(51) Nisin and Lactococcus lactis Disrupt Peri-Implantitis Biofilms on Titanium Discs

Introduction: Peri-implantitis is characterized by a chronic inflammation of the peri-implant mucosa that progressively and irreversibly damages the implant supporting tissues (Smeets et al. 2014). A systematic review including 29 reports found similar pathogens between peri-implantitis and periodontitis lesions, including T. denticola, P. gingivalis, and F. nucleatum (Mombelli and Décaillet 2011), indicating that both diseases may be related.
One new strategy to treat perio disease is the use of bacteriocins and probiotics to suppress oral pathogens and re-establish a healthy microbiome composition (Radaic and Kapila 2021). Studies led by our group demonstrate that nisin and the nisin-producing Lactococcus lactis dose-dependently abrogate the growth of pathogens associated with periodontal disease in their planktonic state, and dose-dependely modulate pathogenic human-derived oral biofilms towards health in vitro without inducing cytotoxicity to human oral cells (Shin et al. 2015) The objective of this study was to test whether nisin and its probiotic form (nisin-producing Lactococcus lactis) can disrupt peri-implantitis pathogenic oral biofilms and promote healthier implant biofilms by decreasing the levels of pathogens within them.

Methods: 10 volunteers with no known health issues consented to donate 5mL saliva each for this study.
T. denticola was cultured in Oral Treponeme Enrichment Broth (Anaerobe Systems, USA), while P. gingivalis and F. nucleatum (ATCC 25586) were cultured in Brain-Heart Infusion broth under anaerobic conditions and split every 3-7 days.

Human saliva-derived oral biofilms were grown by adding 20µL of CCS to 600µL of CFS per well in 24-well plates containing one 13mm diameter by 1mm thick Ti disc Laser-Lok® surface (Biohorizons, USA). The plates were then incubated under aerobic conditions for 48h at 37oC in a humidified incubator

The oral biofilms were challenged with either the nisin-producing L. lactis, non-nisin-producer L. lactis strains (both at 6x103 and 105 CFU/mL) or nisin (1 or 10µg/mL) for 24h. Biofilms were fixed and stained for 15min using LIVE/DEAD BacLight Bacterial Viability Kit, mounted on slides and evaluated using a Confocal Microscope (Leica, Germany).

16S rDNA and Metagenomics Sequencing performed

Results: Innitially, oral biofilms were treated with either nisin or the nisin-producing L. lactis for 24h and the results can be seen in Figure 1. The results show that both nisin and L. lactis disrupted the oral biofilms and significantly decreased their viablity dose dependently, compared to control biofilms.
Next, we spiked the biofilms with known peri-implantitis pathogens and treated them with nisin and L. lactis. The results can be seen in Figure 2. Interestingly, spiking the biofilms with pathogens significantly decreased the overall viability of the biofilms. Similar to the previous results, both nisin and the nisin-producing L. lactis disrupted the pathogenic biofilms and significantly decreased the viability of the pathogenic biofilms dose-dependently.

Conclusions: Nisin and nisin-producing probiotic L. lactis can disrupt biofilms and reduce the viability of both control and pathogenic oral biofilms grown on Grade V titanium discs. Therefore, both of these agents may be useful in promoting healthier oral biofilms in a peri-implantitis setting.

Lead Author’s Contributions to the Research Project: The lead author was responsible for data collection, imaging, data analysis and contributed to authoring the manuscript.