCR). The predictive performance of each bacterial species in distinguishing the severity of disease was evaluated using a receiver operating characteristic curve. Twenty-nine species, including Porphyromonas gingivalis, increased as the severity of disease increased, whereas 6 species, including Rothia denticola, decreased. The relative abundances of P. gingivalis, Tannerella forsythia, Filifactor alocis, and Prevotella intermedia determined by qPCR were significantly different among the groups. The three bacterial species P. gingivalis, T. forsythia, and F. alocis were positively correlated with the sum of the full-mouth probing depth and were moderately accurate at distinguishing the severity of periodontal disease. In conclusion, the salivary microbiota showed gradual compositional changes according to the severity of periodontitis, and the levels of P. gingivalis, T. forsythia, and F. alocis in mouth rinse saliva had the ability to distinguish the severity of periodontal disease. IMPORTANCE Periodontal disease is one of the most widespread medical conditions and the leading cause of tooth loss, imposing high economic costs and an increasing burden worldwide as life expectancy increases. Changes in the subgingival bacterial community during the progression of periodontal disease can affect the entire oral ecosystem, and bacteria in saliva can reflect the degree of bacterial imbalance in the oral cavity. This study explored whether the specific bacterial species in saliva can distinguish the severity of periodontal disease by analyzing the salivary microbiota and suggested P. gingivalis, T. forsythia, and F. alocis as biomarkers for distinguishing the severity of periodontal disease in saliva.

Description and comparative genome analysis of Hallella absiana sp. nov., isolated from pig feces.

OBJECTIVE: The genus Hallella was described within Bacteroidaceae, and then reclassified within Prevotellaceae based on its phenotypic and phylogenetic description. It is associated with degradation of carbohydrate. However, some species of Hallella have pathobiotic properties, and are involved in infections and chronic inflammatory disorders. METHODS: Here, we used a polyphasic taxonomic approach to characterize the two strains: YH-C38T and YH-C4B9b. A detailed metabolic analysis was conducted to compare the two novel isolates with related strains within the genus Hallella. RESULT: Analysis of 16S rRNA gene sequences revealed that the isolates were most closely related to Hallella mizrahii JCM 34422T with 98.5% and 98.6% similarities, respectively. Analysis of the multi-locus species tree based on whole genome sequences of the isolates and related strains revealed that the isolates formed a sub-cluster adjacent to H. mizrahii JCM 34422T. The average nucleotide identity values for YH-C38T and YH-C4B9b, and the most closely related strain H. mizrahii JCM 34422T, were 93.5% and 93.8%, respectively. The main fatty acids were iso C17:0 3OH and anteiso C15:0. The predominant menaquinones were MK-12, MK-11, and MK-13. The cell wall contained the peptidoglycan of meso-diaminopimelic acid. Analysis of comparative metabolic analysis revealed that isolates YH-C38T and YH-C4B9b each contained 155 carbohydrate-active enzymes, and glycoside hydrolase was the largest family. CONCLUSION: Two rod-shaped, obligately anaerobic, Gram-stain-negative bacteria, isolated from pig feces, were designated as strains YH-C38T and YH-C4B9b. Based on the chemotaxonomic, phenotypic, and phylogenetic properties, YH-C38T (=KCTC 25103T = JCM 35423T) and YH-C4B9b (=KCTC 25104 = JCM 35609) represent a novel taxon. The name Hallella absiana sp. nov. is proposed.

Lacticaseibacillus parakribbianus sp. nov., isolated from a pig farm faeces dump.

A lactic acid bacterium isolated from pig faeces was characterized using a polyphasic approach. The strain was Gram-stain-positive, rod-shaped, and facultative anaerobic. Phylogenetic analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the genus Lacticaseibacillus. The multi-locus sequence tree revealed that the strain formed a sub-cluster adjacent to Lacticaseibacillus kribbianus. The main fatty acids were C16 : 0 and C18 : 1ω9c. The average nucleotide identity value, average amino acid identity, and genome-to-genome distance for YH-lacS6T and its most closely related strain, L. kribbianus, were 85.4, 85.2 and 29.2 %, respectively. The G+C content of the genomic DNA was 61.6 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, aminophospholipids and phospholipids. The cell-wall peptidoglycan did not contain meso-diaminopimelic acid. Thus, YH-lacS6T (=KCTC 21186T=JCM 34954T) represents a novel species. The name Lacticaseibacillus parakribbianus sp. nov. is proposed.

