Hyun-Gyun Yuk

Adaptation of Escherichia coli O157:H7 to pH Alters Membrane Lipid Composition, Verotoxin Secretion, and Resistance to Simulated Gastric Fluid Acid

ABSTRACT The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1ω7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 108 CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.

Plant Essential Oils as Active Antimicrobial Agents

Essential oils derived from plants have been recognized for decades to exhibit biological activities, including antioxidant, anticancer, and antimicrobial attributes. Antimicrobial activities of these natural plant materials have been intensively explored in recent years, mainly in response to the overwhelming concern of consumers over the safety of synthetic food additives. Gram-negative organisms are believed to be slightly less sensitive to essential oils than Gram-positive bacteria. Generally, a higher concentration is required to obtain the same efficacy in foods than in synthetic media. The combinations of different types of essential oils or with other food additives have been found to potentially exhibit synergistic if not additive effects. This suggests a cost-efficient and wholesome alternative to both food industry and consumers, at the same time adhering to the hurdle technology in inhibiting proliferation of foodborne pathogens. This review aims to examine the conventional methods commonly used for assessment of antimicrobial activities of essential oils and phytochemicals, the use of these substances as antimicrobials in food products, factors that affect their efficacy, synergism between components or with available food preservatives as well as the challenges and future directions of using essential oils and phytochemicals as natural food preservatives.

Heat Adaptation Alters Escherichia coli O157:H7 Membrane Lipid Composition and Verotoxin Production

ABSTRACT The influence of heat adaptation (growth at 42 and 45°C) on changes in membrane lipid composition and verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57°C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1ω7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion.

Antibacterial effect of light emitting diodes of visible wavelengths on selected foodborne pathogens at different illumination temperatures.

The antibacterial effect of light emitting diodes (LEDs) in the visible region (461, 521 and 642 nm) of the electromagnetic spectrum was investigated on Escherichia coli O157:H7, Salmonella typhimurium, Listeria monocytogenes and Staphylococcus aureus. The irradiances of the 461, 521 and 642 nm LEDs were 22.1, 16 and 25.4 mW/cm², respectively. Bacterial cultures suspended in tryptic soy broth were illuminated by 10-watt LEDs at a distance of 4.5 cm for 7.5h at 20, 15 and 10 °C. Regardless of the bacterial strains, bacterial inactivation was observed with the range of 4.6-5.2 logCFU/ml at 10 and 15 °C after illumination with the 461 nm LED, while illumination with the 521 nm LED resulted in only 1.0-2.0 log reductions after 7.5h. On the other hand, no antibacterial effect was observed using the 642 nm LED treatment. The photodynamic inactivation by 461 and 521 nm LEDs was found to be greater at the set temperatures of 10 and 15 °C than at 20 °C. The D-values for the four bacterial strains at 10 and 15 °C after the illumination of 461 nm LED ranged from 1.29 to 1.74 h, indicating that there was no significant difference in the susceptibility of the bacterial strains to the LED illumination between 10 and 15 °C, except for L. monocytogenes. Regardless of the illumination temperature, sublethal injury was observed in all bacterial strains during illumination with the 461 and the 521 nm LED and the percentage of injured cells increased as the treatment time increased. Thus, the results show that the antibacterial effect of the LEDs was highly dependent on the wavelength and the illumination temperature. This study suggests the potential of 461 and 521 nm LEDs in combination with chilling to be used as a novel food preservation technology.

Overview of Recent Events in the Microbiological Safety of Sprouts and New Intervention Technologies

There has been an increasing trend in consumption of sprouts worldwide due to their widespread availability and high nutrient content. However, microbial contamination of sprouts readily occurs due to the presence of pathogenic bacteria in seeds; and the germination and sprouting process provide optimal conditions for bacterial growth. In recent years, there has been a rise in the number of outbreaks associated with sprouts. These outbreaks occurred mainly in the US, Canada, UK, as well as Europe. More recently in 2011, there were 4 sprout-related outbreaks, with the Escherichia coli O104:H4 outbreak in Germany causing around 50 deaths and 4000 illnesses reported. On top of pathogenic E. coli, Salmonella spp. are often associated with sprout-related foodborne disease outbreaks. The contamination of sprouts has become a worldwide food safety concern. Hence, this review paper covers the outbreaks associated with sprouts, prevalence and characteristics of pathogens contaminating sprouts, their survival and growth, and the source of these pathogens. Physical, biological, and chemical interventions utilized to minimize microbial risks in sprouts are also discussed.

