Limited prior research has shown that inoculation methods affect thermal resistance of Salmonella in low-moisture foods; however, these effects and their repeatability have not been systematically quantified. Consequently, method variability across studies limits utility of individual data sets and cross-study comparisons. Therefore, the objective was to evaluate the effects of inoculation methodologies on stability and thermal resistance of Salmonella in a low-moisture food (wheat flour), and the repeatability of those results, based on data generated by two independent laboratories. The experimental design consisted of a cross-laboratory comparison, both conducting isothermal Salmonella inactivation studies in wheat flour (~0.45 water activity, 80°C), utilizing five different inoculation methods: (i) broth-based liquid inoculum, (ii) lawn-based liquid inoculum, (iii) lawn-based pelletized inoculum, (iv) direct harvest of lawn culture with wheat flour, and (v) fomite transfer of a lawn culture. Inoculated wheat flour was equilibrated ~5 days to ~0.45 water activity and then was subjected to isothermal treatment (80°C) in aluminum test cells. Results indicated that inoculation method impacted repeatability, population stability, and inactivation kinetics (α = 0.05), regardless of laboratory. Salmonella inoculated with the broth-based liquid inoculum method and the fomite transfer of a lawn culture method exhibited instability during equilibration. Lawn-based cultures resulted in stable populations prior to thermal treatment; however, the method using direct harvest of lawn culture with wheat flour yielded different D-values across the laboratories (α = 0.05), which was attributed to larger potential impact of operator variability. The lawn-based liquid inoculum and the lawn-based pelletized inoculum methods yielded stable inoculation levels and repeatable D-values (~250 and ~285 s, respectively). Also, inoculation level (3 to 8 log CFU/g) did not affect D-values (using the lawn-based liquid inoculum method). Overall, the results demonstrate that inoculation methods significantly affect Salmonella population kinetics and subsequent interpretation of thermal inactivation data for low-moisture foods.

Food manufacturers are required to obtain scientific and technical evidence that a control measure or combination of control measures is capable of reducing a significant hazard to an acceptable level that does not pose a public health risk under normal conditions of distribution and storage. A validation study provides evidence that a control measure is capable of controlling the identified hazard under a worst‐case scenario for process and product parameters tested. It also defines the critical parameters that must be controlled, monitored, and verified during processing. This review document is intended as guidance for the food industry to support appropriate validation studies, and aims to limit methodological discrepancies in validation studies that can occur among food safety professionals, consultants, and third‐party laboratories. The document describes product and process factors that are essential when designing a validation study, and gives selection criteria for identifying an appropriate target pathogen or surrogate organism for a food product and process validation. Guidance is provided for approaches to evaluate available microbiological data for the target pathogen or surrogate organism in the product type of interest that can serve as part of the weight of evidence to support a validation study. The document intends to help food manufacturers, processors, and food safety professionals to better understand, plan, and perform validation studies by offering an overview of the choices and key technical elements of a validation plan, the necessary preparations including assembling the validation team and establishing prerequisite programs, and the elements of a validation report.

