Yoon-Kyung Chung

Genes of Escherichia coli O157:H7 That Are Involved in High-Pressure Resistance

ABSTRACT Seventeen Escherichia coli O157:H7 strains were treated with ultrahigh pressure at 500 MPa and 23 ± 2°C for 1 min. This treatment inactivated 0.6 to 3.4 log CFU/ml, depending on the strain. The diversity of these strains was confirmed by pulsed-field gel electrophoresis (PFGE) analysis, and there was no apparent association between PFGE banding patterns and pressure resistance. The pressure-resistant strain E. coli O157:H7 EC-88 (0.6-log decrease) and the pressure-sensitive strain ATCC 35150 (3.4-log decrease) were treated with a sublethal pressure (100 MPa for 15 min at 23 ± 2°C) and subjected to DNA microarray analysis using an E. coli K-12 antisense gene chip. High pressure affected the transcription of many genes involved in a variety of intracellular mechanisms of EC-88, including the stress response, the thiol-disulfide redox system, Fe-S cluster assembly, and spontaneous mutation. Twenty-four E. coli isogenic pairs with mutations in the genes regulated by the pressure treatment were treated with lethal pressures at 400 MPa and 23 ± 2°C for 5 min. The barotolerance of the mutants relative to that of the wild-type strains helped to explain the results obtained by DNA microarray analysis. This study is the first report to demonstrate that the expression of Fe-S cluster assembly proteins and the fumarate nitrate reductase regulator decreases the resistance to pressure, while sigma factor (RpoE), lipoprotein (NlpI), thioredoxin (TrxA), thioredoxin reductase (TrxB), a trehalose synthesis protein (OtsA), and a DNA-binding protein (Dps) promote barotolerance.

Inactivation of Barotolerant Listeria monocytogenes in Sausage by Combination of High-Pressure Processing and Food-Grade Additives

Food-grade additives were used to enhance the efficacy of high-pressure processing (HPP) against barotolerant Listeria monocytogenes. Three strains of L. monocytogenes (Scott A, OSY-8578, and OSY-328) were compared for their sensitivity to HPP, nisin, tert-butylhydroquinone (TBHQ), and their combination. Inactivation of these strains was evaluated in 0.2 M sodium phosphate buffer (pH 7.0) and commercially sterile sausage. A cell suspension of L. monocytogenes in buffer (10(9) CFU/ml) was treated with TBHQ at 100 ppm, nisin at 100 IU/ml, HPP at 400 MPa for 5 min, and combinations of these treatments. Populations of strains Scott A, OSY-8578, and OSY-328 decreased 3.9, 2.7, and 1.3 log with HPP alone and 6.4, 5.2, and 1.9 log with the HPP-TBHQ combination, respectively. Commercially sterile sausage was inoculated with the three L. monocytogenes strains (10(6) to 10(7) CFU/g) and treated with selected combinations of TBHQ (100 to 300 ppm), nisin (100 and 200 ppm), and HPP (600 MPa, 28 degrees C, 5 min). Samples were enriched to detect the viability of the pathogen after the treatments. Most of the samples treated with nisin, TBHQ, or their combination were positive for L. monocytogenes. HPP alone resulted in a modest decrease in the number of positive samples. L. monocytogenes was not detected in any of the inoculated commercial sausage samples after treatment with HPP-TBHQ or HPP-TBHQ-nisin combinations. These results suggest that addition of TBHQ or TBHQ plus nisin to sausage followed by in-package pressurization is a promising method for producing Listeria-free ready-to-eat products.

Characterization and Application of Enterocin RM6, a Bacteriocin from Enterococcus faecalis

Use of bacteriocins in food preservation has received great attention in recent years. The goal of this study is to characterize enterocin RM6 from Enterococcus faecalis OSY-RM6 and investigate its efficacy against Listeria monocytogenes in cottage cheese. Enterocin RM6 was purified from E. faecalis culture supernatant using ion exchange column, multiple C18-silica cartridges, followed by reverse-phase high-performance liquid chromatography. The molecular weight of enterocin RM6 is 7145.0823 as determined by mass spectrometry (MS). Tandem mass spectrometry (MS/MS) analysis revealed that enterocin RM6 is a 70-residue cyclic peptide with a head-to-tail linkage between methionine and tryptophan residues. The peptide sequence of enterocin RM6 was further confirmed by sequencing the structural gene of the peptide. Enterocin RM6 is active against Gram-positive bacteria, including L. monocytogenes, Bacillus cereus, and methicillin-resistant Staphylococcus aureus (MRSA). Enterocin RM6 (final concentration in cottage cheese, 80 AU/mL) caused a 4-log reduction in population of L. monocytogenes inoculated in cottage cheese within 30 min of treatment. Therefore, enterocin RM6 has potential applications as a potent antimicrobial peptide against foodborne pathogens in food.

Inactivation of barotolerant strains of Listeria monocytogenes and Escherichia coli O157:H7 by ultra high pressure and tert-butylhydroquinone combination

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23±2°C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (≥ 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.

Proposed mechanism of inactivating Escherichia coli O157:H7 by ultra‐high pressure in combination with tert‐butylhydroquinone

Aims:  Investigating mechanisms of lethality enhancement when Escherichia coli O157:H7, and selected E. coli mutants, were exposed to tert‐butylhydroquinone (TBHQ) during ultra‐high pressure (UHP) treatment.