Christopher H. Sommers

Suspending Lettuce Type Influences Recoverability and Radiation Sensitivity of Escherichia coli O157:H7†

An outbreak strain of Escherichia coli O157:H7 was inoculated onto closely related but structurally distinct types of lettuce (Lactuca sativa): Boston (butterhead lettuce), iceberg (crisphead lettuce), and green leaf and red leaf (colored variants of looseleaf lettuce). The E. coli O157:H7 was inoculated either onto the surface of cut leaf pieces or into a homogenized leaf suspension. Samples were gamma irradiated, and the radiation sensitivity of the inoculated bacteria was expressed as a D-value (the amount of ionizing radiation necessary to reduce the bacterial population by 90% [kGy]). The recovery of bacteria from nonirradiated leaf pieces was also measured. When inoculated onto the leaf surface, E. coli O157:H7 had significantly stronger radiation sensitivity on red leaf lettuce (D = 0.119 +/- 0.004 [standard error]) and green leaf lettuce (D = 0.123 +/- 0.003) than on iceberg lettuce (D = 0.136 +/- 0.004) or Boston lettuce (D = 0.140 +/- 0.003). When E. coli O157:H7 was inoculated into a homogenized leaf suspension, its sensitivity was significantly stronger on iceberg lettuce (D = 0.092 +/- 0.002) than on green leaf lettuce (D = 0.326 +/- 0.012), Boston lettuce (D = 0.331 +/- 0.009), or red leaf lettuce (D = 0.339 +/- 0.010), with a threefold difference. Significantly fewer bacteria were recovered from the surface of iceberg lettuce than from the surfaces of the other types of lettuce examined. Following radiation doses of up to 0.5 kGy, the texture (maximum shear strength) of lettuce leaves was measured along the midrib and along the leaf edge for each type of lettuce. There was no meaningful change in texture for any type of lettuce for either leaf section examined at any dose up to 0.5 kGy. These data show (i) that relatively subtle differences between lettuce types can significantly influence the radiation sensitivity of associated pathogenic bacteria and (ii) that doses of up to 0.5 kGy do not soften lettuce leaves.

Radiation (Gamma) Resistance and Postirradiation Growth of Listeria monocytogenes Suspended in Beef Bologna Containing Sodium Diacetate and Potassium Lactate

Listeria monocytogenes, a psychrotrophic foodborne pathogen, is a frequent postprocessing contaminant of ready-to-eat (RTE) meat products, including frankfurters and bologna. Ionizing radiation can eliminate L. monocytogenes from RTE meats. When they are incorporated into fine-emulsion sausages, sodium diacetate (SDA) and potassium lactate (PL) mixtures inhibit the growth of L. monocytogenes. The radiation resistance of L. monocytogenes, and its ability to proliferate during long-term refrigerated storage (9 degrees C), when inoculated into beef bologna that contained 0% SDA-0% PL, 0.07% SDA-1% PL, and 0.15% SDA-2% PL, were determined. The radiation doses required to eliminate 90% of the viable L. monocytogenes cells were 0.56 kGy for bologna containing 0% SDA-0% PL, 0.53 kGy for bologna containing 0.07% SDA-1% PL, and 0.46 kGy for bologna containing 0.15% SDA-2% PL. L. monocytogenes was able to proliferate on bologna containing 0% SDA-0% PL during refrigerated storage, but the onset of proliferation was delayed by the addition of the SDA-PL mixtures. An ionizing radiation dose of 3.0 kGy prevented the proliferation of L. monocytogenes and background microflora in bologna containing 0.07% SDA-1% PL and in bologna containing 0.15% SDA-2% PL over 8 weeks of storage at 9 degrees C. Little effect on lipid oxidation and color of the control bologna, or bologna containing SDA-PL mixtures, was observed upon irradiation at either 1.5 or 3.0 kGy.

Radiation sensitization and postirradiation proliferation of Listeria monocytogenes on ready-to-eat deli meat in the presence of pectin-nisin films.

In this study, the ability of pectin-nisin films in combination with ionizing radiation to eliminate Listeria monocytogenes and inhibit its postirradiation proliferation was evaluated. Pectin films containing 0.025% nisin were made by extrusion. The surface of a ready-to-eat turkey meat sample was inoculated with L. monocytogenes at 10(6) CFU/cm2 and covered with a piece of pectin-nisin film. The samples were vacuum packaged and irradiated at 0, 1, and 2 kGy. The treated samples were stored at 10 degrees C and withdrawn at 0, 1, 2, 4, and 8 weeks for microbial analysis. Reductions in L. monocytogenes viability of 1.42, 1.56, 2.85, 3.78, and 5.36 log CFU/cm2 were achieved for the treatments of 1 kGy, pectin-nisin film, 2 kGy, 1 kGy plus pectin-nisin film, and 2 kGy plus pectin-nisin film, respectively. The greatest reduction (5.5 log CFU/cm2) was observed at 1 week for the 2 kGy plus pectin-nisin film treatment, suggesting that nisin was further released from the film to the surface of meat samples. Pectin-nisin films used in this study did not prevent but did significantly slow (P < 0.05) the proliferation of the L. monocytogenes cells that survived irradiation during 8 weeks of storage at 10 degrees C. These data indicate the potential use of pectin-nisin films alone or in combination with ionizing radiation for preventing listeriosis due to postprocessing contamination of ready-to-eat meat products.

