Resistance of acid-adapted Escherichia coli O157:H7 to lactoperoxidase and heat in goat milk

Abstract

Survival of Escherichia coli O157:H7 in fermented dairy products has been attributed to acid-adaptation. Acid-adaptation enhances resistance to extreme acid pH and confers cross-protection to heterologous stresses. This study sought to investigate whether acid-adaptation confers cross-protection to lactoperoxidase (LP) system and lactic acid in Tryptone Soy Broth (TSB), and to determine the mechanism of cross-protection. Subsequently, cross-protection of acid-adapted E. coli O157:H7 to the combination of LP activation, heat and lactic acid treatments was determined in fresh goat milk. Finally, the effect of LP activation and E. coli O157:H7 survival on acid production during fermentation of traditional and commercial goat milk was investigated with indigenous cultures and single strain lactic acid bacteria (LAB) respectively. Acid-adapted E. coli O157:H7 strain UP10 showed high acid-resistance at pH levels 4.0 and 5.0 for up to 24 h in TSB at 25°C compared to non-adapted E. coli O157:H7. Acid-adaptation also conferred cross-protection against activated LP system and lactic acid challenge at pH 4.0 and 5.0. Results from fatty acid analysis and quantitative real time Polymerase Chain Reaction (RT-PCR) indicated that sigma S (RpoS)-independent systems were responsible for acid-resistance and cross-protection in TSB. Increase in the saturation of fatty acids, increased expression of outer membrane porin, OmpC, and activation of the glutamate decarboxylase system contributed to acid-resistance and cross-protection. Growth of acid-adapted E. coli O157:H7 strains UP10 and 1062 were inhibited in fresh goat milk compared to the non-adapted cells. Nonetheless, strain 1062 showed better growth and resistance to activated LP in fresh goat milk compared to strain UP10. LP activation alone did not significantly inhibit either acid-adapted or non-adapted E. coli O157:H7, but it sensitized E. coli O157:H7 cells to sub-lethal heat treatment at 55 and 60°C. The combination of heat treatment at 60°C, LP activation and lactic acid at pH 5.0 had a greater inhibitory effect on both acid-adapted and non-adapted E. coli O157:H7, but the acid-adapted strains displayed cross-protection against combined treatments. This indicates that non-adapted E. coli O157:H7 can survive a certain threshold of stresses unscathed. Below that threshold, acid-adaptation may be detrimental to survival. LP activation did not inhibit growth and acid production by single strain and indigenous LAB in the processing of commercial and traditional fermented goat milk products. LP activation however inhibited E. coli O157:H7 in both the commercial and traditional goat milk products although E. coli O157:H7 had become acid-adapted during the fermentation process. E. coli O157:H7 inhibition could be due to the combination of LP activation, low pH, fermentation time and antimicrobial compounds present in the milk or produced by the LAB during milk fermentation. Results from this study suggest that while acid-adaptation protects E. coli O157:H7 under harsh conditions, it can sensitize E. coli O157:H7 to sub-lethal stresses that does not require acid-adaptation for survival. On the other hand, non-adapted E. coli O157:H7 could become acid-adapted in food at mild acid pH which may enhance prolonged survival in such foods.