Phenol red broth

For phenol red broth (Sigma Aldrich, St Louis, MO, USA) supplemented with 30 mM arginine, culture supernatant at each point was measured the absorbance at 550 nm. A calibration curve was determined in phenol red broth which adjusted to pH values ranging from 4 to 10. The pH values were assessed up to 40 days in the sample which included surviving bacteria. For THY broth, culture supernatant at each point was supplemented with 5 mg/mL phenol red and the absorbance was measured at 550 nm. A calibration curve was determined in THY broth which was supplemented with 5 mg/mL phenol red and adjusted to pH values ranging from 4 to 10.

Streptococcus pyogenes M1T1 strain 5448 (accession: CP008776.1) was isolated from a patient with toxic shock syndrome and necrotizing fasciitis that is genetically representative of a globally disseminated clone associated with invasive S. pyogenes infections (Kansal et al., 2000 (link)). S. pyogenes strains were grown at 37°C in a screw-cap glass tube (Pyrex; Iwaki Glass, Tokyo, Japan) filled with Todd-Hewitt broth (BD Biosciences, San Jose, CA, USA) supplemented with 0.2% yeast extract (BD Bioscience) (THY broth) in an ambient atmosphere and standing cultures. To obtain cultures for experiments and observe pH change, overnight cultures of S.pyogenes were back diluted 1:50 into fresh THY broth or phenol red broth (Sigma Aldrich, St Louis, MO, USA) supplemented with 30 mM arginine. CFUs were determined by plating diluted samples on THY blood agar.
Escherichia coli strain XL-10 Gold (Agilent Technologies, Santa Clara, CA, USA) was used as a host for derivatives of plasmids pSET4s (Takamatsu et al., 2001 (link)) and pQE30 (Qiagen, Hilden, Germany). E. coli strains were cultured in Luria-Bertani medium (Nacalai Tesque, Kyoto, Japan) at 37°C with agitation. For selection and maintenance of strains, antibiotics were added to the medium at the following concentrations: spectinomycin, 100 μg/mL for S. pyogenes and E. coli: carbenicillin, 100 μg/mL for E. coli.