Identification of Fusobacterium Species Using Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry by Updating ASTA CoreDB.

PURPOSE: Fusobacterium species can cause infections, and associations with cancer are being increasingly reported. As their clinical significance differs, accurate identification of individual species is important. However, matrix-assisted laser desorption/ionization-time of flight mass spectrometry has not been found to be effective in identifying Fusobacterium species in previous studies. In this study, we aimed to improve the accuracy and efficacy of identifying Fusobacterium species in clinical laboratories. MATERIALS AND METHODS: In total, 229 Fusobacterium isolates were included in this study. All isolates were identified at the species level based on nucleotide sequences of the 16S ribosomal RNA gene and/or DNA-dependent RNA polymerase ß-subunit gene (rpoB). Where necessary, isolates were identified based on whole genome sequences. Among them, 47 isolates were used for updating the ASTA database, and 182 isolates were used for the validation of Fusobacterium spp. identification. RESULTS: Fusobacterium isolates used for validation (182/182) were correctly identified at the genus level, and most (180/182) were correctly identified at the species level using the ASTA MicroIDSys system. Most of the F. nucleatum isolates (74/75) were correctly identified at the subspecies level. CONCLUSION: The updated ASTA MicroIDSys system can identify nine species of Fusobacterium and four subspecies of F. nucleatum in good agreement. This tool can be routinely used in clinical microbiology laboratories to identify Fusobacterium species and serve as a springboard for future research.

Accuracy of ASTA MicroIDSys, a new matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system, for the identification of Korean reference and clinical bacterial and yeast strains.

This study compared the accuracy of a new MALDI-TOF mass spectrometry system, ASTA MicroIDSys system, with that of MALDI Biotyper system for the identification of reference and clinical bacterial and yeast strains. The identification accuracy of the 2 systems was compared using a total of 406 strains comprising 142 aerobic and 180 anaerobic bacterial strains and 84 yeast strains. The genus and species identification rates were 98.0% and 89.4% using MicroIDSys and 96.1% and 89.4% using Biotyper, respectively. The species identification rates of MicroIDSys and Biotyper for aerobic bacteria were 93.0% and 97.2%, respectively, and those for anaerobic bacteria were 85.6% and 81.7%, respectively. The accuracy of yeast identification at the species level was 91.7% using MicroIDSys and 92.9% using Biotyper. These findings indicate that MicroIDSys could be useful for the accurate identification of bacteria and yeast in clinical microbiology laboratories.

Faecalicatena absiana sp. nov., an obligately anaerobic bacterium from a pig farm faeces dump.

An obligately anaerobic, Gram-stain-positive, spore-forming, short-rod-shaped bacterium, designated strain YH-C36aT, was isolated from a pig farm faeces dump. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Faecalicatena and is most closely related to Faecalicatena contorta KCTC 5831T, Faecalicatena fissicatena KCTC 15010T and Faecalicatena orotica KCTC 15331T, with 96.3, 96.2, and 96.0 % sequence similarity, respectively. The average nucleotide identity values for strain YH-C36aT and the closest related strains were lower than 72 %. The G+C content of the isolate was 43.0 mol%. The cell-wall peptidoglycan was A1γ type and contained meso-diaminopimelic acid. The predominant fatty acids were C16 : 0, C18 : 1 cis 9, C16 : 0 DMA, C18 : 0 DMA and C18:0. The major end products of glucose fermentation were lactate, formate and acetate. Based on its phenotypic, phylogenetic and chemotaxonomic properties, a novel species, named Faecalicatena absiana sp. nov., is proposed for strain YH-C36aT (=KCTC 25106T=NBRC 114768T).