Some thermal processes, such as pistachio roasting, are not yet well characterized with respect to the impact of product and process variables on Salmonella lethality. This study aimed to quantify the effects of process temperature, humidity, and initial product water activity (a), on Salmonella lethality for in-shell pistachios. In-shell pistachios were inoculated with Salmonella Enteritidis PT 30 (∼8.5 log CFU/g), equilibrated (0.45 or 0.65 a), and heated without soaking ("dry") or after a pure-water or 27% NaCl brining pretreatment ("presoaked"). Inoculated pistachio samples (15 g) were heated in a laboratory-scale, moist-air convection oven at 104.4 or 118.3°C, humidities of ∼3, 15, or 30%, v/v (∼24.4, 54.4, or 69.4°C dew point), and air speed of 1.3 m/s. Salmonella survivors were quantified at six times during each treatment, targeting total reductions of ∼3 to 5 log. Survivor data were analyzed using analysis of variance to identify main effects (time, temperature, humidity, and initial a) and two-term interactions with time. As expected, lethality increased ( P < 0.05) with temperature and humidity. For example, the time to achieve a 4-log reduction decreased 50 to 80% when humidity increased from ∼3 to 30%. When the dry and presoaked treatments were analyzed separately, initial product a (0.45 versus 0.65 a or 0.75 versus 0.95 a) did not affect lethality ( P > 0.05). However, when comparing dry against presoaked treatments, the time to achieve a 4-log reduction decreased 55 to 85% ( P < 0.05) for presoaked pistachios subjected to the same temperature-humidity treatment. Salt had no effect ( P > 0.05) on lethality outcomes. These results, relative to initial a, process humidity, brining, and salt effects on process lethality, are critically important and must be considered in the design and validation of thermal processes for Salmonella reduction in pistachio processing.

The enhanced thermal resistance of Salmonella in low-moisture foods (LMFs) presents a challenge when validating pathogen control processes. Product water is recognized as a controlling factor in thermal inactivation of Salmonella in or on LMFs, such as almonds. Water activity (aw) describes the state of water in a product; however, aw is temperature dependent and characterized by hysteresis between sorption states. Moisture content (%MC) describes the amount of water in a product; it is not temperature dependent and might be a more convenient metric than aw to account for water in thermal inactivation processes. To test these two metrics independently, Salmonella-inoculated almonds were equilibrated to two %MC levels but the same aw and to two aw levels but the same %MC. Equilibrated products were vacuum packaged and thermally treated in a water bath at 80°C. Survivors were recovered and enumerated. The resulting inactivation curves were used to fit the log-linear inactivation model, and the inactivation kinetics were compared. D-values ranged from 15.7 to 18.0 min, and the root mean square error was 0.25 to 0.69 log CFU/g. No differentiated (P &gt; 0.05) effect attributable preferentially to aw or %MC was seen in the inactivation kinetics. The separate effects of aw and %MC on the inactivation kinetics of Salmonella in LMFs remain inconclusive, but analyses of data from prior studies strongly suggested an effect of sorption state. Further analysis is needed to identify which metric is best for modeling and validating thermal inactivation processes. HIGHLIGHTS

20Food safety risk assessments and large-scale epidemiological investigations have the potential 21 to provide better and new types of information when whole genome sequence (WGS) data are 22 effectively integrated. Today, the NCBI Pathogen Detection database WGS collections have 23 grown significantly through improvements in technology, coordination, and collaboration, 24 such as the GenomeTrakr and PulseNet networks. However, high-quality genomic data is not 25 often coupled with high-quality epidemiological or food chain metadata. We have created a 26 set of tools for cleaning, curation, integration, analysis and visualization of microbial genome 27 sequencing data. It has been tested using Salmonella enterica and Listeria monocytogenes 28 data sets provided by NCBI Pathogen Detection (160,000 sequenced isolates). 29GenomeGraphR presents foodborne pathogen WGS data and associated curated metadata in a 30 user-friendly interface that allows a user to query a variety of research questions such as, 31 transmission sources and dynamics, global reach, and persistence of genotypes associated 32 with contamination in the food supply and foodborne illness across time or space. The 33 application is freely available (https://fda-riskmodels.foodrisk.org/genomegraphr/). 34 35 Detection 52 (https://www.ncbi.nlm.nih.gov/pathogens) have grown significantly through improvements in 53 technology, coordination, and collaboration of the GenomeTrakr and PulseNet networks in 54 the US. Presently, the two networks sequence and release about 5,000 new bacterial genomes 55 per month [11,12] which makes their analysis and interpretation increasingly demanding. 56Our original goal was to explore what we, as risk assessors and epidemiologists, could learn 57 from the NCBI WGS data. We found, however, that despite extensive efforts by the 58