A quaternary ammonium silane antimicrobial triggers bacterial membrane and biofilm destruction

Abstract

To study the antimicrobial effects of quaternary ammonium silane (QAS) exposure on Streptococcus mutans and Lactobacillus acidophilus bacterial biofilms at different concentrations. Streptococcus mutans and Lactobacillus acidophilus biofilms were cultured on dentine disks, and incubated for bacterial adhesion for 3-days. Disks were treated with disinfectant (experimental QAS or control) and returned to culture for four days. Small-molecule drug discovery-suite was used to analyze QAS/Sortase-A active site. Cleavage of a synthetic fluorescent peptide substrate, was used to analyze inhibition of Sortase-A. Raman spectroscopy was performed and biofilms stained for confocal laser scanning microscopy (CLSM). Dentine disks that contained treated dual-species biofilms were examined using scanning electron microscopy (SEM). Analysis of DAPI within biofilms was performed using CLSM. Fatty acids in bacterial membranes were assessed with succinic-dehydrogenase assay along with time-kill assay. Sortase-A protein underwent conformational change due to QAS molecule during simulation, showing fluctuating alpha and beta strands. Spectroscopy revealed low carbohydrate intensities in 1% and 2% QAS. SEM images demonstrated absence of bacterial colonies after treatment. DAPI staining decreased with 1% QAS (p < 0.05). Fatty acid compositions of dual specie biofilm increased in both 1% and 2% QAS specimens (p < 0.05). Quaternary ammonium silane demonstrated to be a potent antibacterial cavity disinfectant and a plaque inhibitor and can be of potential significance in eliminating caries-forming bacteria.

Conflict of interest statement

The authors declare no competing interests

Figures

Figure 1

Purified sortase A analysis in vitro after incubation with the sorting substrate Dabcyl-QALPETGEE-Edans. Addition of 0.2 M NH2OH increased fluorescence intensity, whereas the addition of pHMB reduced the fluorescence intensity. Marked difference seen in purified sortase A fluorescence intensity (p < 0.05).

Figure 2

(A) Protein secondary structure elements (SSE) including alpha-helices and beta-strands were monitored throughout the simulation. The plot represents SSE distribution by residue index throughout the protein structure. All protein frames were first aligned on the reference frame backbone. Energy minimization was ensured to avoid inappropriate geometry and against steric clashes. Typically, it is observed that the tails (N- and C-terminal) fluctuate more than any other part of the protein. Secondary structure elements such as alpha helices and beta strands are usually more rigid than the unstructured part of the protein, and thus fluctuate less than the loop regions. (B) Restrictive simulation process of the lipid membrane with interaction energy and contact with the membrane. This was presented as the morphology of the upper lipid membrane layer with average z coordinate value of the surface set to 0. (C) The RMSD was calculated based on atom selection. Monitoring the RMSD of a protein provides insights into its structural conformation throughout the simulation. RMSD analysis indicates if the simulation has equilibrated; its fluctuations towards the end of the simulation are around thermal average structure. Since many molecules dock into the binding pocket, the spheres formed within 0.1–0.3 nm root mean square division established the crystal structure and maximum orientations. Changes in the order of 0.1–0.3 nm are acceptable for small, globular proteins. Changes larger than that value, however, indicate that the Sortase A protein undergoes a large conformational change during the simulation. Ligand RMSD (right Y-axis) indicates how stable the ligand is with respect to the protein and its binding pocket. (D) The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand. The plot summarizes the SSE composition for each trajectory frame over the course of the simulation (E) The plot below summarizes the SSE composition for each trajectory frame over the course of the simulation.

