Experimentally Implementing the Linear Nonisothermal Equation for Simultaneously Obtaining D- and z-Values of Salmonella Senftenberg in Skim Milk with a Differential Scanning Calorimeter.

ABSTRACT: For bacteria with log-linear thermal inactivation kinetics in food, D-values are obtained in multiple isothermal inactivation experiments at different temperatures, and the z-value is obtained from these D-values. In a previous work, the cumulative lethality integral was mathematically solved in closed form when temperature in the food increased linearly with time. The solution revealed that each nonisothermal experiment could yield both D- and z-values, eliminating the need for getting multiple D-values to get a z-value. The present study reports on the first experimental implementation of this method of obtaining D- and z-values for Salmonella Senftenberg suspended in skim milk for which a differential scanning calorimeter (DSC) provided the required constant heating rate. The resulting D- and z-values were compared with those obtained from an isothermal method with capillary tubes. No significant differences in z-values were found between the two methods. The D-values also agreed but only after correcting the nonisothermal value for temperature lag in the DSC caused by the large sample size required. A 5 K/min heating rate was used in this comparison. Other rates were also investigated: 1, 3, 7.5, and 10 K/min. Although D- and z-values should be independent of DSC heating rate, heating rates of 1 and 10 K/min yielded values that were significantly different from the others; therefore, these rates cannot be recommended for use in this nonisothermal method.

Comparison of Commercial Test Kits for Detection of Salmonella and E. coli O157:H7 in Alfalfa Spent Sprout Irrigation Water.

BACKGROUND: Sprout growers in the United States are required to test spent sprout irrigation water (SSIW) or in-process sprouts for Escherichia coli O157:H7 and Salmonella species. Pathogen screening kits are commercially available; however, few have been validated for analysis of sprouts or SSIW. OBJECTIVE: This study evaluated AOAC-certified test kits (lateral flow devices [LFDs], enzyme immunoassays [EIAs], and molecular assays) in comparison with culture methods described in the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for detection of Salmonella and E. coli O157:H7 in alfalfa SSIW. METHOD: Twenty-five milliliter aliquots of alfalfa SSIW, either uninoculated or inoculated with Salmonella or E. coli O157:H7 at a low (∼0.5-0.7 CFU/25 mL) or high level (∼10-20 CFU/25 mL), were subjected to the enrichment and assay protocols recommended by each test. Pathogen presence was confirmed following FDA BAM procedures and, if applicable, test kit manufacturer protocols. RESULTS: Twelve of the 13 Salmonella test kits evaluated (except VIDAS UP) performed well and detected Salmonella in 100% of SSIW samples contaminated at 0.61 CFU/mL. Performance varied among E. coli O157:H7 test kits, with four (Reveal, MicroSEQ, GDS, MDA) of 12 kits designed for next-day detection, and four (Reveal, VIP Gold, MicroSEQ, GDS) of seven kits designed for same-day detection capable of detecting the pathogen in 100% samples contaminated at 0.90 CFU/mL. CONCLUSIONS: Enrichment conditions play a key role in determining the performance of test kits and the success of confirmation. HIGHLIGHTS: This study is the first to compare a wide range of commercial test kits for detection of Salmonella and E. coli O157:H7 in SSIW.

Population Dynamics of Listeria monocytogenes, Escherichia coli O157:H7, and Native Microflora During Manufacture and Aging of Gouda Cheese Made with Unpasteurized Milk.

ABSTRACT: Cheeses made with unpasteurized milk are a safety concern due to possible contamination with foodborne pathogens. Listeria monocytogenes and Escherichia coli O157:H7 have been implicated in several outbreaks and recalls linked to Gouda cheese made with unpasteurized milk. The U.S. Food and Drug Administration Code of Federal Regulations requires cheeses made with unpasteurized milk to be aged at a minimum of 1.7°C for at least 60 days before entering interstate commerce. The goal of this study was (i) to assess the population dynamics of L. monocytogenes and E. coli O157:H7 during aging of Gouda cheese when the pathogens were inoculated into the unpasteurized milk used for manufacture and (ii) to compare the native microbial populations throughout manufacture and aging. Unpasteurized milk was inoculated with L. monocytogenes at 1 or 3 log CFU/mL or with E. coli O157:H7 at 1 log CFU/mL, and Gouda cheese was manufactured in laboratory-scale or pilot plant-scale settings. Cheeses were stored at 10°C for at least 90 days, and some cheeses were stored up to 163 days. Initial native microflora populations in unpasteurized milk did not differ significantly for laboratory-scale or pilot plant-scale trials, and population dynamics trended similarly throughout cheese manufacture and aging. During manufacture, approximately 81% of the total L. monocytogenes and E. coli O157:H7 populations was found in the curd samples. At an inoculation level of 1 log CFU/mL, L. monocytogenes survived in the cheese beyond 60 days in four of five trials. In contrast, E. coli O157:H7 was detected beyond 60 days in only one trial. At the higher 3-log inoculation level, the population of L. monocytogenes increased significantly from 3.96 ± 0.07 log CFU/g at the beginning of aging to 6.00 ± 0.73 log CFU/g after 150 days, corresponding to a growth rate of 0.04 ± 0.02 log CFU/g/day. The types of native microflora assessed included Enterobacteriaceae, lactic acid bacteria, mesophilic bacteria, and yeasts and molds. Generally, lactic acid and mesophilic bacterial populations remained consistent at approximately 8 to 9 log CFU/g during aging, whereas yeast and mold populations steadily increased. The data from this study will contribute to knowledge about survival of these pathogens during Gouda cheese production and will help researchers assess the risks of illness from consumption of Gouda cheese made with unpasteurized milk.

