Iron oxide nanozymes stabilize stannous fluoride for targeted biofilm killing and synergistic oral disease prevention

In vitro biofilm model and quantitative analysis

Biofilms were formed using the saliva-coated hydroxyapatite disc (sHA) model as described elsewhere18,33,46. Streptococcus mutans (S. mutans) UA159 (ATCC 700610), a proven virulent and well-characterized cariogenic pathogen, was grown in ultra-filtered (10 kDa, cutoff; Millipore, Billerica, MA) tryptone-yeast extract (UFTYE) broth at 37 °C and 5% CO2 to mid-exponential phase. Briefly, HA discs (surface area of 2.7 ± 0.2 cm2; Clarkson Chromatography Inc., South Williamsport, PA) were vertically suspended in 24-well plates using a custom-made wire disc holder and coated with filter-sterilized human saliva for 1 h at 37 °C. Each sHA disc was inoculated with ~2 × 105 CFU of S. mutans per ml in UFTYE containing 1% sucrose (Sigma-Aldrich, ≥99.5% purity) at 37 °C and 5% CO2. Topical treatment of Fer (AMAG Pharmaceuticals, Inc.) and SnF2 (Sigma-Aldrich, 99% purity) or vehicle control was performed for 10 min at 0, 6, 19, and 29 h. The culture medium was changed twice daily (at 19 h and 29 h). At the end of the experimental period (43 h), the biofilms were placed in 2.8 ml of H2O2 (1%, v/v) for 5 min. After H2O2 exposure, the biofilms were removed and homogenized via bath sonication followed by probe sonication (at an output of 7 W for 30 s). The homogenized suspension was serially diluted and plated onto blood agar plates using an automated EddyJet Spiral Plater (IUL, SA, Barcelona, Spain). The number of viable cells in each biofilm were calculated by counting CFU. The remaining suspension was centrifuged at 5500 g for 10 min. Finally, the resulting cell pellets were then washed, oven-dried, and weighed. SnF2 and NaF (Sigma-Aldrich, 99.99% purity) treatment groups were performed according to the same procedure in a separate experiment (Supplementary Fig. 1).

To visualize the biomass reduction and EPS degradation, SYTO 9 (485/498 nm; Molecular Probes) was used for labeling bacteria and Alexa Fluor 647-dextran conjugate (647/668 nm; Molecular Probes) was used for labeling insoluble EPS. The 3D biofilm architecture was acquired using Zeiss LSM 800 with a 20× (numerical aperture = 1.0) water immersion objective. The biofilms were sequentially scanned using diode lasers (488 and 640 nm), and the fluorescence emitted was collected with GaAsP or multialkali PMT detector (475–525 nm for SYTO 9 and 645–680 nm for Alexa Fluor 647-dextran conjugates, respectively). ImageJ software (version 1.48) was used for biofilm visualization and quantification.

Characterization of Fer & SnF2

Fer (100 µg of Fe/ml) and Fer (100 µg of Fe/ml) + SnF2 (100 µg/ml) prepared in DI water were used for determining hydrodynamic diameter and zeta potential. The measurements were carried out using a Nano-ZS 90 (Malvern Instrument, Malvern, UK) at indicated time points. TEM was performed using a Tecnai T12 (FEI Tecnai) electron microscope at 100 kV. In brief, solutions of Fer and Fer + SnF2 were prepared in 0.1 M sodium acetate buffer (pH 4.5) and incubated for 1 h. After that, 5 µl of the solution of Fer or Fer + SnF2 was dropped onto a TEM grid, and the liquid was dried before microscopy was conducted. 1H NMR spectroscopic data of CMD with or without SnF2 were recorded using a Bruker DMX 500, equipped with a z-gradient amplifier and 5 mm DUAL (1H/13 C) z-gradient probe head, in D2O. The absorption spectra of SnF2 (250 ppm of F) were measured in 0.1 M sodium acetate buffer (pH 4.5) in the presence of various materials (1 mg/ml each) (carboxymethyl-dextran (CMD; Sigma-Aldrich), dextran (Dex, T10; Pharmacosmos, Holbaek, Denmark), citric acid (CA; Fisher Scientific, ≥99.5% purity), L-ascorbic acid (AA; Sigma-Aldrich, ≥99% purity), and poly(acrylic acid) (PAA, average molecular weight: ~2000; Sigma-Aldrich)) initially and after 24 h incubation using a Genesys 150 UV−visible spectrophotometer (Thermo Scientific, Waltham, MA). Similarly, absorption spectra of SnF2 (250 ppm of F) were recorded in the presence of three concentrations of mannitol (Man; Sigma-Aldrich, ≥98% purity) (1, 2, and 10 mg/ml) under the same experimental conditions.

