Briefly, simulated gastric fluid was made as described (Oliveira et al., 2011). Cells were cultured
overnight in LBG medium (pH 7, 37 °C). Subsequently, 30 μL of culture was added to 30 mL of simulated gastric fluid which was adjusted to pH 2.5 with 1 M HCl. Cells were enumerated after 3 and 6 h Dactolisib of incubation at 37 °C by plating serial dilutions on trypticase soy agar (TSA) and overnight incubation at 37 °C. All 11 E. coli O157 strains earlier identified as short to medium–long survivors (i.e. population decline to the detection limit taking < 200 days) in manure-amended soil (Franz et al., 2011) possessed mutations within the rpoS gene, that is deletions, insertions and single nucleotide polymorphisms (SNPs; Table 1). In contrast, the seven E. coli O157 strains earlier identified as long-term survivors (i.e. population decline to the detection limit taking more than 200 days) in manure-amended soil (Franz et al., 2011) all showed absence of mutations in the rpoS gene. The seven strains showing long-term survival with absence of mutations in the rpoS gene had also been characterized before based on an impaired ability to oxidize l-rhamnose, l-glutamic acid
and l-threonine and by an enhanced ability to oxidize propionic acid, α-ketobutyric acid, α-hydroxybutyric acid, methyl β-d-glucoside and l-arabinose (Franz et al., 2011). This is in complete agreement with gene expression studies with rpoS mutants of E. coli O157 showing that these cells have impaired expression regarding fatty acid oxidation PCI-32765 supplier (Dong & Schellhorn, 2009) and that these bacteria have decreased abilities to oxidize propionic acid, α-ketobutyric acid and α-hydroxybutyric acid but an increased ability to oxidize l-threonine (Dong et al., 2009). Recently it was shown that expression of the rpoS gene in E. coli O157 cells in sterile soil was 2.68-fold higher when compared with cells Edoxaban cultured in broth (Duffitt et al., 2011) and that RpoS plays a significant role in the cold stress response of E. coli O157 (Vidovic et al., 2011). Phenotypically, 10/11 short surviving strains with rpoS mutations
showed growth on succinate minimal medium (demonstrating increased nutritional capability). In contrast, 6/7 long-term survivors showed absence of growth on succinate minimal medium. Clearly, the relationship between rpoS status and growth on succinate is not unambiguous, which also has been observed by others (Dong & Schellhorn, 2010). It is likely that some strains use alternative mechanisms to balance stress resistance and metabolic capacity. The acid resistance of the long-term surviving strains without mutations in rpoS was significantly higher than that of the short- to medium-term persisting strains with mutations in rpoS (96.6 vs. 63.5% survival, respectively after 6 h; Student’s t-test, P = 0.0034; Table 1). The results of the current study suggest that E.