Fusobacterium nucleatum (Fn) (ATCC-25586) was obtained from ATCC. Fn was cultured anaerobically at 37 °C for 48 h in the chopped cooked meat media (Anaerobic Systems, Morgan Hill, CA, USA) inside an anaerobic chamber containing an anaerobic gas kit generating system (MGC, AnaeroPACK System, New York, NY, USA). Colon enteroid-derived monolayer (EDM) was infected with (Fn) at a multiplicity of infection (moi) of 100 for 24 h. Escherichia coli K12 strain DH10B (ATCC-PTA5105) was cultured on Luria Broth (LB) agar and LB broth and used to infect EDM at a moi of 100 for 24 h. Adherent-invasive Escherichia coli strain LF82 (AIEC-LF82), isolated from the specimens of Crohn’s disease patients, was obtained from Arlette Darfeuille-Michaud (Clermont-Ferrand, France) and was cultured as previously described [45 (link)]. EDM was infected with LF82 at a moi of 100 for 24 h. H. pylori strain (ATCC-26695) was incubated with the Brucella broth supplemented with 10% fetal bovine serum (FBS) in a shaker at 37 °C in 10% CO2. Colon EDMs were infected with H. pylori at moi 100 for 24 h.
Fusobacterium nucleatum
Manufactured by American Type Culture Collection
Sourced in United States
Fusobacterium nucleatum is a fastidious, anaerobic, Gram-negative bacterium. It is a common inhabitant of the human oral cavity and is associated with periodontal diseases. This product can be used for research purposes, such as studying the biology and pathogenesis of this organism.
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Lab products found in correlation
Porphyromonas gingivalis, by American Type Culture Collection (4 mentions)
Staphylococcus aureus, by American Type Culture Collection (3 mentions)
Streptococcus mutans, by American Type Culture Collection (2 mentions)
Veillonella parvula, by American Type Culture Collection (2 mentions)
Enterococcus faecalis, by American Type Culture Collection (2 mentions)
Escherichia coli, by American Type Culture Collection (2 mentions)
Aggregatibacter actinomycetemcomitans, by American Type Culture Collection (1 mentions)
Sheep blood, by BD (1 mentions)
Vitamin k1, by BD (1 mentions)
Schaedler agar plates, by BD (1 mentions)
Streptococcus oralis, by American Type Culture Collection (1 mentions)
Bacitracin, by Merck Group (1 mentions)
Lactobacillus casei, by American Type Culture Collection (1 mentions)
Pseudomonas aeruginosa schroeter migula, by American Type Culture Collection (1 mentions)
Haemophilus influenzae, by American Type Culture Collection (1 mentions)
Streptococcus pneumoniae, by American Type Culture Collection (1 mentions)
Neisseria meningitidis, by American Type Culture Collection (1 mentions)
Streptococcus mitis, by American Type Culture Collection (1 mentions)
Crystal violet stain, by Merck Group (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using fusobacterium nucleatum
1
Bacterial Infection of Colon Enteroids
2
Pathogenic Bacteria Interactions with Colon Enteroids
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Fusobacterium nucleatum (Fn) (ATCC-25586) was obtained from ATCC. Fn was cultured anaerobically at 37 0 C for 48 hours in the chopped cooked meat media (Anaerobic Systems, Morgan Hill, CA), inside an anaerobic chamber containing anaerobic gas kit generating system (MGC, AnaeroPACK System, Japan). Colon enteroid derived monolayer (EDM) was infected with (Fn) at a multiplicity of infection (moi) of 100. Escherichia coli K12 strain DH10B, (ATCC-PTA¬5105), was cultured on L.B. agar and L.B. broth and used to infect EDM at moi of 100.
Adherent-invasive Escherichia coli strain LF82 (AIEC-LF82), isolated from the specimens of Crohn's disease patient, was obtained from Arlette Darfeuille-Michaud and was cultured as previously described (36) . EDM was infected with LF82 at moi of 100. H. pylori strain (ATCC-26695) was incubated with the Brucella Broth supplemented with 10% FBS in a shaker at 37°C in 10% CO2. Colon EDMs were infected with the H. pylori at moi 100 for 24 h and 48 h.
