Modern Microbiology | Stasiewicz Food Safety Laboratory | Vision: A world where everyone, everywhere is food secure

Validating aggregative soil sampling using bootie and drag swabs hydrated with simple wetting agents in commercial produce fields

Abstract

Bootie and drag swabs may collect more microbiologically representative aggregative soil samples than composite grabs from produce fields. Previous experimental field work has identified some practical wetting agents as potential alternatives to traditionally used skim milk. This study validates the two most promising wetting agents, phosphate-buffered saline (PBS) and buffered peptone water (BPW), by comparing swabs results from 100 m tracks through a melon farm (262,000 m²), a mixed agriculture farm (leafy green, peppers, beets, 4,500 m²), and an apple orchard (114,000 m²). The mean difference between paired samples collected with BPW or PBS ranged from −0.02 ± 0.09 to 0.26 ± 0.09 log₁₀(CFU/g) for aerobic plate count (APC) and total coliforms (TCs). Bootie and drag swabs recovered greater APCs [mean difference 0.63 ± 0.38 to 1.83 ± 0.24 log₁₀(CFU/g) and TCs (mean difference 1.32 ± 0.96 to 5.32 ± 1.03 log₁₀(CFU/g)], and greater prevalence of Escherichia coli compared to soil grabs (90% versus 44% of samples positive by enrichment, P < 0.001). By 16S sequencing, samples collected with PBS had greater within-sample community richness (alpha diversity) than BPW (P-values 0.041 and 0.059) but similarly overlapping taxa. Soil samples had higher within-sample (alpha) diversity (P < 0.05), but lower between-sample (beta) diversity compared to booties and drags. Overall, there was no biologically meaningful difference between the performances of the two wetting agents for bootie and drag swabs, and compared to composite soil samples, these two swab methods recovered more indicator organisms from produce field soil representing five different commodities.

DOI

https://doi.org/10.1128/spectrum.01663-25

Salmonellosis Risk Assessment for Comminuted Turkey under Different Specificities of Concentration-based and Virulence-based Final Product Standards

Highlights

More specifically targeting products with risky contamination is efficient
Concentration- and virulence-based standards capture more risk in less products
Genomic clustering more efficiently than serotype identifies risky contamination

Abstract

Prevalence-based performance standards have guided Salmonella control in poultry industry, but concentration- and virulence-based final product standards could target the most risky contamination more specifically. We adapted our previous risk assessment for chicken parts to comminuted turkey to assess the risk in products implicated by different final product standards, incorporating assumptions from FSIS 2024 risk assessments. We simulated the attributable fraction of illnesses from products contaminated over three level thresholds (0.0031 CFU/g, 1 CFU/g, and 10 CFU/g) and/or containing a serotype in three lists (“Top 3 most prevalent higher-virulence serotypes”, “All higher-virulence serotypes”, and “Higher-virulence proportion of each serotype”). Results showed that 87% of illnesses were attributed to the 0.56% of products with Salmonella exceeding 10 CFU/g. Under more specific criteria of level “AND” serotype, 60% of illnesses were attributed to the 0.14% of products contaminated with Salmonella exceeding 10 CFU/g and one of the three most prevalent higher-virulence serotypes. Further, applying genomic-based clustering information, 75% of illnesses were attributed to slightly more products (0.19%) containing Salmonella exceeding 10 CFU/g and higher-virulence proportion of each serotype. Under the less specific standard, however, 99% of illnesses were attributed to the 5.7% of products containing Salmonella exceeding 10 CFU/g “OR” one of the three most prevalent higher-virulence serotypes. Our study demonstrated that most salmonellosis risk is concentrated in comminuted turkey products with high levels of higher-virulence contaminations. Importantly, more specifically targeting those products could efficiently reduce public health risk while minimizing products implicated.