Influence of Acetic, Citric, and Lactic Acids on Escherichia coli O157:H7 Membrane Lipid Composition, Verotoxin Secretion, and Acid Resistance in Simulated Gastric Fluid

The effect of organic acid (acetic, citric, and lactic acids) adaptation at equivalent initial pH values (6.4 and 5.4) on changes in membrane lipid composition, verotoxin concentration, and acid resistance in simulated gastric fluid (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 ATCC 43895 (HEC) and an rpoS mutant of E. coli O157:H7 ATCC 43895 (RM, FRIK 816-3). For HEC, lactic acid-adapted (pH 5.4) cells had the greatest D-value (32.2 min) and acetic acid-adapted (pH 5.4) cells had the smallest D-value (16.6 min) in simulated gastric fluid. For RM, D-values of citric and acetic acid-adapted cells were similar to those for nonadapted cells grown at pH 7.3, but D-values increased from 13.1 to 27.9 min in lactic acid-adapted cells (from pH 7.3 to pH 5.4). For both strains, the ratio of cis-vaccenic to palmitic acids decreased for citric and lactic acid-adapted cells, but the ratio increased for acetic acid-adapted cells at pH 5.4. Organic acid-adapted cells produced less total verotoxin than did nonadapted cells at approximately 10(8) CFU/ml. Extracellular verotoxin concentration proportionally decreased with decreasing pH for both HEC and RM. Changes in membrane lipid composition, verotoxin concentration, and acid resistance in HEC and RM were dependent on both pH and organic acid. Deletion of the rpoS gene did not affect these changes but did decrease acid resistance in citric acid-adapted cells. Results indicate that decreased membrane fluidity may have caused increased acid resistance and decreased verotoxin secretion.

Growth temperature alters Salmonella Enteritidis heat/acid resistance, membrane lipid composition and stress/virulence related gene expression

The influence of growth temperature (10, 25, 37, and 42 °C) on the survival of Salmonella Enteritidis in simulated gastric fluid (SGF; pH=2.0) and during heat treatment (54, 56, 58, and 60 °C), on the membrane fatty acid composition, as well as on stress-/virulence-related gene expression was studied. Cells incubated at temperatures lower or higher than 37 °C did not increase their acid resistance, with the maximum D-value of 3.07 min in cells grown at 37 °C; while those incubated at higher temperature increased their heat resistance, with the maximum D60 °C-values of 1.4 min in cells grown at 42 °C. A decrease in the ratio of unsaturated to saturated fatty acids was observed as the growth temperature increased. Compared to the control cells grown at 37 °C, the expression of rpoS was 16.5- and 14.4-fold higher in cells cultivated at 10 and 25 °C, respectively; while the expression of rpoH was 2.9-fold higher in those cultivated at 42 °C. The increased expression of stress response gene rpoH and the decreased ratio of unsaturated to saturated fatty acids correlated with the greater heat resistance of bacteria grown at 42 °C; while the decreased expression of stress response gene rpoS at 42 °C might contribute to the decrease in acid resistance. Virulence related genes-spvR, hilA, avrA-were induced in cells cultivated at 42 °C, except sefA which was induced in the control cells. This study indicates that environmental temperature may affect the virulence potential of S. Enteritidis, thus temperature should be well controlled during food storage.

Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings

Conventional culture detection methods are time consuming and labor-intensive. For this reason, an alternative rapid method combining real-time PCR and immunomagnetic separation (IMS) was investigated in this study to detect both healthy and heat-injured Salmonella Typhimurium on raw duck wings. Firstly, the IMS method was optimized by determining the capture efficiency of Dynabeads(®) on Salmonella cells on raw duck wings with different bead incubation (10, 30 and 60 min) and magnetic separation (3, 10 and 30 min) times. Secondly, three Taqman primer sets, Sal, invA and ttr, were evaluated to optimize the real-time PCR protocol by comparing five parameters: inclusivity, exclusivity, PCR efficiency, detection probability and limit of detection (LOD). Thirdly, the optimized real-time PCR, in combination with IMS (PCR-IMS) assay, was compared with a standard ISO and a real-time PCR (PCR) method by analyzing artificially inoculated raw duck wings with healthy and heat-injured Salmonella cells at 10(1) and 10(0) CFU/25 g. Finally, the optimized PCR-IMS assay was validated for Salmonella detection in naturally contaminated raw duck wing samples. Under optimal IMS conditions (30 min bead incubation and 3 min magnetic separation times), approximately 85 and 64% of S. Typhimurium cells were captured by Dynabeads® from pure culture and inoculated raw duck wings, respectively. Although Sal and ttr primers exhibited 100% inclusivity and exclusivity for 16 Salmonella spp. and 36 non-Salmonella strains, the Sal primer showed lower LOD (10(3) CFU/ml) and higher PCR efficiency (94.1%) than the invA and ttr primers. Moreover, for Sal and invA primers, 100% detection probability on raw duck wings suspension was observed at 10(3) and 10(4) CFU/ml with and without IMS, respectively. Thus, the Sal primer was chosen for further experiments. The optimized PCR-IMS method was significantly (P=0.0011) better at detecting healthy Salmonella cells after 7-h enrichment than traditional PCR method. However there was no significant difference between the two methods with longer enrichment time (14 h). The diagnostic accuracy of PCR-IMS was shown to be 98.3% through the validation study. These results indicate that the optimized PCR-IMS method in this study could provide a sensitive, specific and rapid detection method for Salmonella on raw duck wings, enabling 10-h detection. However, a longer enrichment time could be needed for resuscitation and reliable detection of heat-injured cells.

