Two commonly used strains of Salmonella Typhi were used as positive controls in this study, Ty21a and Ty2. Salmonella Typhi strain Ty21a is used in the oral, live attenuated typhoid vaccine and is negative for the Vi antigen. Salmonella Typhi strain Ty2 is a well-characterized, reference strain that is positive for the Vi antigen. To confirm the presence of the Vi antigen in Ty2 prior to experiments, a Vi antigen agglutination test was performed using Difco™ Salmonella Vi Antiserum (Becton, Dickinson and Company, Franklin Lakes, NJ). Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) was used as a negative control in the agglutination test. Ty21a (33459; American Type Culture Collection, Manassas, VA), Ty2, and Salmonella Typhimurium were obtained from Dr. Stephen Libby (University of Washington). For each strain, 10 µL antiserum and 10 µL overnight culture were combined on a glass slide and examined for agglutination after 15 minutes. Agglutination indicated the presence of the Vi antigen in Ty2.
Throughout this work, Ty21a was grown using LB-Miller broth (IBI Scientific, Dubuque, IA), and Ty2 was grown in the dark using LB-Miller broth with a supplemental aromatic amino acid mix and 50 ng/mL ferrioxamine E (Millipore, Burlington, MA). The aromatic amino acid mix was prepared in a 100× stock consisting of L-Phenylalanine (4 mg/mL) (TCI America™, Portland, OR), L-Tryptophan (4 mg/mL) (Acros Organics, Fair Lawn, NJ), 2,3-dihydroxybenzoic acid (1 mg/mL) (TCI America), and para-aminobenzoic acid (1 mg/mL) (Sigma-Aldrich, St. Louis, MO), which were dissolved in deionized water and filter sterilized. To improve our understanding of Ty21a and Ty2 growth and therefore ensure that experiments were seeded during exponential growth, growth curves were determined for Ty21a and Ty2 and measured via optical density at 600 nm and spot plating of 100 µL of relevant dilutions39 on LB-Miller agar or LB-Miller agar with a supplemental aromatic amino acid mix and 50 ng/mL ferrioxamine E. Ty21a and Ty2 inocula for experiments were prepared by growing the organisms for a specified period of time at 37°C, monitoring for exponential growth phase, and storing single-use aliquots of the cultures in 30% glycerol until use (−80°C). Prior to planned experiments, 20 µL of the frozen Ty21a or Ty2 glycerol stock was inoculated in 15 mL of liquid media and incubated with shaking (200 rpm, 37°C, 12–16 hours).
Throughout this work, Ty21a was grown using LB-Miller broth (IBI Scientific, Dubuque, IA), and Ty2 was grown in the dark using LB-Miller broth with a supplemental aromatic amino acid mix and 50 ng/mL ferrioxamine E (Millipore, Burlington, MA). The aromatic amino acid mix was prepared in a 100× stock consisting of L-Phenylalanine (4 mg/mL) (TCI America™, Portland, OR), L-Tryptophan (4 mg/mL) (Acros Organics, Fair Lawn, NJ), 2,3-dihydroxybenzoic acid (1 mg/mL) (TCI America), and para-aminobenzoic acid (1 mg/mL) (Sigma-Aldrich, St. Louis, MO), which were dissolved in deionized water and filter sterilized. To improve our understanding of Ty21a and Ty2 growth and therefore ensure that experiments were seeded during exponential growth, growth curves were determined for Ty21a and Ty2 and measured via optical density at 600 nm and spot plating of 100 µL of relevant dilutions39 on LB-Miller agar or LB-Miller agar with a supplemental aromatic amino acid mix and 50 ng/mL ferrioxamine E. Ty21a and Ty2 inocula for experiments were prepared by growing the organisms for a specified period of time at 37°C, monitoring for exponential growth phase, and storing single-use aliquots of the cultures in 30% glycerol until use (−80°C). Prior to planned experiments, 20 µL of the frozen Ty21a or Ty2 glycerol stock was inoculated in 15 mL of liquid media and incubated with shaking (200 rpm, 37°C, 12–16 hours).
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