Transcriptomic response of Escherichia coli O157:H7 to oxidative stress

Abstract

Chlorinated water is commonly used in industrial operations to wash and sanitize fresh-cut, minimally processed produce. Here we compared 42 human outbreak strains that represented nine distinct Escherichia coli O157:H7 genetic lineages (or clades) for their relative resistance to chlorine treatment. A quantitative measurement of resistance was made by comparing the extension of the lag phase during growth of each strain under exposure to sublethal concentrations of sodium hypochlorite in Luria-Bertani or brain heart infusion broth. Strains in clade 8 showed significantly (P < 0.05) higher resistance to chlorine than strains from other clades of E. coli O157:H7. To further explore how E. coli O157:H7 responds to oxidative stress at transcriptional levels, we analyzed the global gene expression profiles of two strains, TW14359 (clade 8; associated with the 2006 spinach outbreak) and Sakai (clade 1; associated with the 1996 radish sprout outbreak), under sodium hypochlorite or hydrogen peroxide treatment. We found over 380 genes were differentially expressed (more than twofold; P < 0.05) after exposure to low levels of chlorine or hydrogen peroxide. Significantly upregulated genes included several regulatory genes responsive to oxidative stress, genes encoding putative oxidoreductases, and genes associated with cysteine biosynthesis, iron-sulfur cluster assembly, and antibiotic resistance. Identification of E. coli O157:H7 strains with enhanced resistance to chlorine decontamination and analysis of their transcriptomic response to oxidative stress may improve our basic understanding of the survival strategy of this human enteric pathogen on fresh produce during minimal processing.

FIG. 1.

Box plot of extended lag phase of 42 E. coli O157:H7 strains under chlorine treatment. The chlorine concentration was made at 5.0 μl sodium hypochlorite stock solution per 1 ml LB broth. Boxes represent the 25th to the 75th percentile of extended lag phase values (in hours) of E. coli O157:H7 strains belonging to clade 1, clade 2, clades 3 and 4, clade 5, clade 6, clade 7, clade 8, and clade 9, respectively. The triangles in the boxes for clades 1 and 8 indicate the mean values of extended lag phases of Sakai and TW14359, respectively.

FIG. 2.

Heat maps comparing the expression profiles of 284 stress-related genes in Sakai (SK) and TW14359 (TW) under chlorine and hydrogen peroxide treatments. Genes that are functionally associated with bacterial stress response were chosen for hierarchical clustering analysis. The fold change value for each gene was converted to the log₂ value. A pseudocolor scale from red to green shows log₂ values ranging from 3.7 (equivalent to 13-fold upregulation) to −2.3 (equivalent to fivefold downregulation). The genes were divided into six functional groups, A to F. (A) Genes directly involved in environmental stress. (B) Genes encoding the Fe-S assembly and repair system, the thiol-redox system, and other Fe-S cluster containing proteins. (C) Genes involved in cysteine synthesis and transport. (D) Genes encoding heat shock and cold shock proteins. (E) Genes encoding outer membrane proteins and transporters. (F) Virulence genes and biofilm-related genes.

FIG. 3.

Correlation of gene expression fold change generated by the DNA chip and qRT-PCR for 12 selected genes. The fold change value was converted to the log₂ values for comparison.