Bradley P. Marks

Stress, Sublethal Injury, Resuscitation, and Virulence of Bacterial Foodborne Pathogens

Environmental stress and food preservation methods (e.g., heating, chilling, acidity, and alkalinity) are known to induce adaptive responses within the bacterial cell. Microorganisms that survive a given stress often gain resistance to that stress or other stresses via cross-protection. The physiological state of a bacterium is an important consideration when studying its response to food preservation techniques. This article reviews the various definitions of injury and stress, sublethal injury of bacteria, stresses that cause this injury, stress adaptation, cellular repair and response mechanisms, the role of reactive oxygen species in bacterial injury and resuscitation, and the potential for cross-protection and enhanced virulence as a result of various stress conditions.

Inactivation of Salmonella and Listeria in ground chicken breast meat during thermal processing.

Thermal inactivation of six Salmonella spp. and Listeria innocua was evaluated in ground chicken breast and liquid medium. Survival of Salmonella and Listeria was affected by the medium composition. Under the same thermal process condition, significantly more Salmonella and Listeria survived in chicken breast meat than in 0.1% peptone-agar solution. The thermal lethality of six tested Salmonella spp. was additive in chicken meat. Survival of Listeria in chicken meat during thermal processing was not affected by the presence of the six Salmonella spp. Sample size and shape affected the inactivation of Salmonella and Listeria in chicken meat during thermal processing.

A mathematical risk model for Escherichia coli O157:H7 cross-contamination of lettuce during processing.

A stochastic simulation modelling approach was taken to determine the extent of Escherichia coli O157:H7 contamination in fresh-cut bagged lettuce leaving the processing plant. A probabilistic model was constructed in Excel to account for E. coli O157:H7 cross contamination when contaminated lettuce enters the processing line. Simulation of the model was performed using @Risk Palisade© Software, providing an estimate of concentration and prevalence in the final bags of product. Three different scenarios, named S1, S2, and S3, were considered to represent the initial concentration on the contaminated batch entering the processing line which corresponded to 0.01, 1 and 100 cfu/g, respectively. The model was satisfactorily validated based on Standard Error of Prediction (SEP), which ranged from 0.00-35%. ANOVA analysis performed on simulated data revealed that the initial concentration in the contaminated batch (i.e., S1, S2, and S3) did not influence significantly (p=0.4) the E. coli O157:H7 levels in bags derived from cross contamination. In addition, significantly different (p<0.001) prevalence was observed at the different levels simulated (S1; S2 and S3). At the lowest contamination level (0.01 cfu/g), bags were cross-contaminated sporadically, resulting in very low E. coli O157:H7 populations (mean: ≤2 cfu/bag) and prevalence levels (<1%). In contrast, higher average prevalence levels were obtained for S2 and S3 corresponding to 3.05 and 13.39%, respectively. Furthermore, the impact of different interventions on E. coli O157:H7 cross-contamination (e.g., pathogen testing, chlorination, irradiation, and cleaning and disinfection procedures) was evaluated. Model showed that the pathogen was able to survive and be present in the final bags in all simulated interventions scenarios although irradiation (0.5 KGy) was a more effective decontamination step in reducing prevalence than chlorination or pathogen testing under the same simulated conditions.

Quantitative Transfer of Escherichia coli O157:H7 to Equipment during Small-Scale Production of Fresh-Cut Leafy Greens

Postharvest contamination and subsequent spread of Escherichia coli O157:H7 can occur during shredding, conveying, fluming, and dewatering of fresh-cut leafy greens. This study quantified E. coli O157:H7 transfer from leafy greens to equipment surfaces during simulated small-scale commercial processing. Three to five batches (22.7 kg) of baby spinach, iceberg lettuce, and romaine lettuce were dip inoculated with a four-strain cocktail of avirulent, green fluorescent protein-labeled, ampicillinresistant E. coli O157:H7 to contain ∼10(6), 10(4), and 10(2) CFU/g, and then were processed after 1 h of draining at ∼23°C or 24 h of storage at 4°C. Lettuce was shredded using an Urschel TransSlicer at two different blade and belt speeds to obtain normal (5 by 5 cm) and more finely shredded (0.5 by 5 cm) lettuce. Thereafter, the lettuce was step conveyed to a flume tank and was washed and then dried using a shaker table and centrifugal dryer. Product (25-g) and water (40-ml) samples were collected at various points during processing. After processing, product contact surfaces (100 cm(2)) on the shredder (n = 14), conveyer (n = 8), flume tank (n = 11), shaker table (n = 9), and centrifugal dryer (n = 8) were sampled using one-ply composite tissues. Sample homogenates diluted in phosphate or neutralizing buffer were plated, with or without prior 0.45- m m membrane filtration, on Trypticase soy agar containing 0.6% yeast extract supplemented with 100 ppm of ampicillin to quantify green fluorescent protein-labeled E. coli O157:H7 under UV light. During leafy green processing, ∼90% of the E. coli O157:H7 inoculum transferred to the wash water. After processing, E. coli O157:H7 populations were highest on the conveyor and shredder (P<0.05), followed by the centrifugal dryer, flume tank, and shaker table, with ∼29% of the remaining product inoculum lost during centrifugal drying. Overall, less (P<0.05) of the inoculum remained on the product after centrifugally drying iceberg lettuce that was held for 1 h (8.13%) as opposed to 24 h (42.18%) before processing, with shred size not affecting the rate of E. coli O157:H7 transfer.