DOI

https://doi.org/10.1016/j.mran.2025.100359

Quantitative model predicts implementing school cafeteria share tables will not compromise milk safety

Share table model framework, where the cafeteria system consists of (1) the service, (2) the break (or breaks), and (3) overnight refrigeration. A typical day of school cafeteria meal services has n number of services, with n − 1 breaks. All milk cartons served during service or services on a given day are assumed to be in the defined storage condition for the entire day (i.e., throughout services and breaks) until a milk carton is consumed or the day is over and the milk cartons are moved to overnight refrigeration. At the end of each day, leftover milk cartons on the service line or share table can be reserviced on the following day. Each day was simulated 5 times to represent a school week. At the end of the school week, all leftover milk cartons are discarded. Each week is simulated 37 times (a typical school year). This process is repeated 50 times to simulate 50 school years of meal services in cafeterias with share tables.

Abstract

School cafeteria share tables can address food waste and improve food security by allowing students to share unopened items, such as milk. However, unresolved safety concerns present a barrier to recovering milk cartons on share tables. We adapted our previous share table model to study Listeria monocytogenes in pasteurized milk, assuming a concentration distribution that reflects the realistically low prevalence of the pathogen. Student sharing behavior was simulated for 50 yr of school weeks (5 d/wk over 37 wk/yr). Milk safety is assessed by quantifying (1) time to L. monocytogenes growth of 1 log₁₀, (2) L. monocytogenes concentration at consumption, and (3) listeriosis risk. We compare these measures across 23 what-if scenarios to identify potential risk factors and mitigation strategies. Under the baseline scenario (with no share table temperature management), L. monocytogenes increases by 1 log₁₀ after 1 reservice (after service 2). With share table temperature management, improved overnight refrigeration, or shorter services, L. monocytogenes did not increase by 1 log₁₀ until after 2 d of reservice (after service 3). Under excessive time-temperature abuse (inadequate overnight refrigeration or long services), L. monocytogenes increases by 1 log₁₀ before the first day of reservice (before service 2). Comparing the baseline scenarios of share table and no share table, L. monocytogenes concentration at consumption did not substantially differ. Importantly, L. monocytogenes concentration at consumption never exceeded 100 cfu/mL, except under the longest (266 min) service scenario, for only 0.0006% of milk cartons (11/1,794,887). The mean probability of illness per serving (P_Illness) was low across all scenarios. Comparing the baseline share table and no share table scenarios, P_Illness was 3.32 × 10⁻¹³ and 2.72 × 10⁻¹³, respectively, translating to 1 listeriosis illness in every 2,100 (2,000–2,400) and 3,000 (2,700–3,400) yr across all public schools in the United States. These results demonstrate the extremely small predicted risk of listeriosis from consuming milk cartons in cafeterias with share tables, providing an evidence base to support school nutrition staff in conversations with health departments during the approval of share table management practices.

DOI

https://doi.org/10.3168/jds.2025-27069

Comparison of Alternative Wetting Agents for Drag and Bootie Swabs for Agricultural Soil Sampling

Bacterial counts of aerobic plate counts, total coliforms, and generic E. coli for soil collected using drags and booties hydrated by different wetting agents in (A) the field with untreated swine manure and (B) the field with untreated dairy manure. The whiskers represent the interquartile range. Solid jittered points represent the counts of detected samples. Open jittered points represent the LODs/2 of the samples under LODs. Different wetting agents are represented using different color.

Highlights

Wetting agents produced small differences in indicator organism recovery.
Small differences between wetting agents may not be biologically meaningful.
Buffered peptone water is a well-performing nutritious wetting agent.
Phosphate-buffered saline is a well-performing non-nutritious wetting agent.

