Influence of Antibiotic, Acid, and Salt Stress on Resistance of Escherichia Coli O157:H7

Abstract

The objective of this study was to understand the impact of antibiotic, acid, and salt on the stress response of Escherichia coli O157:H7. E. coli cells were exposed to sublethal levels of each stress for 4 hours and post stress tolerance and virulence factor production were evaluated. Heat tolerance (56oC) was determined using the capillary tube method, and simulated gastric fluid (SGF, pH 1.5) survival was used to assess acid tolerance. Stressed and control cells were evaluated in triplicate. In the first part of the study, the impact of sublethal levels of antibiotic on virulence factor production, survival in SGF, and heat tolerance (56oC) were studied. The minimum inhibitory concentration (MIC) for three antibiotics (trimethoprim, ampicillin, and ofloxacin) was determined for two E. coli O157:H7 strains using the dilution series method. Subsequently, virulence factor production (Stx, Hly, EaeA) was evaluated by the creation of lacZ gene fusions followed by use of the Miller assay (a β-galactosidase assay). Heat tolerance and SGF survival following antibiotic exposure decreased compared to control cells (p<0.05). Exposure to ofloxacin increased Stx and Eae production (p<0.05). Exposure to ampicillin or trimethoprim increased Eae production (p<0.05). Hly production decreased when cells were stressed with ampicillin, but increased following trimethoprim stress (p<0.05). Antibiotics can increase E. coli O157:H7 virulence factor production, but do not produce a cross-protective response to heat or decreased pH.

In the second part of the study, the impact of acid and salt exposure on SGF (pH 1.5) survival, heat tolerance (56oC), survival in 14% salt, and survival in 14% salt at pH 5.0 (acidified with lactic acid or HCl) of two E. coli O157:H7 strains (EHEC1, EHEC2) and a nonpathogenic E. coli (NPEC) strain was studied. Acid adaptation of the E. coli cells was achieved by overnight growth in TSB with 1% glucose to a final pH of 4.8. Acid exposure was done in TSB acidified to pH 5.0 with malic, lactic, citric, or hydrochloric acid (HCl). Sub-lethal salt exposure was conducted at 4% (w/v) salt concentration. Acid adaptation of E. coli O157:H7 enhanced SGF survival, heat tolerance, and NaCl survival of the cells with and without additional acid or salt stress (p<0.05). Among the acids used in acid stress studies, lactic acid had the highest impact on the development of resistance to post stress tolerance (p<0.05). Acidification of 14% NaCl TSB to pH 5.0 increased the resistance of cells following sublethal stresses, especially when acidified with lactic acid (p<0.05). Salt stress enhanced SGF tolerance of acid adapted cells of EHEC2 and NPEC (p<0.05) but not EHEC1 (p>0.05). Heat tolerance of EHEC2 and NPEC decreased following salt stress, regardless of acid adaptation (p<0.05). Acid adaptation and acid and salt stress of E. coli produce protection against further challenges, which should be taken into consideration when producing minimally processed foods.