Lactobacillus plantarum

The human intestinal epithelial cell lines HRT18 [American Type Culture Collection (ATCC), CCL-244], Caco-2 (ATCC, HTB-37) and CRISPR/Cas9 knockout derivative cell lines used in this study, as well as HEK293T cells (ATCC, CRL-3216), were routinely grown in 25 cm² flasks in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX (Life Technologies, 31966047) containing 10% fetal calf serum (FCS; Sigma-Aldrich, F7524) at 37°C in 10% CO₂. HEK-Blue Null and HEK-Blue hTLR4 cells were purchased from InvivoGen (hkb-htlr4) and cultured in DMEM containing 10% heat-inactivated FCS, penicillin/streptomycin (BioConnect, ML-105L) and antibiotics from InvivoGen [Zeocin (ant-zn) and Normocin (ant-nr) for HEK-Blue Null cells; zeocin, normocin, blasticidin (ant-bl) and hygromycin (ant-hg) for HEK-Blue hTLR4] at 37°C in 5% CO₂. Cells were detached with 0.25% trypsin (Thermo Fisher Scientific, 25200-072), passaged twice a week in a 1:10 dilution and split before they reached 80% confluency. All cell lines were routinely tested for Mycoplasma contamination. Lactobacillus plantarum (ATCC, 14917) was grown in MRS medium (Millipore, 69966) in aerobic conditions.

The PP mixture comprised various Lactobacillus strains supplied by the American Type Culture Collection (ATCC) (Manassas, VA, USA). The strains included Lactobacillus gasseri (ATCC 33323), Lactobacillus plantarum (ATCC BAA-793), Lactobacillus reuteri (ATCC 23272), Lactobacillus helveticus (ATCC BAA-2840), Lactobacillus fermentum (ATCC 23271), Lactobacillus rhamnosus (ATCC BAA-2836), and Lactobacillus casei (ATCC BAA-2843). Following the supplier’s protocol, we cultured the strains in MRS broth (Beckton Dickinson, Sparks, MD), and preserved them as glycerol stocks at −80°C. To initiate culture growth, we prepared 5 mL of pre-warmed MRS broth with the glycerol stocks and then incubated these starter cultures at 37°C in a 5% CO₂ environment for 2 h. The bacteria were cultured overnight in 1 L of MRS broth under identical conditions until they reached the logarithmic growth phase, confirmed by measuring optical density at 600 nm (OD600). After growth, the bacterial cells were collected through several centrifugation steps at 3000×g and 4°C, followed by a wash in cold PBS. We then resuspended the bacterial pellets in 10% glycerol in PBS and promptly froze them in 1 mL aliquots for future administration to mice. The viability and concentration of the bacteria were verified by serial dilution and colony-forming unit (CFU) enumeration on agar plates.

Non-native bacteria were obtained from ATCC: Bacillus subtilis (ATCC 23857) (Bs), Enterobacter aerogenes (ATCC 13048) (Ea), Lactobacillus plantarum (ATCC 8014) (Lp), Pseudomonas aurantiaca (Pseudomonas chlororaphis subsp. aurantiaca) (ATCC 33663) (Pa), Pseudomonas citronellolis (ATCC 13674) (Pc), Pseudomonas fluorescens (ATCC 13525) (Pf), Pseudomonas putida (ATCC 12633) (Pp), Pseudomonas veronii (ATCC 700474) (Pv), and Serratia marcescens (ATCC 13880) (Sm). Bacillus cereus (Bc) was obtained from Ward’s Scientific Catalog. Escherichia coli MC4100 (CGSC #6152) (Ec) was obtained from the E. coli Genetic Stock Center.
The microbiota strains native to C. elegans were isolated by growing C. elegans N2 for 1 week on individual types of rotten organic material (apples, celery, almonds, and parsnip), followed by washing and sterilizing the worms on the outside, grinding the worms, and plating the resulting bacterial suspension on agar plates (Supplementary Information). The species identity was analyzed by 16S Sanger sequencing (Genewiz, South Plainfield, NJ, USA).

Lactobacillus acidophilus (#4356), Lactobacillus plantarum (#14917), and Lactobacillus fermentum (#14931, all American Type Culture Collection, ATCC, Manassas, VA, USA) were all grown in De Man, Rogosa, and Sharpe broth (MRS; Becton Dickinson, #288130, Sparks, MD, USA) at 37 °C and 5% CO₂. For standard serial dilution plating, bacteria were grown on MRS plates. Bacteria were sub-cultured in MRS from an independent colony, snap-frozen in liquid nitrogen for 30 min during the exponential growth phase, and stored at -80 °C. To determine CFU/mL, bacteria were serially diluted in PBS and spot plated (10 µL) on MRS plates.

Selected lactobacilli strains such as Lactobacillus casei (ATCC 334), Lactobacillus paracasei(ATCC 25303), Lactobacillus plantarum (ATCC 14917) and Lactobacillus rhamnosus (ATCC 7469) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).
The tested strains were cultured in anaerobic conditions at 37 °C for 48 h on de Man-Rogosa-Sharpe MRS agar (Oxoid) broth. All bacterial strains were stored at −80 °C in de Man Rogosa and Sharpe (MRS) medium (Oxoid, Milan, Italy) supplemented with 25% glycerol (v/v). The cultures were propagated three times with about 3% (v/v) of inoculum in MRS and incubated in anaerobiosis (AnaeroGen, Oxoid, Basingstoke, UK) overnight at 30 °C for L. plantarum and 37 °C for L. rhamnosus, L. casei and L. paracasei.
Briefly, bacterial density was adjusted to an OD of 0.06 at a wavelength of 670 nm, i.e., approximately 10⁸ CFU/mL. Extracts were administered at concentrations ranging from 2000 to 250 µg/mL into 96-well plates along with 50 µL of bacterial suspension. The final volume was made up to 100 µL using MRS broth. Negative control included cells treated with solvent/medium and Saquinavir, a known HIV-1 protease inhibitor, was used as reference control. After 24 h incubation at 37 °C, the cytotoxicity of plant extracts on lactobacilli was assessed by the MTT method.