Michelle D. Danyluk

Survival of Salmonella Enteritidis Phage Type 30 on Inoculated Almonds Stored at −20, 4, 23, and 35°C

To evaluate the survival of Salmonella on raw almond surfaces, whole almond kernels were inoculated with Salmonella Enteritidis phage type (PT) 30 collected from a 24-h broth culture or by scraping cells from an agar lawn. Kernels inoculated with lawn-collected cells to 8, 5, 3, and 1 log CFU per almond after a 24-h drying period were stored for 161 days at 23 ± 3°C. Calculated rates of reduction were similar for the four inoculum levels (0.22, 0.28, 0.29, and 0.22 log CFU/month, respectively). Kernels inoculated to 7.1 or 8.0 log CFU per almond after drying were stored for 171 or 550 days, respectively, at selected temperatures, including −20 ± 2°C, 4 ± 2°C, 23 ± 3°C, and 35 ± 2°C. No significant reductions of Salmonella were observed during storage at −20 and 4°C over 550 days. At 35°C, a biphasic survival curve was observed, with calculated reductions of 1.1 log CFU/month from days 0 to 59 and no significant reduction from days 59 to 171. At 23°C, reductions of 0.18 and 0.30 log CFU/month were calculat...

Prevalence and amounts of Salmonella found on raw California almonds.

Data on the prevalence and populations of pathogens in individual foods are critical to the development of product-specific quantitative microbial risk assessments. An outbreak of salmonellosis associated with the consumption of raw almonds in 2000 to 2001 provided an opportunity to evaluate the levels of Salmonella in the recalled product. Duplicate 100-g samples from each of fifty 22.7-kg boxes of recalled almonds were enriched by one of two methods. Salmonella was isolated by at least one method from 42 boxes (84% positive). The levels of Salmonella determined by a three-tube most-probable-number (MPN) method were 8.5+/-1.3 MPN/100 g. In a subsequent study, raw almonds that arrived at almond processors were sampled from 2001 through 2005 to determine the overall prevalence and levels of Salmonella and to characterize the Salmonella isolates obtained. Aerobic plate counts, coliform counts, and MPN levels of Escherichia coli were also determined on positive samples. An isolation frequency for Salmonella of 81 (0.87%+/-0.2%) of 9,274 samples tested (100 g) was determined for raw almonds sampled from throughout California over the 5-year period. Salmonella was not isolated upon retesting in 59 of 65 positive samples. When detected, levels were 1.2 to 2.9 MPN/100 g. Of the 81 total isolates, 35 different serotypes of Salmonella were represented. Aerobic plate counts, coliform counts, and E. coli levels did not correlate with the presence of Salmonella.

Isolation of Salmonella Enteritidis Phage Type 30 from a Single Almond Orchard over a 5-Year Period

In 2001, Salmonella Enteritidis phage type (PT) 30 was isolated from drag swabs of 17 61-ha almond orchards on three farms linked to an outbreak of salmonellosis associated with consumption of raw almonds. The objective of this study was to evaluate the long-term persistence of Salmonella Enteritidis PT 30 in one of the almond orchards associated with the outbreak. Swabs (gauze saturated with full-strength sterile evaporated skim milk and attached to string) were pulled along the orchard floor in a standardized manner for 55 m. At each sample time, two pooled samples (four swabs each) were collected from each orchard quadrant. Swabs were enriched for Salmonella using a delayed secondary enrichment procedure developed for isolation of Salmonella from poultry houses. Suspect Salmonella isolates were selected, confirmed, serotyped, and phage typed, and pulsed-field gel electrophoresis (PFGE) patterns were determined after cleavage with XbaI and BlnI. Salmonella was recovered infrequently from pooled samples collected from January through July (3 of 56 samples, 5.3%). In general, Salmonella isolation frequency per sample time increased during and immediately after the harvest, when large amounts of dust were generated in or near the orchard: August, 4 (20%) of 20 samples; September, 13 (20%) of 64 samples; October, 27 (42%) of 64 samples; November, 4 (25%) of 16 samples; and December, 2 (25%) of 8 samples. All 53 Salmonella isolates during the 5 years were identified as Salmonella Enteritidis PT 30, and two PFGE patterns that differed by the presence of an approximately 40-kb fragment were identified. These data demonstrate the potential for long-term environmental persistence of Salmonella in almond orchards.

Quantitative assessment of the microbial risk of leafy greens from farm to consumption: preliminary framework, data, and risk estimates.