Abstract

Drag and bootie swabs have been used in animal (e.g., poultry litter) and produce (e.g., soil) production for food safety purposes in place of grabs. Skim milk, the industry standard wetting agent for drags and booties, is not ideal for produce soil sampling due to its allergenic properties and animal-based origin, and (depending on preparation) low shelf stability. This study evaluated alternative wetting agents – tryptic soy broth, buffered peptone water, phosphate buffered saline, or deionized water – for hydrating drags and booties. Sampling was performed in fields with untreated swine manure and untreated dairy manure, with a total of 220 drags, 220 booties, and 44 grabs collected along 100 m paths. Indicator organisms including aerobic plate counts (APCs), total coliforms, and Escherichia coli were enumerated. Both wetting agents (p < 0.001) and sampling methods (p < 0.001) significantly affected the recovery of indicator organisms. In the field with swine manure, mean recovery differences between wetting agents ranged from 0.1 to 0.2 log(CFU/g) for APCs and 0.1 to 0.6 log(CFU/g) for total coliforms. In the field with dairy manure, mean recovery differences between wetting agents ranged from 0.0 to 0.2 log(CFU/g) for APCs, 0.1 to 0.4 log(CFU/g) for total coliforms, and 0.1 to 0.4 log(CFU/g) for E. coli. Overall, differences between wetting agents were small and suggest one could select wetting agents for future method development and industry use based on which are most practical for use in produce safety, such as most shelf stable and not animal sourced.

DOI

https://doi.org/10.1016/j.jfp.2025.100573

A Critical Review of Parameters Relevant for Shiga Toxin-producing Escherichia coli and Listeria monocytogenes Risk Assessments of Leafy Greens

Parameter categories under the five defined supply chain stages of a produce supply chain for the reviewed models. Box A shows an example of the two key parameters included under the Storage Conditions parameter category in the Presentation to Consumer supply chain stage.

Highlights

A critical review of contemporary leafy green risk assessments was performed.
Scope is assessments using L. monocytogenes and Shiga-toxin-producing E. coli.
Parameter values and original data sources used are presented in organized tables.
Provides a valuable resource to efficiently develop future risk assessment.

Abstract

In the past decade, several quantitative models and risk assessments for Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes have been developed to guide the management of these pathogens in fresh produce. However, there is a need to collect and critically review the parameters used to guide their potential reuse in future risk assessments. This review (i) identifies 11 and 7 recently published quantitative models and risk assessments for STEC and L. monocytogenes, respectively, in leafy greens, (ii) summarizes parameters, and (iii) reviews the underlying data sources or mathematical formulas used. A total of 70 unique key parameters (55 and 25 for STEC and L. monocytogenes, respectively, 10 shared) were extracted from the reviewed models across five supply chain stages, including: Preharvest, Harvest, Processing, Presentation to Consumer (Retail or Foodservice Locations), and Consumer Handling. Primary growth, secondary growth, and dose-response equations and parameters for STEC and L. monocytogenes were also extracted. Additional literature reviews were performed if (i) certain key parameters were based on a single or a few data sources or (ii) key parameters were identified in a supply chain stage for one pathogen, but missing from the other. The critical summaries of parameters presented here (i) provide a resource for future risk assessments, (ii) help define future data collection needs, and (iii) represent a starting point for similar reviews focusing on other produce commodities and pathogens.

DOI

https://doi.org/10.1016/j.jfp.2025.100497

Aggregative Sampling Performs Similar to Composite Produce Samples to Recover Quality and Safety Indicators Throughout Romaine Lettuce Production