Lacticaseibacillus kribbianus sp. nov., isolated from pig farm faeces dump.

A lactic acid bacteria isolated from pig faeces was characterized using a polyphasic approach. Cells of the strain were Gram-stain-positive, rod-shaped and facultative anaerobic. Phylogenetic analysis of 16S rRNA gene sequence indicated that the isolate belonged to the genus Lacticaseibacillus; however, the similarity to other homologues within the genus was <98 %. Analysis of housekeeping gene sequences (pheS and recA) revealed that the strain formed a sub-cluster adjacent to Lacticaseibacillus absianus and Lacticaseibacillus daqingensis. The main fatty acids of the strain is the C18 : 1ω9c and C16 : 0. The G+C content of the genomic DNA was 62.8 mol %. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, aminophospholipids and phospholipids. The cell-wall peptidoglycan did not contain meso-diaminopimelic acid. Thus, YH-lac21T (=KCTC 21185=JCM 34953) represents a novel species. The name Lacticaseibacillus kribbianus sp. nov. is proposed.

Performance assessment of ASTA MicroIDSys, a new matrix assisted laser desorption ionization-time of flight mass spectrometry system, for identification of viridans group streptococci.

The performance of the ASTA MicroIDSys system (ASTA), a new matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system, was evaluated for the identification of viridans group streptococci (VGS) and compared with the results obtained with the Bruker Biotyper system (Bruker Daltonics). A total of 106 Streptococcus reference strains belonging to 24 species from the bacterial strain bank was analyzed using the two MALDI-TOF MS systems. Of the 106 reference strains tested, ASTA MicroIDSys and Bruker Biotyper correctly identified 84.9% and 81.1% at the species level, 100% and 97.2% at the group level and 100% and 98.1% at the genus level, respectively. The difference between the two systems was not statistically significant (P = 0.289). Out of 24 species, 13 species were accurately identified to the species level with 100% accurate identification rates with both systems. The accurate identification rates at the species level of ASTA MicroIDSys and Bruker Biotyper were 100% and 87.5% for the S. anginosus group; 78.4% and 73.5% for the S. mitis group; 91.7% and 91.7% for the S. mutans group; and 100% and 100% for the S. salivarius group, respectively. The ASTA MicroIDSys showed an identification performance equivalent to that of the Bruker Biotyper for VGS. Therefore, it would be useful for the identification of VGS strains in clinical microbiology laboratories.

Evaluation of antibacterial activity against Candida albicans according to the dosage of various denture cleansers.

PURPOSE: The purpose of this study is to compare the antibacterial activity of currently purchasable denture cleansers against Candida albicans. MATERIALS AND METHODS: This study used tablet-type denture cleansers, Polident®, Coolingdent® and Fittydent®, along with liquid denture cleansers, Hexamedine®, Listerine® and Apple vinegar®. The antibacterial activities of denture cleansers were evaluated based on the number of C. albicans and concentrations of the denture cleansers. RESULTS: In the 0.5 × 106 cfu/ml culture medium, the C. albicans' death rate of Polident® was significantly lower than those of Fittydent®, Hexamedine®, Listerine®, and Apple vinegar®(P<.05). In the 0.5 × 107 cfu/, the C. albicans' death rates of Polident® and Coolingdent® were significantly lower than those of Fittydent®, Hexamedine®, Listerine® and Apple vinegar®(P<.05). The C. albicans' death rates of Polident® and Coolingdent® were significantly decreased at 0.02 g and 0.01 g. The C. albicans' death rate of Fittydent® was significantly decreased at 0.005 g (P<.05). The C. albicans ' death rate of Hexamedine® was significantly decreased at 1/16 dilution. The C. albicans' death rate of Listerine® was decreased at 1/8 dilution, and the antibacterial activity of Apple vinegar® was decreased at 1/4 dilution (P<.05). CONCLUSION: As the number of C. albicans increased, the antibacterial activities of the denture cleansers decrease. In the tablet-type denture cleanser, all denture cleansers showed 100% C. albicans' death rate when used at a dose of 1 tablet. One denture cleanser showed the same antibacterial effect with only 1/3 of a tablet. In the liquid type denture cleanser, the level of dilution required was different for each denture cleanser.

