Exposure of Shigella flexneri to acid stress and heat shock enhances acid tolerance

Abstract

The effects of acid stress and heat shock on changes in acid tolerance of Shigella flexneri were determined. The pathogen was grown at 37°C for 18 h in tryptic soy broth (TSB) containing no glucose (TSBNG) (unadapted cells) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted cells). Cells grown in TSBNG for 18 h then heat shocked at 48°C for 15 min (unadapted heat-shocked cells) were also prepared. The three types of cells were inoculated into TSB (contains 0.25% glucose) acidified with acetic, lactic, or propionic acids to pH 4.5, 4.0, and 3.5 and incubated at 37°C. After incubating for 0.5, 1, 1.5, 2, 4, and 6 h, viable cells were enumerated by plating acidified suspensions on tryptic soy agar (TSA). Populations of all three cell types inoculated into TSB acidified to pH 3.5 with acetic, lactic, and propionic acids rapidly decreased, while a more gradual decline was observed at pH 4.0. Populations of cells remained nearly constant at pH 4.5, regardless of acidulant used. Significantly ( α=0.05) higher numbers of acid-adapted cells and unadapted heat-shocked cells, compared to unadapted cells that were not heat shocked, were recovered from TSB acidified (pH 3.5) with acetic or lactic acids. The population of unadapted heat-shocked cells decreased approximately 3.5 log 10 cfu ml −1, whereas unadapted cells that were not heat shocked decreased 5 log 10 cfu ml −1after 30 min in TSB acidified to pH 3.5 with acetic acid. Chloramphenicol (100 μg ml −1) prevented the development of acid tolerance in unadapted heat-shocked cells, indicating a need for synthesis of heat-shock proteins for the development of acid resistance. Gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) revealed that acid-adapted cells contained more proteins than control, unadapted heat-shocked, and unadapted, chloramphenicol-treated, heat-shocked cells. Results indicate that exposure of S. flexneri cells, unadapted to an acidic environment, to a mild heat shock renders them more tolerant to acidic conditions and may enhance their survival and ability to grow in high acid foods.