This project was undertaken to relate what is known about the behavior of Escherichia coli O157:H7 under laboratory conditions and integrate this information to what is known regarding the 2006 E. coli O157:H7 spinach outbreak in the context of a quantitative microbial risk assessment. The risk model explicitly assumes that all contamination arises from exposure in the field. Extracted data, models, and user inputs were entered into an Excel spreadsheet, and the modeling software @RISK was used to perform Monte Carlo simulations. The model predicts that cut leafy greens that are temperature abused will support the growth of E. coli O157:H7, and populations of the organism may increase by as much a 1 log CFU/day under optimal temperature conditions. When the risk model used a starting level of -1 log CFU/g, with 0.1% of incoming servings contaminated, the predicted numbers of cells per serving were within the range of best available estimates of pathogen levels during the outbreak. The model predicts that levels in the field of -1 log CFU/g and 0.1% prevalence could have resulted in an outbreak approximately the size of the 2006 E. coli O157:H7 outbreak. This quantitative microbial risk assessment model represents a preliminary framework that identifies available data and provides initial risk estimates for pathogenic E. coli in leafy greens. Data gaps include retail storage times, correlations between storage time and temperature, determining the importance of E. coli O157:H7 in leafy greens lag time models, and validation of the importance of cross-contamination during the washing process.

Survival of Salmonella Enteritidis PT 30 on Inoculated Almonds after Commercial Fumigation with Propylene Oxide

Propylene oxide (PPO) is commonly used to reduce microbial populations in U.S. bulk raw almonds, but the process has not been validated for reduction of foodborne pathogens. The reduction of Salmonella Enteritidis phage type (PT) 30 inoculated onto almonds was evaluated after exposure to a standard commercial PPO treatment. Almonds were inoculated with Salmonella Enteritidis PT 30 to approximately 8.0 log CFU/g after drying. Inoculated almonds were placed in bags designed for gaseous sterilization and positioned in the center of 900-kg bins or 22.7-kg boxes of warmed almonds. Almonds were further warmed to an initial temperature of 23 to 34 degrees C, treated with PPO (0.5 kg/m3 for 4 h), and held for 0 or 2 days at 38 to 43 degrees C followed by storage for 2 to 5 days at 15 to 18 degrees C. Salmonella Enteritidis PT 30 was recovered by vigorously shaking 100 g of almonds in 100 ml of Butterfields phosphate buffer, plating onto tryptic soy or bismuth sulfite agar, and incubating at 35 degrees C for 24 or 48 h, respectively. Populations of Salmonella Enteritidis were consistently reduced by > 5.0 log CFU/g (5.2 to > 8.6 log CFU/ g) when initial counts were compared with counts obtained 5 days after PPO treatment. Reductions of 1.2 to 4.4 log CFU/g occurred during post-PPO storage. Reductions were not significantly improved (P < 0.05) when almonds were held at 38 to 43 degrees C after PPO treatment. PPO residues were > 400 ppm immediately after removal from the PPO chamber and declined to < 300 ppm during post-PPO storage. PPO is an effective treatment for reducing populations of Salmonella Enteritidis PT 30 on bulk almonds.

Dispersal of Salmonella Typhimurium by rain splash onto tomato plants.

Outbreaks of Salmonella enterica have increasingly been associated with tomatoes and traced back to production areas, but the spread of Salmonella from a point source onto plants has not been described. Splash dispersal by rain could be one means of dissemination. Green fluorescent protein-labeled, kanamycin-resistant Salmonella enterica sv. Typhimurium dispensed on the surface of plastic mulch, organic mulch, or soil at 10⁸ CFU/cm² was used as the point source in the center of a rain simulator. Tomato plants in soil with and without plastic or organic mulch were placed around the point source, and rain intensities of 60 and 110 mm/h were applied for 5, 10, 20, and 30 min. Dispersal of Salmonella followed a negative exponential model with a half distance of 3 cm at 110 mm/h. Dispersed Salmonella survived for 3 days on tomato leaflets, with a total decline of 5 log and an initial decimal reduction time of 10 h. Recovery of dispersed Salmonella from plants at the maximum observed distance ranged from 3 CFU/g of leaflet after a rain episode of 110 mm/h for 10 min on soil to 117 CFU/g of leaflet on plastic mulch. Dispersal of Salmonella on plants with and without mulch was significantly enhanced by increasing rain duration from 0 to 10 min, but dispersal was reduced when rainfall duration increased from 10 to 30 min. Salmonella may be dispersed by rain to contaminate tomato plants in the field, especially during rain events of 10 min and when plastic mulch is used.

Survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on inoculated almonds and pistachios stored at -19, 4, and 24° C.

The survival of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes was determined on almonds and pistachios held at typical storage temperatures. Almond kernels and inshell pistachios were inoculated with four- to six-strain cocktails of nalidixic acid-resistant Salmonella, E. coli O157:H7, or L. monocytogenes at 6 log CFU/g and then dried for 72 h. After drying, inoculated nuts were stored at -19, 4, or 24°C for up to 12 months. During the initial drying period after inoculation, levels of all pathogens declined by 1 to -log CFU/g on both almonds and pistachios. During storage, moisture content (4.8%) and water activity (0.4) of the almonds and pistachios were consistent at -19°C; increased slowly to 6% and 0.6, respectively, at 4°C; and fluctuated from 4 to 5% and 0.3 to 0.5 at 24°C, respectively. Every 1 or 2 months, levels of each pathogen were enumerated by plating; samples were enriched when levels fell below the limit of detection. No reduction in population level was observed at -19 or 4°C for either pathogen, with the exception of E. coli O157:H7-inoculated almonds stored at 4°C (decline of 0.09 log CFU/g/month). At 24°C, initial rates of decline were 0.20, 0.60, and 0.71 log CFU/g/month on almonds and 0.15, 0.35, and 0.86 log CFU/g/month on pistachios for Salmonella, E. coli O157:H7, and L. monocytogenes, respectively, but distinct tailing of the survival curves was noted for both E. coli O157:H7 and L. monocytogenes.