ROS measurement using 3,3’,5,5’-tetramethylbenzidine (TMB) assay

The catalytic activity of Fer + SnF2 was investigated by a colorimetric assay using TMB (Sigma-Aldrich, ≥99% purity) as a probe, which generates a blue color after reacting with ROS34. Briefly, the stock solution of TMB was made in N,N-dimethylformamide (DMF, 25 mg/ml; Sigma-Aldrich, ≥99% purity). Fer (0.5 mg of Fe/ml) and SnF2 (0.5 mg/ml) were incubated (separately or combined) at room temperature in 0.1 M of sodium acetate buffer (pH 4.5) for 1 h. Afterward, 40 µl of the testing sample (Fer, SnF2, or Fer + SnF2) and 4 µl of TMB (100 µg) were added into 922 µl of 0.1 M sodium acetate buffer (pH 4.5), mixed by pipette and absorbance was recorded at 652 nm. Then, 34 µl of H2O2 (1%, v/v) was added. After 10 min additional incubation in the dark, catalytic activities were monitored at 652 nm. For the control, 40 µl of the buffer solution was taken instead of the testing sample. The effect of pH on the catalytic activity of Fer when exposed to SnF2 was determined at three different pH values (4.5, 5.5, and 6.5) as described above.

The effect of sodium fluoride (NaF) (final concentration 20 µg/ml), barium fluoride (BaF2) (final concentration 20 or 30 µg/ml; Sigma-Aldrich, 99.95% purity) and stannous chloride (SnCl2) (final concentration 20 µg/ml; Sigma-Aldrich, ≥99.99% purity) on the catalytic activity of Fer at pH 4.5 was also investigated as described above, except after adding H2O2, the reaction mixture was incubated only for 5 min. In order to determine the lowest amount of SnF2 required for enhancing the catalytic activity of Fer and to evaluate the effect of various amounts of SnF2 (final concentration 0-80 µg/ml) on the catalytic activity of Fer (final concentration 20 µg of Fe/ml), the stock solution of the mixture of Fer and SnF2 was prepared at pH 4.5 (0.1 M sodium acetate buffer) by using the predetermined amount of SnF2 and then the catalytic activity was assessed at pH 4.5 using the TMB assay (5 min incubation in the presence of 1% of H2O2), as described above. The effect of incubation time on the catalytic activity was investigated after incubating Fer and SnF2 for a predetermined time as described above (5 min incubation in the presence of 1% of H2O2). All the reactions were investigated using the Genesys 150 UV−visible spectrophotometer.

Investigation of ROS generation using o-phenylenediamine (OPD)

The enhancement of the catalytic activity of Fer in the presence of SnF2 was further verified by employing OPD (Sigma-Aldrich, ≥98% purity) as a ROS tracking agent35. Briefly, the stock solution of the combination of Fer (0.5 mg of Fe/ml) and SnF2 (0.5 mg/ml) was incubated for 1 h in 0.1 M sodium acetate buffer (pH 4.5) at room temperature. Afterward, 40 µl of the mixture of Fer (20 µg of Fe) and SnF2 (20 µg) and 4 µl of OPD (100 µg) were added into 922 µl of 0.1 M sodium acetate buffer (pH 4.5) and then mixed via pipetting and absorbance was recorded at 450 nm. After adding 34 µl of H2O2 (1%, v/v), the mixture was further incubated for 1 min, and the absorbance was recorded at 450 nm.

