Chloramphenicol

H. hepaticus strain 3B1 (ATCC 51488) was obtained from the American Type Culture Collection (Manassas, VA). The isogenic mutant 3B1::Tn20 has a transposon inserted near the start of cdtA and no longer produces cytolethal distending toxin (CDT) (29 (link)). Wild-type H. hepaticus 3B1 and 3B1::Tn20 were grown on tryptic soy agar (TSA) supplemented with 5% sheep blood at 37°C for 3 to 4 days in a microaerobic chamber (1 to 2% oxygen; Coy Laboratories). The isogenic mutant 3B1::Tn20 is chloramphenicol resistant and was grown on medium supplemented with 20 μg/ml chloramphenicol (Sigma, St. Louis, MO). Spores of C. difficile reference strain VPI 10463 (ATCC 43255) were prepared and used as previously described by Theriot et al. (22 (link)). Spores were enumerated by plating them on prereduced taurocholate cycloserine cefoxitin fructose agar (TCCFA), prepared as previously described (26 (link)).

ATCC 24067 (American Type Culture Collection, Manassas, VA), C. neoformans 52D strain, was used to infect mice in this study. Cryptococcal cells from frozen stocks (10% glycerol) were plated on Yeast Peptone Dextrose (YPD, BD Difco) agar plates containing 50 mg/ml chloramphenicol (Sigma) at 30°C. Single colonies were subsequently picked and grown overnight in YPD broth (BD Difco) containing 50 mg/ml chloramphenicol at 30°C with 225 rpm shaking. Fungal cells were washed three times in PBS, counted on a hemocytometer with trypan blue, and adjusted to a concentration of 5 × 10⁶ per mL before infection. Mice were infected with 10⁶ yeast (in 200 μl of sterile PBS) via tail-vein intravenous injection. Serial dilutions of the C. neoformans suspension were plated on YPD agar to confirm the number of viable fungi in the inoculum. Weights were monitored three times a week. For analysis of brain fungal burdens, animals were euthanized and organs weighed, brains homogenized in PBS, and serially diluted before plating onto YPD agar supplemented with chloramphenicol (Sigma). Colonies were counted after incubation at 30 °C for 48 hours. For analysis of brain cytokines, brain homogenates were diluted 2-fold in PBS before measurement using the Mouse Cytokine 32-plex Discovery Assay (Mouse Cytokine Array/Chemokine Array 32-Plex Panel; Cat#: MD31; Eve Technologies, Alberta, Canada).

For genetic engineering, E. coli cells were incubated in Lysogeny broth (LB) supplemented, when required, with 100 μg/ml ampicillin (Sigma-Aldrich), 50 μg/ml kanamycin (Sigma-Aldrich), or 34 μg/ml chloramphenicol (Sigma-Aldrich) for plasmid selection, or with 25 μg/ml kanamycin, 20 μg/ml chloramphenicol, or 0.2% sucrose for selection of the genomic insertions of gene cassettes. For imaging strains with fluorescent foci with LacI fusions, we grew cells in liquid M9 minimum medium (Fluka Analytical) supplemented with 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glycerol (Sigma-Aldrich), and 0.01% protein hydrolysate amicase (PHA; Fluka Analytical). For imaging other strains, we grew cells either in liquid M9 minimum medium supplemented with 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glucose (Sigma-Aldrich), and 0.25% PHA, or in LB medium. For imaging, overnight cultures were back diluted into the fresh medium described above to an OD (600 nm, same below) of 0.01 in falcon tubes until an OD of 0.4–0.6 for M9 medium with 0.25% PHA and LB, and OD of 0.1 for M9 medium with 0.01% PHA. The growth conditions for the FtsZ complementation assay are as described in Osawa and Erickson (2005) (link). 0.002% arabinose was used for the induction of ectopic FtsZswTagRFP-T fusion from the plasmids in the presence of the endogenous ftsZ.