Effect of citric acid on the radiation resistance of Listeria monocytogenes and frankfurter quality factors.

Listeria monocytogenes is a common contaminant of ready-to-eat meat products, including frankfurters. Ionizing (gamma) radiation can eliminate L. monocytogenes from frankfurters. Citric acid (CA) is an antioxidant synergist and anti-microbial agent that can be applied to the surfaces of cured meat products prior to packaging. The effect of CA on the radiation resistance of L. monocytogenes that was surface-inoculated onto frankfurters was determined. The D(10) values, the radiation doses required to inactivate 90% of viable L. monocytogenes, were 0.61, 0.60, 0.54, and 0.53 kGy, on frankfurters dipped in 0, 1, 5 or 10% CA solution, respectively. CA, although an antioxidant synergist, did not increase antioxidant activity (AA) on frankfurter surfaces as determined by the ferric reducing antioxidant power (FRAP) assay. Lipid oxidation, as determined by the Thiobarbituric acid reactive substances (TBARS) assay, was not affected by CA or ionizing radiation. Color of frankfurters, determined by Hunter L, a, b, indicated that ionizing radiation induced a small, but visually imperceptible, loss of redness (a-value). Frankfurter firmness, as measured by maximum shear force, was not affected by ionizing radiation or CA. CA enhanced the lethality of ionizing radiation without negatively impacting frankfurter color, lipid oxidation, firmness, or antioxidant activity.

Complex Formation with Damage Recognition Protein Rad14 Is Essential for Saccharomyces cerevisiae Rad1-Rad10 Nuclease To Perform Its Function in Nucleotide Excision Repair In Vivo

ABSTRACT Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1Δ mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.

Irradiation Inactivation of Four Salmonella Serotypes in Orange Juices with Various Turbidities

Reconstituted orange juice inoculated with Salmonella Anatum, Salmonella Infantis, Salmonella Newport, or Salmonella Stanley was treated with gamma radiation at 2 degrees C. To determine the relationship between juice antioxidant power and Dgamma (dose required to achieve 90% mortality), juice solids were removed prior to inoculation by centrifugation and/or filtration to create juice preparations of varying turbidity. In unadulterated orange juice, Salmonella Anatum (Dgamma = 0.71 kGy) was significantly more resistant than the other species tested. Salmonella Newport (Dgamma = 0.48 kGy) and Salmonella Infantis (Dgamma = 0.35 kGy) were significantly different, while Salmonella Stanley (Dgamma = 0.38 kGy) was intermediate between the two. Neither the resistance of each isolate nor the pattern of relative resistance among isolates was altered in reduced turbidity juice preparations. Although total antioxidant power was associated with the level of juice solids resuspended in phosphate buffer, antioxidant power was not significantly associated with turbidity in the juice preparations or with Dgamma of any species. The variable resistance to irradiation of the Salmonella isolates suggests this as a more significant factor than turbidity or antioxidant power in designing antimicrobial juice irradiation protocols.

Ionizing Radiation Sensitivity of Listeria monocytogenes ATCC 49594 and Listeria innocua ATCC 51742 Inoculated on Endive (Cichorium endiva)

Ionizing radiation inactivates the pathogenic bacteria that can contaminate leafy green vegetables. Leaf pieces and leaf homogenate of endive (Cichorium endiva) were inoculated with the pathogen Listeria monocytogenes (ATCC 49594) or Listeria innocua (ATCC 51742), a nonpathogenic surrogate bacterium. The radiation sensitivity of the two strains was similar, although L. innocua was more sensitive to the type of suspending leaf preparation. During refrigerated storage after irradiation, the population of L. monocytogenes on inoculated endive was briefly suppressed by 0.42 kilogray (kGy), a dose calibrated to achieve a 99% reduction. However, the pathogen regrew after 5 days until it exceeded the bacterial levels on the control after 19 days in storage. Treatment with 0.84 kGy, equivalent to a 99.99% reduction, suppressed L. monocytogenes throughout refrigerated storage. Doses up to 1.0 kGy had no significant effect on the color of endive leaf material, regardless of whether taken from the leaf edge or the leaf midrib. The texture of leaf edge material was unaffected by doses up to 1.0 kGy, whereas the maximum dose tolerated by leaf midrib material was 0.8 kGy. These results show that endive leaves may be treated with doses sufficient to achieve at least a 99.99% reduction of L. monocytogenes with little or no impact on the products texture or color.

Gamma irradiation of fine-emulsion sausage containing sodium diacetate.

