Brain heart infusion bhi

Hydrogenated L-α-phosphatidylcholine (Egg PC) and cholesterol were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). LLA was purchased from Ultra Scientific (North Kingstown, RI). Brain Heart Infusion (BHI), Columbia broth, and agar were purchased from Becton Dickinson (Sparks, MD). Horse blood was purchased from Hardy Diagnostics (Santa Maria, CA). BacTiter-Glo microbial cell viability assay kit was purchased from Promega Inc. (Madison, WI). SA, OA, 1-N-phenylnaphthylamine (NPN), and phosphate buffer saline (PBS) were obtained from SigmaAldrich Co. LLC (St. Louis, MO).

Fig 1 depicts the experimental design of this investigation. Planktonic cultures of each reference strain were grown anaerobically at 37°C for 24 h in a protein-rich medium containing Brain-Heart Infusion (BHI) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 2.5 g/L mucin (Oxoid), 1.0 g/L yeast extract (Oxoid), 0.1 g/L cysteine (Sigma), 2.0 g/L sodium bicarbonate (Merck), 5.0 mg/L hemin (Sigma), 1.0 mg/L menadione (Merck) and 0.25% (v/v) glutamic acid (Sigma). By means of spectrophotometry, the late exponential phase growth was verified (optical density at 550 nm). Due to the fact that the bacteria used have different growth rates, and that this biofilm model is static we have adjusted the inocula of the different bacteria as was previously developed and validated by Sánchez et al., [31 (link)], in order to avoid the overgrowth of certain species and an excessive accumulation of waste products, obtaining by dilution in fresh modified BHI medium the following final concentrations:
Using pre-sterilized polystyrene 24-well tissue culture plates (Greiner Bio-one, Frikenhausen, Germany), two types of growing conditions were developed:
The plates were then incubated in anaerobiosis at 37°C for 96 h. To rule out any possible contamination, a set of wells within the same plate were incubated with only culture medium.

Vancomycin-resistant Enterococci (VRE) strains (Table 1) were obtained from the American type culture collection (ATCC) and Biodefense and Emerging Infections Research Resources Repository (BEI Resources). All experiments were carried out in accordance with relevant guidelines and regulations and were approved by the Institutional Biosafety Committee of Purdue University. All chemicals and reagents were purchased from commercial vendors. Auranofin, linezolid (Chem-impex International, Wood Dale, IL), ampicillin (Peosta, IA), vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO), gentamicin sulfate (Fisher Bioreagents, Fairlawn, NJ), and ramoplanin (Sigma-Aldrich, St. Louis, MO) were purchased commercially. Brain heart infusion (BHI), tryptic soya broth (TSB), tryptic soya agar (TSA) and enterococcosel broth were purchased from BD (Becton, Dickinson and Company, Cockeysville, MD) and Phosphate buffered saline (PBS) was purchased from corning (Corning, NY).

Resistance to rifampicin (100 μg/mL) was selected in five L. monocytogenes strains: F2365 (serotype 4b, isolated from Mexican soft cheese) (Linnan et al., 1988 (link); Chen et al., 2016c (link)), ScottA (serotype 4b, sequence type 290, isolated from milk) (Fleming et al., 1985 (link); Briers et al., 2011 (link)), FSL R2-502 (serotype 1/2b, sequence type 3, isolated from chocolate milk) (Dalton et al., 1997 (link); Chen et al., 2016c (link)), LS806 (serotype 4b, sequence type 1, isolated from cheese), and JKS-1, a strain isolated from the process-contaminated ice cream associated with the listeriosis outbreak. The naturally contaminated L. monocytogenes found in the ice cream (including JKS-1) belong to sequence type 5, molecular serogroup IIb, and genetic lineage I (Chen et al., 2017 (link)). All strains were grown separately in Brain Heart Infusion (BHI, Becton Dickinson & Co., Sparks, MD, United States) broth containing rifampicin at 37°C for 16–18 h with 150 rpm shaking. A cocktail of F2365, Scott A, R2-502, and LS806 or JKS-1 by itself was used for artificial inoculation of ice cream.

The strains used in this study are shown in Table 1. Bursa aurealis transposon (Tn) insertion mutations encoding functionally non-redundant TCA- and urea cycle enzymes (Figure 1) in USA300-JE2 were obtained from the Nebraska Transposon Mutant Library (NTML, www.beiresources.org) [16 (link)]. Parental strains UAS391, UAS391-Ery^S (erythromycin resistance cured UAS391), and JE2 are all MRSA belonging to the highly virulent and widespread clonal lineage, USA300. These, as well as Tn insertion mutants, were routinely grown on Brain-Heart infusion (BHI; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 0.1% D(+)-glucose monohydrate (Merck Millipore, Billerica, MA, USA) and BHI Bacto™ agar (Becton, Dickinson and Company, USA) for biofilm, transduction and complementation experiments. Lysogeny broth (LB; Becton, Dickinson and Company, USA) was used for Escherichia coli. For the Tn-carrying S. aureus transductants with the erythromycin resistance marker ermB, 5 or 10 µg/mL erythromycin (Sigma-Aldrich^®, Merck KGaA, St. Louis, MO, USA) was supplemented to the growth medium.

The bile salt hydrolase (BSH)-positive strain Lactobacillus acidophilus DRU, the hydrogen peroxide (H₂O₂) producer Lactobacillus acidophilus ATCC 4356, and the reference strain Lactobacillus rhamnosus GG (ATCC 53103) were routinely cultured in de Man Rogosa and Sharpe (MRS, Biolife, Italy) medium plus 100 mg/L of cycloheximide (Merck, Germany) at 37 °C under anaerobic conditions, using Anaerocult C (Merck, Milan, Italy). The haemolytic positive strains Streptococcus pyogenes ATCC 19615 and Streptococcus pneumoniae ATCC 6303 were cultured on Brain-Heart Infusion (BHI, Becton Dickinson GmbH, Germany) at 37 °C under 5% CO₂ conditions. Escherichia coli 555, E. coli ATCC 25922, E. coli ATCC 700414, and Staphylococcus aureus ATCC 6538 were routinely cultured on Trypticase Soy Broth medium (Oxoid, Milan) at 37 °C, under aerobic conditions. Listeria monocytogenes DSM 12464 strain was reactivated in BHI broth at 30 °C. Gardnerella vaginalis ATCC 14018 was cultured on Casman’s medium base added of 5% of rabbit blood (VWR, Milan, Italy) at 37 °C. Candida albicans ATCC 10231, Candida krusei ATCC 14243, Candida glabrata ATCC 90030, Candida parapsilosis ATCC 90018, Candida lusitaniae ATCC 200951 and Candida tropicalis ATCC 13803 were cultured on Yeast Mold Broth (Conda, Madrid, Spain) at 28 °C in aerobic conditions.