Antimicrobial nisin acts against saliva derived multi-species biofilms without cytotoxicity to human oral cells

Abstract

Objectives: Nisin is a lantibiotic widely used for the preservation of food and beverages. Recently, investigators have reported that nisin may have clinical applications for treating bacterial infections. The aim of this study was to investigate the effects of ultra pure food grade Nisin ZP (>95% purity) on taxonomically diverse bacteria common to the human oral cavity and saliva derived multi-species oral biofilms, and to discern the toxicity of nisin against human cells relevant to the oral cavity.

Methods: The minimum inhibitory concentrations and minimum bactericidal concentrations of taxonomically distinct oral bacteria were determined using agar and broth dilution methods. To assess the effects of nisin on biofilms, two model systems were utilized: a static and a controlled flow microfluidic system. Biofilms were inoculated with pooled human saliva and fed filter-sterilized saliva for 20-22 h at 37°C. Nisin effects on cellular apoptosis and proliferation were evaluated using acridine orange/ethidium bromide fluorescent nuclear staining and lactate dehydrogenase activity assays.

Results: Nisin inhibited planktonic growth of oral bacteria at low concentrations (2.5-50 μg/ml). Nisin also retarded development of multi-species biofilms at concentrations ≥1 μg/ml. Specifically, under biofilm model conditions, nisin interfered with biofilm development and reduced biofilm biomass and thickness in a dose-dependent manner. The treatment of pre-formed biofilms with nisin resulted in dose- and time-dependent disruption of the biofilm architecture along with decreased bacterial viability. Human cells relevant to the oral cavity were unaffected by the treatment of nisin at anti-biofilm concentrations and showed no signs of apoptotic changes unless treated with much higher concentrations (>200 μg/ml).

Conclusion: This work highlights the potential therapeutic value of high purity food grade nisin to inhibit the growth of oral bacteria and the development of biofilms relevant to oral diseases.

Keywords: apoptosis; confocal; dental plaque; human oral cells; nisin; oral diseases; saliva derived multi-species biofilms.

FIGURE 1

Nisin inhibits the growth of cariogenic and periodontal pathogens. Using a broth dilution method, S. gordonii DL1, S. oralis SO34, S. mutans UA159, A. odontolyticus ATCC 17982, S. mutans ATCC 25175, F. nucleatum ATCC 25586, A. actinomycetemcomitans Y4, P. gingivalis W83, P. gingivalis ATCC 33277, P. intermedia clinical isolate and T. denticola ATCC 35405 was cultured with or without nisin (0.1–200 μg/ml) on a microplate for 24 h at 37°C, under aerobic or anaerobic conditions. The determined MIC was the lowest concentration of nisin that inhibited the visible growth (≤0.05 increase in OD₆₀₀ after 24 h growth) of the inoculated bacteria. ^∗P < 0.05: significant differences from the control (nisin-free).

FIGURE 2

Nisin inhibits the formation of multi-species biofilms in a static model system. Cell-containing saliva (CCS) was inoculated in filter sterilized cell-free saliva (CFS) for 20–22 h at 37°C with or without nisin. (A) Confocal microscopy images are represented in the x–y plane. A green signal indicates viable live cells (Syto 9), a red signal indicates damaged/dead cells (propidium iodide), (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate wells (experiments), (C) DNA content of the biofilms was quantified by absorption spectroscopy at fluorescence intensity of 530 nm. ^∗P < 0.05 and ^∗∗P < 0.01: significant differences from the control (nisin-free).

FIGURE 3

Nisin inhibits the formation of multi-species biofilms in a Bioflux controlled flow microfluidic model system. CCS was added, then fed filter sterilized CFS for 20–22 h at 37°C with or without nisin. (A) Confocal microscopy images are represented in the x–y plane. A green signal indicates viable live cells (Syto 9) and a red signal indicates damaged/dead cells (propidium iodide). (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate channels (experiments). ^∗P < 0.05 and ^∗∗P < 0.01: significant differences from the control (nisin-free).

FIGURE 4

Nisin disrupts the maintenance of three-dimensional architecture of pre-formed biofilms. CCS was inoculated in filter sterilized CFS for 20–22 h at 37°C and treated with phosphate buffered saline solution (PBS) solution (control) or nisin at different concentrations and incubation times. (A) Confocal microscopy images are represented in the x–y and x–y–z plane. A green signal indicates viable live cells (Syto 9) and a red signal indicates damaged/dead cells (propidium iodide). (B) Biofilm biomass, thickness, and roughness [mean (SD)] were derived from imaging of at least three separate wells (experiments). (C) An average percentage signal from the biofilms was determined by the Live/viable signal (green) and the Dead/damaged signal (red) in relation to the total signal captured for both. ^∗P < 0.05 and ^∗∗P < 0.01: significant differences from the control (nisin-free).

FIGURE 5

Nisin has minimal cytotoxicity to human cells relevant to the oral cavity. Primary human gingival fibroblast (GF) cells, periodontal ligament (PDL) cells, oral keratinocyte (OK) cells, and osteoblast-like cells were incubated with nisin (1–800 μg/ml) for 24–48 h on a 96-well microplate at 37°C. Cells were then stained with acridine-orange/ethidium-bromide (AO/EB) to evaluate cell viability, apoptosis and necrosis using epifluorescence microscopy. (A) Images of the cells after 48 h incubation period with nisin at different concentrations. (B) The cytotoxicity of nisin was quantified by counting viable, apoptotic and necrotic cells and expressed as bar graphs with heights representing mean % and error bars representing SD. AO (green) stained cells with intact membrane integrity. Early apoptotic cells stained green but contained bright green dots in the nuclei due to chromatin condensation and nuclear fragmentation. Late apoptotic cells stained with EB (orange) with apoptotic phenotypes. Necrotic cells stained orange but the nuclear morphology resembled the viable cells with absence of chromatin condensations. Mean values were calculated with SD. ^∗,#,$ represent P < 0.05: significant differences from the control (nisin free) for viability, apoptosis and necrosis, respectively.

FIGURE 6

Nisin does not effect cell proliferation of human cells. The effect of nisin on cell proliferation was assessed using a Cell Counting Kit-8 measuring the lactate dehydrogenase (LDH) activities in cells. Using a 96-well microplate, GFs, PDL cells, OKs, and osteoblast-like cells were plated at 2 × 10⁴ cells/cm² and incubated for 24–48 h in the presence or absence of nisin (1–800 μg/ml). At 24 and 48 h time points, LDH levels were measured using absorption spectroscopy at 450 nm. Mean values were calculated with SD. ^∗P < 0.05: significant differences from the control (nisin-free).