Streptomyces coelicolor strains and DNA primers used in this study are listed in Table S1 in the supplemental material. Spores of each strain, prepared according to standard procedures (4 ), were inoculated in YEME liquid medium containing 5 mM MgCl₂ and 10% sucrose and grown at 30°C with shaking at 180 rpm. For antibiotic stress conditions, a freshly made solution of tetracycline hydrochloride (Sigma) or stock solutions of chloramphenicol (Sigma) or erythromycin A dihydrate (Sigma) at the indicated concentrations were treated to early exponential cells (optical density at 600 nm [OD₆₀₀] of 0.1 to 0.5). For determining MIC, erythromycin (Sigma), tetracycline hydrochloride (Sigma), lincomycin hydrochloride (Fluka), chloramphenicol (Sigma), fusidic acid sodium salt (Sigma), hygromycin B concentrated solution (Duchefa), linezolid (Sigma), streptomycin sulfate salt (Sigma), thiostrepton from Streptomyces azureus (Sigma), puromycin dihydrochloride from Streptomyces alboniger (Sigma), and spectinomycin dihydrochloride pentahydrate (Fluka) were used. E. coli strains BW25113/pIJ790 and ET12567/pUZ8002 were grown and used as previously described (57 (link)), and E. coli DH5α was grown in LB medium for standard plasmid manipulations.

All strains used in this
study are listed in Table 2. Liquid cultures of S. suis were grown in Todd-Hewitt broth (Oxoid) supplemented with 0.2% Bacto
yeast extract (BD Biosciences) (THY) without agitation. For hemolysis
assays, a complex medium supplemented with 1% (w/v) Pullulan (Sigma-Aldrich)
(CM + Pul) has been prepared as previously described.^{47 (link)} For agar plate cultures, THY supplemented with 1.5% agar
(Thermo Fisher) or Columbia agar plates (Thermo Fisher) supplemented
with 5% sheep blood (Thermo Fisher) were used. Cultures were incubated
in a humidified incubator at 37 °C and a 5% CO₂ level.
Unless otherwise noted, chloramphenicol (Sigma-Aldrich) was added
to a concentration of 5 μg/mL.
E. coliNEBturbo was
used as a general
cloning host. E. coliwas cultured in
a Luria–Bertani (LB) medium (Merck) for liquid cultures. For
agar plate cultures, LB was supplemented with 1.5% agar. When appropriate,
antibiotics were added to the medium at following concentrations:
chloramphenicol (Cml) 25 μg/mL; kanamycin (Merck) (Kan) 50 μg/mL;
and tetracycline (Sigma-Aldrich) (Tet) 10 μg/mL. The cultures
were incubated at 37 °C in a dry incubator without adding CO₂. Liquid cultures were grown in a shaking incubator at 250
rpm.

C. perfringens strain CP1 used in this study was a field isolate from an NE case in Ontario, Canada (41 (link)). Bacterial culture media used throughout this study included tryptone-glucose-yeast (TGY; 3% tryptone, 2% glucose, 1% yeast extract), fluid thioglycolate (FTG; Difco, Sparks, MD), brain heart infusion (BHI) (Thermo Fisher, Burlington, ON, Canada), and cooked meat medium (CMM; Difco), supplemented with 34 μg ml⁻¹ chloramphenicol (Sigma-Aldrich, St. Louis, MO) and 10 μg ml⁻¹ erythromycin (Thermo Fisher) as required. Escherichia coli strain Stellar (TaKaRa Bio, Mountain View, CA) and E. coli strain BL21(DE3) (Sigma-Aldrich) competent cells were used for cloning in Luria-Bertani (LB) broth or agar (Difco), supplemented with 34 μg ml⁻¹ chloramphenicol (Sigma-Aldrich) as required.

The bacterial strains, plasmids and primers used in this study are listed in Table 1 and S1 Table. Porin mutants were constructed in three antibiotic-susceptible K. pneumoniae strains (ATCC 13883, and clinical isolates 10.85 and 11.76 from our laboratory). Bacterial isolates were stored at -80°C in Nutrient broth (NB) with 20% glycerol and recovered on LB agar plates. Unless otherwise indicated, strains were routinely grown in Mueller-Hinton broth (MHB, BD Diagnostics, Franklin lakes, NJ, USA) or Luria-Bertani (LB, Life Technologies, Carlsbad, CA, USA). E. coli and K. pneumoniae strains carrying the chloramphenicol-resistant plasmids pKM200 and pCACtus were grown at 30°C on LB agar or in LB broth supplemented with 20 μg/ml chloramphenicol (Sigma-Aldrich, St. Louis, MO, USA). The growth of bacterial cells was determined by measuring the optical density at 600 nm (OD₆₀₀) in an Eppendorf Biophotometer (Eppendorf AG, Hamburg, Germany).