Lipopolysaccharides of Fusobacterium nucleatum and Porphyromonas gingivalis increase RANKL-expressing neutrophils in air pouches of mice.

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn + Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.

In-house method for direct bacterial identification in positive blood culture broths using microfiltration, bead beating, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Rapid identification of bacterial pathogens facilitates earlier optimization of antibiotic treatment and reduces morbidity and mortality in sepsis patients. The aim of this research was to design an in-house chemical-free method for direct bacterial identification in positive blood culture (BC) broths and to compare the performance of this method with that of the commercial Sepsityper® kit. The overall species identification rates for the in-house and Sepsityper methods were 88.4% and 85.8%, respectively (n = 190). Among 146 facultative anaerobes, 92.5% and 95.9% were identified to the species level using the in-house and Sepsityper methods, respectively. For 32 anaerobic bacteria, the in-house method showed a higher species identification rate (75.0%) than the Sepsityper method (53.1%). The in-house method correctly identified more Bacteroides species (100.0%) than the Sepsityper method (18.2%). Our novel in-house method and the Sepsityper method showed a high accuracy for direct bacterial identification in positive BC broths using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Correction to: Genome-Based Reclassification of Fusobacterium nucleatum Subspecies at the Species Level.

The original version of this article contained errors in the description of novel species. These errors are corrected with this corrigendum.

Anti-inflammatory and in vitro bone formation effects of Garcinia mangostana L. and propolis extracts.

The purpose of this study was to determine the anti-inflammatory and in vitro bone formation effects of Garcinia mangostana L. (mangosteen) and propolis extracts. Immortalized human gingival fibroblasts (hTERT-hNOF) cells were treated with Porphyromonas gingivalis KCOM 2804 lipopolysaccharide followed by treatment with mangosteen and propolis extract alone or in combination. Expression levels of inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay. Effect of mangosteen and/or propolis extracts on mineralization of MG-63 cells was evaluated by alkaline phosphatase activity and alizarin red S staining. Group mangosteen extract complex 1:34 (1 µg/ml mangosteen extract and 34 µg/ml propolis extract) significantly reduced expression levels of IL-6, IL-8, and PGE2. It had higher than other groups in vitro bone formation effect on MG63 cells. These results suggest that mangosteen and propolis extract complex could be used in the prevention and treatment of periodontal disease.

Osmunda japonica Extract Suppresses Pro-Inflammatory Cytokines by Downregulating NF-κB Activation in Periodontal Ligament Fibroblasts Infected with Oral Pathogenic Bacteria.

Periodontal diseases are caused by bacterial infection and may progress to chronic dental disease; severe inflammation may result in bone loss. Therefore, it is necessary to prevent bacterial infection or control inflammation. Periodontal ligament fibroblasts (PDLFs) are responsible for the maintenance of tissue integrity and immune and inflammatory events in periodontal diseases. The formation of bacterial complexes by Fusobacterium nucleatum and Porphyromonas gingivalis is crucial in the pathogenesis of periodontal disease. F. nucleatum is a facultative anaerobic species, considered to be a key mediator of dental plaque maturation and aggregation of other oral bacteria. P. gingivalis is an obligate anaerobic species that induces gingival inflammation by secreting virulence factors. In this study, we investigated whether Osmunda japonica extract exerted anti-inflammatory effects in primary PDLFs stimulated by oral pathogens. PDLFs were stimulated with F. nucleatum or P. gingivalis. We showed that pro-inflammatory cytokine (IL-6 and IL-8) expression was induced by LPS or bacterial infection but decreased by treatment with O. japonica extract following bacterial infection. We found that the activation of NF-κB, a transcription factor for pro-inflammatory cytokines, was modulated by O. japonica extract. Thus, O. japonica extract has immunomodulatory activity that can be harnessed to control inflammation.