Some Like It Hot: Heat Resistance of Escherichia coli in Food

Abstract

Heat treatment and cooking are common interventions for reducing the numbers of vegetative cells and eliminating pathogenic microorganisms in food. Current cooking method requires the internal temperature of beef patties to reach 71°C. However, some pathogenic Escherichia coli such as the beef isolate E. coli AW 1.7 are extremely heat resistant, questioning its inactivation by current heat interventions in beef processing. To optimize the conditions of heat treatment for effective decontaminations of pathogenic E. coli strains, sufficient estimations, and explanations are necessary on mechanisms of heat resistance of target strains. The heat resistance of E. coli depends on the variability of strains and properties of food formulations including salt and water activity. Heat induces alterations of E. coli cells including membrane, cytoplasm, ribosome and DNA, particularly on proteins including protein misfolding and aggregations. Resistant systems of E. coli act against these alterations, mainly through gene regulations of heat response including EvgA, heat shock proteins, σ^E and σ^S, to re-fold of misfolded proteins, and achieve antagonism to heat stress. Heat resistance can also be increased by expression of key proteins of membrane and stabilization of membrane fluidity. In addition to the contributions of the outer membrane porin NmpC and overcome of osmotic stress from compatible solutes, the new identified genomic island locus of heat resistant performs a critical role to these highly heat resistant strains. This review aims to provide an overview of current knowledge on heat resistance of E. coli, to better understand its related mechanisms and explore more effective applications of heat interventions in food industry.

Keywords: Escherichia coli; VTEC; food processing; heat resistance; locus of heat resistance; protein.

FIGURE 1

Heat resistance of Escherichia coli. Data shown are log₁₀ value of D₆₀ (min) of 144 strains collected from past publications: three values of K-12 strains (Chung et al., 2007; Jin et al., 2008; Dlusskaya et al., 2011), 125 of other strains of E. coli (Juneja and Marmer, 1999; Dlusskaya et al., 2011; Enache et al., 2011; Pleitner et al., 2012; Liu, 2015; Mercer et al., 2015), 2 D-values of strains after overexpression of heat shock proteins (HSP) (Hauben et al., 1997; Ruan et al., 2011), 7 D-values of strains after adaptation to salt or acid stress (Buchanan and Edelson, 1999; Pleitner et al., 2012; Garcia-Hernandez et al., 2015), 5 D-values of LHR positive strains (Pleitner et al., 2012; Mercer et al., 2015), and 2 D-values of strains treated by dry heat (Neetoo and Chen, 2011; Kim et al., 2015).

FIGURE 2

Heat effects on cell membranes and attributes to heat resistance of E. coli. Extracellular polysaccharides including colanic acid forms a thick mucoid matrix on cell surfaces and provide protection of cells; disruption of wsc genes required for colanic acid biosynthesis substantially decreased heat resistance when compared to its parental strain (Whitfield and Valvano, 1993; Mao et al., 2001). LPS is a barrier to prevent rapid penetration of hydrophobic molecules, and is stabilized by divalent cations Mg²⁺ and Ca²⁺ against heat or pressure stress (Hitchener and Egan, 1977; Vaara, 1992; Hauben et al., 1998; Li et al., 2016). The solute transport proteins and the outer membrane porin NmpC contribute to heat resistance of E. coli AW1.7 (Ruan et al., 2011). Addition of antimicrobials including chitosan decreased the heat resistance due to the increased permeability of outer membrane (Liu, 2015). The master transcriptional regulator evgA is a cytoplasmic protein that increased heat resistance through activation of genes involved in periplasmic functions (Christ and Chin, 2008). The alternative sigma factors σ^S and σ^E also influence the properties of cell envelope (Lange and Hengge-Aronis, 1991; Bukau, 1993). LPS proteins SurA and PpiD lead to overall reduction in the level and folding of outer membrane proteins, consequently induce the periplamic heat shock response (Missiakas et al., 1996; Dartigalongue and Raina, 1998). Incorporating more saturated fatty acids such as palmitic acid and cyclopropane fatty acids (CFAs) into membrane lipids antagonizes the heat-induced increase in fluidity and achieves an ideal physical state of membrane (Katsui et al., 1981; Ruan et al., 2011; Chen and Gänzle, 2016). Disruption of cfa coding for CFA synthase of E. coli AW1.7 and MG1655 induced accumulation of the unsaturated fatty acid C16:1 and C18:1 in membrane lipids, consequently reducing the heat resistance of them (Chen and Gänzle, 2016).

FIGURE 3

Cytoplasmic determinants of heat resistance in E. coli. (A) Preventions of protein aggregation. Heat enhances misfolding of proteins and consequently induces protein aggregation. General stress response factors σ^S, σ^H, and σ^E, as well as some small HSPs can suppress protein aggregation (Parsell and Lindquist, 1993; Landini et al., 2014). Small HSPs IbpA and IbpB bind to misfolded proteins and thus contribute to disaggregation of during sublethal heat shock (Laskowska et al., 1996; Veinger et al., 1998; Kuczyñska-Wiśnik et al., 2002). The DnaK system acts together with ClpB to prevent protein aggregation induced by heat (Mogk et al., 1999, 2003). (B) Compatible solutes accumulation induced by salt contributes to heat resistance through overcoming osmotic stress and stabilizing ribosomes (Ramos et al., 1997; Lamosa et al., 2000; Pleitner et al., 2012). Accumulation of amino acids including glycine betaine and proline as major cytoplasmic solutes, and the accumulation of carbohydrates including glucose and trehalose occurred in response to the addition of NaCl in E. coli, resulting in increased thermal stability of ribosomes during heat treatment (Pleitner et al., 2012). Mannosylglycerate and diglyerol phosphate protect proteins during heat treatment (Ramos et al., 1997; Lamosa et al., 2000). (C) Mitigation of oxidative stress. Oxidative stress induced by heat damages intracellular components including proteins, ribosomes and DNA. The general stress response factor σ^S and the DNA binding protein dps acts against oxidative stress (Choi et al., 2000; Zhao et al., 2002; Landini et al., 2014). Pyruvate and catalase contribute to recovery of sublethally injured cells after heat treatments (Czechowicz et al., 1996; Mizunoe et al., 2000). (D) Regulation of the locus of heat resistance (LHR). LHR is unique genomic island contributing to extreme heat resistance in E. coli (Mercer et al., 2015). LHR contains 16 predicted ORF encoding small HSPs (sHSP, Orf2, and Orf7), hypothetical proteins yfdX family (Orf8 and Orf9), proteases (Orf3, Orf15, and Orf16), thioredoxin (Orf12), and sodium/hydrogen antiporters (Orf13), accordingly contributing to heat shock response, osmotic stress response, turnover of misfolded or disaggregation proteins, oxidative stress response, osmotic and heat stress response, respectively (Mercer et al., 2015; Lee et al., 2016). Predicted functions of LHR are indicated by dashed lines.