Quantifying the Performance of Pediococcus sp. (NRRL B-2354: Enterococcus faecium) as a Nonpathogenic Surrogate for Salmonella Enteritidis PT30 during Moist-Air Convection Heating of Almonds

Pediococcus sp. NRRL B-2354 was investigated as a potential nonpathogenic surrogate for Salmonella enterica serovar Enteritidis phage type 30 (SE PT30) on the surface of almonds subjected to moist-air heating. Both microorganisms were subjected to various time, temperature, and humidity regimens on almonds processed in a computer-controlled, laboratory-scale, moist-air convection oven. Overall, the mean log reductions for Pediococcus sp. were 0.6 log and 1.4 log lower than those for SE PT30 (P < 0.05) at predicted reductions of 3 and 5 log, respectively. Also, the D(ref)-values for Pediococcus sp., calculated using a modified inactivation model (accounting for moisture) for SE PT30 on the surface of almonds subjected to moist-air heating (30 to 90% moisture by volume) were ~30% larger than those for SE PT30. Based on these findings, Pediococcus sp. NRRL B-2354 can be used as a conservative surrogate for SE PT30 during moist-air heating, and this organism is also likely to be an acceptable surrogate for steam heating.

Effects of Postharvest Parameters on Functional Changes During Rough Rice Storage

ABSTRACT The expansion of value-added uses for rice has created a demand for quantitative models of functional changes during postharvest handling. Consequently, this study evaluated the effects of postharvest parameters on the functional properties of long-grain (cvs. Cypress and Kaybonnet) and medium-grain (cv. Bengal) rice. The experimental treatments included rough rice drying conditions (low vs. high temperature drying), storage moisture content (10, 12, and 14%), storage temperature (4, 21, and 38°C), and storage duration (up to 36 weeks). Milling, cooking, and amylograph pasting properties were analyzed. Polynomial models (up to third-order) were developed to describe the effects of postharvest factors on the functional properties. Drying treatments, storage moisture content, and storage duration affected (P < 0.05) all of the functional properties. Storage temperature influenced (P < 0.01) cooking and pasting properties, but not milling properties. Overall, there were significant interactions amon...

Transfer of Escherichia coli O157:H7 from equipment surfaces to fresh-cut leafy greens during processing in a model pilot-plant production line with sanitizer-free water.

Escherichia coli O157:H7 contamination of fresh-cut leafy greens has become a public health concern as a result of several large outbreaks. The goal of this study was to generate baseline data for E. coli O157:H7 transfer from product-inoculated equipment surfaces to uninoculated lettuce during pilot-scale processing without a sanitizer. Uninoculated cored heads of iceberg and romaine lettuce (22.7 kg) were processed using a commercial shredder, step conveyor, 3.3-m flume tank with sanitizer-free tap water, shaker table, and centrifugal dryer, followed by 22.7 kg of product that had been dip inoculated to contain ∼10(6), 10(4), or 10(2) CFU/g of a four-strain avirulent, green fluorescent protein-labeled, ampicillin-resistant E. coli O157:H7 cocktail. After draining the flume tank and refilling the holding tank with tap water, 90.8 kg of uninoculated product was similarly processed and collected in ∼5-kg aliquots. After processing, 42 equipment surface samples and 46 iceberg or 36 romaine lettuce samples (25 g each) from the collection baskets were quantitatively examined for E. coli O157:H7 by direct plating or membrane filtration using tryptic soy agar containing 0.6% yeast extract and 100 ppm of ampicillin. Initially, the greatest E. coli O157:H7 transfer was seen from inoculated lettuce to the shredder and conveyor belt, with all equipment surface populations decreasing 90 to 99% after processing 90.8 kg of uncontaminated product. After processing lettuce containing 10(6) or 10(4) E. coli O157:H7 CFU/g followed by uninoculated lettuce, E. coli O157:H7 was quantifiable throughout the entire 90.8 kg of product. At an inoculation level of 10(2) CFU/g, E. coli O157:H7 was consistently detected in the first 21.2 kg of previously uninoculated lettuce at 2 to 3 log CFU/100 g and transferred to 78 kg of product. These baseline E. coli O157:H7 transfer results will help determine the degree of sanitizer efficacy required to better ensure the safety of fresh-cut leafy greens.

