Bioactivity Studies of β-Lactam Derived Polycyclic Fused Pyrroli-Dine/Pyrrolizidine Derivatives in Dentistry: In Vitro, In Vivo and In Silico Studies

Abstract

The antibacterial activity of β-lactam derived polycyclic fused pyrrolidine/pyrrolizidine derivatives synthesized by 1, 3-dipolar cycloaddition reaction was evaluated against microbes involved in dental infection. Fifteen compounds were screened; among them compound 3 showed efficient antibacterial activity in an ex vivo dentinal tubule model and in vivo mice infectious model. In silico docking studies showed greater affinity to penicillin binding protein. Cell damage was observed under Scanning Electron Microscopy (SEM) which was further proved by Confocal Laser Scanning Microscope (CLSM) and quantified using Flow Cytometry by PI up-take. Compound 3 treated E. faecalis showed ROS generation and loss of membrane integrity was quantified by flow cytometry. Compound 3 was also found to be active against resistant E. faecalis strains isolated from failed root canal treatment cases. Further, compound 3 was found to be hemocompatible, not cytotoxic to normal mammalian NIH 3T3 cells and non mutagenic. It was concluded that β-lactam compound 3 exhibited promising antibacterial activity against E. faecalis involved in root canal infections and the mechanism of action was deciphered. The results of this research can be further implicated in the development of potent antibacterial medicaments with applications in dentistry.

Fig 1. Synthesis of β-lactam Baylis-Hillman adducts.

Fig 2. Synthesis of β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine cycloadducts.

Fig 3. Time kill assay for E. faecalis.

Time kill assay confirmed growth inhibitory effect of β-lactam compounds (3, 7 and 6a) against E. faecalis. Samples were collected at the indicated times and were evaluated for CFUs. Time kill kinetics of E. faecalis by compound 3 (25 μg/ml) represent dead cells within 1 h. Note: Ut- untreated, Amp- Ampicillin.

Fig 4. Efficacy of β-lactam compounds in ex vivo dentine tubule infection model.

Tooth samples were infected with E. faecalis (1.5x10⁵) cells and were treated with β-lactam compounds (3 and 7) at their MICs. After incubation, dental chips containing resident microbes were harvested at two depths (200 and 400 μm). The mean value of culture O.D was obtained. The percentage of reduction was calculated. Non-parametric test was done for comparisons of significance for test versus control (p< 0.05). Note: Amp- Ampicillin.

Fig 5. In vivo antibacterial efficacy of β-lactam compound.

To determine the in vivo efficacy of compound 3, Balb/C mice were inoculated with E. faecalis and compound 3, Ampicillin and saline was administered once daily for three days. After 120 h, mice were killed and organs were isolated and histopathological examinations were conducted. Histopathological observation of E. faecalis infected mice kidney sections shows A) Kidney section from healthy uninfected Balb/C mice stained with haematoxylin- eosin. B) Moderate degree of tubular degeneration in 120 h after infection with E. faecalis C) Inflammatory cells in the interstitum along with the hyaline cast in kidney cells of high dose (50 mg/kg) of β-lactam treated group D) Interstital infiltration with inflammatory cells in ampicillin treated group.

Fig 6. In silico studies: Binding posses of A(i) Ampicillin A(ii) Compound 7 A(iii) Compound 6a and A(iv) Compound 3 to the active site pocket of PBP (PDB ID: 2Z2M).

Fig 7. Bacterial live/ dead assay: Tooth samples were infected with E. faecalis (1.5x10⁵) cells for 21 days and were treated with 25 μg/ml of β-lactam compound 3 (MIC).

After incubation, the tooth samples were sliced and stained with fluorescein diacetate/ propidium iodide. A) CLSM image of untreated root canal showed green fluorescence indicating live bacteria B) Marked red fluorescence in the root canal treated with β-lactam compound 3 shows dead bacterial cells C) Root canal treated with ampicillin shows red fluorescence. Note: scale bar: A) 20 μm B and C) 100 μm. D) Graph depicting the percentage of bacterial viability after treatment with various agents. Nonparametric test was done for comparisons of significance for test versus control (p< 0.05). Note: Amp- Ampicillin.

Fig 8. Scanning Electron Microscopy.

Scanning Electron Microscope to show membrane damage after treatment with various agents A) untreated cell with intact surfaces B) β-lactam compound 3 induced cell damage in the form of cell swelling (arrows indicate areas of damage) C) damage to the bacterial cell after treatment with ampicillin. Note: Size bars: 100 nm.

Fig 9. Mechanism of action of β-lactam compound 3.

A) FACS based measurement of ROS Production. A(i) Untreated cells showed no fluorescence indicating no ROS production. A(ii) Cells showed fluorescence by compound 3 was monitored by incubation of compound 3 (25 μg/ml) with E. faecalis cells for 1 h. Green fluorescence indicating generation of ROS production after treatment with compound 3. A(iii) Cells showed fluorescence indicating generation of ROS production after treatment with ampicillin. A(iv) Cells showed fluorescence after treatment with hydrogen peroxide (H₂O₂) A(v) Graph indicating the percentage of ROS production after treatment with respective agents. The mean and standard deviations from six triplicates were plotted. B) Flow cytometry analysis to show membrane permeabilization of E. faecalis cells in PI uptake assay. E. faecalis (10⁶ cells) cells were incubated with respective agents for 45 min. B(i) Untreated cells showed no fluorescence indicating no membrane damage B(ii) Cells incubated with β-lactam compound 3 (25 μg/ml) showed increased PI uptake as compared to untreated indicating membrane damage and B(iii) Cells incubated with ampicillin showed increased PI uptake.

Fig 10. Biofilm assay.

Qualitative SEM study to show inhibition of biofilm formation. Images show reduction of biofilm after 24 h treatment with agents A) Large number of viable adherent bacterial cells on the matrix (control without treatment) B) bacterial reduction after treatment with β-lactam compound 3 (25 μg/ml) C) bacterial reductions after treatment with ampicillin. Notes: scale bar: 2 nm. D) Quantitative spectrophotometric measurement to show inhibition of biofilm formation. Graph indicates percentage of biofilm reduction after 24 h treatment with respective agent. Nonparametric test was done for comparisons of significance for test versus control (p< 0.05). Note: Amp- Ampicillin.

Fig 11. Mammalian cell cytotoxicity.

MTT based cytotoxicity assay on NIH 3T3. The cells were treated with various concentrations of β-lactam compounds and incubated for 24 h. After incubation, the percentage of cell viability was calculated. Standard deviations from three observations were plotted. Note: Amp- Ampicillin.