Luria bertani agar

All bacterial strains and plasmids used in this study are listed in Table 1. Y. pestis CO92/pCD1⁻ (YP6) was cultivated on brain heart infusion (BHI) agar (BD Biosciences, Bedford, MA) at 26°C for 48 h and in BHI broth cultures grown with aeration at 26°C or 37°C. E. coli strains were cultivated on Luria-Bertani (LB) agar (BD Biosciences) at 37°C overnight and in liquid cultures with aeration at 37°C or 26°C. When indicated, bacteria were grown in BHI broth that was adjusted and buffered to the appropriate pH. BHI broth was buffered with 100 mM MES [2-(N-morpholino)ethanesulfonic acid; Sigma] and then adjusted to pH 6.3 or 6.7, or it was buffered with 100 mM MOPS [3-(N-morpholino)propanesulfonic acid; Fisher Scientific] and then adjusted to pH 7.3 and filter sterilized. When necessary, antibiotics were added to the growth medium at the following concentrations: kanamycin (Kan), 50 μg/ml; carbenicillin (Carb), 100 μg/ml; and irgasan (Irg), 2 μg/ml. For the expression of genes cloned into pMWO-005, 50 ng/ml anhydrous tetracycline (ATc) was added to the liquid medium when strains were subcultured.

A clinically isolated rough strain of M. mass (M. mass R), reported as M. mass Asan 50594 belonging to the Type II genotype, was used in this study [27 (link), 28 (link), 29 ]. M. mass type II genotype was reported to show loss of genes related to glycopeptidolipid (GPL) biosynthesis and rough type due to an irreversible genetic factor [29 ]. M. mass strain CIP 108297 (M. mass CIP) was obtained from CIP (Collection of Institut Pasteur, Paris, France). M. mass were cultured in Middlebrook 7H9 medium (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 10% OADC (BD Biosciences, Franklin Lakes, NJ, USA), 0.2% glycerol and 0.05% Tween 80 (Sigma-Aldrich, St. Louis, MO, USA). Cultured bacteria were collected by centrifugation. Then, collected mycobacteria were stored at -70°C until use. To prepare single cells of M. mass, cultured bacteria were homogenized with 23G syringe needle and sonicated in 10 min after soft spin centrifugation to exclude bacterial clumping. Nonsonicated M. mass aggregates were prepared by performing only soft spin centrifugation of the same bacterial cultures. The number of viable bacteria in stored bacterial vials was counted on Luria-Bertani (LB) agar (BD Biosciences, Franklin Lakes, NJ, USA).

2-(Methacryloyloxy) ethyltrimethylammonium chloride solution (TMAEMA) were purchased from Alfa Aesar (Ward Hill, MA, USA). Sodium sulfate (SO₄²⁻) was purchased by Showa Chemical Industry (Tokyo, Japan). N,N′-Methylenebis(acrylamide) (MBAA), sodium citrate tribasic dehydrate (Citrate³⁻), sodium hexametaphosphate (PP⁶⁻) were purchased from Sigma Aldrich (St. Louis, MO, USA). Phytic acid (PA), 2-hydroxy-2-methylpropiophenone were purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). LIVE/DEAD BacLight LIVE/DEAD BacLight was bought from Invitrogen (Carlsbad, CA, USA). Tryptic Soy broth, Luria-Bertani agar and broth were bought from Becton Dickinson (Franklin Lakes, NJ, USA) and Neogen (Lansing, MI, USA), respectively. Water used in these experiments was purified by a millipore water purification system with minimum resistivity of 18.0 MΩ·m.

All bacterial strains used in this study appear in Table 1. E. coli strains were cultured at 37 °C in Luria-Bertani (LB) (Becton & Dickinson, Sparks, MD, USA) broth or on Luria-Bertani Agar (Becton & Dickinson, Sparks, MD, USA). Wild-type (WT) strain refers to BW25113.

The three challenge strains (DS1, DS2 and RS) were plated onto Luria-Bertani agar (Becton Dickinson and Company, Sparks, MD, USA) and incubated at 37°C for 24 h. Growth curves were constructed for the three strains to determine the point at which cultures reached logarithmic growth. A colony from each strain was selected and transferred to three sets of 25 mL of LB broth and incubated in a shaker bath at 37°C for 4 h. Concentrations of challenge bacterial broths were adjusted to an optical density reading of 0.1 at OD₆₀₀ with a spectrophotometer (GENESYS™ 20, Thermo Scientific, USA) which correlated to approximately 10⁷ cfu/mL of Salmonella, and 1.5 at OD₆₀₀, which correlated to approximately 10⁹ cfu/mL of Salmonella. The concentrations of inoculum desired to test anti-conjugative effects of flavophospholipol were 10⁷ and 10⁹ cfu/mL for the donor and recipient strains of S. Enteritidis, respectively. Challenge concentrations were adjusted to 10⁹ cfu/mL for the strain used to test plasmid-curing effects of flavophospholipol. This is summarized in Table 2. Suspension concentrations were based on previous successful in vivo colonization and conjugation experiments involving E. coli in broiler chickens.⁷ (link)

Rabbit blood agar (RBA) plates were prepared as follows: Fresh citrated rabbit blood (1 ml) was centrifuged at 5,000 × g for 5 min at room temperature, and sedimented cells were washed three times with 1 ml of 0.9% saline. After the last washing step, cells were suspended in 0.5 ml of 0.9% saline and 500 μl of the erythrocyte suspension was added to 20 ml of sterile Luria Bertani agar (Becton Dickinson) that was cooled to 50°C. Washed erythrocytes and Luria Bertani agar were gently mixed and poured into sterile petri dishes (Greiner Bio-One, Frickenhausen, Germany). If needed, RBA plates were supplemented with 250 μl of a sterile 40% glucose solution (final conc. 0.5%). Single colonies of S. aureus were streaked onto RBA plates and grown for 24 h at 37°C. After 24 h, images of the plates were obtained with a Leica D-Lux 3 in automatic mode.