Efficacy of chlorine and peroxyacetic acid on reduction of natural microflora, Escherichia coli O157:H7, Listeria monocyotgenes and Salmonella spp. on mung bean sprouts

Sprouts-related outbreaks have risen due to increased raw sprouts consumption. To minimize such cases, chemical sanitations are applied. While chlorine is commonly used, concerns with its effectiveness and health implication have prompted researchers to seek alternatives. Peroxyacetic acid (PAA) has shown efficacy in inactivating foodborne pathogens on fresh vegetables, and hence could be considered as an alternative. Thus, the objective of this study was to compare the efficacy of chlorine and PAA in inactivating Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and natural microflora on mung bean sprouts. Resistance of non- and acid-adapted pathogens to these sanitizer treatments was also evaluated. Un-inoculated and inoculated sprouts were treated with chlorine at 106, 130 and 170 ppm and PAA at 25, 51 and 70 ppm for 90 and 180 s at room temperature. Overall, the greater log reductions were obtained with the increase in the sanitizer concentration. For 180 s, chlorine treatment at 170 ppm reduced 2.0, 1.3, 1.5, 0.9-logs and PAA treatment at 70 ppm resulted in 2.3, 1.8, 2.1, 1.1-log reductions for non-adapted E. coli O157:H7, L. monocytogenes, Salmonella spp., and natural microflora, respectively. These results revealed that the efficacy of PAA was significantly better than or similar to that of chlorine. For acid-adapted cells, these sanitizer treatments were less effective with the ranges of 1.0-1.2-log reductions for chlorine and 1.1-1.6-log reductions for PAA compared to non-adapted cells, indicating that acid-adapted cells were more resistant to the sanitizing treatment. These data suggest that PAA may replace chlorine in the disinfection of mung bean sprouts and that acid-adapted pathogens should be used to design an effective sanitizing strategy.

Detection of salmonella by flow-through immunocapture real-time PCR in selected foods within 8 hours.

This study investigated flow-through immunocapture (FTI), using the Pathatrix device, followed by plating on xylose lysine desoxycholate (XLD) agar (FTI-XLD) or analysis by real-time PCR (FTI-PCR) for the detection of Salmonella on smooth tomato surfaces and in potato salad and ground beef within 8 h. Food samples were inoculated with an appropriate dilution of a five-serovar Salmonella cocktail and enriched for 5 h. Following enrichment, samples were analyzed by the FTI-XLD and FTI-PCR methods. Food samples were also analyzed by a modified U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Salmonella culture method for comparison. Salmonella inoculated at 10(0) CFU per tomato or 10(0) CFU/25 g was detected by the FTI-XLD method in 6, 8, and 4 of 10 samples for tomatoes, potato salad, and ground beef, respectively. Salmonella inoculated at 10(0) CFU per tomato or 10(0) CFU/25 g was detected by the FTI-PCR method in 8, 9, and 9 of 10 samples for tomatoes, potato salad, and ground beef, respectively. The FTI-PCR method achieved significantly higher (P < 0.05) detection of Salmonella on tomatoes, whereas the FTI-XLD method achieved significantly lower (P < 0.05) detection of Salmonella in ground beef when compared with the modified BAM Salmonella culture method; however, all other comparisons to the modified BAM method were not significantly different. The FTI-XLD method demonstrated the ability to isolate presumptive Salmonella colonies up to 48 hfaster than did the modified BAM Salmonella culture method.