Thermal Resistance of Heat-, Cold-, and Starvation-Injured Salmonella in Irradiated Comminuted Turkey

To investigate the effects of sublethal stress on Salmonella thermal inactivation kinetics, an eight-strain Salmonella cocktail was subjected to heat shock (30 min at 54 degrees C), cold shock (2 h at 4 degrees C), and starvation stress (10 days in phosphate buffer at 4 degrees C), harvested by centrifugation, and inoculated into irradiated comminuted turkey. Immediately after stressing, the Salmonella cocktails contained 89.1% heat-injured, 44.7% cold-injured, and 67.7% starvation-injured cells, as determined by plating on selective and nonselective media. D60 degrees C-values for the heat-shocked cocktail (0.64 min on Trypticase soy agar containing 0.6% yeast extract [TSAYE], 0.35 min on xylose lysine desoxycholate [XLD] agar) were higher (P < 0.05) than those for the unshocked control (0.41 min on TSAYE, 0.17 min on XLD), whereas D60 degrees -values for the cold-shocked cocktail (0.38 min on TSAYE, 0.17 min on XLD) were not significantly different from those for the control. Starved cells had the same D60 degrees C-value on TSAYE as did the unshocked cocktail, but the D60 degrees C-value on XLD was significantly lower (0.14 min). Although starvation and cold shock were not thermally protective, heat shock increased thermal resistance, indicating that product history and the physiological state of the Salmonella cells should be considered when developing and validating thermal processes. D60 degrees C-values observed on selective media were significantly lower than those observed on nonselective media for all stress treatments and for the control. Therefore, nonselective culture media should be used to assess the response of microorganisms to a thermal challenge when developing performance standards for lethality.

Thermal inactivation kinetics for Salmonella Enteritidis PT30 on almonds subjected to moist-air convection heating.

A traditional thermal inactivation kinetic model (D- and z-value) was modified to account for the effect of process humidity on thermal inactivation of Salmonella Enteritidis PT30 on the surface of almonds subjected to moist-air heating. Raw almonds were surface inoculated to approximately 10(8) CFU/g and subjected to moist-air heating in a computer-controlled laboratory-scale convection oven. Time-temperature data were collected for 125 conditions (five dry bulb temperatures, 121 to 232 degrees C; five process humidity levels, 5 to 90% moisture by volume; and five process durations). Moisture status at the surface of the almond, rather than the humidity of the bulk air, was a primary factor controlling the rate of inactivation; therefore, the D-value could not be a simple function of process temperature. Instead, the traditional D- and z-value model was modified to account for the dynamic water status at the surface of the product under humid heating conditions. The modified model needs only the dew point temperature of the processing air and dynamic surface temperature history of the almonds during moist-air heating. The modified model was more robust and accurate than the traditional model. The accuracy of the modified model was improved by 32 to 44% (in terms of the root mean squared error [RMSE] for the model fit) when compared with the traditional model in all moist-air heating conditions. Also, the prediction error of the modified model (RMSE = 1.33 log reductions) against an independent validation data set was approximately one-half that of the traditional model (RMSE = 2.56 log reduction) in the humidity range of 5 to 90% moisture by volume.

Growth of Escherichia coli O157:H7 and Listeria monocytogenes in Packaged Fresh-Cut Romaine Mix at Fluctuating Temperatures during Commercial Transport, Retail Storage, and Display

Temperature abuse during commercial transport and retail sale of leafy greens negatively impacts both microbial safety and product quality. Consequently, the effect of fluctuating temperatures on Escherichia coli O157:H7 and Listeria monocytogenes growth in commercially-bagged salad greens was assessed during transport, retail storage, and display. Over a 16-month period, a series of time-temperature profiles for bagged salads were obtained from five transportation routes covering four geographic regions (432 profiles), as well as during retail storage (4,867 profiles) and display (3,799 profiles). Five different time-temperature profiles collected during 2 to 3 days of transport, 1 and 3 days of retail storage, and 3 days of retail display were then duplicated in a programmable incubator to assess E. coli O157:H7 and L. monocytogenes growth in commercial bags of romaine lettuce mix. Microbial growth predictions using the Koseki-Isobe and McKellar-Delaquis models were validated by comparing the root mean square error (RMSE), bias, and the acceptable prediction zone between the laboratory growth data and model predictions. Monte Carlo simulations were performed to calculate the probability distribution of microbial growth from 8,122,127,472 scenarios during transport, cold room storage, and retail display. Using inoculated bags of retail salad, E. coli O157:H7 and L. monocytogenes populations increased a maximum of 3.1 and 3.0 log CFU/g at retail storage. Both models yielded acceptable RMSEs and biases within the acceptable prediction zone for E. coli O157:H7. Based on the simulation, both pathogens generally increased <2 log CFU/g during transport, storage, and display. However, retail storage duration can significantly impact pathogen growth. This large-scale U.S. study-the first using commercial time/temperature profiles to assess the microbial risk of leafy greens-should be useful in filling some of the data gaps in current risk assessments for leafy greens.