Short communication: Long-term -20°C survival of Listeria monocytogenes in artificially and process-contaminated ice cream involved in an outbreak of listeriosis.

Listeria monocytogenes was linked to an outbreak of foodborne illness associated with in-process contaminated ice cream in the United States from 2010 to 2015 that sickened 10 individuals and led to 3 deaths. Ice cream obtained from the outbreak was used in this study to examine the population dynamics of L. monocytogenes as in-process contaminants compared with artificially inoculated cells. Because challenge studies of food products generally use artificial contamination, it is necessary to understand the differences in survival, if any, between these 2 types of contaminants. We hypothesized that laboratory-grown cultures of the pathogen that were not exposed to the environmental stresses of the manufacturing facility would show different population dynamics in an ice cream challenge study compared with in-process contaminants. In this study, half of the outbreak-associated ice cream samples were artificially inoculated with a 10 cfu/g cocktail of L. monocytogenes; the other half contained only the in-process contaminants. All samples were stored at -20°C and tested for pathogen levels (n = 10 for each contaminant type at each time point) by the most probable number method at 3-mo intervals for 36 mo. Generally, population levels between the 2 contamination states in the ice cream were not significantly different and L. monocytogenes survived for at least 36 mo, regardless of contamination state. Overall, our results suggest that the use of L. monocytogenes as an artificial contaminant in challenge studies and risk assessment of ice cream during frozen storage give results similar to those shown by in-process contaminants.

Effects of Package Atmosphere and Storage Conditions on Minimizing Risk of Escherichia coli O157:H7 in Packaged Fresh Baby Spinach.

Packaged fresh spinach has been associated with outbreaks of illness caused by Escherichia coli O157:H7. The purpose of this study was to assess the behavior of E. coli O157:H7 in packaged baby spinach in response to storage conditions of temperature and package atmosphere and including effects of inoculation level, spinach leaf damage (cut leaves), internalized or leaf surface contamination, exposure to hypochlorite sanitizer, and package size. Behavior of E. coli O157:H7 inoculated at 2 and 4 log CFU/g on spinach packaged in polymer bags composed of a two-layer laminate (polypropylene and polyethylene) and stored under atmospheres of 20% O2-3% CO2 and 0% O2-15% CO2 (aerobic and anaerobic, respectively) was assessed at 5, 7, 12, and 15°C for up to 14 days. Growth kinetics were calculated using DMFit software. Temperature decreases progressively diminished growth or survival of the pathogen, and an aerobic package atmosphere resulted in longer lag times (4 to 6 days) and lower population levels (0.2 to 1.4 log CFU/g) compared with the anaerobic atmosphere at 15°C. Internalized contamination, leaf cuts, or exposure to 100 ppm of hypochlorite did not result in changes in pathogen behavior compared with controls; however, a growth minimization trend consisting of longer lag times and lower population levels was repeatedly observed in the aerobic compared with the anaerobic package atmospheres. In contrast, growth of indigenous mesophiles and Enterobacteriaceae was unaffected by package atmosphere. Spinach stored at 5 to 7°C in two sizes (5 and 16 oz) of polyethylene terephthalate clamshell packages with ambient air atmospheres was more likely to progress to lower-oxygen conditions in 16-oz compared with 5-oz packages after 7 days of storage (P < 0.05). Practices to maintain aerobic conditions within the package, as well as storage of the package at low temperature, are ways to limit growth of E. coli O157:H7 in packaged spinach.

Metagenomics of pasteurized and unpasteurized gouda cheese using targeted 16S rDNA sequencing.

