Bhi broth

The antimicrobial activity was conducted following the broth microdilution method, following the guidelines of the National Committee on Clinical Laboratory Standards (NCCLS). In detail, E. coli and S. aureus were plated on Brain Heart Infusion (BHI) agar plates (Sigma-Aldrich, Missouri, USA) and incubated at 37 °C overnight (O. N.). A fresh colony of both bacteria was inoculated in BHI-broth (Sigma-Aldrich, Missouri, USA) and incubated at 37 °C under vigorous orbital shaking (180 rpm) for 20 h. The following day, 300 µL of bacterial suspension was inoculated in fresh BHI-broth and incubated at 37 °C at 180 rpm until mid log-phase growth (6 × 10⁸ colony-forming units (CFU/mL). Three serial dilutions were performed to obtain a final bacterial concentration of 1 × 10⁶ CFU/mL. Fifty microliters of bacterial inoculum were added to each well of a sterile 96-well plate. Meanwhile, the 24 compounds were subjected to serial dilutions in phosphate buffered saline 1X (PBS 1X Sigma-Aldrich, Missouri, USA) and 50 µL of each dilution was added to the wells. Ampicillin (10 µg/mL) and vancomycin (5 µg/mL) were used as positive controls for E. coli and S. aureus, respectively. The plates were incubated at 37 °C at 180 rpm for 20 h. Bacterial growth was monitored using a TECAN microplate reader (Sunrise^TM, Männedorf, Switzerland), at an optical density (OD) of 600 nm^{18 (link),19 (link)}.

Enterococcus faecalis (E. faecalis, ATCC 19433), Streptococcus gordonii (S. gordonii, ATCC 10558), Lactobacillus acidophilus (L. acidophilus, ATCC 4356), and Actinomyces naeslundii (A. naeslundii, ATCC 12104) were obtained from State Key Laboratory of Oral Diseases (West China School of Stomatology, Sichuan University, China). Bacteria were incubated individually overnight anaerobically (90% N₂, 5% CO₂, and 5% H₂) at 37°C in brain-heart infusion (BHI) broth (Sigma-Aldrich). 10 μL of overnight grown bacterial suspension was transferred to fresh BHI broth and incubated as described before. But for the starvation test, bacteria were cultured in distilled water, without the supply of glucose or other nutrients in media.

In this study, the strain used was
a mutant of the common Cutibacterium acnes DSM 16379/KPA171202 (DSMZ-German Collection of Microorganisms and
Cell Cultures GmbH). The wild-type strain was genetically modified
to create a strain lacking active restriction modification systems
to prevent DNA degradation and improve protein production. Strains
were commonly grown in brain heart infusion (BHI) broth (Millipore)
to exponential phase. The mutant strain was supplemented with erythromycin
10 μg/mL).

Filter matings were performed with E. coli (EC600, rifampin resistant), S. aureus (RN4220), E. faecalis (JH2-2, rifampin resistant), and B. subtilis (ATCC 6051) as the recipient strains, following the method described elsewhere (Huys et al., 2004 (link)). Overnight cultures of the donor (B. cereus 9i) or recipient strains were diluted to 10⁸ CFU/mL in brain heart infusion (BHI) broth (Land Bridge Technology). Five microliters of each dilution was added into 1 mL BHI broth and incubated at 37°C for 4 h. The cultures of donor (20 μL) and recipients (60 μL) were mixed and filtered through a sterile 0.2 μm membrane filter (Millipore), and placed at 37°C for overnight incubation. After mating, the colonies were washed with 1 mL BHI broth and plated on BHI agars, containing 8 μg/mL TET and further incubated at 37°C for 24 h. For the selection of potential transconjugants, the agar plates contained 8 μg TET /mL; for E. coli EC600 and E. faecalis JH2-2, 25 μg rifampin /mL was added. Potential transformants grown on the selective agar plates were identified by 16S rRNA-gene sequences and tested for the presences of tet(45) by PCR assay.

The experiments were performed on a total of 105 titanium implants (Dentium, Seoul, Republic of Korea), 10 mm in diameter and 4.1 mm thick. S. aureus (IBRC-M10917), E. coli (IBRC-M10698), and C. albicans (ATCC 10231) were used for biofilm formation on implant surfaces. The microorganisms were prepared in brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) at a concentration of 10⁷ colony-forming units (CFUs)/mL. C. albicans was cultured on Sabouraud dextrose (SD) broth (Ibresco, Tehran, Iran). Each implant was then contaminated separately with 1 mL of each bacterial suspension in sterile 12-well microplates. This was followed by incubation for 72 h in a 5% CO₂ atmosphere at 37 °C.

All swabs were tested for the presence of L. monocytogenes, as described by the BS EN ISO 11290–1:1997 + A1:2004. Samples of Phase II and Phase III substrate and casing samples (from substrate and casing producers) as well as samples of spent substrate taken at the end of the mushroom crop, before, and after cookout, were tested for the presence and enumeration of L. monocytogenes, as described by the BS EN ISO 11290‐1 and 11290‐2:1998. Presumptive L. monocytogenes isolates, blue‐green colonies with an opaque halo on Agar Listeria acc. to Ottaviani Agosti (ALOA, BioMérieux, France), were grown in Brain Heart Infusion (BHI) broth (Merck, Darmstadt, Germany) and stored in cryovials containing 20% glycerol at −20°C for further characterization.

After preparation of bacterial suspensions of reference strains S. aureus ATCC BAA-44 and Escherichia coli ATCC 25922 in brain-heart infusion (BHI) broth (Merck, Darmstadt, Germany) with 0.5 McFarland turbidity using a nephelometer (Densimat, bioMérieux, Marcy l’Etoile, France), serial dilutions in BHI were made to produce suspensions with estimated 10-fold concentrations from 1 cfu/ml to 1 × 10⁸ cfu/ml. Additionally, a suspension with 5 × 10⁵ cfu/ml was prepared, which reflects the bacterial density recommended as starting inoculum for AST by the International Organization for Standardization ISO (2006) and the Clinical Laboratory Standards Institute CLSI (2015) guidelines. The real cell concentration in these suspensions was confirmed by vital cell counting after plating of a sample onto tryptic soy agar (TSA) plates in triplicate. Two ml samples of each dilution and a sterile control sample were added to the cuvettes of the BacterioScan instrument, and the bacterial concentration was measured for 24 h at 35°C. Applying different starting inocula, the time until fivefold and 10-fold increase in inoculum was documented to determine the length of time needed to detect the growth by laser scattering.