ROS study using 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFH-DA) probe

In order to further support the enhancement of the catalytic activity of Fer in the presence of SnF2, we used photoluminescence (PL) method using DCFH-DA (Sigma-Aldrich, ≥97% purity) as a ROS probing agent36. First, stock solutions of Fer (0.5 mg of Fe/ml) with or without SnF2 (0.5 mg/ml) were incubated in 0.1 M sodium acetate buffer (pH 4.5) for 1 h at room temperature. Afterward, the working solution (final volume 2 mL) containing DCFH-DA (30 μM) and Fer (20 µg of Fe/ml) with or without SnF2 (20 µg/ml) was prepared in 0.1 M sodium acetate buffer (pH 4.5). Subsequently, PL intensity was recorded at 520 nm with an excitation wavelength of 505 nm. H2O2 (1%, v/v) was then mixed to the reaction mixture to initiate the reaction, and the PL intensity was recorded at 520 nm at different incubation times with the excitation wavelength of 505 nm. For the control, vehicle was used.

Comparison of hydroxyl radical (•OH) production

•OH generated by Fer and Fer + SnF2 in 0.1 M sodium acetate buffer (pH 4.5) was analyzed by a PL technique using coumarin (Sigma-Aldrich, ≥99% purity) as a •OH trapping molecule37,38. First, stock solutions of Fer (0.5 mg of Fe/ml) with or without SnF2 (0.5 mg/ml) were incubated in 0.1 M sodium acetate buffer (pH 4.5) for 1 h at room temperature. Afterward, Fer (20 µg of Fe/ml) with or without SnF2 (20 µg/ml) was mixed with coumarin (0.1 mM) in a 10 mm path length cuvette, and then H2O2 (1%, v/v) was added to the reaction mixture to initiate the reaction. The PL intensity was recorded at 452 nm at different incubation times with an excitation wavelength of 332 nm. Vehicle was used as the control.

Iron release study

The release of soluble iron from Fer, in the presence and absence of SnF2, was investigated using inductively coupled plasma optical emission spectroscopy (ICP-OES, Spectro Genesis). Briefly, 10 ml of Fer (0.5 mg of Fe/ml) was incubated with or without SnF2 (0.5 mg/ml) for 1 h in 0.1 M sodium acetate buffer (pH 4.5, 5.5, or 6.5) at room temperature. Afterward, free iron ions and intact nanoparticles were separated by centrifugation using ultrafiltration tubes (3 kDa MWCO). The pellet was then resuspended in the same volume using 0.1 M sodium acetate buffer. Subsequently, the filtrate and resuspend pellet were digested in nitric acid and finally diluted with DI water before analysis by ICP-OES.

Stability study of SnF2 after catalytic reaction

To investigate the extent of SnF2 oxidation after catalytic reaction, SnF2 (1 mg/ml) was mixed with Fer (1 mg of Fe/ml) in 0.1 M sodium acetate buffer (pH 4.5), and then H2O2 (1 %, v/v) was added to the solution to initiate the reaction. After incubating the mixture for the predetermined time with H2O2, the absorption spectra of the solutions were recorded following a 10-fold dilution. Furthermore, absorption spectra of SnF2 (1 mg/ml) were recorded in the presence and absence of H2O2 (1%, v/v) (10 min incubation in the presence of H2O2) in 0.1 M sodium acetate buffer (pH 4.5). Additionally, the absorption spectrum of the diluted SnF2 solution was measured after 60 min incubation in the presence of H2O2.

Determination of free tin ions after the catalytic reaction

The free tin ions from the combination of Fer + SnF2, in the presence and absence of H2O2, was investigated using ICP-OES. Briefly, SnF2 (1 mg/ml) was mixed with Fer (1 mg of Fe/ml) in 0.1 M sodium acetate buffer (pH 4.5) and then incubated for 24 h at room temperature. The solution was then further incubated for 10 min with or without H2O2 (1%, v/v) to initiate the catalytic reaction. Afterward, free tin ions were collected from the filtrate by centrifugation (1 h; 3184 × g) using ultrafiltration tubes (3 kDa MWCO). Subsequently, the filtrate was digested in nitric acid and diluted with DI water before analysis by ICP-OES.