Adherent-invasive Escherichia coli strain LF82 (AIEC-LF82), isolated from the specimens of Crohn's disease patient, was obtained from Arlette Darfeuille-Michaud and was cultured as previously described (36) . EDM was infected with LF82 at moi of 100. H. pylori strain (ATCC-26695) was incubated with the Brucella Broth supplemented with 10% FBS in a shaker at 37°C in 10% CO2. Colon EDMs were infected with the H. pylori at moi 100 for 24 h and 48 h.
3
Constructing Bacterial Mock Communities
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Bacterial isolates were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Glycerol stocks were prepared upon arrival as recommended by ATCC. Pseudomonas aeruginosa str. PAO1, Burkholderia multivorans (ATCC 17616), Stenotrophomonas maltophila (ATCC 13637), Staphylococcus aureus (ATCC 12600), Fusobacterium nucleatum (ATCC 23726), Streptococcus anginosus (ATCC 33397), Bacillus halodurans (ATCC BAA-125), and Gemella haemolysans (ATCC 10379) were grown (aerobically or anaerobically) overnight in Tryptic-Soy broth (TSB) at 37 °C and subsequently quantified on Tryptic-Soy agar plates. Culture broths were pooled to a density of ~ 8E+ 09 16S copies/ml. A series of seven tenfold dilutions was prepared resulting in sample densities ranging from 8E+ 09 to 8E+ 02 16S copies/ml. Three mock communities were prepared, the first being composed of an equimolar ratio of P. aeruginosa, B. multivorans, and S. maltophilia. The second community was composed of P. aeruginosa and B. multivorans, each accounting for 50% of the community composition. The third community was composed of S. aureus (55%), F. nucleatum (24%), B. halodurans (15%), S. anginosus (5.5%), and G. haemolysans (0.5%).
4
Cultivation of Oral Pathogenic Bacteria
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Oral pathogenic bacteria, viz. Streptococcus mutans (ATCC 35668), Streptococcus sabrinus (ATCC 33478), Fusobacterium nucleatum (ATCC 25586), Aggregatibacter actinomycetemcomitans (ATCC 43718), Enterococccus faecalis (ATCC 29212) and P. gingivalis (ATCC 33277) were obtained from the archival collection at the Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, and used for the experiments. These species were inoculated on horse blood agar plates and incubated in an anaerobic chamber with 5% CO2, 10% H2 and 85% of N2 at 37°C for two days. The bacterial cultures were harvested and suspended in phosphate-buffered saline (PBS) for subsequent microbiological experiments.
5
Antimicrobial Properties of Dental Sealers
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Five bacterial species were used for testing the sealers’ antimicrobial properties: the obligate anaerobes Fusobacterium nucleatum (ATCC 49256), Prevotella intermedia (ATCC 25611), Parvimonas micra (ATCC 33270), and Veillonella parvula (ATCC 17745) as well as the facultative anaerobe Streptococcus oralis (ATCC 35037). All strains were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and grown on Schaedler agar plates supplemented with vitamin K1 and 5% sheep blood (Becton Dickinson, Franklin Lakes, NJ, USA). Cultivation in liquid medium was performed in anaerobic brain–heart-infusion broth (BHI, Becton Dickinson) supplemented with hemin (5 µg/ml) and vitamin K1 (1 µg/ml). The bacteria were incubated at 37 °C in an anaerobic atmosphere (5% H2, 10% CO2, 85% N2).
