Optimum Culture Conditions for Production of Exfoliative Toxin by Staphylococcus hyicus

Abstract

Optimum culture conditions for the production of exfoliative toxin by Staphylococcus hyicus (shET) were examined. High shET activity was obtained from the culture filtrate of HI and TY broth inoculated with S. hyicus. The pH in these two media ranged from 7 to 8.5 during bacterial culture, while the lowest pH in TS and BHI broth was less than 6. shET activity in the culture filtrate from TY broth inoculated with 107 CFU of S. hyicus per ml was higher than that in TY broth inoculated with 106 and 108 CFU of bacteria per ml. When shET activity in the culture filtrate was measured under various shaking conditions, the culture filtrate shaken at 75 oscillations per min had the highest shET activity of the five shaking conditions. shET activity of the culture filtrate of TY broth to which protease inhibitor had been added was the same as that of TY broth without inhibitor. shET activity in a shaking culture in an Erlenmeyer flask was also the same as that in sac culture and that in shaking culture using a shaking (Sakaguchi) flask. shET activity in TY broth supplemented with 100 mM glucose was significantly lower than that in TY broth without glucose. Based on the above results, the optimum culture conditions for the production of shET were as follows: inoculation of 3 × 109 CFU of S. hyicus strain P‐1 into 300 ml of TY broth in a 2,000‐ml Erlenmeyer flask, and incubation at 37 C with shaking at 75 oscillations per min. Then shET activity of the culture filtrate under appropriate culture conditions was measured after various incubation periods. shET activity was detected 6 hr after inoculation, reached the maximum (253 exfoliative unit/0.1 ml) at 16 hr and decreased between 20 and 48 hr. Thus, the optimum incubation period was determined to be 16 hr. Then the optimum concentration of ammonium sulfate for isolation of shET from the culture filtrate under appropriate culture conditions was examined. The greatest shET activity was obtained from the fraction salted out with 90% saturated ammonium sulfate. Thus, the optimum concentration of ammonium sulfate for the isolation of shET was determined to be 90% saturation.

New Exfoliative Toxin Produced by a Plasmid-Carrying Strain of Staphylococcus hyicus

“…In the purification of SHETA (24,27), SHETA activity was highest in the fraction precipitated with a 90% saturation of ammonium sulfate, and the SHETA activity could be detected in the second peak on Sephadex G-75 gel filtration and in the first peak (at 0 to 0.05 M NaCl) on ion-exchange chromatography. The SHETB activity was highest in the fraction precipitated with a 70% saturation of ammonium sulfate, and the SHETB activity could be detected in the second peak on gel filtration and the second peak (at 0.12 to 0.15 M NaCl) on ion-exchange chromatography.…”

“…Bacterial culture for isolation of SHETB. For the isolation of SHETB, S. hyicus was cultured under the optimum conditions described by Watanabe et al (27). S. hyicus cultured on heart infusion agar was suspended in Dulbecco's phosphate-buffered saline without CaCl 2 and MgCl 2 (PBS) at a concentration of 10 9 CFU/ml.…”

Studies on the effect of divalent metal ions on exfoliative toxins from Staphylococcus hyicus: indications of ExhA and ExhB being metalloproteins

“…The biological e¡ect of the exfoliative toxin from S. hyicus was previously studied in vivo in piglet skin [1,3,13,14] and in chickens [15,16]. The use of cell cultures for in vitro detection of partially puri¢ed exfoliative toxin [15] and for detection of toxin in culture supernatants both qualitatively [17] and quantitatively [18] has also been reported. However, the use of in vivo tests for quantifying the toxin would require the use of great numbers of experimental animals.…”

Purification and Characterization of a novel Staphylococcus chromogenes Exfoliative Toxin

“…For the isolation of SCET, the S. chromogenes strains were cultured at the optimum conditions described by Watanabe et al (1996). S. chromogenes cultured on agar Heart infusion (BD) was suspended in Dulbecco's phosphate-buffered saline without CaCl 2 and MgCl 2 (PBS) at a concentration of 10 9 CFU/ml.…”

“…For the isolation of SCET, the S. chromogenes strains were cultured at the optimum conditions described by Watanabe et al. (1996).…”