Toxicity study of the combined treatment of Fer and SnF2 in immortalized human gingival keratinocytes (HGKs)

The in vitro biocompatibility of the combination of Fer and SnF2 was investigated in HGK cells using an MTS [(3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay (CellTiter 96 cell proliferation assay kit; Promega, WI, USA). HGK cells were kindly provided by the laboratory of Dana T. Graves (School of Dental Medicine, University of Pennsylvania) and were cultured in KBM-2 medium (Lonza Group AG, Basel, Switzerland). To determine the cytotoxicity, HGK cells were seeded in 96-well plates at a density of 104 cells per well. Cells were then incubated at 37 °C in a humidified 5% CO2 atmosphere in a cell incubator for 24 h. Afterward, old media was replaced with 100 µl of fresh media with or without Fer (1 mg of Fe/ml) and SnF2 (250 ppm of F), or either alone, and incubated for 10 min. After that, the media was removed, the cells were washed twice with sterile phosphate buffered saline (PBS) and 100 µl of fresh complete cell culture media was added to each well. After 24 h incubation, the cell culture media was removed, and 20 µl of MTS reagent and 100 µl of media were added to each well. After 3 h additional incubation under standard cell culture conditions, the absorbance was recorded at 490 nm using a microplate reader. The cell viability was calculated using the following formula:

$${{{{{\rm{Cell}}}}}}\, {{{{{\rm{viability}}}}}}=\frac{{{{{{{\rm{A}}}}}}}_{490}^{{treated}}}{{{{{{{\rm{A}}}}}}}_{490}^{{untreated}}}\,\times \, 100\%$$

In vivo efficacy of Fer in combination with SnF2

In vivo efficacy was assessed using a well-established rodent model of dental caries, as reported previously44,60. In brief, 15 days-old specific pathogen free Sprague-Dawley rat pups were purchased with their dams from Harlan Laboratories (Madison, WI, USA). Upon arrival, animals were screened for S. mutans by plating oral swabs on mitis salivarius agar plus bacitracin (MSB). Then, the animals were orally infected with S. mutans UA159, and their infections were confirmed at 21 days via oral swabbing. The treatment agents were applied on the tooth surfaces using a custom-made applicator. To simulate a clinical scenario, a topical treatment regimen was used that consisted of a short exposure (30 s) to the agent, followed by another short exposure (30 s) to H2O2 (1%, v/v) (or buffer). All infected pups were randomly placed into five treatment groups, and their teeth were treated twice daily. The treatment groups included: (1) control (0.1 M sodium acetate buffer, pH 4.5), (2) Fer only (1 mg of Fe/ml), (3) SnF2 only (250 ppm of F), (4) 1/4 Fer + 1/4SnF2 (0.25 mg of Fe/ml and 62.5 ppm of F), and (5) Fer + SnF2 (1 mg of Fe/ml and 250 ppm of F). All the samples were prepared immediately before each treatment in 0.1 M sodium acetate buffer (pH 4.5). Each group was provided the National Institutes of Health cariogenic diet 2000 (TestDiet, St. Louis, MO) and 5% sucrose water ad libitum. The experiment proceeded for 5 weeks, and their physical appearance was recorded daily. At the end of the experimental period, all animals were sacrificed, and their jaws were surgically removed and aseptically dissected, followed by sonication to recover total oral microbiota as reported previously61. All of the jaws were defleshed, and the teeth were prepared for caries scoring based on Larson’s modification of Keyes’ system44. Determination of the caries score of the jaws was performed by a calibrated examiner who was blinded for the study by using codified samples. Enamel surfaces were analyzed as described below. Moreover, the gingival tissues were collected for hematoxylin and eosin (H&E) staining for histopathological analysis by an oral pathologist at Penn Oral Pathology. This research was reviewed and approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC #805529).

Preparation of enamel samples for scanning transmission electron microscopy (STEM)