Overall experimental sampling design during preharvest, in-harvest and postharvest stages of commercial romaine lettuce. This sampling process was performed in two replicates. For each replicate, commercial romaine grown on eight beds was sampled preharvest, during harvest and postharvest. For preharvest sampling (1 d before harvest), aggregative, composite produce (60 grabs, 150 g/sample) and high-resolution produce samples were collected (60 grabs, 25 g/sample). During harvest, romaine was sampled by collecting aggregative gloves worn by the harvesting crew, composite produce samples from leftover romaine leaf trims on the ground (60 grabs, 150 g/sample), aggregative swabs of harvested romaine heads as they exited the harvester chute, and aggregative swabs and composite produce samples (60 grabs, 150 g/sample) of romaine heads from the top of transportation bins. For postharvest sampling (∼6 hrs after harvest), ∼800 lb of romaine was sampled by using aggregative gloves to manipulate and place individual heads on a moving conveyor belt; as the heads exited the conveyor belt, they were sampled by an aggregative swab placed at the exit chute, before they fell onto smaller bins. Once there, composite romaine samples (60 grabs, 150 g/sample) were collected from the exterior of the heads. All sampled heads were then chopped in half lengthwise and were sampled by rubbing their interior against an aggregative swab placed on a cartridge and collecting composite romaine samples (60 grabs, 150 g/sample) from the interior of the heads.

Highlights

Aggregative and tissue sampling were compared pre-, during-, and postharvest.
Aggregative glove and swab sampling recovered similar aerobic bacteria to tissue grabs.
Aggregative gloves recovered similar coliforms to tissue grabs.
Aggregative swab recovery could be improved for coliforms and generic E. coli.

Abstract

Aggregative sampling using polymer cloth swabs is a nondestructive, potentially more representative food safety sampling alternative for leafy greens. This study compared aggregative and produce tissue grab sampling to recover aerobic bacteria, total coliforms, and generic Escherichia coli, from commercial romaine grown in 120 m fields, with 5–36 samples at various stages. Aggregative swabs and grab samples were collected preharvest. During harvest, aggregative swabs were collected from romaine exiting the harvester chute, transport bin tops, and as gloves worn by harvesters. Romaine grabs were collected from transport bin tops and trim leftover on the ground. During postharvest, gloves, swabs, and grabs were collected from romaine exteriors, and swabs and grabs from head interiors. In preharvest, swabs had 1.2 log(CFU/g) higher means of aerobic bacteria than romaine tissue grab samples (p < 0.001), but 1.7 log(CFU/g) lower coliforms (p < 0.001). In-harvest, aerobic bacteria means from gloves worn by harvesters and swabs from harvester chute were 0.5 log(CFU/g) higher than romaine samples from leftover trims (p < 0.001) and bin tops (p = 0.01), respectively. Coliform recovery means from gloves was not significantly different from romaine leftover trims (p = 0.99). Swabs from harvester chute and bin tops recovered 1.6 and 1.4 log(CFU/g) lower coliforms means (p < 0.001) than romaine from bin tops, respectively. Generic E. coli was only recovered from one romaine leftover trim grab sample. During postharvest processing, aerobic bacteria (p = 0.25) and total coliforms (p = 0.16) recovery from the exterior of heads was not significantly different between gloves and romaine samples, nor was aerobic bacteria (p = 0.17) and total coliform (p = 0.86) recovery from head interiors. These results suggest that aggregative sampling performs similar to produce grab sampling to recover quality and safety indicators and justifies testing these methods for pathogen sampling in leafy greens.

DOI

https://doi.org/10.1016/j.jfp.2025.100481

Hurdle Approach to Simulate Corn Wet Milling Inactivation of Undesirable Microorganisms: A Pilot Scale Microbial Challenge Study Using Salmonella Surrogate Enterococcus faecium

Highlights

Steeping, peroxide, and flash drying reduced E. faecium during corn-wet milling.
E. faecium did not grow during long-term storage of corn starch.
Corn wet milling unit operations cumulatively reduced E. faecium.
These findings may assist in the development of corn-wet milling food safety plans.

