Time kill assay for <i>E</i>. <i>faecalis</i>.

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

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

<p>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 <i>β-</i>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 (<i>p</i>< 0.05). Note: Amp- Ampicillin.</p

Biofilm assay.

<p>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 <i>β-</i>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 (<i>p</i>< 0.05). Note: Amp- Ampicillin.</p

Efficacy of <i>β</i>-lactam compounds in <i>ex vivo</i> dentine tubule infection model.

<p>Tooth samples were infected with <i>E</i>. <i>faecalis</i> (1.5x10⁵) cells and were treated with <i>β-</i>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 (<i>p</i>< 0.05). Note: Amp- Ampicillin.</p

<i>In vivo</i> antibacterial efficacy of <i>β</i>-lactam compound.

<p>To determine the <i>in vivo</i> efficacy of compound 3, Balb/C mice were inoculated with <i>E</i>. <i>faecalis</i> 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 <i>E</i>. <i>faecalis</i> 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 <i>E</i>. <i>faecalis</i> C) Inflammatory cells in the interstitum along with the hyaline cast in kidney cells of high dose (50 mg/kg) of <i>β-</i>lactam treated group D) Interstital infiltration with inflammatory cells in ampicillin treated group.</p

Minimum Inhibitory Concentration (μg/ml) of <i>β</i>-lactams against pathogens in root canal infection.

<p>-, no activity, Amp- Ampicillin, RS (1–5)—Resistant strains isolated from Root Canal Treatment failure cases.</p><p>Minimum Inhibitory Concentration (μg/ml) of <i>β</i>-lactams against pathogens in root canal infection.</p

Synthesis of <i>β-</i>lactam Baylis-Hillman adducts.

<p>Synthesis of <i>β-</i>lactam Baylis-Hillman adducts.</p

Mammalian cell cytotoxicity.

<p>MTT based cytotoxicity assay on NIH 3T3. The cells were treated with various concentrations of <i>β-</i>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.</p

Synthesis of <i>β-</i>lactam substituted polycyclic fused pyrrolidine/pyrrolizidine cycloadducts.

<p>Synthesis of <i>β-</i>lactam substituted polycyclic fused pyrrolidine/pyrrolizidine cycloadducts.</p

<i>In vivo</i> anti bacterial efficacy of <i>β-</i>lactam in <i>E</i>. <i>faecalis</i> infected mice.

<p>Five mice in each group received three subcutaneous doses of <i>β-</i>lactams (25 and 50 mg/kg) and ampicillin (25 mg/kg) after 4 h of intravenous inoculation of <i>E</i>. <i>faecalis</i>. 120 h after infection and daily treatment with dose indicated, the kidney were harvested and CFU were measured in kidney. Non-parametric test was done for comparisons of significance for test versus control (<i>p</i>< 0.05).</p><p><i>In vivo</i> anti bacterial efficacy of <i>β-</i>lactam in <i>E</i>. <i>faecalis</i> infected mice.</p