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P gingivalis

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P. gingivalis is a laboratory strain of bacteria commonly used in research. It is a Gram-negative, anaerobic bacterium that is known to be associated with periodontal disease. The strain is used in scientific investigations, but a detailed description of its core function is not available while maintaining an unbiased and factual approach.

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Lab products found in correlation

Hemin, by Merck Group (2 mentions) P gingivalis, by American Type Culture Collection (2 mentions) Menadione, by Merck Group (1 mentions) Brain heart infusion broth, by Merck Group (1 mentions) T forsythia, by American Type Culture Collection (1 mentions) N acetylmuramic acid, by Merck Group (1 mentions) F nucleatum, by Merck Group (1 mentions) T denticola, by American Type Culture Collection (1 mentions) As 580, by Anaerobe Systems (1 mentions) Mtge anaerobic enrichment broth, by Anaerobe Systems (1 mentions) Vitamin k, by Merck Group (1 mentions)

5 protocols using p gingivalis

1

Cultivation and Inactivation of P. gingivalis

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P. gingivalis, strain 33277, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cultivation of P. gingivalis was performed under strict anaerobic conditions (85% N2, 5% H2 and 10% CO2) at 37 °C in the Brain-Heart-Infusion (BHI) broth supplemented with menadione (1 μg/mL) and hemin (5 µg/mL), both purchased from Sigma–Aldrich. After harvest, P. gingivalis colonies were rinsed twice with PBS and resuspended in 1mL of PBS. The concentration of P. gingivalis was determined with a spectrophotometer at 600 nm by McFarland turbidity standard No. 0.5, where 0.1 OD refers to 1 × 108 colony-forming units per mL (CFU/mL). To ensure the loss of viability of P. gingivalis prior to GMSCs treatment, P. gingivalis was being heat-inactivated by incubation at 70 °C for 30 min in addition to atmospheric oxygen exposure.
Bekić M., Radanović M., Đokić J., Tomić S., Eraković M., Radojević D., Duka M., Marković D., Marković M., Ismaili B., Bokonjić D, & Čolić M. (2022). Mesenchymal Stromal Cells from Healthy and Inflamed Human Gingiva Respond Differently to Porphyromonas gingivalis. International Journal of Molecular Sciences, 23(7), 3510.
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2

Anaerobic Cultivation of Oral Bacteria

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Bacterial strains and plasmids used in this study are shown in Table 1. A. actinomycetemcomitans (ATCC 43718), F. nucleatum subsp. nucleatum (ATCC 25586), P. gingivalis (ATCC 33277), T. forsythia (ATCC 43037), and T. denticola (ATCC 33521) were obtained from American Type Culture Collection (Manassas, VA, USA). Bacterial cultures were grown under anaerobic growth conditions at 37 °C using an anaerobic growth chamber AS‐580 (Anaerobe Systems, Morgan Hill, CA, USA) supplied with anaerobic gas (90% N2, 5% H2, and 5% CO2). F. nucleatum and P. gingivalis were cultured in brain heart infusion, T. forsythia was cultured using brain heart infusion supplemented with 2.5 mM N‐acetylmuramic acid (Sigma, St. Louis, MO, USA). A. actinomycetemcomitans and T. denticola were cultured in MTGE‐anaerobic enrichment broth (Anaerobe Systems).
Coffey J., Choudhry M., Shlossman M., Makin I.R, & Singh V.K. (2016). Multiplex real‐time PCR detection and relative quantification of periodontal pathogens. Clinical and Experimental Dental Research, 2(3), 185-192.
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3

Cultivation of Oral Bacterial Biofilms

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The use of all bacterial species was approved by University of Maryland Baltimore Institutional Review Board. All four species were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA): S. gordonii ATCC10558; A. naeslundii ATCC12104; P. gingivalis ATCC33277; F. nucleatum ATCC25586.
S. gordonii were grown in brain-heart infusion broth (BHI, Sigma-Aldrich) at 37 °C aerobically (95% air, 5% CO2), following ACTT’s instructions. A. naeslundii, P. gingivalis and F. nucleatum were grown in tryptic soy broth (TSB, Sigma-Aldrich) supplemented with yeast extract (5 g/L), L-cysteine hydrochloride (0.5 g/L), hemin (5 mg/L) and menadione (1 mg/L) at 37°C anaerobically (90% N2, 5% CO2, 5% H2), following previous studies.11 (link),49 (link) For each species, the inoculum was adjusted to 108 colony-forming unit counts CFU/mL for biofilm formation, based on the standard curve of OD600nm vs. CFU/mL for each species.49 (link)
Wang L., Li C., Weir M.D., Zhang K., Zhou Y., Xu H.H, & Reynolds M.A. (2017). Novel multifunctional dental bonding agent for Class-V restorations to inhibit periodontal biofilms. RSC advances, 7(46), 29004-29014.
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4

Culturing Oral Cavity Bacteria

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The major bacteria used in this experiment are the bacteria associated with oral malodor, P. gingivalis ATCC 33277 and T. denticola ATCC 35405, which were purchased from American Type Culture Collection (ATCC). P. gingivalis was cultured in brain heart infusion (BHI) liquid media, which included hemin (0.05 μg/mL; Sigma, St Louis, MO) and Vitamin K (1 μg/mL; Sigma). T. denticola was cultured in tryptone‐yeast extract‐gelatin‐volatile fatty acids‐serum (TYGVS) media in a 37°C anaerobic state (5% H2, 10% CO2, 85% N2) (Ohta, Makinen, & Loesche, 1986). S. salivarius K12 and M18 (Thera Breth; The California Clinics, Los Angeles, CA) were used as probiotics and cultured in BHI media at 37°C. For the coculture of oral cavity bacteria, P. gingivalis was cultured in BHI media containing hemin and Vitamin K. T. denticola was cultured in a mixed media consisting of TYGVS liquid media and BHI liquid media in a one‐to‐one ratio at 37°C in an anaerobic condition.
Yoo H., Jwa S., Kim D, & Ji Y. (2019). Inhibitory effect of Streptococcus salivarius K12 and M18 on halitosis in vitro. Clinical and Experimental Dental Research, 6(2), 207-214.
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5

Inflammatory Gene Expression in hPDLCs

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hPDLCs were treated with 1 μg/ml lipopolysaccharide (LPS) produced from P. gingivalis (Sigma, USA) for 24 h [23 (link)] with or without SB (50 μM, 24 h) preincubation. After treatment, qPCR was used to investigate certain gene expression. The sequences of specific primers are listed in Table 2.
Li X., Zhou R., Han Y., Zeng J., Shi L., Mao Y., Sun X., Ji Y., Zhang X., Chen Y., Jaspers R.T., Wu G., Huang S, & Forouzanfar T. (2023). Silibinin Attenuates Experimental Periodontitis by Downregulation of Inflammation and Oxidative Stress. Oxidative Medicine and Cellular Longevity, 2023, 5617800.
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