Abstract

Corn wet milling (CWM) and corn starch flash drying processing conditions reduce undesirable microorganisms, such as Salmonella. Finished products are historically safe, with intrinsic properties such as low water activity inhibiting microbial growth. Corn processors could use quantified levels of reduction in this study of Salmonella surrogate Enterococcus faecium (E. faecium) to update their food safety plans. Industry-relevant conditions for CWM processes were recreated at pilot or lab scale for 3 unit operations: (1) steeping treatment in sulfur dioxide (SO₂) with low (750 ppm SO₂, 20 hours, 43.3°C), medium (1,500 ppm SO₂, 30 hours, 48.9°C), and high (2,200 ppm SO₂, 40 hours, 53.3°C) treatment conditions; (2) hydrogen peroxide (H₂O₂) treatment tested on bench scale with a factorial design (pH 3.5, 4.0, and 4.5), H₂O₂ concentrations (0.05%, 0.10%, 0.15% (w/w)), and temperatures (32, 38, and 46°C) for 3 and 6 hours; (3) flash drying treatment at 4 different temperatures (149, 177, 204, and 232°C) with 2 different inoculation methods. E. faecium was reduced during each of these unit operations. By the end of each steeping treatment E. faecium was consistently below the limit of quantitation (LOQ), meaning > 6.5 log CFU/mL reduction in steep water, and > 3.7 log CFU/g reduction in ground corn. The peroxide step had a reduction range from 0.03 log CFU/mL in the control group (0% H₂O₂ added) to >6 log CFU/mL observed in the high-intensity treatment of corn starch slurry. Flash drying had a reduction range from 1.7 to 2.7 log CFU/g. There was also no biologically meaningful change (<1 log CFU/g reduction) of E. faecium counts during an 8-week survival study of the dried final product. This hurdle approach study shows that existing CWM conditions are effective for Salmonella surrogate reduction through processing into finished starch and provides quantified E. faecium reductions for use in of food safety plans.

DOI

https://doi.org/10.1016/j.jfp.2024.100432

Aggregative Swab Sampling Method for Romaine Lettuce Show Similar Quality and Safety Indicators and Microbial Profiles Compared to Composite Produce Leaf Samples in a Pilot Study

Abstract

Composite produce leaf samples from commercial production rarely test positive for pathogens, potentially due to low pathogen prevalence or the relatively small number of plants sampled. Aggregative sampling may offer a more representative alternative. This pilot study investigated whether aggregative swab samples performed similarly to produce leaf samples in their ability to recover quality indicators (APCs and coliforms), detect Escherichia coli, and recover representative microbial profiles. Aggregative swabs of the outer leaves of romaine plants (n = 12) and composite samples consisting of various grabs of produce leaves (n = 14) were collected from 60 by 28 ft sections of a one-acre commercial romaine lettuce field. Aerobic plate counts were 9.17 ± 0.43 and 9.21 ± 0.42 log(CFU/g) for produce leaf samples and swabs, respectively. Means and variance were not significantly different (p = 0.38 and p = 0.92, respectively). Coliform recoveries were 3.80 ± 0.76 and 4.19 ± 1.15 log(CFU/g) for produce leaf and swabs, respectively. Means and variances were not significantly different (p = 0.30 and p = 0.16, respectively). Swabs detected generic E. coli in 8 of 12 samples, more often than produce leaf samples (3 of 14 positive, Fisher’s p = 0.045). Full-length 16S rRNA microbial profiling revealed that swab and produce leaf samples did not show significantly different alpha diversities (p = 0.75) and had many of the most prevalent bacterial taxa in common and in similar abundances. These data suggest that aggregative swabs perform similarly to, if not better than, produce leaf samples in recovering indicators of quality (aerobic and coliform bacteria) and food safety (E. coli), justifying further method development and validation.

DOI

https://doi.org/10.3390/foods13193080

Milk spoilage model predicts that share tables would not meaningfully increase spoilage, and improved storage systems can reduce spoilage

Experimental setups of data collection for milk internal temperature under different storage systems and refrigeration condition and external temperature. Both external and internal milk temperature and experimental setup were measured for an hour in duplicate (2 probes) over 4 d resulting in a total of 4 replicates.