We identified the most promising location for focused ion beam (FIB) lift-out as the middle cusp of the buccal side by assessing curvature and roughness using synchrotron micro-computed tomography reconstructions of whole molars (mandibular) and 3D measuring laser confocal microscopy (Olympus LEXT OLS5000 equipped with a laser operating at a wavelength of 405 nm). Whole air-dried M1 molars were attached, with the buccal side facing up, to a scanning electron microscopy (SEM) stub with carbon and copper tape (Electron Microscopy Sciences). Specimens were then coated with carbon (~10 nm, Denton Desk deposition system). The surface of the middle cusp of the buccal side of the tooth was then investigated in detail for microscopic surface roughness, using electron beam imaging at a high tilt angle (52°). Lamellae were lifted out directly from the surface of the tooth in areas that were sufficiently flat (500 nm height modulation), using a dual-beam FIB/SEM (FEI Helios Nanolab 600 FIB/SEM) with a gallium liquid metal source ion source (LMIS) operated at an accelerating voltage of 5–30 kV. Initially, a ~100 nm layer of protective carbon was deposited using the electron beam (5 kV, 1.4 nA) on a 2 µm × 10 µm area of interest using a gas injection system (GIS) through decomposition of a phenanthrene precursor gas. A ~1 µm protective platinum layer was then deposited on top of the carbon using the ion beam (30 kV, 93 pA) through decomposition of a (methylcyclopentadienyl)-trimethyl platinum precursor gas. Next, two trenches were cut (30 kV, 6.5 nA) and edged-cleaned at slightly lower currents (30 kV, 2.8 nA) to allow for a roughly 1.5 µm thick lamella. Following an in situ lift-out procedure, a tungsten micromanipulator (Oxford Instruments) was then welded onto the lamella using platinum, and the sample was cut loose from the bulk material. After mounting the lamella as a flag onto one of the four posts of a TEM Cu half-grid (Ted Pella), the lamella was thinned in a 5 µm wide window (5 kV, 81 pA) and cleaned at low voltage and current (2 kV, 28 pA) until a final thickness of roughly 20–80 nm was achieved near the surface of the lamella.

Scanning transmission electron microscopy (STEM) with energy dispersive spectroscopy (STEM-EDS) and electron energy loss spectroscopy (STEM-EELS)

Imaging of enamel specimens was performed using an JEOL GrandARM 300 F with a cold-cathode field-emission electron gun used at an accelerating voltage of 300 kV, using a probe current of ~204 pA with a dwell time of 10 µs. The collection semi-angle used was 106–180 mrad for high-angle annular dark-field (HAADF) imaging. Elemental maps were recorded using EDS using a windowless 100 mm2 XmaxN 100TLE Silicon Drift detector (SDD) with a solid angle of approximately 0.98 sr (Oxford Instruments NanoAnalysis) with a resolution of 1024 × 1024 pixels with a dwell time of 10 μs per pixel. Elemental maps were binned (4 × 4) and converted to mole fractions, using QuantMap (AZtecTEM). Binned mole fraction maps were then exported for further processing and visualization using Matlab 2022b (Mathworks, Natick, MA).

Line profiles (mole fractions as a function of distance in the direction normal to the external enamel surface) were determined by resampling regions of interest (ROIs) within elemental maps (determined by EDS) on a rectangular query grid rotated such that the y-direction was normal to the interface, as assessed from Ca maps. Resampling by linear interpolation was carried out using the griddedInterpolant() function included in Matlab r2022b (Mathworks, Natick, MA). Resampled ROIs were then averaged in the direction parallel to the interface. The position of the outer surface in treated and untreated samples, of the interface between the Fe and Sn rich layer and underlying enamel were identified manually from line profiles. Profiles were aligned on the outer surface position, and the distance axis was set to zero at the interface between the Fe and Sn rich layer and enamel. Data were plotted as the mean value at the given distance (solid circles), and in smoothed form (lines), as the local 3-point mean (moving average with span 3, using the movmean() function).

EEL spectra were acquired with a GIF continuum system (Gatan) using a K3 IS direct electron detector (Gatan) in counting mode at 300 kV. The high quantum efficiency of this detector (DQE up to 90%) allowed the simultaneous acquisition of the relevant inner shell ionization (core loss) edges and zero loss region at high energy resolution, except for the phosphorous K and L edges, which were outside the selected energy range. The convergence semi-angle of the probe was 19 mrad, and the probe current was ~27 pA, as determined using a Faraday cup. The collection semi-angle of 36 mrad was defined by the EELS entrance aperture (5 mm). The three-dimensional spectrum image dataset was collected using an energy dispersion of 0.35 eV/channel and the probe dwell time was 4 ms/pixel with a pixel size of 6 nm, with sub-pixel scanning enabled (32 × 32) to yield a ~3.8 Å pixel. Simultaneously, ADF images were acquired using a collection semi-angle of 51–115 mrad. In post-processing, the zero-loss peak was aligned in every pixel of the spectrum image using GMS software (Gatan, Inc). Elemental Quantification Analysis was performed in the same software, using a Hartree-Slater cross-section model and including plural scattering corrections.

