Dneasy ultraclean microbial kit

Clay samples were stored at room temperature and DNA extracted from them in the lab using the DNeasy UltraClean Microbial Kit (Quiagen, Düsseldorf, Germany) according to the manufacturer instruction and as previously performed, except that at the cell lysis step, two pulses of 2 min were used in a FastPrep-24 5G homogenizer (MP Biomedicals, Irvine, USA).

Two strains were selected for whole genome sequencing–strains A003/A1 (A. galeatum) and A004/B2 (A. antarcticum). Genomic DNA for whole genome sequencing was extracted from approximately 100 mg of fresh biomass using DNeasy UltraClean Microbial Kit (QuiaGEN, Hilden, Germany) The quality of extracted DNA was checked by agarose gel electrophoresis (1.5% agarose gel, GelRed–Biotium, California, USA) and the DNA was quantified using NanoDrop 1000 (Thermo Fisher Scientific, Waltham, USA). The sequencing was done by commercial Illumina sequencing (Novogene, UK). The whole genome sequences were uploaded to the NCBI database as: BioProject: PRJNA761285, Biosamples: SAMN21250282 (A. galeatum A003/A1) and SAMN21250283 (A. antarcticum A004/B2), accessions: JAIQZM000000000 and JAIQZN000000000.

Samples were stored at room temperature and DNA extracted from them in the lab using the DNeasy UltraClean Microbial Kit (Quiagen, Düsseldorf, Germany) according to the manufacturer instruction and as previously performed^{18 (link),21 (link),41 (link)}.

Genomic DNA was extracted from untreated C. violaceum and E. faecalis cells and cells treated with pre-inhibitory concentrations (pre-MICs) of EOs by using a DNeasy UltraClean Microbial Kit (Qiagen, Milan, Italy), according to the manufacturer’s protocol. Briefly, 3 mL of bacterial cell culture was collected and centrifuged for 10 min. The isolated pellets were resuspended in 300 µL of PowerBead Solution and transferred to PowerBead Tubes. An amount of 50 µL of Solution SL was added to each sample and, after vortexing for 10 min, the tubes were centrifuged at 10,000× g for 30 s. Supernatants were incubated at 4 °C for 5 min in the presence of 100 µL of Solution IRS and centrifugated at 10,000× g for 1 min. After the addition of 900 µL of Solution SB to the supernatants, 700 µL of the sample was loaded into MB Spin Columns and centrifuged at 10,000× g for 30 s. The centrifugation was repeated after adding 300 µL of Solution CB, and the flow-through was discarded. DNA samples were eluted by centrifugation at 10,000× g for 30 s in 50 µL of Solution EB. The DNA concentration and purity were determined spectrophotometrically by using the 260/280 nm absorbance ratio.

To identify the LUZ100 receptor(s), the genomic DNA (gDNA) of four spontaneous phage-resistant PaLo41 colonies was isolated using the Qiagen DNeasy Ultraclean microbial kit according to the manufacturer’s guidelines. The gDNA samples were prepared using the Nextera DNA Flex library preparation kit, and paired-end sequencing was performed on an Illumina MiniSeq device. Next, the quality of the reads was assessed with FastQC (v0.11.8) (55 ), and the adapters and poor-quality reads (Phred score < 33) were removed from the data set using Trimmomatic (v0.39) (56 (link)). Finally, Snippy (v4.6.0) (https://github.com/tseemann/snippy) was used to identify SNPs in the genomes of the LUZ100-resistant PaLo41 clones compared to the PaLo41 reference genome (BioProject no. PRJNA731114).

DNA was extracted from the bacterial isolates using the DNeasy Ultraclean microbial kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Library preparation was done with the QIASeq FX kit (Qiagen). Library quality and fragment size distribution was analyzed by capillary electrophoresis on a fragment analyzer automated CE system (Advanced Analytical Technologies Inc., Heidelberg, Germany). DNA libraries were pooled in equimolar concentrations and paired-end sequencing (2 × 150 bp) was performed using an Illumina MiSeq platform (Illumina, San Diego, CA, USA).

We extracted bacterial genomic DNA by using the DNeasy UltraClean Microbial Kit (QIAGEN, https://www.qiagen.com) according to the manufacturer’s instructions and quantified genomic DNA with a Qubit fluorimeter (ThermoFisher Scientific, https://www.thermofisher.com). We performed Bruce-Ladder and Suis-Ladder multiplex PCRs as described (5 (link),6 (link)).