Abstract

School share tables offer opportunities for food recovery and increased access to healthy foods by allowing students to donate or consume unopened items, such as cartons of milk. However, stakeholders have concerns about temperature abuse potentially causing premature milk spoilage. Although previous research showed short ambient temperature abuse of milk (under conditions representing share tables) does not meaningfully affect microbial milk quality, differences across school cafeterias (e.g., ambient temperatures, storage systems, bell schedules, refrigeration temperature) may limit the generalizability of this conclusion. To address this, the overnight refrigeration temperature and the milk’s initial contamination were predicted to be the main drivers for milk spoilage. Share tables were predicted to only cause inconsequential microbial quality changes (4 spoiled milk per million served, which would be ≤2 milk cartons spoiled per school year) under short and medium bell schedules (≤125 min of total service), even without temperature control during the lunch period. Under long (221 min) and very long (266 min) bell schedules, share tables with ambient temperature storage were predicted to have higher milk spoilage (19 and 42 spoiled milk cartons per million served, respectively), and adding storage systems was predicted to reduce the decline in milk quality (12 and 24 spoiled milk cartons per million served, respectively). These results provide a resource to support science-based decision making for the inclusion of milk in school cafeteria share tables, ultimately working to reduce food waste and address food insecurity.

DOI

https://doi.org/10.3168/jds.2024-25189

Risk Assessment Predicts Most of the Salmonellosis Risk in Raw Chicken Parts is Concentrated in Those Few Products with High Levels of High-Virulence Serotypes of Salmonella

Summary of collected USDA-FSIS publicly available datasets used in this project to characterize Salmonella contamination in chicken parts by presence, level, and serotype

Highlights

Chicken parts are rarely contaminated with high Salmonella levels, above 1 CFU/g.
High-virulence serotypes are frequently found in US chicken parts.
Most salmonellosis risk is concentrated in chicken part products above 1 CFU/g.
Most salmonellosis risk is in products with high levels of high-virulence serotypes.

Abstract

Salmonella prevalence declined in U.S. raw poultry products since adopting prevalence-based Salmonella performance standards, but human illnesses did not reduce proportionally. We used Quantitative Microbial Risk Assessment (QMRA) to evaluate public health risks of raw chicken parts contaminated with different levels of all Salmonella and specific high- and low-virulence serotypes. Lognormal Salmonella level distributions were fitted to 2012 USDA-FSIS Baseline parts survey and 2023 USDA-FSIS HACCP verification sampling data. Three different Dose-Response (DR) approaches included (i) a single DR for all serotypes, (ii) DR that reduces Salmonella Kentucky ST152 virulence, and (iii) multiple serotype-specific DR models. All scenarios found risk concentrated in the few products with high Salmonella levels. Using a single DR model with Baseline data (μ = -3.19, σ = 1.29 Log CFU/g), 68% and 37% of illnesses were attributed to the 0.7% and 0.06% of products with >1 and >10 CFU/g Salmonella, respectively. Using distributions from 2023 HACCP data (μ = -5.53, σ = 2.45), 99.8% and 99.0% of illnesses were attributed to the 1.3% and 0.4% of products with >1 and >10 CFU/g Salmonella, respectively. Scenarios with serotype-specific DR models showed more concentrated risk at higher levels. Baseline data showed 92% and 67% and HACCP data showed >99.99% and 99.96% of illnesses attributed to products with >1 and >10 CFU/g Salmonella, respectively. Regarding serotypes using Baseline or HACCP input data, 0.002% and 0.1% of illnesses were attributed to the 0.2% and 0.4% of products with >1 CFU/g of Kentucky ST152, respectively, while 69% and 83% of illnesses were attributed to the 0.3% and 0.6% of products with >1 CFU/g of Enteritidis, Infantis, or Typhimurium, respectively. Therefore, public health risk in chicken parts is concentrated in finished products with high levels and specifically high levels of high-virulence serotypes. Low-virulence serotypes like Kentucky contribute few human cases.

DOI

https://doi.org/10.1016/j.jfp.2024.100304