High-resolution TEM (HRTEM) imaging

HRTEM imaging of enamel specimens was performed using an JEOL GrandARM 300F at an accelerating voltage of 300 kV. Images (edge length: 4096 pixels, scale factor 0.0328 nm/pixel) were processed using Matlab r2022b (Mathworks, Natick, MA). Two-dimensional Fourier transforms of regions of interest (edge length: 1024 pixels) were determined using fft2() and rearranged using fftshift() to move the zero frequency components to the center of the image. Fourier transform images were unwrapped in the azimuthal direction by interpolation using griddedInterpolant() with a query grid in polar coordinates (radial pitch: 0.0298 nm−1/pixel; azimuthal pitch: 1˚/pixel) and integrated in the azimuthal direction.

X-ray photoelectron spectroscopy (XPS)

Two mandibular (M1) rat molars, one from Fer + SnF2 treated group and one from control group, were dissected and attached using copper tape (Electron Microscopy Sciences). XPS analysis was conducted using a Thermo Scientific Nexsa G2 using an Al-Ka X-ray source, with the following parameters: pressure of 2·10−9 torr (2.5·10−7 Pa), an X-ray gun power of 150 W, a spot diameter of 100 μm, and a takeoff angle of 0°. XPS survey spectra were acquired under a pass energy of 100 eV, using a step size of 1 eV. High-resolution spectra for F, Fe, Ca, P, O, and Sn were acquired under a pass energy of 50 eV, using a step size of 0.1 eV, and averaging over 10 scans. For depth profiling, the surface was excavated using an argon ion beam (4 keV, diameter 500 μm, ‘high current’ mode, 30–300 s increment) between successive spectra. All data were processed using Avantage (Thermo Scientific), and spectra were referenced to adventitious carbon at 284.8 eV.

16S rRNA sequencing

Cells were pelleted from dental plaque by centrifuging at maximum speed for 5 min. DNA was extracted from the pellets using the Qiagen DNeasy PowerSoil htp kit according to the manufacturer’s instructions within a sterile class II laminar flow hood. Mock washes and mock extractions were included as control for microbial DNA contamination arising through the sonication and extraction processes, respectively.

Polymerase chain reaction (PCR) amplification of V1-V2 region of 16S rRNA gene was performed using Golay-barcoded universal primers 27F and 338R. Four replicate PCR reactions were performed for each sample using Q5 Hot Start High Fidelity DNA Polymerase (New England BioLabs). Each PCR reaction contained: 4.3 µl microbial DNA-free water, 5 µl 5X buffer, 0.5 µl dNTPs (10 mM), 0.17 µl Q5 Hot Start Polymerase, 6.25 µl each primer (2 µM), and 2.5 µl DNA. PCR reactions with no added template or synthetic DNAs were performed as negative and positive controls, respectively62. PCR amplification was done on a Mastercycler Nexus Gradient (Eppendorf) using the following conditions: DNA denaturation at 98 °C for 1 min, then 20 cycles of denaturation at 98 °C for 10 s, annealing 56 °C for 20 s and extension 72 °C for 20 s, last extension was at 72 °C for 8 min. PCR replicates were pooled and then purified using a 1:1 ratio of Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN), following the manufacturer’s protocol. The final library was prepared by pooling 10 µg of amplified DNA per sample. Those that did not arrive at the DNA concentration threshold (e.g., negative control samples) were incorporated into the final pool by adding 12 µl. The library was sequenced to obtain 2 × 250 bp paired-end reads using the MiSeq Illumina63.

To analyze 16S rRNA gene sequences, we used QIIME2 v19.464. We obtained taxonomic assignments based on GreenGenes 16S rRNA database v.13_865 and ASV analysis of shared and unique bacterial taxa through DADA266. PCoA was performed using library ape for R programming language67. To test the differences between communities, we used library vegan and UniFrac distances ( R environment (version 4.0.3) was used for statistical analysis. Non-parametrical test Wilcoxon Rank Sum Test was performed for the pairwise comparison between treatment groups for richness and Shannon diversity analysis. PERMANOVA analysis was performed for weighted UniFrac principal coordinate analysis to evaluate the differences between treatment groups. Statistical significance was considered <0.05.

Statistical analysis

The data presented as the mean ± standard deviation were performed at least three times independently unless otherwise stated. One-way analysis of variance (ANOVA) followed by the Tukey test was used to determine the statistical significance between the control and the experimental groups unless otherwise stated. p values < 0.05 were considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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