Listeria monocytogenes, a psychrotrophic foodborne pathogen, is a frequent postprocess contaminant of ready-to-eat (RTE) meat products, including frankfurters and bologna. Ionizing radiation can eliminate L. monocytogenes from RTE meats. Sodium diacetate (SDA) incorporated into fine-emulsion sausages inhibits the growth of L. monocytogenes. Irradiation of L. monocytogenes suspended in SDA solutions resulted in synergistic reductions of the microorganism. L. monocytogenes populations were reduced by > 9 log10 units at a radiation dose of 1.5 kGy when suspended in 0.125% SDA solution. In contrast, the D10-values (the ionizing radiation doses required to reduce the population by 90%) were 0.58, 0.59, 0.57, and 0.53 kGy for L. monocytogenes populations suspended in emulsions containing 0, 0.125, 0.25, and 0.5% SDA, respectively. The D10-values for L. monocytogenes surface inoculated onto frankfurters dipped in 0, 0.125, 0.25, and 0.5% SDA solutions were 0.58, 0.53, 0.54, and 0.52 kGy, respectively. Postirradiation growth of L. monocytogenes suspended in beef bologna emulsion at 9 degrees C was dependent on SDA concentration and ionizing radiation dose. Very small, but statistically significant, changes in bologna redness, lipid oxidation, and shear force were observed for the beef bologna emulsion with the highest SDA concentration (0.5%) and irradiation dose (3.0 kGy). SDA can inhibit the proliferation of L. monocytogenes surviving the irradiation process with minimal impact on fine-emulsion sausage color, lipid oxidation, and firmness when used within regulatory limits.

Effect of native microflora, waiting period, and storage temperature on Listeria monocytogenes serovars transferred from cantaloupe rind to fresh-cut pieces during preparation.

The most recent outbreak of listeriosis linked to consumption of fresh-cut cantaloupes indicates the need to investigate the behavior of Listeria monocytogenes in the presence of native microflora of cantaloupe pieces during storage. Whole cantaloupes were inoculated with L. monocytogenes (10(8)-CFU/ml suspension) for 10 min and air dried in a biosafety cabinet for 1 h and then treated (unwashed, water washed, and 2.5% hydrogen peroxide washed). Fresh-cut pieces (∼3 cm) prepared from these melons were left at 5 and 10°C for 72 h and room temperature (20°C) for 48 h. Some fresh-cut pieces were left at 20°C for 2 and 4 h and then refrigerated at 5°C. Microbial populations of fresh-cut pieces were determined by the plate count method or enrichment method immediately after preparation. Aerobic mesophilic bacteria, yeast and mold of whole melon, and inoculated populations of L. monocytogenes on cantaloupe rind surfaces averaged 6.4, 3.3, and 4.6 log CFU/cm(2), respectively. Only H(2)O(2) (2.5%) treatment reduced the aerobic mesophilic bacteria, yeast and mold, and L. monocytogenes populations to 3.8, 0.9, and 1.8 log CFU/cm(2), respectively. The populations of L. monocytogenes transferred from melon rinds to fresh-cut pieces were below detection but were present by enrichment. Increased storage temperatures enhanced the lag phases and growth of L. monocytogenes. The results of this study confirmed the need to store fresh-cut cantaloupes at 5°C immediately after preparation to enhance the microbial safety of the fruit.

Ultraviolet light (254 nm) inactivation of Listeria monocytogenes on frankfurters that contain potassium lactate and sodium diacetate.

Listeria monocytogenes, a psychrotrophic foodborne pathogen, is an occasional postprocess contaminant on ready-to-eat meat (RTE) products including frankfurters. Ultraviolet C light (UVC) is an FDA-approved technology for the decontamination of food surfaces. In this study, the ability of UVC to inactivate L. monocytogenes on frankfurters that contained potassium lactate (PL) and sodium diacetate (SDA), either before or after packaging, was investigated. UVC irradiation of frankfurters that were surface-inoculated with L. monocytogenes resulted in a 1.31, 1.49, and 1.93 log reduction at doses of 1, 2, and 4 J/cm(2), respectively. UVC treatment had no effect on frankfurter color or texture at UVC doses up to 4 J/cm(2). Frankfurter meat treated with UVC doses up to 16 J/cm(2) did not increase mutagenesis in bacterial or human cells, either with or without exogenous metabolic activation. UVC treatment of single-layer frankfurter packs at a dose of 2 J/cm(2) resulted in a 0.97 (+/- 0.14) log reduction of L. monocytogenes. Following 8 wk of refrigerated storage L. monocytogenes levels decreased by only 0.65 log in non-UVC-treated frankfurter packs compared with 2.5 log in the UVC-treated packs. Because the numbers of L. monocytogenes associated with contaminations of ready-to-eat meats are typically very low, the use of UVC in combination with potassium lactate and sodium diacetate has the potential to reduce the number of frankfurter recalls and foodborne illness outbreaks.