6
Cultivation of Oral Bacterial Strains
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The following standard strains were used: Enterococcus faecalis (ATCC 51299), Actinomyces israelii (ATCC 12102), Lactobacillus casei (IAL#523), Streptococcus mutans (ATCC 25175), and Fusobacterium nucleatum (NCTC 11326) provided by Oswaldo Cruz Foundation—FIOCRUZ, Rio de Janeiro, Brazil. Bacterial suspensions were prepared as described by Caiaffa et al. [29 (link)] from cultures grown in Brain Heart infusion Agar—BHIA (Difco Laboratories, Kansas City, MO, USA) for E. faecalis and A. israelii, MRS Rogosa (Difco) for L. casei, Mitis Salivarius Agar (Difco) with 0.2 U/mL bacitracin (Sigma-Aldrich) for S. mutans and BHI blood agar (Difco) containing 5 mg/mL of hemin, 5 mg/mL of menadione, 0.5% yeast extract powder (YE, Difco), and 5% defibrinated sheep blood for F. nucleatum. All bacteria were grown in an incubator at 37 °C and 5% CO2, except F. nucleatum which grown in an anaerobic chamber (AnaeroGen, Oxoid, Thermo Scientific, Waltham, MA, USA).
7
Cultivation of Oral Bacterial Strains
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Streptococcus mutans Clarke (strain designation NCTC 10449), Pseudomonas aeruginosa (Schroeter) Migula (strain designation PAO1), Fusobacterium nucleatum, subspecies nucleatum (strain VPI 4355), and Porphyromonas gingivalis (strain 2561) were purchased from ATCC (LGC Standards Srl, Milan, Italy). Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis were obtained from clinical isolates and characterized by morphological, biochemical, and molecular typing. S. mutans, S. mitis, S. oralis, and S. sanguinis were maintained in Brain Heart Infusion (BHI) broth or agar under microaerophilic conditions. P. aeruginosa was maintained in trypticase soy (TS) agar or broth. F. nucleatum was maintained in TS agar or broth with defibrinated sheep blood under anaerobic conditions. P. gingivalis was maintained in ATCC Medium 2722 supplemented with TS broth or agar under anaerobic conditions. Before the experiments, bacterial cultures were inoculated in fresh media (dilution 1:100) and grown for 16 h at 37 °C.
8
Culturing Diverse Bacterial Species
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Bacteria, including Azoarcus oleivorans (ATCC2411), Aeromicrobium fastidiosum (ATCC12713), Acidovorax avenae (12530), Capnocytophaga canimorsus (ATCC35979), Fusobacterium nucleatum (ATCC25586), Haemophilus influenzae (ATCC51907), Neisseria meningitidis (ATCC13077), Streptococcus pneumoniae (ATCC6303), Selenomonas noxia (ATCC43541), and Veillonella parvula (ATCC 17742), were purchased from American Type Culture Collection (ATCC, Gaithersburg, MD, USA) and cultured as previously described [4 (link),12 (link)].
9
Cultivation of Oral Pathogenic Microbes
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The following oral pathogens were studied: Candida albicans CBS 562 from “Centraalbureau voor Schimmelcultures” and bacteria Streptococcus sanguis ATCC 10556, Streptococcus mitis ATCC 903, Porphyromonas gingivalis ATCC 33277 and Fusobacterium nucleatum ATCC 25586 from American Type Culture Collection. The microorganisms were stored at −70°C in Sabouraud Dextrose Broth (SDB, Merck® - C. albicans) and Mueller-Hinton Broth (Difco® - bacteria) with 15% glycerol. It was considered the oxygen exigencies of each microorganism (C. albicans - aerobiosis, S. mitis and S. sanguis microaerophilie and F. nucleatum and P. gingivalis anaerobiosis) to choose bacteria growth conditions.
10
Nisin and Oral Bacterial Interactions
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NisinZ®P was purchased from Handary S.A. (Belgium). Nisin-producing L. lactis sp. lactis (Cat# 11454), Treponemadenticola (ATCC 35405), Porphyromonas gingivalis (ATCC 33277), and Fusobacterium nucleatum (ATCC 25586) were purchased from ATCC. The non-nisin producing L. lactis was a gift from Paul Cotter, from the Cork Institute of Technology. Brain Heart Infusion (BHI) media, a crystal violet stain, and all other general chemicals were purchased from Sigma-Aldrich (IL). The LIVE/DEAD BacLight Bacterial Viability Kit and Blocking buffer were purchased from ThermoFisher Scientific (USA). The 16S sequencing Explorer Kit was obtained from uBiome (USA).
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