Brain heart infusion broth bhi

Samples were prepared using C. jejuni subsp. jejuni type strain purchased from DMSZ, DSM number 4688, (ATCC 33560, CCUG 11284, CIP 702, NCTC 11351) and Brain Heart Infusion Broth (BHI) media purchased from Sigma Aldrich (Merck). Tubes containing 9 mL of sterile BHI were inoculated with C. jejuni cells and were aerobically incubated for 24 h at 35 °C in order to produce enough biomass to proceed with the next step of analysis. After the incubation, the culture was used to inoculate tubes of sterile BHI media in order to realize the same optical density (OD) of the number 3 standard of McFarland that corresponds to a concentration of 9 × 10⁸ CFU (Colony Forming Unit) by mL [24 ]. Subsequently, serial dilutions using sterile BHI media were performed until the concentration was decreased by 4 orders of magnitude to 9 × 10⁴ CFU/mL. This concentration was used for the inoculation of the analyzed vials.
Sterile chromatography 20 mL vials containing 4 mL of BHI were inoculated with 100 μL of the 9 × 10⁴ CFU/mL solution reaching a final concentration of 2.20 × 10³ CFU/mL. Control vials were performed as well keeping the vials containing 4 mL of BHI with no inoculum. Furthermore, in order to control the effective number of cells at the beginning and the end of the analysis, a plate count technique was applied using four plates for each time (T0 and T20).

Tissues were mechanically homogenized in 300 μl of Brain Heart Infusion Broth (BHI, Sigma-Aldrich) for 30 seconds from which 10-fold serial dilutions were prepared and spread on BHI solid medium. Following incubation at 37 °C for 24 h, selected colonies were purified and their identity determined by amplifying the16S rRNA gene using 8 F and 1492 R primers followed by Sanger sequencing. Whole genomes from selected strains of Staphylococcus simulans were sequenced and using the PacBio RS II platform as described previously^{30 (link)}. Genome annotation was performed using the NCBI Prokaryotic Annotation Pipeline^{31 (link)} after submission to GenBank. Whole genome sequence comparison and circular map generation was done using BRIG^{32 (link)}. One and two-way average nucleotide identity was calculated using the method of Goris et al.^{33 (link)}. The genome sequences and raw sequence data are archived in Genbank accession numbers CP017428 and SRR4340965, respectively for S. simulans strain MR3; and CP017430 and SRR4340966, respectively for S. simulans strain MR4.

Streptococcus pneumoniae strain 6301, serotype 1 (ATCC, Manassas, VA, USA) was used in all experiments. Prior to use, S. pneumoniae was maintained in 30% glycerol frozen stock solutions and stored at −80°C. For inoculum preparation, S. pneumoniae was grown at 37°C, 5% CO₂ either on Blood Agar plates (TSA w/5% Sheep Blood) (Life Technologies, Carlsbad, CA) or in Brain Heart Infusion broth (BHI) (Sigma Aldrich, St. Louis, MO).

Mueller Hinton Broth 2 (MH broth, Sigma-Aldrich, St. Louis, MO, USA), Malt extract broth (ME broth, Oxoid, Hampshire, UK), Brain Heart Infusion Broth (BHI, Sigma-Aldrich), resazurin (Sigma-Aldrich), methicillin (Sigma-Aldrich), vancomycin (Sigma-Aldrich), citral (Sigma-Aldrich), casamino acids (Sigma-Aldrich), L-arginine (Sigma-Aldrich), Pgp-Glo Assay System (Promega, Madison, WI, USA), Na₃VO₄ (Promega), Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich), doxorubicin (Sigma-Aldrich).

The antibacterial effect of the clary sage extracts on P. aeruginosa (ATCC 27853) and methicillin-resistant Staphylococcus aureus (MRSA 4262) was screened in the laboratory of the Department of Medical Microbiology and Immunology (Medical School, University of Pécs, Hungary). For bioautographic assay, bacteria were grown in 100 mL Brain–Heart Infusion Broth (BHI) (Sigma Aldrich Ltd., Darmstadt, Germany) at 37 °C in a shaker incubator at a speed of 60 rpm for 12 h [53 ]. The bacterial suspension was diluted with fresh nutrient BHI to an OD600 of 0.4, which corresponds to approximately 4 × 10⁷ colony-forming units (cfu)/ mL [16 (link)].

Stool samples were cultured within 24 h of collection on Monday to Friday, or within 48 h if collected at weekends. A 10 μL loopful was added to 5 mL Brain-Heart Infusion Broth (BHI) (Merck, Darmstadt, Germany) containing 3 μg/mL vancomycin (Sigma-Aldrich, St Louis, MO, US) and incubated at 37 °C in air at 100 rpm for 24 h. A total of 200 μL was plated onto Brilliance VRE agar (Oxoid, Basingstoke, UK) and incubated at 37 °C for 48 h. Purple or blue colonies (putative E. faecium or E. faecalis, respectively) were sub-cultured on Columbia Blood agar (CBA, Oxoid, Basingstoke, UK), incubated at 37 °C for 48 h, and the species confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) (Bruker Daltoniks, Bremen, Germany). Antimicrobial susceptibility was determined using the Vitek2 instrument (BioMérieux, Marcy l’Etoile, France) and the AST-P607 card. All stools positive for VRE were cultured for VSE. A 10 μL loopful was plated onto Brilliance UTI agar (Oxoid, Basingstoke, UK). Presumptive enterococci were sub-cultured on CBA containing a 5 μg vancomycin disc (Oxoid, Basingstoke, UK). Colonies growing at the edge of the zone of inhibition were selected for identification and antimicrobial susceptibility testing as above.