BACKGROUND: The microbiome of cheese is diverse, even within a variety. The metagenomics of cheese is dependent on a vast array of biotic and abiotic factors. Biotic factors include the population of microbiota and their resulting cellular metabolism. Abiotic factors, including the pH, water activity, fat, salt, and moisture content of the cheese matrix, as well as environmental conditions (temperature, humidity, and location of aging), influence the biotic factors. This study assessed the metagenomics of commercial Gouda cheese prepared using pasteurized or unpasteurized cow milk or pasteurized goat milk via 16S rDNA sequencing. RESULTS: Results were analyzed and compared based on milk pasteurization and source, spatial variability (core, outer, and under the rind), and length of aging (2-4 up to 12-18 months). The dominant organisms in the Gouda cheeses, based on percentage of sequence reads identified at the family or genus levels, were Bacillaceae, Lactococcus, Lactobacillus, Streptococcus, and Staphylococcus. More genus- or family-level (e.g. Bacillaceae) identifications were observed in the Gouda cheeses prepared with unpasteurized cow milk (120) compared with those prepared with pasteurized cow milk (92). When assessing influence of spatial variability on the metagenomics of the cheese, more pronounced differences in bacterial genera were observed in the samples taken under the rind; Brachybacterium, Pseudoalteromonas, Yersinia, Klebsiella, and Weissella were only detected in these samples. Lastly, the aging length of the cheese greatly influenced the number of organisms observed. Twenty-seven additional genus-level identifications were observed in Gouda cheese aged for 12-18 months compared with cheese only aged 2-4 months. CONCLUSIONS: Collectively, the results of this study are important in determining the typical microbiota associated with Gouda cheese and how the microbiome plays a role in safety and quality.

Listeria monocytogenes Growth Kinetics in Milkshakes Made from Naturally and Artificially Contaminated Ice Cream.

This study assessed the growth of Listeria monocytogenes in milkshakes made using the process-contaminated ice cream associated with a listeriosis outbreak in comparison to milkshakes made with artificially contaminated ice cream. For all temperatures, growth kinetics including growth rates, lag phases, maximum populations, and population increases were determined for the naturally and artificially derived contaminants at 5, 10, 15, and 25°C storage for 144 h. The artificially inoculated L. monocytogenes presented lower growth rates and shorter lag phases than the naturally contaminated populations at all temperatures except for 5°C, where the reverse was observed. At 25°C, lag phases of the naturally and artificially contaminated L. monocytogenes were 11.6 and 7.8 h, respectively. The highest increase in population was observed for the artificially inoculated pathogen at 15°C after 96 h (6.16 log CFU/mL) of storage. Growth models for both contamination states in milkshakes were determined. In addition, this study evaluated the antimicrobial effectiveness of flavoring agents, including strawberry, chocolate and mint, on the growth of the pathogen in milkshakes during 10°C storage. All flavor additions resulted in decreased growth rates of L. monocytogenes for both contamination states. The addition of chocolate and mint flavoring also resulted in significantly longer lag phases for both contamination states. This study provides insight into the differences in growth between naturally and artificially contaminated L. monocytogenes in a food product.

Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.

The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥ 0.5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9-11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.

Thermal resistance of Listeria monocytogenes Scott A in ultrafiltered milk as related to the effect of different milk components.

Pasteurization parameters for grade A milk are well established and set by regulation. However, as solids levels increase, an increased amount of heat is required to destroy any pathogens present. This effect is not well characterized. In this work, the effect of increased dairy solids levels on the thermal resistance of Listeria monocytogenes was examined through the use of ultrafiltered (UF) milk, reconstituted milk powder, and the milk components lactose and caseinate. From the results obtained, lactose and caseinate did not appear to affect thermal resistance. In addition, the level of milk fat, up to 10% of the total solids in UF whole milk, did not result in statistically significant changes to thermal resistance when compared with UF skim milk. Reconstituted skim milk powder at 27% total solids (D6²-value = 1.16 ± 0.2 [SD] min, z = 5.7) did result in increased thermal resistance, as compared with reconstituted skim milk powder at 17.5% (D6²-value = 0.86 ± 0.02 min, z = 5.57) and UF whole milk at 27% total solids (D6²-value = 0.66 ± 0.07 min, z = 5.16). However, that increase appeared to be due to the increase in salt levels, not to increases in caseinate, fat, or lactose. Consequently, total solids, as a single measure, could not be used to predict increased thermal resistance of L. monocytogenes in concentrated milk.

Modification of the submerged coil to prevent microbial carryover error in thermal death studies.

A submerged coil unit generates death rate data for foodborne pathogens through precise computer-controlled sequential sampling rather than the usual manually timed, labor-intensive single sampling associated with other approaches. Our work with Yersinia pseudotuberculosis and Listeria monocytogenes Scott A using the submerged coil unit indicated non-log-linear death rates with large degrees of tailing. Varying degrees of cell adhesion to the surface of the exit port resulted in carryover that was likely the primary cause of these non-log-linear kinetics. This carryover also resulted in erroneously high measured levels of thermal resistance for both organisms. To address the carryover problem, modifications were made to the exit port of the submerged coil unit to ensure continuous and uniform heat treatment. These modifications resulted in a 2-fold decrease in measured D-values for L. monocytogenes Scott A and a 10-fold decrease in measured D-values for Y. pseudotuberculosis. D-values measured with the modified machine for L. monocytogenes Scott A were similar to those found in the literature. Slight tailing in survival curves persisted with the modified method, particularly for Y. pseudotuberculosis. These results indicate that kinetic data for microbial death rates obtained using an unmodified submerged coil unit must be viewed with suspicion in light of the significant potential for carryover.