Figure 3

(A) Results of molecular docking simulation of QAS 1% on crystal structure of SrtA indicating a complex indicating a predicated interaction mode of QAS catalytic center of SrtA. The structure was generated from molecular coordinates from the Protein Data Bank, PDB ID. Subset proposed chemical formula of the QAS molecule. The docking shown in figure is typically performed on the basis of the known Sortase-A crystal structure and the SrtA-quaternary ammonium substrate complexes. The polar capabilities of QAS has enabled it to form charge-charge interactions that can insert with the binding pocket of SrT-A. (B) A schematic of detailed ligand atom interactions with the protein residues. Interactions that occur more than 5.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. (C) Raman spectra of dual specie biofilms grown on demineralized dentine specimens and treated with different concentrations of QAS and CHX disinfectants. Spectral differences of control and treated specimens can be seen in the 484 cm−1 region after normalization. Labelled bands present in the spectra are discussed in the text. Spectra are shifted to avoid overlap between the groups. The spectral lines are quantitative detection with each data point corresponding to the average signal collected from different groups. Raman spectra and the corresponding section corrected for orientation in a side-by-side image. For better comparability of the two measurements, different colours were chosen for the Raman spectrum of the dual specie bacterial biofilms. The molecules within the aromatic and functional groups have polarized electrons as a result of double bonds and free electrons which resulted in increased Raman shifts inside the specimens. The bands refer to the glycosidic link or ring breath of possible polysaccharides which typify the changes seen within the biofilm as a result of disinfectant treatment. These features are specific for polysaccharides (COC stretching and the anomeric C (1)-H deformations of α (1 → 4) glycosidic links) linked by 1–4 glycosidic bonds (amylose, amylopectin, glycogen). This finger print region attributed to bacterial carbohydrate via CO and CC stretching and bending vibrations showing similar changes as per our previous studies, this time also with 1% QAS molecules. (D) Raman image of intact dual specie cells with dark shading representing peak intensities at 484 cm−1 region corresponding loadings plot.

Figure 4

(A) Scanning electron microscope of control specimen showing dentinal tubules covered with dual species biofilm. Bacteria and debris are present on the dentine surface without using standard experimental disinfection. Bacteria blocked the opening of the dentinal tubules; groups displayed singular or multiple deposits on the sample with bacterial cells clumping and chaining to form complex biofilms. (B) SEM showing incomplete removal of bacteria on the dentine surface after using 2% CHX protocol. These dentinal tubules are located in the middle third of the dentine specimen. There were small colony chain formations seen amongst 2% CHX specimens (Fig. 3B) due to slight restructuring as compared to maximum detachment seen in QAS groups. (C, D) Bacterial penetration is limited across the lengths of dentinal tubules and dentinal surface demonstrated in 1% and 2% QAS specimens. (D) Tubule wall of demineralized dentine treated with 1% QAS shows exposed fibrillar collagen network. (E, F) Representative SEM images of etched dentine following application of 1% and 2% QAS respectively showing the QAS molecules did not completely infiltrate into the demineralized collagen matrix forming a crust on the surface. A phase separation is seen due to the presence of water (G). (H) Bacterial biofilms were generally intact within control specimens; (I) the bacterial Lactobacillus within the biofilm showed rough and wrinkled surfaces observed on the membrane after treatment with 1% QAS. There were large damaged areas including the formation of holes inducing significant damage to the membrane of bacterial cells after use of 1% QAS (I).

Figure 5

Fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) assay of (A) control, (B) 2% CHX and (C) 1% QAS specimens; the images shown are consistent with the results of membrane analysis. CLSM images showing (D) 2%CHX and dead bacterial sites around the (E) 1% and (F) 2% QAS molecule attracted due to its surfactant effect. (G) CLSM images and viability of dual specie biofilms treated with different antimicrobial agents, after which, biofilms were stained using the BacLight LIVE/DEAD viability stain.

Figure 6

(A) Tandem mass spectroscopy analysis of extracted membrane lipids showing effective separation of membrane lipids based on hydrophobicity with the different elution time points. Extracted dual species bacterial organism’s membrane lipids with dotted lines indicated the change in gradient and concentration as QAS percentage was increased to 2% with typical elution time for membrane phospholipids of Streptococcus mutans and Lactobacillus acidophilus biofilm. (B) Tandem mass spectroscopy scan showing different ion modes indicating fragmentation of this species to yield different fatty acyl chains in 1% QAS molecules. (C) Time–kill curves of dual specie biofilms treated with different concentrations of antimicrobials. The surviving bacteria were plated at various time points (0–300 min). Both 1% QAS and 2% QAS were able to retard growth for 300 min even after washing during the time-kill assay. After 1 min of QAS treatment, bacterial cells were reduced and showed less survivability. (D) Confocal intensity of area of biofilm in 1% QAS specimens under consideration with excitation performed at λ = 514 nM; QAS quaternary ammonium silane, CHX chlorhexidine, BF biofilm, cps count per second.