Monte Carlo simulations assessing the risk of salmonellosis from consumption of almonds.

Recent outbreaks of salmonellosis associated with raw almonds have raised awareness of this food as a vector for foodborne illness. We performed a quantitative assessment of the risk of contracting salmonellosis from consumption of raw almonds, accounting for factors that become important after almonds reach the processor. We estimated the risk associated with the consumption of raw almonds and the risk reduction associated with almonds treated with a theoretical 5-log reduction process or treated with propylene oxide using a standard commercial process. Probability distributions were chosen to describe the chance of almond contamination and the effects of storage time, storage temperature, and processing from currently available data. A beta-Poisson model for the dose-response relationship for Salmonella was obtained from published literature. The simulation estimated a 78% chance of one or more cases of salmonellosis per year from consumption of raw almonds. The application of a commercial propylene oxide treatment reduced this risk to 0.01%. Hypothetical 5-log reduction treatments with different standard deviations (+/-1, +/-0.5, +/-0.1, and +/-0) reduced the predicted yearly risk of salmonellosis to 0.69, 0.35, 0.30, and 0.21%, respectively. These results suggest that the risk of one or more U.S. cases of salmonellosis per year from consumption of raw almonds can be reduced from 78% to less than 1% by using a process achieving a 5-log reduction in Salmonella with a process standard deviation as large as 1 log unit or by using a commercial propylene oxide treatment.

Fate of Escherichia coli O157:H7 and Salmonella spp. on fresh and frozen cut mangoes and papayas

Escherichia coli O157:H7 and Salmonella infections have been associated with consumption of a number of fruits and vegetables. Although the fate of E. coli O157:H7 and Salmonella on many of these products is well studied, little is known about their behavior on cut mango and papaya. Mangoes and papayas have been associated with four and two documented outbreaks of salmonellosis, respectively. The objective of this study was to evaluate the fate of E. coli O157:H7 and Salmonella on fresh (4 degrees C, 12 degrees C and 23 degrees C) and frozen (-20 degrees C) cut mangoes and papayas. Cut mangoes and papayas were spot inoculated with either a four-strain or five-strain cocktail of E. coli O157:H7 or Salmonella, respectively. Inoculated samples were air dried, placed in containers and stored at 4+/-2, 12+/-2, 23+/-2 and -20+/-2 degrees C. Samples were enumerated following stomaching on nonselective and selective media at days 0, 1, 3, 5, 7, 10, 14, 21 and 28 (4+/-2 and 12+/-2 degrees C); 0, 1, 3, 5 and 7 (23+/-2 degrees C); and 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180 (-20+/-2 degrees C). Population levels (log CFU/g) of fruit were calculated. E. coli O157:H7 and Salmonella have the potential to grow on temperature-abused fresh cut mangoes and papayas held at 23 degrees C. At 12 degrees C, Salmonella populations can grow on cut mangoes and papayas, however E. coli O157:H7 populations only grew on papayas. E. coli O157:H7 and Salmonella survived for 28 days, at 4 degrees C, on refrigerated mangoes and papayas. E. coli O157:H7 and Salmonella can survive on frozen cut mangoes and papayas for at least 180 days. Fresh and frozen cut mangoes and papayas are potential vectors for E. coli O157:H7 and Salmonella transmission.

Predicting Salmonella populations from biological, chemical, and physical indicators in Florida surface waters

ABSTRACT Coliforms, Escherichia coli, and various physicochemical water characteristics have been suggested as indicators of microbial water quality or index organisms for pathogen populations. The relationship between the presence and/or concentration of Salmonella and biological, physical, or chemical indicators in Central Florida surface water samples over 12 consecutive months was explored. Samples were taken monthly for 12 months from 18 locations throughout Central Florida (n = 202). Air and water temperature, pH, oxidation-reduction potential (ORP), turbidity, and conductivity were measured. Weather data were obtained from nearby weather stations. Aerobic plate counts and most probable numbers (MPN) for Salmonella, E. coli, and coliforms were performed. Weak linear relationships existed between biological indicators (E. coli/coliforms) and Salmonella levels (R 2 < 0.1) and between physicochemical indicators and Salmonella levels (R 2 < 0.1). The average rainfall (previous day, week, and month) before sampling did not correlate well with bacterial levels. Logistic regression analysis showed that E. coli concentration can predict the probability of enumerating selected Salmonella levels. The lack of good correlations between biological indicators and Salmonella levels and between physicochemical indicators and Salmonella levels shows that the relationship between pathogens and indicators is complex. However, Escherichia coli provides a reasonable way to predict Salmonella levels in Central Florida surface water through logistic regression.